STUDIES ON KOJIC ACID BIOSYNTHESIS

A Thesis Submitted to
KANPUR UNIVERSITY
for the Degree of
DOCTOR OF PHILOSOPHY

in the
FACULTY OF SCIENCE

. +, By
PRATIMA BAJPAI

S7 °2-!J
4CC Ne ..... ................
~.S.J L - 1ty Librarr

s1ocHEMISTRY 01v1sto~ J un. •

NATIONAL SUGAR INSTITUTE KANPUR
1978

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* *"'1-~~**********************·'E-***-*W
* DEDICATED TO MY LATE FATHER *
* *******************************

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PAGE NO
CHAPTER

Certif icate
..
Acknowledgements 11

List of Abb r evi atio ns l II

Obj ect and Scope of the Pr es ent iV

I nv estig at i on
I INTROWCTION 1
II MATERIALS AND METHODS L,o
III ISOL4TION AND SELECTION OF THE CULTURE 7'-t
OPTIMI ZATION OF F~RMENTATI ON PARAMETERS gq
IV
V STUDIES ON FERMENTATION OF CANE MOLASS ES 117

KOJ IC AC ID PRODUCTION BY RESUSPENDED ISO

VI
FUt-r;AL MYCELIA
VII STUDIES ON THE I NHIBITION OF KOJIC 4CID 17~

PRODUCTION
VIII STUDIES ON SOME RELEVANT ENZYMES
SUMMARY

• •
 I

C E RT I F I C ~ T E

1his is to c ertify that Mrs. Pratima Bajpai , a

candidate for the degree of Doctor of Philosophy in Kannur

Univ e r sity , Kanpur has worked under my guidance f or more

than t wenty f our months , commencing from th e dat e of

r egistrati on and h as put in attendance mor e than two

hu ndred days in the departme nt • 1he r esults ~mbodi ed in

the thesis hav e not be e n submitt ed t o any oth er Univ ersit y

or I nstitut e f or th e awa r d of any deg r ee or diploma .

,...

Dat ed_\! :!_!_-'l~ Dr . L. VIS VANATHAN

Prof ess or and Head
Biochemistry Divis ion
~atio na l Sug ar Institute
Kanp ur .

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II

A C K N O WL E D G E M E N T S
-- - -------------
I wish to express my si nc ere g ratitude to:
- Prof. L. Viswanathan , for his continuous valuable guidance
and co nst ant encouragement thro ughout this investigation.
His clear deep insight into many problems e ncountered during
the period of r es earch has enabled me to ac complish my task
in th e s tipulat ed period of time .

- Dr. P .K. Agrawal for his consta nt help and valuable suqgest-
ions throughout the cours e of this work •

- Dr . K. A. Pr abhu for s everal discussions .

Dr . P .K. Bajpai and Purnima for th eir co ntinuous help in

organization •

- Council of Scient i f ic and Industri al Research (India) for

fi nancial assistanc e •

f\UTHOR

• ••
..

LIST OF ABBREVIATIONS

NAD Nicotinamide ade ~ine dinucleotide

NADP Nicotina mide adenine dinucleotide phosphate
ATP Adenosine triphosphate
ADP Adenosine diphosphate
FAD Flavin adenine dinucleotide
FMN Flavin mononucleotide
YES Yeast extract sucrose
SL Synthetic
AM Glucose salts
PDA Potato dextrose agar
CZ Czap ekdox

• •
•
iv

OBJECT AND SCOP E OF THE PRESENT I NV ESTIGATION

Several secondary metabol i tes of diverse chemical

nature are produces by different strains of the funaus
AspergilluS flavus . These compounds play no obvious role in
the economy of the organism . Whereas pr imary metabolism is
basically the same for all livi ng systems , secondary meta-
bolism is r estricted only to the lower forms of life and
is often strain sp ecific . Factors controlling the secondary
metabol ism of fungi are complex and not fully understood .
Koj ic acid is genera l ly considered as a seco ndary metabolite
of funga l fermentati on . The formation of secondary metabolites
by fungal cells have gathered a new momentum of interest
recently . Several reports have appeared in the last few years
regarding inv estigations on mycotoxins, nntibiotics and other
secondary mPtabolites , but the information available on kojic
acid is scanty at present and not much is known about the
mechanism of its b iosynthesis and significane .
Some studies have be en rep orted o n the mech anism of
kojic acid synthesis by d ifferent workers . However , these stu-
dies pertaining to the biosynthetic mecha nism of koji c acid
14
were mainly restricted to c incorporation studies and some
probable mechanisms were tentatively sugge sted . (i , 2 ) It was
therefore considered desirable to investigate the validity of •
proposed mechanisms on the basis of concrete experimental
evidence . These investigations included studies on the

• •
synth esis and degrad ation of kojic acid on diffe r e nt growth
media , the development of a simp l e r esus pe nded myce lial syst-
e m, inhibition stu dies with sp ecif ic inhi.bitors of key e nzy-
me s that may be involv ed i n ko jic acid synth esis e mploying
growing as we ll as r esuspend ed myc elia , r e l ev a nt e nzvmatic
studies at differ e nt periods of ti me during th e g r owth of
~pe rqillus fl avus and in r esuspe nd ed myc e lia und er diffe r e nt
conditions and chromatographic studi es to i dentify th e pos s-
ibl& int ermedi at es . Thus th e information obtained from th e
proposed studi es would h e l p us to understand some of th e
met abolic pathways op erative inf: . £1~ thus providing basic
information in this area of microbial bioch emistry.
Rec entl y a lot of emphasis is b e ing l a id on prope r
utilization of by pr odu cts of can e s ug ar industry viz. bag-
as se , molasses and pres s mud . Out of thes e , g r eat e r atte ntio n
has been pa i d t o mo la sses on account of larg e a mounts of
c arbohydr at es pr esent t h er e in and av a ilab l e for f er me ntation
by v arious microorganis ms. In India c ane molass es is m~inly
(3)
us ed for f e r me nt ativ e pro duct i on of ethyl alcohol . Howev er,
its exploitation f or production of industrially import a nt
pr oducts like f ood , pha rmac e utical and bake rs y east , citric
acid , lactic acid , vitamins a nd amino ac i ds e t c. h as bee n
ma r kedly hamp e r ed o n account of the de l et e rious ef fe cts of
•
vari ed ty pes of organic and inorg a nic impurities pre s e nt in
. . . (4)p l' . .
mo 1 asses on respec t 1v e microorganisms . r e 1m1 nary stud ies

• •
 \JI

J in our laboratory showed that this kojic acid producing

fungus , h• fl aY.!:!§. had a c ompa ritiv ely o r e at er t oleranc e to
the impurities pr es e nt in can e mola sses than th e citric acid
producing organism , h . ni9Jrr a nd th er efor e cane molasses could
be ea sily us ed for th e productio n of kojic acid wit h littl e
. . t.1a l c l ar1. f.ic a t i. on or pur1. f.ica t i. on(.5 1
or no 1n1 )t was f e lt th at

pretreatment of mola s ses and its supplement ation with y ea st

extract and so me s alts may further e nh anc e kojic ac id pro-
duction . Henc e p r es ent stu d i es were done us ing d i ffe r e n\
concentrations of uncl arif i ed molass es as we ll as mol asses
cl arified by di ffe r e nt chemic a l and physic a l methods in th e
absenc e and pr esence of y eas t extract and salts . It has a lso
been fou nd th at r esuspended myc eli a of ~lip_§rqillus flavuli
could produ c e l ar ge amou nts of kojic acid in a simp l e r esus-
pension medium comp ris ing of phosph ate buffer and sucros e or
g luc ose . Suc h r e susp e nsio n med ia not only economise on sugar
cons umpt ion towa rds fungal g rowth but also he l p in inv estigat-
ing some fundamental metabolic p athwa ys . It was th er efor e
t empting to study if a simil ar fung a l myc elium grown in
y east ext r act sucros e med ium could be successfu lly r esusp e nded
in a molasses med ium and still produc e koj ic acid and whet h er
addit i on of 1%y east e xtract and some sa lts t o th e molas ses
r esu s pension medium exe rt ed s timul atory eff ect on kojic acid
•
production . Hence , th e pres e nt inv estig a tio n wou ld also l ead
to the dev e lopment of a n economic a l l y viab l e process for th e

• •
 \)ii

manufacture of koj ic acid f r om cane molasses . Kojic ac id may

be put t o s evera l uses in i ndus try l i ke in t a nning industry ,
in the prep ar ation of met al ch e l ates , in r es in forma tio n, as
an analyt ic a l r eagent f or iron gold , zinc a nd vanaduim,
in th e preparation of de r ivat iv es for ma nuf acturing ins ect -
icide s , fung icid es , dentifrice , bronchiod il at ors , and anti-
b i otics , in the fie l d of synthetic dy es tuffs and drugs e spe-
c i a lly loc al ana esthetics p articul arl y of th e~ euc a in

. to 15)
type (6

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CHAPTER - L
I NTRODUCTION

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 l

CHAPTER I

INTRODUCTION

Koj ic acid (5- hydroxy-2- hydroxymethyl - 1 -pyrone)

is produced from carbohydrate sources in an aerobic process
by a v ariety of microorganisms . This was first · reported in
1907 by Saito, who isolated it as a crystalline substance .
1
from the mycelia of Aspergillus oryzae grown on steamed rice! )
He thought that it was identical with 0- resorcylcarboxylic
ac id . Shortly thereafter, Yabuta (1913) undertook an ext e nsive
inves tigation of the substance , gave it the name kojic acid
. (2,3)
and fi nally definitely established its constitution in 1924 .
It 1 s chemical synthesis from D- g lucose was ac hieved in l q3Q .( 4 )
Since then, a cons iderabl e amount of study has been devoted
to the biosynthesis of kojic acid and numerous publications
have dealt with its chemic a l and biological properties •

CHEMICAL PROPERTIES
Koj ic acid is a s ubstituted ( -pyrone (I)

HO,~)CH20H
I
Koj ic acid

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 2

1ne activities of the different centres of the

kojic acid molecule are influenced to a considerable ~xtent
by resonance between different forms of the molecule.

]he H!droxyl Groups:

1ne phenolic hydroxyl g roup at C? gives kojic acid
its weakly acidic character and enables it to form salt$
with a number of metals li~e Na, Ba, Ca, Sr, Co, Cu, Ni, Fe ,
(3,4,5,6) . . .
Mn, Cd, Pb, Zn, A1 , La etc. Many derivatives tYP. 1cal
of hydroxy compounds have been prepared from kojic acid. Some
of the known ethers and esters (II) have been listed 'in the
following table.
Table 1. Some kojic Acid Derivatives

Esters and ethers of kojic acid

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 3

r - --
R R' References

H Acetyl 4,7,8
H Benzoyl 8

H Cap royl 7
H Methyl 8

Allyl H 7
Benzoyl H 3,8,9
Acetyl Acetyl 3,4,10,11,12
Benzoyl Benzoy l 3,4,8,10,13
Methyl Acetyl 7
Methyl Benzoyl 8
Benzoyl Acety~ 8

The Unoccupi ed Nu clear Positions

Of the two unsubstitut ed positio~s 3 , 6 in the kojic
acid molecule , posit ion 6 is v ery reactive. Several 6-subst it
uted derivatives are known whereas only one 3-substituted
derivative has be e n prepared . Tilis may be ascribed to the
activating effect of the phenolic function which would be
particularly marked in alkaline media, where koj ic acid
propably exists in the form of a phenoxide ion (III). Ille
negative charge would be shared by C,6 where it would c reate

I
 5

The Heteroatom of the ~u cl~ :

The cleavage of many y
pyrones at the ring oxyg en
atom has been observed in mildly alkaline solutions. (l 4 )1ne
primary products of this r eaction are labile bishydroxy methy-
lene compounds. The initiation of th e basic cleavage at the
ring oxygen atom was furth er indicat ed by the easy formation
of pyridones, when kojic acid and sev eral of i ts deriv~t i ves
. ( 3 17 to 23 ) . . ( 18 , 19 , 22,
were condens ed with ammonia, ' prima~y amines,
.
dif unc t iona l primary
. .
amines ( 21, 22) an d g l yc1ne.
. ( 22)

Oxidation~ctions
Heyns and Vogelsang( 25 ) attempt ed to achieve the
oxidation of kojic acid catalytically. A current of air was
passed through solutions of kojic acid and its 5 methyl ether
0
at 60 C for several hours; the solutions were kept at a pH
close to 5, and a sp ecial platinum activat ed carbon cata l yst
was us ed. Only traces of comenic acid demonstrated by paper
chromatography, were obtained from kojic acid; the yie lds of
comenic acid methyl ether from kojic acid methyl ether we re
close t o 40%. Kojic acid is oxidized by Fehling 1 s solution and
3
by moist silver oxide~ ) Th ese r eactions are propably acco mp-
anied by degrad~tion~

Reduction Reaction:
Jne cataiytic hydrog enation of kojic acid has bee n
• d repea t e dly. Tra et t a- Mosca (lO) and
stu d ie w·iJ kman (ll) us ed
0

•
 6

palladium and platinum catalys ts and found th e uptake of 4 and

6 atoms of hydrogen per molecule r esp ective ly . Th e non- cat a lytic
r eduction of kojic acid has not been r eported , but 6- b enzoylkoji
acid and 6 acetyl koj ic acid hav e been r educed to 6- be nzyl kojic
ac1. d and 6 - eth y 1 kOJ. 1c · 1y .( 26 , 27 )
. ac1· d r espec t 1ve

PHYSICAL PROPERTIES

Kojic acid crystallizes in th e f orm of colourless ,

2
prismatic needles . ( ,lO) Data on its crystal structure derived
from X- ray investigations were provided by Fox and by _~~kinstry
28 29
_g1 _a!. ( • ) It is r ead ily soluble in wat e r , ethanol, ac etone

and is sparingly soluble in eth er , ethyl acetat e , chlorofo~m

3 17
and pyridine and difficulty s oluble in most othe r liquids.( ' )
It has been purified by r ecrys tallizat ion from ac etone (l 7 ),
3
ethanol- eth er( o )and methanol ethyl ac etat e and also by ·sub1i-
mation under diminished pressure at 150 ° to 200°c . (4 , ll, 3 l)
(30 , 3
The melting point has been r eport ed variously a~ +51 to 152 ,
2 4 12
152°. ( ' ' ) 152 . 6 ( 33 ) and 154°C. (lO,ll) The U . V. absorption
sp ectra of koj ic acid and its 5- 7 d iac etate derivativ e exhibit
charact eristic absorption maximum at 315 nm and 255 nm r esp ect-
. l (34)
1ve y •
Pap e r chr~matog raphic data have been r epo rt ed by
Heyns and Vogelsang ! 25 ) Kojic acid had an Rf of 0 .80 in
butanol (saturat ed with wat er) , glacial ac etic acid:

•
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wat er(lB:2:5).

BIOLCG ICAL PROP ERTIES

Antibiotic Activ ity t

2
Yabuta ( ) no t ed th at b act erial growth genera l~y
stopped i n t he pr es ence of mor e t h an 0 .5% of koj i c acid, but
interest in tts antibact erial activity was only r enewed quite
incidentally, when th e disc ov ery of pe nicillin l ed to an i nt e n-
sified search for new antibiot i c substances among mold met a-
bolites. The promisingly high activity of some mo ld cult ur e
media against various bacteria was found t o be due whol l y to
the relatively high conc entration of kojic acid produc ed by
(36 to 40)
t h ese molds, but the inhib i tory power of pure koj ic
acid and some of its derivativ es p roved to be disappointingly
2
low. (l , 38 •41 to 46 ) The pot ency of kojic acid was unaf f ected
by t he number of bact eria pres ent or by i ncub ation i n 50%
s erum at 37°C. Foster and Karow( 47 ) also not ed a slight
inhib ition of pu r e cultures of var ious b acteria by kojic acid.
They obs erv ed t hat gram neg at iv e b acte ri~ we r e mor e s ensitiv e
to sodium koj at e than gram positive ones. With most oth er
(48)
antibiotic s ubstanc e th e r ev ers e is tru e . Kavanagh comp ared
the activity of kojic acid with those of s ev eral of the we ll
known potent antib i otic s ub stanc es. Kojic acid showed by fa r
the lowest activity in tJ:le whole g roup ag ainst s even out of
nine species of bacteria. tee· and Coworkers ( 39 ) found th at
 8

r kojic acid is active agair,st ~uman tuberc le bacilli in vitro

under a variety of cond itions. Complete inhibition of a surface
growth of the ba cilli being caused by 45 mg of koj ic acid per
10 0 ml of liquid medium . However, many othe r act ive substanc es
produce similar effect at much lower concentration. The effect
of koj ic acid at s uch inhibitory l evels is bacteriostatic and
not bact ericidal.
Koj ic acid is not active against virus es . It was
tested, with negative r esults , against one strain of polymyelit
virus and one strain of St. Louis encepha litis virus in mice( 49
and also against sixty races of bacteripphaaes.( 5o)Klein and
Olsen( 5 l) studied the action of kojic ac id on the enzymatic
oxidation of amino acids by rat liver and kidney tissue slices
Low concentrati on of koj ic ac id i nhibited th e in Y,itro oxidatia
of a nurrber of D-amino ac ids, L-phenyl alanine and few r el at ed
compounds, Kojic acid was found to compete with D- amino acid
. (40}
oxidase for its substrate.Bu u Hoi and Rats1mamanga found
that kojic acid pr ot ect ed the adrenal ascorbic acid in test
animals during the reversible period of scurvy with out it-self
showing Vitamin C activity .
(52)
Although McGowa n and Coworkers observed only
a weak activ ity of kojic acid against three sp ecies of fungi,
(53)
Kane and Morey recommended the us e of its complexes with
bivalent heavy metals against several fungal dis e ases of plant!!
Thay found copper, mercury cadmium, lead and tin complexes to~
 9

supe rior to the wel l known Bordeaux mixture in the treatment of

·~
early blight (~lt erania solani) . Thes e c omplexes ar e eas ily
s us pe nded in liquids and thes e spr ays adhere firmly to th e
foliag e and do not inj ure the p lants . Si nc e the metal radicals
are bound to kojic acid in a nonionizable form , thes e complexes
are also much l ess toxic to huma n beings and s af e r to h and l e
than Bo rd eaux mixture its e lf . Thus th e ac tivity of koj ic acid
aga inst c ert ai n fungi may eventu al ly find some pr actic al
application .
Mayer and Cowork ers( 54 ) discov e red that kojic acid is
a moderately effectiv e activator for nicotine ins ecticid es When
treat ed agai nst me lonworm (Diaph ania hy~i nata L. ) and sourth ern
army worm (Prodenia eridan ia cram.) , koj ic acid alo ne was not
toxic, but th e t ox icity of a 5% nicotine sulf at e- pyrophyll it e
dust was increased 35% and th at of a 5% nicotine- bentonite
s pray by so me 50% ,by the addition of 5% of kojic acid . These
workers found kojic acid to be h a rml ess to plants , but others
56
r eported it to b e slightly toxic . ( 55 ) Bastin ( ) inv estigated
the ef fect of kojic acid on th e growth of r oo t cuttings . Further
data on the a ntibioti c activity of kojic acid a r e r eport ed
. ( 57 ) (58 )
in pap ers by Verona and Agelli , Meye r and Coworkers
and No rto n. ( 59 )

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' Toxicity
f indication of the toxicity of koj ic acid to
An
6
mammals was given by Fri edema nn ( o) who obs e:rv ed a definite
r esponse in dogs aft e r i nt e r avenous inj ection of 0 . 15 g of
s odium kojat e per kg of body weight and found th e l ethal dose
to b e about lg per kg of body weight . Pra ctical l y the sa me
symptoms were shown by r abbits and rats . For mic e , t oxicity
of th e sa me orde r was r eport ed by Mort an and Cowork ers(l 2 )
3
and by J ennings and Williams( s ). The l ate r worke rs also
obs erved that human l eucocyt es are killed with i n three hours
by a 1% solution of sodium kojat e at pH 6 . 8 . Twe lv e day chick
e mbryos were also susc eptibl e to ko jic acid, the observ ed LD1oo
being 12 ~ per 100 g of egg weight {_39 ) Mode r ate cardiotoxic
. t onic
an d car d 10 . ac t ivi . . aci.d ar e a 1so r epo rt e d(. 61 •6 ~
· · t i· es o f k OJ1C
1hough the t oxicity was not very great in al l these instar.~ es,
yet i t wa s enough t o discou r age ch emoth erap eut ic inv estigations
and t o c ont r aindicat e the med icina l use of kojic ac id .

MICROBIAL FORMATION OF KOJ IC ACID

.!59..iic Acid F~ entation

Severa l mol ds of the genus Asp erg illus hav e bee n
found to possess th e ability to produce kojic acid from
suitable nu tri ent s olutions . Yabuta( 2 ) i so l ated ko jic acid
from t he nut ri ent solutions of h. a l bus, h . c a ndidus and

•
 11

h.
f nidulan s . Traetta Mosca(lO)claimea to have found it in
63
cultures of h. glaucus. Tamiya and Hida( )reported its
product ion byh. flfilUd.§ , h. gymnosardae, h. awamori, h. ~lavat~
h. fumigatus and h. giqante~ . Birkinshaw and Coworkers( 3o)
showe9 that it i s als o formed by h. par2._siticu~, h, eff!:!fild.§.
and h.
tamarii . Later on , h. l ut_ggvires~~l~) h. lutesc e nf;6)
3
h• went ii ( Jr}d h• alliaceu5'37) wer e added to the list of koj i c
acid produc ers . Most members of the h. flavus - ory ~ i.21D.ru;:ii
group were appa~ently capable of producing kojic acid and
,A. oryzae an~ h.
flav!:lli we re pos sibly most widely used for
this purpose. Oh ara( 64 ) used the produ ction of kojic acid as an
aid in the identificat~on and classification of 111 strains
of the ,A. tamarii-oryzae group . In addition, the formation of
kojic acid was also observed i n the cultures of Penicilluim
~~ ( 30 ), te n s peci es of acetic acid bac il 1 i. , (65 ) and the
66
glu conic acid fermenters Gluc ono bacter opacus var mobilis( )
(67)
and Gluc onobacter !:.Q§_gll§. •

Rice and other c ere a l s were th e prime carbon sources

in the early studies , but soon they were r eplaced by solutions
(1 , 2)
of pur e compounds. Microorganisms wer e grown in med i a
containing a great variety of carbohydrates and related
substrr nces and they produced kojic acid from compounds c ont -
aining two to sev en carbon atmos per molecule . Among them
r educing s ugars , sugar acids , sugar alcohols , di a nd polysa-
ccharides wer e su itable substrates • Table 2 lists a number
of such substrates.

'
 12

t Table 2. Carbon Sourc es f or th e Biosynth esis of Kojic Acid

-------
Number of carbon Compound Ref e r enc es
at oms
2 Ethanol 31,64,66
Glycine 68
Sodium ac etate 68
3 1,3 dihydroxy 2-pro-
pa none
Glyc e raldehyde 71
Glyc erol 11,13,17,30 ,66 ,70 ,7
73,74
Sodium lactate 66
Sodium pyruvate 66,68
4 Tartaric acid 73
5 Ribitol 74
Arabinos e 30,31,64,71,73,75
Xylose 30,31,64,71,73,74,1
6 2 Deoxy glucose 31,71,73,74_

+ Fructose 10,13,30,64,65
66,67,71,73,74
Galact ose 30,64,6 6 ,71,74
Gluconic acid 71
Gluconolactone 74
Glucose 3,10 ,11,13, ~0 , 33 ,6
66,69,71,72 ,73 ,74,
75,77,78
 13

.
co ntinu ed Tab l e 2/
t
Inosito l 71,73
Mann i tol 11, 30 , 64,65 , 73
74,79
Ma nnos e 64 ,71 ,73 ,74
Sorbit ol 71,74
Sorbos e 80
7 Ouini c acid 72
Shikimic acid 72
12 Lactobionic ac id 72
Lactose 30 , 64
Maltos e 38 ,64 ,71
Sucros e 10 ,ll ,13 , 30 , 32 ,64 ,
71 ,74 ,79 , 81
Tr eh alos e 72
18 Raff inos e 64
6n Dext r in 64
I nulin 64,71
+ Pecti n 72
St arc h 30 ,64
 14

The concentration of carbon sources varying from

5 to 30% have been used but the best r es ults have been
obtained with 20% g l ucos e concentration. Media of different
compositions have been used for kojic acid produ ct io n, s uch
79
as med ium K~ ) Czap ekdox medium, yeast extract sucrose
medium( 35 ) and th e med ium us ed by KatAgiri a nd Kitahari (?l)
It was shown th at th e yeast e xtract sucrose medium supported
bett e r growth of ,h. flavus than a glucose salts mediumi 82 )
Stationary cultures proeuc ed higher yi elds of kojic acid
than shake fl ask cultures . Inorganic salts lik e KCl, NH4N03 ,
M;JS04 7H20 have been us ed in different media. Peoptone,
yeast- extract and coba lt amines hav e bee n found to be more
. f ac t ory as n1. t rogeno us sourc es . ( 83 ' 84 ) Recen t 1y Sh oh e1.
sa t is
(85)
and Coworkers h ave r epo rt ed th at 50% of this complex
org anic nitrogen s ource i. e . y east extract, can be subst-
itut ed by ur ea or ammonium nitrat e without sacrificing kojic
acid yields.
The pH r ang e of 2 t o 5 and the temperature rang e
of 30° to 350c have bee n fo und to be optimum for koj ic acid
f ermentation. 1he f erme ntation generally r equires 9 to 20
days for completion, depending upon the tyP e of substrate,
pH, temp e rature~ and other factors. After th e complete
exhaustion of sugar in the medium, koj ic acid has bee n
r eported to be metaboliz ed further by the mold r esulting in
a decrease in its conc e ntration.
 15

A number of su~stan ces have been found to promo-

te kojic acid production. In a survey of forty organic
compounds, it was found that ethylene chlorohydrin (l g/1)
produced a mark ed increase in koji c acid yield , in a period
of 10 days . (86 ) Besid es this methyl alcohol , ethyl alcohol ,
propyl alcohal , isopropyl alcohal and p almitic ac id were
found to be stimulants for ko jic acid pr oduction. (87 t o so )
Phosphate as KH2P04 was also found to be necessary for both
g rowth and koj ic acid pr oduction(?O) High phosphate l evels
r es ult ed in rapid kojic acid synthes is and deg r adation while
at lower phosphate leveJ s both the formation and degradation
of kojic acid were slow. ( 9 l) Sodium fluoride , iodoacetic .
ac id, ars enate , malonate , potassiu m cyanide, sodium azide ,
pentachlorophenol , dinitrophe nol and sodium thiosulfate
inhibited kojic acid production . Though inhibition by flu-
oride and iodoacetic acid was reversed whe n an org anic acid
like citrate , pyrtNat e or succinate was added to the medium ,
(92)
th e inhibition by arsenate could not be revers ed .

Iso l ation of Ko j i c Acid:

After comp l etion of fermentation , kojic acid is
r ecov er ed from the broth by one of the follo wing methods :-
Precipitation as copper salt, ( 2 , 3 i ) ext ract ion with ethyl
acetate , (l7 ) continuous extraction with ether , ( 33 , 6 9)
2
evaporation to small volume leading to crystallization( o )
 19

oxidation to a ke t o ni c intermed iat e which is the n dehy-

drated to g iv e kojic acid . 1his was frequently called the
carving out theory of th e format i o n of kojic acid

D glucose Keto nic intermedi ate Ko j ic aci.d

Oppo s ed to it was the fission theory , which cou l d

be dated from the first convers i on of pe ntoses to kojic
. (13 , 7 1,75 ) (75)
acid . Challenger and Coworkers had hoped to
obtai n pyromeconic acid f rom pe ntoses with h- Oryz ae , by
analogy wi t h the formation of koj ic -acid from hexoses .

- H'2. ).

Pentose Pyr omeco nic acid

 20

Inst ead , ko jic acid was produced fr om L. arabi-

r • . . ( 13 ) t oo
. • an d Gr egor 1n11
nos e an d D• Xy 1 ose, Cor b e 11 1n1
observed the pr oduction of ko jic acid by ,h. fl aY.1J~ from
t he se two p ent os es and from D- fructose . They not ed that
Haworth ' s sch e me would r equir e· the formation of an is omer
of koj ic acid f r om D- fructose .

HO~ / H
H, / ~/ H
I

;
H~9 )?t
Ui,;P "-. H
-\\ 'l
- 'ZJ\"l0
~

D- rhMc..toJ.ie

It was th er ef ore suggest ed th at kojic acid was

f ormed dir ectly by th e conde nsatio n of two triJs e molec u-
l es wi thout prior f ormatio n of a hexose . Glycer aldehy de and
g lyceric dialdehyde , which co~ld be produ ced from sug ars
by fission and from g lyc e r ol by oxidation were assumed t o
....J-
b e th e triose precurso rs .
0

HO~H HD
f-l-C + ).
II
0

\
 22

sourc e they have, to a r es erve carbohyd r ate and lat er hydro-

lyze it to a h exose which is th en conv erted to kojic acid.
. a (73 ) r each ed a s1m1
Tam1y . ·1 ar cone 1usion and consi-

dering the cus t omary long cu ltur i ng periods, i t seemed

entire ly r e asonab l e . Th e ir vi ew was shared l at er by Kluyv er
. (78)
and Perquin when these wo rkers found that, with short
incubation periods in s pec i al r eplac ement cult ures, h• i~~

produced practically no koj ic acid in media cont aining .

pentoses, sugar alcohals or hexoses other than glucose . Under
identical c onditions, pr o~~ction of koj ic acid from glucose is
at optimum l evels. Negative r esults obta ined with other
car bon sourc es sugg est ed that only glucose i s c onverted
directly to kojic acid and that all oth er compounds are first
assimilated in t o a res erve carbohydrate.
Trios es, particularly dihydroxyac etone were again
consider ed t o be one of th e most important intermediates
(72}
in th e formation of kojic acid , when Katagi ri and Kit ahari
who worked with h• o ~ , r eported a 55% yie ld of kojic
acid from di.hydroxy ac etone , traces from g lyc eraldehyde but
none from acet aldehyd e or diethyl acetal. Gould(74 ) found
that the production of kojic acid f r om glucose by h. tamarii
is not affect ed by the pre senc e of aldehyde trapping reag ents
like dimedone or bisulphite in the growth medium arP that
no kojic acid is produc ed by the fungus if mold subst a nc e
i. e. r es erv e carbohydrat e is the only sourc e of c arbon .
 23

f Sev e ral contradict ory theories were thus pr esent ed

in some papers; however it may be stress ed th at the extreme
sensitivity of this microbiological pr ocess to seemingly
minor change s i n fermentation c ond i tio ns should be kept in mind
Gould furt her observed that the formation of kojic acid
probably did not pro ceed t hrough the phosphoric esters , as
it t ook place readily in the phosph ate free media cont ain-
ing g lucose , xylo se or g l yce r ol .
Sakagu chi and Coworkers(66 ) suceeded in produc-
ing kojic acid from ethanol by means of h. oryzae and t hereby
provided the first experime nt a l evidenc e supporting the
diose intermed iate theory . Bar nard and Challenger( 3 l) devo-
ted considerable attention to this problem. 1hey obt ained
0
12 to 17% yie lds of koj ic acid at 32 C from a c ulture of
,h. or yzae grown on basal salts medium containing 1 . 3 t o 2 . 1%
of ethanol , but none at lower temperatures or higher ethano l
conc entratio ns . 1he carefully washed fungus was g r own on
th e basal s alt s medium alo ne for six weeks , and its failure
t o produce koj ic acid e liminat ed the possib il ity of kojic
ac id pr odu ction from r eserve car bohydrate material i n the
exp eriments with eth anol . All fungal cultures g r own o n
ethanol so lutions , were shown to contain ac eta~dehyde and
the add ition of dimedon , but no t of bisulphite , r educ ed
the yield of koj ic ac i d to 5% and delayed it s formations .
Th ey could get further evidence for ' fission theory' and
 24

ag ainst ' c a:rving out ' th e ory as r ev eal ed by the fact that
three deriv at iv es of g lucose which we r e not expect ed to
undergo fis s ion gave no-f - pyrone derivatives and th at a
20% yield of kojic acid was obtained from 2- deoxy- glucose
which by the ca:rving out process should have given r i s e to

- '2.. 1-\'2..D
> ~
l / Cl-t,_DH
i H~~lh-ox~ rne.rnt 1
?"Q/1.-0ne
2 hyd roxymethyl - y - pyrone . Clearly th e accumul ated infor-
mation r evealed many details , but the e xact nature of the
biosynthetic process r emained a matt er of conj ecture .
Arnst ein and Bentley(l0 6 ) applied the isotopic
trac.er t echniqu.e in their studi es on kojic acid biosyneth-
sis . As the first s tep , they inv estigat ed th e me chanism of
alkaline Cleav age and deg radation of di- 0-methyl kojic
ucid (;0 6 ) Yabuta ( 3 ) had f ound that treatment of di- 0- met hyl
kojic acid with barium hydroxide yie lds equimolar quanti-
ties of fo rmic acid , methoxyac etone and methoxyac etic aci d .
The s ame three compounds we r e obta ined from di- 0- me t hyl
14
kojic acid lab e ll ed with c in one of th e methyl group s .
1he formic acid was comple t e ly non- radioactive and all th e
 25

r ad ioactivity was distributed bet ween th e other t wo frag -

me nts, the share of methoxyac etic ac id being about 20%
high er than th at of methoxyac et one ( ! 06 ) These r e sults
indic ated that the c l eav age occurred in t wo diffe r e nt
ways.

Sche:rnc. A

0
/°'
CH9-di2 CH.? C..H~O - ,
Me.tr'tll)(..d~Q~h~\ _ _ - - H - - - ~ - > HC'OC-CH,z.OCH3
HCOOfl < 7 l1-12 0CJ\c, lv\e.\r, ox~ <l.l.e.t.c. o.C:,d.
~0"""~1.c a.c ~cl O
 26

According to scheme A, C2 and C3 give methoxy-

acetic acid and C4 C5 and C6 g iv e methoxyacetone. Accord-
ing to scheme B, C5 and C6 appear in methoxyacet i c acid
and C2 , C3 and C4 in methoxyaceto ne . TI1e great er radio-
activity of methoxy acetic acid po int ed to a sli~ht predo-
minance of s cheme B. For mic acid was fo rmed in al l cases.
Furth er Arnst ein a nd Bentley( 69 ) produced kojic
acid with h.oryzae and ,h. flay!d§.::oryzac grown on media
14
containing D- g lucose l ab ell ed with c on Cl, D-glucose
l ab ell ed on C3 and C4 and dihydroxyacetone labelled on C2 .
14
Be -tween 2 and 20% of C was r ecover ed in kojic ac id. Kojic
ac id was conv ert ed to s ame et hyl methyl ether in each case
and degradation of t his unsymmetrical eth er by hot aqueous
barium hydroxide followed by the separation of the frag-
ments , and their assay for radioactivity gave a n exact
measure of the amou nt of 14 c inc orpor at ed into each of th e
six c arbo n atoms of kojic acid . TI1e schemes for th is degra-
dation is given in the next page . . .
 Sc hene J'.

Sch ene B

Et

of k oj ic ocid
/
 28

After treatment of ethyl methyl ether of kojic

acid with hot aqueous barium hydr oxide the liquor conta-
ined formic , methoxyac etic and ethoxyacet ic acids , methoxy-
ac etone and eth9xy acetone , produced by the t wo modes of
cleavage A and B. Fo r mi c acid was conv e rt ed with r ed mer-
curie oxide to carbo ndi oxide ; this was r ecovered as barium
c arbo nate , the radioactivity of wh ich was a measu r e of
14
the c inc orporated into Cl of koj ic acid in th e b iosyn-
thetic process . 1he alkoxyac etones we r e r emoved from the
liquor by steam distillation and c onve rt ed t? i odoform and
a mixture of ethoxy and methoxy ac etic ac ids . Th e i odoform
was r ecover ed by filtrati on , its rad ioactivity indicated
t he proportion of 14 c incorporat ed into C4 . 1he a lkoxy
acetic acids we re isolated by continuous extract i on of the '

f ilt er ate wi th ether , a nd th9se i n the o rio ina l liquor

we re r emov ed in the same way . These acids we r e separat ed
chromatog raphic ally and c onverted to th e ir silv er salts .
Methoxyac etic acid from both the sourc es co ntai ned C5 and
C6 of ko jic acid , wh il e the whole of the ethoxy acetic ac id
.was c ompo s ed of C2 and C3 of koj ic acid . As say of the
radioact ivity contained i n the s i lver salts gav e the amount
14
of C incorporat ed in C2 + C3 and in C5 + C6 of kojic ac id
r espec tive ly. Deg r adat ion of the silver salts with br omine
libe rat ed c arbo n di oxide containing C3 from ethoxyac et ic,
and carbondioxide c ont a ining C5 from methoxy ac etic acid .
 29

TI-ie s e porti ons of co2 we r e also r ecov e red and assayed in

14
t he fo r m of barium carbo nate . Th e amo unt of c which
was inc or porat ed in to C2 and C6 was finally c a lculated
by diff er enc e .
Th e r esults of t hes e experiments showed th at in
14
the cas e of kojic acid produc ed from D- g lucos e- 1- c,
70 to 90% of the 14c was locat ed in Cl and 6 to 16% in C6,
14
in the cas e of kojic acid produc ed from D-g luc os e-3, 4- c2
14
90% of the c was loc at ed i n C3 and C4; and in th ~ c as e
14
of kojic a cid produc ed from dihydr oxyac eton~- 2- c, 60 to
70% was to found in C2 and C5 of koj ic aci d. Thes e distri-
butions of r ad i oactivity v e ry stro ng ly indicat e th at kojic
acid is f or med from D- g l uc os e , l arge ly by direct conve rsion
in which no s p litting of c ar b on ch a in takes pl ac e . The
14
dist r ibution of c in t he kojic acid pr oduc ed fro m l abe lled
dihydroxyacet one , mor eov er , showed cl early tha t if f r ee
dihydroxyac et one or dihy dr oxyac etone phosph at e were an
14
i mport ant inter medi ate , t h e conv ers i on of D- glucos e- 1- C
14
wou 1d h ave 1 ed to a more e xtens iv· e incorpor
· a t·ion of c
in t o C2 to C6 of kojic ac i d . The pr es enc e of 6 to 16%
14
of the c in C6 nev ert hel e ss point ed to a minor pathway
in th e f ormat i on of kojic acid , which involv es th e spli-
tting of t he g l ucos e mol ecu l e and th e r eco mbinat ion of
trios es thus formed .
 30

lhe abov e r esult s were a l so c o nfirmed by Kit ada

107
and Fukimbara.5 ) lhey inv ~stig a t ed the conv e rsion of
glu cos e to kojic acid , by h • Q r ~ in a subme rg ed cult-
14 14 3 3
ure , using 1- C, 6- C, 3- H, and 5- H g luc ose as s ubs t-
r ates and examined the distributio n of r ad io activ ity in
koji c _acid formed. Between 80 and 90% of t ota l r ad io act-
ivit y, cont a ined in th e isol at ed kojic ac id , was found in
c arbon atoms corres ponding to t hose l abell ed in g l uc ose
3 3
mol ecul e . When 3- H g l ucos e and 5- H g l ucose we r e us ed
3
as su~strat es , t he incorpor ation of H in t o koj ic acid
was 3.5% and 1 . 35% r es pectiv ely. Th es e r esults indi cate
that major pathway of koj ic acid formation is th e direct
conversion of g lucos e without any s pl itti ng of ca rbon chai n.
>- '
In t he ir s ubseque nt studi es , Arnst ein and
Bentley ( 9 i) demo nstrat ed the presence of a l dolase a nd
triose phosphat e isomeras e i n fungi , producing kojic acid
They ~lso found th at both production a nd destruction of
kojic acid were r apid i n med i a cont a ining high phosphate
l evels a nd slow at lowe r phosphat e l eve ls . lhey pref err ed
t o consider koji c acid as a normal met abolite of fu ngi
r ather than as an e nd product .
6
Lastly Arnst e in and Bentley ( S ) inv estivat ed the
i nc orpo r ation of small mo l ecul es in to koj ic ac id . Buff-
ered s olut io ns of pyruvic acid , ac eti c acid , glycine and
14
s ome other r e l ~ted compou nds , al l l abelled with c were
 31

added to As.....12erg illus cu lt ur es grown initially on med ium

cont aining unlabell ed g l ucose~ The recovery of 14c 1n
. k ..
OJ1C
acid was usually l ess than 1% and labelled c a rbon atoms
were distribut ed ove r the who l e mo l ecu l e pr edominating
however , in all c ases in C4, C5 and C6. A si mi l ar predomi-
nance of r ad i oac tivity in the lower half of kojic acid mole -
cul e was observed during stud i es with l abe ll ed dihydroxy
. t h e presence of nonradioactive
ac etone 1n . .
g l ucose (69)
•
These observations might be e xplained in both instanc es
by the condens ati on of a labell ed th r ee c arbon inte r rned-
i ate e . g . pyruvic acid or dihydroxy acetone , with a n un-
(68)
l abe lled triose or triose phosphate de riv ed from glucose .
In neither case wer e major quantities of g luc os e converted
to k~jic acid by this pathway. Desp it e the above explanat -
ions , the exact mechanism of kojic ac id biosynthesis is
still not known . Hence it would be a v e ry inter e st ing prob-
lem to inv estigat e .
 32

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Ch ern., JJ , 1 223 (1969) .
TCL.tS4.:m 1 C ·,
102 . Irnose. J . and No nornura s . A Agr . Bio l . Chern., ~
1443 (1970 ) .

103 . Kit ada M. ' Hakko Kagaku Zassh i. , 12 , 343 (1971) !

104 . Kitada M. ' Hakko Kagaku Zassh i. , 46 437 (1968),

105 . Kit ada M. ' Hak~o Kagaku Zasshi. , ~{! 411 (1970) .
106 . Arnst ein H. R. v. , and Bentley R. , r. Ch em. Soc . ,

3436 (1951) .
107 . Ki t ada M. and Fu kirnbara .T. ,Hakko Kag aku Zas sh i. , 49 ,

847 (1971 ) ~
 ~HAPTE!L::_II
M~TERI ALS \ND METHODS
 40

CHAPTER - II

MATERI ALS AND METHODS

Org anis.m...Jls ed
The org anism us ed in t he present inves t igation
was a nontox i genic strain of Aspe.niillus fl avus i s o lated in
our laboratory fr~m at mos phe ric dust and maintained by
mo nthl y transfers on potato dext r os e ag ar sl a nts.

Iso l ation and maint enance of the organism

Petri dishes containing st e rile potato dext r os e
ag ar medi um supp l emented with 0.5 ml of 0 . 2% bromocres ol
g r e en we re us ed for the i s ol ation of t he strains . The p etri
d ish es were opened and exposed to t he at mos phere for a few
hours and th en clos ed and incubat ed aseptically at r oom temp-
er at ur e for a few days . In t h is way fungal strains capable
of producing acid wer e obta i ned f r om the atmos ph e ric dust .
For the i so l at i on of c ult ur es f r om s oil, lg r epresentative
s oi l s ampl es we re s us pend ed i n s t er i l e water and 0 .5 ml
al i quots of the appropriately diluted suspension we re uni-
form ly spre ad over surf ace of the potato dextros e agar
medium contained in p etri dishes. The soil s uspension was
dil ut ed with sterile wat er i n such a way t hat about 5 to 10
colonies we re obtained per p lat e o n incubat ion at r oo m
temper ature .
 41

t Cultures producing large amounts of acid were

identified by the large size of colour zone around col J ny
formed by the change in indicator's colour from green to
yellow. These cultures were t r ansferred to potato dextrose

•
aga r ·slants and were maintained by monthly transf ers . At
suitable time inteivals , th e ir acid producing ability was
checked again by plating on the above medium containing
bromoc res~l green . For isolation of kojic acid producing
organisms , the cultures were pl at ed on pot~to dextros e agar
medium containing 0 . 1% FeC1 3 solution in O. l N HCl . Strains
y i elding l arge amounts of ko jic acid were maintained and
us ed for s ubs equent studies •

~ultural Conditions
Growth media
Al l medi a were st erilis ed by autoc laving at
15 p . s . i . g for 15 minut es . The following media were used .

I Potato d extros e agar (P. D. A. ) Med ium

Constituents Concentration
(g/1)
Glucose 20
Potato extract 100 ml/1
( extract of 20 g
potato in 100 ml)
~ ar Agar
 42

The potato extract was ·prepared by boiling 20 gms

of washed sliced potatoes in 100 ml of distilled wate r f or
half an hour . The extract was filtered and other ingredients
of the medium we r e added to it.
(1)
II Yeast Extract Sucrose (YES ) Medium (Davis et al . 1966)
Co nstitu ents Conc e ntration
(g/1)
Sucrose 200
Yeast extract 20
pH of the medium was adjusted to 6 . 5
2
III Synthetic (SL) Medium (Reddy· et al• l97 1)( )
Co nstitu ents Conc e ntr)t ion
(g/1
Sucrose 85
As parag ine 10
(NH4 )2S04 3 . 50
KH2 P04 0 .750
~so4 . 7820 0 .350
CaC1 2 . 21120 0 .075
ZnS04 .7H20 0 .010
MnC1 2 . 4820 o.oo~
FeS04 .7H2 0 0 . 002
(NH4 )6 M0.,024 .. 4H 0 0 .002
2
H3 so3 0 ,002
pH of the medium was adju s ted to 6 . 5
 43

IV Glucose Salts (AM) Medium (Adye and Mateles 1964)( 3 )

Constitue nts Conc e ntratio.n
(g/ 1)
Glucose 50
( NH4 \so4 3
1<82P04 10
Mgso4 • 7H2 0 0 . 20
(NH4)6~024• H20 0 . 50
CuS0 - 5H2 0 0 . 0003
4
MnS04 !~0 0 . 00011
ZnS04 .7H20 0 . 0176
Fe2 (so4 ) 3 . 6H 0 , 0 . 010
2
Na2 B4 0., 0 .0007
pH of the medium was adjusted to 6 . 5 .

(4)
V Czapekdox (CZ) Medium (Thom and Church 1930)
Constituents Concentration
(g/1)
Glucose 50
NaN~ 2
KH2P04 l
KCl 0 .50
WgS04 .7~0 0 . 50
FeS0 • 7820 0 .01
4
pH of the medium was adjust ed to 6 . 5
·1'
 44

Fermentation Conditions I

Most of the studies we re ca r ried with stationary

cultur es . Diff erent fermentation parameters viz g rowth
media , pH, temperature , s pore inoculum size , carbo n and
nitrogen s our ces , substrate c onc entration and other growth
facto r s were standard ized by va r ying different parameters .
Subsequent studies were do ne employing the optimum fer-
mentation ~onditions thus determined . According ly a pH
value of 6 . 5 , a temperature of 30°C and a spore inoculum
size of 5x 106 spores per 50 ml medium were used . For
further studies , in general YES medium cont ai ning 20%
sucrose and 2% yeast extract was used exc ept in resus -

,'
pension studies . In case of static cultures , inord er to
prov ide suff i cient aeration to the mycelial mats, the
liquid volume i n fermentation flasks was r estricted to
1/5 of the t ota l volume . In the case of shake flask stud i es ,
t he culture flasks we re incubated on a rotary shaker at a
speed of 150 r . p . m.

§rowth_~nd Res uspension of the Fbngus

Preparation of the inoculum
.b flavus spores from the stock culture tube ,
we re first g r own as eptically on a potato dextros e agar
slant for four days at 30°C ,Spores fr om this slant were
suspended in 5 ml sterile distilled wat er and used to
 46

this perood. After growing the cultures i n YES medium for

eight days, mycelial pads we re separated from the medium
and washed thrice with 50 ml aliquots of double distilled
wat er. 'Ibey were then transferred to 250 ml Erlenmeyer
_f
flasks containing 50 ml of the st eril e resus pension medium
and incubated under either shaking or stationary conditions.
In some of the experiments, resuspension studies were done
with six days old mycelia.

Buffers used for resus pension

In addition to YES medium, a number of buffers
(with ~r without glucose) lik e phosphate buffer, citrate
buffer, citrate phosphate buffer, tris buffer, tris maleate
buffer, glycyl glycine buffer, tri et hanolamine hydrochlo-
ride buffer and acetate buffer were employee for r esuspen-
sion of myc e lia . Samnles were drawn intermittantly to esti-
mate kojic acid lev els. 1he composition of the buffers
used is as f ollows. Al l the buff ers used had a pH of 6.5
(5 )
I 0.2M Acetate buffer

A. 0.2M solution of acetic acid

(11.55 ml of ac etic acid per liter)
B. 0 .-2M solution of sodium acetate
(16.4 g of c2 H302 .Na per liter)
 47

4 . 8 ml of A and 53 . 2 ml of B was ta ke n and

diluted to a total of 100 ml.

II Q.._lM Citrate buffer (6 )

A. O.lM s o l utio n of anhydrous c it ric acid
(21 . 01 9 Of &nhydrous cit r ic acid pe~ l it er)
B. O. l M s ol ut ion of s od ium citrate
(29.41 g C6850.,Na 3 . 2H2 0 per lit er)
5 . 1 ml of A and 44 . 9 ml of B was ta ~en and
dilut ed to a t otal volume of 100 ml .

III . _Citrate- phosphate buffe r(?)

A. O.lM solutio n of anhydrous citric acid
(21 . 01 g of anhydrous citric acid per liter)
B. 0 . 2M s olut i on of dibasic sodium Dho sphate
(53 . 65 g of Na2HP04 . 7H2 0 per liter)
15 . 4 ml of A and 34 . 6 ml of B was t ake n and

diluted to a t otal of 100 ml .

IV Q~Phosphate buff!il:(s)
A. 0 . 2M s olutio n of mo nob asic potass ium phosphat e
(27 . 2 g of anhydrous K82P04 per l i~ er)
B. o . 2M s olution of dibasic potassium phos phate
(34 . 8 g of anhydrous ~HP04 per lit er)
68 . 5 ml of A and 31 . 5 ml of B was t aken and

diluted to a total of 200 ml .

 57

(3) Peroxidase solution , 2 rrg/ml.

(4 ) Enzyme solution in O.lM phosphate buffer (pH 7 . o )

Proceduro
To 2.4 ml of the dye in buffer, 0.5 ml of D-glucose
0.01 ml of perox ida se and O.l ml of the enzyme solution were
added. The reaction mixture was incubated for 10 minutes
at 37°C and the absoroance was r ead at 4 36 nm •
One unit of en zyme activity was defined as that
amount of e nzyme which catalysed the co nversion of one
micromole of D-glucose to D gluconic acid.
This was calculated as follows :-

Ch an~n 0, D, per minute x 3.01

Activity = 8 .3
Where 8 .3 is t h e extinction coefficient of oxidi-
zed 0- dianisidine hy drochloride at 436 nm.

Glucose Dehydrogenase (EC 1.1.1.47)

The activity of glucos e dehydrogenase was measured
(17) .
by the method of Hauge 1n crude homogenate prepared i n
O.lM potas sium phos phat e buffer (pH 6.0)

Principle
The reduction in optical density of 2 ,6-dic hlo-
ropheno l indophenol at 600 nm is used as a measure of
enzyme activity.
 58

Reagents
(1) O. lM Phosphat e buffer (pH 6.0)
(2) 0.6M Glucose solution
(3) Dichloroph eno l i ndophe nol solution, 1 . 4 micromole per ml
in water.
(4) Enzyme s olution in O.lM potassium phosphate buffer
(pH 6 . O)

Procedur e
The assay mixture comprised of 1.5 ml of 1:1 dilu-
t ed phosphat e buffer , 0 . 1 ml of glucose solution, O. l ml of
the dye , enzyme solution and water to bring the final volume
'
to 1. 2 ml. A one cm cuvette fill ed with distilled wat er
s erv ed as a blank . Th e r eaction was started by the addition
of O.l ml of en zyme solution and th e change in optical deni-
sity per minute was r ecoraed at 600 nm •
One unit of enzyme activity was defined as that
amount of enzyme which r educed one micromole of the dye per
minute or brought . about a change of 0 . 6 in the optical
density at 600 nm.

Gluconate Dehydrogenase (EC l. l. 99 . 3 )

The activity of this enzyme was measured by the
method of Wood Fett ing and Hertlein (ls) in C'.!'.'ude homogenat e
prepared in 0.5M sodium acetate buff e r (pH 5.5) .
 59

Principle

The rate of g l uconate oxidation to ketog luconate

is equivalent to the amount of fe rrocyanide formed by redu-

ction of fe rricyanide and is determined by colorimetric

measurement of stabilized Pruss i a n blue formed on adding

ferric sulfate Dupanol reagent .

Reagent s

(1) 0 .2M Postassium gluconate

{2) O.lM Potassium ferricyanide

(3) o.SM Sodium Acet at e buffer (pH s.s)

(4) Ferric Sulfate Dupanol reagent . This reagent contained

5 g of ferric sulfate, 3 g of Dupanol (sodium l auryl sul-

fate) and 95 ml of 85% phosphoric acid d iluted with distil led

water to a final volume of one litve .

Procedure

A known vo l ume of suitably diluted enzyme was

added to 0 . 2 ml of sodium acetate buffer and enough dist-

illed water to bring the final volume to 1 ml . The reaction

was started by adding 0.1 ml of the substrate (reagent 1) .

After keep ing the reaction mixture at 37°c for 10 minutee·

0.5 ml of ferric sulfate- Dupanol reagent was added and the

mixture was allowed to stand at room temperature for one

minute. Distilled water was added to a total volume of 10 ml

and the optical dens i ty was read at 660 nm. 'Ihe amount of
 60

f erricyanide r educed was det ermined from a ca lib rat ion

CUIVe.

One unit of enzyme activ ity was defin ed as that

amount of enzyme wh i ch caus es the oxidation of one micro-
mole of gluconate or the r educt i on of two micromoles of
f erricyanide per minut e under the assay conditions desc r ibed .

Pap er Chromatography
Paper chromatography was performed on Whatman Nol
chromatographic paper at 30°C using the desc end ing chro-
matographic t echnique in all cases. Developing s olvents and
spray r e ag~nts us e~ were as follows :-

F ..
K011c .d(19)
....Qr ac1
Solv ents
(1) n- Butanol: ac etic acid: wat er (50:25: 25, v/v)
(2 ) Upper phas e f rom a mixture of et hyl ac etate, wat er,
f or mic acid (60:35 : 5 , v/v)
(3) Is opropanol: wat er: cone. HCl (65:18 .4:16.6 , v/v)

Spray r eagents
One percent f crric ch l oride in o'. l N HCl (2o)
lhis was prepared by dissolving 5 g of anhydro-
ous f erric chloride in 500 ml of O.lN HCl.
 Ani l ine hydrogen pthalate ( 2 l )
Thi s reaoent was pr epar ed by dissolving <no rrg

of aniline and 1 . 6 g _of ptha li c acid to 100 ml of butanol

saturated with wat er .

'\
For glu coni c acid- ~ - lactone
Sol vents
(1 ) n- Butanol : glacial acetic acid : water (50:25 : 25 , v/v)
(2) Up per phase from a mixture of water: secondary
butanol: tertiary butano l (48 . 4:34 : 17 . 6 ,v/v)
(3 ) Is opr opanol : water: cone HCl (65 : 18 . 4 : 16 . 6,v/v)

Sp r ay reagent
The r eagent was pr epared by the method of Abdel
(22 )
and Akher by mixing eq~a l volumes of normal hydroxy-
lamin e hydroc hloride a nd l .l N potassium hydroxide , both
in methanol soluti on . The mixture was sprayed on the dried
c hromatogram. The pap er wa5 agai n dried i n air for 10 min-
utes and spr ayed with a solution of 2% FeC1 3 in 1% HCl .
Bl ue or matNe spots were formed by the lac t ones .

( 19 )
For Gl ucose- 6~p hos ph ate
Sol vents
(1) n- Butanol: gl acia l acetic ac id: water (50:25 : 25 , v/v)
(2 l Upper phase from a mixtur e of water , secondary
but anol and tertiary butanol (48 . 4 : 34: 17 . 6 ,v/v)
 62

(3) Isopropanol: Water Cone . He! (65:18 . 4 : 16 . 6,v/v)

Spray r eagent
The r e agent was prepared by the method of Han~
23
and Isherwood . ( ) This method is based on the hydrolysis
J of the phosphate esters,,followed by formation of phos -
phomolybdic acid and i ts subsequent r eduction t o moly-
bdenum blue . The r eagent was prepared by mixing 5 ml of
60% (w/w) perchloric acid , 25 ml of 4% ammonium mo lybd ate ,
10 ml of lN HCl and 60 ml of distilled water . The mixture
was spr ayed on the dried chromatogram. Inorganic orthophos -
phates appeared immediately as yellow spots . The paper wa s
dried at ss 0 c for 5 minut es a nd then irradiated with a U. V
la~ for 10 minutes . Organic phosphat e compounds gave blue
s pot s while inorganic phosphates appear ed yellowish gr een.'

£Q.L..§,- Phosphogl uconic acid(l 9 )

Solvents
(1) n- Butanol : glacial acetic ac id: Water (50 : 25 : 25 , v/v)
(2) Opp er phase from a mixtur e of ~ater: s econdary
butanol : tertiary butanol (48 . 4: 34:17 . 6 , v/v).
(3) Isopropanol: Wate r : Cone . HCl (65 :18.4: 16 . 6,v/v)

Spray Reagent
Hans and Ish erwood reagent as described abov e
 Studies on~ Ferme ntatiQD.._Qf Cane Mo lass es
The fo llowing types of molas s es solutio ns were
used as f ermentation medi a e ithe r as su ch or aft er sup ple-
ment at ion with 1% y east ex tract. Unclarif i ed molas ses med-
\ ium was us e d as a control.
-
/A

Unclarif.ied Mo las s ~s
Vaccum p a n s ug a r fact ory c an e mola sses was dilu-
t ed to 10 , 20 and 30° Brix conc e ntration.

Chemically Clarified WK> las~

Mola sses was clarif i ed by following methods
C1 ari. f ica
. t ion
. . (24 )
wi. th egg a l b umin
Powd ered egg albumin (1. 4%, w/v) was added t o
60° Brix molas s es solution, which was then steamed at pH 7. 0
for 20 to 25 minutes, and f i ltered through cott on Wool. The
clear filt erate was di lut ed to 10° Bri x conc e ntration bef or e
use.

Cl ari.f ica
. t·1 QQ.~~·th ammq nium
· l h ate ( 25 )
su_D
The molas s es wais dilut ed to _30° Brix and he-
ated to boiling ~ Ammonium s u lfat e (33. 3% , w/w) was added
to the boil i ng mol as s es s ol ution a nd the boiling was co nt-
inu ed f or 15 minut es . Aft er cooling, it was c ent rif uged
and the clear supernatant was dilut ed to 10° Brix conc ent-
I

rat i on.
 64

Clar ific ation with ammonium sulphate and superphosphate (26)

Ammoni um sulphate (0 . 5%, w/v) and superphosphate
(0 . 05%, w/v) were added to the 60° Brix molasses solution .
The pH of the mo l asses solution was adjusted between 4 . 8 and
5 . 0 by addition of sulphuric acid (about 0 . 5%, v/v) . 1ne
0
mi xture was then heat ed to 80 C and maintained at that temp-
erat ure for 6 hours . After cooling , it was centrifuged a nd
the clear supernatant was diluted to 10° Brix concentrat ion.

Cl arific ation with lime and superphosphate( 27 )

Quic k lime (CaO) (0 . 5%, w/w) was added t o the 60°
Brix molasses solution followed by superphosphate (1 %, w/w) .
The mixtur e was kept boiling for 10 minutes , c ooled , centri-
fuged and the supernatant wa~ diluted to 10° Brix concent-
ration .
C1 ari. f ica
. t ion
. wi. th pot as s ium
· f errocyani· d e ( 28 )

The molasses was diluted to 60° Brix concentration

and its pH was adjusted to 7.0 by the addition of quick
lime (CaO) . It was then heated to so0 c and to this, potas s:
ium fer rocyanid e (1 . 5%, w/v) and 85% phosphoric acid ·(Q.05%,
v/v ) were added . The mixture was boiled for 45 minutes,
cool ed , centrifuged and the c lear supernatant was dilut ed t o
10° Brix concentration .
 65

Cl ari.f.ica t.ion b y su l p h uric ' (29 )

. acid

To 60° Brix mo las ses, sulphuric acid was added

in quantities just sufficie nt t o lower down the pH by 0.5
unit. It was heated to so 0 c and ma intained at t ha t temper-
ature for 6 hours. Th e n it was cooled , centrifuged and the
clear supernatant was diluted to 19° Brix conc entration.
JQn Exchange Resin Treated Mola sses
The molas ses solutions were tre ated with the
anion and cation exchange resins, Duo lite A-100 and Duolita
C-300 r espectiv e ly as follows -
Regeneration and pac ki ng of ion excha nge resin columns
The ion exchange res i ns were re~e nerat ed prior
to th eir packing into the r espective glass columns. To a
slurry of Duolite A-100 r esin in distilled water, 4N HCl was
added and kept for 24 hours with occasional stirring. Aft er
draining of th e acid, 4N NaOH solution was adde d and again
left for 24 h?urs with occassional stirring. Aft er draining
of th~ alkali, the r esin was thor ough ly washed with distilled
water,till it was free from any alkali and the pH of the
effluent was 7.0. Th e n the resin was car efully packed in to
a glass c olumn (6 x 24 cm) and equilibrated with deioniz ed
distilled water prior to use.
 70

Materials Used
Kojic acid, 6 phosphogluconic acid, g luconic
acid and sodium lauryl sulphate we r e purchas ed from sigma
Chemicals Co. U.Sft A~, Yeast extract was obtained from Difeo
Labo ratories, u .s.A. , Glucose-6-phosphat e and NADP we r e
purchas ed from Centron Res earch Laboratories, Bomb ay. ATP
was obtained fro m Biochemical Units, V. P. Ch est Institut e ,
De lhi. Iodoacetic acid was a pro~uct of E. Merc k, West
Germany. Th e ion exch ange r esins,'Duolite A-100 and C-300
were the products of Monte Cautie, Mil ano , It a ly. All the
other chemicals were purchased from B. D.H. (Indi 9 ) or
Sarabhai Ch emicals (India) an9 wer e of A.R. or L.R. grade
according to th e r equ irements.
I•

71

RE FEREI\ICES

1. Davis N.D., Di ener U.L. and Eldridg e D. W., Appl.

MicrobioL, l l , 378 (1966).
2. Reddy T. V., Vis wa nath an L. and Ve nkit asubramanian
T.A., ~pl. Microbial., . 22 , 393 {1971 ).
3, Adye J, and Mat el es R.I., Bi ochim. Biophys. Act a ,
.6§. , 418 (1964 ).
4. Thom c. and Church M. B., "The Aspe rgilli", Th e.
Williams and ll'ilkins Company, Ba ltimore (1930 ),
5. Wapol e G.s., J. Ch em. Soc., 105 , 2501 (1904).
6. Lillie R.D., "Hist cpathologic TE?chniqu e ", Bl akiston,
Philade lphia and Toronto (1948).
7, Yellvaine T.C., J. Biol. Chem.,~ , 183 (1921)~
8. Gomori G., Methods in Enzymology , Vo l. I, Edit ed b y
Colowick S. P. and Kaplan N.O., Ac ademic Pr ess,
New York (1955). p 14 3 .
9. King J., Methods of Enzymatic An alysis, Vol. II,
Edit ed by Bergmeyer H. U., Acad emic Press, New York
( 196 3 ) , p • 6 3 3.
10. Gomori G., Proc. Soc. Exptl. Biol. Med., §.a, 354
(1948 ),
11. Lohr G. w. and Waller H. D., Methods of Enzymatic
Analysis, Vol. II, Edit ed by Bergmey er H. V., Ac ademic
Press, New York (1963)., p.638.
 72

12 . Gomori G., Methods in Enzymol ogv , Vol . Is Edit ed by

Colowick S . P. and Kap l a n N. O., Acad emic Pr ess ,
New York (1955 ) •
'
13. Darrow R. A. and Col owick S . P ., Methods in Enzymo l ogy ,
Vo l V, Edi t ed by Colowick S . P. ~nd Kapl an N. O. ,
Ac ade mic Pr e ss , New York (196 2) ,p,226·
14 . Kuby S. A. a nd Nol tmann E. A., Methods i ~ Enz y~ology,
Vo l IX , Edit ed by Col owi ck S . P. and Kapla n N. O.,
Ac ademic Pr ess , New York (1966 ), p .116 ~
15. Pont remol i s. Ard Graz i E., Methods in Enzymol~gy,
Vol IX , Ed i t ed by Co l owi ck S. P. a nd Kapla n N. O. ,
Ac ade mic Pr es s , New Yor~ (1966 ), p . 137 ~
I

16 . Hugg et A. S . and Nixon D. A. , Bioc he m. J . , 66 , 12 p

(1957 )~
17. Hauge J . G., M~t ~ods in Enzymology , Vol IX , Ed i t ed
by Co l owic k S. P. a nd Kap l a n N. O. , Academic Pr ess ,
New Yor k (1966 ) , p . 107 •
18. Wood w. A., Fettin;J R. A. and He rtl e in B. c . , M~thods
in Enzymo l ogy , Vol V, Ed it ed by Co l owi c k S.P. and
Kapla n N. o ., Academi c Pr ess , New York . (196 2 ) ,
. .
p. 287~
19 . Fink K. And Cl i ne R~E., J. Ana l . Che m~ ,-~ , 389
(196 3 ).
20 . Be ntl ey R., Met hods i n Enzym9l og y, Vol III , Ed i t ed by
Col owic k S. P. and Kap l a n N. O., Ac ade mic Pr ess,
New York (1957), p . 240 ~
 73

21 . · Block R. J ., Durram E.L. and Zw~i g G$ , Paper chro-

matography and Ele~trophoresis~ Ac ademic Press ,
New York (1957 ), p.181.
22. Abde l Akher M. a~d Smith F., J. Am. Ch em. Soc .,
11, 5859 (1951) .
23 . Hanes ~.s . and Ish erwood F. A., N2tur e , l~-• 1107,
(1950 ).
'
24 . Hans en A. and Jorgenson A., Microorganisms and
Fermentations, Ch arles Gr iffon ?nd Co., London,
(1939) , p .72 ~
25 . Shu kla J . P. and Kapoor B. D., Proc . 26th Annu al Con-
v ent ion of Sugar Technologists Association (Indi a) ,
·,
G- 39 (1958 ).
'
26 . Wa lt er F. G., Manufacture of Compr essed Yeas t, Ch apman
and Ha ll (1956 ), p . 131.
27. A;rroy R., Int. Suga r J ., 45 , 250 (1943 )!
'
28 . Waller C. J ., BI NS Final Report No . 489 , Item No . 22
(1946 )~
29 . Reich G. T., Tr ans. Inst. Ch em. Eng ., ~ , 1049
(1943 )~
30 . Lowry O. H., Rosenburgh N. J ., Farr A.L. and Randall
R~J., J~ Biol. Che m . , ~ , 256 (i931 ).
 CHAPTER - III

ISOLATION AND SELECTION OF THE CUL1UR E

 74

CHAPTER III

-IS OLATION AND SELECTION OF THE CULTIJRE

-
1he ill1)ortance of a suitable organism , in~ fer-
mentation study, can not be over emphasized . Studies,
whether related to fundame ntal or commercial aspects of a
microbial fermentation, need to be performed on a specific
org anism, which is fast growing, has s imple and well defi ned
growth require ments is stab le in its biochemica l, morpholo-
gical and physiological characteristics and produces high
yield of the desired met abo lic end product. With this in
view , suitable strains need to be isolat ed from its natural
habitat, screened fo r yield of the r equired product , and
mainta ined in the l abor atory for subseque nt studies . Some-
times it becomes imperative t o do some mut agenic studies
on the selected organis m, inorder to improve its perfor-
mance . A nu mbe r of reviews are av ail able on isolation and
selection techniques .
It has been r eported that earlier studies on
kojic acid fermentation were done mainly on ~ergil.11!§
strains. Saito (l) first isolated the acid as a by proeuct.
of t he fermentati on of steamed ric e by As pg_rgillus oryz~ .
When ferric chloride wa s added to the fermented br oth , an
(2)
intense red colour was produced . Yabuta report e~ the
production of koj ic acid in nutrient solutior$ of b . albus ,
 75

3
1. eand idus and 1. nidulans . Traetta- Tvbsca ( ) showed that
kojic acid was produced by 6. 91:aucus from sucrose, glucose,
fructose and glycerol . Wijkman (4 ) obtained the acid by
the fermentation of sucrose usi ng an unnamed st r ain of
5
As-pergi l lus . In 1929 Tamiya and Hida ( ) p oint ed out that
several different Aspergilli had the aBility to produce
kojic acid from sucrose, Among the species mention~d were
h! oryza e , h• flavus, h• gymnosard ae , h! .filY.affiQ~~ h. candidus,
h• clav atus, h• f umig at us, .,b. gig anteus . Birkinsh aw and
Coworkers( 6 ) r epo rt ed that kojic acid coul~ be produced from
sugars by h.
~arasiticus, h. effusus and h. tamarii. Later
on h. luteoviresce.!1§. ~7 ) h. lutescens(g) and h• we nti i( 9 )
were a lso included in th e list of mic r oorganisms p~ducing
kojic acid . Most of the members of h. flavus oryz ae-t amarii
gr oup were found to produce kojic acid but amongst them
b. ~!!§. and b• .Q£Y~ we re mainly employ ed for the pro-
duction of kojic acid. In ~ddit i on , cultures of Penicillium
(6)
Jig~ were also f ound to produce kojic acid under fav -
ourable conditions . Among the bacteria , the formation of
kojic acid was obseIVed in several species of acetic acid
bacilli~lO)Gluconobacter Opacus v ar mobi lis(ll) and
j

(12)
Gluconobacter .tQ.§_eus. Further, s everal wo r kers have
r eported the isolation of koj ic acid producing organisms ,
almost all of them being fu ngi of the genera h§pergillus
 76

and £eoicillium(~ 3 ) There are no reports available to

demonstrate kojic acid production by yeasts .
With a view to obtain a high kojic ac id yielding
organism, suitable for fundamental and commercial fer-
mentation studies , cultures were isolated from different
natural sources and suitable ones were selected and ma i n-
tained in the laboratory for subse quent studies .

MATERIALS AND METHODS

In the present study fungal cultur es, capable

of producing kojic acid were isolated from ai r and from
garden soil. 15 ml portions of potato dextrose agar were
aseptically poured tn petri dishes . Some of these were
supplemented with 0 . 5 ml of 0 .2% bromocresol green solu-
tion while 0 . 1 ml of 1% ferric chloride solution was added
on some other petri dishes .

Isolation from air

Petri dishes containing potato dextrose agar
medium were opened and exposed to the atmosphere for six
hours . Then they were closed and incubated at 30°C for
five days, High kojic acid producing strains we re identi-
fied by the change in the indicator's colour and were
examined microscopically . Then , they were transferred on
 77

pot ato dextrose agar slants and we r e mai nt ained by

subcu]turing at monthly intervals .

Isolation from soil

A number of r ep r ese ntative f erti le gard en so it
samples , were coll ected from different p laces in Ka npur.
l g portions of the samp l es were suspended i n 10 ml of
st erile water. After shaking the suspensions thoroughly
for 10 minutes, they we re suitably diluted with st erile
wat er by serial dilution so as t? get 5 to 10 colonies per
pl ate on incubation. 0 . 5 ml aliquots of the suitable dilu -
tions were th e n spread uniformly with the help of a st erile
g l ass rod on agar media i n petr i dishes. Th e petri dishes
were then incubated at 30°C for fiv e days . The high kojic
acid pro ducing stra ins we re identified by change in the
indicator's colour and were examined unde r the microscope.
Then they were transferred on pot ato dextrose aga r s l a nts
and were maintained by subcultu ring at monthly int ervals.
The strains produ cing large amounts of acic
were identifi ed by the cha nge i n the indicator's from green
to y ellow in case of bro mocresol gr een. High kojic acid
producing microorganisms we re specifically identified by
change in colour from yellow to r ed in the agar medium
cont ai ning f e rric chloride.
 78

The amount of koj ic acid produced by the s elect ed

fungal strains grown on semisynthetic medium containing
sucrose and yeast extract (YES mediu m)(l 4 ) was det e rmined
as f ollows •
Fungal s pores from the stock cultures wer e first
inoculat ed on potato dextrose agar slants a nd incubat ed
at 30°c f or four days. The spores harvest ed from th e
slants were th en used t o inoculat e 100 ml of the pot ato
dextrose agar med ium contained in diff e rent one lit er Rou x
bottles and incubat ed again at 30°c f or fiv e days. The
spores were harvested as eptically with 100 ml of st erile
distilled wat e r containing .001% Tween 80 from the each
Roux bottle. 5 ml porti ons of the wel l disp ers ed s por e sus-
6
pens i on conta ining 5 x 10 s pores wa s us ed to inoculate
50 ml of th e st eri l e medium contai ned in different 250 ml
Erlenmeyer flasks. The pH of the medium was adjust ed to 6 .5
before st er ilization. The inoculated medium was i ncubated
as stationary cultures at a t empe rature of 30 0 c. From each
cultur e flas k , s u itabl e aliquots of th e f ermentation broth
we r e drawn daily starting from fourth day up to t we lfth day
and kojic acid c ontent was determined. Koj ic acid wa s
(15)
estimated by the method of Be ntley ·• Th e proc edure f or
media sterilization and kojic acid assay have already been
described in Chapter II. At the end of the experiment, the
mycelial pads we re separat ed from th e medium and the growth
 79

was measured by following the method• mentioned in

Chapter II .

RESULTS AND DISCUSSION

It has bee n reported that high koj ic acid produ-

cing strains are wide ly distributed in nutritionally rich
cultivat ed fi elds and are rare in rutrit ionally poor barren
lands and air . Therefore a large number of strains were
isolated from garden soils for investigation. The cultu r e~
isolated in the present study h ave been list ed in Table l.
together with their respective sources . All the cultur es
c apab le of pr oducing kojic acid were identified as Aspergillus
flaY.1!§, on the basis of their morphologica l characteristics.
Atte mpts to isolate bacterial or yeast cultures c apable of
pr oducing koj ic acid prov ed futile.
Table 2 presents the amount of ko jic acid f ormed
by s ome of the isolated cultures. It would be noted th at
in the ca se of all these cultures, kojic acid level i n
fermentation medium rap i dly increased with the pe r iod of
incubation and r ea ched the maximum around 7 to 11 days ,
after which it decreased rapidly. Thus, bo th synth esis and
degradation of ko jic acid is occuring i n these cultures .
Their relativ e values varied with the strain and the period
of incubation. Considerable variation was also obse:r:ved in
 80

TABLE ::..J.
.§OVRCE§_ OF THE ISOLATED f l]l\[; AL CUL1URES

Cu l ture Source

A, B, C and D Atmosph er i c dust

Nat i onal Sugar I nst i t ute
Kanpu r.

E, F, G and H Gar den So il

In d i an Institu t e of Technology
Ka npu r.

I , J , K and L Gar den Soil

Nati ona l Sugar Inst itut e
Ka npur.

M, N, 0 and P Garden So il, Radha Kr ish na

Temple , Pandu Nagar
...
Kanpu r.

Q, R, S and T Ga rd en Soil
Moti Jh eel , Swaroop Nagar
Ka npur.
 ~ ~
._ y
~
•
J~BLE--=-.1
KOJIC ACID PRODJCTION BY SELECTED FUN3AL CUI. TIJRES ON YES MEDIUM
-- - ·
Cu l ture P e rio d of incubation (d ays )
4 5 6 7 8 9 10 11 12
Koji c acid produc ed (rrg / ml)
- -- -·-- - ---------·-· ------- --
A 7 . 40 15 . 30 28 . 20 38 .41 54 . 90 60 . 25 62 . 32 58 . 12 54 . 20

B 11.23 18 . 74 38 . 22 44 . 20 66 . 40 70 . 20 75 .61 62 . 02 52 .42

C 8 . 21 15 . 30 16 . 23 20 . 24 26 . 21 21 .54 16 . 28 12 .02 8 .34

D 16 . 02 28 .40 32 . 92 39 . 23 45 . 12 37 . 23 35 . 53 26 . 24 16 . 94

E 13. 64 23 . 42 27 . 23 32 . 23 37 . 26 31 . 02 26 . 24 20 . 12 13 .64

F 12 .63 23 .02 24 . 84 30 . 23 40 . 80 31 . 43 24 . 23 18 . 53 12 .63

G 5 .64 8 .04 16 . 30 35 . 32 36 . 40 37 . 23 43 . 20 46 . 85 36 .02

H R.02 11 . 23 25 . 02 54 .63 57 . 01 60.12 63 . 02 67 .60 57.21

I 22 . 40 27 .02 31 . 42 35 . 40 36 . 21 40 . 07 43 . 23 40 .02 36 . 03

J '.?() . 62 34 . 23 41.24 46 . 83 44 . 80 40 . 80 36 .04 30 . 90 26 . 20

K 4 .02 9 . 26 12 . 60 16 . 23 20 .02 24 . 23 21 . 24 18 . 36 15 . 23

co
I--
 ,i-- ~ )f ¥' )r-
,...

continued •..••••• T3ble 2/

Culture
- -
Per iod of incubation (days)
4 5 6 7 8 9 10 11 12
Kojic acid produc ed (mg/ml}
-------
L 6 . 21 9 . 82 24 . 23 42 .53 48 . 93 52 . 36 58 . 29 46 . 43 40 . 23
M 10 .61 18.62 25 . 23 32 . 42 45 .63 34 . 20 26 . 43 20 . 24 15 . 43
N 4 . 32 12.46 17 ~24 21.45 25.23 36 . 46 28 . 23 25 ,36 21.29
0 17. 23 24 .60 32. 36 38 .43 42 .65 56 . 23 43 . 29 39 . 43 31 . 56
p 4 . 20 9 .43 14. 26 18.43 22 .64 30 .02 26 . 43 22 . 26 18 .23
Q 8 . 43 18 . 29 28 .33 32 , 45 38 . 29 42 . 5 3 37 .34 32 .53 28 ~33
R 8 . 88 12 . 20 28 . 24 32 . 29 38 . 24 45 . 43 40 .26 36 . 4 3 32 .53
s 5.2 10 . 43 16 .73 23 . 52 31.23 40 . 52 48.23 42 . 46 38 .10
T 2 .2 8 . 20 14 . 40 20 .50 26 .70 33 . 4 1 40.99 34 . 23 30 . 52
·-

co
rv

~
 83

the rate of producti on of ko jic acid. It may be observed

that culture B produced the maximum amoun~ of kojic acid
(75~6 rrg/ml) c orresponding to 37. 8% yield, on the basis of
sugar initially present i n the medium. In this case, maximu~
koj ic acid production t ook p la ce on 10th da y of incubatio0 .
Cultures Hand A also yie lde d somev.hat c omp arab le r es ults,
producing maxi mum kojic acid (67.6 rrg/ml) on 11th and
(62 . 3 mg/ml) on 10 th day, respectively. The kojic acid y ield
calculated on the basis of sugar initially pr esent i n the
medium, was 33. 8% in the case of culture H' a~d 31 . 5% in the
case of cultur e A. The lowest amount of kojic acid
(24 ~2 rrg/ml) was produced by culture Ko n the 9th day of
incubation r epr esenting only 12% y ield . In general , the
amount of kojic acid produced by dif~erent cultures, r a ng ed
f r om 24 rrg to 75 . 6 rrg/ml. Howev er, it may be stressed that
actual total yi e lds of kojic acid would be cons iderably higher
th an the v alues reported , since a s ing i f icant amount of
koj ic acid, has bee n r eported to be pr esent i n myc e lia~ pads,
which may also be r ecover ed. For the sc ree ning purpose,
kojic acid was det ermined only in the medium as the amount
pres ent in myc elium was comp aratively quite small .
The mycelial wet we i ghts of all the culture iso-
lates we re r ecorded o n the 12th day of incubation and have
been pres ent ed in Tab l e 3. It would be not ed that YES
medium used for this experiment supported good g rowth of the
 84

TABLE - 3
MYC ELIAL GROWTH OF SELECTED FUN3AL CUL TURES ON YES MEDIUM
--
Culture Wet wei ght of mycelium Maximum kojic
on 12th day in grams acid p roduced
( ID9/ml)

B 6 . 98 75 .61
H 6 . 23 67 .60
A 5 . 80 6 2 . 32
L 5.40 58.29
0 5 . 23 56 . 23
s 4 . 59 48 . 23
G 4 . 39 46 . 85
J 4 .26 46 .83
M 4 . 20 45 .63
R 4.18 45 . 43
D 4 . 12 45 . 12
I 4 .02 43.23
Q 3 . 92 42 . 53
T 3 . 86 40 . 99
F 3 . 73 40 . 80
E 3 .59 37 . 26
N 3 . 46 36 . 46
p 3 . 10 30 .02
¥ C 2.99 26 . 21
K 2 . 90 24 . 23
--A ---
 85

fungus . Higher rnycelial weights on YES medium are appar-

ently du e to high l eve l of carbohydrat e and the pr es ence
of undefined growth fact ors in yeast extract used a s a
constituent of this medium. 1he data in Tab le 3 are arranged
in the order of decr easing mycelial we ights. It i s appar-
e nt that there is very good corr e lation , between the levels
of kojic acid produc ed and myc e lial growth . The maximum
growth 6 . 98 g was obtained i n case of cultur e B which
incidentally happened to produce th e maximum amount of
kojic acid. Similarly cu ltur e Hand 4 which h ad also pro-
duced comparitiv ely high amounts of kojic acid (67. 6 and
62 !3 mg/ml r esp ective ly) also exhibit ed good myc el ial g rowth
(6 . 23 and 5 . 8 g resp ectively) . This clos e corr elationship is
surprising in view of th e fact that different strains of
h~ f lav1!§. wer e involved . It is pos sib l e that both th e mass
and the surface area of the myceliurn play a rol e in determin-
ing kojic acid yields . It has been sugg est ed th at a la~ae r
myc e Jial surfac e area would fav our the production of ko jic
acid , part icul arly in th e c ase of stat ionary cultur es . Th is
may be attribut ed to a g r eat er uptak e of sugars and excretio n
of kojic acid.
Mo,:phological charact eristics of the selected cul-
tur es c apable of producing ~oj ic acid .
All the seleet ed cultur es wer e tentatively iden-
tifi ed as A§p e:rgillus flavus , based on the morphological
 86

characteristics(l6 )listea below :-

1) Rather high and wid ely separated conidiophores ter-
I

minating in yellowish gree n globular spore heads .

2) The conidiophores are spiny and septate and arise
from large and prominent foot cells.
3) The conidiophore t er minat es in a large sphericle
vessicl e , from which arise r adially dispos ed pri-
mary stalk or sterigmata , each of which bears s ev eral
secondary sterigmata.
I

4) The st alks are pitted and the conidia are smooth.

Sinc e amo ng all the cultures tested , cu ltur e

B, gav e good myce lia l growth and maximum yi e ld of kojic
acid , all subs equent studi es were p erf ormed on this organism.
n

87

RE FERENCES

l~ Saito K., Bot an. Mag . (Tokyo), 11, 240 (1907).

2. Yabuta T., Orig . Com. 8th Int ern. Congr. Appl. Che m.
(Appendix) , 25 , 455 (1912 ) ; Ch em. Abstracts, 1 ,
2191 (1913 ) •
3, Traetta- Mosc a F., Ann. Chi m. App l; l , 477 (1914 ).
4. Wij kman N., Ze it. Physiol • . Che m., ~2 104 (1924 )~
5. Tamiya H. and Hid a T. , Act a Phytoch im (Japan), ~ . ,
343 (1929 ); Ch em. Abstracts, .21 , 2496 (1930 ) •
6. Birkins h aw J • H. , Cha rl es J. H. V . , Lilly c. H. and
Raistri c k H., Tra ns . Roy. Soc. ( Lo ndo n), B220 , 127
(1931).
7. Morto n H. E., Ko chol aty W., Junowi c z-Kocholaty R. a nd
Ke Jn er A., ~. Bact eriol., 50 , 579 (1945 ).
8. Marston R. Q., Nature , 161 , 961 (1 949) .
9. Dorothy G.c., Brit J . Exptl. Pathol., ]Q , 119 (1 949) .
10 . Takahash i K. and Asai T., J. Ag r. Chem. Soc. J apa n,
.2 , 55 , 369 (1933 ) ; Che m. Abstracts, 27 , 1026 ,
·3 735, (1933 ) •
11. Sakaguch i K., As a i T., a nd I keda Y., J . Ag P. Chem . Soc .
Japan, 19 , 711 (1943); Ch em. Abst r acts, 1 i ,
550 8 (1 948 ) •
12. Ikeda Y., J. Agr. Che m. Soc. J apa n, 26 , 90 (1952 ) ;
Chem. Abst r acts, 48 , 10113 (1954) •
 CHAPTER - IV
OPTIMIZATION OF FERMENTATION PARAMETERS
 89

CHAPTER IV

OPTIMIZATION OF FERMENTATION PARAMETERS

For prop er growth and development of any organis m,

a c e rtain optimum l eve l of evniromental c o nditions is
requir ed. In the case of microorganism, t hese include growth
media containing c ar bon and ni trogen s ources and growth
fact ors in a p articular concentration, pH , temper at ure ,
aeration etc. In the ca se of fung i, especially Asp erg i llus
species, these conditions have been found to v a ry not only
from organism to organims, but different strains of the
same organis m may require diff erent optium growth and f er ment-
ati on condit io ns. Some strai0s of ,h. niq er produce max i mum
citric acid at a pH around 2.0, while others do so at about
2
pH 6 .0• (l, ) Further , addit io n of 0 .3% Wg(N03 ) s ti mulat ed
2
citric ac i d product ion by a strain of h• niger, but decre-
as ed the acid p r odu ctio n by anoth er ct rain (~) Again t he
eff ect of increasing th e l ev e l of iron or zinc on th e medium
( , 5 , 6 ) s imi
· f r om stra i· n to strain.
·
4 · ·1 ar s t ud'i es h ave
v aries
been r ep ort ed in case of koj ic acid producing microorg a nis ms.
Tamiya(?) fo und that pH 5 .5 was optimum for the f er me nt-
ation of sucrose by ,h. fl_g~, whi l e Barham(s) and S~its( 9 )
obtained high est y i el d s i n th e pH r ang e of 2 t o 3. 5 •
Further, a t emp erature of 35 0 C was found best for ko j i c
acid fer me ntation by Barham(s ) and Smits( 9 ) while Katagiri

•
 90

. . ( 10) ( 11)
and K1tahar1 and Gould found that maximum kojic
0 0 ·,
acid producti on took place at 31 C a nd 20 C respectively .
Similarly , the preference for a particular carbo n source
has also bee n r qported to va ry.
In vi ew of the above facts , the sele cted strain
of h • flav us was s ubject ed to different fermentation para-
meters in order to det er mine th eir optimum l ev els . The
investigations we re aimed at s electingth e optimum growth
medium, _ni troge n sour ce , carbo n s ource and their co nc ent-
rations , pH , temperature , s por e inoculum size a nd g rowth
factor s l i ke vitamins and amino acids . The optimum conditions
det e r mined on the basis of these studies were used in al l
subs equent studies .

MATERIALS AND METHODS

All the stud i es we re carr i ed out on a strain of

Asperqillus flavus isolated in this laboratory and termed
as culture Bin Chapt er II I . It was maintained on potato .
dext ros e agar slants by s ubculturing at monthly int ervals .
To determine optimum fermentation parameters following
proce duie was generally adopted :-
A number of 250 ml conical flasks , each containi nc;
50 ml of sterile YES medium , wer e inoculat ed aseptically
6
with 5 ml each of a uniform spore suspe ns io n containg 10
 91

s por es per mJ . 1hese flasks were incubat ed as static cult-

o
ures at 30 C. Su it able aliquots of the culture media were
removed at r egular intervals of time a nd analysed fo r kojic
acid content. Th e following mod i fications to this procedure
wer e app lied .
To det e rmine an optimum growth and fermentation
medium , a number of culture medi a wer e used inlcuding yeast
12 3
extract sucrose (YES) medium( ) synthetic med iym(l ), a
( 14) . ( 15 )
g lucose salts medium, and Czapekadox medium. The
pH of all med ia was adjusted t o 6 .5 wit h O.lN NaOH solut i on
and we r e s terili zed . The proc edure for med i a st erlization
has been given in Chap t er II.
To study the effect of pH on koj ic ac i d produ-
ction, th e pH of diff erent flasks cont ain ing 50 ml each
of YES medium was adjusted to diff e r ent values ranging f r om
3 to 10 by additio n of O.lN HCl or O.lN NaOH solution. Then
the f l a! kS we re st eriliz ed as usu al .
Simi l arly to st~dy the effect of temperature on
koj ic acid production , differe nt s ets of cultur e flasks ,
were incubated as static c ultu r es at differ ent t emper atures
0
varying from 20 to 38 C.
1he optimum spor e inoculum size was determi ned by
aseptically inoculating 5 ml ea ch of uniform spore suspensions
2
of differ ent s pore concentratio n r anging from 10 s pores to
8
10 spores/ml.
 92

The effects of diff erent ca r bon sources were

studied by substituting 20% sucrose of YES medium with 20%
concent ratio n of different sugars viz glucose, fructose ,
sucr ose and maltose . The optimum substrate concentration
was determined by v arying sucrose content of YES medi um from
5 to 20% keeping all other const ituents constant .
TI1e effects of substituting the yeast extract
of YES medium with 2% concentration of diff e rent complex
mater ial s viz malt extract , casein hydrolysate , peptone ,
beef extract , liver extract and t:ryptone were also studi ed .
To study the effects of vitamins 50 mg/1 con-
centrations of eac h of thiamine , riboflavin , pantothenic
acid , nicotinic acid , inositol , pyridoxine , folic acid ,
as corbic acid and retinal were added to YES medium . Simi-
larly to study th e influ ence of amino acids , 200 mg/1 con-
centrations of each of histidine , lysine , cystine , t y rosine,
histidine , g lycine , alanine , leucine , threonine and aspartic
acid were added to YES medium .
Al l experiments were do ne in duplic ate and the
average values have been reported here .
Glucose , sucrose , fructose , maltose we re pur-
chased from B. D. H. (India) . Malt extract , peptone , c as e in
hydrol ys at e , tryptone , beef extract and l iv e r extract we re
obtained from c e ntron Researc h Laboratories , Bombay. Yeast
extract (c ertified grade) was purchas ed from Difeo
 93
•
laboratories U. S. A. All the amino acids and vitamins were
the products of B. D. H. (England) . other chemtcal were pur-
cha sed from Sarabhai Chemicals (India) and E. Merck West
Germany and were of A. R. grade .

RESUL IS AND DISCUSS ION

The growth pattern of the organism on different media

and rate of increase in kojic acid content is apparent from
Table 4 . It may be noted that YES medium containing 20%
sucrose and 2% yeast extract support ed the maximum mycelial
growth . other medi a like czapekdox medium , synthetic medium
and glucose salts medium supported much less growth . Table 4
also indicates that kojic acid content in YES medium incre-
ased rapidly with the period of incubation and reached the
maximum on 10th day; after that it declined . The production
of kojic acid on YES medium was 80 . 02 mg/ml representing
a 40% conversion of sucrose to kojic acid while other media
supported the production of only s~all amounts of kojic
acid . The yie l ds were 20 , 16 and 5 . 7% in case of czapekdox
medium , glucose salts medium and synthetic medium resp ect-
ively on the basis of available sugar in the medium. Hence
in all subsequent studies , YES medium was employed for
studying different parameters for growth of the culture and
kojic acid production . The large mycelial growth and
 94

I
,,... TABL.£..1

EFF ECT OF DIF FER8'JT GROWTH MEDIA ON KOJIC ACID PRODUCTION

Growth
medium
Period of incubation (days)
I wet wei-
ght of

I
4 6 8 10 11 13 mycelium
(g )
Koj ic acid produced mg/ml
-- ~ f

Cza~ekdox O . 08 o .. 94 2.16 10 .. 16 8 . 42 4.52 3~60

(CZ medium

Glucose-s alts '

(AM) Medium 4.96 8.02 5.08 1.65 2 . 85

Synthetic (s L)
medium 0 . 25 4.25 4 , 83 1.2 1~76

Yeast e{tra t
sucrose YES
medium
1 45 . 20 76 . 03
8 . 30 80 . 02 68 .80 36.04 7.10
koj i c ac i d pr oduction in YES medi um may b e due to t h e h igh
c ar bohy dr at e cont e nt and th e pr es e nc e of undefi ned sti mu-
l at o ry gr owth f acto r s i n yeart extract . Also i t ha s been
obs erv ed that s econda ry meta bo lit e pr oductio n by fung i is
g r eater on a high c ar bohyd rat e low ni trogen med ia . For examp l e
. ( 12 ) ( 16 )
Dav 1s et _al. and Gupt a fil ,al. obta ined high yie lds
of af lat ox i ns on YES med i um. The yeast e xt r act poss i bly
stimul ates g r owth as well as the kojic acid biosy nth esis by
pr ov i di ng a number of cofact or s a nd t r ac e el eme nts . Ar nst ei n
(17 )
and Be ntl ey r eport ed t hat kojic acid produc tion i~
g r eat er in pr es enc e of hig her conce nt r at ions of phosphat e .
Howev er , th ey us ed cz apekdox medium . Appar ently high l eve l s
of phos ph ate are no t r equired i n th e cas e of YES medium .
Di f fe r e nt work ers hav e us ed wid e l y vary ing media and subst -
.
r at es f ors t u d 1es . . ac1' d f er me nt a t·i on. (10 , 16 ,17 ,18 )
o n kOJ1C
The yie lds obtai ned with YES medi a compare fav our ably with
those r eported in l it er at ur e . I n all th e media test ed , the
koj ic acid lev e ls dec r eas ed aft e r pro l ong ed incub at ion . This
i ndic at es the degrad at ion of koj ic ac id by the f ungus . Im o se
Nonomur a and Tatsu mi h av e iso l at ed pur ifi ed a nd c haract e:rized
• . ac 1• d ox1. d as e f rom
k OJic t1 ....
~
t h r ob ac t er · ns . ( 19 , 20 ·)
ur e af ac1e
Th is enzyme conv erts koj i c acid to come naldehyde by ox idat i.on
of th e hydroxy methyl g roup wh en Arthr obact e r ur eafa ci ~ns
str a i n (K-1) i s grown with koj i c ac id as a s ol e carbo n s our ce .
Th i s e nz yme (a new t ype of no nh eme iro n prot e i n ) is assumed
 %

to catalyze the dehydroa enation of kojic acid and the ferric

ion contained in the enzyme is consider ed to serve as an
,
acceptor of hydrogen r eleased from kojic acid . The ferrous
ions so formed are oxidized either by molecu lar oxygen under
aerobic conditions or by NAD under anaerobic conditions, with
the formation of hydrogen peroxide or reduced NAD res pect-
. 1y . A1 so , 14 -c koj1c
1ve · acid has been reported to be con-
v erted to acetate and incorporat ed in to aflatoxins by
(21 , 22)
,h. f l av us .
Table 5 and Fig . l show the effect of pH on my-
celial growth and kojic acid production. It is obvious that
both 5- 0 and 6 . 5 are favourable for mycelial growth . Kojic
acid production was low whe n the initial pH was 10 and was
maximum .when initial pH was 6 . 5 or 5 . 0; this was follow ed
by _pH 8. 0 an9 3 . 0 . The maximum kojic 9cid production was
80 . 92 and 80 . 22 mg/ml at pH 6 . 5 and 5 . 0 r esp e ctively. It may
hence be inf erred that with the organism under study , an
initial pH of 6 . 5 favours myc e lial growth a nd kojic acid
produ ction. There is a considerable variation in the pH
optima r eported i n the literature for koj ic acid ferment-
. (7.,8,9)
ation. There are seve ral possible mechanisms by
which pH may affect the production of a metabolite . Thes e
include affects on ionization of the subst r ates , th eir
uptake by the cells , th e activ ity of different metabolic
pathways , ex~retion of the metabolite etc . In th e present
 97

TAB.1,E -~
EFFECT OF pH ON KOJIC ACID PRODUCTI ON

-- wet
Initial pH Period of i ncubat ion (days) we i ght
of the of mv.ce-
med ium 4 6 8 10 11 13 15 luim(g)
Kojic acid production( mg/ml)

3.0 4.80 17. 80 27 .so 34.42 38 . 6 1 40 . 80 40 . 62 3. 6

5 .0 6 .50 39 .6 2 78.91 80 . 22 73 . 60 44.20 7.10

6.5 8 . 30 45 . 20 76 . 02 80 . 92 68.80 36.02 - 7 . 02

8.0 8 . 02 26 .60 62. 41 68 . 80 56.60 32.80 - 6 . 03

10 . 0 2 . 20 4 . 91 13.92 24 . 40 20 . 80 26 . 51 26 . 20 2 . 40

-----
 EFFECT OF pH ON KOJIC ACID PRODUCTION

3·0 0

s·o ""'
6'5 ,c

80 s·o •
10·0 •

60
--E
~
--
Cl
E
0
u
<( 4
u
-.
0
~

4 6 8 10 1 14 16
PERIOD OF INCUBATION (DAYS)
Fig. 1
 98

cas e , pH s eems to act indirect ly as a r esult of its affect

on g rowth . 1here is a clos e corre latio n between mycel ial wet
we i ght and koj ic acid l evels . 1he kojic acid degrading syst em
is ap parently comp l et e ly inactivat ed at pH 3 . 0 and 10 . 0 , as
the usual de cr eas e in kojic ac i d lev e ls aft er prolonged
incubation was not s ee n at th es e pH values .
Growth and kojic acid l ev els of cultures g r own at
four differe nt t emper atures are pr es ent ed in Table 6 a nd
Fi g. 2. Growth was maximal at 30°C and decrea s ed at higher
and lower t emperatu r es . There was a difference in the r es -
pons es of kojic acid synthesis and deg rad ation to t empe rature .
0
Both the proc esses were max imal at 30 Cat wh i ch max i mum
yi elds were obtained on the 10th day . Koj ic ac id degradation
wa s not ap par ent upto the l ~th day at 20°c where as it s et in
around th e 13th day at 26 °c. 1hus a higher yi eld was obtained
0 0
at 20 C than at 26 c . The effects of t emperatur e on meta-
bo lic process could be quite complex . A simplified model
would s ugg est an incr eas e i n product f ormatio n till a t emp-
erature is r eached where inactivation of enzyme s yst ems and
or r etardation of growth becomes signific ant . The dat a i n
Table 6 are consist ent with such a mod e l . A close corre l at-
ion betwe e n kojic ac id production and g rowth may nl s o be
'
noted . The effec t of temperature on ko jic acid production
appare ntly r es ults from its effect on growth of the
I

fungus.
•,,.. EFFECT OF TEMPERATURE ON KOJIC ACID PRODUC'flON

2o•c 0

26'c •
30'c •
BO 38'c •

6('

--E
......
en
§.
0
u
<f 40
u
-

20

6 8 10 12 14 16
PERIOD OF INCUBATION (DAYS)
' l
 ~
-- >- y
,,,.. ,,,, ..
TABLE - 6
:FFECT OF TEMP ERA1URE ON KOJIC ~CID PRODUCTI ON

--------- --
Temperature i 4 6
Period of i==~ti=-~:ays

8 10 11
Koj ic acid produc ed ( mg/ml)
)------T
13 15 I Wet wei~
ght of
mycelium
(g )
-
I
- - - - - - - - - - - - -- - I
20°c 0 . 60 1 . 35 2.90 12 . 50 17 . s o 42 . 02 6 1 . 60 5 . 39

26°C 4. 72 14. 20 18 . 32 23 . 05 20 . 42 32 . 41 20 . 60 2 . 80

30°C 8 . 31 4 . 20 7 6 .02 80 . 02 68 . 80 36 . 02 - 7 . 02

38°C 2 . 20 4 . 40 7 .10 7.32 7 . 75 7 . 50 - 1.02

---- --

'°'°
 100

The effects of inoculum size on mycelial growth.

I

a nd kojic ac id production are shown in Table 7 and Fig. 4.

It may be s een that both mycelial 9 rowth and kojic acid
production were maximal whe n the inoculum conta;ned 10 6
spores/ml . Tnere was a slight decrease with inocula con-
7 8
taining 10 or 10 spores/ml. Howev er the effect of s pore
inoculum size was not as marked as the effects of some of
the other parameters. Even at the lowest inoculum level
used , a complete mycelial mat was obtained. The ch anges in
kojic acid production seem to be corre lat ed with the changes
in the growth of the mycelium. Th e slight decrease in
growth and in kojic acid formation at th e two hioher inoculum
levels may be due to ~n overcrowding of the spores o n the
surface of the medium. At 107 spor es/ml of inoculum, the
average spacing between spores on the surface of the medium
may be of th e order of I ·~ • Thus there is not enough
space for unhindered germination and .g rowth .
The best carbon source for mycelial growth and
kojic ac id production was glucose (T?ble 8) . The maximum
amount of kojic acid produced was 86 . 85 ~/ml r epr esenting
43 . 4% conversion of glucose to kojic acid. This was
followed by sucrose, fructose , and maltose . These r esults
(23)
are in agree ment with the findin9s of May a nd Associates
who obtained the highest yields of kojic ac id with glucose•
Sucrose which may be expected to be hydrolys ed to glucose

\
 r
"'
)- ¥
. •

TABLE-=--1

EFFECT OF SPORE INOCULUM S I -ZE ON KOJIC ACID PR0D1JCTI0N

Numb e r of ~ - - - - - - - - - - ; : r i o d of incubation (d ays) - 1--;: t we ight

spore s inocu-
l at ed in t o Y
j 4 6 8 10 11 13 of mycelium
50 ml of me- 0 Koj ic acid produced ( mg/ ml } (g)
dium _ _ _ _ ~ - - -- -------------------------L
2
5 X 10 5 .18 30 . 34 54 .76 59.40 48 . 84 23 . 42 5 .0 2

3
5 X 10 5. 62 34 . 03 6 1 . 42 66 . 20 50 .78 25 . 04 5.76

5 X 10 4 5 . 95 34. 20 64 .50 68 . 90 53. 20 27 . 04 6 . 02

5
5 X 10 6 .16 36 . 08 65 .12 70. 40 56 .08 29 . 04 6 .10
6
5 X 10 7. 45 41. 20 74 . 32 7 9 . 20 66 . 40 33. 20 6.95

5 X 10 7 6 . 51 38 .13 68.82 74.0 61 . 38 30.6 9 6 . 40

8
5 X 10 6 .37 37 . 30 6 7. 40 71. 60 60 . 0 6 30 . 03 6 . 28

----- --------
--·----
I-'
0
I-
 EEF~T QF SPORE INOCULUM SIZE ON KOJIC ACID
' PRODUCTION
1. 102 Spores/ml of lnoculum
2. 103 .. " "
3. 10 I. ,, • •
4. 105 " " "
80 5. 10 6 " • •
6. 107 " " "
7. 108 .. ,, •

60
--E.
......
0)

-
0
E

u
<t 1.0
u
-

6 8 10 12 14 16
PERIOD OF INCUBATION (DAYS)

Fig. t..
 )'- -J.. J. t
y

TABLE - 8

_____J________ - - -----~----
Ca rbohy dr -
EFFECT OF DI FFERENT CARBOHYDRATES al KOJ IC ACID PRO[UCTION

P~ riod of i n cub ation (da ys ) Wet we i g ht

at e us ed 4 6 8 10 11 13 of myc e lium
(20 % co nc e n-
tr at io n ~ Koj ic a cid produc ed (mg/ml ) I (g)
- _l_ - _ L ____ _

Gluc ose 10 . 21 46 . 25 7 8 . 20 86 . 85 76 . 02 40 . 20 7. 43

Fr uct o s e 8 .15 39 . 0 2 60 . 60 70 . 05 66. 02 35 . 0 2 6 . 10

Sucrose 8 . 90 40 . 90 6 8 . 21 76 . 0 2 68 . 65 38 . 0 2 6 . 65

Ma ltose 6 . 35 2 9. 90 5 3. 02 60. 4 1 50 . 82 28 . 40 5 . 20

--------------- ------------------------------

1--
0
f\.)
 103

and fru cto se , exhibited yields intermediate between those

given by these two hexoses ~ 1ne reason for the lower yield
with maltose is not clear. However there was a close corr-
elation between growth and kojic acid production . Commer-
cially sucrose may be a su itabl e subst r ate for kojic acid

), f e r me nt at ion .
1ne dependance of myce lial g r owth and kojic acid
pr oduction on substrate concentration i s apparent from
Table 9 and Fig . 3. It may be observed that using sucrose
as a carbon s ource , the best yields of kojic ac i d were
obtained at a subst r ate concentration of 20% wh ere as the
mycel ial g r owth was maximum at 25% conc entration . 1ne yield
of koj ic acid as perc ent of sucrose add ed to the medium
increased with s ucrose concentration upto 20% sucrose and
there was a sligh t fall at the higher concent r ation • It may
be noted that at lower concent r at i ons of sucrose, t he max i-
mum levels of koj ic acid we r e obtained at shorter incubat-
io n periods . 1nis is probably due to the init iatio n of koj ic
acid deg radation around the 8th day its e lf, since the
koj ic acid l evels beca me very low by the 10th day. 1ne
r e l ation bet ween concentration and product yields may be
expect ed to vary with the strai n and the experimental
. . (23)
conditions used. Ma y and associates used substrate
co ncentration in the r ange of 15 to 33% and obtained the
highest yield of kojic acid with h • flavus at 20% g l ucose
 y i. >- '

TABLE - 9
EFFECT OF SUBSTRATE CONC ENTRATION ON KOJIC ACID PRODUCTION
-----
Percent
cone ent-
ration of
sucrose
--------------
4 6 8 10 11 13
0 yield as
Koj ic acid produc e d (mg/ml) 0 percent as
I
Perio d of incuba-ti-on-(-da~:-;--IM-ax-im-u m-,;-et wei--
0 koj ic acid ght of
myce lium
(g)
0 of suc r -
~ _ _ _l__Q se added f

5 0 . 54 0 . 58 0 . 46 0 .45 0 .38 o. 30 11 . 60 1. 04

10 2 . 32 12 . 05 16 . 82 0 . 76 0 . 60 0 . 45 16 . 82 3. 0

15 3. 80 1 8 . 20 30 . 41 3.12 1 . 25 0 . 65 20 . 27 5.2

20 8 .• 9.2 40 . 20 68 . 20 76 . 02 65 . 6 1 32 . 20 38 . 0 1 6 . 65

.
25 4 . 50 38 . 20 75 . 02 90 . 02 82 . 50 38.01 36 . 00 7 . 87

---- --

.....
0
~
EFFECT OF SUCROSE CONCENTRATION ON KOJIC ACID
PRODUCTION
10 1 s•,.
2 10•1.
5 3 1s•1.
4 20·1.
5 2s•1.

80

-E
-m 60
E
-
0
u
<{

~
-,
0
~ 40

20

l
6 8 \0 12 14 16
4
PERIOD OF INCUBATION {DAYS)

Fig.
 105

24
concentration . Barham and Smits( ) found 15% concentration
of xylose to be mos t suitable for ko jic acid productio n .
On the oth er hand , Katagiri and Kitahari (lO) obtained sat-
isfactory results with 5% concentration of a large number of
subst r ates.
The effects of r eplacing th e ye ast extract in the
YES medium by other complex substances are presented in
Table 10 . It is obvious that the best nutrient source for
mycelial growth and kojic acid production is yeast e xtract .
Ma l t ext ract supported very little mycelial growth and the
kojic acid productio n was also v e ry litt l e . Cas e in hydro-
lysate and tryptone also provid ed l ow g rowth and kojic
acid yields . Shohei ( 25 ) has also obtained good yields with
yeast extr act . It ha s f urth er been r eport ed that 50% of
this complex org anic nitroge n s ourc e can be substitut ed
by ur ea or ammonium nitrat e without sacrificing kojic
26
acid yi e ld ! ( ) The s e compl ex substanc es c ontain s ev eral
components . It is difficult to asc e rtain the exact c ompo-
nents which ar e stimulatory. Thes e are appare ntly not amino
acids or peptides as case in hydrolys at e and pepto ne are not
so effective .
To increas e the kojic acid pr oduction furth er ,
it was felt desirable to supple ment th e yeast e xtract
sucrose medium with ce rtain amino acids and vitamins . The
'y
-(. ... ¥
• •

I ABL]_~ _10
EFFECT OF REP LACI NG YEAST EXTRACT BY OTHER COMPLEX MA.TERI ALS

Subst anc e
I
Pe riod of incubati on (d ays) - - ,Wet -:ei -
added at
2% lev el 4 6 8 10 12 14 16
f ght myce-
lium
(g )
Koj ic ac id pr oduced (mg/ml)
_____ _L_ _ _______ _J _ _ _ _

Yeast 7 . 90 38 . 02 65 . 62 73 . 52 48 . 89 36 . 42 25 . 82 6 . 68
extract
Malt 0 . 35 0 . 89 1.58 1. 95 2 . 52 3. 82 4 . 65 1 . 60
extract
Cas i en 4 . 21 4 . 85 14 . 02 17 . 82 20 . 25 19 . 21 19. 80 2 . 20
hydrolysate
Peptone 5 .12 14 . 02 20 . 02 34. 6 1 46 . 02 50 ! 81 43 . 20 4 . 50
Beef extract 1 .15 7.15 23.02 38 . 62 6 1 . ?.2 46 . 20 35 . 61 5 , 30
Liver ext r ac t 0 132 2 . 06 19. 25 24 . 62 48 . 02 38 , 82 26 . 65 4 , 28
Tryptone 1 . 15 4 . 02 9 .15 12 . 80 19 . 02 17 . 81 17.02 2 . 04

--
f-
0
°'
 107

eff ects of amino acid supp l ement ation on myc e li a l growth

and kojic acid production ar e g iv e n in Tabl e 11 . The
maximum stimul at ory eff ect was ob s er ved in the ca se of
cystine . It increased th e y i e ld of kojic acid by 17.34%
This was f ollowed by lys i ne , aspa rtic acid and glycine
respectiv ely . The st imulatory eff ect of _aspartic acid is
reminisc ent of th e findigns of Reddy gt · a l. ( 27 ) a nd Gupta
(28) .
et fil • who obt a ined t he hig hest yie lds of af lat ox in
and incorporation of 14 C ac etat e in t o afl atoxins wh e n
aspartic acid wa s added to the medium .
Table 12 shows th e eff ect of adding vitamins
to YES medium on g rowth of~ . flavuc myc e lium a nd kojic
acid production. It is appa r e nt fr om t h is tabl e that ascor-
bic acid , folic acid and pyridoxine st i mul at e both th e
myc e lial g rowth and kojic acid f ormatio n. On t h e other hand
thiamine , ribofl avin and p antothe nic ac i d e xhibit ed a
marked inhibitory ef fe ct. This is r ath er unexp ect ed . It is
difficult to assign at pres ent any sat i sfactory explanation
to this inhibit ory eff ect . The mode of action of ribo-
flavin will b e d iscus sed in the l at e r chapt e rs . However
there was a good correlation bet wee n th e eff ects on
growth and kojic acid yie lds.
From above studie s, it is evide nt that with
the strain of h. flavus us ed , the optimum conditio ns f or
 )-- -.
)r
'"" '

- TABLE~ ~l
EFFECT OF AMINO ACIDS ON KOJIC ACID PRODUCTION
____ _.,,,.--
- - --
Period of incubat ion (days) ' Wet weight
o..rnino acid 4 6 8 10 12 14 of mycelium
added Kojic acid· produced (mg/ml) (g )
(200 mg/1)
-------------- - - - ~ - - ~ - --
Control 7 .. 12 30.52 58 . 02 70~28 38p92 28 .50 6 . 12
(No addition)
Glycine 7. 26 40 . 65 73.81 68 . 82 39 . 21 29,52 6.60

Alanine 6.50 l 36 .10 52.06 64 . 82 28 . 08 20 . 72 5 .. 68

Leucine 6 . 76 28 . 52 44 . 02 66 . 82 36 . 18 26 . 64 5 , 86
.
Threonine 5 . 50 29.52 53 . 96 58,82 29 . 72 22 ,21 5 ,14

Asp art ic acid 7 .61 32 . 20 58,60 76 . 20 41.04 30 . 82 6 . 60

Lysine 9 . 32 46 . 62 72.05 77 . 62 42 . 80 30 . 92 6,82

Cystine 8 . 16 38.24 58.98 82 . 42 48 . 52 33 . 52 7 . 10

T'jn.0S1ne 5 . 32 22, 82 40 . 8 1 69 . 52 37 . 21 26~92 6 ,10

Histidine 5 . 82 25.62 44 . 82 62 . 40 33 . 6 1 24 . 20 5 . 50

---- - --- ---

......
0
0)

..
 ~ -------1111111111111111111111111111111111111111
..._
'y
--(
"' )<
•
TABLE - 12
EFFECT OF VITAMI NS ON KOJ IC ACID PROCUCTION

Vitamin
add ed
(50 mg/1 )
T-:-
_l_
6 8
- ---- - -
Pe riod of incubation (days )
10 12 14
Eight
f
of yc elium
(g )
-- Ko i ~--S£lg__Qroduc ed (mg/ml) _J__~
Control 8 . 30 36 . 20 6 1 . 50 72 . 81 49 . 92 30 . 50 6 . 40
(No addit io n )
Thi ami ne 1.12 2. 20 3 . 65 4 . 62 1, 25 o. 99 L. 75
Ribofl avin 3. 70 13. 51 25 . 60 35 . 61 25 . 18 15 . 02 3 . 04
Nicotinic acid 10 . 10 24 , 6 2 41. 81 48 . 02 38 , 42 20 , 25 4 . 20
Pa ntot he nic 1 . 30 12. 82 24 . 0 2 2& .91 18. 95 10 . 50 2 . 69
acid
I nosito l 10 . 0 2 29. 81 50 . 51 58 . 50 32. 45 24 . 75 5 .16
Py ridox in e 13. 30 40 .. 5 1 68 . 60 79. 20 53. 45 33 . 02 6 . 70
Felic acid 12 . 80 46 , 21 78 . 02 89 . 14 59 . 50 37 . 50 7 . 60
Ascor b ic aci d 10 . 50 47 . 80 80 . 02 90 . 01 59 . 86 38. 25 7 . 80
Retinol 5. 20 28 . 6 2 40 . 80 46 . 0 2 35 . 50 19. 52 4 . 02

- -· - - - --
----
f--
0

'°
 110

koj ic acid production are the YES medium containing 20%

0
sucrose and 2% yeas t extrac t, a t e mper ature 30 C, pH 6 . 5
and an inoculum siz e of 10 6 spores/ml . The amino acids
cystine , l ysine , aspa r tic acid and glycine and the vit a-
mi ns ascorbic ac id , foli c acid a nd pyr idoxi ne we r e stimu-
latory .
The dat a present ed in Tables 4 to 13 sugg est ed
a clos e corr el ation betwee n koj ic acid production and
g r owth . Therefore the data in the indivi du al tables as well
as s ome comb inations of the tables we r e s ubject ed to st at -
istical analysis . The valu es we re fitt ed to a :-egressio n
line y = ax+ b , where y is the maximum amount of kojic
acid produced expr es sed as mg/ml of th e medium and xis
th e wet weight of the mycelium in grams . Sinc e both x
and y are subject to variation du e to experimental errors
th e following formulae wer e us e d to calculate a and b : -

a = / ~ ~ 't~ - C~ !J:) ~ , - - - - - -- - - - U)
'YI ~ x ~ - CEx )~

a_ (_ ~ )< ~ E.. { - ~ X ~ X £') C ~ r £ )( r -~ x_ ~t<) - - - - {Ii)

[ <?? J
~x~ -(;Ex)~ ['">i ~ t~- CE lf Y·J
Th ~ sig ns of a and b had to be ascertained (11)
separat e ly as their values ar e obt ai ned as squ a r e roots in
the abov e formulae . The sign of a was obviously positiv e .
Th e sign of
 111

b was det ermined from the fo rmul a

b = <Ev - n a E x (III)

Where n is th e nunt> e r of obs e rvations .

lne data from Tab l e 11 gav e an irrational value
for b when the formul a (ii) was us ed . So its actu a l value

• was also obt ained from formul a (iii) •

The r es ults of this a na lysis are pres ent ed in Table
13 and Fig . 5 . It is obvious that th er e is a close corre-
lation between kojic ac id productio n and growth . The v a lue
of the regression co effici ent a v arie9 f~om 10 . 42 to 15 . 70
and the v alue of b from - 33. 65 to + 6 . 61 . Th is v ari ation was
mostly du e to th e f act th at consid er able ch ang es in the
growth media were made in Tables 4 , 9 , 10 and this c a n be
e xp ected to aff ect th e abov e corre l ation. In Table 4 ., diff-
e rent media were us ed; in Tabl e 9 the conc e ntration of
sucros e was alt er ed and in Tabl e 10 y eas t extrac t was r ep l ac ed
by other substances . If the dat a from th es e t ables ar e
omitted , a f a lls in th e r ang e 10 . 42 t o 13 . 13 and b v ari es
from - 9. 46 to 6 . 61 . lne dat a a lso ar e close to t h e r e l e-
v a nt r eg r ession line (dott ed line ) in Fig . 5 .
However the v alu es from Table 12 sugg est th at
supplementation with some of the vitamins is consider ab ly
a ltering th e above correl at i on. With th e v a lues from Tab l es
2 , 3 , 4 , 5 and 8 th e correl ation is v ery good and th e calculated
 112

TABLE - 13
CORRELATION BETWEEN MYCELIAL WEIGHT AND KOJIC ACID
PROWCTION

Data from Va lue of Va lu e of

Table No. a b

4 15.70 -33!65
5 11.58 - 1!09
6 11. 94 - 3.02
7 10 . 42 + 6 .61
8 11.80 1.52
9 13. 99 - 23 , 09
10 13.27 -10.74
11 11. 60 - 1.57
12 13.13 - 9 .46
Total data 13.76 -14 . 64
(Tables 4 t o 12)
Total data minus 12.33 - 5.32
(Tables 4,9,10)
Total data minuc 11.77 - 2.03
(Tables 9,10,12)

The data we re fitted to the regression line

Y =ax~ b where y is maximum kojic acid yield
in mg/ml and xis the wet weight of the mycelium
in grams.
- CORRELATION BETWEEN MYCELIAL WEIGHT & KOJIC
ACID PRODUCTION

'

80

-E
"'~
-
e

'I'
'I'
'/

12 /
1i1 . '/
'/
C
- I
I

.,9 1Q
12
• I
I

1
/
.'
/
6 ,"
•
I
I

.
/
/
,"10
2 Ao
/
I ' .
9
/

2"03tr",,,...._.......
9. _---:!_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
I 2 ' 6 8
5•32 I
WET WEIGHT OF MYCELIUM (grams)
 113

• regression line has 9 slope of 11.77 and i s s hown in Fi g . 5

as a c ontinuous line . The v a l ues of b 9 re s ma ll and neg-
ativ e exc ept for the dat a f rom Table 7. The i ndi v idua l
T ab l es g ive ' a' v al ues f r om 10 . 42 to 11 .94 and ' b' va l ues
f r om - 3. 02 t o + 6 . 6 . The dist ributi~n of the indiv i du al
va lu es about th is r eg r es sio n l in e i s s hown in Fi g. 5 . The
c los e corr el at i on obt ained ind i cat es th at t he effects of pH ,
t emper ature , s pore inoculum, ca rbohydrat e s ourc e and supp l e-
me nt ation wi t h amino acids on kojic ac id pr oduct io n r es ult
ma inly from their eff ect on th e growth a nd henc e on the mas s
of fungal cel l s pres e nt. Ch anges in t he concentrat i on of
sucros e and r ep l acement of y east extract by ot her s ub st a nces
s eem to act directly on the koj ic acid pr oducing s yst em and
not t hrough their eff ect on growth.
 11'1-

RE FERENCES

11 Prescott s . c. and Dunn c. G., Indus t ri a l Microb i o-

l ogy , 3rd edit io n, Mcg raw Hi l l Book Co. New Yor k
1 959, p .537 ·•
2. Paturau J . M., Byproducts of t he Ca ne Sugar Ir,dustr :· .
An Introduction to the ir Industri al Uti lizatio~ ,
Els evi er Publishing Co., New York , 1969 .
3. Pr escott s.c. and Dunn C.G., Industrial Mi crobi ol ogyt
3rd edition, Mcgraw Hill Book Co . New Yo r k
1959, p.536.
4. Erkama J., Hag erst rand H.B. an d Jun kkone n S ~,Acta
Chem. Scand., 3 1 862 (t949).
5. Qu ilco A. and Dicapua A., Chimica and Industri a
(Mi lan ), ~ , 289 (1932 ).
6. Perl man D., Dorrell w. w. 9nd Johnson M. J ., Arch.
Biochem., .lQ , 131 (1946 ).
7. Ta miya Ht, Acta ~hytoch im. ~ap an., 3 , 51 (1927 )9
8. Barha m H. t:,J . , Ind. Eng. Ch em. (Anal. Ed., ) , l l ,
31 (19 39 ) .
9. Smits B.L., Trans. Kansas ~cad. S9i., ~2, 91 (19~4 ) .
10. Katagiri H. and Kitah~ra K., Bul l . Ag r. Che m. Se c ~
Japan.,~, 38 (1 929 ).
11. Gould B. S., Biochem. J ., ~ , 797 (1939 ).
 115

12 . Davis N. D., Biener U. L. and Eldridg e D. W., Appl.

Mic r obiol ., ~ , 378 (1966) .
13 . Reddy T.V., Viswanathan L. and Ve nkitasubramanian
T. A. ,Appl . Microb i ol ., 2i , 393 (1971 ) .
14 . Ady e J . and Mate l es R. I ., Bi ochlm. Biophys . Acta ,
86 , 418 (1 964 ).
15. Thom C. and Church M. B. ' The Aspergilli', The Willi ~rns
and Wilkins Company , Baltimore (1 930 ) .
16 . Gupt a S . R. Viswanathan L. a nd ve nketasubra manian T. A. 1
J. Ge n. Microbio l., ~ 243 (1971) .
17. Arnst ein H. ~. v . and Bent l ey R., Biochem . J ., 54 ,
508 (1953).
18 . Kinoshita K., Acta . Phvtoch im . Japan ., .]_, 31 (1927) .
19 . I mose J ., Nonomura s . and Tatsumi C. lAgr . Riol . Ch em.,
3 3 , 12 23 ( 1 96 9 ) •
20 . Imos e J ., Nonomura S . and Tatsumi C., Agr . Biol .
Ch em . 34 , 1443 (1970) .
21. Annual Report , Nutritional Research Laboratori es ,
HydGrabad , India (1966- 1967) .
22 . Tulpule P. G., Ind . J. Med . Res ., 21 , 102 (196 q ).
23 . May O. E., Moy er J . A. Wells P . A. and Herrick H. T.,
J . Am . Chem . Soc ., 2~ , 774 (1q31) .
24 . Bar ham H. N. and Smits B. L. Ind . Eng . Chem ., ,2
38 (1929) .
 116

25 . Shohei K., Kogy o Kagalru Zas shi , 72 , 513 (196 9) .

26 . Shohei K., Tsuyoshi F. and Tsujimoto K., Ha kko
Kyokaishi , .~ , 341 (1975 )~
27 . Re~dy T. V., Metbolic Stud i es on .,6§.Q~g illus flavus ,
Ph . D. t h esis, Univ ersity of Delhi , India (1972}~
28. Gupta S. R., Metabolism of ~ erg illu s with Ref er-
enc e to Aflatoxin Pr oductio n, Dh. D. th esis, Unive=sity
of Delhi , Ind i a (197 3) .
 )

fH~PTER - V
STUDIES ON FERMENTATION OF CANE MOLASSES

• J
 117

CHAPTER V

S1UDIES ON FE RMa-JTATION OF CANE MOLASSES

A f air]y large number of c a rb on sourc e s h av e b e en

fermented to ko jic acid by different microorganisms. Th e se
includ es sta r ches, dextrins, disaccha rides like sucrose and
maltose, hexoses like glucos e , fructos e, manno s e and galactos e,
pentoses like x ylose and arabinose, sugar a lcohols like
sorbitol, dulcitol, inositol and glyc e r o l and many oth e rs
like inulin, glycerophosphate, dihydroxyacetone, gluconi c
acid and t art aric acid. Out of th ese , howev e r t he best yie l ds
(1)
have in gene r al b een r eport ed from g luc ose and Xylose.
The different carbon sourc es have b een used for
kojic acid fermentation in c once ntrations v a rying f rom 5 t o
(2 )
30%•May and associ ates us ed sugar concentrations in the
rang e of 15 to 3 3% and obt a ined th e highest yie ld at 20%
glucose c onc entration with Aspergillu~ flavus • Barham a nd
3
Smits( ) found 15% xylos e to be most s u it abl e for kojic acid
4
ferment ation. On th e oth er h a nd, K3tagiri and Kit ahara( )
obtained satisfactory r esults with 5 % c o ncentratio ns of a
large nu rrb er of &Ubsto.nc es •
Howev er, v ery little information is av a il able on
th e fermentation of crud e c arbohydr ate sourc es like c a ne
juice, gur (jaggery) and molass e s t o kojic acid. Cane mo la-
sses has been ext e nsive ly us ed as an inexpe nsive sourc e of
)-
 118

ca r bohydrat es f or several industrial f ermentations such as

fo r the production of ethyl alcohol,baker ' s yeast, fo od
and fod de r yeast etc . (5 ) Its ave r age composition is o iven in
Tab 1 e 14 (6 ) •.

TABLE 14
COMPOS ITION OF CANE MOLA.SS ES
----------
Constituents Percent Ma in compo nents Perc ent com-
compo- position of
sit i on. ma in comr.o ne
ts.

Water 26 . 00
Silica (as S102) 0 !19
Pot as h ( as K20) 3. 50
~ Cale ium (as CaO) 1. 97
Ash 9 . 40 Magnes ia (as MgO) 0.18
Phosphorous ( as P2°'5 ) 0.10
Chlorine ( as Cl) 0 . 90
Soda (as Na20) 0 .30
Iron (a s Fe 2 03)
Simple carbo- [ Sucrose 35.45
hydrates
52 .5 Dextrose
and Levulose 15 .09
Nitrogenous [ Albuminoids 0 . 30
bodies
-( Amides (as aspa ra-
gine) 0 . 30

f
 119

Amino aci d (as l .7r::

aspartic acid)
Nit rogenous 2 .9 Nitric acid 0 .15
bo dies
Ammonia 0 . 02
Xanthine bodies 0 . 30

Other nitrogenous 0 .13

bodies
Soluble gums 2 .0 Xyl an , Pe ctin
and Araban
Free acids 2 .0
Combined acids 5 .0 Aconitic, Saccha-
rinic acid etc
Lip ids 0 . 15

It may be obs erved that cane molasses contains abou t 52.5%

of simple carbohydrates . This can potentially be fermented to
several industrially important products such as citric acid,
lactic acid , g l ucon i c acid , koj ic acid, glutamic acid, lysine
vitamin B12, acetone , butanol, amylase , dias ta se , glucose
o xidase etc . Its use for such microbial fermentations has been
mar ked ly hampered o n account of the deleter iGus effects of
i mpurit ies present in mo la sses on the concerned microor ga-
. 7
nisms . ( ) Pre 1 1m1nary
· · s t ud ies
. . our 1 ab ora t ory h aves h own
in
that Indian cane molasses can be easily used for the produ-
ction of kojic acid withou t any initial clarific ation and
purification~B) Kojic acid fermentat io n by Asg eraillus flfilLld.§.

)-
 120

strains appeared to exhib it a compart i v e ly great e r tolerance

to th e i mpuriti es pres e nt in molas s es than citric acid fer-
mentati on by b spe rgillus nioer.
Therefore, th e present investigations we re undert a ke
towards the dev e l opment of an economically viab l e process for
the manufacture f rom molas s es of kojic a~id , whi ch may be
put to several potential uses in industry .

MATERIALS AND METHODS

All the studies were carried out on a strain of
h. f l aY1!§. isolated in this laborat ory and t ermed as cultur e
Bin Chapter III . The methods pertaining to organism and
cultur al conditions have already b~en described in Chapt e r II
Aliquots were drawn from the f ermentation v ess e l at differ-
ent time intervals and analysed for kojic acid content after
diluting th e sample s uitably . Kojic acid was ass ay ed by
the method of Bentley( 9 ) . This method has also b ee n des -
cribed in Chapt er II •
The use of resuspended myc e lia for kojic acid
production appears to be a potentially useful t echnique .
The mycelia were grown on y east extract sucrose medium(lO)
for 10 days and we re wa5h ed thrice with 50 ml aliquots of
distilled water each time a nd then r esuspended in st eril e
molass es medium.
 121

The following types of molasses solutions were

used as fermentation med ia either as such or after supple-
mentation with 1% yea st extract • A medium containing un-
clarified mol asses s olution was used as a control .
a) Uncl arified Mol as ses
Va cc um pan sugar factory cane molasses was diluted
to 10, 20 and 30°C Brix concentration •
b) Chemically Clarified Mo la sses
lvb las ses was clarified chemically by treatment with
l . Su l phuric acid (ll)
(12)
2. Ammonium sulphate
(13)
3. Ammonium sulphate and superphosphate
4. Lime and superphosphate(l 4 )
5. Potassium ferrocyanide (i5 )
6. Egg albumin ( 16 )
The clarification proc edures have already been desctibed
in Chapt er II •
c) Ion Exchange Resin Treated Molasses
Mol as s es was clarified by treatment with
1) Anion exchange resin
2) Cation exchange resin
3) Anion and cation excha nge resins
The r e levant procedu~es have al s o been descri-
bed in Chapter II •
 122

.
d) Unclarified Molasses So lutions Supplemented With Salts
Vaccum pa n sugar factory cane molasses was dil uted
to 10° Brix and salts were added as in Doelger and Presc ott
(17)
medium . The sa lts used and their c oncentrations are given
below:
Salt Concentration
(g/1)

NH4No3 2 . 23

K2 HP0 1.00
4
WgS04. 7H20 0 .23 '

RESULTS AND DISCUSSION

Pr eliminary studies i n ou r l aboratory had shown

th at dilute s ol ut i ons of vacc um pa n sugar fact ory cane mol-
asses yield quite significant a mounts of kojic ac id. It
was felt that pr etreatment of molasses and its supplement-
ation with yeast extract and some s alts may further enhance
kojic acid produ ct io n. Henc~ present studies we re done using
different c oncentrations of unclarified molasses as well
as molasses clarified by different methods in the abs enc e
and pre s e nce of yea s t extract and s alts . It has a l so been
observed that resus pended mycelia of Asp erail l Y.§. flavu~
could produce larg e amounts of kojic acid in a simp le
 123

resusp e nsion medium c omprising of phosphate buffer and

sucrose or g lucose. This is described in detail in Chcp-
ter VI. Such r es us pension med ia not only economise on sugar
consumption towards fungal growth but also help in invest-
igat ing some funda me ntal metabolic pathways. It was there-
fore t empting to stud y if a similar fungal myc e lium grown
in YES medium could be successfully r esuspended in a mol-
asses medium and still produce kojic acid and whether addit-
ion of 1% yeast extract t o the mo la sses r esuspe nsion mediu m
exerted stimulatory effect on kojic acid production. Als o ,
it is well known that low nitrogen and hig h carbohydrate
media leads to higher production of secondary metabolites .
. (18 )
For example, YES medium g iv es high yi elds of afl atoxins •
This fact has sev eral i mportant implications for indus trial
fermentations. In view of the for e- go ing , the effect> of sup-
plementation with yeast e xtract and some salts on kojic
acid yi elds from molasses we re als o investigated.
It may be noted from Table 15 and Fi g . 6 that koj ic
acid pr oduction on unclarified mola sses start ed from third
day of incubation and its level increased upto 20 days •
The yields of kojic acid a lso increas ed with an incr ease
in molasses concentration fro m 10 to 30° Brix. No myc el ial
growth was obs e rv ed on 40° Brix molas ses solution even up
to ten days; however , it appeared on 11th day aft er which
it start ed forming kojic acid up to a maximum of 22 mg/ml
 124

}ABLE - l?
KOJ IC ACID PRODUCTION ON UNCLARIFIED MOLASSES SOLUTIONS

Period of incubation (days)

SL Med ium 3 6 9 11 14 20
No.
Koj ic ac id produced · (mg/ml)

l . 10° Brix 1.82 2 .40 3.02 4.72 6 .10 7.50

molass es

2 20° Brix 2 .15 3 .55 4.55 7 .. 02 8 .25 12.60

mo lass es

0
3 30 Brix 10.81 11.80 12.70 20.02 22 .42
mol as ses *

* Very little gr~wth was obseIVed, kojic acid was

not estimated •
KOJtC ACID PRODUCTION DURING GROWTH ON
UNCLARIFIED MOLASSES SOLUTION WITH &
WITHOUT 1°/o YEAST EXTRACT

o o o WITH YEAST EXTRACT

.... .. __ _. WITHOUT YEAST EXTRACT

80

_6
-
E
......
0\

-E
0
u
<t 40
-
u
30 BRIX

20 BRIX
__ ... 30 BRIX
20
,,, /
-- --
/

.---- _.. . --·

/
_.. 20 BRIX
.. -
.......- --- ~ ---- -- - 10 BRIX
..... ---...,- " _.r- ---
a::.---_-_.. - - - 1
o----~---~------~----~-----
' ..

3 6 9 12 15 18 21
RERIOO OF INCUBATION (DAYS,

Fig. 6
 I

125

by th e 22nd day. In th e ca se of 50° Brix mola s s es solut io n,

no mycelial growth was noted ev e n up to 20 days. He nce it
may be concluded that mola ss es solutions up to 30° Brix
are suitabl e for kojic acid fermentation . The r easons for
th e l ack of gr owth in t he more conc e ntrat ed mola s s es solut-
i on wer e not investigat ed in detail. This could be du e e i t her
to th eir high e r osmotic pressures or du e to the inhibit io n
by th e impuriti es or by th e inorganic compounds pr es e nt in
molasses .
The effects of supplementinq unclarified molass es
solutions of diff erent conc entrations with 1% yeast extract
are illustrated in Table 16 a nd Fig. 6 . It may be observed
t hat the presence of yeast extract in the medium not only
stimulated kojic acid production in all cases but also redu-
c ed the fer mentation pe riod (required to achiev e maximum
l ev e l s of kojic ac i d) f r om 20 to 14 days. Table 17 summarises
the r esults depicted in Tables 15 and 16 . It is evident from
it th at by a simple additi on of 1% yeast extract to the
0
unclarified molasses of 10 ,20 and 30 Brix conc e ntration,
p€rcent yield of kojic acid calculated on the basis of
initial sugar concentration increased from 15, 12 .6 and
16 . 13% to 46 . 3 , 40 . 02 , 53 . 3% respectively . This amounts t o
208 , 217 and 230 percent increase in kojic acid yields,
respec tively . Hence it may be safely co ncluded that a v ery
good medium for the commercial production of koj ic acid
 126

T.@LE - 16
KOJIC ACID PRODUCTION ON Uf\CLARIFIED MOLASSES SOLU-
TIONS SUPPLEM8'JTED WITH 1% YEAST EXTRACT

sL Period of incubation (days)

No . Medium 3 6 9 11 14 20
supplemen-
ted with 1% Ko jic ac id produced (mg/ml)
yeast extract
0
l 10 Brix 9 .. 52 15.20 16.02 17.21 23.10 7 .15
mol asses

0
2 20 Brix 16.40 22 .41 25 . 20 25 . 82 40 .02 26 . 82
mo lass es

0
3 3 o Bri x * 33 .8 35 .40 73.21 80 . 02 35 .60
molasses

* Very littl e gr~wth was observed; kojic acid was

not estimated ,.
 127

T.@LES - 17
EFFECT OF YEAST EXTRACT ON MAXIM!)M KOJIC ACID YIELDS
FROM UNCLARIFIED MOLASSES SOLUTIONS

SL Medium Maximum kojic Perc ent yield* Perc ent in-

No. acid produced of kojic acid cre ase in
(mg/ml) kojic acid
~~:-:T~---m----~----m:rri=~:z:--~--Production
Withou~ vvith ye- intnou1:. W1l-n due to
yeast ast ext- yeast yeast yeast
extract tract extract extract ·ex-tract
-------------·- -
l 10 o Brix
. 7.50 23.10 15,0 46 .2 208
molasses

2 20° Brix lZ.6 40.02 12.60 40 .02 217

molasses

3 30° BriK 2 2 .42 80 .02 16 .13 53 .30 230

molass es

* Calculat ed assuming a total sugar concentration

of 50% in molasses .
 128

i s 30° Brix mo l as ses s ol ut i on suppl emented with 1% y east ext-

r act. The above not ed stimulatoxy effec.,t of yeast extract on
kojic acid pr oduction is in agree me nt with th e find ings of
Gupt a ..e1 a L(l8 ) who obt ained high yields of aflatoxin by toxi-
g enie strain of Asll§rqillus flavus on a medium cont a ining
y east e xtract. 'The y east extract possibly sti mul at es g r owth as
we ll as the kojic acid synthesizing enz ymes by providing a
number of nutrie nts, cofactors and trac e e l e me nts. 'In es e s t imu-
latory factors pres ent in the yeast e xtract, eve n if th ey are
identified, would be quite costly to be add ed in th e pur i fi ed
form in a comme rcial f e rmentation proc es s . Yeast e xtract app-
arently stimulat es both th e synthesis and degrad ation of kojic
acid causing their occurre nc e in a c omparitiv e ly shorter p er-
iod of ti me.
Certain f ung al ferm entations ar e v ery s e nsitive to
the impurities pr esent in mola sse s and the yie lds ar e incre-
ased conside rably by t he clarificatio n of mola sses. A well
known examp l e of this is th e citric acid f e r me ntation b y
h• niger. Th er efore , th e eff ects of clarification of mol ass es
by chemical methods on kojic acid p roduc t ion, in th e prese nc e
and abs enc e of yea s t extract were inv estigat ed (Tabl e 18
and 19). It may be obs e rv ed that in med ia 1/1.hich are not supp-
l e me nt ed with y east extract, clarific at i on of mola s s es by
ammonium sulphat e or by egg albumin increas ed kojic acid pro-
duction from 6 .1 t o 6.42 mg/ml and 7.45 mg/ml r es pectiv e ly.
 129

TABLE - 18
KOJIC ACID PRODUCTI ON ON CHEMICALLY CLARIFI ED 10° BRIX
MOL ASS ES SOLUTIONS
------ --
Sl. Clarificat- Period of incubation (days)
No . ion proced- 3 6 9 11 4 20
ure used Koji c acid p r oduced (mg/ml 1
--- -- -----
l Cont r ol No 1.82 2 .40 3 . 02 4.72 6 .10 7.50
clarif icat-
ion
2 Sulphuric 1.20 2 .08 2 .45 2 .50 3 .45 5 . 35
acid
3 Ammonium 1.52 3.25 3.32 3.55 6.42 4.52
sulphate
4 Ammonium 1.55 1.92 4.55 4.80 5.02 5 .65
sulphate and
superphosphate
5 Lime and 1 . 65 2 . 80 3.95 4.65 5 . 85 5.05
superphosphate
6 Potassium 1.22 1.40 1.65 4 .45 5.02 4 . 35
ferrocyanide
7 Egg albumin 2 .31 3 .36 5.02 5.56 7.45 5 .02

--
 130

I~BLL.::_19
KOJIC ACID PRODUCTION ON CHEMICALLY CLARIFI ED 10° BRIX
MOLA-SS ES SOLUTIONS SUPPLEMENTED WITH 1% YEAST EXTRACT
----- ---------
Sl. Clarification Period of incubation (days)
No . procedure 3 6 9 11 14 20
used Kojic acid produced (mg/ml)

l Control No 9 . 52 15.20 16 .02 17.21 23.10 7 .15

clarification

2 Sulphuric 2.82 4.35 5 .62 5.65 6.65 10 .78

acid

3 Ammonium 1.15 1.42 1 .6 5 2.86 3 . 28 5 . 86

sulphate

>- 4 Ammonium sul- 2.95 4 .55 5.02 9 .56 13.50 9.02

phate and
superphosph ate

5 Lime and 4 . 55 6.50 8 .35 11.35 17.75 7.15

superphosphate

6 Potassium 5 .45 6 .20 6.65 8 . 85 16 .78 10 .56

ferrocyanide

7 Egg albumin 6 .15 8 .02 12.48 15 .26 19.02 4 .65

-- --
 130

Howev er, clarific at ion by ammonium sulphate 1nd s upe rphos phat e
or lime and superphosphat e or potass i um fe rrocyanide or s ul-
phuric acid r es u lt ed in a decline i n koj ic acid pr oduct i on.
On the othe r hand , al l the clarific ation proc edur es decre ased
the yi eld obt ained on th e mo la sses mecHum supp l ement ed \,dth
y east extract. The maximum yi e l d of 23 .l mg of koj ic acid/ml
was obtain ed on th e unclarifi ed mola sses medium £Upp l emented
with ye ast e xtract.
The ef fe cts of yeas t extract on th e uncla rifi ed
mol asses med ium and the diffe rent cl arified molas ses med ia
ar e r epres e nt ed in Table 20 , which compares th e data of t he
previous t wo Tabl es. Yeas t extract increas ed the yeilds on
t he unclarified mo las ses media by 93 to 234%, exc ept in the
case of clarificatio n by ammonium sulph at e alone , where yeast
extract caus ed an inhibition.
Both molass es and yeast extract are complex mat er-
i als containing s ev e ral inorganic ~nd or ga nic constitu e nts .
The cl arification proc edur es adopt ed may b e expect ed to r em-
ove part of t h e th es e constitu e nts f r om molasses a nd at the
same time introduce c ert a in substanc es like ammonium , s ul-
phate and f errocyanid e ions etc. In view of the complexity
of th e compone nts pres e nt in molasses and y ea st extract and
th e nature and amount of components remov ed from the same
duri ng cl arific ati on it is v e ry dif ficult to provid e comp l ete
theoritical explanation for t h e diffe r e nt effect s that h ave
 1 31

TABLE - 20

EFF ECT OF YE AS T EXTRAC T ON KOJIC ACI D PRODUCTI ON ON CHEM-

I CALL Y CLARIFI ED 10° BRIX MOLASS F.S SOLUTI ONS

Sl. Clarification Yeast ext- Ko jic acid Pe rc ent ch anc

No. proc e dure ract (1%) produce d in koj i c ac ic
used (mg/ml) in product i on du
14 days t o yea st ext-
ract

l Control No clarification 6 .10

Control No clarification + 2 3.10 + 27 8

2 Sulphuric acid 3.45

Sulphuric acid + 6 .65 + 93

3 Ammonium sulphate 6 . 42
Ammonium sulph ate + 3. 28 49

4 Ammonium sulphate 5 .02

and superphosphate
Ammonium sulphate + 13 .50 + 169

and suoerphosphate

5 Lime and s uperphosph ate 5.85

Lime and s uperphosphate + 17.75 + 203

6 Potassium ferrocyan id e 5 .02

Potas sium ferrocyanide + 16 .78 + 234

7 Egg albumin I 7.45

I

Egg albumin + 19.02 + 155

----------------------
 132

been r eported i n the abov e Tables .

Kojic acid production , similar to that of many
oth er s econdary metabolites , is high on a high carbohydrate ,
low nitrogen medium and th e yields are high on a sucrose
yeast e xtract medium. Some kojic acid ts pr oduced on a mo l -
as ses medium not supp l e me nt ed with yeast extract , sinc e mol-
asses itself co nt ains s everal g rowth factors lik e amino acids ,
vitamins etc i n varying amou nts. Apparently thes e are not
pres e nt in optimal amo unts and supp lementation wi t h yea st
extract causes a marked increase in kojic acid yields . It ha s
also been noted that c ertain vitamins and amino acids stimu-
late kojic acid formati on wh il e some vitamins oreatly inhibit
this proc ess .
The s light increase in kojic ac i d formation obs er -
v ed in unsu pple ment ed media by clarification with ammonium
sulphate or egg albumin may be du e t o th e r e moval of inhi-
bitory compounds . Suppleme ntation w1 ~yeast extract in almost
all the cas es appare ntly provides several stimulatory fa ct-
ors and increases yi e lds . The data on th e yeast e xtract
supple ment ed media sugoe st that th e clarification proc ed-
ures are probably r e moving some of the stimulat ory fact ors .
Cl arifi c ation by ammo ni um sulphat e introduc es ammonium i o ns
in the medium and this alt ers the ratio of nitrog e n and
carbohydrate and thus r educ es koi ; c acid productio n . It has
already b een mentio ned that kojic acid yi elds are high on a
 133

high carbohydrate and l ow nitrogen medium.

The compone hts of y east extract ar e affecting not
only th e synthesis but al s o the degradation of kojic acid.
Whereas koj ic aci~ l ev e ls go on incre asing even upto 28 days
in mola s ses media, suppl ementation with yeast extract leads
to a fall in ko jic acid l ev els afte r th e 14th day.
Irresp ectiv e of the mechanisms, involv ed in the
effects described upto this point, it is quit e obvious that
a 30 Brix molasses medium supple mented with 1% yeast ext ract
is a v ery satisfactory medium for kojic acid formation a nd
gives more th a n 50% conversion of the carbohydrat e pres ent
to kojic acid. But, the f e rmentation period is rather long ,
about 14 days.
Kojic acid production from ion exchang e r esin tre-
ated mol ass es solutions without and with yeast extract supp-
l ementation ha s been pres ented in Tabl es 21 and 22. It may
be obs erv ed from Table 21 that with unsuppleme nt ed mola s ses
media no significant incre ase in th e kojic acid product io n
is_ achiev ed by treatment of molasses either with anion (Duolit
A-100) or cation (Duolit e C-300) or with both the r esins.
Kojic acid production increased with th e p eriod of incub at-
ion in all the cases reaching th e maximum valu e in 28 days.
The r e moval of cations only lower ed th e initial rat e of ko jic
acid production, but th e final yields on 28th day we r e com-
parable with that of control. But the r e moval of anions
 '
j
... • ~ t

TABLE - 21
KOJIC ACID PROilJCTION FROM ION EXCHANGE RESIN TREATED 10° BRIX MOLASSES S011.JTIONS

Sl . Period of incubation (days)

No . Treatment 3 6 9 12 18 20 24 28
Kojic acid produced (mg/ml)
------
l Control No tre at- 1.82 2 .42 3.27 4.97 6 . 75 7 . 55 9.86 12 .75
ment

2 With anion exch- 2 . 48 2 .72 3.55 4 .26 5 .04 5 .46 6 . 67 7 . 82

ange resi_n

3 With cation exch- 1.04 1. 85 2.72 3.55 6 .30 7 . 45 9 .73 13 . 70

ange r esin

4 With both anion 0.55 1 . 03 1.37 1 .77 3.08 3 . 52 5.53 7.45

and cat ion exc h-
ange resin s
--

......
w
~
 ,.._,
~ ~

-. ~ ~
*'
TABLE - 22

KOJ IC ACI D PF.0 DUCTI°'1 FROM I ON EXCHANGE RES I N TREATEE 10° BRIX MOLASS ES
SOLUTIONS SUPPLEMENT ED WITH 1% YEAS T EXTRACT

S l. Tr e at me nt Per iod of incubatio n (d ays)

No . 3 6 9 12 14 16 18 20
Ko j ic acid p roduc ed (mg/ ml)

l Co nt r ol No 9 . 52 1 5 . 20 16 . 02 - 23 . 10 - - 7 . 15
tre atmen t

2 With an io n exch-
a nge r esin
6 . 92 8 . 62 5 . 35 3 . 12 - 3 .05 2 . 55 1 . 12

3 With c 2ti on exch-

ange r es in
7 . 60 9 . 21 4 . 65 1.72 - '
1. 50 1.15 0 . 75

4 1J.1it h both an ion

and cat -to n exch-
8 . 85 18 . 82 1 8 . 35 8 . 36 - 8 .12 3 . 92 1 . 75

ange r esi ns .
----- - - - - - - - - --- --

f---
w
(.JI
 136

or of both anions and c ations r educ ed t h e yie ld by about 50% .

Thus in~h e unsupp l ement ed mo l asses medi a , th e anio ns ar e more
important t han c at i ons fo r ko jic acid p roduction .
The molasses medium supp l eme nt ed with y east extract
gav e somewhat differ ent r esults , p r obab ly du e to t h e ef f e ct
of an added paramet er , that is the deg r adati on of kojic acid
(Tab l e 2 2) . He r e also i o nexchang e treat me nts d id not incr ease
kojic acid yie lds. Tr eatment with anion exchang e r esin r edu-
c ed th e yie lds as in th e previous e xperiment. Howev e r th e
yie lds we r e low a l so on molass es f r om which c ations had been
r e moved . On r emoval of both anions and cations , a peculiar
eff ect was ob s erv ed . Though th e l ev e ls of kojic a cid we re
comparabl e with th e untreat ed control , th er e was a Sudde n
drop in th e conc entration of kojic acid by th e 12th day •
Appar ent ly th e d egradati on of kojic acid was stimulat ed under
th es e conditions . On the ba sis of Tabl es 21 and 22 , it may
b e concluded that lik e che mical cla rific ation , ion exchange
r esi n treatment of mol asses too , could not e nhanc e kojic
acid production, wh eth er th e medium was supple ment ed with
ye as t extract or not.
Fr om the for egoing discussion , it is appare nt th at
anionic and cationic c ompone nts of mo lasses p lay a signi-
ficant rol es in cont ro lling kojic acid f ermentation. Thes e
would include inorg anic components also . Th e r efor e it was
conaidered desirabl e to add to the me dium thos e salts
 137

including inorganic nitrogen sources which ar e the major

compone nts of synth e tic media for fungal growth . The s alt
mixture used contained ammo nium nitrate , magnesium sulph ate
~nd dibasic potassium phosphate .
Supplement ation with t his s a lt mixture incre a sed
kojic acid yields on a molasses medium ( Table 23 and Fig . 7)
from 7 . 5 to 15 . 43 mg/ml. At the same time , it a l so incre ased
the r ate of kojic ac id degradation at l at er st ages of growth ,
that is after the 14th day . The increase in koj ic ac id proc ,1- 1

ction could be attribut ed to the increased growth of the

fungus due to the extra nitrogen sourc e ~vai l able . The d~t a
obtained on a y east extract suppl ement ed mol asses medium were
just opposit e . Th e add ition of the s alt s mixture decreased
kojic acid yields by about 50%. In this c ase the molasses
plus yeast extract medium was close to the optim~l medium
for kojic acid ferment ation. Addition of th e salts mixture
prob ab ly acted by a lt ering the nitroge n to c arbohydrate r atio
as the salts mixture cont a ined ammonium nitrate .
The e xp erime nts described till now in t his ch apter
utiliz ed growth medium in wh i ch both 0 r owth of the funa us as
wel l as kojic acid synthesis were occurring . It is quit e
likely that many of the effects observed on koji c acid yie lds
weresecondary their primary effect being on th e growth
process its e lf a nd not on kojic 1cid synthesis. It has a lr eady
b ee n pointed out in earlier chapt e rs that th er e is a close
 . - . •.&'
l~ ,
i
,t
"'
TABLE .=__23
EFFECT OF SALTS AND YEAST EXTRACT ~N KOJIC ACID PRODUCTION ON
UNCLARIFIED 10° BRIX MOLASSES SOLUTIONS
-----------------------------·-----·- ---- ---------------
Sl. Supplements Period of i ncubati on (days ) Maximum koj ic
No. added t o the 3 6 9 11 14 20. acid yield as
molasses sol- Koji c acid produced (mg/ml) perc e nt of
_ _ _ __Jdt i o._n_ _
--·------ ~ar-2.2d e d _

l Control No add- 1 . 82 2 . 40 3 . 02 4 .7 2 6 .10 7 . 50 15.0

ition

2 Salts * 5 .56 6 . 82 8 .12 10.92 15 .43 4 .12 30 . 8

3 1% y east 8 .81 12.61 14 .02 15 .62 21 .. 50 6 .32 43 .o

e xtract

4 s~1ts• ~ 1% 4 .12 5.06 5 .56 7.02 11.25 3 . 05 2 2.50

yeut ~t~~~
--------------------
* As describ ed in the s ectio n on ' Materials and Methods'

f--
w
00
-EFFECTS OF SALTS & YEAST EXTRACT ON
tSQJIC ACID PRODUCTION ON UNCLARIFIED 10
as1x MOLASSES SOLUTION

1. NO ADDITION
2. SALTS + 1•1. YEAST EXTRACT
l SALTS
4. 1•1. YEAST EXTRACT

25

-......-2
E
CJ'

--oe
u
15
4'
u
a1
~

9 12 15 18 21
PERIOD OF INCUBATION (DAYS)

Fig. 7
 139

correlation betwee n growth and koj ic acid production. Ther e-

fore , it w~ s f elt desir able to r epeat some of th e above experi
ments using already grown and r esusp ended myc elia of ,b. f l avus
The conditions for obt a ining kojic acid synthesis by such a
r esusp ended myceli a·, as we ll as consider ab l e amou nt of data
on this syst em hav e been described in Ch apt er VI . The fungu s
was first grown f or 10 days on an y east extraet sucros e med-
ium and then r esusp ended in dif fe r ent tyP eS of mo l asses med i a .
It was found th at unc l arifi ed unsupplement ed mola-
s ses s olutions support ed fairly high kojic acid product ion by
r esuspended myceli a (Tabl e 24 and Fig. 8 ) • This is in c ont r ns t
to th e low yi elds obtained W"l en these s olutio ns were us ed
as growth media (Tab l e 15). S'inc e th e r esusp ended myc elia c ould
produ ce kojic acid from buf f er plus glucose or s ucros e as
shown in Ch apt er VI ~ the r equirements fo r this proc ess are
not as el abo rate as for gr owth, Thus good yi elds are obt ai ned
on molass es alo ne . Mo lass es solutions whe n us ed for r esus-
pension, a llowed the degradation of koj ic acid a lso. This is
also in contrast with th e r esults given by mol asses s olutions
as growth media . Th e period of ons et of degradation s ee ms to
-
be r elat ed to the initial carbohydrate conc entration. With an
increase in the Brix of molasses, th e degradation start ed at
lat er periods of incubation. Th e exact medh anism by which
kojic acid degradation is tontrolled is not clear.
...
. •
~
~
-
TABLE - 24
KOJIC ACID PRODUCTION BY MYCELIA RESUSPENDED ON UNCLARIFIED
MOLASSES SOLUTIONS
-
S l. Medium Peri od of incubatio n (days )
No . l 3 5 7 9 11
Kojic acid produced (mg/ ml )

0 .
l 10 Br1x 8 .60 12 . 20 lQ . 26 18 . 86 4 . 28 2 . 20
mo l asses

2 20° Brix 6 . 50 25 . 20 39 . 92 42 . 41 10 . 81 5 .10

molasses

0 •
3 30 Brix 7. 02 8 .70 29 . 20 55 . 21 72 . 02 32 . 0 4
mol ass es

~
~
0
 l<.OJIC ACID PRODUCTION BY MYCELIA RESUS-
PENDED ON UNCLARIFIED MOLASSES SOLUTION
WITH & WITHOUT 1°/o YEAST EXTRACT

o o o WITH YE AST EXTRACT

... _ --•- --.. WITOUT YEAST EXTRACT

_ 60

-E
0\
E
._,
I
\
\
\
0 I

u \
<{ \
40
-•u
0
:it:

}'

PERIOD OF INCUBATION

Fig. 8
 A
~
~

-. • -"fL_ .

T~L~ ~

KOJIC ACID PRODUCTION BY MYCELIA RESUSDE~DED ON UNCLARIFIED MOLASSES SOLUTIONS

SUPP LEMENTED WITH 1% Y~AST EX TRACT

Sl. Med ium Period of incubation (day s)

No. l 3 5 7 9 11
Koj ic acid produced (mg/ml)
-------------
l 10° Brix 10 .60 11.65 11.95 5 . 02 0 . 55 0 . 24
mo l asses

2 20 ° Brix 7.60 13 . 52 36 .29 36 .02 2 . 81 1.20

molasses

3 30° Brix 9 . 60 10 . 02 19 .21 40.04 62 . 43 26 .02

rno l 3s ses

1--
~
I\)
 ~
~ ~
~ • '"'Yi...

TABLE- 26

KOJIC ACID PRO[UCTION BY MYCELIA RESUSPENDED ON I ON EXCHANGE RES I N TREATED

10 o BRIX MOL ASS ES SOLUTIONS
-
Sl-. Treatment Period of incubation (days)
No. l 3 5 7 9 11
Kojic acid produced (mg/ml)
------
l r;ontrol No 8 .6 12 . 20 19 . 26 18 . 86 4 . 28 2 . 20
treatment
·'
2 livith anion ex- 2 . 40 8 .10 13.92 15 .7 2 16 . 30 13 . 92
change ·resin

3 V"ith cation ex- 4 .30 10 .42 15 . 82 · 16 . 92 14 .60 12 . 02

change r esin

4 With both anio n 1.40 6 .20 · 8 .41 ·., 10.40 18 . 92 12.60

a nd cat ion exch-
ange res ins

f-
.i::,.
w
 ....
),'~
... ""'- ~

TAB LE - 27
KOJ IC ACID PRODUCTION BY MYCELIA RESUSPENDED ON ION EXCHANGE RESIN TREATED

10° BRI X MOLASSES SOLUTIONS SUPPLEMEN TED lflITH 1% YEAST EXTRA.CT

------------------
Sl . Treatment Pe ri od of incubatio n (days)
No . l 3 5 7 9 11
Ko jic acid p r oduced (mg/ml )
- - - - -- ----
l Contro l No 10 . 6 11 . 65 11 . 95 5 . 02 0 . 55 0 . 24
t r eatment

2 With anio n ex- 2 . 50 10 . 60 9 . 62 2 . 65 2 . 42 1 . 55

change res in

3 Wi th cat io n ex- 5 . 02 12 . '4 1 7 . 42 0 . 42 0 . 15 0 . 11

chr1 nge resin

4 With both a n io n 1.02 7 . 12 8 . 32 14 .7 2 18 . 82 14 . 42

a nd cat io n
exchange r es i ns

.......
+::,.
+::,.
 145

with yeas t extract , th e degradation was reduced to a large

extent and the ko jic acid l eve ls were comparable with those
obtained on unsu pp lemented mol asses or unsunplemented mola-
sses treated with both the r es ins. 1hough th e eff ects desc~i-
bed are quite c omp lex, it wou l d appear that at least t hr ee
different g r oups of substancre-s are necessary for obtaining
a fast deg r adat ion of koj i c ac id. These are a) some a nionic
components of mo l asses b) some c ationic components of mol-
asses and c) s ome c ompo nents of yeast ext r act.
Some of the above findings h ave been s umma ris ed i n
Tab le 28 fr om which it is evident that usi ng 10° Brix unclari-
fied molasses s olution only 15% of sugars ar e conv erted int o
kojic acid , which may be rai sed upto a three fold leve l
either by usi ng th e same molasses soluti on with r esusDended
myceli a (38 .5%) or by addi ng 1% y east extract to it and
usi ng it as growth and fermentation medium (46 . 2%) • It may
also be r evea l ed from Table 28 that tr eatme nt of mo l asses sol-
utions with ion exchange res ins or additio n of yeast extract
to the r esuspe nsion media could not enhance the kojic acid
production. 1he r esu spe nsion sys tem is partic ula r ly interest -
ing f r om th e point of view of l arge scale fermentatio ns.
Good kojic ac i d yie lds ar e ob t a ined during th e init i al gr owth
on the yeast extract sucrose medium. Probably a mol asses
yeast extract medium can also be used for this purDos e . The
-same mycelium ca n be r es uspended i n a mo lasses med ium and
 147

furth er kojic acid can be obtained. The mycelium s eems to

maintain its activity even aft e r a very long per iod a nd it
is hop ed that it can be used again a nd aga in with several
batches of molasses s olutions.
 148

REFERENCES

1. Pr escott s.c. and Du nn C.G., I ndustri al Microbiology,

3rd Editio n, McGraw Hill Book Co ., New York , (1959)

2. May O. E., Moyer A. J . Wel ls, P. A. and Herrick H. T.,

J. Arn . Chern. Soc ., ~J, 774 (1931).
3. Barham H. N·. and Smits B. L., Ind. Eng·. Ch em., 28 , 567
(1936 ).
4. Katagiri H. and Kitahara K., Bu ll. Agr . Chem. Soc.
Japan, 2, ~8 (1929).
5. Paturau J.M., By products of the Ca ne Sugar Industry.
An Introduction to th eir Industrial Vtilization , Else-
vier Publishing Co ., New York (1969 ).
6. Olbrich H., Princ i ples of Sug ar Technology , Vol III,
Frlited by Honig P., Elsevier Publishing Company,
New York (1963).
7. Banerjee N. and Viswanathan L., Proc. 5th Joint Conv en-
tion of Sugar Technologists' Association (India ), Decc an
Sug ar Techno logists ' Associ atio n and South Indian Suga~
Cane and $ugar Technologists' Associatio n, G-77 (1975).
8. Ag r awa l P.K. and Viswanathan L., Ibid, G-115 (1q75).
9. Bentley R., Methods in Enzymology, Vol. III , Edited
by Colowick S . P. an~ Kapl~n N. O., Academic Press,
,, New York , (1957), p . 238 •
 149

10. Dav is N. D., Di e ne r U. L. a nd Eldridge D. W., Appl.

Mic robiol ., 14 , 378 (1966).

11 . Re ich G.T., Trans . Inst . Chem . Eng ., ~ , 1049 (1942).
12 . Shukla J . P . and Kapoor B. D., Proc . 26th Annual
Convention of Suga~ Tech nologists ' Association (India),
G-39 (1958 ) •
13·. Walter F .G., Manufac ture of Compressed Ye ast , Chap ma n
and Ha ll (1956) , p . 131 •
14. Arroy R., Int. Sugar J., 45,250 (1 943).
\

15. Walle r C. J ., BINS Fina l Report No . 48 9 , Ite m No . 22,

(1946) •
16. Hans e n A. and Jorgens o n A., Microor ganisms and Fer-
men tations , Charl e s Griffon and Co ., London (1939),

p . 72 •
17. Do e lger W. P . and Prescott s .c ., Ind . Eng . Ch e m.,

2~ , 1142 (1934) .
18. Gupta S . R., Viswanatha n L. a nd Venk it a subramanian
T.A., J . Gen . Mi crobiol., 2~ , 243 (1971) •
 CHAPTER-=--.Yl.
KOJIC ACID PRODUCTION BY RESUSPENDED FUNG~L MYCELI ~

,
 150

CHAPTER - VI
KOJIQ_ACID PROI:UCTION BY RESUSPENDED FUNGAL MYCELIA

Like antibiotics and aflatoxins etc, kojic acid is

generally considered to be a secondary metabolite. Not much is
known about the signifi cance of secondary metabolites and the
mechanism of their synthes i s by fungi. Some studies have
been reported by different wo rkers on the mechanism of bio-
( l to 6 ) (7 to 13)
synthesis of aflatoxins and ko jic acid
However, the studies pertaining to the mechanism of kojic
14
acid synthesis were mainly r estricted to c incorporation
studies and some probable mechanisms were tentatively sugg-
ested . It was therefore considered desirable to investigate
the v al idity of proposed mechanisms on the basis of concrete
experimental data . An ideal system for such investigations
wou ld be a c el l free enzymatic system. Preliminary efforts
in this direction were not successful . As such, the next
best alternative, namely , a g rown and resuspended mycelial
system was resort ed to . I t has been pointed out i n earlier
chapters , (Chapter IV) that several factors affect growth
primarily and koj ic acid production is altered as a secondary
result of the effect on the growth of the fungus. There is
also a clos e correlation between the wet weight of the my-
cel ium and the amount of kojic acid producee. Thus, to obtain
clear data on factors primarily affecting kojic acid sy-
nth es is and degradation , a system bas ed on resusp e nded
 151

mycelia is ideal. The deve l opment of such an experimental

system as well as th e effe cts of sev eral parameters on its
effici ency are dealt with in this chapt e r. Although YES med-
ium has been r eport ed to b e the most favourable medium for
kojic acid production, it is unsuitabl e for carrying out th e .
fundamental studies on th e mechanism of kojic acid synth esis,
on account of its undefined composition . (l 4 )The pr esenc e of
yeast extract in this medium leads to th e probab l e int erplay
of its effects on a number of r elat ed metabolic pathways, t h us
making it difficult to give reasonable int erpretationsof t h e
results . Hence, it was conside r ed desirabl e to s earch for a
ch emically defin ed medium for r esuspension of th e myc e lium
so as to facilitate fund amental studies regarding the mech-
anism of kojic acid b iosynthesis.

MATERIALS AND METHODS

All th e studi es were performed on a nontoxigenic

strain of Asp_grqillus flav!d§. isolat ed in our laboratory a nd
named as culture B, as me ntion ed in Chapt er III . 1n e methods
pertaining to organism and cultural conditions have already
bee n describ ed in Chapt er II , 5 ml of th e uniform spore sus-
6
pension containing 10 spor es/ml was used to inoculate 50 ml
of the growth medium contained in 250 ml Erl e nmeye r flasks .
All the fungal myc elia meant for use in resus-
pension studies we r e grown on YES medium whos e pH was
 152

previously adJust ed to 6 ;5 • The flasks wer e incubat ed at

0
30 C as static cultur es. Su itabl e al i quot s wer e dr awn from
t be f e r mentation v essel at differ e nt time int erv als and ana-
lys ed for koj ic acid cont e nt aft er diluting th e sample suit-
ably . On the 10th day , whe n maximum amou nt of koj ic acid was
produced , whole intact myc e lia were taken out of fe r ment at-
ion vess el , and r esuste nded as follows. Th e pr oc edur e for
r es us pe nsion has been describ ed in 01apter II •
l. Resusp ended as such in 50 ml of YES med ium, 0 . 2M phos-
phat e buffer pH 6 .5 with or without th e followi ng supp-
l ements:
20% glucose , 20% sucros e kojic acid (12mg/ml ) or kojic
acid p l us g l ucos e (12 mg/ml each)
2. Th e myc elia wer e prei ncubat ed i n b uf fe r a lone for seven
days and t he n r esuspende d in 50 ml st eril e buffer + 20%
glucos e .
3. The myc el ia grown in YES med ium fo r three weeks, were
resus pended i n 50 ml sterile buffer+ 20% glucose.
4. The mycelia were cut into two , fo ur or ma ny fi ne pieces
and the n resuspendee i n buffer+ 20% g lucose and buffer
+ kojic acid (12 mg/ml) or they were resusp ende d together
t

with a full intact mycelium ih 10 0 ml buffer + 20% glucos e .

5. The mycelia were r es us pend ed i n st erile buffer and homo-
genised with a pestle and a mortar or were disinteqrated
by treatment in a Waring blender fo r l or 5 minutes , then
 153

the volume was made upto 50 ml and a ca lc ulated amount of

gluc ose or s ucro se (20 0 mg/ml each) or kojic acid (12 mg/ml)
was add ed to th is mycelial suspension a nd incubat ed as usual .
In most of the experi ments, the resus pe nded mycelia we r e in-
cubated under s tatic conditions. But i n some ca ses , espec i-
ally where fragmented mycelia were used the res uspe nded rrry-
celia were incubated also on a rotary shake r. The pH of the
r esuspension mediu m was adjusted to 6 .5 in all t he cases . The
samp l es from the resus pe nsion sy stems we re a naly sed for kojic
acid cont ent periodic ally. The procedures for med ia sterili-
zation and kojic acid assay hav e already been described in
Chapter II.

RESULTS AND DISCUSSION

It has been alread y s hown that the yeast extract
sucrose (YES ) medium is a n i dea l medium for g r owth and kojic
acid product ion by }.s12,grgillus flaY,.!!S (Chapt er IV) . Th er efore ,
this medium was us ed for initial l y growing the fu ngal rrryc el ia.
Table 29 demonst rat es the rate of kojic ac i d production during
th e g rowth of Asperqill us f lsf:LQ§. on YES medium. It woul d be
observed that kojic _acid levels increased with the period of
inc ub ation markedly , r eaching a maximum in 10 days(81 . 5 mg/ml)
after which kojic acid content decreased r apidly . Therefore
10 days old mycelia were us ed for resuspe nsion experiments.
Diffe r e nt exp erimental conditions were test ed for carrying out
the r esuspension. Vfu en intact mycelia of~ . flavu~ g rown
 154

TABLE -_22
KOJIC ACID PROIVCTION DURING THE GROWTH OF ASPERGILLUS
FLAWS ON YEAST EXTRACT SUCROSE (YES) MEDIUM

Period of in)ubation Ko jic acid ~roduc ed

(da ys (mg/ml
---
4 9 .98
5 18 . 26
6 36 . 98
7 42 . 82
8 66 .30
9 72. 22
10 81.50
11 66 .20
13 32 .6 8
15 20 .96
17 12.52
20 8 .53

---
 IB5

on YES medium for lO _days were r esuspe nded in 50 ml buffer

after pr oper washing , very little kojic acid was fo rmed
(Fig . 9 ). It was observed that only 0 .14 mg/ml of koj ic acid
was pr oduc ed in 14 hours and after that the levels of kojic
acid decreased rapidly. The small amounts of kojic acid detec-
ted wer e probably intracellular koj ic acid diffusing out of
mycelia during incubat ion . This was du e to total absence of a
carbon sou rce in the r esus pe nsio n medium. However, whe n r es -
us pe nsion of the washed mycelia was done in 50 ml of sterile
YES medium or in st eril e buffer co ntain i ng 20% glucose or
sucrose, koj ic acid was produced almost to the same ext e nt
as in case of g rowth medium (YES ). It wo uld be noted from
Fig . 9 that in all such cases koj ic acid levels increas ed with
the period of incubation and r eached the maximum in 8 days and
decreased thereaft er . Thus, the intact mycelia could produce
80 . 2 , 78.4 and 70.4 mg kojic acid/ml whe n r esuspe nd ed in YES
medium, buffer + gluc ose and buffer+ sucrose respectively.
The abi lity of different buffers to s erve as resus-
pension buffers , after supplementation with glucose was stu-
died. Table 30 r epres ents koj ic acid synth esis by int act my-
celia r esus pended in different buff e rs containing 20% glucos~
The buff ers used were O.l M citrate buffer, 0 .15 M citrate pho-
sphate buffer, 0 . 2M acetate buffer, O.lM tri ethanolamine
 KOJIC ACID PRODUCTION BY MYCELIA OF A f
RESUSPENDED IN DIFFERENT MEDIA

1. • • YEAST EXTRACT SUCROSE (YES) MEDIUM

2. • • 0"2M PHOSPHATE BUFFER t,H s·s
,3. - 0·2 M ,, " pH 5·5 + 20°/o GLUCOSE
4. --- 0·2M " ~, pH 5·5 + 20°/. SUCROSE

80

--E
60 .
I

...... ''
'
~,
O'\
E '' '
I

-
0
(J I
I

ct J. I

~
"'""
0
~

•
2

0-----........,_.~--------..---------
J. 8 12
PERIOD OF INCUBATION (DAYS)

Fig. 9
 ... + • • . .~

I6BLI_=- 30
KOJ I C ACi u PF.ODUCTION BY A. FLAWS MYCELIA RESUSDENDED IN DIFFERENT BUFFERS OF
PH 6 . 5 SUPPLEMENTED WITH 20% GLUCOSE

Buffer s Molari t y Period of inc ubatio n (days )

( M) 1 2 3 4 5 6 7 8
Kojic acid pr oduct ion (mg/ml )
------------------
Tris ma leatc . 20 3 . 88 5 . 20 13 . 30 17 . 62 41 . 23 71 . 54 81.73 69 .7 3
Phos phate . 20 6 . 40 8 . 38 1 3 .06 17. 32 40 . 20 69 . 26 79 . 80 68 . 20
Tris . 20 4 . 28 5 .60 12. 53 15 . 40 38 .02 65 .62 76 .02 64 .62
Cit rate . 10 4 . 39 5 . 80 10 .75 14 .02 33 .02 56 . 92 66 . 92 68 . 20
Triethano l am- . 10 3 . 39 4 . 52 9 . 82 13 .72 30 .02 52 . 36 58 .90 51.02
ine hydroch-
loride
Citrate .15 3.70 4 . 92 9 . 23 12 .10 28 . 50 49 . 20 57.02 48 . 42
phosph ate
Acet ate . 20 3 .78 5 .02 9 .02 12 . 02 27 .73 48 . 10 55 . 12 46 . 92
Glycyl glycine .10 1 . 59 2 .10 3 . 43 4 . 52 10 . 53 18 . 32 20 . 91 17 . 93

- -----------------
.....
(Jl

°'
 157

hydrochloride buff e r , O.lM glycyl glycine buffe r, 0 ,2M

potassium phosphat e bu ff er , 0 . 2M trisbuffer and 0 .2M tris-
maleate buffe r . All t he buf fe rs had a pH of 6 . 5 . It would be
observ ed that the bes t yie lds of kojic acid were a iven by
t r ismaleate, phosph ate and trisbuffe r. In all s ubseque nt
studies, pho sphate buffer ha s bee n used r outinely,
It is apparent that h• .flsl~ mycel ium can be fir s t
g rown in a favourable gr owth medium like YES medium till it
ma r ked l y develops the ability to synthesize kojic acid a nd
then it can be resuspended into a simp le fermentation mediu·.1
for successful produc tio n of kojic acid ev en on a commercial
sc ale . Although Kitada and Fuk i mbara (l 3 ) have employ ed the

• resuspension technique for investigating the mechanism of

co nv ersion of glucose t o kojic acid , no attempt as yet has
been made t o exploit i t s fea s ibility f or commerc i al appli-
cation . The use of a cheap er r esuspensio n med ium containing
ca ne mola ss es for commerc ia l pur poses is described in
Chapt er V. On th e basis of data pr es ented in Fig . 9 , it may
be calculat ed that an intact mycelium r esuspended in YES
mediu m or i n buffer containing glucose or sucro se may yie ld
40 .l, 39 . 2 and 35 . 2% koj j c ac id respectively on the bas is
of i nitial sugar co nc entratio n as compa r ed to 40 .75~ y ield
obtained duri ng th e grovrt h o n YES medium • It s hows that res-
uspension of the intact myce lium does not impair its ability
to synthesize kojic acid on YES medium or buffer containing
 158

glucose or sucrose .
Th e data presented indicate the need for external
substrate for the production of ko jic acid . 11:e endogenous
material p r es ent in the mycelium i s not converted in to kojic
acid . The ex act quantity of e ndogenoussubstrates present in
the myc elia have not bee n determi ned in this case . On the
contrary resuspe nded myc elia of Aspergillus niq er produce near-
ly the same a~ou nis of citric acid IAhether phosphate buffer
used for resuspension is supp lement ed with g lucose or not(:6 )
Appare ntly c itric acid is produced from endogenous substanc es
and not fr om the add ed glucose .
An important feature of this r esus pension system
is the lag period of some days b efor e ko j i c acid production
rate picks up , though the mycelium had b een producing kojic
acid at a fast rat e just before r emoval f r om the original
growth medium . The r eas on f or this lag is not cl ear . Probably
some chang es have to t a ke p lace int he external medi um , such
as for example , the accumu l ation of some c ontro l ling metabo-
lit e , before kojic acid can be produc ed at a considerable
r ate . Sev eral attempts to devis e procedures for r educing the
lag period were not successful .
• The r esusp ension of myc e lia in buff er alone even
upto sev en days did not inactivate the kojic acid synth esizing
enzyme system, sinc e on r esusp endi ng this mycelium again i n
fresh buffer c o ntaining g l ucose , large amounts of kojic ac id
 159

were synthesized . fith such preincubat ed mycelia kojic actd

production increased slowl y with the period of incubation,
reached the maximum on 10th day and then decreased rapidly
(Fig . 10 ) . The maximum kojic acid yield was 73.62 rrg/ml repre-
senting 36 . 81% cornrersion of glucose to kojic acid . ¥~en fungal
mycelia or microbial c ells are r esus pended in buffer , in the
abs e nce of a carbon source, usually endogenous substrates are
metabol is ed over the period of sev e ral hours and are ev e ntu ally
exhausted . Some enzyme systems are also inactivated and
mycelia incubat ed in buffer for many hours are usually inc2
pable of carrying out many o~ the synthetic processes. For ex-
ample , h• ~rasiticus mycelia preincubated in phosphate buffer

• 14
for 6 hours loose their ability to incorporate acetate-1 c
into aflatoxins . (l?) Therefore , it is surprising to note that
b• f l,ay!J.§ myc elia r etai.n tr e i ! ability to form koj ic acid from
glucose even aft er pr eincubation in buffer for 7 days in the
absence of g lucose . The concerned enzyme syst em appears to be
unusually stab le. Also , it is possible that the formation of
koji c ac i d from glucose is not energy depende nt and thus the
integrity of . the e nergy produ ction syst em is not so important
in this c ase .
It is int e r esting to obs e rve that even thre e weeks
old mycelium (when kojic acid content in the fermentation
medium has fallen to v ery low levels) could s till form kojic
 KOJIC ACID PRODUCTION BY DIFFERENT TYPES
OF A. f LAVUS MYCELIA ON RESUSPENSION IN
0·2M PHOSPHATE BUFFER, r:2H 6.5 + 20°/o GLUCOSE
TYPES OF MYCELIA
GROWN FOR 10 DAYS IN YEAST EXTRACT SUCROSE (VES) MED,
..
o •

•---- ,, ,, ,, " 9J •

8. PAE INCUBATED IN O 2M PHOSPHATE SUFFER pH 6'5 ALONE

FOR 7 DAYS
• • GROWN FOR 3 WEEKS IN YEAST EXTRACT SUCROSE {VES}MED.

80

\
:::60 '
E ' '•,
"""Ol ''
E '~

0
u
<t
u4

20

-+

oL..:~-------4----~-----8--------~~12~---=-------::,G
~ - ~ - ~ - - - - - ~ - -- --,...._- " c ,i.., c uo L!L:\ hl J..nA'iS l ~ - - ~ - - - J
 160

acid to the same extent (75 .62 mg/ml) if it is resuspended

in buffer containing glucose . In this case , maximum Droduction
of kojic acid took place on 12th day and the yield was 37 . 81%
o n th e basis of initial sugar concent ration (Fig . 10) . This
provides another example of the stability of the kojic ac id
synthes i zing enzyme system . It ' s activity is still maintained
intact ev e n after growth on YES medium for 3 weeks . By this
period , koj ic acid l evels in the origi nal orowth medium fall to
low l evels and one would expect th e kojic acid synthesizing
enzymes to be inactive and th e koj ic acid degrading syst e m to
be act ive . But this is not so . Apparently th e synthesis or
degradatio n of koj ic acid is determined by some environmental
conditions in the surrounding medium rath e r tha n by the pr es-
ence or abse nce of the r e levant enzyme systems . It has not yet
been possible to id entify th e controlling factors or condit-
ions i n th e medium .
The above fi ndi ngs are significant not only from
the fu ndamenta l point of view but a l so from the point of

• view of i ndust r ial app licati on. Th e use of resusp ended my-
celia for commercia l manufacture of koji c acid would not only
make crude and inexpensiv e carbohydrate s ources useable for
the production of kojic acid , but would also provide the
basis for the l ater dev e lopment of similar syst e ms for
comme rcial manufactu r e of citric and lactic acid also .
Secondly, it would a lso lead to increas ed yie l ds since the
carbohydrate which would otherwise hav e bee n utilised for
 161

mycelia l growth would be f,dved for conversion to koj ic acid .

It wou l d also lead to the development of a commercial process
for continuous fermentation of carbohydrates to kojic acid
by us ing the same mycelium for lon~ periods of time, thus
resulting into a saving on raw materials , fermentor volume,
labour and other costs . Thus the cost of koj ic acid manufact-
ur e may be substantially reduced . Further it has been noted
that after completion of the fe r mentation, in addition to
the kojic acid pres ent in the fermented medium some amount of
it is present in the mycelial pad itself which may be effici-
ently extracted without damaging the mycelium , thus increas-
i ng the final yields .
In the experiments described till now, the mycelia
were resus pended under stationary conditions . In contrast
to the static resus pension studies , when Aspergillus fl~~
myc e lia were resuspended in flasks placed on a rotary sha ker ,
much small er amounts of kojic acid were synthesized . It may
be noted from Fig . 11 that only 20 . 82 rng/ml of kojic acid was
synthesized in 8 days . This represents 10 . 41% conversio n of
glucose to kojic acid as compared to 39.2% yield given by
the i nt act mycelium resusp ended under static conditions .
Two possible reasons c an be suggested for this finding . Under
aerobic conditions , g lucose may be oxidized to a greater
-+- extent and thus less of clucose und er aoes conversion to
kojic ac id . Alternatively, the degradatio n of kojic acid may
 KOJIC ACID PRODUCTION BY A. fLAyU5- MYCELIA
RESUSPENDED ON [2M PHOSPHATE BUF~ER,
RH 6.5 UNDER STATIONARY & SHAKING CONDIT IONS

• 0 STATIONARY CONDITION
• • SHAKING "

80

60

-E
.......
C7I
E
-
0 1.0

u
.,
0
~

8 l2
PERIOD OF INCUBATION (CAYS)

Fig. 11
 162

be fast er und e r ae r obi c co nditio ns . Howev er, this expe riment

was carri ed ou t to serv e a s a contro l for comparison with the
exp e rime nts wit h r es us pended myc elia l fragme nts to be des -
cribed next .
With a vi ew t o deve l op e a c e ll fr ee syst em capable
of p r odu cing kojic acid from glucos e , homoge nates of ,h. flaY:1!.§.
myc elium wer e t est ed but t h ey fail ed to de monst r at e any acti-
vity. In order to find out th e r easons for this failur e ,
s ev eral experiments were carried out. These are describ ed be low
Vhen th e mycelia wer e homogeniz ed in th e bl e nder
or ground i n pestl e and mortar, only v ery small amou nts of
koji c acid were synthesized upo n r esuspension in buffer or
buff er + 20% glucos e medium under stationary or shaking cond-
itions (Fig 12 and Tabl es 31 and 32). Th e bl e nding or th e
grinding of th e myc elium h ad compl et e ly brok en it up as obs er-
ved in th e microscop e . Howev e r th e pres e nc e of a small number
of spor es or ev en of int a ct mycel i a l c e lls could not b e fully
ruled out. Th es e might have grown furth e r during r esusp e nsion
and produced small amounts of koj ic acid. It is like ly that
disrupture of the myce lium might caus e a physical damag e to
th e structure and or to th e activity of th e koj ic acid syn-
th esizing enzyme syst em. Anoth er possible r e ason might be
th e r e l ease of c ertain substanc es or degradative enzy mes
that might inactivate th e kojic acid synthesizing syst em or
 .. + )po-
>- ... I
OMPARISON OF KOJIC ACID PRODUCTION BY INTACT MYCELUIM AND BY
MYCE LIAL HOMOGENATE
.__ INTACT MVCELUIM RESUSPENDED IN 0·2M F i g. 12
PHOSPHATE BUFFER pH 5·5 + 20•1. GLUCOSE
8 8 1 MYCELIAL Ho.10GENATE IN 0"2M PHOSPHATE BUFFER
•--- pH 5·5 + 20•1. GLUCOSE

_ MYCEUAL HOMOGENATE IN 0·2 M PHOSPHATE

BUFFER pH 5·5

--E
6 6

• I ~

-
--
GI
E
-E
..- ,
Cl
0
E ( .)

c4 ,,,..
-
u Sil ,
, '\
<(
0'""" , \
\
X I '
~
~
,'. \
I '

i I
0,

,,
\
'' \
/ \
2 2 ' ''

,
I
,'
I
I '
I
' \

.
\
\
\
\

,If ',
/ \
~ \
Oi...._____________________.,..___________ I

4 8- 12 - 4 8 12
PERIOD OF INCUBATION (DAYS)

•
 16::i

J'\BLE- 31
KOJIC ACID PRODUCTION BY INTACT , FRAG 1ENTED AND HOMCGENISED
MYCELI A OF A. FLAVUS DURI NG RESUSPENS I ON ON O . 2M PHC6PHATE
BUFFER, pH 6 .5 PLUS 20% GLUCOS E UNDER STATIONARY COND ITIONS

SI . No . l 2 3 4

Type of One full One h a lf Two quart- One full cut

myc e li a l int act pie c e e r p i ec es i nto fine
prep ar at - pie c e bits
ion us ed
Wet we ight
of myc e li-
um (g) - ~ ·o - - - - 4 . 5 4 . 45
- - - - - - -9 .-20- - -
Per i od of
incub ation
(days ) Kojic acid produced (rrg/ml)
------- -
0 0 . 35 0 .10 0 .12 0 .30
l 1 . 96 0 .68 0 .72 1 . 12
2 4 . 45 1 . 53 1.62 2 . 83
3 9 .79 3AO 3.60 6 .17
4 16 . 91 5 .78 6 . 12 10 .71
5 2P . 70 q. 18 q .7 2 17 .02
6 48 . 06 16 . 32 17 . 28 30 . 24
7 55 . 60 19 . 04 20 .02 35 . 28

8 70.42 23 . 30 25 .62 44 . 30
9 59 .63 20 .6 1 21.60 37 . 72

10 53 . 42 16 .02 18 .08 33 . 36

11 48 . 95 11 .66 13.64 30 . 87
Max imum 440 . O 25 8 . 9 287 . 6 240 .8
koj i c acid
production
mg/g of rny-
£ e lium ---~
 164
c o ntinu ed ..... 7ab l e 31/

Sl.No . 5 6 7 8
•
Type of One fu ll One full One f ul 1 Che full
mycelial well ground int a ct p i e c e int ac t p i ece intact piec e
prepara- with sand piu3 2 h a lf plus 4 qua r- plus o n e full
ti o n us ed and buf f er pie c es t er pieces cut i nto fine
bits
Wet weigh-
t of myc e-
lium (g ) 9 . 12 16 . 92 17 .0 1 16 . 81
Period of
incubation Kojic ac id produced (mg/ml)
(d a ys)
- - - - ·-- --
0 0 . 27 0 . 21 0 .18 o .18
1 0 .60 0 . 93 0 . 98 1.03
2 1.02 1.96 2 . 25 2 . 38
3 1 .62 4 .45 5 . 23 5 . 80
4 2 . 88 8. 32 8 . 56 o .0 1
5 3 . 20 12 .74 13 .52 15 .32
6 4 .43 20 .52 22.26 22 . 42
7 3 . 60 27 .44 28 . 83 2q .68
8 3 . 18 33.82 35 . 52 38 . 21
9 2 . 94 29 .12 30 . 89 32 .75
10 24 . 38 26 . 52 27 .65
11 28,54 22 . 59 24 . 97
Max imum 24 .18 200 . 0 208 . 8 227 . 3
kojic a cid
production
~/g of .
myc e lium
 165

}~LE- ~i
KOJIC ACID PRODUCTION BY INTACT , FRAG MEI\JTED AND HOMcx:;E-
NI S ED MYCELIA OF A. FL!l,VUS DURING RESUSPENSION ON O . 2M
PHOSPHATE BUFFER ,pH 6":°5PLUS 20% GLUCOSE ON A ROTARY
SHAKER

SI . No . l 2 3 4 5
Type of One full O~e half Two qua- One full One fulJ.
myc e li- intact piece rter cut in well grou nd
al pr ep- piec e p i eces to fine with sand
aration b its and buffer
~~ ·~--------~~
Wet 8 .62 4 . 29 4 . 36 8 . 52 8 .78
weight of
mycelium
(g)
Period of
inc ubat- Kojic acid produced (mg/ml)
ion (daw___ _

0 0 .10 0 .02 0 .05 0 .17 0 . 28

l 0 . 54 0 .16 0 . 21 0 . 37 0 .58
2 1.32 0 . 3( 0 . 48 0 . 85 0 . 92
3 2 . 86 0.83 1 . 04 l . 86 1 .45
4 5 . 94 1 . 44 1. 82 3 . 21 2 . 22
5 8 . 82 2 . 29 2 . 85 5 . 07 :3 .64
6 13 . 04 4 . 08 4 . 63 8.02 3 .1 2
7 16 . 12 5 .10, 5 . 94 10 . 20 2 .42
8 20 . 82 6 . 28 7. 56 12 .53 2 .02
9 17 . 42 4 . 76 5.82 11 . 22 1.65
10 15 .64 3 . 53 4 . 61 9 . 68
11 14.32 2 . 16 3 . 44 8 .62
Max i mum 120 .77 73 .19 86 .69 73 .53 20 .72
koj ic ac id
production
mg/g of my-
celi~u=m___________~ ____
 M6

degrade ko jic acid it se l f . The lat er pos sibility appears to

be more plausibl e as s hown b y data obtained with partly h roken
myce lia (Tab l es 31 and 32) . The presence of broke n myc e lia
r educ ed the koj ic acid l ev e ls attained by intact myc elia •
Kojic acid production und e r static an d sha king
conditions by fragm ent ed myc e lia resus pe nded in buff er+
glucose is r epr esent ed in the Tabl es 31 and 32 . It may be
observ ed from th e Tabl e 31 that half p i ec e of one full myc e lium
produced 258 . 9 mg kojic acid/ g of myc elium as compared t o
440 mg kojic acid/g of myce lium synt hes ized by th e int act

myc elium r esuspende d in buffer+ glucos e . Similarly two quar-

ter pi eces out of one full myc elium and fine bits of one full
myc elium p roduc ed 287 .6 a nd 240 . 8 mg ko jic acid/g of mvc elium
respectiv ely only .
In the c ase of £haken co ndit io n 6 . 25 , 7 . 56 and 12 .53
mg kojic acid per ml wer e pr oduc ed by ha lf pi ec e , two quart er

pi ec es and f ine bits of one full myce lium r esp ectively when
r e susp e nd ed in buf fe r+ g luc os e (Table 32 ) whil e th e intact
r e susp ende d myc elium in buffe r+ glucos e , produc ed 20 .82 mg
kojic ac id/ml unde r similar c o nditi ons . Furth er whe n t wo
piec es , four piec es and fin e bits of one full myc e lium were
r esuspended together with one intact myc e lium, kojic acid
yi elds we re ag ain lowe r ed to 200 to 2 Z7 . 3 rng/g of myc e lium
(Table 31) • It may thus be s een that the pres e nc e of brok en
 167

myc elia degraded ev e~ th ~ ko jic acid produc ed by normal

intact myce lium . Thi s s eems to be the r easo n for th e l ow
yie l ds obtained du:-cii ,.J t h e r es us pe nsion of br oken or bl ended
myc e lial fragments,
Ev e n with th e intact myc e lium on a g r owth mediu m,
koj ic acid deg r adatio n usually s et in aft er a c e rtain period
of time . Therefor e th e deqradation of koj ic ac i d by intact
and by brok en myc elia was inv estigat ed . Fig . 13 shows ko jic
acid degradation by int act fungal myc e lium r esuspended in
buffer+ kojic acid . It wou ld be observ ed th at koj ic acid
deg radation in creased r apid ly wit h the period of i ncubation
and the degradation was mo r e than 90% i n about a we ~k's
time . It appea rs t hat th e organism is utilizing kojic acid
as a s econdary carbon or e ne rgy sourc e . Whe n intact myc e lium
was r esus pended in buff er suppl e me nted with koj ic ac id a nd
glucose , fu r th er amou nt s of koj ic ac id wer e synth esized up
to 48 hours., t hen it ·Nas degraded by the organism (Fig, 13 ),
It may be further r ev ea l ed from th e data pr ese nt ed in Fig . 14
that fragment ed myc e lia also deg r aded koj ic acid ef f iciently
It i s int e r esting to obs e rv e that ev e n th e compl et e ly homo-
genised myc el i a were able to degrad e kojic acid very effect-
iv ely (Fig , 15) in static or shak e n condition . Actually th e
r at e of degradation was much fast e r tha n in intact or frag-
ment ed mycelia (Fig . 14) . Henc e it may be concluded that
disruption of myc elium inactivat es koji c acid synthesis
DEGRADATION OF KOJIC ACID BY INTACT MYCELIUM
OF A; f LAVUS RESUSPENDED UNDER STATIONARY
CONDITION
20
RESUSPENSION MEDIA
• • 0"2M PHOSPHATE BUFFER pH 6°5 • KOJIC ACID ( 12mg/ml)
• • 0·2M ,, ,, ,, ,, + GLUCOSE
(12 m9 / ml tach)

_J2
E
'-
Cl

-
0
E

u
ct

..,
~8
0
~

0,-.--------:-----------,.---------.....----------
8 · 12 16
PERIOD OF INCUBATION (DAYS)

Fig. 13
DEGRADATION OF KOJIC ACID BY INTACT A ND
FRAGMENTED A. f LAV US MVCELIA RESUSPEN
0·2M PHOSPHATE BUFFER, RH s·s + KOJIC ACID
(12mg/ml) UNDER STATIONARY CONDIT~ON

• • ONE FULL INTACT MYCELIUM

16 •---.. TWO HALF PIECES OF MYCELIUM
• • ONE FULL MYCELIUM CUT INTO FINE BITS

--E
-Ol

-
0
E

u 8
<(

u
~
0
::-::

o-----------~--~~----~----.. .
I. 8 12
PERIOD OF INCUBATION (DAVS t
Fig. 14
DEGRADATION OF KOJIC ACID BY HOMOGENISED
MYCELIA OF A. fLAVUS ON 0"2M PHOSPHATE
BUFFER, pH 6'5 + KOJ~C ACID (12 mg /ml) UNDER
SHAKING & STATIONARY CONDITIONS

16 1. HOMOGENISED FOR 15 SEC.

SHAKE CONDITION. { . ,,
2
3.
,, " 5 MINUTES
,, 15 SEC.
STATIONARY CONDITIO..J { .
4 '' 5 MINUTES

12
--E
.......
0,
E

-a
u
<{
u 8
~
0
~

o---------~---------------------
2 4 6
PERIOD OF INCUBATION (DAYS)

Fig 15
 168

and stimulates the abilit y t o degrade koj ic acid .

Ver:y litt l e informatio n is availab l e on the path-
way of degradation of kojic acid by h • flavus and the rele-
vant enzyme systems . It has been reported that a kojic acid
decompos ing bacterium Arthrob~ter .Y.r~afac iens strain K- 1
degraded kojic acid by the following path way (lB , l 9 )

~oj 1C. 0-C, to l

(.I-\ D y Hi.DH CH~

H-1-oH - D . ';;\ a.fa c.~i..0 G--o r
> CDA- ~ - CO C:t-\~
I J~a.{c ~e.{_c,f. I co
1-\ c,.....G- H ..th,'l rru'"'hA,9>~ k O- C- H toc H Ac.eJ,j co A.
I I
HO-f-i-\ HO- c- 1-\
H-r--Oli H-{-oH
COOH COOH
 169

Some int ermed iat es of this pathway have been identifi ed and
some enzymes like kojic ac id oxidase , comenic aldehyde
dehydrog enas e a nd D-ga lacturonat e ketol i s omerase have been
purif i ed and charact e rized . Evidence is also available for
14 14
th e conversion of c kojic ac id to c aflatoxins i n AspeI-
gillus parasiticus, probably through the intermediate for-
14 (20)
mation of C ko jic acid •
 170

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418 (1964) •
2. Do nke rsloot J . A. a nd Mat e l e s R. I ., J . Am. Che m. Soc .,
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Mo ody D.P ., Natur e , 202 , 1 88 (1964) •

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subramania0 T. A., Experentia , 25 , 1141 (1 Q69) .

5. Holk er U. S . E. and Underwood J . G. , Chem . Ind . , 1 865
(1965) •

6. Basappa s .c., Sre e nivasamurthy V . an d Parpia H. A. B.,

J. Gen . Microbial ., .£1_, 81 (1 970) .

7• Ch a 11 e ng er F • , K1 e in L • and Wa 1 k e r T • K • , J • Ch em •

S oc., 1498 (1929) .

8. Corb ellini A. and Gr egorinii B., Gaz z . Chim. Ital .,

..§Q., 244 ( 1 930 ) •
9. Birkinshaw J.H ., Charl es J .H.V., Lilly C. H. and

Raistrick H., Trans . Roy . S oc . (London) , B220 , 127

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10 . Ar nstein H. R. V . and Be nt l ey R., Natur e 166 , 948 (1q40)

11 . Arns tein H. ffil . V . and Bentley R. , J . Chem. Soc .,
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12 . Ar nstein H. R. V. a nd Bentl ey R., Bioch em. J ., .21

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 17 1

13 . Kitada M. , and Fukimbara T., Hakko Kagaku 7asshi ,

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15 . Davis N. D. , Di ener U. L. and El dridge D. W. , App l.

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to Aflatoxin Producti on, Ph . D. thesis , Univ e rsity of
Delhi , Indi a (1973 ) .
18 . Imose J. , No no mu r a S . and Tatsumi C., J. Agr . Chem .
Soc . Japan ., .11 , 94 (1967 ) .
19. I mos e J ., No nomura S . and Tatsu mi C. , Agr . Biol. Ch em . ,
11 , 1 223 (1969 ); 34 • , 1443 (1Q70 ) ; 35 , 2025
(1971) .

20 . Tulpul e P. G. , Ind . J . Med . Res . , 2I , 10 2 (1969) .

ISOLATION AND CHARACTERIZATION OF KOJIC ACID
FROM ASPERGILLUS FLAVUS AND OPTIMIZATION OF
BIOPROCESS FOR ITS ECONOMIC PRODUCTION

Thesis Submitted to the GITAM University

In Partial fulfillment for the award of the Degree of
Doctor of Philosophy in Biotechnology

By
K. BALA DURGA DEVI, M.Phil.

Under the guidance of

Dr. B. VEERENDRA KUMAR, M.Sc., Ph.D.
Department of Biotechnology
GITAM Institute of Science
GITAM University

DEPARTMENT OF BIOTECHNOLOGY
GITAM UNIVERSITY
VISAKHAPATNAM
MARCH- 2015
 DECLARATION

I certify that
a. The research work contained in the thesis is original and has been done by me
under the guidance of my supervisor.
b. The work has not been submitted to any other University for any degree or
diploma.
c. I have followed the guidelines provided by the University in writing the thesis.
d. Whenever I have used materials from other sources, I have given due credit to
them by citing them in the text of the thesis and giving their details in the
references..
e. Whenever I have quoted written materials from other sources, I have put them
under quotation marks and given due credit to the sources by citing them and
giving required details in the references.

(K. BALA DURGA DEVI)

Date:

i
 CERTIFICATE

This is to certify that the thesis entitled Isolation and characterization of

Kojic acid from Aspergillus flavus and optimization of bioprocess for its
economic production, submitted by Mrs. K. Bala Durga Devi to the department of
Biotechnology, GITAM UNIVERSITY, is a record of bona fide research work
carried out under my supervision.

Dr. B. VEERENDRA KUMAR, M.Sc., Ph.D

Assistant Professor
Department of Biotechnology
GITAM Institute of Science
GITAM University
Date:

ii
 ACKNOWLEDGEMENT

The work presented in this thesis would not have been possible without my
close association with many people who were always there when I needed them the
most. I take this opportunity to acknowledge them and extend my sincere gratitude for
helping me to make this thesis a possibility.

I am highly indebted to my research director, Dr. B. VEERENDRA

KUMAR, Assistant Professor, Department of Biotechnology, GITAM Institute of
Science, GITAM University for his immense help, cooperation, scholarly inspiration
and valuable guidance that he had extended to me for the successful completion of
this investigation. I am grateful to him for his constructive criticism and moral support
throughout the research period. I wish to express my sincere thanks to him for his
continuous support and help in successful completion of this thesis.

I would like to express my heartfelt gratitude and sincere thanks to

Prof. S. V. RAJAGOPAL, Head of the Department of Biotechnology, GITAM
Institute of Science, GITAM University for his valuable suggestions and
encouragement for completion of this thesis.

I am thankful to Dr. N. LAXMANA DAS, Dean and Principal, GITAM

Institute of Science, GITAM University for giving me an opportunity to work in this
prestigious institute and making all the facilities available for carrying out my
research work successfully.

I express my sincere thanks to Prof. K.V. RAO, Dr. M. ANITHA, Dr. P.

SHANMUKH ANAND, Dr. T. SRINIVASAN, Dr. N. SAI KISHORE, Department
of Biotechnology, GITAM Institute of Science, GITAM University for their constant
support and assistance throughout the research work.

I express my sincere thanks to Dr. G. SRINIVASA RAO, Department of

Chemistry, GITAM Institute of Science, GITAM University for his continuous
support and encouragement throughout the research work.

iii
 ACKNOWLEDGEMENT

The work presented in this thesis would not have been possible without my
close association with many people who were always there when I needed them the
most. I take this opportunity to acknowledge them and extend my sincere gratitude for
helping me to make this thesis a possibility.

I am highly indebted to my research director, Dr. B. VEERENDRA

KUMAR, Assistant Professor, Department of Biotechnology, GITAM Institute of
Science, GITAM University for his immense help, cooperation, scholarly inspiration
and valuable guidance that he had extended to me for the successful completion of
this investigation. I am grateful to him for his constructive criticism and moral support
throughout the research period. I wish to express my sincere thanks to him for his
continuous support and help in successful completion of this thesis.

I would like to express my heartfelt gratitude and sincere thanks to

Prof. S. V. RAJAGOPAL, Head of the Department of Biotechnology, GITAM
Institute of Science, GITAM University for his valuable suggestions and
encouragement for completion of this thesis.

I am thankful to Dr. N. LAXMANA DAS, Dean and Principal, GITAM

Institute of Science, GITAM University for giving me an opportunity to work in this
prestigious institute and making all the facilities available for carrying out my
research work successfully.

I express my sincere thanks to Prof. K.V. RAO, Dr. M. ANITHA, Dr. P.

SHANMUKH ANAND, Dr. T. SRINIVASAN, Dr. N. SAI KISHORE, Department
of Biotechnology, GITAM Institute of Science, GITAM University for their constant
support and assistance throughout the research work.

I express my sincere thanks to Dr. G. SRINIVASA RAO, Department of

Chemistry, GITAM Institute of Science, GITAM University for his continuous
support and encouragement throughout the research work.

iii
 I express my sincere thanks to Mr. V. V. S. L. PRASAD TALLURI,
Ms. V. SHILPA, Mrs. P. VIJAYALAKSHMI, Research Scholars, GITAM
University for their help and encouragement.

I am thankful to the non-teaching staff of Department of Biotechnology,

GITAM Institute of Science, Mr. K. SRINIVASU, Mrs. K. SWATHI,
Mr. P. RAMA RAO for their cooperation during my course of work.

I specially thank M/s. ORANGE LIFE SCINECES, Visakhapatnam for

providing facilities for carrying out some of the experiments of my investigation.

Finally, I would like to express my indebted gratitude to my husband

G.V.V.S. Gopi Chand, my sweet little kids, Tejesh & Devesh, in-laws
Sri. Golagani Apparao & Smt. Venkata Ramanamma, my parents Sri. Kayitha
Appalaraju & Smt. Yerraji for their cooperation and encouragement for successful
completion of this research work.

(K. BALA DURGA DEVI)

iv
 I express my sincere thanks to Mr. V. V. S. L. PRASAD TALLURI,
Ms. V. SHILPA, Mrs. P. VIJAYALAKSHMI, Research Scholars, GITAM
University for their help and encouragement.

I am thankful to the non-teaching staff of Department of Biotechnology,

GITAM Institute of Science, Mr. K. SRINIVASU, Mrs. K. SWATHI,
Mr. P. RAMA RAO for their cooperation during my course of work.

I specially thank M/s. ORANGE LIFE SCINECES, Visakhapatnam for

providing facilities for carrying out some of the experiments of my investigation.

Finally, I would like to express my indebted gratitude to my husband

G.V.V.S. Gopi Chand, my sweet little kids, Tejesh & Devesh, in-laws
Sri. Golagani Apparao & Smt. Venkata Ramanamma, my parents Sri. Kayitha
Appalaraju & Smt. Yerraji for their cooperation and encouragement for successful
completion of this research work.

(K. BALA DURGA DEVI)

iv
 THESIS ORGANIZATION

Chapter I of the thesis is the introductory chapter and deals with the derivatives and
applications of kojic acid.
Chapter II (Section-A) describes the literature review on various methods of kojic
acid fermentation, biosynthesis of kojic acid, structure, properties of kojic acid,
various fermentation techniques, microorganisms and carbon, nitrogen sources.
Chapter II (Section-B) this chapter reports the market potential of kojic acid,
Rationale for the study, aims and objectives of the present research.
Chapter III describes the screening of soil samples and taxonomical identification of
fungal isolates. It deals with the selection of promising isolates for the production of
kojic acid, effect of agitation, types of fermentation on the production of kojic acid by
the promising isolate. It also deals with the aflatoxin production by the soil isolate.
Chapter IV deals with optimization of cultural parameters on kojic acid production
using One-factor-at-a-time method.
Chapter V describes about the statistical analysis of optimized parameters using
Response Surface Methodology.
Chapter VI deals with the studies on effect of inducers on promising isolate for the
enhanced production of kojic acid.
Chapter VII describes the biophysical analytical methods for the structural
elucidation of kojic acid.
Chapter VIII reports the antimicrobial and antiproliferative activity of isolated kojic
acid crystals.
Chapter IX describes about the production economics of the present investigation.
Chapter X describes the conclusion of the present investigation.
Chapter XI describes the References and List of Publications

v
 CONTENTS

S.No Description Page No.

CHAPTER – I
1. Introduction
1.1 Derivatives of kojic acid 3
1.2 Applications of kojic acid 4

CHAPTER – II
Section – A
2. Review of literature
2.1 Introduction 8
2.2 Fermentation methods for kojic acid production 8
2.3 Microorganisms used for kojic acid production 11
2.4 Production of kojic acid from different carbon sources 12
2.5 Review of literature on kojic acid production 14
2.6 Biosynthesis of kojic acid 21
2.7 Properties of kojic acid 23
Section – B
2.8 Rationale of this investigation 24
2.9 Aims and objectives of the present investigation 25

CHAPTER – III
3. Isolation and identification of the promising isolate
capable of producing kojic acid
3.1 Introduction 26
3.2 Objectives 29
3.3 Material and methods 29
3.4 Results and discussion 33
3.5 Conclusion 38

vi
 CHAPTER - IV
4. Optimization of cultural parameters for cost effective
production of kojic acid
4.1 Introduction 39
4.2 Objectives 41
4.3 Materials and methods 43
4.4 Results and discussion 51
4.5 Production of kojic acid after optimization of
cultural parameters 99
4.6 Conclusion 103

CHAPTER – V
5. Statistical optimization of cultural parameters using
response surface methodology
5.1 Introduction 104
5.2 Objectives 104
5.3 Response surface methodology for optimized carbon
source Palmyra sap 105
5.4 Response surface methodology for optimized carbon
source Muntingia calabura fruits 117
5.5 Response surface methodology for the optimized carbon
source Sago starch 129
5.6 Conclusion 141

CHAPTER – VI
6. Effect of enhancers on kojic acid production
6.1 Introduction 142
6.2 Objectives 143
6.3 Materials and methods 143
6.4 Results and discussion 143
6.5 Conclusion 145

vii
 CHAPTER – VII
7. Isolation, purification and structural elucidation
of kojic acid
7.1 Introduction 146
7.2 Objectives 146
7.3 Materials and methods 146
7.4 Results and discussion 154
7.5 Conclusion 162
CHAPTER – VIII
8. Investigations for the bioactive properties of kojic acid
8.1 Introduction 163
8.2 Objectives 164
8.3 Materials and methods 164
8.4 Results and discussion 166
8.5 Conclusion 172
CHAPTER – IX
9. Production economics
9.1 Introduction 173
9.2 Objective 173
9.3 Materials and Methods 174
9.4 Results and Discussion 174
9.5 Conclusion 174
CHAPTER – X
10. Conclusion of the present investigations 175

CHAPTER – XI
11. References 177
List of publications 195

LIST OF TABLES ix
LIST OF FIGURES xi
LIST OF PLATES xviii
LIST OF FLOW CHARTS xix
ACRONYMS AND ABBREVIATIONS xx

viii
 LIST OF TABLES

Table No. Title of the Table Page No.

Table 2.1 Microorganisms used in the kojic acid production 11
Table 2.2 References pertained for the production of kojic acid from 12
various carbon sources
Table 3.1 CZA mediun 29
Table 3.2 Lacto phenol cotton blue stain 29
Table 3.3 Ariff’s medium 30
Table 3.4 Czapek’s agar solution in absence of nitrogen source 30
Table 3.5 Fungi isolated from soil samples 33
Table 4.1 Ariff’s medium 49
Table 4.2 Kojic acid concentrations at optimized cultural parameters 100
Table 5.1 Independent variables in the experimental plan 109
Table 5.2 CCD matrix having real values along with the experimental and 110
predicted values of kojic acid concentration
Table 5.3 Model coefficients estimated by multiple linear regression 111
(significance of regression coefficients)
Table 5.4 ANOVA for the entire quadratic model 112
Table 5.5 Independent variables in the experimental plan 120
Table 5.6 CCD matrix having real values along with the experimental and 121
predicted values of kojic acid concentration
Table 5.7 Model coefficients estimated by multiple linear regressions 122
(significance of regression coefficients)
Table 5.8 ANOVA for the entire quadratic model 123
Table 5.9 Independent variables in the experimental plan 132
Table 5.10 CCD matrix having real values along with the experimental and 133
predicted values of kojic acid concentration
Table 5.11 Model coefficients estimated by multiple linear regressions 134
(significance of regression coefficients)
Table 5.12 ANOVA for the entire quadratic model 135
Table 6.1 Effect of enhancers on kojic acid production 145
Table 7.1 Analytical conditions of HPLC 149
Table 7.2 Analytical conditions of XRD 150

ix
 LIST OF TABLES

Table No. Title of the Table Page No.

Table 2.1 Microorganisms used in the kojic acid production 11
Table 2.2 References pertained for the production of kojic acid from 12
various carbon sources
Table 3.1 CZA mediun 29
Table 3.2 Lacto phenol cotton blue stain 29
Table 3.3 Ariff’s medium 30
Table 3.4 Czapek’s agar solution in absence of nitrogen source 30
Table 3.5 Fungi isolated from soil samples 33
Table 4.1 Ariff’s medium 49
Table 4.2 Kojic acid concentrations at optimized cultural parameters 100
Table 5.1 Independent variables in the experimental plan 109
Table 5.2 CCD matrix having real values along with the experimental and 110
predicted values of kojic acid concentration
Table 5.3 Model coefficients estimated by multiple linear regression 111
(significance of regression coefficients)
Table 5.4 ANOVA for the entire quadratic model 112
Table 5.5 Independent variables in the experimental plan 120
Table 5.6 CCD matrix having real values along with the experimental and 121
predicted values of kojic acid concentration
Table 5.7 Model coefficients estimated by multiple linear regressions 122
(significance of regression coefficients)
Table 5.8 ANOVA for the entire quadratic model 123
Table 5.9 Independent variables in the experimental plan 132
Table 5.10 CCD matrix having real values along with the experimental and 133
predicted values of kojic acid concentration
Table 5.11 Model coefficients estimated by multiple linear regressions 134
(significance of regression coefficients)
Table 5.12 ANOVA for the entire quadratic model 135
Table 6.1 Effect of enhancers on kojic acid production 145
Table 7.1 Analytical conditions of HPLC 149
Table 7.2 Analytical conditions of XRD 150

ix
Table No. Title of the Table Page No.
Table 7.3 Analytical conditions of LC/MS 151
Table 7.4 Analytical conditions of FTIR 152
Table 8.1 Composition of Mueller-Hinton agar 165
Table 8.2 Effect of kojic acid on MDAMB435S cell lines 167
Table 8.3 Effect of kojic acid on K562 cell lines (Leukemia) 168
Table 9.1 Market potential of kojic acid 173
Table 9.2 Production cost for the present investigation 174

x
Table No. Title of the Table Page No.
Table 7.3 Analytical conditions of LC/MS 151
Table 7.4 Analytical conditions of FTIR 152
Table 8.1 Composition of Mueller-Hinton agar 165
Table 8.2 Effect of kojic acid on MDAMB435S cell lines 167
Table 8.3 Effect of kojic acid on K562 cell lines (Leukemia) 168
Table 9.1 Market potential of kojic acid 173
Table 9.2 Production cost for the present investigation 174

x
 LIST OF TABLES

Table No. Title of the Table Page No.

Table 2.1 Microorganisms used in the kojic acid production 11
Table 2.2 References pertained for the production of kojic acid from 12
various carbon sources
Table 3.1 CZA mediun 29
Table 3.2 Lacto phenol cotton blue stain 29
Table 3.3 Ariff’s medium 30
Table 3.4 Czapek’s agar solution in absence of nitrogen source 30
Table 3.5 Fungi isolated from soil samples 33
Table 4.1 Ariff’s medium 49
Table 4.2 Kojic acid concentrations at optimized cultural parameters 100
Table 5.1 Independent variables in the experimental plan 109
Table 5.2 CCD matrix having real values along with the experimental and 110
predicted values of kojic acid concentration
Table 5.3 Model coefficients estimated by multiple linear regression 111
(significance of regression coefficients)
Table 5.4 ANOVA for the entire quadratic model 112
Table 5.5 Independent variables in the experimental plan 120
Table 5.6 CCD matrix having real values along with the experimental and 121
predicted values of kojic acid concentration
Table 5.7 Model coefficients estimated by multiple linear regressions 122
(significance of regression coefficients)
Table 5.8 ANOVA for the entire quadratic model 123
Table 5.9 Independent variables in the experimental plan 132
Table 5.10 CCD matrix having real values along with the experimental and 133
predicted values of kojic acid concentration
Table 5.11 Model coefficients estimated by multiple linear regressions 134
(significance of regression coefficients)
Table 5.12 ANOVA for the entire quadratic model 135
Table 6.1 Effect of enhancers on kojic acid production 145
Table 7.1 Analytical conditions of HPLC 149
Table 7.2 Analytical conditions of XRD 150

ix
 LIST OF TABLES

Table No. Title of the Table Page No.

Table 2.1 Microorganisms used in the kojic acid production 11
Table 2.2 References pertained for the production of kojic acid from 12
various carbon sources
Table 3.1 CZA mediun 29
Table 3.2 Lacto phenol cotton blue stain 29
Table 3.3 Ariff’s medium 30
Table 3.4 Czapek’s agar solution in absence of nitrogen source 30
Table 3.5 Fungi isolated from soil samples 33
Table 4.1 Ariff’s medium 49
Table 4.2 Kojic acid concentrations at optimized cultural parameters 100
Table 5.1 Independent variables in the experimental plan 109
Table 5.2 CCD matrix having real values along with the experimental and 110
predicted values of kojic acid concentration
Table 5.3 Model coefficients estimated by multiple linear regression 111
(significance of regression coefficients)
Table 5.4 ANOVA for the entire quadratic model 112
Table 5.5 Independent variables in the experimental plan 120
Table 5.6 CCD matrix having real values along with the experimental and 121
predicted values of kojic acid concentration
Table 5.7 Model coefficients estimated by multiple linear regressions 122
(significance of regression coefficients)
Table 5.8 ANOVA for the entire quadratic model 123
Table 5.9 Independent variables in the experimental plan 132
Table 5.10 CCD matrix having real values along with the experimental and 133
predicted values of kojic acid concentration
Table 5.11 Model coefficients estimated by multiple linear regressions 134
(significance of regression coefficients)
Table 5.12 ANOVA for the entire quadratic model 135
Table 6.1 Effect of enhancers on kojic acid production 145
Table 7.1 Analytical conditions of HPLC 149
Table 7.2 Analytical conditions of XRD 150

ix
Table No. Title of the Table Page No.
Table 7.3 Analytical conditions of LC/MS 151
Table 7.4 Analytical conditions of FTIR 152
Table 8.1 Composition of Mueller-Hinton agar 165
Table 8.2 Effect of kojic acid on MDAMB435S cell lines 167
Table 8.3 Effect of kojic acid on K562 cell lines (Leukemia) 168
Table 9.1 Market potential of kojic acid 173
Table 9.2 Production cost for the present investigation 174

x
Table No. Title of the Table Page No.
Table 7.3 Analytical conditions of LC/MS 151
Table 7.4 Analytical conditions of FTIR 152
Table 8.1 Composition of Mueller-Hinton agar 165
Table 8.2 Effect of kojic acid on MDAMB435S cell lines 167
Table 8.3 Effect of kojic acid on K562 cell lines (Leukemia) 168
Table 9.1 Market potential of kojic acid 173
Table 9.2 Production cost for the present investigation 174

x
 LIST OF FIGURES

Figure No. Title of the Figure Page No.

Figure 2.1 Kojic acid biosynthetic pathway 22
Figure 2.2 Structure of kojic acid 23
Figure 3.1a-b Study area 28
Figure 4.1 Effect of different nitrogen sources on the production of 51
kojic acid
Figure 4.2 Progress of starch hydrolysis for various substrates 53
Figure 4.3a Effect of substrate concentration on kojic acid production 54
with the substrate Ipomea
Figure 4.3b Effect of substrate concentration on kojic acid production 54
with the substrate Cassava
Figure 4.3c Effect of substrate concentration on kojic acid production 55
with the substrate Sago starch
Figure 4.3d Effect of substrate concentration on kojic acid production 55
with the substrate A.macrorrhiza
Figure 4.3e Effect of substrate concentration on kojic acid production 56
with the substrate Palmyra sap
Figure 4.3f Effect of substrate concentration on kojic acid production 56
with the substrate Paneer whey
Figure 4.3g Effect of substrate concentration on kojic acid production 57
with the substrate Coconut water
Figure 4.3h Effect of substrate concentration on kojic acid production 57
with the substrate M.calabura fruits
Figure 4.3i Effect of substrate concentration on kojic acid production 58
with the substrate Cashew apple
Figure 4.3j Effect of substrate concentration on kojic acid production 58
with the substrate Sugar cane baggase
Figure 4.3k Effect of substrate concentration on kojic acid production 59
with the substrate Rice bran
Figure 4.3l Effect of substrate concentration on kojic acid production 59
with the substrate Wheat bran
Figure 4.4a Effect of Peptone concentration on kojic acid production 60
with the substrate Ipomea
Figure 4.4b Effect of Peptone concentration on kojic acid production 61
with the substrate Cassava
Figure 4.4c Effect of Peptone concentration on kojic acid production 61
with the substrate Sago starch

xi
Figure No. Title of the Figure Page No.
Figure 4.5k Effect of Time on kojic acid production with the substrate 72
Rice bran
Figure 4.5l Effect of Time on kojic acid production with the substrate 72
Wheat bran
Figure 4.6a Effect of pH on kojic acid production with the substrate 74
Ipomea
Figure 4.6b Effect of pH on kojic acid production with the substrate 74
Cassava
Figure 4.6c Effect of pH on kojic acid production with the substrate 75
Sago starch
Figure 4.6d Effect of pH on kojic acid production with the substrate 75
A.macrorrhiza
Figure 4.6e Effect of pH on kojic acid production with the substrate 76
Palmyra sap
Figure 4.6f Effect of pH on kojic acid production with the substrate 76
Paneer whey
Figure 4.6g Effect of pH on kojic acid production with the substrate 77
Coconut water
Figure 4.6h Effect of pH on kojic acid production with the substrate 77
M.calabura fruits
Figure 4.6i Effect of pH on kojic acid production with the substrate 78
Cashew apple
Figure 4.6j Effect of pH on kojic acid production with the substrate 78
Sugar cane baggase
Figure 4.6k Effect of pH on kojic acid production with the substrate 79
Rice bran
Figure 4.6l Effect of pH on kojic acid production with the substrate 79
Wheat bran
Figure 4.7a Effect of Temperature on kojic acid production with the 80
substrate Ipomea
Figure 4.7b Effect of Temperature on kojic acid production with the 81
substrate Cassava
Figure 4.7c Effect of Temperature on kojic acid production with the 81
substrate Sago starch
Figure 4.7d Effect of Temperature on kojic acid production with the 82
substrate A.macrorrhiza
Figure 4.7e Effect of Temperature on kojic acid production with the 82
substrate Palmyra sap

xiii
Figure No. Title of the Figure Page No.
Figure 4.7f Effect of Temperature on kojic acid production with the 83
substrate Paneer whey
Figure 4.7g Effect of Temperature on kojic acid production with the 83
substrate Coconut water
Figure 4.7h Effect of Temperature on kojic acid production with the 84
substrate M.calabura fruits
Figure 4.7i Effect of Temperature on kojic acid production with the 84
substrate Cashew apple
Figure 4.7j Effect of Temperature on kojic acid production with the 85
substrate Sugar cane baggase
Figure 4.7k Effect of Temperature on kojic acid production with the 85
substrate Rice bran
Figure 4.7l Effect of Temperature on kojic acid production with the 86
substrate Wheat bran
Figure 4.8a Effect of Potassium dihydrogen phosphate on kojic acid 87
production with the substrate Ipomea
Figure 4.8b Effect of Potassium dihydrogen phosphate on kojic acid 87
production with the substrate Cassava
Figure 4.8c Effect of Potassium dihydrogen phosphate on kojic acid 88
production with the substrate Sago starch
Figure 4.8d Effect of Potassium dihydrogen phosphate on kojic acid 88
production with the substrate A.macrorrhiza
Figure 4.8e Effect of Potassium dihydrogen phosphate on kojic acid 89
production with the substrate Palmyra sap
Figure 4.8f Effect of Potassium dihydrogen phosphate on kojic acid 89
production with the substrate Paneer whey
Figure 4.8g Effect of Potassium dihydrogen phosphate on kojic acid 90
production with the substrate Coconut water
Figure 4.8h Effect of Potassium dihydrogen phosphate on kojic acid 90
production with the substrate M.calabura fruits
Figure 4.8i Effect of Potassium dihydrogen phosphate on kojic acid 91
production with the substrate Cashew apple
Figure 4.8j Effect of Potassium dihydrogen phosphate on kojic acid 91
production with the substrate Sugar cane baggase
Figure 4.8k Effect of Potassium dihydrogen phosphate on kojic acid 92
production with the substrate Rice bran
Figure 4.8l Effect of Potassium dihydrogen phosphate on kojic acid 92
production with the substrate Wheat bran

xiv
Figure No. Title of the Figure Page No.
Figure 4.9a Effect of Magnesium sulphate on kojic acid production 93
with the substrate Ipomea
Figure 4.9b Effect of Magnesium sulphate on kojic acid production 94
with the substrate Cassava
Figure 4.9c Effect of Magnesium sulphate on kojic acid production 94
with the substrate Sago starch
Figure 4.9d Effect of Magnesium sulphate on kojic acid production 95
with the substrate A.macrorrhiza
Figure 4.9e Effect of Magnesium sulphate on kojic acid production 95
with the substrate Palmyra sap
Figure 4.9f Effect of Magnesium sulphate on kojic acid production 96
with the substrate Paneer whey
Figure 4.9g Effect of Magnesium sulphate on kojic acid production 96
with the substrate Coconut water
Figure 4.9h Effect of Magnesium sulphate on kojic acid production 97
with the substrate M.calabura fruits
Figure 4.9i Effect of Magnesium sulphate on kojic acid production 97
with the substrate Cashew apple
Figure 4.9j Effect of Magnesium sulphate on kojic acid production 98
with the substrate Sugar cane baggase
Figure 4.9k Effect of Magnesium sulphate on kojic acid production 98
with the substrate Rice bran
Figure 4.9l Effect of Magnesium sulphate on kojic acid production 99
with the substrate Wheat bran
Figure 4.10 Kojic acid concentration at initial and after optimization 101
Figure 4.11 Kojic acid crystal yields at initial and after optimization 102
Figure 5.1 Response surface plot for kojic acid production versus 112
Peptone and substrate concentration
Figure 5.2 Response surface plot for kojic acid production versus 113
Peptone and pH
Figure 5.3 Response surface plot for kojic acid production versus 113
Temperature and Peptone
Figure 5.4 Response surface plot for kojic acid production versus 114
Temperature and pH
Figure 5.5 Response surface plot for kojic acid production versus 114
Temperature and Substrate concentration
Figure 5.6 Response surface plot for kojic acid production versus pH 115
and Substrate concentration

xv
Figure No. Title of the Figure Page No.
Figure 5.7 Response surface plot for kojic acid production versus 115
Incubation time and Peptone
Figure 5.8 Response surface plot for kojic acid production versus 116
Incubation time and pH
Figure 5.9 Response surface plot for kojic acid production versus 123
Peptone and Substrate concentration
Figure 5.10 Response surface plot for kojic acid production versus pH 124
and Peptone
Figure 5.11 Response surface plot for kojic acid production versus pH 124
and Substrate concentration
Figure 5.12 Response surface plot for kojic acid production versus 125
Incubation time and Substrate concentration
Figure 5.13 Response surface plot for kojic acid production versus 125
Temperature and pH
Figure 5.14 Response surface plot for kojic acid production versus 126
Temperature and Incubation time
Figure 5.15 Response surface plot for kojic acid production versus pH 126
and Incubation time
Figure 5.16 Response surface plot for kojic acid production versus 127
Temperature and Peptone
Figure 5.17 Response surface plot for kojic acid production versus 127
Incubation time and Peptone concentration
Figure 5.18 Response surface plot for kojic acid production versus 128
Temperature and Substrate concentration
Figure 5.19 Response surface plot for kojic acid production versus 135
Temperature and pH
Figure 5.20 Response surface plot for kojic acid production versus 136
Temperature and Peptone concentration
Figure 5.21 Response surface plot for kojic acid production versus pH 136
and Peptone concentration
Figure 5.22 Response surface plot for kojic acid production versus 137
Temperature and Incubation time
Figure 5.23 Response surface plot for kojic acid production versus pH 137
and Incubation Time
Figure 5.24 Response surface plot for kojic acid production versus 138
Temperature and Substrate concentration
Figure 5.25 Response surface plot for kojic acid production versus pH 138
and Substrate concentration

xvi
 LIST OF FIGURES

Figure No. Title of the Figure Page No.

Figure 2.1 Kojic acid biosynthetic pathway 22
Figure 2.2 Structure of kojic acid 23
Figure 3.1a-b Study area 28
Figure 4.1 Effect of different nitrogen sources on the production of 51
kojic acid
Figure 4.2 Progress of starch hydrolysis for various substrates 53
Figure 4.3a Effect of substrate concentration on kojic acid production 54
with the substrate Ipomea
Figure 4.3b Effect of substrate concentration on kojic acid production 54
with the substrate Cassava
Figure 4.3c Effect of substrate concentration on kojic acid production 55
with the substrate Sago starch
Figure 4.3d Effect of substrate concentration on kojic acid production 55
with the substrate A.macrorrhiza
Figure 4.3e Effect of substrate concentration on kojic acid production 56
with the substrate Palmyra sap
Figure 4.3f Effect of substrate concentration on kojic acid production 56
with the substrate Paneer whey
Figure 4.3g Effect of substrate concentration on kojic acid production 57
with the substrate Coconut water
Figure 4.3h Effect of substrate concentration on kojic acid production 57
with the substrate M.calabura fruits
Figure 4.3i Effect of substrate concentration on kojic acid production 58
with the substrate Cashew apple
Figure 4.3j Effect of substrate concentration on kojic acid production 58
with the substrate Sugar cane baggase
Figure 4.3k Effect of substrate concentration on kojic acid production 59
with the substrate Rice bran
Figure 4.3l Effect of substrate concentration on kojic acid production 59
with the substrate Wheat bran
Figure 4.4a Effect of Peptone concentration on kojic acid production 60
with the substrate Ipomea
Figure 4.4b Effect of Peptone concentration on kojic acid production 61
with the substrate Cassava
Figure 4.4c Effect of Peptone concentration on kojic acid production 61
with the substrate Sago starch

xi
Figure No. Title of the Figure Page No.
Figure 4.4d Effect of Peptone concentration on kojic acid production 62
with the substrate A.macrorrhiza
Figure 4.4e Effect of Peptone concentration on kojic acid production 62
with the substrate Palmyra sap
Figure 4.4f Effect of Peptone concentration on kojic acid production 63
with the substrate Paneer whey
Figure 4.4g Effect of Peptone concentration on kojic acid production 63
with the substrate Coconut water
Figure 4.4h Effect of Peptone concentration on kojic acid production 64
with the substrate M.calabura fruits
Figure 4.4i Effect of Peptone concentration on kojic acid production 64
with the substrate Cashew apple
Figure 4.4j Effect of Peptone concentration on kojic acid production 65
with the substrate Sugar cane baggase
Figure 4.4k Effect of Peptone concentration on kojic acid production 65
with the substrate Rice bran
Figure 4.4l Effect of Peptone concentration on kojic acid production 66
with the substrate Wheat bran
Figure 4.5a Effect of Time on kojic acid production with the substrate 67
Ipomea
Figure 4.5b Effect of Time on kojic acid production with the substrate 67
Cassava
Figure 4.5c Effect of Time on kojic acid production with the substrate 68
Sago starch
Figure 4.5d Effect of Time on kojic acid production with the substrate 68
A.macrorrhiza
Figure 4.5e Effect of Time on kojic acid production with the substrate 69
Palmyra sap
Figure 4.5f Effect of Time on kojic acid production with the substrate 69
Paneer whey
Figure 4.5g Effect of Time on kojic acid production with the substrate 70
Coconut water
Figure 4.5h Effect of Time on kojic acid production with the substrate 70
M.calabura fruits
Figure 4.5i Effect of Time on kojic acid production with the substrate 71
Cashew apple
Figure 4.5j Effect of Time on kojic acid production with the substrate 71
Sugar cane baggase

xii
Figure No. Title of the Figure Page No.
Figure 4.5k Effect of Time on kojic acid production with the substrate 72
Rice bran
Figure 4.5l Effect of Time on kojic acid production with the substrate 72
Wheat bran
Figure 4.6a Effect of pH on kojic acid production with the substrate 74
Ipomea
Figure 4.6b Effect of pH on kojic acid production with the substrate 74
Cassava
Figure 4.6c Effect of pH on kojic acid production with the substrate 75
Sago starch
Figure 4.6d Effect of pH on kojic acid production with the substrate 75
A.macrorrhiza
Figure 4.6e Effect of pH on kojic acid production with the substrate 76
Palmyra sap
Figure 4.6f Effect of pH on kojic acid production with the substrate 76
Paneer whey
Figure 4.6g Effect of pH on kojic acid production with the substrate 77
Coconut water
Figure 4.6h Effect of pH on kojic acid production with the substrate 77
M.calabura fruits
Figure 4.6i Effect of pH on kojic acid production with the substrate 78
Cashew apple
Figure 4.6j Effect of pH on kojic acid production with the substrate 78
Sugar cane baggase
Figure 4.6k Effect of pH on kojic acid production with the substrate 79
Rice bran
Figure 4.6l Effect of pH on kojic acid production with the substrate 79
Wheat bran
Figure 4.7a Effect of Temperature on kojic acid production with the 80
substrate Ipomea
Figure 4.7b Effect of Temperature on kojic acid production with the 81
substrate Cassava
Figure 4.7c Effect of Temperature on kojic acid production with the 81
substrate Sago starch
Figure 4.7d Effect of Temperature on kojic acid production with the 82
substrate A.macrorrhiza
Figure 4.7e Effect of Temperature on kojic acid production with the 82
substrate Palmyra sap

xiii
Figure No. Title of the Figure Page No.
Figure 4.7f Effect of Temperature on kojic acid production with the 83
substrate Paneer whey
Figure 4.7g Effect of Temperature on kojic acid production with the 83
substrate Coconut water
Figure 4.7h Effect of Temperature on kojic acid production with the 84
substrate M.calabura fruits
Figure 4.7i Effect of Temperature on kojic acid production with the 84
substrate Cashew apple
Figure 4.7j Effect of Temperature on kojic acid production with the 85
substrate Sugar cane baggase
Figure 4.7k Effect of Temperature on kojic acid production with the 85
substrate Rice bran
Figure 4.7l Effect of Temperature on kojic acid production with the 86
substrate Wheat bran
Figure 4.8a Effect of Potassium dihydrogen phosphate on kojic acid 87
production with the substrate Ipomea
Figure 4.8b Effect of Potassium dihydrogen phosphate on kojic acid 87
production with the substrate Cassava
Figure 4.8c Effect of Potassium dihydrogen phosphate on kojic acid 88
production with the substrate Sago starch
Figure 4.8d Effect of Potassium dihydrogen phosphate on kojic acid 88
production with the substrate A.macrorrhiza
Figure 4.8e Effect of Potassium dihydrogen phosphate on kojic acid 89
production with the substrate Palmyra sap
Figure 4.8f Effect of Potassium dihydrogen phosphate on kojic acid 89
production with the substrate Paneer whey
Figure 4.8g Effect of Potassium dihydrogen phosphate on kojic acid 90
production with the substrate Coconut water
Figure 4.8h Effect of Potassium dihydrogen phosphate on kojic acid 90
production with the substrate M.calabura fruits
Figure 4.8i Effect of Potassium dihydrogen phosphate on kojic acid 91
production with the substrate Cashew apple
Figure 4.8j Effect of Potassium dihydrogen phosphate on kojic acid 91
production with the substrate Sugar cane baggase
Figure 4.8k Effect of Potassium dihydrogen phosphate on kojic acid 92
production with the substrate Rice bran
Figure 4.8l Effect of Potassium dihydrogen phosphate on kojic acid 92
production with the substrate Wheat bran

xiv
Figure No. Title of the Figure Page No.
Figure 4.9a Effect of Magnesium sulphate on kojic acid production 93
with the substrate Ipomea
Figure 4.9b Effect of Magnesium sulphate on kojic acid production 94
with the substrate Cassava
Figure 4.9c Effect of Magnesium sulphate on kojic acid production 94
with the substrate Sago starch
Figure 4.9d Effect of Magnesium sulphate on kojic acid production 95
with the substrate A.macrorrhiza
Figure 4.9e Effect of Magnesium sulphate on kojic acid production 95
with the substrate Palmyra sap
Figure 4.9f Effect of Magnesium sulphate on kojic acid production 96
with the substrate Paneer whey
Figure 4.9g Effect of Magnesium sulphate on kojic acid production 96
with the substrate Coconut water
Figure 4.9h Effect of Magnesium sulphate on kojic acid production 97
with the substrate M.calabura fruits
Figure 4.9i Effect of Magnesium sulphate on kojic acid production 97
with the substrate Cashew apple
Figure 4.9j Effect of Magnesium sulphate on kojic acid production 98
with the substrate Sugar cane baggase
Figure 4.9k Effect of Magnesium sulphate on kojic acid production 98
with the substrate Rice bran
Figure 4.9l Effect of Magnesium sulphate on kojic acid production 99
with the substrate Wheat bran
Figure 4.10 Kojic acid concentration at initial and after optimization 101
Figure 4.11 Kojic acid crystal yields at initial and after optimization 102
Figure 5.1 Response surface plot for kojic acid production versus 112
Peptone and substrate concentration
Figure 5.2 Response surface plot for kojic acid production versus 113
Peptone and pH
Figure 5.3 Response surface plot for kojic acid production versus 113
Temperature and Peptone
Figure 5.4 Response surface plot for kojic acid production versus 114
Temperature and pH
Figure 5.5 Response surface plot for kojic acid production versus 114
Temperature and Substrate concentration
Figure 5.6 Response surface plot for kojic acid production versus pH 115
and Substrate concentration

xv
Figure No. Title of the Figure Page No.
Figure 5.7 Response surface plot for kojic acid production versus 115
Incubation time and Peptone
Figure 5.8 Response surface plot for kojic acid production versus 116
Incubation time and pH
Figure 5.9 Response surface plot for kojic acid production versus 123
Peptone and Substrate concentration
Figure 5.10 Response surface plot for kojic acid production versus pH 124
and Peptone
Figure 5.11 Response surface plot for kojic acid production versus pH 124
and Substrate concentration
Figure 5.12 Response surface plot for kojic acid production versus 125
Incubation time and Substrate concentration
Figure 5.13 Response surface plot for kojic acid production versus 125
Temperature and pH
Figure 5.14 Response surface plot for kojic acid production versus 126
Temperature and Incubation time
Figure 5.15 Response surface plot for kojic acid production versus pH 126
and Incubation time
Figure 5.16 Response surface plot for kojic acid production versus 127
Temperature and Peptone
Figure 5.17 Response surface plot for kojic acid production versus 127
Incubation time and Peptone concentration
Figure 5.18 Response surface plot for kojic acid production versus 128
Temperature and Substrate concentration
Figure 5.19 Response surface plot for kojic acid production versus 135
Temperature and pH
Figure 5.20 Response surface plot for kojic acid production versus 136
Temperature and Peptone concentration
Figure 5.21 Response surface plot for kojic acid production versus pH 136
and Peptone concentration
Figure 5.22 Response surface plot for kojic acid production versus 137
Temperature and Incubation time
Figure 5.23 Response surface plot for kojic acid production versus pH 137
and Incubation Time
Figure 5.24 Response surface plot for kojic acid production versus 138
Temperature and Substrate concentration
Figure 5.25 Response surface plot for kojic acid production versus pH 138
and Substrate concentration

xvi
Figure No. Title of the Figure Page No.
Figure 5.26 Response surface plot for kojic acid production versus 139
Incubation Time and Substrate concentration
Figure 5.27 Response surface plot for kojic acid production versus 139
Peptone concentration and Substrate concentration
Figure 5.28 Kojic acid yields after RSM optimized parameters 140
Figure 7.1 Elution profile of kojic acid using sephacryl S-200 154
Figure 7.2 Elution profile of test kojic acid sample by HPLC 156
Figure 7.3 Elution profile of standard kojic acid sample by HPLC 156
Figure 7.4 X-ray diffractogram of test kojic acid sample 157
Figure 7.5 X-ray diffractogram of standard kojic acid sample 158
Figure 7.6 Mass spectrum of kojic acid 159
Figure 7.7 FTIR spectrum of Kojic acid test sample 160
Figure 7.8 FTIR spectrum of kojic acid standard sample 160
Figure 7.9 Proton NMR of test kojic acid sample 161
Figure 7.10 Proton NMR of standard kojic acid sample 162
Figure 8.1 Zone of inhibitions of different bacteria to the 167
antimicrobial compound kojic acid
Figure 8.2 Inhibition activity of kojic acid on MDA MB435S cell 168
lines (Breast cancer)
Figure 8.3 Inhibition activity of kojic acid on K562 cell lines 169
(Leukemia)
Figure 8.4 Activity of kojic acid on MDA-MB435S (Breast cancer) 170
cell lines before and after addition
Figure 8.5 Activity of kojic acid on K562 (Leukemia) cell lines 171
before and after addition

xvii
 LIST OF PLATES

Plate No. Title of the Plate Page No.

Plate 3.1 Morphological structure of A.flavus 34
Plate 3.2 Morphological structure of A.sojae 35
Plate 3.3 Morphological structure of A.nidulans 35
Plate 3.4 Morphological structure of A.fumigatus 35
Plate 3.5 Morphological structure of A.niger 35
Plate 3.6 Morphological structure of A.oryzae 35
Plate 3.7 Morphological structure of Fusarium 35
Plate 3.8 Morphological structure of Mucor 36
Plate 3.9 Morphological structure of Penicillium 36
Plate 3.10 Morphological structure of A.terrus 36
Plate 7.1 Kojic acid crystals after evaporation 155
Plate 7.2 Kojic acid crystals under microscope 155

xviii
 LIST OF FLOW CHARTS

Flow chart No. Title of the Flow chart Page No.

Flow chart 4.1 Optimization of kojic acid 42

xix
 ACRONYMS AND ABBREVIATIONS

Notation Abbreviation

3D Three dimensional
CFU/ml Colony forming unit/millilitre
cm-1 Centimeter per second
g/cc Gram per cubic centimeter
g/g Gram / gram
g/L Gram / litre
g/l/d Gram per litre per day
h Hour
IU International Unit
KCal Kilo calories
KJ Kilo joules
KNU Unit of enzyme activity
Kw Kilo watt
kα Constant
kd Retardation constant
mA Mega ampere
mM Milli molar
MHz Mega hertz
Min Minute
Ml/h Millilitre per hour
mW Mega Watt
m/z Mass by charge ratio
Nm Nanometer
O.D Optical density
ppm Parts per million
Psi Pressure per square inch
R2 Determination coefficient
rpm Revolution per minute
UV Ultra violet
v/v Volume by volume
vvm Void volume
w/v weight / volume
‘p’ value Probability value
A.flavus Aspergillus flavus
A.sojae Aspergillus sojae
Agilent QQQ LC/MS Agilent triple quadrupole liquid
chromatography mass spectroscopy
ANOVA Analysis of variance
CCDM Central composite design matrix
CRISP Center for research on interface
structure and phenomenon

xx
Cu (I)-B2 Copper monovales riboflavin
Cu (I)-B3 Copper monovales niacin
CYA Czapekdox yeast extract agar
CZA Czapk-Dox Agar
DHAP Dihydroxy acetone phosphate
DMSO Dimethyl sulphoxide
D2 O Deuterium oxide
DOPS Direct optical positioning
DOT Dissolved oxygen tension
ELISA Enzyme linked immuno sorbent
assay
ESI Electron spin ionization
FBS Fetal bovine serum
FTIR Fourier transform infrared spectrum
FT-NMR Fourier transform Nuclear magnetic
resonance spectroscopy
F-test Fischer test
HeNe Helium Neon
1
H-NMR Proton-nuclear magnetic resonance
spectroscopy
HPLC High pressure liquid chromatography
Leu Leucine
LPCB Lacto phenol cotton blue
Lys Lysine
MEM Minimal essential medium
MTT 3-(4,5-dimethyl thiazol-2-41)-2,5
diphenyl tetrazolium bromide
NADH Nicotinamide adenine dinucleotide
NADPH Nicotinamide adenine dinucleotide
phosphate
OFAT One-factor-at-a-time method
PBS Phosphate buffer solution
RF Radio frequency
RSM Response Surface Methodology
TLC Thin layer chromatography
TMS Trimethyl silane
t-test Turkey test
XRD X-ray diffractometry
YES Yeast extract sucrose medium

xxi
 CHAPTER – I
INTRODUCTION
 CHAPTER 1

1. INTRODUCTION

Industrial microbes have the potential to become good economic resources. They were
used in different fields of industrial, clinical, agricultural, pharmaceutical etc. on which,
in some manner, a monetary value can be positioned despite of whether involves a
fermentation product or some form of deterioration, disease or waste disposal. Industrial
microbiology deals itself with the segregation and explanation of microorganisms from
normal environments like soil or water and with the cultural stipulations desired for
achieving rapid and massive growth of these cultures in the laboratory and in fermentors
which were frequently known as large scale cultural vessels. Microrganisms have the
capability to transform invaluable raw materials or substrate to economically worthful
organic compounds apparently is of prominent concern to the industrial microbiologists
Casida (2008).

Fermentation yields may be constituents of the microbial cells, the whole cells, both
extracellular and intracellular enzymes or chemicals which are generated or altered by the
cells. The competitive position and prospective profits from a fermentation outcome are
nearly linked to the economy of the various components of the production medium.
Depending on the cost basis inoculum medium is normally cost effective because less is
needed and it is consumed to provide rapid growth of cells and not to transform a huge
amount of carbon substrate into microbial cells or fermentation outcome. In contrast, an
elevated single cost medium constituent for the production medium can honestly
command the retail price for the fermentation product Crueger and Crueger (1984).

Apparently all trials should be made to find a convertible or a modified less cost
substituent for such a component of a medium. Any content of the medium nevertheless
may be subject to market in constancy in prices and availability. The efficiency of a
fermentation to generate eminent yields and to allow excellent product recovery is the
first regard in fermentation economics and in this view, products and product recovery

1
must be studied. Simultaneously since a superior yield is of less importance if the
recovery of the product was not done accurately for marketing.The overall cost of the
fermentation products was due to its fractionation procedures, solvent extraction,
consecutive recrystallization and other needs for purification. The cost of the product lies
not only with the expenditure of the product purification process but also with the loss of
fermentation product takesplace in every step of purification procedure Patel (1984).

During the centuries ago, it was found that divergent types of natural sources like
animals, microorganisms, insects and some plants were capable of producing numerous
active metabolites. Out of these sources, the microorganisms producing the metabolites,
occupy promptly the utmost industrial applications because they are chemo-
organotrophic in nature, possess a maximum growth rate within a short period of life
cycle so produces high amounts of biomass in short time. Hence generation of fungal
metabolites industrially claims minor complex operational control process Bracarense
and Takahashi (2014). Different types of microorganisms have the capability to convert
carbohydrates into organic acids. For example Clostridium produces acetic and butyric
acids. Lactobacillus and Streptococcus produce lactic acid, Acetobacter produce acetic
acid, gluconic acid etc. The fungal acids which have wide commercial applications are
extensively investigated include citric acid, itaconic acid, fumaric acid, gluconic acid,
gibberellic acid and kojic acid Casida (2001). These isolated natural products, one such
compound, kojic acid covers a wide range of applications in various fields like food,
pharmaceutical, cosmetics, medical, chemical industries etc.

In the kojic acid the word koji was the name of the starter culture which was widely used
in the preparation of oriental fermented food products. Chemically it is called 5-hydroxy-
2-hydroxymethyl-γ-pyrone Yabuta (1924). The chemical compound Kojic acid presents
naturally in fungus (or) mushrooms and produced through fermentation of matting of rice
for the preparation of wine sake. Numerous fungal strains have been used for the
production of kojic acid which includes 19 different Aspergillus species and five
Pencillium species and few bacteria like Bacterium xylinoides, Glucono-acetobacter
opacus var. mobilis and Gyrinium roseum Arnstein (1952). Terabayashi et al. (2010) had
concluded that nearly 14 genes A0090113000132 to A0090113000145 including a

2
transcription factor gene KojR, an enzyme gene KojA and a transporter gene KojJ which
form a cluster were involved in the biosynthesis of kojic acid. The kojR gene encodes a
fungal-specific Zn (II)Cy6 transcription factor that is situated between kojA (upstream
743bp) and kojT (downstream 383bp). A mutant strain with transcription factor gene
cannot able to secrete kojic acid in any way Marui et al. (2011).

A search on kojic acid biosynthesis mechanism utilizing different carbon sources by

various researchers have unveiled that the kojic acid synthesis was occurred from
phosphorylated intermediates. The two major areas strongly associated with the
development on kojic acid production was strain development and fermentation
technology. Very few studies have attempted in the strain development process using
mutational methods and recombination techniques. The Ultraviolet or Gamma irradiated
mutagenic genes in the fungi causes them to secrete high amounts of kojic acid which
was more beneficial for kojic acid manufacturers Abd El-Aziz (2013). Though extensive
research was carried out in the optimization of nutrients and cultural conditions especially
in utilizing inexpensive carbon sources like agro waste materials, industrial wastes etc,
using different nitrogen sources under aerobic fermentation may leads to lesser quantities
of kojic acid. Till date pure glucose was the only source reported maximum yield of kojic
acid.

1.1 DERIVATIVES OF KOJIC ACID:

Kojic acid though having antimicrobial activity it has weak bacteriostatic action hence
many researchers they worked on the novel group of antibiotics and more over from
perspective fungal metabolites Beelik (1956). The soluble property of kojic acid in water,
ethyl acetate, ethanol or acetone helps to synthesize one hundred and fifty different types
of kojic acid derivatives like kojic acid laureate and kojic acid dipalmitate Brtko et al.
(2004), Lee et al. (2006), Khamaruddin et al. (2008), Ashari et al. (2009) and some novel
chemical compounds which were not synthesized formerly Uher et al. (1997), Hudecova
et al. (1996), Hasizume et al. (1968). These derivatives are highly stable and soluble in
oily cosmetic products Smith and Lindsay (2001). The kojic acid derivatives possess

3
ethylene inkage of phosphonate with aldehyde are 8 times more effective than kojic acid
Lee et al. (2006). The derivatives of kojic acid exhibit three major characteristics-
antibacterial, antifungal and anti-inflammatory properties Brtko et al. (2004), Kayahara et
al. (1990), Balaz et al. (1993). Both the gram positive and gram negative bacterial growth
was inhibited by 0.5% W/V kojic acid and its derivatives Kotani et al. (1976), Beelik et
al. (1956), Reddy et al. (2010). The work at Slovak Academy of Sciences Institute of
Experimental Endocrinology had revealed that the derivatives of Kojic acid bear anti-
leukemic property. Some of the derivatives of kojic acid were used as a major ingredient
in the preparation of some antibiotics like Cephamycin. So, many prospective studies
have been developed to produce novel group of kojic acid derivatives which may be used
as a potent pharmaceutical agents Rosfarizan (2010). Due to its specific physico-chemical
properties like hydrophobicity, acidity and chelating nature with metal ions, it was
expected that it performs a prominent role in biological activity of derivatives of kojic
acid. Some of the important derivatives of kojic acid are azidometal kojates, nickel salts
of 5-hydroxy-2-azidomethyl-4H-pyran-4-one and sulphur containing kojates used to treat
fungal skin diseases of humans and animals Cho et al. (2012), Uher et al. (1997), Brtko et
al. (2004), Wei et al. (1991), Fickova et al. (2008).

1.2 APPLICATIONS OF KOJIC ACID:

1.2.1 Applications in food industry:

Kojic acid along with the combination of vitamin ‘C’ is utilized as an anti-browning
agent or antispeck activity Uchino et al. (1988) in food stuffs reported by Food
Agriculture Organization. Utilized as a food additive to impede enzymatic browning and
used during the preparation of foods like miso, soy sauce, sake etc Wood (1998). It is
also used as an anti-browning agent in raw noodles for the preservation and processing
purposes. The kojic acid producing fungi were used commonly in the production of
traditional staple Japanese fermented foods like amazake is a sweet beverage; shochu is a
distilled liquor and mirin a sweet alcoholic seasoning. Most of the healthy foods
consumed in japan contain kojic acid Niwa and Akamatsu (1991). As the kojic acid has a
powerful anti-oxidizing activity Wehner et al. (1978), it is used widely as a natural

4
preservative and flavour enhancer. It prevents the formation of nitrosopyrrolidine in fried
bacon Theiler et al. (1984). In unripe strawberries as it produce reddening Curtis (1977).
It prevents browning of fruits like apples after cut Son et al. (2000) and some of the food
items like sea food-shrimp, to protect their natural colour. It can also be used for the
ripening of unripened tomatoes. It also acts as a precursor for flavour enhancers like
maltol and ethyl maltol Szklarzewicz et al. (2012). Comenic acid is produced from kojic
acid which is the intermediate in maltol synthesis Tatsumi et al. (1969), Le Blanch et al.
(1989). Maltol is used widely as flavour enhancer in food products; constituent in
perfumes etc Ichimoto et al. (1965), McNail et al. (1992). Burdock et al. (2001) evaluated
the health phase of kojic acid in food products. If the kojic acid is used as a preservative,
the food seems fresh for a longer period of time. It inhibits the rottening and spoiling of
foods. Due to its anti-bacterial activity, the colours red and pink of the sea food, meat and
vegetables remain viable. Recently it was discovered that, it inhibit the
polyphenoloxidase (PPO) activity of crustaceans, which include grass prawn, white
shrimp, Florida spiny lobster, Mushroom, potato and apples Saruno et al. (1978), Chen et
al. (1991).

1.2.2 In chemical industry:

It is used as a precursor in the manufacture of various chemical compounds like ethers,

metal chelates, pyridines, azo dyes, pyridines mannich base which were used commonly
in the chemical industry Ichimoto et al. (1965) and Wilson (1971). The presence of
hydroxyl group at carbon 5 positions in kojic acid helps it to produce salts with the metals
like Zn, Cu, Ni, Na, Ca and Cd Crueger and Crueger (1984). In combination with
chitosan produce kojic acid-chitosan conjugates which have wide applications in
chemical industry Guibal (2004), Synytsya et al. (2008). Kojic acid is used as a building
block for the manufacture of biodegradable plastics Tomita et al. (1996), Ochiai et al.
(2012). It is considered as a first pyrone derivative and used for the analysis of iron in
ores Toso et al. (2013). Metal chelating agent so used as a reagent for the analysis of iron
in chemical industries Wilson (1971). When it reacts with 0.1ppm ferric ion and gives
intense red colour due to the formation of coordination ferric complex which cannot be
reversibly oxidized or reduced Buchta (1982), Mohammadpour et al. (2012). This reddish

5
purple complex has maximum absorption at 500nm and this property is useful in the
quantitative analysis of kojic acid Crueger and Crueger (1984), Bentley (1957), Sadeghi
et al. (2013).

1.2.3 In medicine:

It can be taken as an oral medicine against ageing and cancer. According to one study at
Bioscience and Biotechnology Institute, Korea, the Kojic acid and its derivatives possess
anti-inflammatory characters, hence inhibit the proliferation of cancerous cells Novotny
et al. (1999). The halogenated derivatives of kojic acid are used for the treatment of
leukemia Bransova et al. (1997) for eg. 5-OH-2-Chloromethyl-4-pyran-4-one inhibit
DNA, RNA and protein synthesis Bransova et al. (1998) and 5-benzyloxy-2-thrio
cyanatomethyl-4-pyran-4-one prevent DNA synthesis and cytoplasmic phosphorylation
hence reduces the cell growth of neoplastic cells Vachalkova et al.(1996), Yoo et al.
(2010). According to University of Maryland Medical Centre, a cream which contains
kojic acid, tretinion and azelaic acid used to treat the conditions like melasma because
kojic acid reduces the melanin synthesis since it represses the catecholase activity of
tyrosinase enzyme Kaatz et al. (1999). It is also used in the preparation of anti-
inflammation and pain relieving drugs Sansho (1980). Kojic acid in combination with
vanadium is an anti-diabetic agent Wei et al. (2011, 2012), Ragaa (2006). The acidic
nature of kojic acid found to act like effective anti-microbial agent against numerous
bacteria and fungi comparatively than antibiotics like nitrofurantoin and ceftazidime El-
Aasar (2006), Hussein et al. (2014).

1.2.4 In cosmetics:

Kojic acid was used as a key ingredient in the preparation of skin care products because it
acts as a skin lightening and de-pigmenting agent Ahn et al. (2003). It suppresses the
melanin formation in human skin by inhibiting the activity of tyrosinase enzyme
responsible for the synthesis of melanin Ohyama and Mishima (1990), Noh et al. (2009),
Lajis et al.(2012). It is useful in the manufacture of beauty products like creams, gels,
bath salts, bubble bath products, baby lotions, sun screens etc., to enhance the skin
complexion Masse et al. (2001), Cabanes et al. (1994). Soaps which contain kojic acid
6
ingredients as well as exfoliant will soften the skin. Some items when the skin is severely
exposed to the sun cause the damage of melanocytes which produce melanin. As a result
high amount of melanin is deposited in the dermis which in turn produces brown spots
termed as freckles. Kojic acid dipalmitate helps in fighting against freckles and
eliminates the darkening areas Ohyama and Mishima (1990), Lee et al. (2006). It
functions as bleaching and anti-oxidizing factor in cosmetic products. It replaces the
hydroquinone usage in the cosmetic products which bleaches the skin. The manganese
and Zinc complex derivative of kojic acid was used as a radio protective agent against γ-
radiations Reelfs et al. (2010), Emami et al. (2007). It has anti-bacterial and anti-fungal
activity and used extensively in the acne and melasma, chloasma disorder therapy Lim
(1999), Jimbow et al. (2001). It is also used to remove scars remains after acute acne on
the skin Kim et al. (2002). It is used in the healing of some skin conditions where it
removes toxins from the body. It heightens the phagocytic phenomenon of neutrophils
and proliferation of lymphocytes with phytohemagglutinin. It increases the activity of
number of leukocytes while scavenging reactive oxygen species propagated in tissues of
blood Niwa and Akamatsu (1991). 0.2-1% is the range of kojic acid concentration used in
the preparation of skin products. Garcia and Fulton (1996) concluded that, kojic acid
along with glycolic acid and hydroquinone used to treat melasma and related conditions.
Kojic acid loaded nanotechnology based drug delivery systems can modulate drug
permeation through the skin and improve the drug activity for the treatment of skin
ageing Goncalez et al. (2013).

1.2.5 In agriculture:

Kojic acid was used in the preparation of insecticides and pesticides Beard and Walton
(1969). In agricultural sector it is used as chelating agent and activator for production of
insecticides. Two ligand complexes contain vaniline and O-vaniline molecules each with
two kojic acid molecules linked with methylene group act as a potent chelators of Fe and
Al Nurchi et al. (2011). The toxic effect of nicotine insecticide was enhanced from 5-
35% by the addition of 5% kojic acid Beelik et al. (1955), Buchta (1983).

7
 CHAPTER – II
REVIEW OF LITERATURE
(Section – A)
 CHAPTER 2

2. REVIEW OF LITERATURE

2.1 INTRODUCION:

The fungi are ubiquitous in nature include diverse types of microorganisms which
produce hundreds of primary and secondary metabolites and out of them only few have
industrial significance. Generally chemical processes are employed for their production
because through fermentation the fungus produce desired product in little quantities or
the separation step of the product in downstream processing is too expensive. Due to
these reasons only few products with more commercial uses were produced industrially.
Industrial production of organic acids was eventuated by employing various
microorganism using different fermentative techniques. The specific fungi belongs to the
genus Aspergillus were familiar in excess production of different organic acids like Citric
acid, Itaconic acid, Gluconic acid etc. However the organic acids like Malic acid,
Gibberellic acid, Kojic acid, Lactic acid etc were produced in little quantities. These acids
are highly useful in therapeutics, research and commercial purposes. Kojic acid is the
secondary metabolite produced by the fungi like Aspergillus, Penicillium, Acetobacter
and it is identified in the year 1903 Yabuta (1916), Prescott and Dunn (1940), Kitamoto
(2002), Corbellini and Gregorini (1930). A number of biologically active compounds and
pharmaceuticals have been produced from kojic acid as it showed lesser side effects and
bearing a polyfunctional groups in its structure Aytemir (2012), Brtko (2004).

2.2 FERMENTATION METHODS FOR KOJIC ACID PRODUCTION:

Many attempts have been made in producing kojic acid through different types of
fermentation methods like solid state fermentation (SSF), submerged fermentation
(SMF), surface fermentation (SF), batch fermentation, fed batch fermentation, continuous
fermentation etc., based on the way through which the fermentor will be operated, Kitada
et.al (1967), Ariff et.al (1996), Futamura et al. (2001), Correia et al.(2004).
8
 CHAPTER – II
REVIEW OF LITERATURE
(Section – A)
 CHAPTER 2

2. REVIEW OF LITERATURE

2.1 INTRODUCION:

The fungi are ubiquitous in nature include diverse types of microorganisms which
produce hundreds of primary and secondary metabolites and out of them only few have
industrial significance. Generally chemical processes are employed for their production
because through fermentation the fungus produce desired product in little quantities or
the separation step of the product in downstream processing is too expensive. Due to
these reasons only few products with more commercial uses were produced industrially.
Industrial production of organic acids was eventuated by employing various
microorganism using different fermentative techniques. The specific fungi belongs to the
genus Aspergillus were familiar in excess production of different organic acids like Citric
acid, Itaconic acid, Gluconic acid etc. However the organic acids like Malic acid,
Gibberellic acid, Kojic acid, Lactic acid etc were produced in little quantities. These acids
are highly useful in therapeutics, research and commercial purposes. Kojic acid is the
secondary metabolite produced by the fungi like Aspergillus, Penicillium, Acetobacter
and it is identified in the year 1903 Yabuta (1916), Prescott and Dunn (1940), Kitamoto
(2002), Corbellini and Gregorini (1930). A number of biologically active compounds and
pharmaceuticals have been produced from kojic acid as it showed lesser side effects and
bearing a polyfunctional groups in its structure Aytemir (2012), Brtko (2004).

2.2 FERMENTATION METHODS FOR KOJIC ACID PRODUCTION:

Many attempts have been made in producing kojic acid through different types of
fermentation methods like solid state fermentation (SSF), submerged fermentation
(SMF), surface fermentation (SF), batch fermentation, fed batch fermentation, continuous
fermentation etc., based on the way through which the fermentor will be operated, Kitada
et.al (1967), Ariff et.al (1996), Futamura et al. (2001), Correia et al.(2004).
8
The production of kojic acid by Solid state fermentation was not studied extensively
because of poor yields obtained with solid substrates. The reports of Nurashikin et al.
(2013) revealed that, 70% moisture content during SSF produce opulent yields of kojic
acid. Comparitively SSF produce lesser yields than SMF and SF. Kharchenko (1999)
isolated 8.5-9.5g kojic acid/Kg of substrate. The study used grains, grain forages, maize,
oats, ryes and barley grains. Nurashikin et al. (2013) reported 0.263g/g kojic acid from
pine apple peel. Chaves et al. (2012) produce 0.31g/g kojic acid from 20% sucrose.
Hazzaa et al. (2013) through SSF produce maximum yield 0.602g/g kojic acid from 100g
glucose.

Surface fermentation takes place in absence of agitation or shaking. It was complicated

to manage the cultural conditions like pH, temperature and dissolved oxygen tension in
SF. Submerged fermentation takes place under agitation conditions and commonly
employed for the production of kojic acid Coninck et al. (2000). The microbial growth
was restricted by the accessibility of carbon sources, enzymes and energy. For the
production of kojic acid through SMF and SF various types of carbon sources like
glucose, sucrose, acetate, ethanol, arabinose and xylose have been used. But it was
identified that glucose was the most suitable source of carbon for its production because
of the structural similarity between glucose and kojic acid. The production of kojic acid
was initiated only when the microbial growth obtained the stationary phase and hindered
when glucose in the fermentation medium was exhausted. Due to high starch
concentration in the fermentation medium there was a dramatically increase in its
viscosity. As a result there was a poor oxygen transport to the culture. This causes very
low yield of kojic acid at extreme concentrations of starch solutions. Hence in order to
produce mycelia mat with more enzymatic activity require high aeration rate throughout
its growth phase. This might be responsible for changing the glucose in to kojic acid,
Ariff et al. (1996). In submerged fermentation the fungus exhibits pulpy or pellet like
morphology Braun and Vecht-Lifshitz (1994).

In membrane surface liquid culture technique (MSLC technique) the fungi were
cultivated on the surface of the porous membrane opposite to the air like surface

9
The production of kojic acid by Solid state fermentation was not studied extensively
because of poor yields obtained with solid substrates. The reports of Nurashikin et al.
(2013) revealed that, 70% moisture content during SSF produce opulent yields of kojic
acid. Comparitively SSF produce lesser yields than SMF and SF. Kharchenko (1999)
isolated 8.5-9.5g kojic acid/Kg of substrate. The study used grains, grain forages, maize,
oats, ryes and barley grains. Nurashikin et al. (2013) reported 0.263g/g kojic acid from
pine apple peel. Chaves et al. (2012) produce 0.31g/g kojic acid from 20% sucrose.
Hazzaa et al. (2013) through SSF produce maximum yield 0.602g/g kojic acid from 100g
glucose.

Surface fermentation takes place in absence of agitation or shaking. It was complicated

to manage the cultural conditions like pH, temperature and dissolved oxygen tension in
SF. Submerged fermentation takes place under agitation conditions and commonly
employed for the production of kojic acid Coninck et al. (2000). The microbial growth
was restricted by the accessibility of carbon sources, enzymes and energy. For the
production of kojic acid through SMF and SF various types of carbon sources like
glucose, sucrose, acetate, ethanol, arabinose and xylose have been used. But it was
identified that glucose was the most suitable source of carbon for its production because
of the structural similarity between glucose and kojic acid. The production of kojic acid
was initiated only when the microbial growth obtained the stationary phase and hindered
when glucose in the fermentation medium was exhausted. Due to high starch
concentration in the fermentation medium there was a dramatically increase in its
viscosity. As a result there was a poor oxygen transport to the culture. This causes very
low yield of kojic acid at extreme concentrations of starch solutions. Hence in order to
produce mycelia mat with more enzymatic activity require high aeration rate throughout
its growth phase. This might be responsible for changing the glucose in to kojic acid,
Ariff et al. (1996). In submerged fermentation the fungus exhibits pulpy or pellet like
morphology Braun and Vecht-Lifshitz (1994).

In membrane surface liquid culture technique (MSLC technique) the fungi were
cultivated on the surface of the porous membrane opposite to the air like surface

9
fermentation in absence of agitation. However the yields were lesser than the yields of
batch fermentation under nitrogen limited environment. The reason was that, most of the
consumed glucose molecules were diverted to microbial growth in the production phase
and very less to kojic acid conversion. These studies concluded the fact that, to achieve
greater yields of product the nitrogen supply should be restricted to the culture so that, the
growth was limited and more conversion of glucose to kojic acid takes place.

In Fix volume fed-batch fermentation the limiting substrate was fed continuously
without culture dilution. As the feeding prolongs, the growth was declined steadily, as
biomass increases it access to maximum sustainability in the vessel once again, at that
moment the culture dilution was performed again Yee et al. (1992).

In Variable volume fed-batch fermentation the volume of the culture changes with the
fermentation time period in response to the substrate fed. Rosfarizan et al. (2002) used
this method to produce kojic acid from gelatinized sago starch and reported 16.43g/L
yield. The author employed DOT strategy in fix volume fed-batch fermentation where
DOT was controlled at elevated levels in active growth phase which was essential to
secrete the most important enzymes for better productivity. The yield resulted was
31.23g/L higher than variable volume fed-batch technique.

Continuous fermentation mode was rarely operated for kojic acid production than batch
type. Two-stage continuous fermentation was used by Kitada et al. (1970), Kitada et al.
(1971) to signify the production rate by A.oryzae in presence of a growth limiting factor
peptone. The yield obtained was 9g/L. This method was done only under nitrogen limited
conditions to get higher yields.

Kwak and Rhee (1991, 1992) employed calcium alginate immobilization method
through repeated batch fermentation for the enhanced production of kojic acid by
A.oryzae. Two important factors which influence this method include bead size and
growth limiting nutrient. Kojic acid precipitates in the form of long colourless prismatic
crystals during down-stream processing in the fermented broth and they sublime in
vacuum without undergoing any changes.

10
2.3 MICROORGANISMS USED FOR KOJIC ACID PRODUCTION:

Kojic acid production was first harnessed in Japan, using Aspergillus species. However
nearly 58 diverse strains of kojic acid producing microorganisms like Aspergillus,
Penicillium have been used Abd El-Aziz (2013) and it was reported recently that even
some plants like Kingella africana can also produce kojic acid Eyong et al. (2012). For
better development in the production of kojic acid, an extended research was done in the
strain development procedures involving various mutational methods, genetic
engineering etc Wu et al. (2004) (Table 2.1).

Table 2.1: Microorganisms used in the kojic acid production:

Microorganisms References
A.flavus Basappa et al. (1970)
Ariff et al. (1996)
Saad et al. (1996)
Chang et al.(2011)
Prabu et al.(2011)
A.oryzae Kwak and Rhee (1992)
Takamizawa et al. (1996)
Wan et al. (2005)
Terabayashi et al. (2010)
A.oryzae MK107-39 Futamura et.al (2001)
A.oryzae var.effusus Kumeda and Asao (1996)
Lee et al. (2006)
A.tamarii Gould (1938)
A.tamarii CCM-F-780, CCM-F-781 Brtko et al. (2004)
A.parasiticus Lin et al. (1976)
Nandan and Polasa (1985)
Coupland and Niehaus (1987)
A.fumigatus Karachenko et al. (1986),
Moubasher et al. (1979),
El-kady et al. (1984)
A.candidus Manabe et al. (1984),
Wei et al. (1991)
A.awamori, A.clavus, A.ustus, A.wentii, Manabe et al. (1984)
A.nidulans
A.lutescens Marston (1949)

11
 2.4 PRODUCTION OF KOJIC ACID FROM DIFFERENT CARBON SOURCES:
Different types of raw materials were used by the earlier researchers to obtain better
yields of kojic acid which include various synthetic carbon sources like glucose, sucrose,
maltose, xylose, alcohols Barnard and Challenger (1949) agrowaste by-products, fruit
wastes, vegetable wastes, industrial wastes etc (Table 2.2). Till now it was reported that
the better source for the production of kojic acid was glucose. Kojic acid directly
produced from glucose without the breakage of its carbon skeleton into shorter
fragments. The production does not takesplace through the break down of earlierly
synthesized storage products like mould polysaccharides. In the following order sucrose,
glucose, galactose and fructose the organism utilizes sugars rapidly Gould (1938).

Table 2.2: References pertained for the production of kojic acid from various
carbon sources

‘C’ Source ‘N’ source Mineral Microorgani Yield Reference

salts sms
Glucose Peptone K2HPO4 A.flavus 57g/L May et al. (1931)
MgSO4
Maltose Ammonium K2HPO4 A.oryzae 41g/L Katagiri and
sulphate MgSO4 Kitahara (1933)
Glucose NaNO3 K2HPO4 A.tamarii 24g/L Gould (1938)
MgSO4, KCl,
FeSO4
Sucrose Yeast extract KH2PO4 A.candidus 60g/L Wei et al. (1991)
MgSO4
Glucose 0.1% yeast No mineral A.oryzae 45 g/L Wakisaka et.al
extract salts NRRL- 484 (1998)
Glucose Peptone, Ammonium A.oryzae 24.2g/L Kitada et al. (1967)
nitrate,
Ammonium
sulphate
Glucose Yeast KH2PO4 A.oryzae 83 g/L Kwak and Rhee
extract, MgSO4 (1992)
Ammonium K2HPO4
sulphate
Glucose Yeast extract KH2PO4 A.oryzae 20 g/L Ogawa et.al
MgSO4 NRRL 484 (1995)
Glucose Ammonium KH2PO4 A.flavus 32.5 g/L Ariff et.al
nitrate MgSO4 44 – 1 (1996)
Corn starch Yeast extract KH2PO4 A.flavus 19.2g/L Rosfarizan et al.
MgSO4 (1998)
Glucose Yeast extract KH2PO4 A.flavus 62g/L Rosfarizan (2000)

12
 MgSO4 Link 44 – 1
Corn starch with Wheat germ, MgSO4 A.oryzae 40 g/L Futamura et.al
Corn steep liquor NaNO3 MK-107-39 (2001)
Gelatinized sago Yeast extract KH2PO4 A.flavus 31 g/L Rosfarizan &
starch MgSO4 Link 44 – 1 Ariff (2002)
Glucose Rice bran KH2PO4 A.oryzae 41g/L Wan et al. (2005)
MgSO4

Glucose Yeast KH2PO4 A.parasiticus 34.38g/L El-Aasar (2006)

extract, MgSO4
peptone,
ammonium
sulphate,
ammonium
nitrate
Sucrose Yeast extract KH2PO4 A.flavus 40.2g/L Rosfarizan &
MgSO4 S44 – 1 Ariff (2006)

Glucose Yeast extract KH2PO4 A.flavus 45.3g/L Rosfarizan et al.

Sucrose MgSO4 Link 44 – 1 33.4g/L (2007)

Sucrose Yellow corn No additional Trichoderma 0.06g/g Saleh et al. (2011)

meal, yeast mineral salts viridae
Sucrose extract Trichoderma 0.03g/g
reseei

Sucrose Yeast xtract No additional A.oryzae 0.31g/g Chaves et al. (2012)

mineral salts
Glucose Ammonium KCl, H3PO4 A.oryzae var 25.5g/L, Hazzaa et al. (2013)
nitrate MgSO4 effuses 42g/L
NRC 14
Glucose Yeast extract KH2PO4 A.flavus 50.7g/L Abd-El-Aziz (2013)
MgSO4

Pine apple peel No addition No addition A.flavus 0.263g/g Nurashikin et al.

(2013)
Corn stalk Peptone KH2PO4 A.oryzae 33.1g/L Yan et al. (2014)
hydrolysate MgSO4 M866

Molasses, Ammonium KH2PO4 A.flavus -7, 18.3 – El-kady et.al

Cheesewhey,CS nitrate MgSO4 23, 24 strains 34.4g/L ( 2014)
L,fruit waste of
orange, peech,
apple and
apricot.Vegetabl
ewaste, Agro-
waste
byproducts-
Wheat
bran,ricefragmen
ts and rice husk.
13
2.5 REVIEW OF LITERATURE ON KOJIC ACID PRODUCTION:

In the year 1907, Saito isolated the kojic acid from the mycelial mat of Aspergillus
oryzae. This contaminant was grown in steamed rice. Later the rice was popularly called
as Koji and the name Kojic acid was invented in the year 1913 by the scientist Yabuta.
He also proposed the kojic acid structure and described it as 5-hydroxy-2-hydroxy
methyl-δ-pyranone Yabuta (1912).

Kojic acid was produced by using Raulin-Thom medium with glucose concentration
400g/L by Aspergillus luteo-virescens. 50g of absolute kojic acid crystals were obtained
from 1L unrefined culture filtrate. The mother liquor still contains 20 to 50 g of kojic acid
Morton et al. (1945).

The research report by Arnstein and Bentley had stated that, mould have enzymes like
aldolase and Triose phosphate isomerase and DHAP is formed in some levels and it is in
equilibrium with glyceraldehyde phosphate. In the presence of high phosphate
concentration 0.25%, the production of kojic acid is rapid and maximum with in 10d of
fermentation and the kojic acid breakdown completely with in 30d Arnstein and Bentley
(1953).

It was reported by Arnstein and Bentley that D-Ribose and D-xylose gave greatest
product of kojic acid, out of the eight stereo isomeric pentoses that were given as
substrates for Aspergillus flavus. No production was observed by A.flavus from L-xylose
or L-ribose as they hardly favor the growth. D-ribose and D-xylose were benefit sources
for prolonged kojic acid formation in case of replacement cultures where mycelia were
previously grown on glucose. L-Rhamnose is an excellent source for production by
A.flavus. Neither A.flavus nor A.oryzae was grown on L-fucose Arnstein and Bentley
(1955).

14
Norman D Davis studied the microbial growth and fermentation by Aspergillus flavus
MTCC10124 when peanut oil was used as a sole source of carbon. Physicochemical
parameters which effect the growth and kojic acid production by the organisms like effect
of pH, time, temperature and surface area-to-volume ratio, nitrogen source and KH2PO4
concentration were examined Norman D Davis (1962).

An investigation report was submitted in the year 1965 by Parrish et al. regarding the
species of Aspergillus and Penicillium collected from the Natick laboratories culture
collection which produce aflatoxins and kojic acid. 166 strains belong to 14 species of
Aspergillus and 8 species of Penicillium were tested for kojic acid and aflatoxin
production. All strains of A.parasiticus and 26 strains of A.flavus generate aflatoxins and
their amounts differ with the fungal strain per ml of culture broth. 5mg/ml of kojic acid
was recovered from culture medium except one strain of A.flavus and all strains of
A.parasiticus. Nine species of Aspergillus and four species of Penicillium isolates yield
kojic acid Parrish et al. (1965).

Basappa et al. had investigated to facilitate the resting cells of A.flavus produced both
kojic acid and aflatoxins with substrate glucose. All the strains of A.flavus produce Kojic
acid but may or may not produce aflatoxin. Only kojic acid production but no aflatoxin
synthesis is noted with D-xylose and ethanol. It was found that kojic acid production and
aflatoxin synthesis had different pathways. Kojic acid production with substrate acetate
was optimum at pH 6 and 7 and at temperature 370C. Aflatoxin production was optimum
at pH 5, 6 and at temperature 250C Basappa et al. (1970).

It was reported by the Lin et al. that one of the Kojic acid and aflatoxin producing strain,
A.parasiticus UNBF A12 was obtained from the air samples of Federal district of Brazil.
The organism produces very higher yields of yellowish- brown kojic acid crystals in the
culture flasks and also able to produce elevated amounts of aflatoxins. The study also
determines that the optimum pH for the production of kojic acid crystals was pH 4.5 to
6.2 Lin et al. (1976).

15
The main aim of this study was to compare the kojic acid accumulation in three different
culture media by Aspergillus candidus. YES medium produced excellent production of
kojic acid around 60mg/ml on 9-12 days and it was three times greater than in medium B.
40mg/ml of yield was obtained in medium A on day 12-15 and it was preferable than
medium B in kojic acid production. Enhanced yield was observed in stationary cultures
than shaken cultures at 100rpm. In all three media no aflatoxin was produced by
A.candidus. All these observations were mentioned by Wei et al. (1991).

Ogawa et al. used two different techniques; shake flask culture method and membrane-
surface liquid culture (MSLC) method for kojic acid fermentation using A.oryzae NRRL-
484. While comparing the results they noticed that maximum production 30mg/ml was
obtained with MSLC technique utilizing 0.25 – 0.5% yeast extract while surface culture
technique produced 20mg/ml of kojic acid with 0.05 – 0.25% yeast extract. The
productivity identified was 14.2g/l/d through repeated fed-batch MSLC technique which
accounts for 6 times greater than the rate of shake culture methods Ogawa et al. (1995).

A fungal strain which was separated from morning glory flower (Bixa Orellana) was
capable on growing cooked starch and produced elevated levels of kojic acid. This fungus
was recognized as A.flavus. Its ability to grow and produce kojic acid was studied on
various kinds of starch like sago, potato and corn starch in shake flasks. Though it grows
adequately on all starch types, an eminent level of kojic acid was produced with corn
starch. When 75g/L corn starch was fermented, elevated kojic acid production 19.2g/L
was achieved. This was reported by Rosfarizan et al. (1998).

Using different carbon sources like glucose, xylose, sucrose, starch, maltose, lactose or
fructose and nitrogen sources like NH4Cl, (NH4)2S2O8, (NH4)2NO3, yeast extract or
peptone, the kinetics of kojic acid fermentation were resoluted with models established
on logistic and Luedeking-Piret equations by Aspergillus flavus Link 44-1 (when 100g/L
glucose was used, elevated kojic acid yield 39.90g/L was achieved in submerged batch
fermentation). The suitable carbon to nitrogen (C/N) ratio was 93.3 for kojic acid
fermentation. This was reported by Rosfarizan and Ariff (2000).

16
Rosfarizan et al. in the year 2000 had reported that the effect of pH in both submerged
fermentation and resuspended cell system on kojic acid fermentation by Aspergillus
flavus Link 44-1. At starting pH 3.0 the production rate was examined and elevated
growth was achieved at pH 6 and 7 in submerged fermentation. In resuspended cell
system, culture growth and maximum yield of kojic acid was occurred at pH 3.
Production in 50L stirred tank fermenter with pH control strategy was better by 20% than
fermentation without pH control. Highest production of 62.0g/L of kojic acid was
obtained in fermentation with pH control strategy Rosfarizan et al. (2000).

It was concluded by Futamura et al. that no kojic acid production was observed in a 3L
airlift bioreactor when glucose or wheat germ medium was used. Whereas elevated kojic
acid production was acquired in a jar fermentor by Aspergillus oryzae MK-107-39.
Partially hydrolyzed corn starch with little amount of corn steep liquor was appropriate
medium for culture in an airlift bioreactor which yields 40g/L of kojic acid. Energy cost
was also one-fourth less to that of jar fermentor which comprises glucose or wheat germ
medium Futamura et al. (2001).

The ability of various fermentation modes were operated in an 8L stirred tank fermentor
using gelatinized sago starch for optimum production of kojic acid by Aspergillus flavus
Link 44-1. Fermentation with primal starch concentration of 60g/L followed by addition
of huge quantity of sago starch (140g/L) yields elevated levels of kojic acid (16.43g/L).
The difference in culture pH during the growth phase is crucial and important for kojic
acid production which indicates the significance of pH control strategy. This was
revealed by Rosfarizan et al. (2002).

El-Aasar reported that A.parasiticus produced elevated levels of kojic acid among the
five regional Aspergillus species employed for kojic acid production on four intended
synthetic medium. A.parasiticus produced eminent levels of kojic acid 34.38g/L by
optimizing the different cultural characteristics like glucose 6%, yeast extract 1%, initial
pH 5, temperature 280C, time 10d in shake flask conditions (220 rpm). Three Gram –ve,

17
three Gram +ve and two strains of Candida were used to illustrate the antimicrobial
activity of kojic acid and some antibiotics El-Aasar (2006).

It was reported by Rosfarizan and Ariff that kojic acid fermentation was performed in
250ml Erlenmeyer flask and a 2L stirred tank fermenter with sucrose as carbon source
using Aspergillus flavus strain S44-1. Shake flask fermentation was employed for kojic
acid production using different carbon sources like glucose, sucrose, fructose and a
mixture of glucose and fructose. The yield was 25.8g/L, 23.6g/L, 6.4g/L, and 13.5g/L
respectively. Elevated levels of kojic acid 40.2g/L was acquired from 150g/L sucrose in a
2L fermenter while the least with fructose 10.3g/L as the only carbon source Rosfarizan
and Ariff (2006).

Rosfarizan et al. in the year 2007 reported the fermentation by A.flavus Link 44-1 in
resuspended cell system by the efficiency of cell-bound enzyme. 2L stirred tank
fermenter was used for culture growth in batch fermentation. The culture was then
transferred in to different carbon source solutions containing shake flasks. Based on the
carbon source consumed, superior production was occurred with glucose (0.365g/g)
succeeded by sucrose (0.279 g/g), starch hydrolysate (0.212 g/g) and fructose (0.195g/g).
With increasing growth, the biotransformation rate was also enhanced. 45.3g/L and
33.4g/L of kojic acid was achieved with 100g/L of glucose and 100g/L of sucrose
respectively Rosfarizan et al. (2007).

Prabu et al. concluded the importance of random mutational methods in the enhancement
of kojic acid yields by A.flavus Link S44-1 in the year 2011. N-methyl-N-nitro-N-
nitrosoguandine (NTG), Ultra violet (UV) and γ-irradiation were used to impel random
induced mutations in A.flavus Link S44-1. By using fed-batch fermentations, the induced
mutant yields kojic acid which is about 2.3 to 2.7 fold greater than the parent strain Prabu
et al. (2011).

18
Abd El-Aziz isolated 58 different types of strains belonging to 6 species of Aspergillus, 1
species of Mucor, 2 species of Penicillium chrysogenum. Among them A.flavus produce
highest yield 0.234g/L/h at 100g/L glucose, 5g/L yeast extract, temperature 300C,
180rpm agitation for 9d. Though different carbon sources used maximum production
50.27g/L was obtained with glucose followed by sucrose 48.95g/L. 50.21g/L of kojic
acid was obtained with yeast extract compared to other nitrogen sources. The study also
produces a mutant strain AFUV8 mutated with UV and Gamma radiations. The mutant
strain produce 40.67g/L of kojic acid from potato starch, 32.31g/L from molasses,
23.58g/L from sugarcane baggase and 16.45g/L from barley grains at 300C, 180rpm
agitation on 9d Abd El-Aziz (2013).

In one study, 10 Aspergillus species were found to produce kojic acid out of the 20 fungal
strains which were cultured on a solid glucose salts medium. Fermentation was carried
out at 280C, pH 4 for 18 days shaken or static. Among the other isolates like Aspergillus
flavus NRC13, Aspergillus tamarii NRC18 and A.parasiticus elevated levels of kojic acid
0.626 and 0.602g/glucose consumed were produced under static and shaking condition by
Aspergillus oryzae var.effusus NRC14. The study also concluded that no aflatoxin
production was identified with Aspergillus oryzae var.effusus NRC 14 and in addition to
that a static culture (42g/L of kojic acid) yielding better amounts of kojic acid while
comparing with the shake flask culture (25.5g/L of kojic acid). This was concluded by
Hazzaa et al. (2013).

The study by Yamada et al. invented the potent and cost effectual production of kojic
acid from invaluable substrate like cellulose and also eports that in the production of
kojic acid 14 genes A009013000132 to A009011300045 including a transcription factor
gene (Koj R; A0090113000137), an enzyme gene (Koj A; A0090113000136) and
transporter gene (Koj T; A0090113000138) form a cluster. The transcription of two
genes Koj A and Koj T was regulated by the Koj R gene. A fungal strain with a defective
Koj R gene will lose the capability of producing kojic acid Marui et al. (2011). It pretends
that Koj R was the main important factor in kojic acid fermentationYamada et al. (2014).

19
An eminent kojic acid yielding strain M866 was originated from a wild strain A.oryzae
B008 by combined mutations of ion beam implications and ethyl methane sulfonate
manipulation. The strains M866 produce kojic acid 1.7 times (40.2g/L) greater than wild
strain (23.58g/L) from 100g/L of glucose in a shake flask. Kojic acid production achieved
90.8% when glucose and xylose were given as carbon source with concentration at 75g/L
and 25g/L respectively and this yield was narrowly lesser than with glucose as whole
carbon source. The maximum kojic acid acquired at the end of fermentation was 33.1g/L
when concentrated corn stalk hydrolysate was used. This was communicated by Yan et
al. (2014).

Mutagenesis of Aspergillus flavus No. 193 was carried out to produce a mutant which
yields kojic acid greater than its parent strain. The efficient mutants were screened with
1% Fecl3 and used for fermentation in a 250ml Erlenmeyer flask with 100ml of 10% w/v
YES medium at 300C and 180 rpm. Spectrophotometry and TLC densitometry were used
to analyze kojic acid. Mutant N40C10 yields (8.15g/L) kojic acid which was twenty
times greater than the yield produced by the parent strain. This was concluded by Suryadi
(2014).

El-kady et al. used 278 different isolates for kojic acid production; five superior kojic
acid producers were used for fermentation on 15 agro industrial byproducts. Aspergillus
flavus No.23 had the capability to utilize all 15 byproducts and yields kojic acid 0.1 –
5.1g/L and the superior byproduct was molasses (5.1g/L kojic acid). Aspergillus flavus
No.7 and 24 generate kojic acid in only 12 byproducts and production ranges between
0.1-12.1g/L and 0.4-9.3g/L respectively. Two isolates of A.flavus var. columnaris (No.36
and 41) will capitulate kojic acid on 13 waste products and the production ranges from
0.1-21.2g/L and 0.1-18.2g/L respectively. The most suitable substrates for the isolates for
kojic acid production were rice fragments and molasses. An elevated yield of 53.5g/L of
kojic acid was achieved (after eight days) on molasses medium by Aspergillus flavus
No.7 by employing 1.5L laboratory fermentor. This was revealed by El-Kady et al.
(2014).

20
Hassan isolated A.oryzae var.effusus NRC14 from Egyptian soil. The highest yield was
obtained with glucose 49g/L followed by 38g/L by sucrose, 34g/L by fructose, 26g/L
from starch, 3g/L by lactose and no production was obtained from cellulose, xylose,
maltose and arabinose. The fermentation conditions maintained were initial pH 4.0,
temperature 300C, and time 12d under static conditions. The reducing sugar levels were
decreased as the concentration of kojic acid and biomass increases Hassan (2014).

Sago hampas was utilized for kojic acid production by the researcher Siti Nuraishah binti
Salehhudin by using different strains of fungus A.flavus. The cultural conditions
maintained were inoculum level 10%, 10g sago hampas, 20% mineral salt solution, 70%
moisture content, temperature 300C, incubation time 360h and the maximum yield
obtained was 2.65g/10g Nuraishah binti Salehhudin (2013).

From these findings, it was found that static fermentation and submerged fermentation
gave higher yields of kojic acid while comparing to solid state fermentation. Only few
studies have explored the possibility of cheap carbon sources being utilized for kojic acid
production. Rosfarizan et al. (1998), Futamura et al. (2001) used corn starch as carbon
source, Rosfarizan and Ariff (2002) reported by using gelatinized sago starch, Abd El-
Aziz (2013) used potato starch and molasses for production, Nurashikin et al. (2013)
reported the production from pine apple and recently El-kady et al. (2014) used different
types of carbon sources like industrial wastes, agro waste by-products and fruit wastes.
The yield obtained was low. Research trials were continued to evaluate a cost-effective
process of kojic acid in exploiting a novel and economical carbon sources to meet the
demand levels for its commercial applications. The present study is an attempt to search
for an alternate and cheap carbon sources to reduce the cost of the fermentation process.

2.6 BIOSYNTHESIS OF KOJIC ACID:

The biosynthesis of kojic acid through fermentation by Aspergillus often involves the
direct conversion of glucose through multistep enzymatic reactions. The following
enzymes Glucose-6-phosphate dehydrogenase, Hexokinase and Gluconate

21
dehydrogenase which constitutes a cell bound enzyme system was involved in the kojic
acid biosynthesis Arnstein (1953), Basappa et al. (1970). The pathway involves the
intermediates Gluconic acid-δ-Lactone
acid Lactone and one of the three compounds Gluconic acid
lactone, 3-keto
keto glucose and oxy kojic acid (Figure 2.1). Glucose act as a precursor for
kojic acid biosynthesis and production ceases when all the glucose molecules were
depleted in the medium Rosfarizan et al. (2002). Terabayashi et al. (2010) identified that,
three genes KojR, KojA, KojT which encode transcription
ption factor, kojic acid synthesizing
synthe
enzyme and a transporter were responsible for kojic acid biosynthesis.

Kojic acid synthesizing fungus Aspergillus oryzae was studied by Ikeda in 1958 for
genetic analysis to know how the sugars are metabolized into a compound
compound which may be
or may not be a precursor of kojic acid. The heterocaryons are generated using induced
mutation. The results concluded that heterocaryons generated by wild type strain (Lys-
(Lys
22) and hetero fermentative type strain (Leu-1)
(Leu yielded some concentration
oncentration of kojic acid
like parental strain (Lys-22).
(Lys 22). Meanwhile the heterocaryons produced by Lys-22
Lys wild
strain and Leu-31
31 a respiratory type strain extracted very less amounts of kojic acid. So
this reveals that three alternate metabolic pathways were
were involved in sugar metabolism
for kojic acid fermentation.

Figure 2.1:: Kojic acid biosynthetic pathway

22
2.7 PROPERTIES OF KOJIC ACID: (Friedmann, 1934)

The chemical name of kojic acid is 5-hydroxy-2-hydroxymethyl-4-pyrone Kahan et al.

(1995) Nandan and Polasa (1985) (Figure 2.1). The molecular formula is C6H6O4. It is an
odourless white crystalline powder combustible at high temperature. The molecular
weight of kojic acid is 141.1 Uchino et al. (1988). The melting point of kojic acid is
1510C-1540C Ohyama and Mishima (1990). It is highly soluble in water, ethanol and
ethyl acetate and weakly soluble in ether, alcohol ether mixture, chloroform and pyridine
Wilson (1971), Bentley (1957). It is weakly acidic and combine with metals like Na, Zn,
Cu, Ni, Ca, Cd and forms salts Crueger and Crueger (1984). It produces Colourless
prismatic needles.

Figure 2.2: Structure of kojic acid:

23
 CHAPTER – II
REVIEW OF LITERATURE
(Section – B)
2.8 RATIONALE OF THIS INVESTIGATION

The outstanding applications of kojic acid commercially in various industries and its
growing demand in the world-wide, an extensive research has been done by a number of
researchers to found out the most applicable cost-effective production of kojic acid. In
view of the fermentation economics any bioproduction at the industrial level was
significantly influenced by the raw material used, the current research provides a cost-
effective production of kojic acid in terms of using novel and cheap carbon sources to
reduce the production cost less than $10/kg which serves a market with strong prospects
of growth.

Commercially, there was an expanded growth in the kojic acid production from
the past 40 years and it was noticed that Charles Pfizer and Company, USA manufactured
the kojic acid for the first time. Later the company patented the kojic acid production
methodology and methods of its derivatives which were used as pesticides. In India, kojic
acid was manufactured by nearly 15 small scale industries. The current price of the kojic
acid is US$ 2500/kg. According to Futamura et al. (2001) the cost for the kojic acid
production was $10/kg for cosmetic purposes. It should be reduced to less than $2/kg. As
its demand is increasing day-by-day there is a need to explore various possibilities for
cost-effective bioproduction of kojic acid to fulfill its requirement in various industries.

24
2.9 AIMS AND OBJECTIVES OF THE PRESENT INVESTIGATION

Collection of soil samples for mycological analysis.Screening of soil samples and

taxonomical identification of the fungal isolates. Selection of promising isolates
which produce kojic acid.

Optimization of cultural parameters for cost-effective production of kojic acid.

Statistical optimization of kojic acid production.

Effect of enhancers on kojic acid production

Purification, characterization and structural elucidation of kojic acid

Investigation for the bioactive properties of kojic acid

25
 CHAPTER – III
ISOLATION AND IDENTIFICATION OF
THE PROMISING ISOLATE CAPABLE
OF PRODUCING KOJIC ACID
 CHAPTER 3

3. ISOLATION AND IDENTIFICATION OF THE PROMISING

ISOLATE CAPABLE OF PRODUCING KOJIC ACID

3.1 INTRODUCTION:

These fungi are ubiquitously present in nature. Aspergillus is the best known and most
frequently used economically important fungi for the production of various chemicals,
enzymes and even used for biosynthetic transformations. Current taxonomies identify
185 species of Aspergillus Kirk et al. (2001). From the past 30 years it was observed that,
various fungal species and few specific bacteria were used for the production of an
important secondary metabolite and a natural organic acid kojic acid Zhiqiang (2005),
Papagianni (2004), Kinoshita (1927), Collins (1949).

Fungus belonging to the genus Aspergillus especially A.flavi group (flavus-oryzae-

tamarii) was capable of secreting very high levels of kojic acid during the stationary
phase. The glucose with its six carbon ring behaves as a precursor for the synthesis of
kojic acid Megalla et al. (1986). The production of kojic acid was initiated only when the
microbial growth attained the stationary phase and hindered when glucose in the
fermentation medium was exhausted Rosfarizan et al. (2002). The carbon source glucose
was not only used for the biomass built-up but also act as a precursor for kojic acid
production Arnstein and Bentley (1956), Kitada and Fukimbara (1971), Balint et al.
(1988). Both wild type strains and mutated strains were employed for the kojic acid
production. Mutational techniques and recombination methods were used to isolate high
yielding strains of kojic acid Fantini (1975), Crueger and Crueger (1984) Abd El-Aziz
(2013), Oda et al. (2011), Chang et al. (2011). Parrish et al. (1966) isolated 14 species of
Aspergillus and recorded the kojic acid production from A.clavatus, A.flavus,
A.fumigatus, A.oryzae, A.parasiticus, A.tamarii, A.ustus and A.nidulans. El-Aasar (2006)
used A.flavus, A.glaucus, A.oryzae, A.parasiticus and A.tamarii for the production of

26
kojic acid. These organisms were isolated from soil samples and seed cultures were
prepared used Ariff’s medium. Hazzaa et al. (2013) isolated 12 different strains of
A.oryzae from soil, water and air and used for the kojic acid fermentation. El-kady et al.
(2014) studied the fungi A.flavus var. columnaris, A.terrus, A.versicolor, A.phoenicis,
A.sclerotiorum, Eurotium amstelodami and reported for the first time that they are the
kojic acid producers. The authors isolated 135 cultures belonging to 18 species of
Aspergillus and stated that, A.flavus and A.flavus var. columnaris produce higher yields
of kojic acid. It should not be ignored about the risk of aflatoxin production by the kojic
acid producing fungus though the production of kojic acid and aflatoxins utilized
different pathways Basappa et al. (1970).

Most of the Aspergillus flavus group microorganisms which include A.flavus, A.oryzae,
A.sojae and A.parasiticus were able to produce aflatoxins Sargeaut et al. (1961),
Sakaguchi and yamada (1944), Raper and Fennel (1965), El-Khadem (1976), Diener and
Davis (1966) concluded that nearly 40% of isolated microorganisms of A.flavus among
the world were not able to produce aflatoxins. Kojic acid producers A.flavus and
A.parasiticus have a common trait of aflatoxin production. Some of the non-toxigenic
organisms were also able to produce aflatoxins Parrish et al. (1966). Not many studies
have compared the aflatoxin positive producers and aflatoxin negative producers Gupta et
al. (1970), Schmidt et al. (1977), Saad et al. (1996). However it was reported that
Heathcote et al. (1965), Roze et al. (2013) kojic acid act as a precursor for the aflatoxin
production.

The present investigation was planned to conduct for the production of kojic acid using
soil isolated fungi instead of culture collections to make the fermentation process
economical. Hence different soil samples were collected in and around areas of
Visakhapatnam (Figure 3.1a-b). The promising isolates were also tested for aflatoxin
production.

27
Figure 3.1a-b: Study area

17042”31’N

83002”36’E

28
3.2 OBJECTIVES:

To isolate and identify various soil fungi from different soil samples.

To screen the promising fungal isolate for kojic acid production.

To identify the aflatoxin production by the promising isolate.

3.3 MATERIALS AND METHODS:

Table 3.1: CZA medium

Ingredients Composition (g/L)

Sucrose 30
Sodium nitrate 2.0
Dipotassium phosphate 1.0
Magnesium sulphate 0.5
Potassium chloride 0.5
Ferrous sulphate 0.01
Agar 15
Distilled water 1l

Table 3.2: Lacto phenol cotton blue stain:

Ingredients Composition
Phenol crystals 20g
Lactic acid 20ml
Glycerol 40ml
Distilled water 20ml
Cotton blue or methyl blue 0.075g

29
Table 3.3: Ariff’s medium

Ingredients Composition (g/L)

Glucose 50
Yeast extract 5.0
Potassium dihydrogen phosphate 1.0
Magnesium sulphate 0.5

Table 3.4: Czapek’s agar solution in absence of nitrogen source

Ingredients Composition (g/L)
Potassium dihydrogen phosphate 1.0g
Magnesium sulphate 0.5g
Potassium chloride 0.5g
Ferrous sulphate 0.01g
Sucrose 30g
Agar 20g
Distilled water 1L

Preparation of Fecl3 reagent:

Reagent was prepared by dissolving 1g of Fecl3 in 100ml of distilled water. To this 10ml
of 0.1N Hcl (0.896ml of Hcl mixed in 91.04 ml of water) was added.

3.3.1 Collection of soil samples:

Samples of different types namely paddy soil, peanut soil and garden soil were collected
from different places superficially in a screw capped tubes Dubey and Maheshwari
(2003).

3.3.2 Isolation of fungi:

Serial dilution technique Dubey and Maheshwari (2003) was employed to isolate the soil
fungi. From the dilution, 0.5ml volumes were pippeted out into Czapek Dox agar media

30
(CZA) plates (Table-3.1). Spread plate technique Dubey and Maheshwari (2003) was
performed and the plates were incubated at 280C for one week. After one week, the
plates were examined for colony morphology. The isolated fungal strains were
maintained on Czapek Dox agar medium slants at 40C and sub cultured for every 15 days
in Czapek Dox agar medium Pitt (2009).

3.3.3 Identification of fungi:

Lacto phenol cotton blue staining was used for fungal species identification. A drop of
95% alcohol was placed on the slide and then a small fragment of the culture was gently
teased and placed in an alcohol with the help of a needle. When the above suspension
was spread most of the alcohol was evaporated. Now a drop of LPCB stain (Table -3.2)
was applied and covered with a cover slip to avoid air bubbles. A gentle pressure was
exerted if the fungus fragments do not lie flat. By using a piece of blotting paper, excess
stain was removed from the edges of the cover slip. After few minutes, the preparation
was examined under low power and high power compound microscopy Mackey and
McCartney (1996).

3.3.4 Kojic acid production:

The seed medium designed by Ariff et al. (1996) was utilized for the inoculum
preparation (Table-3.3). The spore suspension (1x107) for inoculation was prepared by
adding two drops of 0.1% Tween 80 solution into a test tube containing 10ml of sterilized
distilled water. Now the spores were carefully scraped from the CZA slants using an
inoculation loop and transferred into a test tube. 5ml of spore suspension of each isolated
fungal strain was added to 250 ml conical flask have 50 ml of seed medium. All the
flasks were incubated at 280C for 12 days. After fermentation, the broth was filtered and
the supernatant was estimated for kojic acid production by Bentley’s colorimetric method
Bentley (1957). Two different fermentation techniques, surface fermentation and
submerged fermentation were used for the kojic acid production. Submerged
fermentation was conducted in an orbital shaker (Remi instruments Ltd.) at different
agitation speeds of 100, 150, 200, 250 rpm. The isolated fungal organisms which showed

31
positive response for the kojic acid production were selected for further fermentation
processes at various physicochemical conditions by using the production medium
described above Ariff et al. (1996) but with replacement of glucose with starch
substrates, liquid substrates, agro waste by-products and bran substrates.

3.3.5 Quantitative estimation of kojic acid production by the culture supernatant:

Standard curve of kojic acid: The standard solution of kojic acid was prepared by adding
50mg of kojic acid in 100ml of water. 0.2ml to 1.6ml of standard sample solution was
pipette out into a series of test tubes. Now distilled water was added to test tube to make
the total volume to 2ml. Later 1ml of Fecl3 reagent was added to each tube and boiled for
20min in a waterbath. Finally 7ml of water was added and the intensity of the colour was
measured in a spectrophotometer at 505nm. The concentration of kojic acid in the test
samples was determined by using standard graph of kojic acid.

3.3.6 Checking for Aflatoxin production:

The aflatoxin production from the isolated fungal organism was detected by the
fluorescent-agar medium procedure followed by Hara et al. (1974), Mayura and
Sreenivasamurthy (1969).The isolated fungal organism was inoculated into the petridish
at the centre of the solidified agar medium (Table-3.4). The plates were incubated at 280C
under dark conditions. Intensity of the fluorescence was found out individually. The
presence or absence of blue fluorescence in the agar plates surrounding the isolated
colonies was observed under UV (366nm) illumination. For extraction of aflatoxins 30g
of agar medium exhibiting blue fluorescence was combined with 75ml of water in a
warring blender for 5min. By using 25ml of chloroform the aqueous slurry was extracted
for 5min by blending. Subsequent centrifugation eliminates the chloroform layer. The
retained chloroform extraction of the aqueous layer was repeated. Now the two
chloroform fractions were merged, filtered and concentrated to dryness. The residues
were tested for TLC using 10ml of chloroform.

32
3.4 RESULTS AND DISCUSSION:

3.4.1 Morphological identification of fungi:

Ten different fungal cultures were isolated from soil samples. The fungal organisms were
identified based on their morphological characteristics like diameter, colour and texture
of the colony as well as conidia size, texture and the conidiophores structure Pitt (2009),
Olutiola (1976) (Table 3.5), (Plates 3.1-3.10). The isolated cultures were preserved in the
form of agar slants in a refrigerator for futher experiments.

Table 3.5: Fungi isolated from soil samples

Morphological features Fungi

Colonies are Velvety, textured loosely with greenish to yellow Aspergillus
shades. flavus
Conidial heads are Columnar yellowish green in colour.
Conidia are thin walled, variable in size and shape. Very long
chains of blue coloured spores were observed. Growth is rapid
on Czapek-Dox agar plates.
Velutinous colonies with white mycelium with pale or dull Aspergillus
brown to yellowish brown colouration. Conidial production is terrus
heavy.
Colonies are wrinkled, dense and velutinous green or bluish Aspergillus
green in colour with clear exudates. Conidial heads are densely fumigatus
packed.
Black or dark brownish black coloured colonies. Spherical Aspergillus
black conidia. Colonies are plain, velutinous, subsurface niger
mycelium, closely packed with dark brown or black conidial
heads.
Cottony like growth is observed on CZA plates. Size of the Fusarium
colony was 60mm. White mycelium often covered with fine solani
droplets of clear exudate. Macroconidia are abundant, stout,
slightly curved, thick walled with septa usually sickle shaped
like structures.
Growth is low, moderately dense, powdery, and brown in Mucor
colour spreading across the petridish. The colour of the colonies piriformis
is brown on CZA plates. Brush like conidiophores is seen borne
from surface hyphae. Irregular epical projections of columellae,

33
 brown sporangiospores with rough or spiny walls. Clear
exudate is seen on the colonies.
Colonies are dark green in colour on CZA plates and can be Aspergillus
easily identified from other cultures. Conidia with echinulate sojae
surfaces. Conidia colour is olive brown
Colonies are plain green in colour. Reverse is olive green in Aspergillus
colour. Conidial heads are short and columnar. Conidiophores nidulans
are short and smooth walled. Conidia are globose and rough
walled
The size of the colony is 37mm in diamt. The mycelium is Penicillium
yellowish white. Conidial production is moderate and bluish chrysogenum
green in colour. Conidiophores borne from surface hyphae and
smooth walled.
The dia mt. of the colony is 75mm. Colony spreading quickly, Aspergillus
floccose, white centered green. The texture of the colony is wet oryzae
and the colour of the conidia is yellowish-green and powdery.
Conidia smooth to finely roughened, globose to ellipsoidal.

Plate 3.1: Morphological structure of A.flavus

A.flavus was isolated from garden soil 10-5 dilution

34
Plate 3.2: A.sojae Plate 3.3: A.nidulans

A.sojae was isolated from garden soil A.nidulans was isolated from paddy soil
at 10-5 dilution at 10-3 dilution

Plate 3.4: A.fumigatus Plate 3.5: A.niger

A.fumigatus was isolated from garden soil A.niger was isolated from garden soil
at 10-5 dilution at 10-5 dilution

Plate 3.6: A.oryzae Plate 3.7: F.solani

A.oryzae was isolated from paddy soil Fusarium was isolated from peanut soil soil at
10-5 dilution at 10-5 dilution

35
Plate 3.8: M.piriformis Plate 3.9: P.chrysogenum

-4
Mucor was isolated from peanut soil at 10 Penicillium was isolated from paddy
dilution soil at 10-5 dilution

Plate 3.10: A.terrus

-5
A.terrus was isolated from peanut soil at 10 dilution

3.4.2: Selection of promising isolates which produce kojic acid and checking the
isolate for Aflatoxin production:

The isolated fungal organisms when tested for the kojic acid production in Ariff’s
glucose medium using two different fermentation techniques namely surface
fermentation and submerged fermentation, only two fungi A.flavus and A.sojae were able
to produce kojic acid in surface fermentation technique. The yields obtained were

36
37.9g/L by A.flavus and very low yield by A.sojae 11.3g/L. In submerged fermentation
technique, A.flavus produce 5.53g/L of kojic acid at 100 rpm agitation speed and no
production was observed with A.sojae because the mycelia growth was confined only to
the walls of the conical flask and no growth was identified in the centre of the flask.

In surface fermentation the fungal growth takes place on the surface of the solid or liquid
substrate Wei et al. (1991). The study reported by Wei et al. (1991) that, high amounts of
kojic acid were produced by A.candidus ATCC 44054 under static conditions than
shaking conditions at 100 rpm. Saad et al. (1996) concluded that, more production by
A.parasiticus was takes place at stationary conditions than at shaking conditions 150rpm.
The results of Hazzaa et al. (2013) indicated that, the yield of kojic acid was higher at
static conditions than shake flask at 150rpm by A.oryzae var.effusus NRC14 and A.flavus
NRC13. The results of the present research were in-line with many earlier findings which
reported that surface fermentation was more effective for kojic acid production than
submerged fermentation Hara et al. (1974), Lin et al. (1976), Nandan and Polasa (1985),
Wei et al. (1991), Ogawa et al. (1995). During the fermentation the fungi utilizes initially
the glucose molecules for its growth and later synthesizes the kojic acid during its early
stationary phase and decline phase Kitada et al. (1967). Upon prolonging the
fermentation time period, the cells were still capable of secreting kojic acid or possess a
stable cell bound enzymes which were involved in kojic acid biosynthesis Bajpai et al.
(1982). The isolated fungal cultures were identified as non-aflatoxin producers because
no blue fluorescence was observed surrounding the colonies in a fluorescent agar plates.
From the literature it was revealed that, most of the kojic acid producers were non-
aflatoxin producers Basappa (1970), Madihah (1996), Bracarense and Takayashi (2014).
The present results were in-line to the past studies.

37
3.5 CONCLUSION:

Ten different species of soil fungi A.flavus, A.sojae, A.niger, Penicillium chrysogenum,
A.terrus, Fusarium solani, A.fumigatus, A.nidulans, Mucor piriformis, A.oryzae were
isolated and identified from three different soil samples paddy soil, peanut soil and
garden soil. Out of these different isolated fungal cultures A.flavus and A.sojae were
found to be producing kojic acid. A.flavus produce higher yield compared to A.sojae in
surface fermentation and there was no or little production in submerged fermentation
method by both the fungi. Hence for further studies A.flavus was selected. The isolated
kojic acid producing fungal cultures were identified as non-aflatoxin producers.

38
 CHAPTER – IV
OPTIMIZATION OF CULTURAL
PARAMETERS FOR COST EFFECTIVE
PRODUCTION OF KOJIC ACID
 CHAPTER 4

4. OPTIMIZATION OF CULTURAL PARAMETERS FOR

COST-EFFECTIVE PRODUCTION OF KOJIC ACID

4.1 INTRODUCTION:

It is still uncertain for kojic acid production about the efficiency and production
economics because the nutritional media plays a more significant role in the betterment
of such fermentation process. Now-a-days, research trials were done to identify the novel
and potential nutritive sources and most advanced fermentative methods to achieve very
higher yields of product and at the same time high conversion rate of the substrate
molecules. Usage of pure sugars like glucose, sucrose, arabinose, xylose, fructose,
mannose, and galactose Arnstein and Bentley (1956), Burdock et al. (2001), Rosfarizan
and Ariff (2007) resulted in the production of pure product subsequently the cost of the
purification process reduced. But economically this was not feasible because of the high
cost of the pure sugars. Hence these expensive sources were substituted by the usage of
low cost agricultural resources. Literature was available on kojic acid production by
using various industrial and agro-waste by products cocoa juice El-Sharkawy (1995), pea,
kidney bean, vegetable waste, fruit waste of apricot, orange and peach, carrot waste,
turnip waste, cornsteep liquor, molasses, cheese whey, wheat bran, rice husk, rice
fragments by El-kady et al. (2014), corn starch, sago starch, potato starch by Rosfarizan
et al. (1998), gelatinized sago starch Rosfarizan and Ariff (2002), corn stalk hydrolysate
Yan et al. (2014), pine apple peel by Nurashikin et al. (2013). Kitada et al. (1967)
reported that, excellent yields of kojic acid were produced by the sugars glucose, sucrose
and fructose and no production was identified from starch and xylose. The present study
is one such to provide an alternative and economically feasible carbon sources and cost-
effective process of kojic acid production. Optimization of process parameters is the most
valuable step in industrial production methodology, during which even a slight progress
may lead to success in the fermentation process commercially. It was important that, the
secondary metabolites which of high economic importance were not or little produced by

39
fungal species at unfavourable cultivation conditions Bills et al. (2008). Hence it is
necessary to manipulate the cultivable conditions to optimum so as to get maximum
yield. Until 2003, the optimization of medium composition and cultural conditions for
kojic acid production was not discovered Futamura et al. (2001), Lin (2001), and Gad
(2003). But during the year 2006, El-Aasar first reported the optimum cultural conditions
for the production of kojic acid. Variation in the Carbon source concentration, Nitrogen
concentration, Mineral salt concentration, pH, Time, Temperature shows an immense
effect on the production of kojic acid. The fermentation medium composition had main
effect on the production of kojic acid and generally differs for each and every
microorganism and nearly 30-40% is the estimated cost of the production medium in the
total production cost of a fermentation process. So it is essential to optimize the nutrient
composition and fermentation conditions accordingly.

In the present study, empirical optimization strategy or traditional ‘one-factor-at-a-time’

(OFAT) method was used for the kojic acid production. This method involves varying
one factor at the same time keeping the other factors constant under a specific set of
conditions (Flow chart-4.1) Ahamed et al. (2006), Alexeeva et al. (2002) Patidar et al.
(2005). The method is considered as a closed ended system used for optimization of
medium ingredients as well as physical conditions of the fermentation. The method is
simple, easy and also practical to study the influence of process parameters in the form of
graphs Kar et al. (1999) and Kumar et al. (2003). Most of the past works related to kojic
acid production were carried out using OFAT method but recently an experimental
design called Response surface methodology (RSM) was introduced to study the main
and interaction effects of various factors. The current report presents exclusively in
utilizing fourteen different carbon sources or raw materials categorized into four types
were used in the optimization process of kojic acid production by A.flavus selected from
the preliminary screening experiment. After performing the OFAT experiments the high-
yielding substrates were selected and subjected to statistical optimization by RSM. From
the literature review it was found that, till now there were no studies on kojic acid
production involving RSM methodology. The present study was the first attempt of using
RSM technology for kojic acid production.

40
4.2 OBJECTIVES:

To select a better nitrogen source which gives the highest production of kojic acid

To optimize the process parameters for cost-effective production of kojic acid like
Carbon source concentration, peptone concentration, pH, Time, Temperature,
KH2PO4 concentration and MgS04 concentration.

To determine the statistical significance of each independent variable on the
production of kojic acid using One-way-ANOVA.

41
 Flow chart 4.1: Optimization of kojic acid

Ipomea starch
Cassava starch
Sago starch
Alocasia macrorrhiza starch
Palmyra sap
Carbon sources Paneer whey
Coconut water
Muntingia calabura fruits
Cashew apple
Pine apple peel
Papaya waste
Sugar cane baggase
Wheat bran
Rice bran
Chemical
parameters
Peptone

Nitrogen source Yeast extract

Ammonium
sulphate

Mineral salts KH2PO4

MgSO4

pH

Physical
Temperature
parameters

IncubationTime

42
4.3 MATERIALS AND METHODS:

Different physico-chemical factors include carbon source concentration, peptone

concentration, pH, time, temperature, potassium dihydrogen phosphate concentration and
magnesium sulphate concentration were choosen for the optimization of kojic acid
production.

4.3.1 Selection of better nitrogen source:

Peptone and yeast extract were commonly used for the kojic acid production. They
promote the better growth of the fungus because of the presence of certain oligoelements
and vitamins Gad (2003), stabilizing the pH of the medium and act as a precursor for
kojic acid production. Peptone was the best nitrogen sources for the production of kojic
acid compared to yeast extract Kitada et al. (1967), Coupland and Niehaus (1987). As the
nitrogen source play one of the key role in kojic acid production it is necessary to select a
suitable nitrogen source before performing optimization process. Hene two different
nitrogen sources inorganic salts and organic nitrogen sources were tested for maximum
production. Inorganic nitrogen source was ammonium sulphate and organic nitrogen
sources were peptone and yeast extract. Ariff’s medium was used as a testing medium
contain 5g/L of nitrogen source.

4.3.2 Carbon sources:

All the carbon sources were collected from the local areas of Visakhapatnam. Fourteen
different types of carbon sources were chosen for the optimization of kojic acid
production which includes solid starch substrates Ipomea, Cassava, Alocasia and Sago
starch, liquid substrates Palmyra sap, Paneer whey and waste Coconut water, fruit and
fruit based sources Muntingia calabura fruits, Cashew apple, Papaya waste and Pine
apple peel, agro waste by-products Sugarcane baggase, Rice bran and Wheat bran.

43
A criterion of selection of carbon sources in the current study was based on:
 Most of them were novel sources and commercially not exploited for kojic acid
production.
 They were available in huge quantities.
 The collection of the raw materials was so easy.
 The substrates were present all over the tropical areas some were available at free
of cost.
 Most of the raw materials were accessible throughout the year and some were
available seasonally.
 As they are rich in carbohydrate content make them to use as carbon sources.
 The raw materials also contain proteins, vitamins, fatty acids, minerals etc. hence
fermentation with these sources need very little quantities of additional factors i.e.
mineral salts, growth factors etc. for the favourable production of kojic acid.
 The raw materials do not show any adverse effects on the environment.
 Most of them are not staple foods

a. Alocasia macrorrhiza tuber: Very large stem tubers of Alocasia macrorrhiza were
collected from the road side plants. A.macrorrhiza L. also called as Gaint Taro belonging
to the family Araceae Boyce (2008). The plant produces a thick cylindrical stem
originated from a basal corm which was used as one of the raw material in the current
study. The plant was commonly grown as ornamental foliage plant Kay (1987). The
edible portion of the raw material possess an energy value of 293-599 KJ/100g, water 63-
81%, crude protein 0.6-3.3%, fat 0.1-2%, carbohydrate 17-27%, ash 1.1-1.3% and
contains little quantities of mineral ions and vitamins. The stem tuber also contains
calcium oxalate crystals Harley Manner (2011) and Kay (1987). The amount of starch
present in the tuber ranges from 16-21%, Foliaki et.al (1990).

b. Ipomea batatas: The dicotyledonous plant, Ipomea batatas commonly called as sweet
potato was related to the family, Convolvulaceae. The plant possesses large sweet starch
tuberous roots as a root vegetable. 100 grams of raw tuber contains an energy value of

44
359 KJ (86 Kcal). Its composition is carbohydrates 20.1g, starch 12.7g, sugar 4.2g,
dietary fibre 3g, fat 0.1g, protein 1.6g and also contains vitamins like Vitamin A (89%),
Vitamin B1 (7%), Vitamin B2 (5%), Vitamin B3 (4%), Vitamin B5 (16%), Vitamin B6
(16%), Vitamin B9 (3%), Vitamin C (3%), Vitamin E (2%). The traces of metals like Ca
(3%), Fe (5%), Mg (7%), Mn (12%), P (7%), K (7%), Na (4%), and Zn (3%) are also
present.

c. Cassava tuber: Cassava is commonly known as Manihot esculenta or tapioca root. It is

a woody shrub belongs to the family Euphorbiaceae and a common inhabitant of South
America. It is an annual plant and widely cultivated in the tropical and sub-tropical areas
due to its large edible tuber, containing abundant starch. The tuber contains 62-65%
moisture, 20-31% carbohydrates, 1-2% crude protein and very low amounts of vitamins
and minerals. 100 grams of cassava contains 670 KJ of energy. Its composition is protein
1.4g, fat 0.28g, carbohydrates 38g, fibre 1.8g, sugar 1.7g, Ca-16mg, Fe-0.27mg, Mg-
21mg, P-27mg, K-271mg, Na-14mg, Zn-0.34mg, Cu-0.1mg, Mn-0.38mg, Se-0.7µg,
Vitamin C-20.6mg, Vitamin B1-0.09mg, Vitamin B2-0.05mg, Vitamin B3-0.85mg,
Vitamin B5-0.11mg, Vitamin B6-0.09mg, Vitamin B9-27 µg, Vitamin A-13IU, Vitamin
E-0.19mg, Vitamin K1-1.9 µg, beta carotene-8 µg Anonymous (1956).

d. Sago starch: Metroxylon Sago or Sago palm is a starch containing palm pith Jong
(1995). Maximum amount of starch content was accumulated in the trunk of the plant just
prior to the flowering stage of the plant. It belongs to the family Palmae. It is found
commonly in hot humid tropics.150-300 kg of starch has been obtained from a single
palm tree. 100g of dry sago contains 94% of carbohydrates, 0.2% of proteins, 0.5% of
dietary fibre and little quantities of minerals, fats, vitamins etc.

e. Palmyra sap: The robust tree Borassus flabellifer or Palmyra palm or toddy palm or
sugar palm is inhabitant to the Indian subcontinent and Southeast Asia. It produces one of
the most significant product sap have a dominant sugar sucrose in proportions 10.36% -
16.94% which was selected as a carbon source in the present study. The nutritional

45
composition of Palmyra sweet sap (g/100cc) is protein – 0.35, total sugar – 10.93,
reduced sugar – 0.96 and other minerals and sugars.

f. Paneer whey: Whey is the by-product from dairy products since has no added value
and it is dumped in to the environment. It was determined by Aneja et al. (2002) that
paneer production in India was high and is 1,50,000 tonnes. This may result in the
production of 2 million tonnes of whey which might contain 1,30,000 tonnes/annum of
desired milk nutrients. The amount of nutrients present in the whey depends on the
ingredient composition of milk from where the whey was derived and also based on the
milk processing methods. The major sugar present in whey was lactose within the range
of 70%. The nutritional composition of whey was Na (mg/L) – 350, K (mg/L) – 1300, Ca
(mg/L) – 480, Mg (mg/L) – 59, Cl (mg/L) – 1349, Citrate (mg/L) – 6750, Zn (mg/L) –
280, Total proteins (%) – 0.41, Fat (%) – 0.01, Lactose (%) – 4.5, Total solids (%) – 5.8.

g. Coconut water: It is the liquid which is present in the endosperm of coconut taken as
a healthy and nutritional drink by the human beings worldwide. The water is clear, sterile
and contains unique compound like sugars, vitamins, electrolytes, amino acids, enzymes,
minerals, phytoharmones and cytokine. Its scientific name is Cocos nucifera belonging to
Atecaeceae family. The tree grows commonly in coastal tropical areas. The liquid is rich
with sugars and amino acid and consists of very less amounts of sodium and chlorides.
The composition of coconut water per 100g is energy – 19Kcal, carbohydrates – 3.71g,
protein – 0.72g, total fat – 0.2g, dietary fiber – 1.1g.

h. Muntingia calabura L: The plant is the inherent species of Northern South America,
Central America, Great Antilles, and Southern Mexico and can be seen as a road side
plant generally in South-East Asian countries. It is commonly called as Jamaica cherry or
Panama berry from Elaeocarpaceae family. The plant contains round berries whose
diameter varies from 1-1.25cm and are usually red, sometimes produces yellow coloured
berries and the fruit have a thin, tendered, smooth skin. The berry inside contain a pulp
which is light brown, soft and juicy possess fig like flavour and also have many minute
dark brown or black coloured seeds. Fruits are borne nearly throughout the year and ripen

46
in 6-8 weeks from the stage of anthesis. 100g of berries nearly contain 73.63g of H2O,
2.1g protein, 2.3g fat, 17.9g carbohydrates, 6.0g fibre, 1.4g ash, 125mg Ca, 94mg P,
0.015mg Vit-A, 90mg Vit-C. The energy value is 380KJ/100g Morton J (1987), Rama
rao (1914).

I. Cashew apple: It is scientifically called as Anacardium occidentale L from

Anacardiaceae family and also called pseudo fruit treated as a by-product in cashew nut
industry. It is Pan-tropical in distribution.The shape of the fruit nut is kidney shaped and
during the maturation phase, the stalk above it becomes swollen, fleshy and becomes a
pear shaped accessory fruit which is 2-4 inches contain yellow or red coloured juice. The
mature fruit was often characterized by a good aroma, more sugar content and acidity and
less astringent. The fruit contains 84 – 88% H2O, 0.1 – 0.16g protein, 0.05 – 0.5g fat, 9.1
– 9.8g carbohydrates, 0.4 – 1.0g dietary fibre, 0.9 – 5.4mg Ca, 0.2 – 0.7mg Fe, 0.02 –
0.03mg Vit-B1, 0.1 – 0.4mg Vit-B2, 0.1 – 0.5mg Vit-B3, 147 – 372mg Vit-C.
Carotenoids, quercetin, anacardic acid, tannins, and organic acids, anti oxidants were also
present Morton J (1987), Verheij, E.W.M (1992).

j. Pine apple peel: Ananas comosus L. (Bromeliaceae) or pine apple added greater than
20% of the world production of tropical fruits Coveca (2002). Ketnava et al. (2009)
revealed that the pine apple peel is a huge waste and a good source for extraction of
valuable bioactive compounds. The waste still have a considerable quantity of soluble
sugars, in addition to high fiber and low protein content. Correia et al. (2004), Rosma et
al. (2005) reported that the pine apple waste which consists of peel, core and unwanted
parts of pine apple contain upto 6.14% of carbohydrate, mineral especially Mg and 0.6%
of crude protein.

k. Papaya waste: Papaya fruit (Carica papaya L.) is a common fruit of tropical America,
Southern Mexico, and Northern Nicaragua. It found frequently in all tropical areas. Brazil
is the second largest producer of papaya. As a result of processing it produces a waste
papaya waste which is rich with calcium, Vit-A and Vit-C. The chemical composition

47
(g/100g) of peel Ash 11.8g, lipids 2.44g, protein 18.18g, total fiber 33.05g, carbohydrates
9.67g.

l. Sugarcane baggase:
In the form of feed stock, sugarcane was utilized worldwide for the production of sugar
and bio-ethanol. Through the milling process, the juice was extracted from the sugarcane
and left out of the baggase as a by-product which comprises 60 – 80% of carbohydrates.
Though it is a good source of carbohydrates, it is often treated as an agricultural waste
and discharged in the environment which makes pollution. Now-a-days this by-product
baggase was used as a renewable feed stock for the production of various industrial
compounds like ethanol, citric acid, bio-fuel production and in pulp industry etc. The
carbohydrates cellulose and hemicelluloses embedded in the matrix of lignin were
present in the baggase. The baggase contains 35.2% cellulose, 24.5% hemicelluloses,
22.2% lignin and 20.9% ash.

m. Wheat bran: The scientific name of wheat is Triticum aestivum L. It contributes the
significant source of dietary fiber and contains ample quantities of minerals and few
vitamins. Wheat bran is the external hardy layer of the wheat grain. Majority of the foods
were made with wheat flour and the procedures making the wheat flour get rid of the
bran. The processing procedures however cause the loosing of vitamin compounds and
fibre material from the wheat bran. The raw material was preserved at a low temperature
to prevent the rancidity. The wheat bran possess an energy value of 1216KJ, protein
16.2g, fat 5.3g, carbohydrate 65.1g, dietary fibre 40.2g, ash 5.4g, moisture 8.2g, vitamin
E 1.6mg and vitamin K 83µg .

n. Rice bran: Rice bran is one of the byproduct obtained through paddy milling process.
The rice bran was not utilized as a food supplement for humans. It is necessitate to
exploit the complete possible of the accessible rice bran in the country, equally as a
source of healthy edible oil and as a food supplement for supporting our population’s
nutrition and health. Now-a-days the rice bran made in the rice mills contain endosperm
starch and rice germ. The nutritional value of rice bran per 100g is protein 16.5g, fat

48
 CHAPTER – IV
OPTIMIZATION OF CULTURAL
PARAMETERS FOR COST EFFECTIVE
PRODUCTION OF KOJIC ACID
 CHAPTER 4

4. OPTIMIZATION OF CULTURAL PARAMETERS FOR

COST-EFFECTIVE PRODUCTION OF KOJIC ACID

4.1 INTRODUCTION:

It is still uncertain for kojic acid production about the efficiency and production
economics because the nutritional media plays a more significant role in the betterment
of such fermentation process. Now-a-days, research trials were done to identify the novel
and potential nutritive sources and most advanced fermentative methods to achieve very
higher yields of product and at the same time high conversion rate of the substrate
molecules. Usage of pure sugars like glucose, sucrose, arabinose, xylose, fructose,
mannose, and galactose Arnstein and Bentley (1956), Burdock et al. (2001), Rosfarizan
and Ariff (2007) resulted in the production of pure product subsequently the cost of the
purification process reduced. But economically this was not feasible because of the high
cost of the pure sugars. Hence these expensive sources were substituted by the usage of
low cost agricultural resources. Literature was available on kojic acid production by
using various industrial and agro-waste by products cocoa juice El-Sharkawy (1995), pea,
kidney bean, vegetable waste, fruit waste of apricot, orange and peach, carrot waste,
turnip waste, cornsteep liquor, molasses, cheese whey, wheat bran, rice husk, rice
fragments by El-kady et al. (2014), corn starch, sago starch, potato starch by Rosfarizan
et al. (1998), gelatinized sago starch Rosfarizan and Ariff (2002), corn stalk hydrolysate
Yan et al. (2014), pine apple peel by Nurashikin et al. (2013). Kitada et al. (1967)
reported that, excellent yields of kojic acid were produced by the sugars glucose, sucrose
and fructose and no production was identified from starch and xylose. The present study
is one such to provide an alternative and economically feasible carbon sources and cost-
effective process of kojic acid production. Optimization of process parameters is the most
valuable step in industrial production methodology, during which even a slight progress
may lead to success in the fermentation process commercially. It was important that, the
secondary metabolites which of high economic importance were not or little produced by

39
fungal species at unfavourable cultivation conditions Bills et al. (2008). Hence it is
necessary to manipulate the cultivable conditions to optimum so as to get maximum
yield. Until 2003, the optimization of medium composition and cultural conditions for
kojic acid production was not discovered Futamura et al. (2001), Lin (2001), and Gad
(2003). But during the year 2006, El-Aasar first reported the optimum cultural conditions
for the production of kojic acid. Variation in the Carbon source concentration, Nitrogen
concentration, Mineral salt concentration, pH, Time, Temperature shows an immense
effect on the production of kojic acid. The fermentation medium composition had main
effect on the production of kojic acid and generally differs for each and every
microorganism and nearly 30-40% is the estimated cost of the production medium in the
total production cost of a fermentation process. So it is essential to optimize the nutrient
composition and fermentation conditions accordingly.

In the present study, empirical optimization strategy or traditional ‘one-factor-at-a-time’

(OFAT) method was used for the kojic acid production. This method involves varying
one factor at the same time keeping the other factors constant under a specific set of
conditions (Flow chart-4.1) Ahamed et al. (2006), Alexeeva et al. (2002) Patidar et al.
(2005). The method is considered as a closed ended system used for optimization of
medium ingredients as well as physical conditions of the fermentation. The method is
simple, easy and also practical to study the influence of process parameters in the form of
graphs Kar et al. (1999) and Kumar et al. (2003). Most of the past works related to kojic
acid production were carried out using OFAT method but recently an experimental
design called Response surface methodology (RSM) was introduced to study the main
and interaction effects of various factors. The current report presents exclusively in
utilizing fourteen different carbon sources or raw materials categorized into four types
were used in the optimization process of kojic acid production by A.flavus selected from
the preliminary screening experiment. After performing the OFAT experiments the high-
yielding substrates were selected and subjected to statistical optimization by RSM. From
the literature review it was found that, till now there were no studies on kojic acid
production involving RSM methodology. The present study was the first attempt of using
RSM technology for kojic acid production.

40
4.2 OBJECTIVES:

To select a better nitrogen source which gives the highest production of kojic acid

To optimize the process parameters for cost-effective production of kojic acid like
Carbon source concentration, peptone concentration, pH, Time, Temperature,
KH2PO4 concentration and MgS04 concentration.

To determine the statistical significance of each independent variable on the
production of kojic acid using One-way-ANOVA.

41
 Flow chart 4.1: Optimization of kojic acid

Ipomea starch
Cassava starch
Sago starch
Alocasia macrorrhiza starch
Palmyra sap
Carbon sources Paneer whey
Coconut water
Muntingia calabura fruits
Cashew apple
Pine apple peel
Papaya waste
Sugar cane baggase
Wheat bran
Rice bran
Chemical
parameters
Peptone

Nitrogen source Yeast extract

Ammonium
sulphate

Mineral salts KH2PO4

MgSO4

pH

Physical
Temperature
parameters

IncubationTime

42
4.3 MATERIALS AND METHODS:

Different physico-chemical factors include carbon source concentration, peptone

concentration, pH, time, temperature, potassium dihydrogen phosphate concentration and
magnesium sulphate concentration were choosen for the optimization of kojic acid
production.

4.3.1 Selection of better nitrogen source:

Peptone and yeast extract were commonly used for the kojic acid production. They
promote the better growth of the fungus because of the presence of certain oligoelements
and vitamins Gad (2003), stabilizing the pH of the medium and act as a precursor for
kojic acid production. Peptone was the best nitrogen sources for the production of kojic
acid compared to yeast extract Kitada et al. (1967), Coupland and Niehaus (1987). As the
nitrogen source play one of the key role in kojic acid production it is necessary to select a
suitable nitrogen source before performing optimization process. Hene two different
nitrogen sources inorganic salts and organic nitrogen sources were tested for maximum
production. Inorganic nitrogen source was ammonium sulphate and organic nitrogen
sources were peptone and yeast extract. Ariff’s medium was used as a testing medium
contain 5g/L of nitrogen source.

4.3.2 Carbon sources:

All the carbon sources were collected from the local areas of Visakhapatnam. Fourteen
different types of carbon sources were chosen for the optimization of kojic acid
production which includes solid starch substrates Ipomea, Cassava, Alocasia and Sago
starch, liquid substrates Palmyra sap, Paneer whey and waste Coconut water, fruit and
fruit based sources Muntingia calabura fruits, Cashew apple, Papaya waste and Pine
apple peel, agro waste by-products Sugarcane baggase, Rice bran and Wheat bran.

43
A criterion of selection of carbon sources in the current study was based on:
 Most of them were novel sources and commercially not exploited for kojic acid
production.
 They were available in huge quantities.
 The collection of the raw materials was so easy.
 The substrates were present all over the tropical areas some were available at free
of cost.
 Most of the raw materials were accessible throughout the year and some were
available seasonally.
 As they are rich in carbohydrate content make them to use as carbon sources.
 The raw materials also contain proteins, vitamins, fatty acids, minerals etc. hence
fermentation with these sources need very little quantities of additional factors i.e.
mineral salts, growth factors etc. for the favourable production of kojic acid.
 The raw materials do not show any adverse effects on the environment.
 Most of them are not staple foods

a. Alocasia macrorrhiza tuber: Very large stem tubers of Alocasia macrorrhiza were
collected from the road side plants. A.macrorrhiza L. also called as Gaint Taro belonging
to the family Araceae Boyce (2008). The plant produces a thick cylindrical stem
originated from a basal corm which was used as one of the raw material in the current
study. The plant was commonly grown as ornamental foliage plant Kay (1987). The
edible portion of the raw material possess an energy value of 293-599 KJ/100g, water 63-
81%, crude protein 0.6-3.3%, fat 0.1-2%, carbohydrate 17-27%, ash 1.1-1.3% and
contains little quantities of mineral ions and vitamins. The stem tuber also contains
calcium oxalate crystals Harley Manner (2011) and Kay (1987). The amount of starch
present in the tuber ranges from 16-21%, Foliaki et.al (1990).

b. Ipomea batatas: The dicotyledonous plant, Ipomea batatas commonly called as sweet
potato was related to the family, Convolvulaceae. The plant possesses large sweet starch
tuberous roots as a root vegetable. 100 grams of raw tuber contains an energy value of

44
359 KJ (86 Kcal). Its composition is carbohydrates 20.1g, starch 12.7g, sugar 4.2g,
dietary fibre 3g, fat 0.1g, protein 1.6g and also contains vitamins like Vitamin A (89%),
Vitamin B1 (7%), Vitamin B2 (5%), Vitamin B3 (4%), Vitamin B5 (16%), Vitamin B6
(16%), Vitamin B9 (3%), Vitamin C (3%), Vitamin E (2%). The traces of metals like Ca
(3%), Fe (5%), Mg (7%), Mn (12%), P (7%), K (7%), Na (4%), and Zn (3%) are also
present.

c. Cassava tuber: Cassava is commonly known as Manihot esculenta or tapioca root. It is

a woody shrub belongs to the family Euphorbiaceae and a common inhabitant of South
America. It is an annual plant and widely cultivated in the tropical and sub-tropical areas
due to its large edible tuber, containing abundant starch. The tuber contains 62-65%
moisture, 20-31% carbohydrates, 1-2% crude protein and very low amounts of vitamins
and minerals. 100 grams of cassava contains 670 KJ of energy. Its composition is protein
1.4g, fat 0.28g, carbohydrates 38g, fibre 1.8g, sugar 1.7g, Ca-16mg, Fe-0.27mg, Mg-
21mg, P-27mg, K-271mg, Na-14mg, Zn-0.34mg, Cu-0.1mg, Mn-0.38mg, Se-0.7µg,
Vitamin C-20.6mg, Vitamin B1-0.09mg, Vitamin B2-0.05mg, Vitamin B3-0.85mg,
Vitamin B5-0.11mg, Vitamin B6-0.09mg, Vitamin B9-27 µg, Vitamin A-13IU, Vitamin
E-0.19mg, Vitamin K1-1.9 µg, beta carotene-8 µg Anonymous (1956).

d. Sago starch: Metroxylon Sago or Sago palm is a starch containing palm pith Jong
(1995). Maximum amount of starch content was accumulated in the trunk of the plant just
prior to the flowering stage of the plant. It belongs to the family Palmae. It is found
commonly in hot humid tropics.150-300 kg of starch has been obtained from a single
palm tree. 100g of dry sago contains 94% of carbohydrates, 0.2% of proteins, 0.5% of
dietary fibre and little quantities of minerals, fats, vitamins etc.

e. Palmyra sap: The robust tree Borassus flabellifer or Palmyra palm or toddy palm or
sugar palm is inhabitant to the Indian subcontinent and Southeast Asia. It produces one of
the most significant product sap have a dominant sugar sucrose in proportions 10.36% -
16.94% which was selected as a carbon source in the present study. The nutritional

45
composition of Palmyra sweet sap (g/100cc) is protein – 0.35, total sugar – 10.93,
reduced sugar – 0.96 and other minerals and sugars.

f. Paneer whey: Whey is the by-product from dairy products since has no added value
and it is dumped in to the environment. It was determined by Aneja et al. (2002) that
paneer production in India was high and is 1,50,000 tonnes. This may result in the
production of 2 million tonnes of whey which might contain 1,30,000 tonnes/annum of
desired milk nutrients. The amount of nutrients present in the whey depends on the
ingredient composition of milk from where the whey was derived and also based on the
milk processing methods. The major sugar present in whey was lactose within the range
of 70%. The nutritional composition of whey was Na (mg/L) – 350, K (mg/L) – 1300, Ca
(mg/L) – 480, Mg (mg/L) – 59, Cl (mg/L) – 1349, Citrate (mg/L) – 6750, Zn (mg/L) –
280, Total proteins (%) – 0.41, Fat (%) – 0.01, Lactose (%) – 4.5, Total solids (%) – 5.8.

g. Coconut water: It is the liquid which is present in the endosperm of coconut taken as
a healthy and nutritional drink by the human beings worldwide. The water is clear, sterile
and contains unique compound like sugars, vitamins, electrolytes, amino acids, enzymes,
minerals, phytoharmones and cytokine. Its scientific name is Cocos nucifera belonging to
Atecaeceae family. The tree grows commonly in coastal tropical areas. The liquid is rich
with sugars and amino acid and consists of very less amounts of sodium and chlorides.
The composition of coconut water per 100g is energy – 19Kcal, carbohydrates – 3.71g,
protein – 0.72g, total fat – 0.2g, dietary fiber – 1.1g.

h. Muntingia calabura L: The plant is the inherent species of Northern South America,
Central America, Great Antilles, and Southern Mexico and can be seen as a road side
plant generally in South-East Asian countries. It is commonly called as Jamaica cherry or
Panama berry from Elaeocarpaceae family. The plant contains round berries whose
diameter varies from 1-1.25cm and are usually red, sometimes produces yellow coloured
berries and the fruit have a thin, tendered, smooth skin. The berry inside contain a pulp
which is light brown, soft and juicy possess fig like flavour and also have many minute
dark brown or black coloured seeds. Fruits are borne nearly throughout the year and ripen

46
in 6-8 weeks from the stage of anthesis. 100g of berries nearly contain 73.63g of H2O,
2.1g protein, 2.3g fat, 17.9g carbohydrates, 6.0g fibre, 1.4g ash, 125mg Ca, 94mg P,
0.015mg Vit-A, 90mg Vit-C. The energy value is 380KJ/100g Morton J (1987), Rama
rao (1914).

I. Cashew apple: It is scientifically called as Anacardium occidentale L from

Anacardiaceae family and also called pseudo fruit treated as a by-product in cashew nut
industry. It is Pan-tropical in distribution.The shape of the fruit nut is kidney shaped and
during the maturation phase, the stalk above it becomes swollen, fleshy and becomes a
pear shaped accessory fruit which is 2-4 inches contain yellow or red coloured juice. The
mature fruit was often characterized by a good aroma, more sugar content and acidity and
less astringent. The fruit contains 84 – 88% H2O, 0.1 – 0.16g protein, 0.05 – 0.5g fat, 9.1
– 9.8g carbohydrates, 0.4 – 1.0g dietary fibre, 0.9 – 5.4mg Ca, 0.2 – 0.7mg Fe, 0.02 –
0.03mg Vit-B1, 0.1 – 0.4mg Vit-B2, 0.1 – 0.5mg Vit-B3, 147 – 372mg Vit-C.
Carotenoids, quercetin, anacardic acid, tannins, and organic acids, anti oxidants were also
present Morton J (1987), Verheij, E.W.M (1992).

j. Pine apple peel: Ananas comosus L. (Bromeliaceae) or pine apple added greater than
20% of the world production of tropical fruits Coveca (2002). Ketnava et al. (2009)
revealed that the pine apple peel is a huge waste and a good source for extraction of
valuable bioactive compounds. The waste still have a considerable quantity of soluble
sugars, in addition to high fiber and low protein content. Correia et al. (2004), Rosma et
al. (2005) reported that the pine apple waste which consists of peel, core and unwanted
parts of pine apple contain upto 6.14% of carbohydrate, mineral especially Mg and 0.6%
of crude protein.

k. Papaya waste: Papaya fruit (Carica papaya L.) is a common fruit of tropical America,
Southern Mexico, and Northern Nicaragua. It found frequently in all tropical areas. Brazil
is the second largest producer of papaya. As a result of processing it produces a waste
papaya waste which is rich with calcium, Vit-A and Vit-C. The chemical composition

47
(g/100g) of peel Ash 11.8g, lipids 2.44g, protein 18.18g, total fiber 33.05g, carbohydrates
9.67g.

l. Sugarcane baggase:
In the form of feed stock, sugarcane was utilized worldwide for the production of sugar
and bio-ethanol. Through the milling process, the juice was extracted from the sugarcane
and left out of the baggase as a by-product which comprises 60 – 80% of carbohydrates.
Though it is a good source of carbohydrates, it is often treated as an agricultural waste
and discharged in the environment which makes pollution. Now-a-days this by-product
baggase was used as a renewable feed stock for the production of various industrial
compounds like ethanol, citric acid, bio-fuel production and in pulp industry etc. The
carbohydrates cellulose and hemicelluloses embedded in the matrix of lignin were
present in the baggase. The baggase contains 35.2% cellulose, 24.5% hemicelluloses,
22.2% lignin and 20.9% ash.

m. Wheat bran: The scientific name of wheat is Triticum aestivum L. It contributes the
significant source of dietary fiber and contains ample quantities of minerals and few
vitamins. Wheat bran is the external hardy layer of the wheat grain. Majority of the foods
were made with wheat flour and the procedures making the wheat flour get rid of the
bran. The processing procedures however cause the loosing of vitamin compounds and
fibre material from the wheat bran. The raw material was preserved at a low temperature
to prevent the rancidity. The wheat bran possess an energy value of 1216KJ, protein
16.2g, fat 5.3g, carbohydrate 65.1g, dietary fibre 40.2g, ash 5.4g, moisture 8.2g, vitamin
E 1.6mg and vitamin K 83µg .

n. Rice bran: Rice bran is one of the byproduct obtained through paddy milling process.
The rice bran was not utilized as a food supplement for humans. It is necessitate to
exploit the complete possible of the accessible rice bran in the country, equally as a
source of healthy edible oil and as a food supplement for supporting our population’s
nutrition and health. Now-a-days the rice bran made in the rice mills contain endosperm
starch and rice germ. The nutritional value of rice bran per 100g is protein 16.5g, fat

48
21.3g, minerals 8.3g, crude fibre 11.4g, carbohydrate 49.4g, starch 24.1g, free sugar 50g
and also contains minerals and vitamins. The energy value is 359Kcal.

4.3.3 Minerals:

Potassium dihydrogen phosphate and magnesium sulphate were used as the mineral salts
for the preparation of fermentation medium. Phosphate was the essential element helpful
for the growth of the microorganism Coupland and Niehaus (1987). Phosphate was one
of the atom of nucleic acids, phospholipids, sugar phosphates and suphur atom in amino
acids like cysteine hence these minerals play a important role in fungal energy
metabolism.

Table 4.1: Ariff’s medium

Ingredients Composition (g/L)

Glucose 50
Yeast extract or peptone or ammonium 5.0
sulphate
Potassium dihydrogen phosphate 1.0
Magnesium sulphate 0.5

4.3.4 Preparation of starch powder:

The solid starch substrates require some preliminary treatment methods like preparation
of starch powder and enzymatic hydrolysis by amylase enzyme. The tubers were washed
with water to get rid of soil particles and then peeled; the peeled tubers were cut, grated
and made into powders according to Lodha and Nemade (2012). Sago starch powder was
purchased from the market.

4.3.5 Enzymatic hydrolysis of various starches: Padmaja et al. (2001)

All the four different starch substrates were subjected to preliminary starch hydrolysis
procedure before used for the production. Initially 100g of all powdered starchy samples

49
21.3g, minerals 8.3g, crude fibre 11.4g, carbohydrate 49.4g, starch 24.1g, free sugar 50g
and also contains minerals and vitamins. The energy value is 359Kcal.

4.3.3 Minerals:

Potassium dihydrogen phosphate and magnesium sulphate were used as the mineral salts
for the preparation of fermentation medium. Phosphate was the essential element helpful
for the growth of the microorganism Coupland and Niehaus (1987). Phosphate was one
of the atom of nucleic acids, phospholipids, sugar phosphates and suphur atom in amino
acids like cysteine hence these minerals play a important role in fungal energy
metabolism.

Table 4.1: Ariff’s medium

Ingredients Composition (g/L)

Glucose 50
Yeast extract or peptone or ammonium 5.0
sulphate
Potassium dihydrogen phosphate 1.0
Magnesium sulphate 0.5

4.3.4 Preparation of starch powder:

The solid starch substrates require some preliminary treatment methods like preparation
of starch powder and enzymatic hydrolysis by amylase enzyme. The tubers were washed
with water to get rid of soil particles and then peeled; the peeled tubers were cut, grated
and made into powders according to Lodha and Nemade (2012). Sago starch powder was
purchased from the market.

4.3.5 Enzymatic hydrolysis of various starches: Padmaja et al. (2001)

All the four different starch substrates were subjected to preliminary starch hydrolysis
procedure before used for the production. Initially 100g of all powdered starchy samples

49
were weighed separately and then 1L of 0.02M sodium phosphate buffer solution of pH
6.9 was added and the contents were thoroughly mixed. They were subjected to starch
gelatinization procedure by keeping them in a boiling water bath shaker (M/s.Remi
instruments Ltd.,) to prevent lump formation for 3h. Now liquefaction was performed
with α-amylase enzyme (purchased from M/s.Coastal Chemical Enterprises Ltd.,
Visakhapatnam) at a concentration of 9.0KNU/100g suspension. The contents were
incubated at 300C. The hydrolysis was performed for 240min-300min. Throughout the
incubation time, the samples were withdrawn for every 50min interval and the quantity of
reducing sugars liberated were estimated using 3,5 Dinitrosalicylic acid method Miller
(1959). Finally all the beakers are kept in boiling water bath for 5 min to prevent the
activity of α-amylase enzyme Sadasivam and Manickam (1996).

4.3.6 Optimization of cultural parameters for the production of kojic acid through
one-factor-at-a-time method (OFAT):

The production medium at initial conditions contain 1000ml starch hydrolysates or liquid
substrates or 1000g/L of fruits or fruit waste or 100g/L of agro waste by-products,
Peptone 1.0g/L, KH2PO4 1.0g/L, MgSO4.7H2O 0.5g/L. Fermentation was conducted with
all the substrates at incubation time 12d. Optimization was carried out at varying
physical conditions like Temperature (200C-350C), pH (4.0-8.0), Time (11d-37d) and
chemical conditions like substrate concentration (100ml-1000ml starch hydrolysate for
tuber and liquid substrates, 100g/L-1000g/L for fruits, 10g/L-100g/L for agro waste by-
products), Peptone concentration (1-5g/L), KH2PO4 (0.5g/L-2.5g/L),
MgSO4concentration (0.1g/L-0.9g/L) using one-factor-at-a-time method. At the end of
the fermentation, the fermented broth was filtered and the supernatant was subjected to
quantitative analysis of kojic acid by Bentley’s colorimetric method. The mycelial mat
was dried in hot-air oven at 800C for 24h and Dry cell weight was estimated.

4.3.7 Production of kojic acid after optimization:

When once the optimized conditions were established with each cultural parameter from
all the carbon sources, final production was performed at these conditions. The resulted

50
fermented broth was subjected to evaporation in a refrigerator at 50C for 24h for the
extraction of kojic acid crystals.

4.3.8 Statistical analysis:

The results were analyzed by one-way ANOVA at 95 % confidence level and the sample
means were analysed by Tukey’s test at significance level of p≤0.05 using STATISTICA
6.0 (Stat-Ease Inc., Tulsa, 130 OK, USA).. Each experiment was performed 3 times. ‘p’
value determined the significant effect of each factor on kojic acid production.

4.4 RESULTS AND DISCUSSION:

4.4.1 Selection of better nitrogen source:

Nitrogen source was one of the most potent factors for the production of kojic acid.
Though two different nitrogen sources inorganic nitrogen source-ammonium sulphate
and organic nitrogen sources-yeast extract and peptone concentrations were tested the
highest production was observed with peptone (Figure 4.1). Hence peptone was chosen
for further optimization process. The low production in response to inorganic salts was
due to lack of some essential growth elements in them like vitamins and oligo compounds
Parrish et al. (1996), Bazaraa and Al-Dagal (1999), Gad et al. (2003).

Figure 4.1: Effect of different nitrogen sources on the production of kojic acid

Effect of different nitrogen sources on kojic acid production by

A.flavus
50
45
40
35
30
Conc. of kojic 25
acid (g/L) 20
15 Concentration of kojic acid
10 to different nitrogen
5 sources
0
yeast peptone Ammonium
extract sulphate
Nitrogen source

51
4.4.2 Hydrolysis for various starches:

After extraction of starch powder from tuber substrates they were subjected to amylosis.
Rapid hydrolysis of starch molecules was taken place within the first 120 min of
incubation time and then after 120 min, the rate of hydrolysis was decreased till 300 min.
It was also observed that starch samples of four different origins used were susceptible to
α-amylase action differently and released reducing sugars at different concentrations. The
concentration of reducing sugars released were 47.1 g/L for Cassava, 42.95 g/L for
Ipomea, 38.2 g/L for A.macrorrhiza and 46.2g/L for Sago starch (Figure 4.2). The
difference in the susceptibility and mode of enzyme action was based on starch source,
enzyme system and botanical origin. The degree of digestibility depends on crystalline
polymorphic forms of starch molecules Jane et al. (1997), Planchot et al. (1997) and
Valetudie et al. (1993). The starch which exhibit A-type X-ray diffraction spectrum was
more susceptible to hydrolysis than with starch of B-type X-ray spectrum Williamson et
al. (1992). It was reported that the starch hydrolysis was also based on the size and
arrangement of starch residues in two different forms, amorphous and crystalline lamellae
forms and also on the interaction with non-starch compounds. A-type starch contain more
A-chains and branch points in their crystalline lamellae structure hence produced weak
points which were susceptible to amylosis very easily whereas in the B-type starch there
were many branch points in their amorphous region thus producing a superior crystalline
structure that was resistant to amylosis.

52
Figure 4.2:
Progress of starch hydrolysis for various substrates

50

Reducing sugar concentration (g/L)

40

30

20

10

0
0 50 100 150 200 250 300 350

Time (min)
Concentration of reducing sugars released from Cassava
Concentration of reducing sugars released from Ipomea
Concentration of reducing sugars released from Alocasia
Concentration of reducing sugars released from Sago starch

4.4.3 Optimization of carbon source concentration for kojic acid production:

It was observed that the production was higher at carbon source concentration 1000ml or
1000g/L for starch, liquid and fruit substrates and 10-30g/L for agro wastes (Figure 4.3a-
L). No production was obtained with pine apple peel and papaya waste. Beyond the
crucial levels of substrate concentration the water activity has been diminished which
leads to plasmolysis and causes the decrease in the rate of fermentation Roukas (1993).
Enhanced initial sugar levels can cause significant increase in the residual sugar moieties,
possibly due to incapability of microorganisms to metabolize very higher amounts of
sugar. The lesser consumption of sugar encountered with the higher levels may be due to
osmosis effect.

53
Figure 4.3a:

Effect of substrate concentration on kojic acid production with the substrate Ipomea

Concentration of kojic acid (g/L) & Biomass dry weight (g/L)

50

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

From the Figure 4.3a it was observed that, substrate concentration 1000ml was highly
significant (’p’value 0.00002) for the production of kojic acid from Ipomea.

Figure 4.3b:

Effect of Substrate concentration on kojic acid production with the substrate Cassava
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0
0 200 400 600 800 1000 1200

Substrate concentration(ml))

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was showed from the Figure 4.3b that, substrate concentration 1000ml highly
influences (’p’ value 0.00007) the production of kojic acid from Cassava.

54
Figure 4.3c:

Effect of Substrate concentration on kojic acid production with the substrate Sago starch

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

80

60

40

20

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.3c represented that, substrate concentration 1000ml was highly significant (‘p’
0.00002) for the production from Sago starch.

Figure 4.3d:

Effect of substrate concentration on kojic acid production with the substrate

A.macrorrhiza
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)
Biomass dry weight

Figure 4.3d showed that 1000ml substrate concentration highly influences (‘p’ 0.00017)
the productivity rate of kojic acid from the substrate A.macrorrhiza.

55
Figure 4.3e:
Effect of substrate concentration on kojic acid production with the substrate Palmyra sap

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was noticed from the Figure 4.3e that significant production (’p’ value 0.00001) was
occurred at 1000ml substrate concentration with palmyra sap.

Figure 4.3f:
Effect of Substrate concentration on kojic acid production with the substrate
paneer whey
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

0
0 200 400 600 800 1000 1200

Substrate concentration(ml))

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.3f showed that, substrate concentration 1000ml highly influences (‘p’ 0.00003)
the production from paneer whey.

56
Figure 4.3g:
Effect of substrate concentration on kojic acid production with the substrate
Coconut water

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

35

30

25

20

15

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.3g substrate concentration 1000ml was significantly influences (‘p’
0.0007) the kojic acid production from coconut water.

Figure 4.3h:

Effect of Substrate concentration on kojic acid production with the substrate

M.calabura fruits
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

70

60

50

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Substrate concentration 1000g/L influences (‘p’ 0.0001) greatly the production rate of
kojic acid from M.calabura fruits (Figure 4.3h).

57
Figure 4.3i:
Effect of substrate concentration on kojic acid production with the substrate
Cashew apple

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

18

16

14

12

10

2
0 200 400 600 800 1000 1200

Substrate concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight

From the above Figure 4.3i it was noticed that 500g/L substrate concentration was highly
significant (‘p’0.003) for the production from Cashew apple.

Figure 4.3j:

Effect of substrate concentration on kojic acid production with the substrate

Sugarcane baggase
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

25

20

15

10

0 20 40 60 80 100 120

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was deduced from the Figure 4.3j that, 30g/L substrate concentration significantly
influences (’p’ 0.002) the production from sugarcane baggase.

58
Figure 4.3k:

Effect of substrate concentration on kojic acid production with the substrate

Rice bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0 20 40 60 80 100 120

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.3k it was observed that 10g/L produces significant yield (’p’ 0.002)
from Rice bran.

Figure 4.3l:

Effect of substrate concentration on kojic acid production with the substrate

Wheat bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

0 20 40 60 80 100 120

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.3l represented that, substrate concentration 20g/L significantly enhances ’p’
value 0.01) the yield from Wheat bran.

59
4.4.4 Optimization of nitrogen source concentration for kojic acid production:

From the Figure 4.4a-4.4l it was indicated that, nitrogen source concentration 2.0-4.0g/L
was the optimium concentration for the production of maximum yields of kojic acid from
all the 12 carbon sources. At increased concentration of peptone, the sugars were not
converted into kojic acid rather than utilized for the excess growth of the fungus. Hence
the supply of nitrogen source should be restricted for excess production of kojic acid and
less mycelia growth. In such conditions, the non-growing mycelia cells can able to
convert excess glucose to kojic acid Ariff et al. (1996).

Figure 4.4a:

Effect of Peptone concentration on kojic acid production with the substrate Ipomea
Concentration of kojic acid (g/L) & Biomass dry weight (g/L)

60

50

40

30

20

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.4a indicated that, significant production (‘p’ 0.00006) takes place with peptone
concentration 3g/L.

60
 CHAPTER – IV
OPTIMIZATION OF CULTURAL
PARAMETERS FOR COST EFFECTIVE
PRODUCTION OF KOJIC ACID
 CHAPTER 4

4. OPTIMIZATION OF CULTURAL PARAMETERS FOR

COST-EFFECTIVE PRODUCTION OF KOJIC ACID

4.1 INTRODUCTION:

It is still uncertain for kojic acid production about the efficiency and production
economics because the nutritional media plays a more significant role in the betterment
of such fermentation process. Now-a-days, research trials were done to identify the novel
and potential nutritive sources and most advanced fermentative methods to achieve very
higher yields of product and at the same time high conversion rate of the substrate
molecules. Usage of pure sugars like glucose, sucrose, arabinose, xylose, fructose,
mannose, and galactose Arnstein and Bentley (1956), Burdock et al. (2001), Rosfarizan
and Ariff (2007) resulted in the production of pure product subsequently the cost of the
purification process reduced. But economically this was not feasible because of the high
cost of the pure sugars. Hence these expensive sources were substituted by the usage of
low cost agricultural resources. Literature was available on kojic acid production by
using various industrial and agro-waste by products cocoa juice El-Sharkawy (1995), pea,
kidney bean, vegetable waste, fruit waste of apricot, orange and peach, carrot waste,
turnip waste, cornsteep liquor, molasses, cheese whey, wheat bran, rice husk, rice
fragments by El-kady et al. (2014), corn starch, sago starch, potato starch by Rosfarizan
et al. (1998), gelatinized sago starch Rosfarizan and Ariff (2002), corn stalk hydrolysate
Yan et al. (2014), pine apple peel by Nurashikin et al. (2013). Kitada et al. (1967)
reported that, excellent yields of kojic acid were produced by the sugars glucose, sucrose
and fructose and no production was identified from starch and xylose. The present study
is one such to provide an alternative and economically feasible carbon sources and cost-
effective process of kojic acid production. Optimization of process parameters is the most
valuable step in industrial production methodology, during which even a slight progress
may lead to success in the fermentation process commercially. It was important that, the
secondary metabolites which of high economic importance were not or little produced by

39
fungal species at unfavourable cultivation conditions Bills et al. (2008). Hence it is
necessary to manipulate the cultivable conditions to optimum so as to get maximum
yield. Until 2003, the optimization of medium composition and cultural conditions for
kojic acid production was not discovered Futamura et al. (2001), Lin (2001), and Gad
(2003). But during the year 2006, El-Aasar first reported the optimum cultural conditions
for the production of kojic acid. Variation in the Carbon source concentration, Nitrogen
concentration, Mineral salt concentration, pH, Time, Temperature shows an immense
effect on the production of kojic acid. The fermentation medium composition had main
effect on the production of kojic acid and generally differs for each and every
microorganism and nearly 30-40% is the estimated cost of the production medium in the
total production cost of a fermentation process. So it is essential to optimize the nutrient
composition and fermentation conditions accordingly.

In the present study, empirical optimization strategy or traditional ‘one-factor-at-a-time’

(OFAT) method was used for the kojic acid production. This method involves varying
one factor at the same time keeping the other factors constant under a specific set of
conditions (Flow chart-4.1) Ahamed et al. (2006), Alexeeva et al. (2002) Patidar et al.
(2005). The method is considered as a closed ended system used for optimization of
medium ingredients as well as physical conditions of the fermentation. The method is
simple, easy and also practical to study the influence of process parameters in the form of
graphs Kar et al. (1999) and Kumar et al. (2003). Most of the past works related to kojic
acid production were carried out using OFAT method but recently an experimental
design called Response surface methodology (RSM) was introduced to study the main
and interaction effects of various factors. The current report presents exclusively in
utilizing fourteen different carbon sources or raw materials categorized into four types
were used in the optimization process of kojic acid production by A.flavus selected from
the preliminary screening experiment. After performing the OFAT experiments the high-
yielding substrates were selected and subjected to statistical optimization by RSM. From
the literature review it was found that, till now there were no studies on kojic acid
production involving RSM methodology. The present study was the first attempt of using
RSM technology for kojic acid production.

40
4.2 OBJECTIVES:

To select a better nitrogen source which gives the highest production of kojic acid

To optimize the process parameters for cost-effective production of kojic acid like
Carbon source concentration, peptone concentration, pH, Time, Temperature,
KH2PO4 concentration and MgS04 concentration.

To determine the statistical significance of each independent variable on the
production of kojic acid using One-way-ANOVA.

41
 Flow chart 4.1: Optimization of kojic acid

Ipomea starch
Cassava starch
Sago starch
Alocasia macrorrhiza starch
Palmyra sap
Carbon sources Paneer whey
Coconut water
Muntingia calabura fruits
Cashew apple
Pine apple peel
Papaya waste
Sugar cane baggase
Wheat bran
Rice bran
Chemical
parameters
Peptone

Nitrogen source Yeast extract

Ammonium
sulphate

Mineral salts KH2PO4

MgSO4

pH

Physical
Temperature
parameters

IncubationTime

42
4.3 MATERIALS AND METHODS:

Different physico-chemical factors include carbon source concentration, peptone

concentration, pH, time, temperature, potassium dihydrogen phosphate concentration and
magnesium sulphate concentration were choosen for the optimization of kojic acid
production.

4.3.1 Selection of better nitrogen source:

Peptone and yeast extract were commonly used for the kojic acid production. They
promote the better growth of the fungus because of the presence of certain oligoelements
and vitamins Gad (2003), stabilizing the pH of the medium and act as a precursor for
kojic acid production. Peptone was the best nitrogen sources for the production of kojic
acid compared to yeast extract Kitada et al. (1967), Coupland and Niehaus (1987). As the
nitrogen source play one of the key role in kojic acid production it is necessary to select a
suitable nitrogen source before performing optimization process. Hene two different
nitrogen sources inorganic salts and organic nitrogen sources were tested for maximum
production. Inorganic nitrogen source was ammonium sulphate and organic nitrogen
sources were peptone and yeast extract. Ariff’s medium was used as a testing medium
contain 5g/L of nitrogen source.

4.3.2 Carbon sources:

All the carbon sources were collected from the local areas of Visakhapatnam. Fourteen
different types of carbon sources were chosen for the optimization of kojic acid
production which includes solid starch substrates Ipomea, Cassava, Alocasia and Sago
starch, liquid substrates Palmyra sap, Paneer whey and waste Coconut water, fruit and
fruit based sources Muntingia calabura fruits, Cashew apple, Papaya waste and Pine
apple peel, agro waste by-products Sugarcane baggase, Rice bran and Wheat bran.

43
A criterion of selection of carbon sources in the current study was based on:
 Most of them were novel sources and commercially not exploited for kojic acid
production.
 They were available in huge quantities.
 The collection of the raw materials was so easy.
 The substrates were present all over the tropical areas some were available at free
of cost.
 Most of the raw materials were accessible throughout the year and some were
available seasonally.
 As they are rich in carbohydrate content make them to use as carbon sources.
 The raw materials also contain proteins, vitamins, fatty acids, minerals etc. hence
fermentation with these sources need very little quantities of additional factors i.e.
mineral salts, growth factors etc. for the favourable production of kojic acid.
 The raw materials do not show any adverse effects on the environment.
 Most of them are not staple foods

a. Alocasia macrorrhiza tuber: Very large stem tubers of Alocasia macrorrhiza were
collected from the road side plants. A.macrorrhiza L. also called as Gaint Taro belonging
to the family Araceae Boyce (2008). The plant produces a thick cylindrical stem
originated from a basal corm which was used as one of the raw material in the current
study. The plant was commonly grown as ornamental foliage plant Kay (1987). The
edible portion of the raw material possess an energy value of 293-599 KJ/100g, water 63-
81%, crude protein 0.6-3.3%, fat 0.1-2%, carbohydrate 17-27%, ash 1.1-1.3% and
contains little quantities of mineral ions and vitamins. The stem tuber also contains
calcium oxalate crystals Harley Manner (2011) and Kay (1987). The amount of starch
present in the tuber ranges from 16-21%, Foliaki et.al (1990).

b. Ipomea batatas: The dicotyledonous plant, Ipomea batatas commonly called as sweet
potato was related to the family, Convolvulaceae. The plant possesses large sweet starch
tuberous roots as a root vegetable. 100 grams of raw tuber contains an energy value of

44
359 KJ (86 Kcal). Its composition is carbohydrates 20.1g, starch 12.7g, sugar 4.2g,
dietary fibre 3g, fat 0.1g, protein 1.6g and also contains vitamins like Vitamin A (89%),
Vitamin B1 (7%), Vitamin B2 (5%), Vitamin B3 (4%), Vitamin B5 (16%), Vitamin B6
(16%), Vitamin B9 (3%), Vitamin C (3%), Vitamin E (2%). The traces of metals like Ca
(3%), Fe (5%), Mg (7%), Mn (12%), P (7%), K (7%), Na (4%), and Zn (3%) are also
present.

c. Cassava tuber: Cassava is commonly known as Manihot esculenta or tapioca root. It is

a woody shrub belongs to the family Euphorbiaceae and a common inhabitant of South
America. It is an annual plant and widely cultivated in the tropical and sub-tropical areas
due to its large edible tuber, containing abundant starch. The tuber contains 62-65%
moisture, 20-31% carbohydrates, 1-2% crude protein and very low amounts of vitamins
and minerals. 100 grams of cassava contains 670 KJ of energy. Its composition is protein
1.4g, fat 0.28g, carbohydrates 38g, fibre 1.8g, sugar 1.7g, Ca-16mg, Fe-0.27mg, Mg-
21mg, P-27mg, K-271mg, Na-14mg, Zn-0.34mg, Cu-0.1mg, Mn-0.38mg, Se-0.7µg,
Vitamin C-20.6mg, Vitamin B1-0.09mg, Vitamin B2-0.05mg, Vitamin B3-0.85mg,
Vitamin B5-0.11mg, Vitamin B6-0.09mg, Vitamin B9-27 µg, Vitamin A-13IU, Vitamin
E-0.19mg, Vitamin K1-1.9 µg, beta carotene-8 µg Anonymous (1956).

d. Sago starch: Metroxylon Sago or Sago palm is a starch containing palm pith Jong
(1995). Maximum amount of starch content was accumulated in the trunk of the plant just
prior to the flowering stage of the plant. It belongs to the family Palmae. It is found
commonly in hot humid tropics.150-300 kg of starch has been obtained from a single
palm tree. 100g of dry sago contains 94% of carbohydrates, 0.2% of proteins, 0.5% of
dietary fibre and little quantities of minerals, fats, vitamins etc.

e. Palmyra sap: The robust tree Borassus flabellifer or Palmyra palm or toddy palm or
sugar palm is inhabitant to the Indian subcontinent and Southeast Asia. It produces one of
the most significant product sap have a dominant sugar sucrose in proportions 10.36% -
16.94% which was selected as a carbon source in the present study. The nutritional

45
composition of Palmyra sweet sap (g/100cc) is protein – 0.35, total sugar – 10.93,
reduced sugar – 0.96 and other minerals and sugars.

f. Paneer whey: Whey is the by-product from dairy products since has no added value
and it is dumped in to the environment. It was determined by Aneja et al. (2002) that
paneer production in India was high and is 1,50,000 tonnes. This may result in the
production of 2 million tonnes of whey which might contain 1,30,000 tonnes/annum of
desired milk nutrients. The amount of nutrients present in the whey depends on the
ingredient composition of milk from where the whey was derived and also based on the
milk processing methods. The major sugar present in whey was lactose within the range
of 70%. The nutritional composition of whey was Na (mg/L) – 350, K (mg/L) – 1300, Ca
(mg/L) – 480, Mg (mg/L) – 59, Cl (mg/L) – 1349, Citrate (mg/L) – 6750, Zn (mg/L) –
280, Total proteins (%) – 0.41, Fat (%) – 0.01, Lactose (%) – 4.5, Total solids (%) – 5.8.

g. Coconut water: It is the liquid which is present in the endosperm of coconut taken as
a healthy and nutritional drink by the human beings worldwide. The water is clear, sterile
and contains unique compound like sugars, vitamins, electrolytes, amino acids, enzymes,
minerals, phytoharmones and cytokine. Its scientific name is Cocos nucifera belonging to
Atecaeceae family. The tree grows commonly in coastal tropical areas. The liquid is rich
with sugars and amino acid and consists of very less amounts of sodium and chlorides.
The composition of coconut water per 100g is energy – 19Kcal, carbohydrates – 3.71g,
protein – 0.72g, total fat – 0.2g, dietary fiber – 1.1g.

h. Muntingia calabura L: The plant is the inherent species of Northern South America,
Central America, Great Antilles, and Southern Mexico and can be seen as a road side
plant generally in South-East Asian countries. It is commonly called as Jamaica cherry or
Panama berry from Elaeocarpaceae family. The plant contains round berries whose
diameter varies from 1-1.25cm and are usually red, sometimes produces yellow coloured
berries and the fruit have a thin, tendered, smooth skin. The berry inside contain a pulp
which is light brown, soft and juicy possess fig like flavour and also have many minute
dark brown or black coloured seeds. Fruits are borne nearly throughout the year and ripen

46
in 6-8 weeks from the stage of anthesis. 100g of berries nearly contain 73.63g of H2O,
2.1g protein, 2.3g fat, 17.9g carbohydrates, 6.0g fibre, 1.4g ash, 125mg Ca, 94mg P,
0.015mg Vit-A, 90mg Vit-C. The energy value is 380KJ/100g Morton J (1987), Rama
rao (1914).

I. Cashew apple: It is scientifically called as Anacardium occidentale L from

Anacardiaceae family and also called pseudo fruit treated as a by-product in cashew nut
industry. It is Pan-tropical in distribution.The shape of the fruit nut is kidney shaped and
during the maturation phase, the stalk above it becomes swollen, fleshy and becomes a
pear shaped accessory fruit which is 2-4 inches contain yellow or red coloured juice. The
mature fruit was often characterized by a good aroma, more sugar content and acidity and
less astringent. The fruit contains 84 – 88% H2O, 0.1 – 0.16g protein, 0.05 – 0.5g fat, 9.1
– 9.8g carbohydrates, 0.4 – 1.0g dietary fibre, 0.9 – 5.4mg Ca, 0.2 – 0.7mg Fe, 0.02 –
0.03mg Vit-B1, 0.1 – 0.4mg Vit-B2, 0.1 – 0.5mg Vit-B3, 147 – 372mg Vit-C.
Carotenoids, quercetin, anacardic acid, tannins, and organic acids, anti oxidants were also
present Morton J (1987), Verheij, E.W.M (1992).

j. Pine apple peel: Ananas comosus L. (Bromeliaceae) or pine apple added greater than
20% of the world production of tropical fruits Coveca (2002). Ketnava et al. (2009)
revealed that the pine apple peel is a huge waste and a good source for extraction of
valuable bioactive compounds. The waste still have a considerable quantity of soluble
sugars, in addition to high fiber and low protein content. Correia et al. (2004), Rosma et
al. (2005) reported that the pine apple waste which consists of peel, core and unwanted
parts of pine apple contain upto 6.14% of carbohydrate, mineral especially Mg and 0.6%
of crude protein.

k. Papaya waste: Papaya fruit (Carica papaya L.) is a common fruit of tropical America,
Southern Mexico, and Northern Nicaragua. It found frequently in all tropical areas. Brazil
is the second largest producer of papaya. As a result of processing it produces a waste
papaya waste which is rich with calcium, Vit-A and Vit-C. The chemical composition

47
(g/100g) of peel Ash 11.8g, lipids 2.44g, protein 18.18g, total fiber 33.05g, carbohydrates
9.67g.

l. Sugarcane baggase:
In the form of feed stock, sugarcane was utilized worldwide for the production of sugar
and bio-ethanol. Through the milling process, the juice was extracted from the sugarcane
and left out of the baggase as a by-product which comprises 60 – 80% of carbohydrates.
Though it is a good source of carbohydrates, it is often treated as an agricultural waste
and discharged in the environment which makes pollution. Now-a-days this by-product
baggase was used as a renewable feed stock for the production of various industrial
compounds like ethanol, citric acid, bio-fuel production and in pulp industry etc. The
carbohydrates cellulose and hemicelluloses embedded in the matrix of lignin were
present in the baggase. The baggase contains 35.2% cellulose, 24.5% hemicelluloses,
22.2% lignin and 20.9% ash.

m. Wheat bran: The scientific name of wheat is Triticum aestivum L. It contributes the
significant source of dietary fiber and contains ample quantities of minerals and few
vitamins. Wheat bran is the external hardy layer of the wheat grain. Majority of the foods
were made with wheat flour and the procedures making the wheat flour get rid of the
bran. The processing procedures however cause the loosing of vitamin compounds and
fibre material from the wheat bran. The raw material was preserved at a low temperature
to prevent the rancidity. The wheat bran possess an energy value of 1216KJ, protein
16.2g, fat 5.3g, carbohydrate 65.1g, dietary fibre 40.2g, ash 5.4g, moisture 8.2g, vitamin
E 1.6mg and vitamin K 83µg .

n. Rice bran: Rice bran is one of the byproduct obtained through paddy milling process.
The rice bran was not utilized as a food supplement for humans. It is necessitate to
exploit the complete possible of the accessible rice bran in the country, equally as a
source of healthy edible oil and as a food supplement for supporting our population’s
nutrition and health. Now-a-days the rice bran made in the rice mills contain endosperm
starch and rice germ. The nutritional value of rice bran per 100g is protein 16.5g, fat

48
21.3g, minerals 8.3g, crude fibre 11.4g, carbohydrate 49.4g, starch 24.1g, free sugar 50g
and also contains minerals and vitamins. The energy value is 359Kcal.

4.3.3 Minerals:

Potassium dihydrogen phosphate and magnesium sulphate were used as the mineral salts
for the preparation of fermentation medium. Phosphate was the essential element helpful
for the growth of the microorganism Coupland and Niehaus (1987). Phosphate was one
of the atom of nucleic acids, phospholipids, sugar phosphates and suphur atom in amino
acids like cysteine hence these minerals play a important role in fungal energy
metabolism.

Table 4.1: Ariff’s medium

Ingredients Composition (g/L)

Glucose 50
Yeast extract or peptone or ammonium 5.0
sulphate
Potassium dihydrogen phosphate 1.0
Magnesium sulphate 0.5

4.3.4 Preparation of starch powder:

The solid starch substrates require some preliminary treatment methods like preparation
of starch powder and enzymatic hydrolysis by amylase enzyme. The tubers were washed
with water to get rid of soil particles and then peeled; the peeled tubers were cut, grated
and made into powders according to Lodha and Nemade (2012). Sago starch powder was
purchased from the market.

4.3.5 Enzymatic hydrolysis of various starches: Padmaja et al. (2001)

All the four different starch substrates were subjected to preliminary starch hydrolysis
procedure before used for the production. Initially 100g of all powdered starchy samples

49
21.3g, minerals 8.3g, crude fibre 11.4g, carbohydrate 49.4g, starch 24.1g, free sugar 50g
and also contains minerals and vitamins. The energy value is 359Kcal.

4.3.3 Minerals:

Potassium dihydrogen phosphate and magnesium sulphate were used as the mineral salts
for the preparation of fermentation medium. Phosphate was the essential element helpful
for the growth of the microorganism Coupland and Niehaus (1987). Phosphate was one
of the atom of nucleic acids, phospholipids, sugar phosphates and suphur atom in amino
acids like cysteine hence these minerals play a important role in fungal energy
metabolism.

Table 4.1: Ariff’s medium

Ingredients Composition (g/L)

Glucose 50
Yeast extract or peptone or ammonium 5.0
sulphate
Potassium dihydrogen phosphate 1.0
Magnesium sulphate 0.5

4.3.4 Preparation of starch powder:

The solid starch substrates require some preliminary treatment methods like preparation
of starch powder and enzymatic hydrolysis by amylase enzyme. The tubers were washed
with water to get rid of soil particles and then peeled; the peeled tubers were cut, grated
and made into powders according to Lodha and Nemade (2012). Sago starch powder was
purchased from the market.

4.3.5 Enzymatic hydrolysis of various starches: Padmaja et al. (2001)

All the four different starch substrates were subjected to preliminary starch hydrolysis
procedure before used for the production. Initially 100g of all powdered starchy samples

49
were weighed separately and then 1L of 0.02M sodium phosphate buffer solution of pH
6.9 was added and the contents were thoroughly mixed. They were subjected to starch
gelatinization procedure by keeping them in a boiling water bath shaker (M/s.Remi
instruments Ltd.,) to prevent lump formation for 3h. Now liquefaction was performed
with α-amylase enzyme (purchased from M/s.Coastal Chemical Enterprises Ltd.,
Visakhapatnam) at a concentration of 9.0KNU/100g suspension. The contents were
incubated at 300C. The hydrolysis was performed for 240min-300min. Throughout the
incubation time, the samples were withdrawn for every 50min interval and the quantity of
reducing sugars liberated were estimated using 3,5 Dinitrosalicylic acid method Miller
(1959). Finally all the beakers are kept in boiling water bath for 5 min to prevent the
activity of α-amylase enzyme Sadasivam and Manickam (1996).

4.3.6 Optimization of cultural parameters for the production of kojic acid through
one-factor-at-a-time method (OFAT):

The production medium at initial conditions contain 1000ml starch hydrolysates or liquid
substrates or 1000g/L of fruits or fruit waste or 100g/L of agro waste by-products,
Peptone 1.0g/L, KH2PO4 1.0g/L, MgSO4.7H2O 0.5g/L. Fermentation was conducted with
all the substrates at incubation time 12d. Optimization was carried out at varying
physical conditions like Temperature (200C-350C), pH (4.0-8.0), Time (11d-37d) and
chemical conditions like substrate concentration (100ml-1000ml starch hydrolysate for
tuber and liquid substrates, 100g/L-1000g/L for fruits, 10g/L-100g/L for agro waste by-
products), Peptone concentration (1-5g/L), KH2PO4 (0.5g/L-2.5g/L),
MgSO4concentration (0.1g/L-0.9g/L) using one-factor-at-a-time method. At the end of
the fermentation, the fermented broth was filtered and the supernatant was subjected to
quantitative analysis of kojic acid by Bentley’s colorimetric method. The mycelial mat
was dried in hot-air oven at 800C for 24h and Dry cell weight was estimated.

4.3.7 Production of kojic acid after optimization:

When once the optimized conditions were established with each cultural parameter from
all the carbon sources, final production was performed at these conditions. The resulted

50
fermented broth was subjected to evaporation in a refrigerator at 50C for 24h for the
extraction of kojic acid crystals.

4.3.8 Statistical analysis:

The results were analyzed by one-way ANOVA at 95 % confidence level and the sample
means were analysed by Tukey’s test at significance level of p≤0.05 using STATISTICA
6.0 (Stat-Ease Inc., Tulsa, 130 OK, USA).. Each experiment was performed 3 times. ‘p’
value determined the significant effect of each factor on kojic acid production.

4.4 RESULTS AND DISCUSSION:

4.4.1 Selection of better nitrogen source:

Nitrogen source was one of the most potent factors for the production of kojic acid.
Though two different nitrogen sources inorganic nitrogen source-ammonium sulphate
and organic nitrogen sources-yeast extract and peptone concentrations were tested the
highest production was observed with peptone (Figure 4.1). Hence peptone was chosen
for further optimization process. The low production in response to inorganic salts was
due to lack of some essential growth elements in them like vitamins and oligo compounds
Parrish et al. (1996), Bazaraa and Al-Dagal (1999), Gad et al. (2003).

Figure 4.1: Effect of different nitrogen sources on the production of kojic acid

Effect of different nitrogen sources on kojic acid production by

A.flavus
50
45
40
35
30
Conc. of kojic 25
acid (g/L) 20
15 Concentration of kojic acid
10 to different nitrogen
5 sources
0
yeast peptone Ammonium
extract sulphate
Nitrogen source

51
4.4.2 Hydrolysis for various starches:

After extraction of starch powder from tuber substrates they were subjected to amylosis.
Rapid hydrolysis of starch molecules was taken place within the first 120 min of
incubation time and then after 120 min, the rate of hydrolysis was decreased till 300 min.
It was also observed that starch samples of four different origins used were susceptible to
α-amylase action differently and released reducing sugars at different concentrations. The
concentration of reducing sugars released were 47.1 g/L for Cassava, 42.95 g/L for
Ipomea, 38.2 g/L for A.macrorrhiza and 46.2g/L for Sago starch (Figure 4.2). The
difference in the susceptibility and mode of enzyme action was based on starch source,
enzyme system and botanical origin. The degree of digestibility depends on crystalline
polymorphic forms of starch molecules Jane et al. (1997), Planchot et al. (1997) and
Valetudie et al. (1993). The starch which exhibit A-type X-ray diffraction spectrum was
more susceptible to hydrolysis than with starch of B-type X-ray spectrum Williamson et
al. (1992). It was reported that the starch hydrolysis was also based on the size and
arrangement of starch residues in two different forms, amorphous and crystalline lamellae
forms and also on the interaction with non-starch compounds. A-type starch contain more
A-chains and branch points in their crystalline lamellae structure hence produced weak
points which were susceptible to amylosis very easily whereas in the B-type starch there
were many branch points in their amorphous region thus producing a superior crystalline
structure that was resistant to amylosis.

52
Figure 4.2:
Progress of starch hydrolysis for various substrates

50

Reducing sugar concentration (g/L)

40

30

20

10

0
0 50 100 150 200 250 300 350

Time (min)
Concentration of reducing sugars released from Cassava
Concentration of reducing sugars released from Ipomea
Concentration of reducing sugars released from Alocasia
Concentration of reducing sugars released from Sago starch

4.4.3 Optimization of carbon source concentration for kojic acid production:

It was observed that the production was higher at carbon source concentration 1000ml or
1000g/L for starch, liquid and fruit substrates and 10-30g/L for agro wastes (Figure 4.3a-
L). No production was obtained with pine apple peel and papaya waste. Beyond the
crucial levels of substrate concentration the water activity has been diminished which
leads to plasmolysis and causes the decrease in the rate of fermentation Roukas (1993).
Enhanced initial sugar levels can cause significant increase in the residual sugar moieties,
possibly due to incapability of microorganisms to metabolize very higher amounts of
sugar. The lesser consumption of sugar encountered with the higher levels may be due to
osmosis effect.

53
Figure 4.3a:

Effect of substrate concentration on kojic acid production with the substrate Ipomea

Concentration of kojic acid (g/L) & Biomass dry weight (g/L)

50

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

From the Figure 4.3a it was observed that, substrate concentration 1000ml was highly
significant (’p’value 0.00002) for the production of kojic acid from Ipomea.

Figure 4.3b:

Effect of Substrate concentration on kojic acid production with the substrate Cassava
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0
0 200 400 600 800 1000 1200

Substrate concentration(ml))

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was showed from the Figure 4.3b that, substrate concentration 1000ml highly
influences (’p’ value 0.00007) the production of kojic acid from Cassava.

54
Figure 4.3c:

Effect of Substrate concentration on kojic acid production with the substrate Sago starch

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

80

60

40

20

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.3c represented that, substrate concentration 1000ml was highly significant (‘p’
0.00002) for the production from Sago starch.

Figure 4.3d:

Effect of substrate concentration on kojic acid production with the substrate

A.macrorrhiza
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)
Biomass dry weight

Figure 4.3d showed that 1000ml substrate concentration highly influences (‘p’ 0.00017)
the productivity rate of kojic acid from the substrate A.macrorrhiza.

55
Figure 4.3e:
Effect of substrate concentration on kojic acid production with the substrate Palmyra sap

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was noticed from the Figure 4.3e that significant production (’p’ value 0.00001) was
occurred at 1000ml substrate concentration with palmyra sap.

Figure 4.3f:
Effect of Substrate concentration on kojic acid production with the substrate
paneer whey
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

0
0 200 400 600 800 1000 1200

Substrate concentration(ml))

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.3f showed that, substrate concentration 1000ml highly influences (‘p’ 0.00003)
the production from paneer whey.

56
Figure 4.3g:
Effect of substrate concentration on kojic acid production with the substrate
Coconut water

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

35

30

25

20

15

10

0 200 400 600 800 1000 1200

Substrate concentration (ml)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.3g substrate concentration 1000ml was significantly influences (‘p’
0.0007) the kojic acid production from coconut water.

Figure 4.3h:

Effect of Substrate concentration on kojic acid production with the substrate

M.calabura fruits
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

70

60

50

40

30

20

10

0 200 400 600 800 1000 1200

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Substrate concentration 1000g/L influences (‘p’ 0.0001) greatly the production rate of
kojic acid from M.calabura fruits (Figure 4.3h).

57
Figure 4.3i:
Effect of substrate concentration on kojic acid production with the substrate
Cashew apple

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

18

16

14

12

10

2
0 200 400 600 800 1000 1200

Substrate concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight

From the above Figure 4.3i it was noticed that 500g/L substrate concentration was highly
significant (‘p’0.003) for the production from Cashew apple.

Figure 4.3j:

Effect of substrate concentration on kojic acid production with the substrate

Sugarcane baggase
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

25

20

15

10

0 20 40 60 80 100 120

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was deduced from the Figure 4.3j that, 30g/L substrate concentration significantly
influences (’p’ 0.002) the production from sugarcane baggase.

58
Figure 4.3k:

Effect of substrate concentration on kojic acid production with the substrate

Rice bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0 20 40 60 80 100 120

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.3k it was observed that 10g/L produces significant yield (’p’ 0.002)
from Rice bran.

Figure 4.3l:

Effect of substrate concentration on kojic acid production with the substrate

Wheat bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

0 20 40 60 80 100 120

Substrate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.3l represented that, substrate concentration 20g/L significantly enhances ’p’
value 0.01) the yield from Wheat bran.

59
Figure 4.4b:

Effect of peptone concentration on kojic acid production with the substrate Cassava

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was observed from the Figure 4.4b that, 2g/L peptone concentration significantly
produces (’p’ value 0.0001) kojic acid from Cassava.

Figure 4.4c:

Effect of peptone concentration on kojic acid production with the substrate Sago starch
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

100

80

60

40

20

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Peptone concentration 4g/L produces significant yield (’p’ value 0.0001) from sago
starch (Figure 4.4c)

61
Figure 4.4d:

Effect of peptone concentration on kojic acid production with the substrate

A.macrorrhiza

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

14

12

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

With the substrate A.macrorrhiza peptone concentration 2g/L produces enhanced yield
significantly (‘p’ 0.0004) Figure 4.4d.

Figure 4.4e:

Effect of peptone concentration on kojic acid production with the substrate Palmyra sap
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

100

80

60

40

20

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.4e indicated that, significant yield (‘p’ 0.0001) was obtained with palmyra sap at
3g/L peptone concentration.

62
Figure 4.4f:

Effect of peptone concentration on kojic acid production with the substrate

Paneer whey

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.4f it was deduced that, peptone concentration 2g/L significantly
produces (‘p’0.0001) kojic acid from paneer whey.

Figure 4.4g:

Effect of peptone concentration on kojic acid production with the substrate

Coconut water
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Peptone concentration 4g/L produces significant yield (’p’ value 0.0003) with the
substrate coconut water (Figure 4.4g).

63
Figure 4.4h:

Effect of peptone concentration on kojic acid production with the substrate

M.calabura fruits

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

70

60

50

40

30

20

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.4h it was noticed that, highest production takes place at 4g/L peptone
concentration significantly (’p’ 0.0001) with M.calabura fruits.

Figure 4.4i:
Effect of peptone concentration on kojic acid production with the substrate
cashew apple
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

50

40

30

20

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

It was showed from the Figure 4.4i that, 4g/L peptone concentration produces significant
yield (’p’ value 0.0002) with Cashew apple.

64
Figure 4.4j:
Effect of peptone concentration on kojic acid production with the substrate
Sugarcane baggase

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

16

14

12

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Peptone concentration 4g/L was significant (‘p’ 0.01) for kojic acid production with
sugarcane baggase (Figure 4.4j).

Figure 4.4k:
Effect of peptone concentration on kojic acid production with the substrate
Rice bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

18

16

14

12

10

2
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.4k it was noticed that maximum production was occurred with 4g/L
peptone concentration significantly (‘p’ 0.004) with rice bran.

65
Figure 4.4b:

Effect of peptone concentration on kojic acid production with the substrate Cassava

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was observed from the Figure 4.4b that, 2g/L peptone concentration significantly
produces (’p’ value 0.0001) kojic acid from Cassava.

Figure 4.4c:

Effect of peptone concentration on kojic acid production with the substrate Sago starch
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

100

80

60

40

20

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Peptone concentration 4g/L produces significant yield (’p’ value 0.0001) from sago
starch (Figure 4.4c)

61
Figure 4.4d:

Effect of peptone concentration on kojic acid production with the substrate

A.macrorrhiza

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

14

12

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

With the substrate A.macrorrhiza peptone concentration 2g/L produces enhanced yield
significantly (‘p’ 0.0004) Figure 4.4d.

Figure 4.4e:

Effect of peptone concentration on kojic acid production with the substrate Palmyra sap
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

100

80

60

40

20

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.4e indicated that, significant yield (‘p’ 0.0001) was obtained with palmyra sap at
3g/L peptone concentration.

62
Figure 4.4f:

Effect of peptone concentration on kojic acid production with the substrate

Paneer whey

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.4f it was deduced that, peptone concentration 2g/L significantly
produces (‘p’0.0001) kojic acid from paneer whey.

Figure 4.4g:

Effect of peptone concentration on kojic acid production with the substrate

Coconut water
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Peptone concentration 4g/L produces significant yield (’p’ value 0.0003) with the
substrate coconut water (Figure 4.4g).

63
Figure 4.4h:

Effect of peptone concentration on kojic acid production with the substrate

M.calabura fruits

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

70

60

50

40

30

20

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.4h it was noticed that, highest production takes place at 4g/L peptone
concentration significantly (’p’ 0.0001) with M.calabura fruits.

Figure 4.4i:
Effect of peptone concentration on kojic acid production with the substrate
cashew apple
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

50

40

30

20

10

0
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

It was showed from the Figure 4.4i that, 4g/L peptone concentration produces significant
yield (’p’ value 0.0002) with Cashew apple.

64
Figure 4.4j:
Effect of peptone concentration on kojic acid production with the substrate
Sugarcane baggase

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

16

14

12

10

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Peptone concentration 4g/L was significant (‘p’ 0.01) for kojic acid production with
sugarcane baggase (Figure 4.4j).

Figure 4.4k:
Effect of peptone concentration on kojic acid production with the substrate
Rice bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

18

16

14

12

10

2
0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.4k it was noticed that maximum production was occurred with 4g/L
peptone concentration significantly (‘p’ 0.004) with rice bran.

65
Figure 4.4l:
Effect of peptone concentration on kojic acid production with the substrate
Wheat bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

8

0 1 2 3 4 5 6

Peptone concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.4l showed that, peptone concentration 4g/L produce highest yield significantly
(‘p’ 0.02) with the substrate wheat bran.

4.4.5 Optimization of Time period for kojic acid production:

The cultural flasks were incubated under appropriate conditions and the aliquots of the
samples were collected at different time periods and examined for kojic acid production.
It was found out from the Figure 4.5a-4.5l that, incubation time 16-28d except for
palmyra sap 32d was favourable for the production of kojic acid from the 12 carbon
sources used. According to Ariff et.al (1996, 1997) the effect of time on fermentation
production of kojic acid by using A.flavus was considered to be a non-growth associated
process and often involves two important phases called growth phase and production
phase. Probably the growth of the fungus attains maximum after 120h of duration and the
production was initiated after 48h-72h of incubational period. During this phase the
production was occurred continuously in a linear fashion till the glucose was exhausted
completely in the fermentation medium. In 1967, Kitada et.al have established that the
kojic acid fermentation using A.oryzae was called as mixed process because the kojic
acid production takes place in both growth and non-growth phases.

66
Figure 4.5a:
Effect of Time on kojic acid production with the substrate Ipomea

Concentration of kojic acid (g/L) & Biomass dry weight (g/L)

35

30

25

20

15

10

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was observed from the Figure 4.5a that, highest production takes place significantly
(‘p’ 0.0004) at incubation time period 28d with Ipomea.

Figure 4.5b:
Effect of Time on kojic acid production with the substrate Cassava
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0
5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.5b showed that, time period 28d was highly significant (‘p’0.0001) for the
production with Cassava.

67
Figure 4.5c:
Effect of Time on kojic acid production with the substrate Sago starch

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

70

60

50

40

30

20

10

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Incubation time 28d produces significant yield (’p’ 0.0002) with the substrate sago starch
(Figure 4.5c).

Figure 4.5d:
Effect of Time on kojic acid production with the substrate A.macrorrhiza
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

40

30

20

10

5 10 15 20 25 30 35 40

Time (d)
Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

From the Figure 4.5d it was noticed that incubation time 21d significantly produces (‘p’
0.0003) highest yield with the substrate A.macrorrhiza.

68
Figure 4.5e:
Effect of Time on kojic acid production with the substrate Palmyra sap

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

80

60

40

20

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was observed from the Figure 4.5e that, significant production (‘p’ value 0.00002)
takes place at time period 32d.

Figure 4.5f:
Effect of Time on kojic acid production with the substrate Paneer whey
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

5 10 15 20 25 30 35 40
Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.5f it was noticed that, time period 21d was highly significant (’p’
0.0002) for the production with substrate paneer whey.

69
Figure 4.5g:
Effect of Time on kojic acid production with the substrate Coconut water

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Incubation time 16d showed significant influence (‘p’0.0004) on production with

coconut water (Figure 4.5g).

Figure 4.5h:
Effect of Time on kojic acid production with the substrate M.calabura fruits
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

80

60

40

20

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Incubation time 28d influences (‘p’ 0.0001) greatly the production rate of kojic acid from
M.calabura fruits (Figure 4.5h).

70
Figure 4.5i:
Effect of Time on kojic acid production with the substrate Cashew apple

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

50

40

30

20

10

5 10 15 20 25 30 35 40

Time (d)
Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

From the above Figure 4.5i it was noticed that incubation time 16d was highly significant
(‘p’0.0006) for the production from Cashew apple.

Figure 4.5j:
Effect of Time on kojic acid production with the substrate Sugarcane baggase
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

0
5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was deduced from the Figure 4.5j that, time period 21d significantly influences (’p’
0.009) the production from sugarcane baggase.

71
Figure 4.5k:
Effect of Time on kojic acid production with the substrate Rice bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.5k it was observed that incubation time 16d produces significant yield
(’p’ 0.0005) from Rice bran.

Figure 4.5l:
Effect of Time on kojic acid production with the substrate Wheat bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

5 10 15 20 25 30 35 40

Time (d)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.5l represented that, time period 16d significantly enhances (’p’ value 0.03) the
yield from Wheat bran.

72
4.4.6 Optimization of initial pH for kojic acid production:

It was represented from the Figure 4.6a-4.6l that, pH 4.0-6.0 was the optimium value for
maximum production by the carbon sources. The results were agreed to earlier studies on
kojic acid production. Most research was carried out to study the influence of initial pH
on the growth and production Lin et al. (1976), Clevstrom and Lundjgren (1985). The
influence of pH on kojic acid production by A.flavus was examined by setting the pH of
the culture medium to their respective values with 0.1N NaOH or 0.1N HCl. The
optimum pH required for the production of kojic acid is different from the optimum pH
required for the growth of microorganisms. Katagiri and Kitahara (1933) in their studies
observed that, at pH 2.4 favourable production of kojic acid takes place where as at pH
5.0 the growth of the fungus A.oryzae was favourable. The production of kojic acid was
takes place at very low pH of 1.9 – 3.0 Wood (1998), Clevstrom & Liunggren (1985).
The optimum production also occurs at pH of 5.0 – 7.0 El-Aasar (2006), Basappa et al.
(1970). Rosfarizan et al. (2002) observed that, at pH 4.5-6.0 the fungus shows growth
phase during which production of enzymes was takesplace used for the conversion of
glucose to kojic acid and at pH 2.0-3.0 the fungus exhibits stationary phase during which
production of kojic acid takesplace. Rosfarizan et al. (2000) used double phase pH
strategy to obtain maximum yield of kojic acid. However the optimum pH value was also
based on the source of carbon and nitrogen utilized for the production of kojic acid.

73
Figure 4.6a:

Effect of pH on kojic acid production with the substrate Ipomea

Concentration of kojic acid (g/L) & Biomass dry weight (g/L)

18

16

14

12

10

3 4 5 6 7 8 9

pH

From the Figure 4.6a it was observed that, pH 5.5 was highly significant (’p’value 0.001)
for the production of kojic acid from Ipomea.

Figure 4.6b:
Effect of pHon kojic acid production with the substrate Cassava
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

70

60

50

40

30

20

10

3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was showed from the Figure 4.6b that, pH 6.0 highly influences (’p’ value 0.00004) the
production of kojic acid from Cassava.

74
Figure 4.6c:
Effect of pHon kojic acid production with the substrate Sago starch

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

60

50

40

30

20

10

0
3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight

Figure 4.6c represented that, pH 6.0 was highly significant (‘p’ 0.0001) for the
production from Sago starch.

Figure 4.6d:
Effect of pHon kojic acid production with the substrate A.macrorrhiza
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

16

14

12

10

0
3 4 5 6 7 8 9

pH
Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

Figure 4.6d showed that pH 6.0 highly influences (‘p’ 0.0003) the productivity of kojic
acid from the substrate A.macrorrhiza.

75
Figure 4.6e:
Effect of pHon kojic acid production with the substrate Palmyra sap

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

60

50

40

30

20

10

0
3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was noticed from the Figure 4.6e that significant production (’p’ value 0.0008) was
occurred at pH 4.0 with palmyra sap.

Figure 4.6f:
Effect of pHon kojic acid production with the substrate Paneer whey
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

12

10

3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.6f showed that, pH 4.0 highly influences (‘p’ 0.0002) the production from
paneer whey.

76
Figure 4.6g:
Effect of pHon kojic acid production with the substrate Coconut water

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

40

35

30

25

20

15

10

0
3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.6g pH 6.0 was significantly influences (‘p’ 0.0003) the kojic acid
production from coconut water.

Figure 4.6h:
Effect of pHon kojic acid production with the substrate M.calabura fruits
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0
3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight

pH 6.0 influences (‘p’ 0.00005) greatly the production rate of kojic acid from M.calabura
fruits (Figure 4.6h).

77
Figure 4.6i:
Effect of pHon kojic acid production with the substrate Cashew apple

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

22

20

18

16

14

12

10

0
3 4 5 6 7 8 9

pH
Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

From the above Figure 4.6i it was noticed that pH 6.0 was highly significant (‘p’0.0004)
for the production from Cashew apple.

Figure 4.6j:
Effect of pHon kojic acid production with the substrate Sugar cane baggase
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

14

12

10

2
3 4 5 6 7 8 9
pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was deduced from the Figure 4.6j that, pH 5.5 significantly influences (’p’ 0.009) the
production from sugarcane baggase.

78
Figure 4.6k:

Effect of pHon kojic acid production with the substrate Rice bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

25

20

15

10

0
3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.6k it was observed that pH 6.0 produces significant yield (’p’ 0.002)
from Rice bran.

Figure 4.6l:
Effect of pHon kojic acid production with the substrate Wheat bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

10

3 4 5 6 7 8 9

pH

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.6l represented that, pH 5.5 significantly enhances (’p’ value 0.04) the yield from
Wheat bran.

79
4.4.7 Optimization of Temperature for kojic acid production:

Temperature is one of the most important vital factors which effect the fungal growth,
metabolism and its survivellance. It was deduced from the Figures 4.7a-4.7l that,
Incubation temperature 28-300C was optimum for kojic acid production by all the carbon
sources.Temperature was one of the most critical factors of microbial fermentation. El-
Aasar (2006) reported that, 24-280C were the optimum ranges of temperature for kojic
acid fermentation. The decrease in the concentration of kojic acid after the optimized
period of time was due to degradation of kojic acid to oxalic acid and acetic acid by the
fungal mycelium under depleted condition of glucose concentration Bajpai et al. (1982),
Madihah et al. (1993), Ariff et al. (1997), Rosfarizan and Ariff (2007).

Figure 4.7a:

Effect of Temperature on kojic acid production with the substrate Ipomea

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

35

30

25

20

15

10

0
18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.7a indicated that, significant production (‘p’ 0.0001) takes place at incubation
temperature 280C with the substrate Ipomea.

80
Figure 4.7b:
Effect of Temperature on kojic acid production with the substrate Cassava

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

12

10

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was observed from the Figure 4.7b that, temperature 280Csignificantly produces (’p’
value 0.0004) kojic acid from Cassava.

Figure 4.7c:
Effect of Temperature on kojic acid production with the substrate Sago starch
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

60

50

40

30

20

10

0
18 20 22 24 26 28 30 32 34 36

Temperature (oC)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Temperature 280C produces significant yield (’p’ value 0.0002) from sago starch (Figure
4.7c).

81
Figure 4.7d:
Effect of Temperature on kojic acid production with the substrate A.macrorrhiza

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

25

20

15

10

0
18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)
Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

With the substrate A.macrorrhiza, temperature 280C produces enhanced yield

significantly (‘p’ 0.0002) Figure 4.7d.

Figure 4.7e:
Effect of Temperature on kojic acid production with the substrate Palmyra sap
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

10

0
18 20 22 24 26 28 30 32 34 36

Temperature (oC)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.7e indicated that, significant yield (‘p’ 0.0001) was obtained with palmyra sap at
temperature 280C.

82
Figure 4.7f:
Effect of Temperature on kojic acid production with the substrate
Paneer whey

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

25

20

15

10

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.7f it was deduced that, temperature 280C significantly produces
(‘p’0.0007) kojic acid from paneer whey.

Figure 4.7g:

Effect of Temperature on kojic acid production with the substrate

Coconut water
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

16

14

12

10

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Temperature 280C produces significant yield (’p’ value 0.004) with the substrate coconut
water (Figure 4.7g).

83
Figure 4.7h:
Effect of Temperature on kojic acid production with the substrate M.calabura

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

35

30

25

20

15

10

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.7h it was noticed that, highest production takes place at temperature
280C significantly (’p’ 0.001) with M.calabura fruits.

Figure 4.7i:
Effect of Temperature on kojic acid production with the substrate Cashew apple
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

40

30

20

10

18 20 22 24 26 28 30 32 34 36

Temperature (oC)
Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

It was showed from the Figure 4.7i that, temperature 280C produces significant yield (’p’
value 0.007) with Cashew apple.

84
Figure 4.7j:
Effect of Temperature on kojic acid production with the substrate
Sugarcane baggase

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

18

16

14

12

10

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Temperature 280C was significant (‘p’ 0.001) for kojic acid production with sugarcane
baggase (Figure 4.7j).

Figure 4.7k:
Effect of Temperature on kojic acid production with the substrate
Rice bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

12

10

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.7k it was noticed that maximum production was occurred at
temperature 300C significantly (‘p’ 0.0008) with rice bran.

85
Figure 4.7l:
Effect of Temperature on kojic acid production with the substrate
Wheat bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

8

18 20 22 24 26 28 30 32 34 36
o
Temperature ( C)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.7l showed that, temperature 280C produce highest yield insignificantly (‘p’ 0.12)
with the substrate wheat bran.

4.4.8 Optimization of Potassium dihydrogen phosphate concentration for kojic acid

production:
It was found out from the Figures 4.8a-4.8l that, KH2PO4 concentration 1-2g/L produce
maximum yield from all the 12 carbon sources. Phosphate shows stimulated effect on the
enhanced production of kojic acid Arnstein and Bentley (1953).

86
Figure 4.8a:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Ipomea

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

10

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.8a it was observed that, 2g/L potassium dihydrogen phosphate
concentration was highly significant (’p’value 0.001) for the production of kojic acid
from Ipomea.

Figure 4.8b:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Cassava
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

30

25

20

15

10

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was showed from the Figure 4.8b that, KH2PO4 concentration 2g/L highly influences
(’p’ value 0.004) the production of kojic acid from Cassava.

87
Figure 4.8c:

Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Sago starch

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

12

10

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.8c represented that, KH2PO4 1g/L was highly significant (‘p’ 0.0001) for the
production from Sago starch.

Figure 4.8d:
Effect of potassium dihydrogen phosphate concentration on kojic acid production
with the substrate A.macrorrhiza
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

4.5

4.0

3.5

3.0

2.5

2.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

Figure 4.8d showed that KH2PO4 concentration 1g/L highly influences (‘p’ 0.004) the
productivity rate of kojic acid from the substrate A.macrorrhiza.

88
Figure 4.8e:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Palmyra sap

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

8

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was noticed from the Figure 4.8e that significant production (’p’ value 0.0009) was
occurred at 2g/LKH2PO4 concentration with palmyra sap.

Figure 4.8f:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Paneer whey
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.8f showed that, KH2PO4 concentration 1g/L highly influences (‘p’ 0.0002) the
production from paneer whey.

89
Figure 4.8g:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Coconut water

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.8g KH2PO4 concentration 1g/L was significantly influences (‘p’
0.0006) the kojic acid production from coconut water.

Figure 4.8h:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate M.calabura fruits
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

35

30

25

20

15

10

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Potassium dihydrogen phosphate concentration 2g/L influences (‘p’ 0.001) greatly the
production rate of kojic acid from M.calabura fruits (Figure 4.8h).

90
Figure 4.8i:
Effect of potassium dihydrogen phosphate concentration on kojic acid production
with the substrate Cashew apple

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

25

20

15

10

0
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

From the above Figure 4.8i it was noticed that KH2PO4 concentration 1.5g/L was highly
significant (‘p’0.003) for the production from Cashew apple.

Figure 4.8j:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Sugarcane baggase
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

11

10

3
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was deduced from the Figure 4.8j that, KH2PO4 concentration 1.5g/L significantly
influences (’p’ 0.009) the production from sugarcane baggase.

91
Figure 4.8k:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Rice bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

22

20

18

16

14

12

10

2
0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.8k it was observed that KH2PO4 concentration 2g/L concentration
produces significant yield (’p’ 0.001) from Rice bran.

Figure 4.8l:
Effect of potassium dihydrogen phosphate concentration on kojic acid production with the
substrate Wheat bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Potassium dihydrogen phosphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.8l it was observed that KH2PO4 concentration 1.5g/L concentration
produces significant yield (’p’ 0.001) from wheat bran.

92
4.4.9 Optimization of Magnesium sulphate concentration for kojic acid production:
It was evident from the Figures 4.9a-4.9l that, MgS04 concentration 0.3-0.7g/L was
optimum for the production of kojic acid from all the carbon sources. Mineral salts were
one of the key ingredients of fermentation medium which promote the kojic acid
production in a favourable forward direction and also enhance the growth of the kojic
acid synthesizing fungus Basappa et al. (1970) Wei et al. (1991).

Figure 4.9a:

Effect of magnesium sulphate concentration on kojic acid production with the

substrate Ipomea
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

14

12

10

0
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.9a indicated that, significant production (‘p’ 0.001) takes place at MgSO4
concentration 0.5g/L with the substrate Ipomea.

93
Figure 4.9b:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Cassava

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

25

20

15

10

0
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

It was observed from the Figure 4.9b that, 0.5g/L MgSO4 concentration significantly
produces (’p’ value 0.0004) kojic acid from Cassava.

Figure 4.9c:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Sago starch
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

14

12

10

0
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Magnesium sulphate concentration 0.5g/L produces significant yield (’p’ value 0.001)
from sago starch (Figure 4.9c)

94
Figure 4.9d:
Effect of magnesium phosphate concentration on kojic acid production with the
substrate A.macrorrhiza

Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium phosphate concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

With the substrate A.macrorrhiza MgSO4 concentration 0.5g/L produces enhanced yield
significantly (‘p’ 0.0007) Figure 4.9d.

Figure 4.9e:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Palmyra sap
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

18

16

14

12

10

0
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.9e indicated that, significant yield (‘p’ 0.0001) was obtained with palmyra sap at
MgSO4 0.5g/L concentration.

95
Figure 4.9f:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Paneer whey

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

6

0
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.9f it was deduced that, MgSO4 concentration 0.3g/L significantly
produces (‘p’0.00007) kojic acid from paneer whey.

Figure 4.9g:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Coconut water
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

40

30

20

10

0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

MgSO4 concentration 0.3g/L produces significant yield (’p’ value 0.0004) with the
substrate coconut water (Figure 4.9g).

96
Figure 4.9h:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Muntingia calabura fruits

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

50

40

30

20

10

0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.9h it was noticed that, highest production takes place at 0.7g/L MgSO4
concentration significantly (’p’ 0.0004) with M.calabura fruits.

Figure 4.9i:
Effect of magnesium phosphate concentration on kojic acid production with the
substrate Cashew apple
Concentration of kojic acid (g/L)& Biomass dry weight (g/L)

22

20

18

16

14

12

10

2
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium phosphate concentration (g/L)

Concentration of kojic acid (g/L)
Biomass dry weight (g/L)

It was showed from the Figure 4.9i that, 0.7g/L MgSO4 concentration produces
significant yield (’p’ value 0.003) with Cashew apple.

97
Figure 4.9j:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Sugarcane baggase

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

12

11

10

4
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Magnesium sulphate concentration 0.7g/L was significant (‘p’ 0.002) for kojic acid
production with sugarcane baggase (Figure 4.9j).

Figure 4.9k:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Rice bran
Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

18

16

14

12

10

2
0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

From the Figure 4.9k it was noticed that maximum production was occurred with
Magnesium sulphate concentration 0.7g/L significantly (‘p’ 0.005) with rice bran.

98
Figure 4.9l:
Effect of magnesium sulphate concentration on kojic acid production with the
substrate Wheat bran

Concentration of kojic acid (g/L) &Biomass dry weight (g/L)

5

0.0 0.2 0.4 0.6 0.8 1.0

Magnesium sulphate concentration (g/L)

Concentration of kojic acid (g/L)

Biomass dry weight (g/L)

Figure 4.9l showed that, MgSO4 concentration 0.7g/L produce highest yield significantly
(‘p’ 0.0004) with the substrate wheat bran.

4.5 Production of kojic acid after optimization of cultural parameters:

Table 4.2 showed the yields of kojic acid obtained at optimized conditions from the 12
carbon sources. The results of One-way ANOVA indicated that significant effect of these
factors (‘p’< 0.05) on productivity yields of kojic acid. Figure 4.10 showed the
concentration of kojic acid obtained at initial and final conditions with Bentley’s method.
Figure 4.11 represented the yields at initial and final production in terms of kojic acid
crystals. More than 50%-75% increase in the kojic acid crystal yield was identified after
optimization. Maximum production was obtained with M.calabura fruits 86.06g/L, Sago
starch 79.47g/L and Palmyra sap 76.31g/L. The yield obtained with the sago starch
79.47g/L in the present research was higher than the yield reported by the earlier studies.
Potato starch, corn starch and sago starch were used for kojic acid fermentation with the
kojic acid producing fungal strain A.flavus S33-2 isolated from morning glory flower.

99
 Table 4.2: Kojic acid concentrations at optimized cultural parameters

Substrate peptone Time pH Temp. MgSO4 KH2PO4 Final ‘p’value

‘C’ concentration production
source (g/L)
Ipomea 1000 ml 3g/L 28d 5.5 280C 0.7 g/L 1.5g/L 53.9±0.2 0.000005
44.2±0.36 48.9±0.6 29.7±1.12 8.27±0.59 30.4±0.6 7.31±0.42 5.16±0.3
Cassava 1000ml 2g/L 28d 6.0 280C 0.5g/L 2g/L 65.59±0.5 0.00002
25.18±0.36 25.9±0.55 27.3±0.55 59.8±0.66 10.15±0.37 6.81±0.25 3.46±0.4

Sago 1000ml 4g/L 28d 6.0 280C 0.5g/L 1g/L 79.47±0.8 0.00004
71.38±0.59 82.5±1.55 66.5±1.81 50.6±1.09 52.6±1.57 12.68±0.87 4.62±0.09
Alocasia 1000ml 2g/L 21d 6.0 280C 0.5g/L 1g/L 40.13±0.9 0.00019
34.73±0.8 3.86±0.14 36.8±1.1 4.52±0.14 22.4±0.55 3.64±0.17 3.65±0.4
Palmyra sap 1000ml 3g/L 32d 4.0 280C 0.5g/L 2g/L 76.31±0.5 0.00001
47.21±0.31 78.5±1.65 75.91±0.68 50.9±2.58 6.46±0.15 5.59±0.11 3.84±0.19
Paneer 1000ml 2g/L 21d 4.0 280C 0.3g/L 1g/L 42.7±0.3 0.00002
whey 6.84±0.06 3.53±0.07 42.2±1.21 3.84±0.1 19.3±0.94 4.23±0.2 3.81±0.1
Coconut 1000ml 4g/L 16d 6.0 280C 0.3g/L 1g/L 48.13±1.1 0.0001
water 30.8±1.42 46.7±1.5 46.1±1.75 36.4±1.2 15.2±1.81 37.4±1.43 3.76±0.16
M.calabura 1000g/L 4g/L 28d 6.0 280C 0.7g/L 2g/L 86.06±0.9 0.00003
65.32±1.45 60.47±1.36 73.6±1.66 28.63±0.36 28.8±2.01 42.5±1.64 31.17±1.77
Cashew 500g/L 4g/L 16d 6.0 280C 0.7g/L 1.5g/L 46.3±1.41 0.0003
apple 16.59±1.77 40.65±1.03 42.67±1.91 19.2±0.67 34.1±5.07 20.4±2.13 21.5±2.1
Sugar cane 30g/L 4g/L 21d 5.5 280C 0.7g/L 1.5g/L 17.8±0.7 0.0005
baggase 19.6±1.67 8.56±1.67 1.32±0.22 12.69±2.1 15.52±0.97 9.5±0.86 6.72±1.15
Rice bran 10g/L 4g/L 16d 6.0 300C 0.7g/L 2g/L 30.7±1.2 0.0005
25.5±2.07 15.6±1.83 28.3±1.13 21.41±1.89 10.96±0.55 15.86±1.97 21.04±1.56
Wheat bran 20g/L 4g/L 16d 5.5 280C 0.7g/L 1.5g/L 0.95±0.19 0.01
0.77±0.14 0.64±0.16 0.84±0.29 0.803±0.28 0.82±0.54 0.4±0.01 0.38±0.02
± :Standard deviation ‘p’: probability value

100
The yield obtained was 1.7g/L with potato starch, 19.2g/L with corn starch and 0.3g/L
with sago starch Rosfarizan et al. (1998). It was reported that, 40g/L of kojic acid was
obtained with A.oryzae MK-107-39 strain using partially hydrolyzed corn starch
supplemented with little amount of corn steep liquor Futamura et al. (2001). To enhance
the production rate of kojic acid, fermentation was conducted in 8L stirred tank fermentor
using gelatinized sago starch by A.flavus Link 44-1 and reported 16.43g/L of kojic acid
Rosfarizan and Ariff (2002). It was revealed that, maximum kojic acid production
40.67g/L from potato starch using a potato mutant strain AFUV8 Abd El-Aziz (2013).
The fruits of M.calabura produce an opulent yield of kojic acid 86.06g/L higher than the
formerly used fruit based substrates by the earlier researchers. El-Kady et al. (2014) used
various fruit wastes, and reported maximum production 5.9g/L with oranges, 3.1g/L with
apricot, 4.2g/L from apple waste. Nurashikin et al. (2013) used pine apple waste and
reported maximum production 26.3g/L of kojic acid. The differences in the production
may be either due to the culture conditions or due to species differences El-Aasar (2006).

Figure 4.10:
Kojic acid concentrations at initial and after optimization

100
F
F
F
80
F
Conc. of kojic acid (g/L)

60 F
F I F
F
I F
I
40 F
I I
I I
I I F
20 I
I
I F
0
Cash
0 Ipom Cassa 5 Aloca 10 Pane coco 15 20 Rice wheat25
ea va sia Palm er nut M.calaew Sugar bran
bran
Sago yra 'C' source bura
cane
bagasse
Error means for concentartion of kojic acid (g/L)
I: initial, F:final

101
Figure 4.11: Kojic acid crystal yields at Initial and after optimization

Kojic acid yields at initial and after optimization

25

20

15
Conc. of kojic
10
acid crystals (g/L) Conc. of kojic acid crystals at
5 initial conditions
Conc. of kojic acid crystals
0 after OFAT optimization
Palmyra sap
Ipomea
Cassava

Coconut water

Sugarcane baggase
Paneer whey
Sago starch

Wheat bran
Alocasia

Cashew apple
M.calabura

Rice bran

Carbon source

102
4.6 CONCLUSION:

From the results of OFAT method, the optimized cultural parameters were established
with A.flavus and found to be in the ranges of Substrate concentration 1000ml or 1000g/L
for starch, liquid and fruit substrates except for cashew apple 500g/L and 10-30g/L for
agrowastes, Nitrogen source concentration 2.0-4.0g/L, Time 16-28d except for palmyra
sap 32d, Temperature 28-300C, pH 4.0-6.0, KH2PO4 concentration 1-2g/L, MgS04
concentration 0.3-0.7g/L. From the results it was also found that, among the 12 different
carbon sources used for the optimization studies, M.calabura fruits, Palmyra sap and
Sago starch showed maximum yields with A.flavus.

103
 CHAPTER – V
STATISTICAL OPTIMIZATION OF
CULTURAL PARAMETERS USING
RESPONSE SURFACE METHODOLOGY
 CHAPTER 5

5. STATISTICAL OPTIMIZATION OF CULTURAL

PARAMETERS USING RESPONSE SURFACE
METHODOLOGY

5.1 INTRODUCTION:

Recently, statistical optimization was commonly employed in the fermentation process

because it quickly screens a number of multiple parameters and their interactions, and
reflects the function of individual factor or component. It constitutes a set of empirical
procedures employed for evaluating the connecting link between the group of controlled
experimental parameters and measured response. The biotechnological and biochemical
processes concerned to food systems have utilized the RSM model successfully for the
optimization of process parameters De Lima et al. (2010). RSM is a sequential procedure
with an initial objective to lead the experiments rapidly and efficiency to the general
vicinity of the optimum. Coelho et al. (2010) used glycerol as the substrate for the
production of kojic acid using CCD matrix. Different carbon and nitrogen sources were
screened for production using A.flavus NRRL 626. The yield obtained was 18.8g/L at 22d
incubation. It was determined that, 29g/L of kojic acid after 20d incubation among the
runs. The present research use OFAT and RSM for statistical optimization of kojic acid
production using a soil fungus A.flavus. Based on the preliminary optimization
experiments through OFAT method, the most significant factors substrate concentration
(or) carbon source concentration, peptone concentration, pH, time, temperature were
selected to find the optimal concentrations for higher kojic acid production using CCD
and RSM Coninek et al. (2000), Shirai et al. (2001), Gorrect et al. (2004).

5.2 OBJECTIVES:

To enhance the kojic acid yields of Palmyra sap, Sago starch and M.calabura
fruits.

To study the parameter interactions of independent variables which influence the
production rate of kojic acid.

104
5.3 Response surface methodology for optimized carbon source Palmyra sap

The present report assess on the way of kojic acid production from inexpensive ‘C’
source Palmyra sap and on the process of variables optimization through OFAT method
and response surface methodology (RSM). This way assists in drawing a cost-effective
methodology for the production of kojic acid. Literature survey has revealed that no
studies have been reported for statistical optimization of kojic acid production by RSM.
Bracarense & Takahashi (2014) have used fractional-factorial design to produce the bio-
active metabolite kojic acid by A.parasiticus using two different synthetic media CYA
and YES media. One-factor-at-a-time method is a traditional method used for the
optimization of process variables. However this method was not able to detect the factors
which contribute for the optimum response since the effect of interaction among the
factors was not taken as a criteria in such strategies Deepak et al. (2008).

5.3.1 MATERIALS AND METHODS:

5.3.1a Kojic acid production:

The basal fermentation medium was prepared to contain 50ml of Palmyra sap, 0.5g
peptone, 0.5g KH2PO4 and 0.25g MgSO4.7H2O was taken into a conical flask. The design
of the fermentation medium was based on Ariff et al. (2006). Now 5ml of spore
suspension of A.flavus was added to the flask and incubated at 280C for 12d under static
conditions. When the fermentation was finished, the broth was filtered and the mycelia
mat was washed with water and kept in a hot air oven at 800C for 1d and the dry weight
mass was estimated. The concentration of kojic acid in the supernatant was determined
by Fecl3 method Bentley (1957) and further subjected to crystallization process (Hazzaa,
2013). Finally the weight of the crystal mass was determined.

5.3.1b Optimization of parameters by OFAT strategy:

Experiments were performed by conventional and traditional method called OFAT

strategy to choose the appropriate factor for getting maximum yield of kojic acid. The
ranges of Substrate concentration (100-1000ml), Peptone concentration (1-5g/L),

105
KH2PO4 (0.5-2.5g/L), MgSO4 concentration ((0.1-0.9g/L), pH (4-8), Time (11-37d),
Temperature (20-350C) were selected for the optimization of kojic acid production. In
order to acquire very higher amounts of kojic acid, the optimal concentrations of the
chosen key factors were furthermore determined.

5.3.1c Optimization by Response Surface Methodology:

In the present days, the highly advanced optimization strategy, RSM was usually applied
for the microbial production of both primary and secondary metabolites. It was well
suited to study the main and interaction effects of various factors and the production of
kojic acid. Hence five-factor-three-level was used in the current study Adinarayana and
Ellaiah (2002). On the basis of the best results obtained in the preliminary optimization
experiments, the ranges and the levels of the most significant variables in enhancing kojic
acid production were mentioned in the Table-5.1. In order to expand the regression
equation, the test factors were coded according to the below equation (1).

xi = (Xi - Xi*) / ∆Xi - (1)

th
xi is the coded value of the i independent variable,
Xi is the uncoded value of the ith independent variable,
Xi* is the uncoded ith independent variable at the centre point and
∆Xi is the step change value
Minitab version 16.0 was used to perform the design matrix. The CCD matrix with five
variables X three levels (-1 0 +1) was represented in the Table-5.2. The matrix comprises
32 runs. A second order polynomial equation (2) which is shown below contain all linear,
quadratic and interaction terms was used to calculate the predicted response.
Y = b o + ∑ b i Xi + ∑ b i 2 Xi 2 + ∑ b ij Xi Xj - (2)
Y is the concentration of kojic acid (g/L)
bo is the intercept
bi is the coefficient for linear direct effect
bi 2 is the coefficient for quadratic effect and is responsible for curvatures of the model
bij is the coefficient for interaction effect a positive (or) negative significant value
indicates possible interactions between the variables.

106
Statistical analysis of the models was utilized to determine the ANOVA i.e., analysis of
variance. The quality of fit of the polynomial model was inferred by the coefficient of
determination R2 and its statistical significance was ascertained by the F-test whereas
student’s t-test was performed to determine the significance of the regression coefficients.

5.3.2 RESULTS AND DISCUSSION:

The results of one-factor-at-a-time method reveals that the key process parameters which
effects the kojic acid production were Substrate concentration (1000ml), Peptone
concentration (3g/L), KH2PO4 (2g/L), MgSO4 concentration (0.5g/L), pH (4.0), Time
(32d) and Temperature (280C). The kojic acid production obtained at these optimized
conditions was 75.9g/L.

5.3.2a Central composite design matrix:

In order to find the optimized conditions of physicochemical factors and interactions

between them a five-variable-three-level CCD matrix was used in the present study. The
concentrations of kojic acid (g/L) for each and every single run along with their
experimental and predicted response were illustrated in Table-5.2.The maximum
concentration of kojic acid obtained runs of experimental value was 78.6g/L whereas in
the predicted response the highest production obtained was 71.9g/L This result
corroborated the validity and effectiveness of this model. However it was noted that the
same conditions of optimization was observed with CCD like OFAT method.

By employing RSM, the below regression equation has been obtained and it accounts for
the empirical connecting link between the kojic acid and the test variables in coded units.

Y = 9447.02 + 31.70 x1 + 462.15 x2 + 316.36 x3 – 629.45 x4 – 162.19 x5 – 0.37 x1 x2 –

0.09 x1 x3 – 0.10 x1 x4 + 0.48 x1 x5 – 1.78 x2 x3 + 1.06 x2 x4 +3.46 x2 x5 – 5.45 x3 x4 + 0.25
x3 x5 – 1.70 x4 x5 + 0.10 x12 + 34.60 x22 + 23.00 x32 – 9.26 x42 – 3.01 x52

‘Y’ is the response i.e., the concentration of kojic acid obtained as function of x1, x2, x3, x4
and x5 test variables. For the above equation, F-test was performed to its statistical
significance.

107
The result of the second order response surface quadratic model fitting in the form of
ANOVA was shown in Table-5.4. The ‘F’ value of the model was 11.97 and the
probability value (P ~ 0) showed that the model terms were significant. It was indicated
by the regression equation was that R2 was 0.9561 Table-5.4 which specified the fitness
of the model and designates that roughly 95% of the variability in the response can be
interpreted by the model. The value of R2 is in between 0 and 1. If the R2 value is nearer
to 1.0, the stronger the model, the better it predicts the response. The adjusted
determination coefficient value was also high (0.8962) supporting the significance of the
model. There was no evidence of ‘lack of fit’. The signal to noise ratio was determined
by adequate precision value and it was 10.425. The ratio should be greater than 4. Hence
the present model could be used to navigate the design space.

The ‘F’ test and the correlating ‘p’ values along with the parameters were evaluated and
showed in Table-5.4. The calculated coefficients of the regression model were listed in
Table-5.3 and that contains 5 linear, 10 quadratic, 5 interaction terms and 1 block term.
For each coefficient term its significance was measured by Student’s t-test and p-value.
The results from the Table-5.3 revealed that two linear coefficients (pH, peptone
concentrations) and one cross product coefficient (peptone x peptone concentration)
showed significant effects on kojic acid production (p<0.05).

5.3.2b Response Surface plots and Contour plots:

In order to examine the interaction between different parameters and to find the optimal
amount of each factor which influences in yielding higher concentrations of kojic acid,
response surface plots should be traced out. The 3D surface curves were plotted across
any two independent variables while keeping other variable at its control (0) level. Figure
5.1 indicates the interdependence of kojic acid production on peptone concentration and
substrate concentration. The kojic acid production enhanced with increase in peptone
concentration to about 2-3g/L and afterwards the production rate was declined with
furthermore increase in peptone concentration. The similar propensity was noticed in
Figure 5.2. Increase in the incubation temperature 27-280C ensued increase in the kojic
acid production. This was apparent from Figures 5.3-5.5. Figure 5.6 presents the

108
dependency of kojic acid production on pH. The effect of pH resembles to peptone
concentration. The increase in pH from 4.0-5.0 enhances the production of kojic acid.
Increased kojic acid production was identified on Time 32d was depicted in the Figures
5.7-5.8. The optimum conditions established for the production of kojic acid were
Substrate concentration (1000ml), Peptone concentration (3g/L), KH2PO4 (2g/L), MgSO4
concentration (0.5g/L), pH (4.0), Time (32d) and Temperature (280C). While comparing
the predicted values obtained by regression model equation to that of experimental model
the results were closely related to each other. To check the validity of the experimental
model, production was once again done at optimized conditions in triplicates. The result
was compared with the predicted response, so that the validity of the model was proved.

The validity of the model was verified by fitting the values of the independent variables
into the regression model equation and by conducting experiments using these values.
The optimization of the evaluated responses confirmed that the optimum results for kojic
acid production 78.6g/L were obtained with Substrate concentration (1000ml), Peptone
concentration (3g/L), pH (4.0), Time (32d) and Temperature (280C). Nearly 7% increase
in the kojic acid yield was identified by RSM. Each and every point was positioned near
the central point of the design. Upon crystallization the broth was concentration and
produced 21.94 g/L of dry crystals. It was finally concluded that, the Central Composite
Design and Response Surface Methodology facilitated in resolving the optimized
parameters for the excess production of kojic acid from the economical carbon source
Palmyra sap through surface fermentation with the soil isolate Aspergillus flavus.

Table 5.1: Independent variables in the experimental plan

Independent Coded Levels

Variables Symbols
-1 0 +1

Substrate concentration (ml) X1 90 100 110

Peptone concentration (g/L) X2 2 3 4
Incubation time (d) X3 31 32 33
pH X4 3 4 5
Temperature (0C) X5 27 28 29

109
Table 5.2: CCD matrix having real values along with the experimental and
predicted values of kojic acid concentration

Run substra pH Pepton Incubati Temp Experimen Predicted

Ord te conc e on time era tal values of
er (ml) (g/L) (d) ture values kojic acid (g/L)
0 of kojic
( C)
acid
(g/L)

1 110 3 2 31 27 2.68 1.727

2 90 3 2 33 27 5.07 0.684
3 100 4 4 32 28 51.9 48.152
4 100 4 3 32 28 78.6 71.916
5 110 5 4 33 29 3.11 6.257
6 100 4 3 32 29 70.4 72.661
7 110 5 2 31 29 5.39 6.333
8 110 3 4 31 29 3.91 5.835
9 90 5 2 31 27 39.5 36.308
10 90 4 3 32 28 45.1 63.900
11 100 3 3 32 28 15.7 25.546
12 100 4 3 32 28 78.6 71.916
13 100 4 3 33 28 76.4 81.359
14 110 3 2 33 29 10.54 10.288
15 90 3 2 31 29 5.59 1.878
16 90 5 4 33 27 23.7 22.712
17 110 3 4 33 27 5.98 7.231
18 100 4 3 31 28 75.93 80.997
19 100 4 2 32 28 35.9 49.673
20 90 5 2 33 29 44.7 42.210
21 90 3 4 31 27 6.45 4.240
22 90 5 4 31 29 35.4 35.086
23 100 4 3 32 27 69.43 77.194
24 90 3 4 33 29 6.92 5.412
25 100 4 3 32 28 78.6 71.916
26 110 4 3 32 28 68.4 59.626
27 100 4 3 32 28 78.6 71.916
28 100 4 3 32 28 78.6 71.916
29 100 5 3 32 28 48.9 49.080
30 110 5 4 31 27 36.13 38.575
31 110 5 2 33 27 37.82 38.089
32 100 4 3 32 28 78.6 71.916

110
Table 5.3: Model coefficients estimated by multiple linear regression (significance of
regression coefficients)

Term Coefficient Standard error T-value P-value

coefficient
Constant 9447.02 7741.90 1.220 0.248
Substrate conc 31.70 17.36 1.827 0.095
pH 462.15 125.92 3.670 0.004
Peptone 316.36 121.10 2.612 0.024
Incubation time -629.45 432.54 -1.455 0.174
Temperature -162.19 382.52 -0.424 0.680
Substrate conc*substrate -0.10 0.07 -1.528 0.155
conc
pH*pH -34.60 6.65 -5.207 0.000
Peptone*Peptone -23.00 6.65 -3.461 0.005
Incubation 9.26 6.65 1.394 0.191
time*Incubation time
Temperature*Temperature 3.01 6.65 0.453 0.659
Substrate conc*pH -0.37 0.26 -1.437 0.179
Substrate conc*Peptone 0.09 0.26 0.362 0.724

Substrate conc*Incubation 0.10 0.26 0.381 0.710

time
Substrate -0.48 0.26 -1.860 0.090
conc*Temperature
pH*Peptone -1.78 2.61 -0.682 0.509
pH*Incubation time -1.06 2.61 -0.407 0.692
pH*Temperature -3.46 2.61 -1.327 0.211
peptone*Incubation time -5.45 2.61 -2.090 0.061
Peptone*Temperature -0.25 2.61 -0.098 0.924
Incubation 1.70 2.61 0.652 0.528
time*Temperature

111
Table 5.4:
.4: ANOVA for the entire quadratic model

Source DF Seq SS Adj SS Adj MS F P

Regression 20 26027.4 26027.4 1301.37 11.97 0.000

Linear 5 2678.1 2841.5 568.31 5.23 0.011
Square 5 21937.1 21937.1 4387.43 40.37 0.000
Interaction 10 1412.2 1412.2 141.22 1.30 0.336
Residual Error 11 1195.6 1195.6 108.69
Lack-of-Fit 6 1195.6 1195.6 199.26 * *
Pure Error 5 0.0 0.0 0.00
Total 31 27223.0

R-Sq:95.61%, R-Sq
Sq (pred): 0.00%, R-Sq
R (adj): 89.62%
DF:Degree of freedom, SS: sum of squares

Figure 5.1:
.1: Response surface plot for kojic acid production versus Peptone and
substrate concentration

112
Figure 5.2:
.2: Response surface plot for kojic acid production versus Peptone and pH

Figure 5.3:
.3: Response surface plot for kojic acid production versus Temperature and
Peptone

113
Figure 5.4:
.4: Response surface plot for kojic acid production versus Temperature and
pH

Figure 5.5:
.5: Response surface plot for kojic acid production versus Temperature and
Substrate concentration
ion

114
Figure 5.6:
.6: Response surface plot for kojic acid production versus pH and Substrate
concentration

Figure 5.7:
.7: Response surface plot for kojic acid production versus Incubation time
and Peptone

115
Figure 5.8:
.8: Response surface plot for kojic acid production
production versus Incubation time
and pH

116
5.4 Response surface methodology for optimized carbon source Muntingia calabura
fruits

The optimized physical and nutritional parameters obtained by non-statistical method

one-factor-at-a-time method (OFAT) using M.calabura fruits as carbon source were
selected for further optimization through statistical design model called Response Surface
Methodology (RSM) to study their interactions which may influence the yield of kojic
acid.

5.4.1 MATERIALS AND METHODS:

5.4.1a Kojic acid production:

Surface fermentation was done with the production medium containing 100g of
M.calabura fruits, Peptone 1.0g, KH2PO4 1.0g and MgSO4.7H2O 0.5g was added to 1L
conical flask. After addition of 10ml of spore suspension of A.flavus, the flask was
incubated at 250C for 12d. After completion of the fermentation, filtration was done and
the mycelia dry weight was estimated. The supernatant was subjected to Bentley’s
colorimetric method (Bentley, 1957) and crystallization (Hazza, 2013). Finally the weight
of the crystal mass was determined.

5.4.1b Optimization of parameters by OFAT strategy:

Through OFAT method different physico-chemical parameters which influence the kojic
acid production were tested. The parameters tested were Substrate concentration (100g/L
– 1000g/L), Peptone concentration (1 – 5g/L), KH2PO4 (0.5 – 2.5g/L), MgSO4
concentration (0.1 – 0.9g/L), pH (4 – 8), Time (11 – 37d) and Temperature (20 – 350C).
The screened optimized fermentation conditions obtained by OFAT method were further
optimized by central composite design and RSM analysis.

117
5.4.1c Optimization by Response surface methodology:

The process parameters substrate concentration, peptone concentration, pH, time and
temperature were selected as significant variables for CCD analysis while the optimum
phosphate concentration by OFAT method was kept constant throughout the experiment.
Based on the following equation, the testing variables were coded and represented in
Table-5.5.

xi = (Xi - Xi*) / ∆Xi - (1)

The kojic acid concentration (g/L) with 32 experimental runs comprising different
combinations of five variables was shown in Table-5.6. Minitab version 16.0 was used
for the statistical analysis. For calculating the predicted response for the model terms the
second order polynomial equation was used.

Y = b o + ∑ b i Xi + ∑ b i 2 Xi 2 + ∑ b ij Xi Xj - (2)

ANOVA was employed for determination of significant variables. ANOVA implicated

Fischer’s test to estimate the overall significance of the model, associated ‘p’ values and
determination coefficient R2 is used to determine the regression model’s goodness of fit.
Then fitted polynomial equation was furthermore represented in terms of 3D and contour
plots where the interactions can be shown in the form of graphs. At last, the model was
also validated.

5.4.2 RESULTS AND DISCUSSION:

The preliminary studies of the present research was conducted using one-factor-at-a-time
technique and the results revealed that the optimum process parameters Substrate
concentration 1000g/L, Peptone concentration 4g/L, KH2PO4 concentration 2g/L, MgSO4
concentration 0.7g/L, pH 6.0, Time 28d and Temperature 280C causes the maximum
production of kojic acid of 85.1g/L. Among these the phosphate concentration was kept
constant and the remaining five variables were screened to statistical optimization
through RSM to study the combined effects of the factors on the response. Table-5.6
depicted the experimental and predicted values of kojic acid concentrations in 32

118
experimental runs with various combinations of the factors. The highest production
obtained at Run 32 in the predicted response and the optimal conditions were Substrate
concentration 1000g/L, Peptone concentration 4g/L, pH 6.0, Time 28d and Temperature
290C and the maximum kojic acid production was 88.8 g/L.

5.4.2a Central composite design matrix:

The second order regression equation presents the concentration of kojic acid as the
function of five variables substrate concentration, peptone concentration, pH, time and
temperature which can be expressed as a coded terms in the following equation.

Y = 10904.3 + 788.5 x1 + 28.3 x2 - 36.3 x3 + 401.3 x4 – 1106.4 x5 + 2.5 x1 x2 – 0.1 x1 x3 –

0.4 x1 x4 – 1.4 x1 x5 + 0.1 x2 x3 – 0.6 x2 x4 - 0.6 x2 x5 + 0.2 x3 x4 + 0.7 x3 x5 + 1.3 x4x5 +
41.8 x12 + 9.7 x22 – 0.9 x32 + 31.6 x42 – 20.0 x52

Table- 5.8 showed the ANOVA for the response surface. The ‘F’ value for the model is
2.98 and it is greater than probability ‘p’ value 0.03 proves that the model terms are
significant. The results of multiple regression analysis in Table-5.7 reveals that the model
terms x1, x12 and x42 were significant for the production of kojic acid. The calculated
determination coefficient R2 for the kojic acid production was 84.43%. R2 was generally
used to measure the goodness-of-fit of the model. The R2 value implies that 84.43% of
the variability in the response could be explained by the model and only15.57% of the
variation was not explained. The adj R2 value was 56.13%.

5.4.2b Response surface plots and Contour plots:

The 3D graphs and contour plots describe the interaction effects occur in between the
physicochemical factors with relation to the kojic acid production. The 3D response
surface plot of kojic acid production against substrate concentration and peptone
concentration Figure 5.9 showed the kojic acid production increases with increase in the
substrate concentration and reached a maximum of 60g/L. In the mid-value region of
peptone concentration of 4g/L, Figure 5.10 represents the surface plot for the interaction

119
between pH and peptone concentration. The concentration of kojic acid increases 60g/L
at the mid-value at pH 6.0. The interaction between the substrate concentration and pH
Figure 5.11 depicted that while the mid-value of substrate continue to give maximum
production, high pH 6.0 was required to produce maximum yield 60g/L. The
concentration of kojic acid reached to maximum 80g/L at the mid-value of substrate
concentration with increase in incubation temperature 280C Figure 5.12. The interaction
effect between the other factors was depicted in Figures 5.13-5.18. In order to validate
the experimental model, surface fermentation in triplicates was performed under optimal
operation conditions and kojic acid production obtained was 88.8 g/L which indicates
that 3% increase in the production yield was observed with the statistical optimization
strategy. The experimental values were compared with the actual predicted values
obtained by regression model which proved the satisfactory validity of the model. After
crystallization the fermented broth yields 24.3g/L of kojic acid crystals.

Table 5.5: Independent variables in the experimental plan

Independent Coded Levels

Variables Symbols
-1 0 +1

Substrate concentration (g) X1 90 100 110

Peptone concentration (g/L) X2 3 4 5
Incubation time (d) X3 27 28 29
pH X4 5 6 7
Temperature (0C) X5 27 28 29

120
Table 5.6: CCD matrix having real values along with the experimental and
predicted values of kojic acid concentration

Run Substrate Peptone Incubation pH Temperature Experimental Predicted

Order Conc. Conc. time values values
(g/L) (g/L)

1 90 3 27 7 27 11.69 9.293
2 110 4 28 6 28 18.42 28.544
3 90 5 29 7 27 9.58 2.703
4 100 4 28 6 28 86.75 69.052
5 100 4 28 6 28 86.75 69.052
6 100 4 28 5 28 42.06 39.448
7 110 3 29 5 29 6.38 4.381
8 110 5 27 5 29 15.15 19.786
9 100 3 28 6 28 40.5 58.082
10 100 5 28 6 28 51.64 60.604
11 110 5 27 7 27 12.94 12.407
12 90 5 27 5 27 5.16 7.812
13 100 4 28 6 28 86.75 69.052
14 110 3 27 5 27 4.91 7.272
15 100 4 28 6 28 86.75 69.052
16 110 5 29 7 29 13.06 8.167
17 90 4 28 6 28 9.49 25.912
18 100 4 28 6 27 71.06 89.121
19 110 3 29 7 27 7.65 0.483
20 110 3 27 7 29 5.53 5.117
21 90 3 27 5 29 10.16 12.931
22 100 4 29 6 28 31.5 67.002
23 90 5 29 5 29 4.47 2.761
24 100 4 28 6 28 86.75 69.052
25 90 3 29 7 29 3.52 -3.237
26 110 5 29 5 27 9.42 7.302
27 100 4 28 6 28 86.75 69.052
28 90 3 29 5 27 12.04 8.057
29 100 4 28 7 28 6.31 35.469
30 90 5 27 7 29 3.66 3.537
31 100 4 27 6 28 81.9 72.944
32 100 4 28 6 29 80.4 88.886

121
Table 5.7: Model coefficients estimated by multiple linear regression (significance of
regression coefficients)

Term Coefficient Standard error T-value P-value

coefficient
Constant 10904.3 14066.6 0.775 0.455
Substrate conc 788.5 362.6 2.175 0.052
Peptone Conc. 28.3 257.0 0.110 0.914
Incubation time -36.3 814.9 -0.045 0.965
pH 401.3 285.7 1.405 0.188
Temperature -1106.4 814.9 -1.358 0.202
Substrate conc*Substrate -41.8 14.2 -2.939 0.013
conc
Peptone*Peptone -9.7 14.2 -0.682 0.509
Incubation 0.9 14.2 0.065 0.950
time*Incubation time
pH*pH -31.6 14.2 -2.220 0.048
Temperature*Temperature 20.0 14.2 1.402 0.188
Substrate conc*Peptone 2.5 5.6 0.455 0.658
Conc.
Substrate conc*Incubation -0.1 5.6 -0.011 0.992
time
Substrate conc*pH 0.4 5.6 0.075 0.942
Substrate 1.4 5.6 0.245 0.811
conc*Temperature
Peptone Conc.*Incubation 0.1 5.6 0.026 0.980
time
Peptone Conc.*pH 0.6 5.6 0.114 0.912
Peptone 0.6 5.6 0.111 0.913
Conc.*Temperature
Incubation time*pH 0.2 5.6 0.034 0.973
Incubation -0.7 5.6 -0.124 0.904
time*Temperature
pH*Temperature -1.3 5.6 -0.232 0.821

122
Table 5.8: ANOVA for the entire quadratic model

Source DF Seq SS Adj SS Adj MS F P

Regression 20 29724.0 29724 1486.20 2.98 0.033
Linear 5 290.2 4095.1 819.01 1.64 0.228
Square 5 29249.9 29249.9 5849.98 11.74 0.000
Interaction 10 184.0 184.0 18.40 0.04 1.000
Residual Error 11 5480.4 5480.4 498.22
Lack-of-Fit 6 5480.4 5480.4 913.40 * *
Pure Error 5 0.0 0.0 0.00
Total 31 35204.4

R-Sq:84.43%, R-Sq (pred): 0.00%, R-Sq (adj): 56.13%

DF:Degree of freedom, SS: sum of squares

Figure 5.9: Response surface plot for kojic acid production versus Peptone and
Substrate concentration

Surface Plot of Result vs Peptone Conc, Substrate Conc (g/100 ml)

Hold Values
Incubation Time 28
pH 6
Temperature 28

60

Re sult
40
5
20
4
P eptone C onc
9
10 3
11
Substr ate C onc ( g/1 0 0 ml)

123
Figure 5.10: Response surface plot for kojic acid production versus pH and Peptone

Surface Plot of Result vs pH, Peptone Conc

Hold Values
Substrate C onc (g/100 ml) 10
Incubation Time 28
Temperature 28

60
Result
40
7

20 6 pH
3
4 5
5
P eptone C onc

Figure 5.11: Response surface plot for kojic acid production versus pH and
Substrate concentration

Surface Plot of Result vs pH, Substrate Conc (g/100 ml)

Hold Values
Peptone C onc 4
Incubation Time 28
Temperature 28

60

40
Result
20
7
0
6 pH
9
10 5
11
Substr ate C onc ( g/1 0 0 ml)

124
Figure 5.12: Response surface plot for kojic acid production versus Incubation time
and Substrate concentration

Surface Plot of Result vs Incubation Time, Substrate Conc (g/100 ml)

Hold Values
Peptone C onc 4
pH 6
Temperature 28

80

60
Result

40 29

20 28
Incubation T ime
9
10 27
11
Substr ate C onc ( g/1 0 0 ml)

Figure 5.13: Response surface plot for kojic acid production versus Temperature
and pH

Surface Plot of Result vs Temperature, pH

Hold Values
Substrate Conc (g/100 ml) 10
Peptone Conc 4
Incubation Time 28

80
Result
60
29
40
28
T emper atur e
5
6 27
pH 7

125
Figure 5.14: Response surface plot for kojic acid production versus Temperature
and Incubation time

Surface Plot of Result vs Temperature, Incubation Time

Hold Values
Substrate C onc (g/100 ml) 10
Peptone C onc 4
pH 6

90

Result
80
29
70
28
T emper atur e
27
28 27
29
Incubation T ime

Figure 5.15: Response surface plot for kojic acid production versus pH and
Incubation time

Surface Plot of Result vs pH, Incubation Time

Hold Values
Substrate C onc (g/100 ml) 10
Peptone Conc 4
Temperature 28

70

60
Result
50
7
40
6 pH
27
28 5
29
Incubation T ime

126
Figure 5.16: Response surface plot for kojic acid production versus Temperature
and Peptone

Surface Plot of Result vs Temperature, Peptone Conc

Hold Values
Substrate C onc (g/100 ml) 10
Incubation Time 28
pH 6

90

80
Result
70
29
60
28
T emper atur e
3
4 27
5
P eptone Conc

Figure 5.17: Response surface plot for kojic acid production versus Incubation time
and Peptone concentration

Surface Plot of Result vs Incubation Time, Peptone Conc

Hold Values
Substrate C onc (g/100 ml) 10
pH 6
Temperature 28

70
Result 65

60 29

55 28
Incubation T ime
3
4 27
5
P eptone C onc

127
Figure 5.18: Response surface plot for kojic acid production versus Temperature
and Substrate concentration

Surface Plot of Result vs Temperature, Substrate Conc (g/100 ml)

Hold Values
Peptone C onc 4
Incubation Time 28
pH 6

80

Result 60

40 29

20 28
T emper atur e
9
10 27
11
Substr ate C onc ( g/1 0 0 ml)

128
5.5 Response surface methodology for the optimized carbon source Sago starch

The following steps were involved in the potent statistical optimization of kojic acid
production by A.flavus using sago starch as carbon source;

1. Screening of process parameters through OFAT strategy which influence the kojic acid
production

2. Optimization of most significant factors or variables by using CCD matrix

3. The developed model was validated underneath the optimized conditions.

Though there was an available literature in using sago starch as a carbon source
Rosfarizan et al. (2002) the present research differs in using sago starch hydrolysate as a
carbon source instead of using sago starch directly as a substrate and optimization of
process conditions was done with a combinational non-statistical and statistical approach.

5.5.1 MATERIALS AND METHODS:

5.5.1a Kojic acid production:

Kojic acid fermentation was done with the production medium containing 50ml of starch
hydrolysate, Peptone 0.5g, KH2PO4 0.5g and MgSO4.7H2O 0.25g was added to 250ml
conical flask. Five ml of spore suspension of A.flavus was added and the flask was
incubated at 250C for 12d. Starch hydrolysate was prepared from 10g of starch powder
dissolved in 100ml of sodium phosphate buffer and hydrolyzed with α-amylase enzyme
with enzyme activity 9.0KNU/100g. When fermentation was finished, filtration was done
and the mycelia dry weight was estimated. The supernatant was subjected to Bentley’s
colorimetric method Bentley (1957) and crystallization Hazza (2013). At the end, weight
of the crystals was determined.

5.5.1b Optimization of parameters by OFAT strategy:

For optimization with OFAT method the following physico-chemical parameters which
effect the kojic acid production were examined. The parameters include Substrate

129
concentration (100ml – 1000ml starch hydrolysate), Peptone concentration (1 – 5g/L),
KH2PO4 (0.5 – 2.5g/L), MgSO4 concentration (0.1 – 0.9g/L), pH (4 – 8), Time (11 – 37d)
and Temperature (20 – 350C). Among these, the most significant factors which influence
the production were further optimized by central composite design and RSM analysis.

5.5.1c Optimization by RSM:

CCD and RSM was used to optimize the 5 important variables for increasing the kojic
acid production rate which was examined at 3 different coded levels Table-5.9 and 32
experimental runs were performed Table-5.10. By using second order polynomial
equation (Equation 1), the effect of individual variable, their interactions and statistical
analysis to calculate predicted responses were interpreted.

Y = b o + ∑ b i Xi + ∑ b i 2 Xi 2 + ∑ b ij Xi Xj - (1)

Y is the concentration of kojic acid (g/L)

bo is the intercept
bi is the coefficient for linear direct effect
bi 2 is the coefficient for quadratic effect and is responsible for curvatures of the model
bij is the coefficient for interaction effect a positive (or) negative significant value
indicates possible interactions between the variables.
For validation of the experimental model, the fermentation was carried out at the
optimized conditions, for verifying the results from the response surface.

5.5.2 RESULTS AND DISCUSSION:

A group of experiments were performed serially to study the physical factors pH, Time,
Temperature and chemical factors like Carbon source concentration, Nitrogen source
concentration, KH2PO4 and MgSO4 concentration on kojic acid production by A.flavus
with OFAT experiments. It was observed that the production was higher at carbon source
concentration 1000ml (71.2g/L of starch hydrolysate), peptone concentration 4g/L
(82.6g/L), KH2PO4 1g/L (4.62g/L), MgSO4 concentration 0.5g/L (12.05g/L), pH 6.0
(50.6g/L), Time 28d (66.8g/L) and Temperature 280C (52.9g/L). The kojic acid yield

130
78.9g/L was obtained in a conical flask under these optimized conditions. From the
OFAT results it was found out that 5 variables Carbon source concentration, Peptone
concentration, pH, Time, Temperature play a significant role in the kojic acid production.
Hence these variables were chosen for further optimization studies by RSM using 5-
factor-3-level-CCD. Hence a set of 32 experiments were done with different
combinations of 5 variables Table-5.10. The highest production 90.8g/L was observed at
Run 2. The predicted response was 82.142g/L.

5.5.2a Central composite design matrix:

A second-order polynomial function was fitted to the experimental kojic acid production
may results in the generation of the below regression equation in terms of actual factors.

Y = -1469.08 + 97.29 x1 + 211.10 x2 -357.31 x3 +220.47x4 +354.73 x5+0.85x1 x2+1.73x1

x3 + 2.82 x1 x4 + 0.80x1 x5 +0.50x2 x3 - 1.63 x2 x4 +1.53x2 x5 +0.28 x3 x4-0.02x3 x5 -3.65x4
x5 + 0.33 x12 + 25.83x22-6.62x32 +26.38x42+6.48x52

The F-value of the model was 10.87 and the probability value (P ~ 0) indicates the
significant nature of the model. Subsequent ANOVA analysis and generation of
regression equation implies that the R2 value of 95.18% (R2adj: 86.43%) assured an
adequate adjustment of the quadratic model to the experimental data Table-5.12. The
Lack of fit F-value was not evident and the adequate precision value was 10.0 specifies
that an adequate signal to noise ratio. As the precision value is greater than 4.0 so the
model could be used to navigate the design space. The estimated regression coefficients
for the model terms were represented in Table-5.11 which revealed that, two interaction
or cross product terms (Peptone concentration*Peptone concentration and pH*pH)
showed significant effect on kojic acid production (p<0.05).

5.5.2b Response surface plots and Contour plots:

3D response surface plots aided in recognizing the main and the interaction effects of five
factors. The plots were illustrated with pair wise combinations of five different variables,

131
 whereas the rest were held at middle level. Figure 5.19 shows the kojic acid production as
a result of interaction between Temperature and pH with substrate concentration
10g/100ml, peptone concentration 4g/L and incubation time 28d respectively. The
production was increased with increase in pH to 5.0 and temperature to 280C and later
decreased. The similar fashion was also exhibited in Figure 5.20, when peptone
concentration increases to 4g/L and Temperature to 280C the production enhances
significantly. Figure 5.21 showed the interaction between between pH and peptone
concentration. The kojic acid production reached to maximum 80g/L at the mid-value of
peptone concentration 4g/L with increase in pH 6.0. Figures 5.22-5.27 represents the
interactions among the other factors.

A verification experiment in triplicates was done to validate the statistical results

using predicted optimal medium in conical flasks under static conditions. The
experimental yield 90.8g/L was in reasonable agreement with the predicted yield
82.14g/L. Finally from the results of combinational approach it was assessed that, the
optimized medium which can yield maximum production was Substrate concentration
110ml, Peptone concentration 4g/L, KH2PO4 1g/L, MgSO4 concentration 0.5g/L, pH 6.0,
Time 28d and Temperature 280C. After crystallization of fermented broth 24.91g/L of dry
crystals were produced. Response surface statistical analysis used in the current research
proved that, optimization of kojic acid production by A.flavus resulted in the significant
enhancement of yield by 12% higher than the result obtained by one-factor-at-a-time
traditional method.

Table-5.9: Independent variables in the experimental plan

Independent Coded Levels

Variables Symbols
-1 0 +1

Substrate concentration (ml) X1 90 100 110

Peptone concentration (g/L) X2 3 4 5
Incubation time (d) X3 27 28 29
pH X4 5 6 7
Temperature (0C) X5 27 28 29

132
Table-5.10: CCD matrix having real values along with the experimental and
predicted values of kojic acid concentration

Run Substrate Peptone Incubation Temp. Experimental Predicted

order Conc. Conc. Time pH values values

(g/L) (g/L)

1 90 3 27 7 27 11.58 8.969
2 110 4 28 6 28 90.8 82.142
3 90 5 29 7 27 9.61 6.759
4 100 4 28 6 28 82.6 76.073
5 100 4 28 6 28 82.6 76.073
6 100 4 28 5 28 60.5 57.552
7 110 3 29 5 29 41.2 43.176
8 110 5 27 5 29 28.3 29.338
9 100 3 28 6 28 53.7 56.240
10 100 5 28 6 28 37 44.251
11 110 5 27 7 27 14.3 14.488
12 90 5 27 5 27 16.49 15.251
13 100 4 28 6 28 82.6 76.073
14 110 3 27 5 27 49.2 51.938
15 100 4 28 6 28 82.6 76.073
16 110 5 29 7 29 17.9 17.326
17 90 4 28 6 28 50.9 69.349
18 100 4 28 6 27 68.7 69.151
19 110 3 29 7 27 15.6 16.726
20 110 3 27 7 29 29.4 29.066
21 90 3 27 5 29 30.1 28.339
22 10 4 29 6 28 79.8 83.299
23 90 5 29 5 29 14.5 12.499
24 100 4 28 6 28 82.6 76.073
25 90 3 29 7 29 29.4 26.027
26 110 5 29 5 27 36.1 38.598
27 100 4 28 6 28 82.6 76.073
28 90 3 29 5 27 33.5 33.199
29 100 4 28 7 28 29.1 41.839
30 90 5 27 7 29 11.58 7.269
31 100 4 27 6 28 75.8 82.092
32 100 4 28 6 29 60.7 70.040

133
Table-5.11: Model coefficients estimated by multiple linear regression (significance
of regression coefficients)

Term Coefficient Standard error T-value P-value

coefficient

Constant -1469.08 6342.88 -0.232 0.821

Substrate Conc. (ml) 97.29 163.49 0.595 0.564
Peptone Conc. (g/L) 211.10 115.88 1.822 0.096
Incubation time (d) -357.31 367.46 -0.972 0.352
pH 220.47 128.82 1.711 0.115
0
Temperature ( C) 354.73 367.46 0.965 0.355
Substrate conc*Substrate -0.33 6.42 -0.051 0.960
conc
Peptone*Peptone -25.83 6.42 -4.025 0.002
Incubation 6.62 6.42 1.032 0.324
time*Incubation time
pH*pH -26.38 6.42 -4.111 0.002
Temperature*Temperature -6.48 6.42 -1.010 0.334
Substrate conc*Peptone 0.85 2.52 0.338 0.742
Conc.
Substrate conc*Incubation -1.73 2.52 -0.687 0.506
time
Substrate conc*pH -2.82 2.52 -1.122 0.286
Substrate -0.80 2.52 -0.318 0.756
conc*Temperature
Peptone Conc.*Incubation 0.50 2.52 0.199 0.846
time
Peptone Conc.*pH 1.63 2.52 0.646 0.531
Peptone -1.53 2.52 -0.607 0.556
Conc.*Temperature
Incubation time*pH 0.28 2.52 0.110 0.914
Incubation 0.02 2.52 0.009 0.993
time*Temperature
pH*Temperature 3.65 2.52 1.450 0.175

134
Table-5.12:
.12: ANOVA for the entire quadratic model

Source DF Seq SS Adj SS Adj MS F P

Regression 20 22020.6 22020.6 1101.0 10.87 0.000

Linear 5 2504.5 979.7 195.94 1.93 0.168
Square 5 19021.0 19021.0 3804.20 37.550 0.00
Interaction 10 495.1 495.1 49.51 0.49 0.865
Residual Error 11 1114.3 1114.3 101.30
Lack-of-Fit 6 1114.3 1114.3 185.72 * *
Pure Error 5 0.0 0.0 0.00
Total 31 23134.9

R-Sq:95.18%, R-Sq
Sq (pred): 0.00%, R-Sq
R (adj): 86.43%
DF:Degree
Degree of freedom, SS: sum of squares

Figure-5.19:
.19: Response surface plot for kojic acid production versus Temperature
and pH

135
Figure-5.20:
.20: Response surface plot for kojic acid production versus Temperature
and Peptone concentration

Figure-5.21:
.21: Response surface plot for kojic acid production versus pH and Peptone
concentration

136
Figure-5.22:
.22: Response surface plot for kojic acid production versus Temperature
and Incubation time

Figure-5.23:
.23: Response surface plot for kojic acid production versus pH and
Incubation Time

137
Figure-5.24:
.24: Response surface plot for kojic acid production versus Temperature
and Substrate concentration

Figure-5.25:
.25: Response surface plot for kojic acid
acid production versus pH and
Substrate concentration

138
Figure-5.26: Response surface plot for kojic acid production versus Incubation
Time and Substrate concentration

Figure-5.27: Response surface plot for kojic acid production versus Peptone
concentration and Substrate concentration

139
Figure 5.28: Kojic acid yields after RSM optimized parameters

Kojic acid yields after optimization

100
90
Conc. of kojic acid (g/L) and

80
kojic acid crystals (g/L)

70
60
50 Conc. of kojic acid (g/L)
40
30 Conc. of kojic acid crystals
20 (g/L)
10
0
Palmyra sap M.calabura Sago starch

Carbon sources

140
5.6 CONCLUSION:

From the OFAT and RSM results it was concluded that, the fungal organism A.flavus
produce varied kojic acid amounts from the 12 carbon substrates. Among them maximum
kojic acid yield 90.8g/L was obtained with Sago starch with static fermentation. The
result was similar to Yan et al. (2014) who reported that, the highest percentage of kojic
acid 90.8% when the fermentation medium containing glucose and xylose as carbon
source with A.oryzae M866 strain. The study reported higher yields of kojic acid while
comparing to May et al. (1931) reported 57g/L from glucose under static conditions. Wei
et al. (1991) reported 60g/L from YES medium. Kojic acid yield 21.4g/L reported by El-
kady et al. (2014) with static fermentation using agro wastes. After crystallization the
fermented broth from sago starch was crystallized to give 24.91g/L of kojic acid crystals
(Figure 5.28). A high concentration of kojic acid in the broth causes kojic acid to
crystallize in to fine needles Rosfarizan et al. (2010), Kwak and Rhee (1992). Morton et
al. (1945) reported results in terms of grams of kojic acid crystals. Fifty grams of absolute
kojic acid crystals were obtained from 1L culture filtrate. The mother liquor still contains
20-50 g of kojic acid. The fermentation medium contains glucose as carbon source. The
yield was higher with the results of the current research and it is found that maximum
amount of kojic acid crystals 24.91g was obtained with Sago starch. Comparitively with
the glucose the sago starch was the cheaper carbon source to produce a value-added
product like kojic acid which may ultimately leads to considerable decrease in the
production economics.

141
 CHAPTER – VI
EFFECT OF ENHANCERS ON
KOJIC ACID PRODUCTION
 CHAPTER 6

6. EFFECT OF ENHANCERS ON KOJIC ACID

PRODUCTION

6.1 Introduction:

Different types of enhancers were generally used to increase the yields of kojic acid by
the fungal cultures and they include Copper-monovales-nicotinic acid complex and
Copper-monovales-riboflavin complex Megalla et al. (1987), Cycasin or Methyl-
azoxymethyl-β-D-glucose Tadera et al. (1985), Methanol Madihah et al. (1996). Addition
of Cu (I)-B3 complex to the fermentation medium enhances the yield by 47% with the
fungus A.flavus Megalla et al. (1987). The complex was utilized by the fungus
biochemically in a manner related to that of niacin utilized naturally. According to Bajpai
et al. (1981) the enzymes glucose dehydrogenase and gluconate dehydrogenase were
involved in the kojic acid biosynthesis were NAD and NADP dependent produced from
nicotinic acid or niacin. The Cu (I)-B2 complex enhances the kojic acid yield at
concentration of 75µg/100ml of fermentation medium. This chelating complex is a heavy
metal derivative and can be precipitated in certain enzymatic reactions. It shows
biochemical effects in the biochemical pathway of kojic acid. Cycasin was produced by
Japanese Cycad plant Cycas revoluta is a toxic metabolite has carcinogenic and
neurotoxic property stimulates the production of kojic acid by kojic acid producers. It
decreases the activity of Triose phosphate isomerase and stimulates the production. It
also suppresses the growth of the fungus and elevates the pH of the culture medium.
0.2% of cycasin inhibits the spore formation of A.oryzae and enhances the yield to 6-fold.
Hence the molds were able to produce greater yields of kojic acid on cycasin treatment.
When methanol was added to the culture it will reduce the bubble size especially in
stirred tank fermentors and increases the aeration rate. This may lead to increase in the
production rate of the compound.

142
6.2 OBJECTIVES:

To enhance the yields of kojic acid from 12 different carbon sources using
Methanol and two different copper-vitamin coordination complexes

6.3 MATERIALS AND METHODS:

6.3.1Fermentation media and Fermentation conditions:

Fermentation was performed with 12 carbon sources with their optimized production
media compositions and fermentation conditions obtained after optimization experiments.

6.3.2 Addition of enhancers:

In the current research, three different enhancers were tested for the enhanced production
of kojic acid. Methanol at a concentration of 4% v/v, Cu (I)-B2 complex and Cu (I)-B3
complex at a concentration of 0.1mg/ml were used. The samples were examined for the
enhanced production after 24h of addition of enhancer. The concentration of kojic acid
was determined by Bentley’s colorimetric method and the resulted broth samples were
subjected to crystallization and purification processes. The results were expressed in g/L
of dry crystals for each and every carbon source.

6.4 RESULTS AND DISCUSSION:

Table 6.1 depicted the yields of kojic acid crystals obtained in response to the addition of
3 different enhancers. The results were also compared to yields obtained without the
addition of enhancers. The cultures showed enhanced production to methanol rather than
the copper complexes. With methanol the yield was increased to 1-12% where as for the
copper complexes the yield was increased up to 1-3%. However Megalla et al. (1987)
reported the yield was enhanced by 47%. Aspergillus flavus when copper-monovalent-
nicotinic acid complex were incorporated to a synthetic medium unique for kojic acid
production. Carriers like NAD and NADP are forecasted. According to this prophecy, the
model proposed by Bajpai et.al (1981) is the basis to explain the biosynthetic pathway of
kojic acid. NAD and NADP dependent enzymes will take part in this model. In this

143
report, particularly the production rate was increased in starch substrates followed by
bran substrates in response to enhancers than other substrates used. The maximum yield
was showed by the substrate Sago starch 28.5g/L of kojic acid crystals with methanol
followed by M.calabura fruits 24.71g/L and Palmyra sap 22.83g/L.

Morton et al. (1945) extracted 50g/L of kojic acid crystals with Aspergillus luteo
virescens from glucose supplemented medium. Lin et al. (1976) used A.parasiticus
UNBF A12 strain for the production of kojic acid crystals with YES medium and
concluded that 16.6g of kojic acid crystals were precipitated out for 1L broth. The other
two isolated cultures A.parasiticus NRRL 2999 and A.flavus NRRL 3251 were unable to
produce kojic acid crystals. The investigative report of Kwak and Rhee (1992) had
revealed that, highest accumulation of kojic acid crystals 83g/L was takesplace in glucose
medium by A.oryzae. Saleh et al. (2011) isolated 3-5g/L of crystals from Trichoderma
spp by using sucrose as the carbon source. Hazzaa et al. (2013) had concluded that, 39g/L
of kojic acid crystals was obtained in a solid glucose salt medium with the fermenting
microorganism Aspergillus oryzae var.effusus NRC14. Hassan et al. (2014) reported
49.5g/L of kojic acid crystals by using glucose salt medium from the culture Aspergillus
oryzae var.effusus NRC14 at different process conditions of Hazzaa et al. (2013).

From these findings, it was observed that, the yield obtained in the current study 28.5g/L
was higher than the yields reported by Lin et al. (1976), Saleh et al. (2011) and lower
than the yields of Morton et al. (1945), Kwak and Rhee (1992), Hazzaa et al. (2013) and
Hassan et al. (2014).

144
Table 6.1: Effect of enhancers on kojic acid production

Substrates Before Addition of Addition of Addition of Cu-

addition of methanol Cu-Vitamin Vitamin
enhancer B2complex B3complex
M.calabura 24.3 24.71 24.4 24.52
Palmyra sap 21.94 22.83 22.05 22
Sago starch 24.91 28.5 23.6 23.82
Cassava 17.94 20.3 24.14 23.87
Coconut water 16.8 17.9 17.1 17.05
Ipomea 16 19.3 17.53 17
Cashew apple 15.9 16.87 16.2 16.39
Paneer whey 14.2 14.5 14.31 14.35
A.macrorrhiza 12.9 16.28 14 14.62
Rice bran 9.62 14.9 10.9 11.01
Sugarcane 3.95 4.85 4.01 4.16
baggase
Wheat bran 0.04 2.06 1.72 1.35

6.5 CONCLUSION:

Among the 3 different enhancers used maximum enhancement of yield was obtained with
methanol. The yield was increased by 1-12% with all the carbon sources.

145
 CHAPTER – VII
ISOLATION, PURIFICATION AND
STRUCTURAL ELUCIDATION
OF KOJIC ACID
 CHAPTER 7

7. ISOLATION, PURIFICATION AND STRUCTURAL

ELUCIDATION OF KOJIC ACID

7.1 INTRODUCTION:

After the fermentation was completed, the broth samples were subjected to Gel filtration,
evaporation followed by crystallization and purification. The structure of kojic acid was
elucidated by using different biophysical analytical techniques like Fourier transform
infrared spectroscopy (FTIR), Proton nuclear magnetic resonance spectroscopy (1H-
NMR) and X-ray diffraction spectroscopy. High pressure liquid chromatography (HPLC)
was used to characterize the purity of the compound Upadhyay and Upadhyay (2009),
Stryer (2002). These methods were done in the Dept. of Chemistry, Dept. of Pharmacy,
GITAM University and Eisai Pharmaceutical Company, Visakhapatnam.

7.2 OBJECTIVES:

Isolation and purification of kojic acid crystals from the fermented broth using
solvent extraction methods, gel chromatography etc.

To check the purity and mass of the isolated kojic acid compound with HPLC and
LC/MS.

To elucidate the structure of kojic acid through biophysical analytical methods
like XRD, FTIR and Proton NMR.

7.3 MATERIALS AND METHODS:

7.3.1 Isolation and purification of kojic acid:

Various types of polar compounds like ethyl acetate, chloroform etc., have been used for
the kojic acid extraction from the fermented liquid. While comparing with water, the

146
solubility of kojic acid takes place highly in ethyl acetate. There it forms a separate layer
with the solvent used initially so that it can be easily separated through the opening of the
stopcock funnel. Hazzaa et al. (2013) and Chaves et al. (2012) have used ethyl acetate for
purification of kojic acid. Parrish et al. (1966) and Hazzaa et al. (2013) used chloroform
for the separation of kojic acid from the fermented broth.

7.3.1a Solvent extraction:

In the present work, the fermented broth samples were filtered and the mycelia mat was
separated, dried in a hot air oven at 1600C for 6-7h, weighed and then measured for each
flask. Charcoal treatment was done to remove coloured substances from the supernatant.
Initially 10ml of ethyl acetate was added to the supernatant to 100ml of fermented broth.
The suspension was subjected to agitation at 100rpm. Inorder to remove solvents from
the concentrated kojic acid, the suspension was evaporated at 500C for 10h.

7.3.1b Gel-chromatography:
Gelfiltration was used to separate the specific biomolecule of interest from other
compounds in a biological extract. The gel chromatography works on the basis of
partition coefficient or distribution coefficient (kd) which explains the way through which
the molecule distributes itself between the two immiscible phases. The molecules were
separated based on their sizes and shapes. The chromatographic column (90x1.6cm i.d)
bottom was covered with glass wool and the column was kept in an up-right position to a
stand. Later it was loaded with stationary phase sephacryl S-200 media equilibrated with
20mM Tris-HCl buffer (pH 7.2). The gel settles down to a specific height by gravitational
force. The sample was applied through buffer pipeline in column buffer on the top of bed.
The bufferline was connected to elution buffer for the development of chromatogram.
The resultant effluent was monitored at 280nm in a spectrophotometer. The effluent was
collected in a fraction collector. The elution volume and the retention time were
determined. The elution was continued 2-3 times until the O.D reached baseline value.
Then the column was eluted with the same buffer at a flow rate of 60 ml/h and 3.0 ml
fractions were collected and the kojic acid concentration was determined in each fraction

147
and active fractions were pooled for further HPLC analysis to check the purity of the
compound.

7.3.1c Crystallization:
The collected fractions were kept in a refrigerator at 50C for 24 h. Upon evaporation, the
extractant yields kojic acid crystals in the form of needles. The crystals were collected
and dried at 800C for 8 h. For purification of the crystals, they were repeatedly washed
with a mixture of water and acetone. Later the dry weight of the crystals from each
sample was determined Hazzaa et al. (2013).

7.3.2 High pressure liquid chromatography (HPLC)

The system used intensely high pressure up to 8000Psi, then the flow rate was high and
the experimental time was decreased consequently. The supporting particles were
incredibly small and more or less uniform in size. The lateral diffusion was less due to
two factors, the smaller particle size and huge pressure, which decreases the time that a
solute spends in the column. As a result, band broadening was decreased. It has a fast
speed of resolution. Pico or femto gram levels of samples were analyzed.

7.3.2a Instrumentation and sample analysis:

The major components involved in HPLC are: A solvent reservoir contain mobile phase,
High pressure pump to push the mobile phase through the column, Injector to inject the
sample into the mobile phase, Column in which the separation will takes place, Detector
used to detect the concentration of the sample components as they come out of the
column and Recorder to generate chromatogram. 20 µl of test sample with a dilution of
50 mg/ml was prepared and injected. Standard sample was dissolved in a concentration of
10 mg/ml and injected (Table 7.1).

148
Table 7.1: Analytical conditions of HPLC

System Shimadzu prominence (Model LC-20 AD)

Mobile phase Water
Detector Refractive Index Detector (RID- 10 A)
Flow rate 0.3 ml/min
Column Varian MetaCarb 87P (300 mm × 7.8 mm)
Run time 6 min
Oven temperature 600C

7.3.3 X-ray crystallography or X-ray diffractometry (XRD):

It is a versatile analytical technique used to identify different crystalline forms or phases

of compounds. In XRD, the X-rays irradiated on the sample set on the axis of the
spectrometer or Goniometer was diffracted by the sample. The alterations in the
diffracted X-ray intensities were computated, traced and plotted against the rotation
angles of the sample. The result obtained was X-ray diffraction pattern of the sample. The
Goniometer unit permits analysis of samples present in different states. The drive
mechanism characteristics an independent dual axis θ-2θ linkage drive, and independent
2θ and θ axis drives, freely selectable for competent thin film and a variety of types of
analysis. Counter monochromator convert X-rays which have passed through the
entrance slit into monochromatic X-rays, allowing only the characteristic X-rays (kα
radiations) to be detected.

7.3.3a Instrumentation and sample analysis:

In the present research, Xpertpro powder XRD (Plate 6.4) was employed to found out the
structure of kojic acid crystal. The instrument was provided with CRISP technology,
3Kw generator, pneumatic shutters and beam attenuators. Goniometer utilizes the Direct
Optical Positioning (DOPS) system and X’celerator detector and also bears
interchangeable X-ray modules. The instrument also have three sample stages, standard
4" wafer mount, solid sample holder, incident beam optics, Anton paar DHS 900 domed
hot stage for data collection. After inserting the sample, intially X-rays were emerged

149
from X-ray copper tube and passed into soller slit then divergence slit to sample and get
diffracted by the sample and then enter into anti-scatter slit, soller slit to beta filter,
detector and finally to data processing software which displays the diffractogram of the
sample (Table 7.2).

Table 7.2: Analytical conditions of XRD

Model Panalytical Xpertpro powder XRD

Source of radiation Cukα radiation
Spectrometer Goniometer with DOPS system
Detector X’celerator with 128 channels of 70µm
Sample holder Glass sample holder (25x1mm dia mt.)
X-ray tube
Focus size 1x10mm
Tube voltage current 45Kwx40mA
Target Cu 2Kw
X-ray filter Ni filter
Minimum step angle 0.0020 (2θ), 0.0010(θ)
Scanning angle range -60 to 1630 (2θ)
-180 to 1800 (θ)

7.3.4 Agilent 6410 Triple Quadrupole Liquid chromatography-mass spectroscopy

(Agilent 6410 QQQ LC/MS):
In the mass spectra the preferred molecule produces a molecular ion peak situated at m/z
parallel to its molecular mass. Agilent 6410 Triple Quadrupole Liquid chromatography-
mass spectroscopy (Agilent 6410 QQQ LC/MS) was used because it analyzes the target
compounds in complex matrices. Agilent 6410 QQQLC/MS employed advanced
nebulization technology and contain three consecutive quadrupoles arranged in series to
incoming ions.

150
7.3.4a Instrumentation and sample analysis:

In the present study, ESI was used which generates the ions in solution prior to the
analyte reaches the mass spectrometer. In the presence of a strong electrical field and
heated drying gas, the test sample was nebulized into a chamber at atmospheric pressure.
The electrostatic field causes more dissociation of the analyte molecules and the heated
drying gas causes the solvent in the droplets to evaporate. The shrinkage of droplets
causes the increase in charge concentration in the droplets. Later the repulsive force
among the ions with like charges exceeds the cohesive forces and ions are ejected into the
gas phase. These ions were attracted to and pass through a capillary sampling orifice into
the mass analyzer. The m/z range for a typical LC/MS is around 3000m/z. Applying
voltage to the rods produce electromagnetic fields. These fields determine which m/z
ratio of ions can pass through a filter at a known time. In the present investigation
electrospray mass spectroscopy was used to determine the molecular weight (Table 7.3).

Table 7.3: Analytical conditions of LC/MS

Model Agilent 6410 Triple QQQ LC/MS

spectrometer
(Agilent technologies, Santa clara, USA)
Derivatization PVCF
Ionization ESI
Injection volume 15
Vial position 31
Fragmentor voltage 135
Collision energy 0
ESI Scan 0.170-0.273min
Number of scans 15

7.3.5 Fourier transform infrared spectroscopy (FTIR)

For the recognition of functional groups in a finished product FTIR is used widely. In
order to get the infrared spectrum of a sample by this method, primarily interferogram of

151
a sample signal was collected from the interferometer, and later FT was carried out on the
interferogram to obtain the spectrum. The function of FTIR spectrometer was to gather
and digitalizes the interferogram, doing the FT function, and displays the spectrum.

7.3.5a Instrumentation and sample analysis:

The FTIR spectrometer has an infrared light as the light source, interferometer, sample
chamber, detector, electrical system circuits and a computer. The electronic system was
supported on Motorola 68340 integrated processor. High signal to noise ratio helps FTIR
for more useful for difficult samples.The sample was prepared according to potassium
bromide pellet method (KBR pellet method). One milligram of kojic acid crystals were
weighed and added to 100mg of KBR pellets in the motor. Now the mixture was grinded
to a fine mass in the micro scale motor with a pestle for 2min.

An ample quantity of the mixture was put into a 2 piece open metal chamber and the
chamber bottom surface was covered properly. The lid was kept on the chamber
clutching the finely crushed mixture. Later this 3 piece metal set was put inside the qwik
Handi press and press hard and then released the press. The 3 piece metal set taken out
from the qwik Handi press and observe the pellet appeared in the middle metal piece. It
had somewhat clear and uncracked pellet formed and screw to the pellet holder. Now IR
was performed on KBR pellet (Table 7.4).

Table 7.4: Analytical conditions of FTIR

Model Perkin-Elmer
Spectrum RX1
Source IR
Beam splitter KBR beam splitter
State solid (KBR pellet)
Alignment excitation Source HeNe laser, 633nm, 1mW
Sampling procedure Transmission
Spectral range 450-4000cm-1

152
 Resolution 1cm-1
Detector DTGS
7.3.6 Proton nuclear magnetic resonance spectroscopy (1H NMR):

In proton NMR the unknown sample to be tested was suspended in a liquid solvent and
kept under magnetic field and the absorption of radio-frequency waves by definite nuclei
like protons was determined. Protons on dissimilar atoms present in various molecular
environments absorb energy of different levels. The absorbing nucleus produce chemical
shift which was calculated in parts per million (ppm), was the spectral location of
resonance comparative to a standard signal, frequently tetramethyl silane (TMS). The
proton NMR signal was “split” by interactions with adjacent protons. This feature was
called spin-spin coupling and helps to determine the positions and numbers of equivalent
and non equivalent protons. NMR is a demonstration of nuclear spin angular momentum,
a quantum mechanical property of atomic nuclei.

7.3.6a Instrumentation and sample analysis:

Fourier transform Nuclear magnetic resonance (FT-NMR) was operated in the present
research. Small change in the magnitude of the energy was entailed in this spectroscopy
which implies that, sensitivity was the main limitation. In the spectrometer, each and
every frequency in a spectrum was irradiated concurrently with a radio frequency pulse
(RF pulse). Single RF pulse would excite all the types of hydrogen simultaneously. TMS
was used as the reference compound. Subsequently the pulse and the nuclei revisit to
thermal equilibrium. A time domain emission signal was recorded by the instrument as
the nuclei release. Fourier transformation would produce frequency domain spectrum.

Twenty milligram of the sample was measured and dissolved in 0.7ml of D2O solution in
a clean vial and swirl gently till the sample melts. The sample was now pipette out in a 5-
mm NMR tube with the help of a Pasteur pipette. It was necessary that, the tube must
contain 5cm of solvent. Now the NMR tube was enclosed with a cap. The test sample
was placed by removing the standard reference sample into the spinner and corrected its
depth by means of paper gauge on the right magnet leg. Kim wipe was used to clean the

153
lower part of the sample tube. Now the sample was injected. 10-20m was the spinning
rate. JEOL JNM EX-90FT-NMR was the model used. The frequency used was 90MHZ.
7.4 RESULTS AND DISCUSSION:

7.4.1 Gel chromatography:

Figure 7.1 represented the elution profile of kojic acid from sephacryl S-200 gel filtration
chromatography. The kojic acid was eluted in fractions 54-56 as a single peak.

Figure 7.1: Elution profile of kojic acid using sephacryl S-200

7.4.2 Crystallization:

The isolated kojic acid crystals resulted after the process of evaporation (Plate 7.1) and
purification were observed under the microscope (Plate 7.2). Very long needle shaped
crystals were obtained after purification.

154
Plate 7.1: Kojic acid crystals after evaporation

Plate 7.2: Kojic acid crystals under microscope

155
7.4.3 High pressure liquid chromatography:

From the results of HPLC (Figure 7.2) it was evaluated that, the purity of the compound
achieved was 92% where as the standard sample shows 97.2% purity (Figure 7.3). The
retention time of the test compound was 6.901 min where as for standard it was 6.621

Figure 7.2: Elution profile of test kojic acid sample by HPLC

Figure 7.3: Elution profile of standard kojic acid sample by HPLC

156
7.4.4 X-ray crystallography:

The X-ray diffraction spectrum of test kojic acid sample showed seven characteristic
peaks at 2θ angles of 90, 220, 260, 310, 330,380 and 420 (Figure 7.4). The X-ray diffraction
spectrum of the standard kojic acid sample showed (Figure 7.5) seven distinctive peaks
were appeared at 2θ angles 90, 220, 260, 310, 380, 420.

Figure 7.4: X-ray diffractogram of test kojic acid sample

157
Figure 7.5: X-ray diffractogram of standard kojic acid sample

7.4.5 Liquid chromatography mass spectroscopy:

In the resultant mass spectra of kojic acid crystals, the molecular ion peak was located at
142.07 (Figure 7.6), which confirmed the presence of kojic acid because the molecular
weight was similar to standard kojic acid (kojic acid mass spectrum, Mass bank record
JP003237). More peaks were visible larger than the molecular ion peak due to solvent
used or impurity or an isotope of the molecular ion or may be due to the presence of
more than one carbon atom in a compound. However peaks with mass less than the
molecular ion were the result of fragmentation of the molecule.

158
Figure 7.6: Mass spectrum of kojic acid

7.4.6 Fourier transform infrared spectroscopy:

The FTIR spectrum of test kojic acid sample obtained after purification (Figure 7.7)
showed the peak wave number values for the functional groups similar to that of standard
kojic acid. The bands appear for functional groups at 3270.8 cm-1, 3179.43 cm-1 (- OH),
2925.17 cm-1, 2854.05 cm-1 (aliphatic - CH), 1660.59 cm-1 (cyclic -C=O), 1611.11 cm-1
(C = C), 1472.61 cm-1 (deformation of -CH2), 1074.04 cm-1 (cyclic C-O-C), 943.58 cm-1,
863.66 cm-1 and 775.65 cm-1 (1, 4 α -disubstituted ring). Where as the standard sample
showed peaks at 3271 cm-1, 3178 cm-1 (- OH), 2926 cm-1, 2866 cm-1 (aliphatic - CH),
1661 cm-1 (cyclic -C=O), 1611.11 cm-1 (C = C), 1474 cm-1 (deformation of -CH2), 1076
cm-1 (cyclic C-O-C), 944 cm-1, 866 cm-1 and 778 cm-1 (1, 4 α -disubstituted ring) (Figure
7.8).

159
Figure 7.7: FTIR spectrum of Kojic acid test sample

Figure 7.8: FTIR spectrum of kojic acid standard sample

160
7.4.7 Proton NMR:

The1H NMR of kojic acid crystals showed (Figure 7.9)) 4 characteristic protonic signals
of varying size intensity and few smaller signals. The four peaks ob
obtained at 9.03 (s, 1H),
6.332 (s, 1H), 4.286 (s, 2H), 3.340 (s, OH). The result was simila
similar to standard sample
(Figure 7.10).. Peaks appeared at 9.03 (s, 1H), 6.24 (s, 1H), 4.16 (s, 2H), 3.65 (s, OH).

Figure 7.9:: Proton NMR of test kojic acid sample

161
 Figure 7.10: Proton NMR of standard kojic acid sample

7.5 CONCLUSION:

Based on these observations, the functional groups –OH, C=O, C=C, -CH, C-O-C and –
CH2 were identified by FTIR and four protonic groups were identified by proton NMR
and the entire structure of kojic acid crystalline phases were observed by XRD and
molecular weight 142.07 with LC/MS. This confirms the presence of kojic acid and its
purity was 92% by HPLC.

162
 CHAPTER – VIII
INVESTIGATIONS FOR THE
BIOACTIVE PROPERTIES OF
KOJIC ACID
 CHAPTER – VIII
INVESTIGATIONS FOR THE
BIOACTIVE PROPERTIES OF
KOJIC ACID
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
 CHAPTER – VIII
INVESTIGATIONS FOR THE
BIOACTIVE PROPERTIES OF
KOJIC ACID
 CHAPTER – VIII
INVESTIGATIONS FOR THE
BIOACTIVE PROPERTIES OF
KOJIC ACID
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
CN24, A.fumigatus AF293, MAPK mutant strains were sensitive to these co-applied anti
fungal agents. The heightened activity often involves the change in the function of fungal
anti oxidation systems. The growth of Bacillus megaterium was inhibited by kojic acid
extracted from moldy cheese Moubasher et al. (1979). It also inhibits the swarming
movements of Azospirillum and Proteus mirabilis. Some of the zinc derivatives of
Azidometal kojates showed cytotoxic effect on Hela cells Hudecova et al. (1996).

Most of the natural occurring metabolites have afforded a good source of compounds
which initiated numerous applications in cancer chemotherapy. Globally the incident rate
of cancer will increase to 50% to 15 million by 2020 (WHO, 2010). Chemotherapy is one
of the effective treatments for extending the patient’s life Sirinet (2010). More than 70%
of the available anticancerous agents are either natural products or natural product
derived substances Karikas (2010) and the therapeutic applications of microbial
metabolites afford the opportunity for the invention of anticancer agent for example kojic
acid possesses an anti-cancerous activity.

8.2 OBJECTIVES:

To determine the antimicrobial microbial property of the kojic acid against

different bacteria

To determine the antiproliferative activity of kojic acid using Breast cancer cell
lines and leukemia cell lines.

8.3 MATERIALS AND METHODS:

8.3.1 Antimicrobial activity of kojic acid:

The agar-slants of pathogenic bacteria suspensions were collected from M/s.Doctor’s

Diagnostic Centre, Gajuwaka, Visakhapatnam. Individual bacterial suspensions were
prepared using a sterile distilled water containing 108CFU/ml. By applying spread plate
technique Dubey and Maheshwari (2003), 0.5ml of bacterial inoculum was spread
uniformly on a petridish containing Mueller Hinton agar (Table 8.1). The isolated kojic

164
acid crystals were made to 0.1ml suspension with sterile distilled water at a concentration
of 250µg/ml was inoculated into one of the well created in the petridish. Into other two
wells negative control was maintained with distilled water and a positive control with a
standard drug Cefrazidime prepared like test sample.The plates were incubated at 370C
for 16-24 hrs. After 24 h, zone of inhibitions surrounding the wells were identified and
measured in millimetres Saleh (2011).

Table 8.1: Composition of Mueller-Hinton agar:

Ingredients Composition (g/L)

Beef infusion 300ml

Casein hydrolysate 17.5g
Starch 1.5g
Agar 10g
Distilled water 1L

8.3.2 Antiproliferative activity of kojic acid:

8.3.2.1 Cell culture:

Human cancer cell lines used in this study were obtained from National Centre for Cell
Science, Pune. They were grown in a Minimal essential medium (MEM, GIBCO)
supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 5% fetal bovine serum (FBS)
(growth medium) at 37°C in 5% CO2 incubator.

8.3.2.2 MTT assay:

The standard MTT assay followed by Mosmann (1983) was modified and used to
determine the inhibitory effects of test compound kojic acid on cell growth in vitro. The
trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue
culture plate at a density of 5x103 cells/well in growth medium and cultured at 370C in
5% CO2 to adhere. After 48h incubation, the supernatant was removed and the cells were
pre-treated with growth medium and afterward mixed with different concentrations of

165
 CHAPTER – VIII
INVESTIGATIONS FOR THE
BIOACTIVE PROPERTIES OF
KOJIC ACID
 CHAPTER – VIII
INVESTIGATIONS FOR THE
BIOACTIVE PROPERTIES OF
KOJIC ACID
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
 CHAPTER 8

8. INVESTIGATIONS FOR THE BIOACTIVE

PROPERTIES OF KOJIC ACID

8.1 INTRODUCTION:

Antibiosis is the microbial growth inhibition phenomenon in presence of some

metabolites, lytic factors or enzymes which were secreted by other organisms in very low
concentrations usually less than 10ppm Irtwange (2006). Kojic acid is one of the notable
secondary metabolite synthesized by very rare microorganisms like Aspergillus oryzae,
A.flavus, A.tamarii etc., as well as some Penicillium species and few bacteria during the
stationary phase of growth cycle of bacteria (Wilson 1971, Blumenthal 2004, Bentley
2006, Terabayashi et al. 2010). The major biological functions of kojic acid are
antibacterial, antifungal, insecticidal activities etc. Both gram positive and gram negative
bacteria are inhibited by kojic acid Kotani et al. (1976). More than 0.5% concentration of
kojic acid inhibits the bacterial growth Beclik (1956). Bacteria of distinct genera
including Proteus, Staphylococcus, Streptococcus, Pseudomonas, Bacillus,
Corynebacterium, Clostridium, Aerobacter, Escherichia, Klebsiella, Salmonella,
Chromobacterium, Gaffkya, Micrococcus, Neisseria, Pasteurella, Vibrio etc are inhibited
by kojic acid Morton et al. (1945). The growth of the Mycobacterium tubercle bacilli was
inhibited by kojic acid at a concentration of 45mg/100ml Lee et al. (1950). The study
reported by Kim et al. (2012) that the minimum inhibitory (MIC) or fungicidal (MFC)
concentrations of amphotericin B and strobilurin has greatly lowered by kojic acid
against pathogenic filamentous fungi and yeasts. Kojic acid shows the phenomenon of
chemo sensitization hence in the present investigation, the researchers tested this
chemosensitizing property by co-applying kojic acid to some commercially available
anti-fungal agents like Amphotericin-B, fludioxonil, strobilurin also called kresoxim
methyl and hydrogen peroxide and they found that the fungal strains like A.fumigatus,
A.terrus, C.albicans CAN276, S.cerevisiae, Candida krusei ATCC6258, C.neoformans

163
CN24, A.fumigatus AF293, MAPK mutant strains were sensitive to these co-applied anti
fungal agents. The heightened activity often involves the change in the function of fungal
anti oxidation systems. The growth of Bacillus megaterium was inhibited by kojic acid
extracted from moldy cheese Moubasher et al. (1979). It also inhibits the swarming
movements of Azospirillum and Proteus mirabilis. Some of the zinc derivatives of
Azidometal kojates showed cytotoxic effect on Hela cells Hudecova et al. (1996).

Most of the natural occurring metabolites have afforded a good source of compounds
which initiated numerous applications in cancer chemotherapy. Globally the incident rate
of cancer will increase to 50% to 15 million by 2020 (WHO, 2010). Chemotherapy is one
of the effective treatments for extending the patient’s life Sirinet (2010). More than 70%
of the available anticancerous agents are either natural products or natural product
derived substances Karikas (2010) and the therapeutic applications of microbial
metabolites afford the opportunity for the invention of anticancer agent for example kojic
acid possesses an anti-cancerous activity.

8.2 OBJECTIVES:

To determine the antimicrobial microbial property of the kojic acid against

different bacteria

To determine the antiproliferative activity of kojic acid using Breast cancer cell
lines and leukemia cell lines.

8.3 MATERIALS AND METHODS:

8.3.1 Antimicrobial activity of kojic acid:

The agar-slants of pathogenic bacteria suspensions were collected from M/s.Doctor’s

Diagnostic Centre, Gajuwaka, Visakhapatnam. Individual bacterial suspensions were
prepared using a sterile distilled water containing 108CFU/ml. By applying spread plate
technique Dubey and Maheshwari (2003), 0.5ml of bacterial inoculum was spread
uniformly on a petridish containing Mueller Hinton agar (Table 8.1). The isolated kojic

164
acid crystals were made to 0.1ml suspension with sterile distilled water at a concentration
of 250µg/ml was inoculated into one of the well created in the petridish. Into other two
wells negative control was maintained with distilled water and a positive control with a
standard drug Cefrazidime prepared like test sample.The plates were incubated at 370C
for 16-24 hrs. After 24 h, zone of inhibitions surrounding the wells were identified and
measured in millimetres Saleh (2011).

Table 8.1: Composition of Mueller-Hinton agar:

Ingredients Composition (g/L)

Beef infusion 300ml

Casein hydrolysate 17.5g
Starch 1.5g
Agar 10g
Distilled water 1L

8.3.2 Antiproliferative activity of kojic acid:

8.3.2.1 Cell culture:

Human cancer cell lines used in this study were obtained from National Centre for Cell
Science, Pune. They were grown in a Minimal essential medium (MEM, GIBCO)
supplemented with 4.5 g/L glucose, 2 mM L-glutamine and 5% fetal bovine serum (FBS)
(growth medium) at 37°C in 5% CO2 incubator.

8.3.2.2 MTT assay:

The standard MTT assay followed by Mosmann (1983) was modified and used to
determine the inhibitory effects of test compound kojic acid on cell growth in vitro. The
trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue
culture plate at a density of 5x103 cells/well in growth medium and cultured at 370C in
5% CO2 to adhere. After 48h incubation, the supernatant was removed and the cells were
pre-treated with growth medium and afterward mixed with different concentrations of

165
kojic acid (12.5, 25, 50, 100 and 200 µg/ml) to achieve a final volume of 100µl and then
cultivated for 48h. The compound was prepared as 1.0mg/ml concentration stock
solutions in PBS. Culture medium and solvent were used as controls. Each well then
received 20µl of fresh MTT (0.5mg/ml in PBS) followed by incubation for 4hr at 37°C.
The supernatant growth medium was removed from the wells and replaced with 200µl of
DMSO to solubilize the colored formazan product. After 30 min incubation, the
absorbance (O.D) of the culture plate was read at a wavelength of 492nm on an ELISA
reader and Anthos 2020 spectrophotometer.

8.4 RESULTS AND DISCUSSION:

8.4.1 Antimicrobial activity:

From the Figure 8.1 it was observed that maximum zone of inhibition (9mm) was
observed with the cultures S.aureus and E.coli followed by B.subtilis (8mm) indicates
that these organisms were highly sensitive to the antimicrobial compound kojic acid.
Others showed least sensitivity to kojic acid. However no zone of inhibition was
observed with negative control where as the positive control showed highest zone of
inhibition 12mm with S.aureus. This indicated that, standard antibiotic Cefrazidime
showed more inhibitory activity than isolated kojic acid. These findings were similar to
Saleh (2013) reported 10mm zone of inhibition to MRSA strains and El-Aasar used
Minimum inhibitory assay method to determine the antimicrobial activities of the
synthesized kojic acid from A.parasiticus. The MICs of bacterial isolates were 176-
285µg/ml. Whereas Aytemir and Erol (2003) reported that, the bacterial growth was
inhibited at 128-256µg/ml. The present results agreed well with the previous studies and
the inhibitory concentration used was 250µg/ml.

Hence so many clinical trials were in progress in using kojic acid derivatives to enhance
the antimicrobial activity of kojic acid because it is non-toxic and naturally occurring
secondary anti-metabolite.

166
kojic acid (12.5, 25, 50, 100 and 200 µg/ml) to achieve a final volume of 100µl and then
cultivated for 48h. The compound was prepared as 1.0mg/ml concentration stock
solutions in PBS. Culture medium and solvent were used as controls. Each well then
received 20µl of fresh MTT (0.5mg/ml in PBS) followed by incubation for 4hr at 37°C.
The supernatant growth medium was removed from the wells and replaced with 200µl of
DMSO to solubilize the colored formazan product. After 30 min incubation, the
absorbance (O.D) of the culture plate was read at a wavelength of 492nm on an ELISA
reader and Anthos 2020 spectrophotometer.

8.4 RESULTS AND DISCUSSION:

8.4.1 Antimicrobial activity:

From the Figure 8.1 it was observed that maximum zone of inhibition (9mm) was
observed with the cultures S.aureus and E.coli followed by B.subtilis (8mm) indicates
that these organisms were highly sensitive to the antimicrobial compound kojic acid.
Others showed least sensitivity to kojic acid. However no zone of inhibition was
observed with negative control where as the positive control showed highest zone of
inhibition 12mm with S.aureus. This indicated that, standard antibiotic Cefrazidime
showed more inhibitory activity than isolated kojic acid. These findings were similar to
Saleh (2013) reported 10mm zone of inhibition to MRSA strains and El-Aasar used
Minimum inhibitory assay method to determine the antimicrobial activities of the
synthesized kojic acid from A.parasiticus. The MICs of bacterial isolates were 176-
285µg/ml. Whereas Aytemir and Erol (2003) reported that, the bacterial growth was
inhibited at 128-256µg/ml. The present results agreed well with the previous studies and
the inhibitory concentration used was 250µg/ml.

Hence so many clinical trials were in progress in using kojic acid derivatives to enhance
the antimicrobial activity of kojic acid because it is non-toxic and naturally occurring
secondary anti-metabolite.

166
Figure 8.1: Zone of inhibitions of different bacteria to the antimicrobial compound
kojic acid

Zone of inhibitions of different bacteria to kojic acid

10
9
8
7
Zone of 6
5
Inhibition (mm) 4
3
2
1
0 Diameter of Zone of
inhibition (mm)

Bacteria

8.4.2 Antiproliferative activity:

The compound kojic acid exhibits antiproliferative activity on the breast cancer cell lines
MDAMB435S (Table-8.2), (Figure-8.2, Figure-8.4) and Leukemia cell lines K562
(Table-8.3), (Figure-8.3, Figure-8.5). The percentage of inhibitory activity for the Breast
cancer cell line was 22.32% at 100 µg/ml concentration and for the Leukemia it was
67.07% at 200 µg/ml concentration.

Table 8.2: Effect of kojic acid on MDAMB435S cell lines

Control 0.693
Blank - 0.030
Conc. In µg/ml OD at 492nm % Cell Survival % of Inhibition
activity
12.5 0.613 87.93 12.6
25 0.61 87.48 12.51
50 0.599 85.82 14.17
100 0.545 77.67 22.32
200 0.565 80.69 19.3
IC50 = 863.029471 µg/mL

167
Table 8.3: Effect of kojic acid on K562 cell lines (Leukemia)

Control 0.850
Blank - 0.030
Conc. In µg/ml OD at 492nm %Cell Survival % of Inhibition
activity
12.5 0.717 83.79 16.21
25 0.507 58.17 41.82
50 0.506 58.04 41.95
100 0.466 53.17 46.82
200 0.3 32.92 67.07
IC50 =112.0212 ug/mL

Figure 8.2: Inhibition activity of kojic acid on MDA MB435S cell lines (Breast
cancer)

Inhibition activity of kojic acid on MDA MB435S cell line

25

20
% of inhibition activity

15

10 % of inhibition activity

0
12.5 25 50 100 200
Conc. in µg/ml

168
Figure 8.3: Inhibition activity of kojic acid on K562 cell lines (Leukemia)

Inhibition activity of kojic acid on K562 cell line

80

70

60
% of inhibition activity

50

40

30 % of inhibition activity

20

10

0
12.5 25 50 100 200
Conc. in µg/ml

169
Figure 8.4: Activity of kojic acid on MDA-MB435S (Breast cancer) cell lines before
and after addition

A- Control B- 12.5 µg/ml

C-25 µg/ml D- 50 µg/ml

E- 100 µg/ml F – 200 µg/ml

170
Figure 8.5: Activity of kojic acid on K562 (Leukemia) cell lines before and after
addition.

A- Control B- 12.5 µg/ml

C-25 µg/ml D- 50 µg/ml

E- 100 µg/ml F – 200 µg/ml

171
8.5 CONCLUSION:

The higher antimicrobial activity of kojic acid was observed with Staphylococcus aureus
and Escherichia coli. The Minimum inhibitory activity was less compared to standard
antibiotic Cefrazidime. It showed antiproliferative activity on the breast cancer cell lines
MDAMB435S and Leukemia cell lines K562. The percentage of inhibitory activity for
the Breast cancer cell line was 22.32% at 100µg/ml concentration and for the Leukemia it
was 67.07% at 200 µg/ml concentration. It was concluded that, inhibitory effect was
more on the cell line K562 (Leukemia) when compared to the MDAMB435S (Breast
cancer) cell line.

172
 CHAPTER – IX
PRODUCTION ECONOMICS
 CHAPTER 9

9. PRODUCTION ECONOMICS OF THE COMPOUND

9.1 INTRODUCTION:

Till now the raw materials used in the present study have not been commercially utilized
for kojic acid production. The raw materials which give the highest yield have the
potential to be used for industrial production of kojic acid. The market potential of the
kojic acid was described in Table 9.1.

Table 9.1: Market potential of kojic acid

Product Kojic acid

By-products
Present market price
Indigenous production
Specifications of the product
Nil
2,500 US$/Kg
2300 INR/kg (Futamura et al. (2001)
Nearly fifteen small-scale industries manufacture
kojic acid in India. Most of is imported from
Japan and USA.
FTIR, Proton NMR, XRD and HPLC were done
to test for its purity

9.2 Objective:

To estimate the production cost of the present investigation

9.3 Materials and methods:

The cost of the materials used in the production of kojic acid in the present study was
estimated in the Table 9.2 below.

173
Table 9.2: Production cost for the present investigation

Materials Cost details

(per 100g substrate in Rs.)
Sago starch powder 2.00
Culture -
KH2PO4 4.00
MgSO4 3.00
Peptone 12.00
Water 8.00
Acetone 7.00
Ethyl acetate 5.00
Total (Rs.) 41.00

9.4 Results and Discussion:

Approximately the production cost of the kojic acid was Rs.410/- with 1kg of substrate in
the present study. According to Futamura et al. (2001) the production cost was Rs.620/-
with the substrate corn starch. So the production cost was reduced by 15% compared to
Futamura et al. (2001). From corn starch the author reported 40g/L of kojic acid with
Bentley’s method where as the present research reported 90.8g/L. So the production rate
was twice of Futamura et al. (2001). According to Indian market price the cost of kojic
acid was Rs.230/- per gram of kojic acid (M/s. Himedia laboratories Pvt. Ltd.,). From
these findings it was concluded that, 285g of kojic acid was produced with the production
cost of Rs.410/-. This interms proved that, the present method was the most reliable and
cost-effective method for the production of kojic acid.

9.5 Conclusion:
From the present study it was concluded that, the production cost of kojic acid was
reduced by 15%.

174
 CHAPTER – X
CONCLUSION OF THE
PRESENT INVESTIGATIONS
 CHAPTER 10

10. CONCLUSIONS OF THE PRESENT INVESTIGATION

Mycological analysis of soil samples found that, ten different species of fungi were
isolated and identified. These include Aspergillus flavus, Aspergillus sojae, Aspergillus
oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus
terrus, Mucor, Fusarium and Penicillium.

A promising isolate of kojic acid production A.flavus was screened from other isolated
soil fungi. The fungus produce better yields of kojic acid with surface fermentation
technique than submerged fermentation. The organism was identified as a negative
producer of aflatoxin.

For enhancing the yield of kojic acid a combinational optimization methodology i.e.
OFAT and RSM have been used consecutively. It was observed that, the yield was
enhanced more than 12% using RSM.

The possibility of using waste carbon sources as profitable substrates for kojic acid
production using Aspergillus sps. were studied for the first time and can be used
commercially for the large-scale production of value added products like kojic acid. Out
of 14 different raw materials used, enhanced yields of kojic acid crystals was noticed
with Sago starch followed by M.calabura and Palmyra sap by the soil isolate A.flavus.

The optimized conditions established with Sago starch Substrate concentartion 1000ml,
pH 6.0, Time 28d, Temperature 280C, Peptone concentration 4g/L, KH2PO4
concentration 1g/L, MgSO4 concentration 0.5g/L. The yield obtained was 28.5g/L. For
M.calabura fruits the optimized conditions were Substrate concentartion 1000g/L, pH
6.0, Time 28d, Temperature 280C, Peptone concentration 4g/L, KH2PO4 concentration

175
2g/L, MgSO4 concentration 0.7g/L. The yield obtained was 24.71g/L. The optimized
conditions with Palmyra sap were found to be Substrate concentartion 1000ml, pH 4.0,
Time 32d, Temperature 280C, Peptone concentration 3g/L, KH2PO4 concentration 2g/L,
MgSO4 concentration 0.5g/L. The yield obtained was 22.83g/L.

Kojic acid crystals were isolated and purified. The structural characterization of kojic
acid was confirmed by Proton NMR, FTIR and XRD. XRD elucidated the entire
crystalline phases of kojic acid. Where as the molecular weight and purity of kojic acid
was confirmed by LC/MS and HPLC.

The isolated kojic acid crystals shows high antimicrobial activity against pathogenic
bacteria Staphylococcus aureus and Escherichia coli. Maximum zone of inhibition
(9mm) was observed with the cultures S.aureus and E.coli followed by B.subtilis (8mm).
The inhibitory activity was less while comparing with standard antibiotic Cefrazidime
(12mm). The inhibitory effect of kojic acid was more on the cell line K562 (Leukemia)
when compared to the MDAMB435S (Breast cancer) cell line.

Finally it can be concluded that, the carbon sources used in the present study have proved
the auspicious potentiality in exploiting the alternate sources for higher production of
kojic acid by Aspergillus flavus through surface fermentation. The present study helps to
scale-up the kojic acid fermentation to a large-scale level in order to produce expensive
chemical like kojic acid from economical raw materials.

176
CHAPTER – XI
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194
 LIST OF PUBLICATIONS

“Response surface methodology for the optimization of kojic acid production by

Aspergillus flavus using Palmyra sap as a carbon source”. European journal of
Biotechnology and Bioscience”. Vol.2, Issue 5, 52-57, 2014. Impact factor 1.7.

Statistical optimization of kojic acid production through Response surface methodology

by Aspergillus flavus using Sago starch hydrolysate as a carbon source”. Asian journal of
applied science and engineering”. Vol.3, No.4, Issue 8, 2014. Impact factor 2.04.

“Response surface methodology for the optimization of kojic acid production by

Aspergillus flavus using Muntingia calabura fruits as a carbon source”. Accepted for
publication in Indian Journal of Science and Technology.

Under processing:

Optimization of cultural parameters for cost effective production of kojic acid by fungal
species isolated from soil.

Surface fermentation of kojic acid production using soil fungal isolates from inexpensive
nutritive sources.

195
 CHAPTER- VII
STUDIES ON THE INHIBITION OF KOJIC ACID PRODUCTION
 172

CHAPTER.. VII
STUDI ES ON THE I NHI BITION OF KOJIC ACID PRODLC TION

Inhibitors are useful in the delineation of meta-

bolic pathways. He nc e th e effects of a number of me tabol i-
ties and metabolic inhibit ors on kojic acid synthesis have f,:j~en
studied employing growth medium as we ll as r esusp e nded my-
celia inorder to get an idea about the possible enzymes in-
volved in kojic acid bio synt hes is. Most of the findinas
reported in literature on the effects of diff erent facto rs
on kojic acid production are based either on alte r ation of
experimental conditions of growth or that of growth medium
constituents. The effe cts obse.IVed could have been secondary ,
resulting from changes in spore germination and mycelial gro-
wth. Therefore it was thought that a system in wh i ch kojic
acid was produced aft er resus p ensi on of fully grown mycelium
would be of greater us e in this study. It has been shown in
the previous chapt e r that such a sys tem is exper i mentally
feasible. Interpretations of results obtained with diffe rent
inhibitors is, however, rather diffic ult , because only a few
inhibitors at a particular concentration have been shown to
be inhibitory for any spe cific enzyme. Hence, the ef fec ts of
different concentrations of potential inhibitors of enzymes
of the relevant pathways of interest were studi ed •
 173

MATEfi.IALS AND METHODS

All such studi es were performed on a strain of

bspergj.~ flrn_, isolated in this l .aboratory and termed
as culture Bin Chapter III. 1he methods pertaining to organ-
ism and cultural conditions have been described in Chapter I I.
5 ml of the well dispersed spore suspension containing 106
spores/ml was used to inoculate 50 ml of the g rowth medium.
Yeast extract sucrose (YES) medium was used as growth mediu m.
For resuspension studies, rnycelia were grown in YES medium
for six days and were then r esuspended in 50 ml st erile 0 . 2M
phosphate buffer pH 6.5, containing 20% glucose. 1he proced-
ures for media sterilization and r esuspension hav e already
been mentioned in Chapter II. Different concentrations of
inhibitors used were added aseptically to th e st erile medium
before inoculating the spores or r esusp ending the mycelium.
1he flasks were incubat ed as usual at 30°C as stationary
cultures. Suitable aliquots of the media were drawn from the
fermentati on vess el at djfferent time intervals and analys ed
for kojic acid cont ent aft er diluting the sample suitably.
M{celial growth and kojic acid were determined by followi ng
standard procedures mentioned in Chapt er II.

RES UL TS AND DISCUSS ION

1his chapter deals with the ef fect of differ ent
inhibitors on kojic acid synthesis during growth and during
 174

resuspens1on of a lready g r own mycel ia . The eff e cts s t udied

s eem to be conc e ntration depe nde nt . It may be ob s eIV ed from
3
Tab l es 33 and 34 that at 10- and 10- 4 M conc e ntratio ns , r ibo-
flavi n i nh ib it ed g r owth and ko jic acid product io n to a mar ked
-5
ext e nt whil e , at 10 M conc e ntrat ion it s t imul ated sligh tly
g r owth and ko jic ac id p r oduction . The f act th at s ever a l inh i -
bitors stimu l at e met abo lic p r oc esses at low c oncent ratio ns
(1 )
and inhibit at h ig h conc entrations ha s be en wel l do cume nted •
.
Th e mechanism of act i vat ion at low c o nc e ntrations is not
cle arly u nde rst ood . Th e exte nt of s t i mu l at o ry or i nh i b itor y
eff e ct v ?r i es depend i ng o n t he inhib i to r and th e p r oc ess
c onc ern ed . Sev er a l hYPothese s hav e b een put forward t o exp-
lain th e ob s e r ved variat i o ns . Low conce nt r ati ons of inh ibitor
may mop off c ompounds inh ib itory t o the e nzy me s ystems , fo r
e xa mp l e , EDTA fo r ms ch e l at e s with trac es of heav y met a l i ons .
St imulat ory effect of an inhib itor may also be due t o its
eff ect on s ome met abo lit e or st ep involv ed in the r egul ation
of metabolis m. 1/h en a c omp ou nd is a substra t e for t wo or more
co mp etitiv e r e actions , an inhibition of one of t hese r e act-
i o ns ma y r esult i n a n appare nt stimul at ion of th e other
r e act i on . Thus , ribof lav i n inhibit ed koj ic acid synth es i s by
73 . 5% and 68 . 8% at 10- 3 and 10- 4 M conc e ntrati ons r es pe ct iv e ly
but s t imu l at ed its s ynt hesis by 6 . 6% at 10- 5 M c o nc e ntrat i o n .
On the oth er h and , it inh i bit ed ko j ic acid s y nthesis i n
3
r e suspe nsion medium at 10- and l 0- 4 M co nc e ntrat ions by 4(%
 ,._
-~
'~
..- .. t

TAB LE- 33

EFFECT OF RIBOFLAVIN ON GROWTH AND KOJ I C '\C I D PRODUCTION ON GR0 11vrH MEDIUM

Percent Wet we i a ht
Con centr at- Pe r iod of i nc ub at i on ( day s ) cha nge of myc el i-
ion 4 5 6 7 8 9 10 11 i n t he um o n 11th
(M) Koj ic acid produced (mg/ml) maximum day
koiic
a cid le-
----· vel __ _ __ _ _ . _ _
- ------ ---
Contro l
. 10 . 65 17 . 23 36 . 21 42 . 02 65 . 05 6q . 92 75 .02 6 1 . 02 - 6 . 88

10 - 5 8 . 12 14 . 25 22 . 05 40 . 60 56 .8 1 70 . 20 80 . 0 8 66 . 7 2 +6 .6 6 . 23

10 - 4 2 . 90 4 . 82 7 . 80 1 2 .1 2 16 .qo 23 . 40 20 .7 2 16 . 20 - 68 . 8 3.q8

10· 3 3 . 42 5 . 41 5 . 85 10 . 60 1 3 . 68 16 . 92 1 9 . 88 12 . 62 -7 3 . 5 3 .7 3

·- -
,. ____________

~
-..J
(JI
 '{
.,.
.
),--
... ~
•

TAB LE- 34
EFFECT OF RIBOFLAVI N ON KOJ IC ACID PRODUCTION DURI NG RESUS DENSION

Pe r ce nt
Conce n- Pe riod of i nc ub at ion (days ) cha nge
t rat io n 0 l 2 3 4 5 6 7 8 i n t he
(M) maximum
Ko j ic ac i d produced ( mq/ml) ko j ic ac i d
____l ~ e l _ _

Control 0 . 68 4 .10 5 . 96 11. 30 18 . 40 37 . 05 6 2 .71 7 2 . 08 61 .70

10- 4 0 . 44 2 .6 1 3 . 20 7.18 11. 50 23 .02 4 1.15 47 . 28 39 . 84 - 34. 4

10 - 3 0 . 40 2 . 30 2.92 6.0 5 10 . 5 20 . 82 37.62 43 . 2 1 36 .47 - 40

--------------------

,-....
-.J
(J\
 177

and 34 .4% rQspectively (Tab le 34) . The. comparitively greater

inhibition of kojic acid synthesis by 10- 3 and 10- 4 M concent-
rati ons of riboflav in i n case of growth (YES) medium may b e
due to some secondary effects . Inhibition of kojic acid nro-
duct ion and myce lia l gmwth by riboflavi n suggests the poss-
ible ro l e of flavoprotei n enzymes as it has been shown to be
2
a pot ent inhibitor of flavoprotei n ele ctro n transport syste~ )

Flavopr ote in enzymes are of the two types (i) thos e which have
dissociable prosthetic coenzymes FMN or FAD and (ii) thos e
whe r e pr os t het i c coenzymes FMN or FAD are tight ly bound to the
protein component of the enzymes . In form e r case , an inhibi-
tio n of competitive natur e is not unexp ected . In latter case ,
thr ee mechan i sms may possibly be sugqested : I n the first pl ace
it i s now known that various flavins and their nucl eotides
form mol ecular compl exes with one another . The formation of
such compl exes with bound FMN or FAD may prevent the normal
interact ions of coenzyme during oxidation r e actions. Th e
s eco nd possible mec hanism sugges t s that i n some instanc es ,
the inhibitors may interfere with th e experimental e l ectron
acceptor pqrticularly when this is a dye . Lastly in case of
third mechanism , there is a possibility of nonspecific binding
of these polyh eterocyclic compounds to the enzymes, Blake ly
3
and Ciff e ri ( ) have reporte d that glu~onic acid dehydrogenase
of Aerobact er ~ero~~ns is inhibit ed by riboflavin at 10- 4 M
 178

concentration. The conside rable inhibition observed during

r esuspension sugg e sts a possible role of g l uconic acid dehy-
drogenase in kojic acid biosynthesis.
It would be not ed that at higher conc e ntrations
glucose-6-phosphat e inh i bit ed myc 0lial growth and kojic acid
s ynthesis markedly while at lower conc e ntrations th e inhi-
bition was les s (Tabl e 35). It inhibit ed kojic acid synthes is
4 5
by 68.4%, 23.5% and 19% at 10- , 10- and 106 M conce ntrat-
ions, r espectively, in the case of growth medium · while i n
the case of resus pension medium, the perc ent inhibit ion of
kojic acid synthesis was 65 . 5% and 5.2% at 10- 4 and 10· 5 M
concentrati ons r esp ective ly (Table 36). Fructos e-1,6-diphos-
phate was found to be inhibitory at all th e l evels tried in
case of growth medium as well as in r esusp e nsion medium
(Tables '37 and 38). Data available for the h exokina se acti-
vity of a numb er of animal tissues, of which brain is perhaps
th e best example, show th at r at brain hexokinase is strongly
•
inhibited by hexos e monophosphates; glucose-6-phosphate be ing
about three times mo r e ef fe ctive than fructose-1,6-diphos-
4
phate . ( ) An equilibrium mixture of the two gave an int ermed-
iate inhibition value. These r esults were later confirmed and
extended to a variety of other animal tissues by Crane and
Sols. ( 5 ) On the other hand , it has been r eport ed that yea st
hexokinase is not affected by the product of its r e action
 • .. ~
}

TABLE- 35

_fil'FECT JF GLl 'COS E- 6 - PHOSP HATE ON GRO'. JTH AND KOJIC ACI D PRODUCTION ON GROWTH MED I UM

Con ce nt- Per i od of incubatio n · (da ys ) ,i .

r at ion 4 5 6 7 8 9 10 11 Percent Wet Wei-
( M) ch anc e ght of
Kojic ac id produced (mg/ ml) in t h e mycel i um · .
maximum ·on 11th
koj i c ac- d ay
--.- ---. - - - - - _i_d_l~y e 1

Co nt ro l 1. l . 20 18 .75 I 38 . 21 44 .02 66 . 02 70 . 20 75 .6 1 62 .0 6 . 88

10- 6 10.42 18 . 17 23 . 80 43 . 20 47 . 45 56 . 90 61. 21 44 . 20 - 19 6 .30

10-5 9 . 36 17 .02 22 . 10 42.50 45 . 30 54 .40 57 . 80 42 . 50 - 23 . 5 5 . 55

-4 .., .
10 3 . 36 6 .08 8 . 50 11 . 92 13 .65 22 .12 23 . 82 20.1 2 - 6 8 .4 5 . 85

-- --- -

1--
....J
'°
 y _., 'y
.... l
r•

TABLE - 36
Err ECT OF GllJCOS E-6 - PHOSPHATE ON KOJIC 4CID PRO~ucTI ON DURING RESUSPENS I ON
---------- Percent
Con c~ntrat - Per iod of incubat i on (days ) ch a nge
i on 0 l 2 3 4 5 6 7 8 in th e
(M) max imum
Kojic acid produc ed (mg/ml) koj ic
acid
·- --- l e\f~l

Control 0 .68 4 .10 5 . 96 11 . 30 18 . 46 37 .02 62 .72 7 2 .02 6 1.76

10- 5 0 . 30 2 .60 3 . 82 8 .62 14 .23 32 . 28 58 . 20 68 .23 57 . 80 - 5 .2

10- 4 0 . 25 1 . 30 1.72 3 . 81 5 . 89 13 .05 21 . 80 24 . 81 21 . 12 - 65 . 5

----- - - - - - ----- --

1--
(X)
0
 .. -4, ~
}

l hfil..§-=.~
EFFECT OF FRUCTOSE 1-6 DIPHOSPHATE ON GROWTH AND KOJ!C ACID PROOUCTION ON GRGWTH
MED IUM
- ----------- --~---- ~~-~~~-
Perce nt Wet we i ght .
Conc e nt- Per i od o f 1 nc ubat i o n (da ys ) c ha nge of myc e l iu m
r at i on 4 5 6 7 8 9 10 11 in the o n 11th
(M) ma~imum day
Koj ic a cid produced ( mg/ml) kojic
aei d
_ _ __ _ _ _ __ _____ _ -1.,ey:e l,.....__~~
- ·- -
:Cont r ol 11 . 20 1 8..7 5 38 . 21 44 . 02 66 . 06 7 0 . 20 7 5 . 60 6 2 ,0 6 .88

10-5 9 . 42 17~82 2 9.42 .39_.20 5 2. 30 59.52 64 . 6 0 55;, o •14,.5 7,60

J:(J-4 8 .70 16.15 26 . 40 33.95 40.90 49. 62 56 .14 46 -85 • 25 . 8 6 , 45

-3
10 P .10 15 .32 22 .10 28 .90 35.70 44 .92 54 .. 46 45 .. 90 .28 5 ,. 85

----------- -- ---------~ ~--- ----~

- -~--~ -~ -----,.-.-.~

.....
(1)
.....
 y 'r-
. 4- ~
}

J,6BLL- 38
EFFECT OF FRUCTOSE 1-6 - DIPHOSPHATE ON KOJIC ACID PRODUCTION DURI NG RESUSPENS I ON .
-----·- --------------- Perce nt
Con cent- Period of incubation (days ) ch ang e in
rati on 0 l 2 3 4 5 6 7 8 th e max i-
(M) Koj ic acid p roduced (mg/ml) mum koj ic
--- ----·------ __ ~cid level

Control 0 . 68 4 • .ll 5 .96 11. 30 18 .40 37 . 05 62 .71 7 2 .08 6 1.70

10- 4 0 .51 3 .08 4 .71 8 • 20 13 . 20 2 8 •0 l 47 • 21 5 3 •6 0. 46 , 2 - 25 .5

10- 3 0 . 36 2 . 10 2.62 5 . 82 8 . 96 19 . 80 33 . 20 37 . 70 32 .10 - 47.6

-- --

......
co
f\)
 183

. . (5 ;6 ) Gl
though it is inhibited by adenylic acid . ucose dehy-
drogenase of liver has bee n r eported to be strongly inhibited
in a competitive manner by glucose-6 - phosphate with a ki of
6
2 .5 x 10 - M and by fruct ose- 1 , 6 - diphosphate with a ki of 6 .2 x
6
10- 5 M. ( , 7 ) The results obtained in the present investig at-
ions suggest that hexokinase and glucose dehydrogenase may be
involved in kojic acid biosynthesis .
The effects of sodium azide on kojic acid f ormat-
ion on g r owth and . resuspensio n media are presented in Tahles
39 and 40. It may be seen that growth and ko jic acid product-
io n were inhibited completely at l0- 4 M and higher conc e nt-
rations of sodium azide while there wa s very little inhibit-
-5
ion in kojic acid sy nthesis and qrowth at 10 M conc e ntration .
In resuspension medium s od ium azide inh ibited kojic acid pro-
duction by 90.9% and 11.4% at 10- l and 10- 2 M concentrations
resp ectively . The more drastic effect of sodium azide obser-
ved in case of growth medium might be due to some secondary
effects . Tissieres has refer red to the effects of azide as
an inhibitor of oxidative phosphorylation. (s) An uncoupling
of oxidati on from phosphorylation often r esults in increased
oxygen uptake . It is relevant that whereas cyanide inhibits
growth and r egeneration of some microorganisms , azide may
inhibit only regeneration and this aspect of metabolism is
certainly dependent on phosphorylation . ( 9 ) Rosenbars found
that azide inhibits the dea mination of glucosamine by g. Goli
 y
..
\

~ ~
}

TAB LE - 39
EFFECT uF S0CIUMAZIDE ON GROWTH AND KOJIC ACID PRODUCTION ON GROWTH MEDIUM
Pe r ce nt Wet- weio h
Conc ent- Period of i nc ubat io n (days) c hange in t of my-
r ation 4 5 6 7 8 9 10 11 the max i- c el i um
(M) Koj ic acid produced (mg/ml ) mum kojic on 11th
acid day
leve. L _._ __
--------------------------
Control 11 . 20 18 .75 38 . 2 1 44 .02 66 •.06 7 0 . 20 7 5 . 60 62 .0 6 . 88

10-5 6 . 92 11.7 8 17 . 25 39 .76 57 . 68 6 1. 32 70 . 05 5 9 . 08 6 . 47

- 7 .3

-1 2
10 , 10: No koj ic acid for mation No g r owth
10~3 10- 4

......
co
~
 y )"
}
~
~
.~

TABLE - 40
EFFECT OF SODIUMAZ IDE ON KOJIC ACID PRODUCTION I:URING RESUSPENSI ON
- Pe r c ent -
Conc ent- Pe r i od of incubation (d ays ) ch a nge i n
r at ion 0 l 2 3 4 5 6 7 8 t he max i-
( M) mum kojic
Koj ic ac id p r oduc ed (mg/ml ) ac id leve l
- -- - - - ----
Control 0 . 68 4 .15 5 ~96 11 . 30 18 .40 37 . 80 6 2 .72 7 2 .02 6 1 .75

-2 3 . 50 4 . 40 9 .70 15 . 30 33 .02 55 . 91 6 3. 80 56 . 21 - 11. 4

10 0 .6 2

10- 1 0 .05 0 . 33 0 . 41 0 .91 1.25 3 .05 5 . 20 6 . 52 5 .05 - 90 . 9

--- - - -- - --

1---
0)
(J1
 1 86

but not its oxidation. (lO) Whe n phosphate is omitted, the

glucosamine is not deaminated . These r esults point out towards
an effe ct of on oxidative phosphoryl ation . The inhibitory
effects of azide on phosphory l atio n have been describ ed by
u _ tc h k iss
,~ . ' ( 12 ) L oomis
( l l ) , C ase an d Mc 11wain, . an d L ipman
' ( 13 )
4
and Sp iege lman JU al(7 ) The last g r oup concluded that triose
phosphate dehydrogenas,e may be inv olved . As th is enzyme P.ro-
. . (15)
bably cont ai ns zinc, the azide may react wit h t his metal .
Triose phosphate deh ydrogenase is also a key enzyme in fer-
mentatio n. Robe rts on and Bayer howev er r egard the effects
of azide on phospho·:z.ylation .as indirect a nd i ndependent of
phosph ate or ac cepter availability and acco rding to Rakestraw
and Roberts, (l6 ) phosphorylatio n may be increased or d~creased
without affecting r espiration by h12tobacter . Recently,
(17) .
Suelt er ~1 al. have s hown that azide inhibits th e accumul-
ation of an unidentified intermed i ate of oxidative pho spho:ry-
lation by r at liv er mitochondria. It is r eported that in case
of Ac_etopact er § Uboxy_da~ and Pseudo monas auercitopyrogall i ca ,
the ox idatio n of glucose by oxyge n is inhib it ed more than
(18)
90% by O.5mM cyanide ( at pH 7 . O) or az i de (at pH 6 .O or 7 .o) .
Thus inhibition by azide could r esult fro m an i nh ibition of
glucose dehy dr ogenase and also from a n uncoupl ing of oxidat-
ive phosphorylation and the consequent decrease in ATP l evels .
The azide inhibition of kojic acid formation of r esus pended
myc el ia either indicat e the nee d for energy fo r this pro ces s
 187

or th e involve ment of glucose dehy drogenase in kojic acid

biosynth esis .
Fluorid e inhibited growth and kojic acid p roduct-
ion at all th e l evels tried , i n growth medium as well as in
r esuspension medium. Thus in growth medium , th e pe rc e nt inhi-
bition i n kojic acid synth esis was 32 . 8%, 29 . 3% and 26 . 9% at
10- 2 , 10- 3 and 10- 4 M conc entrations r esp ectively, whil e i n
resus pension med ium the p erc ent inhibition in kojic ac id f or -
2
mation was 36 .9% and 32 .3% at 10- and 10- 3M conc e ntration
respectiv e ly tTables 41 and 42 ). The inhibition by f luoride
ion might be du e to its ability to form complexes with sev eral
metal enzyme syst e ms including thos e dep e nd e nt o n Fe , Ca or
(19 , 20 , 21 )
rig , Enolas e which catalyz es th e r e action b etwe en 2-
phosphoglyc eric acid and phos phoenol pyruvic acid r equir ~
+2 +2
Wg for activ ity. I n th e pr esenc e of ~~ and phosphate ,
fluoride ions strongly inhibit th e e nzyme . Th e effect is r e l-
at ed to th e formation of a magnesium fluorophosph at e co mplex
which is only slightly dis sociat ed and the r eby eff ectiv ely
22
r emoves lvlg +2 fr om th e r eact ion mixture ( ). It is rathe r
difficult to e xplain the inhibition of kojic acid f ormation
by fluorid e as glucose is be liev ed to be directly convert ed
into kojic acid without undergoing g lycolysis . Howev er, the
involvement of phosphorylat ed int ermediat es cannot be r ul ed
out .
 r ~
}
~
"'

TABLE - 4J.
EFFECT OF FLUORIDE ON GROWTH AND KOJIC \CID PROIXJCTION ON GROWTH MEDIUM
---- - - -- - - ~- --

Percent Wet weight

Conc ent- Period of incubation (days) change of mycelium
ration I
in the on 12th
(M) 4 5 6 7 8 9 10 11 12 maximum day
Kojic acid produced (mg/ml) koj ic
acid
--------- level

Contro l 10 . 65 17 . 23 36 . 21 42 .02 65 .05 69.92 75 .02 61 .02 31 . 25 6 . 88

-4
10 3.32 5 . 40 6 . 52 13.02 28.21 41 . 20 54 . 80 32 . 90 12 . 60 .. 26 . 9 6 . 84

10- 3 2 .05 4 . 30 5 . 36 12 . 06 24 .60 40 . 20 53 .05 31 . 20 11 .62 - 29 . 3 6 .7 8

10- 2 1 . 90 2 . 81 3 . 85 10 . 94 22.80 38 . 08 50 . 41 30 . 02 11 .05 - 32 . 8 6 .62

-- -- - --- - --

1--
(X)
(X)
 _,._
""'y ~ }
~
~- -~

TABLE - 42
EFFECT OF FLUORI DE ON KOJIC ~CID PROIJJCTION DURING RESUSPSNS I ON
Period of incubation ( da ys ) - p ere ent ch a.ng e in-
Con cent- 0 1 2 3 4 5 6 7 8 the J7\.Q>t i..tru111, KOj ic
r at ion Koj i c acid produced (mg/ml ) acid. level
_J.ML __ _

Co ntrol 0 .70 4 .2:) 5 . 89 11 . 65 18 . 84 37.89 64 . 56 74 . 89 63 . 50 •

-3
10 0 . 47 2 .78 3 . 30 8 .10 10 . 85 25 . 92 43 . 20 50 .62 42 . 50 - 32 . 3

-2
10 0 . 44 2 . 50 3 . 17 7.60 10 .15 24 .02 41.28 47 . 25 40 . 82 - 36 . 9

1--
0)

'°
 • 190

1h e prese nc e of iodoac etic acid in growth medium

inhib ited kojic acid production by 35 . 3% , 15 . 2% and 10 . 4%
at 10 - 2 , 10 - 3 and 10 -4 M concent rations
. ( Table 43 ) . Simil arly ,

in resuspension med ium ,iodoace t t c ·acid decreased kojic acid

product ion by 37 .7 % "a nd 18 . 7 % at 10 -2 and 10 -3M concentrat-
io ns (Tab le 44) . Iodoacet ic acid is a well known inhibitor of
· ( 23
g l yco l ysis · h 1· b.1t ory ac t i. on of
• ) Th e in . d oace t.ic ac1. d is
10 .

due to the covalent and irreversible binding of this reage nt

with SH groups of dehydrogenases , thereby blocking the cata-
l ytic act ion . 6-phosphogluconic dehydroqe nase also DOssess es
sulphydryl groups which ar e r equired for catalytic activity
a nd hence it is also i nh ibited by iodoacetic acid in the same
(24)
manner. The obseIVed inh ibition by iodoacetic acid sugge .
sts a possible r ole of 6 - phosphogluconic dehydrogenase in
kojic acid biosynthesis.
Arsenate inhibited growth and kojic acid pr oduction
almost completely at 10- 2 and 10- 3 M concen trations while i t
caused a slight stimu lat ion of kojic acid p r oduction at the
lower c oncentratio n tried (Table 45) . In resuspension medium ,
the percent decrease in kojic acid productio n was 82.3% and
2
42 . 9% at 10- and 10- 3 M concentrations respectiv ely (Ta ble 46)
The inhibition c aused by arse nate at h i gher concentrat io ns
may be due to th e r e aso n that ars enat e uncouples th e subst-
rate linked phosphory l ation during th e oxidatio n of ph os pho-
(25 26 )
g lyceraldehyde and ~-ketoq lutarate ' Crane and Lipman
 y
~ J '~ l

'

I.6BLE =-~
EFFECT JF I ODOACETIC ACID ON GROWTH AND KOJIC AC ID PRODUCTION ON GROVnH MEDIUM
Percent Wet weight
Con cent- Period of incubation (days ) chanae of mycelium
rat ion 4 5 6 7 8 9 10 l.i. 13 in the on 13th
( M) maximum day
Kojic acid produ ced (mg/ml) kojic
ac id
lgveL ___
Control 10. 65 17 . 23 36 . 21 42 .02 65 .05 69 . 92 75 . 02 61 ~OS 31 . 20 6.88

10- 4 ':} .66 13 .6 2 18 . 90 32 .92 45.81 58.07 67 . 21 50 . 12 26 . 31 - 10 . 4 6 .58

10- 3 9 .05 12 . 32 16 . 95 28 .75 40 . 20 50 . 95 63 .60 49 . 20 24 .7 2 - 15 . 2 6 .43

10- 2 7 . 20 10 .12 13.52 23 .75 32 .62 40.32 48.~2 38.02 19 .08 - 35.3 6 .02
------

.....
'°.....
 _. ;J r ~
}

Jt, BLE_:: 44
EFFECT OF IODOACETIC ACID ON KOJ IC ACID DRODUCT ION BURH,TG rlESUSDENSION
--------------- ------------------------------·-·--------------- --- 0 e rc ent
Con c e nt- Period of inc ubatio n (d ays ) ch ange in
rati on 0 l 32 4 5 6 7 8 the maximu m
( M) Kojic ac id p r oduced (mg/ml) koj ic ac id
- - - - -- - -- - -- - - - -- - - - - -- - - -- · -· ____ _ __ l evel ____

Contro l C' . 70 4 . 20 5 .89 11.65 18 . 84 37 . 89 64 . 56 74 . 89 63 . 58

10 - 3 0 . 56 3 . 20 4 . 98 8.92 14.94 30 . 84 52 .7 5 60 . 7 4 51 . 20 - 18 .7

10- 2 0 . 47 2 . 30 3 . 10 7.05 10 . 92 23 . 52 40 . 89 46 . 6 1 3Q . 92 - 37 .7

--------------- - -------------- ----------------- "·--· __ ...

.......
'°
l'v
 '
'( )'r
~
}
-~ ....

J~BL1= - 45
l= Ff ECT OF A.RS !:NA.TE ON GROWTH A.ND K0JIC ACID DRODUCTi nN ON ~R01t'TH Ml::DIUM
- - -- - - -- - - · ·- - - - - -- - Pe riod of incubati on \daySJ
Conce nt - 4 5 6 7 8 9 Pe rcent VTet weiah t
10 11 13
rati on cha nae of myce l i -
( N; ) Koj ic ac id p r oduced (mg/m l ) in the um o n 13th
maximum day
koj ic
~c id
- - - - - -- - - - - - - - - - - -- -------------------- - - - ·- ------------------
level
Contro l 10 .65 17 . 23 36 . 21 42 .0 2 65 .05 6 9 . 92 7 5 . 25 61 . 80 31 . 20 6 . 93

10 - 4 7 .5 13 .5 22 . 5 32 .70 50 .40 75 .60 80 .12 58 . 42 30 .51 + 6 .5 6 . 95

10 - 3 and 10- 2 No kojic aci d f o rmat ion No growth

f--
'°w
 V
-J
'y
~
}
),

1 1121.L.::_46
EFFECT OF ARSENATE ON KOJIC ACID DRODUCTION DIJRING RES USP ENSION

Period of i ncubati on ,days

-,--T-----, - -- - -- - - - - -
Concent- 0 l 2 3 4 5 6 7 8 Perc e nt change
ration Kojic acid produced (mg/ml) in the maximum
( M) . koj ic ac i d
· - - - - - - - - - - - - - - - - - - - -- - ·- - - - - leve l _____

Control 0 .70 4 . 28 5 . 89 11. 61 18 . 85 37 . 89 64 .56 74 . 89 63 . 50

10- 3 0 . 39 2 . 28 2 . 84 6 . 24 9 . 89 21.75 36 . 84 42 .75 35 .7 6 - 42 .9

l o- 2 0 .12 0 .70 0 . 86 1. 92 2 . 81 6 ,05 11.48 13 . 26 11 . 85 - 82 .3

----------

I--

'°
~
 195

showed t hat it also uncoupled r espirat ory chain phosphory-

latio n. It ind uc es an ATPase and inhibits the Pi- ATP
exchange r e act ion, the /\DP- t\TP exchang e r eaction and the
excha ng e of oxygen atoms b e tween Pi and water . ( 27 , 2 8 , 2 9 , 3 0)
Ars enate has also been r epo rted to inhibit phos phatas es
a lthough t hese ar e gene rally inhibit ed by the product of
r eact ion. Most interesting are th e inhib itio ns by v ar ious
a nio ns th at may be c onside r ed as a nalogues of e ith e r phos-
phates or th e substrate phosphates.' Thus ars e nate inhibits
various phos phatases almost as pot e ntially as phosphat e
·
an d p ro b a b 1 y comb ine wi· th e nzymes in
· a simi
· . l ar manner(31
. , 3 2 , 3 3 , 34 )
Th e strong inhibition obs e rved in koj ic acid formation by r e-
susp ende d myc e li a would indicate th e involv e me nt of some phos-
phat e deriv atives a nd henc e phosph atas es in kojic ac id synth-
esis .
The p r e s enc e of p , hydroxyqu i noli ne in growth med-
ium inhibited g r owth and henc e kojic acid p r oduction almost
compet1 e 1y at 10 - 2 and 10 - 3 M c onc e ntrat i. ons, whil
. e t h e decr-

ea s e in koj ic acid production was 57. 1% and 38 . 8% at 10- 4

5
and l0- M c once ntrations r es pectiv e ly (Tab l e 47) . At 10- 2 a nd
10 -3M concentrations,
.
it a l most compl etely inhibited the ko jic
acid p r oduct ion by r esus pended mycelia of .,b. . fl avus (Table 48 ).
The inhibito ry action of p- hyd roxyquinoline may be due t o
i. ts a b i. 1 1ty
. · 1 trac e e 1 ements , ( 35 )
to c h e 1 ate ess e ntia
 ,. y'
'_.
y
~
'
I

TABLE- 47

EF FECT OF p- HYDROXYQU I NOLINE ON GROWTI-I AND KOJIC ACID PRODUCTI ON

ON GROWTH MEDI UM
------------------·- - ----------
Perc ent Wet
Conc e nt- Period of i n cubation (day s ) ch ang e We i qht
r at i on 4 5 6 7 8 9 10 11 12 i n t he of myce-
(M) I
maximum lium on
Ko jic acid pr oduc ed (mg/ml) koj i c 12t h
acid day
- - - - - - - -- ----~-- l ev el

Control 10 . 02 17. 25 36.02 41 .7 2 63 . 6 1 67. 20 70 . 20 59 . 81 38 . 90 6 . 66

10 - 5 6 . '37 9 .38 19 , 95 30 . 9 2 4 1.7 2 42 . 92 40 . 52 32 . 42 23 . 4 - 38 . 8 6 . 06

10- 4 3 . 08 5 .62 7.72 13 . 50 17 .10 1 8 . 25 20 . 40 25 . 21 30 . 02 - 57.l 5 . 97

-3
10 · and No kojic acid fo rmation No g r owth
-2
10
---------------------------·-·------- ----------

......
'°°'
 "(
r~
r ~
}'
~~

TABLE - 48
EFFECT OF p- I-NDROXYQUINOLI NE ON KOJIC ACID PRODUCTION DURING RESUSPENSION
Percent cha-
Conc ent r- Period of incubation (days) nge in the
at ion l 2 3 4 5 6 7 8 maximum
(M) Koj ic acid produced (mg/ml) ~ojic acid
l~vel
------
Control 0 .66 3 .70 5.65 10 .72 18 . 27 35 .02 60 . 81 70 . 58 60 . 89

10- 3 0 . 45 1.10 1.55 1.80 1.85 1 . 50 - - - - 97 .3

10- 2 0 .70 1.64 2 . 23 2. 42 2 . 58 2 .12 - - - - 96 .4

-------- - - --------------

......
.0
...J
 198

p . Hydroxyquinoline analogues must have two properties inorder

to be highly fungitoxic; they must be able to chelate and
they must be lipoid soluble . ( 36 ) Rich p roposes that highly
toxic copper oxinate acts at vita l sites which can only be
at t acked by compounds with hioh lipoid solubility . Once within
these sites e.g . microsomes , the half chelate may block funct -
ional g roups by being bound to cellular constituents through
the uns atisfied metallic atom . The less toxic and more water
soluble , unchelated p . hydroxyquinoline may poisin by removing
37
essential metals from water soluble constituents of the cell ~ )
p . Hydroxyquinoline at l 0- 2 M concentration inhibits fungal
3
glucos e oxidase . ( s ) The data obtained suggest the involvem~nt
of some metalloenzymes , pe rhaps even glucose oxidase itself ,
in koj ic acid biosynthesis .
The effects of semicarbazide on mycelial g rowth and
kojic acid synthesis have been repres e nted in Tables 49 a nd
50. It may be observed that in growth medium , semicarbazide
-1
inhibited growth and kojic acid production completely at 10 M
concentration , while at 10 - 2 and 10 - 3 M concentrations , it
dec reased kojic acid production by 42.3% and 25.7% respectively
In the resuspension medium , the decrease in kojic acid pro-
duction was 33 .7% and 26 . 3% at 10 -1 and 10 -2M concentrations
.

r espe ctively . Carbonyl r eagents are r eported to inhibit vit-

amin B 6- dependent enzymes , usua l ly reversibly through binding
to pyriodoxal phosphate . It is r eported that glucose ox idase
2
activity of Penicillium not atlli!l is inhibited 20% by l0 - M
 '( y
•
.)-- -~

'
TABLE- 42
EFFECT OF S EMI CARBAZIDE ON GROWTH AND KOJ I C AC I D PRODUCTION ON GROWTH
MEDI UM

Perc ent Wet

Conc en- Period of incubation (days) chang e we ight
tration 4 5 6 7 8 9 10 11 12 in th e of my-
( M) Kojic acid produced (mg/ml) maximum celium
koj ic on 12th
acid day
- ·- --- l ev el

Control 10 .0 2 17 . 25 36 .02 41.72 63 .6 1 67. 20 70 . 20 59 . 8 1 38 . 90 6 .66

10 - 3 6 .0 3 13 .05 20 . 5 30 . 45 3p .05 40 . 51 45 .62 52 . 22 41 .70 - 25 .7 6 . 24

10- 2 5 . 51 7 .7 2 17. 55 23 .10 27.02 30 . 91 33 . 42 37 . 40 40 . 50 - 42 . 3 4 . 91

10 - 1 No koj ic ac i d f ormat i on No
g rowth
----------------- --- ----·- ---------

.....
'°'°
 y
. )r
.~ '
'
JAB.1.§---=. 50
EFF ECT OF S EMICARBAZIDE ON KOJIC ACI D P ROD! TCTION DURI NG RESUSPENSION
Per ce nt c hange
Conc e nt - Pe riod of i ncubation (d ays ) i n t h e ma xi mu m
ration 0 l 2 3 4 5 6 7 8 koji c ac id
(M) Koj i c ac i d produced ( mg/ml ) l ev e l
- ---
Contro l 0 . 66 3 .70 5 . 65 10 .7 2 18 . 27 35 . 02 60 . 8 1 70 . 58 60 . 89

10- 2 0 . 49 2 . 97 3 .7 5 8 .12 11.10 26 . 20 45 . 32 52 . 02 44 . 50 - 26 . 3

10- 1 0 . 43 2 . 62 3 .35 7 . 24 9 . 81 23 .0 2 40 . 85 46 . 82 39 . 25 - 3 3 .7

~
0
 201

·
semicar b az1. d e ( 38
. ) Th us in
. h 1.b 1. t.10 n 1n
. kOJ1C
. . aci. d synth es1s
' by

both p-hy droxy quino lin e and semicarbazide may possibly result
f r om an i nh i bition of g l ucose oxidase which may be a part
of the kojic acid synthesizing system .
In g r owth medium, sodium nitrate and potassium
nitrate strongl y inhibited kojic acid p roduction at high con-
centrations , while at lower concentrations , the i nh ibition WMS

comparitiv ely less (Tables 5 1 a nd 53) . The effect on g rowth

was less than that on koji c acid producti on . Similar r esu l ts
were obtained in resuspension med ium (Tab l es 52 and 54) . In
r esuspension medium , the inhibition was less as compared with
the g rowth medium . Nitrate ions h ave been r eport ed to inhibit
3
glucose oxidase . ( s)Henc e inhibition cou ld r es ult from a n
inhibition of g lu cose oxidase . Also th e inhib it ion in kojic
ac id production may be due to the r easo n that Secondary meta-
bolite production is often less in media containing high
perc e ntage of nitroge nf 39 )
In order t o asc ertain whether the decreased ko jic
acid format io n was due to an increase in nitroqe n content of
the medium, the effect of ammo niu m sulphate was also investi-
gated . Th e effect of an equ ivale nt conce ntration of potas s-
ium sulphat e was al so studied.
Addit i on of ammonium sulphate to th e g r owth medium
inhibited kojic acid productio n by 57. 8% at 10-lM concent-
ratio n, while in r esuspension medium th e decr ease in kojic
 y ,,.-
~
... ~

'
JABLE - 51
EFFECT OF SODIUM NI TRATE ON GROWTH AND KOJIC ACID PRODUCTION ON GROWTH MEDI UM
Perce nt Wet weight
Concent- Period of incubation (d ays ) cha nge of mycelium
r atio n 4 5 6 7 8 9 10 11 i n the on 11th
(M)
maximum day
Kojic acid produced (mg/ml) 1<oj ic
acid
---- l eve l

Contro l 9 .75 17 . 26 37 . 2 42.81 64 . 21 68 . 91 7 2 . 02 60.52 6 .78

10- 3 8 . 45 16 . 92 19 .74 41.36 55.46 60 . 16 68 . 60 52 . 60 - 4.7 5 . 85

10- 2 8 .12 14.94 17.43 33.52 48.92 53.12 60 .75 48 . 14 - 15 .6 5.65

10- 1 3 . 85 8 .64 10.98 21 . 12 28.82 30 .72 35 .32 26. 88 - 50.9 5 .20

-- -- - - - - - - - - -- -----------

~
[\)
 )' ... ..... r
:.- ~

T\BLE- 52
EFFECT OF SODI UM NI TRATE ON KOJIC ACID PRODUCTION DURING RESUS PENSION
~~-~~--~--~- Perce nt cha-
Concent- Pe r iod of incub ation (days) nge in the
ration 0 l 2 3 4 5 6 7 8 maximum koj ic
(M) . . Kojic acid produced (mg/ml) acid level

Control 0 .7 2 4 .10 5 . 92 11.30 18 . 42 35 . 90 62.70 7 2 . 56 6 1.70

10- 2 0 .63 3 .09 4 . 52 9 . 82 13.52 32 . 41 56 .72 65 . 52 55 . 80 - 9 .7

10- 1 0.44 2 .06 3 .66 7.42 10 . 86 23 .68 46 .62 54 . 20 45 . 81 - 25 .3

------ -

~
w
 -., f'
~
" ... '!

TABLE-= 53
EFFECT OF PCT ASS I UM NITR!'.I.TE ON GROWTH A.ND KOJIC A.CID PRODUCTION ON GROWTH MEDI UM

Pe rc e nt Wet weight
Conce nt - Perio d of i nc ubat i o n (days ) cha nge of myc e-
ration 4 5 6 7 8 9 10 11 in the l ium o n
(M) max i mu m 11th d ay
Koj ic a cid p r oduc ed ( mg/ml ) koj ic
ac id
------ - ~ -- ~ - - --- leve l

Control 9 . 75 17 . 26 37 . 20 42 . 80 64 .2 1 68 . 9 1 7 2 . 02 60 . 52 6 . 80

10 - 3 9 . 68 1 5 . 84 18 . 48 36 . 72 54 . 92 56 . 32 63 . 90 49 . 28 - 1 1 .. 2 5 . 92

-2
10 8 . 90 14 . 82 17 . 42 33 . 50 4g . 02 52 . 9 1 60 . 71 47 . 10 - 15 . 7 5 . 70

-1 14 . 08 18 . 80 20 . 48 22 . 50 1 8 . 56 - 68 ,7 5 .12
10 3 . 20 6 .76 8 .7 2

--
I\)
0
~
 ,- .. r
:.- ~
•

TABLE - 54
EFFcCT OF POTASSIUM NITRATE ON KOJI C ACID PRODUCTION DURING RESUSPENSION
-- - ----- - - - ---Percent change
· Conc en- Period of incub atio n (days ) in the max imum
trat ion 0 l 2 3 4 5 6 7 8 koj ic acid
(M) Kojic acid produced (mg/ml) level
- - ---
Control 0 .7 2 4 . 10 5 . 92 11 . 30 18 . 42 35 . 90 62 .70 72 . 56 61 . 70

-2 56 . 26 65 . 90 54 . 80 - 9 .2
10 0.53 3 . 02 3 . 98 8 . 95 14 . 26 31.28

10- 1 0 .34 1 . 96 2.45 5 . 89 7 . 85 17 . 64 31 . 02 35 . 52 30 .38 - 51 .04

- - ·- --·-

~
(J1
 206

acid productio n was 47 . 3% at the same concentration . Growth

was inhibit ed to a much l es ser extent tha n kojic ac id format-
io n (Tabl es 55 and 56 ) . Similarly 10-lM concentration of
potas s ium sulphate inhibited kojic acid production by 17.2%
and 13 . 4% in g r owth and r es uspension medi a (Tabl es 57 a nd 58) .
The inhibito r y effect of po tassium sulphate on growth was
of the same order as that on kojic acid production. In this
r es pect potassium sulphat e differed from ammo nium sulph at e
or the nitrates . Ammo nium sulphate caused inhib itions of the
same order as potassium nitrat e and sodium nitrate . Thus it
wou ld ap pear that the inhibition due t o th e latt er do not
r esult from any action on a sp ecific e nzyme . such as glucose
oxidase , but due to a chanqe in th e r elative amounts of nit-
rogen in th e medium . Though potassium sulphate also i nhib it ed
ko jic acid formation , the effect was of much l ess magnitude .
Thus th e potas s ium or sulphate ions may not ac count fu lly
f or the inhibition observed with potassium nitrate a nd with
ammonium sulphat e . However , th e possibility of an invo lv e -
ment of glucose oxidas e in kojic acid biosynthesis can not
be rul ed out me r e ly on th e bas is of these data •
In growth medium phosphat e at 0 . 2M conc e ntration
inhibited kojic acid product ion almost completely . ~ls o , it
supported very littl e growth at this conc e ntration while at
.05M and O. l M c oncentrations , it inh ibit ed ko jic acid pro-
duction by 23 .06% and 98 . 5% r es pe ctive ly . The r esults are
 +-·
Ji.
t

" ~ r

T ~~ 55
EFFECT OF AMMONIUM SULFATE ON GROWTH AND KOJIC ACI D PRODUCTI CN ON GROWTH MEDIUM
Pe rc e nt Wet we i ght
Con ce nt- Period of i nc ub ation (d ay s ) ch ang e of myc e l i um
r at ion 4 5 6 7 8 9 10 11 in the on 11th
( M) Ko j ic acid produc ed (mg/ml) maximum day
kojic
ac i d
l ev e l

Cont r ol 9 . 75 17 . 26 37 . 20 42 . 80 6 4 . 21 68. 91 7 2 . 02 60 .52 6 . 82

10- 3 9 . 12 16 . 56 22. 92 40 . 20 54 .60 60.01 6 8 .6 1 53 . 50 - 4 .7 6 .1 0

10 - 2 8 .5 15 . 2 22 . 32 38 . 48 53.28 58 . 80 6 7.52 51. 50 - 6 .2 6 .0 1

10- 1 4 .1 7 .38 8 .61 16 .64 24 .99 26 . 24 30 .40 22 . 90 - 57. 8 5.19

-------·-----

rv
0
.....J
 )..
~ ·

...
'fr
... r

-TABLE-
- --56
EFFECT OF AMMON IUM SULFATE ON KOJIC ACID PRODUCTION DVRIN3 RESUSPENSION
- -.-- Perc e nt
Con ce nt- Period of incubation (days) chang e in
ration 0 1 2 3 4 5 6 7 8 the maximum
(M) Kojic acid produced (mg/ml ) kojic acid
--- lev e l

Control o. 72 4 . 10 5 . 92 11.30 18 . 40 35 . 90 62 .70 72 . 51 61.50

-2
10 0 .60 3 . 84 4 . 82 10.56 16 . 42 34 . 56 60 . 48 70 . 20 59 . 52 . - 3 .2

10- 1 0 . 36 2 .08 2 . 80 5.72 8 . 80 18 .72 32 .76 38 . 20 32 . 24 - 47.3

. ...
_

~
00
 ~ ,,....
), ~ :...

JA.BLE - 57
EFFECT OF POTASS !UM SULPHATE ON SROWTH AND KOJIC ACID PROOOCTION ON GROWTH
.. MEDIUM
- - --Percent Wet
Concent- Period of incubation (days) change weight
ration 4 5 6 7 8 9 10 11 in the of my-
: (M) Kojic acid produced (rrg/ml) maximum celium
koj ic on 11th
acid day
-- - -- lev~--

Control 9.75 17. 26 37 .20 42.80 64.19 68.91 72.02 60 .52 6.82

10- 3 9.21 16 .28 23.96 41.20 56 .16 61.40 69 .72 54.71 - 3.2 6.32

10- 2 8 . 80 14.98 20 .20 38.81 54.82 60.25 68.16 51. 50 - 5.3 6.10

10- 1 8.02 13.72 19.21 36 .08 48.38 52.48 59.60 45.90 -17 .2 5.82
---- ---·

~
\{)
 ),
~
~
~
~ :r

TABLE_:_~
EFFECT OF POTASSIUM SULPHATE ON KOJIC ACID PRODUCTION DURING RESUSPENSION
-- ---
Period of -incubation
- - - -nld-
daysT Percent
--,

Concent- change iri

ration 0 I l 2 3 4 5 6 7 8 th e maxi-
(M) Kojic acid produc ed (mg/ml) mum koj ic
_ _ _ _ acid leve l

Control 0 .72 4 . 10 5 . 92 11 . 30 18 . 42 35 . 90 62 .72 72 . 52 61 . 50

10- 2 o . 68 3 . 88 4.85 10 .67 16 . 80 34 . 92 61.12 71 . 28 60 . 14 - 1 .7

-1
10 0 .60 2 .04 3 . 35 8 . 46 12 . 90 30 . 96 54 .18 62 . 78 58 . 82 - 13 .4

--

I\)
1--
0
 211

pres ented in Tab l e 59 . Th e i nhibition of g rowth at h igh er

co nc e ntrations by s ulph at e and phos ph at e co uld r esu l t f ro m
an inhibit ion of glyc olysis . As it has bee n r eport ed i n
lit erature th at multiv a l ent a nio ns in g e ner a l ar e the inhi-
bit ors of g lyc ol ys i s . The anae rob ic f ormation of l act at e f r om
glu cos e in pige on he mo lysates h as been r eport ed t o b e depress-
ed 84% by 40 mM sulphat e , 64% by 20 mM phos ph Mte , 10 0% by
1
4 . 2 mM oxa l ate and 48% by O . 1% r ibonucle at e:{ ~ The r eact ions
f rom glu cos e to l actat e a re inhibit ed more strong ly t ha n f r om
fructose-1,6- diphosph at e t o l actat e; therefore there i s i nhi-
bition previou s t o fruct ose-1,6 - diphosph ate . The sequence
f r om g luc os e t o g lyc eraldehyde- 3- phosph at e is inhib it ed stro-
ng ly by sulph ate and rib onucleate . Al do l ase is inhib i t ed about
20% by 40 mM s ulph at e , but is not aff ected by ox alat e . It was
cond luded that there are t hree sit es of action ; t h e s tro ng est
on hexok inas e , next on glyc era ledhyde- 3- phosph ate dehydro-
gena se and the la st poss i b ly on pyruvate ki nase , th e inhihit-
ions being competitiv e with ATP or NAD. However , it is diff -
icult to attribut e th e inh ib ition of ko jic acid f ormatio n
by sulphate and pho s ph at e ions to th eir ef f ect o n g l ycolysis
as it h as been r epo rted t hat gluco s e is dir ectly conv er ted in
to koj ic aci d with out und er going g lyc olys is . The i nh ib it i on
in kojic acid product ion by phosphat e a l s o could r es ul t f rom
or
an inh ibit ion of g luc ose- 6- phosph at e dehydrogenase/pho sphat ases .
 ~
~-
'' (
..__
,. r

TABLE--=-22
EFFECT CF I NORGANIC PHOSP HATE ON GROVITH AND KOJIC ACID P RODUCTION ON GROWTH
MEDIUM
- - - ------·
Pe rc ent We t we i g ht
Conc ent- Peri od of incub at io n (day s ) ch a ng e of myc e lium
rat~ on 4 5 6 7 8 9 10 11 in th e on 11th
(MJ Koj ic acid p roduc e d (mg/ml) max i mum day
koj ic
ac id
l ev e l
--------------------· ·- - -
Co ntrol 10 . 65 17 . 23 36 . 21 42 . 02 65 .0 5 69 . 92 7 5 . 02 61 .08 6 . 88

.05 6 . 30 10. 80 15 . 45 25 . 84 36 . 80 47 . 62 57 . 70 47 . 04 - 23 . 06 6 .65

.01 0 .11 0 .21 0 . 225 0 . 45 0 .56 0 .78 1 . 12 0 .78 - 98 .5 3 . 95

.2 No ko jic acid formation No growth

l'0
I-"
l'0
 213

As it has been r epo rted that phosph ate inhib its phos phatases
a nd g l ucose- 6- phosph ate dehydrogenase at a concentration ,
above O. l M :i.n a competitive manner.
The findings of this series of experiments and the
probab le infe renc es derived are summarised in Table 60 •

TABLE - 60

Inference
Inhibitor Enzymes or processes probably
involved i.n.__koiic ~id bio~nthesis
l . Riboflavin Flavoprotein enzymes , perh aps glu-
conic acid dehydrog e nase
2. Glucose-6 - phosphatel Hexokinase and glucose
de~ydrogenase
3. Fructose 1 ,6 diphos~
phate
4. Sodium azide Need for energy or th~ involve-
ment of glucose dehydrogenase
5. Fluoride Role of phos phorylat ed int ermed-
iates
6. Iodoacetic acid 6 Phosphogluconic acid dehydro-
ge nase
7. Ar~enate Involvement of some phosphate de r-
ivatives and he nce phosphatases
8. p . Hydroxyquinoline Metalloenzymes perhaps glucose
oxidas e
9. Semicarbazide
10 . Sodium nitrate

11 . Potassium nitrate
l Glucose oxidase

Importance of r elative amounts

of nitrogen in the medium
12 . Ammonium sulphate
 214

13. Pot as s i um su l ph at e No cl ear infere nce

14. Phos phat e Gluc ose-6-phosphate dehydroge-
na ses a nd phospha tases

It s eems like ly that one or more ·of the e nzymes

. \

g l ucos e ox idas e , g lucos e dehydrog e na s e, g luconic acid dehy-

drog enas e, hexokinase >g lucose- 6-phosphate dehydrog e nase and
6-phosphogl uconic acid dehydrogenase as we ll as s ome phos-
phorylat ed intermediates may be involv ed in koj i c ac id bio-
synthesis. Some of the s e pos s ibiliti es h av e been furth e r
inves tigat ed by det e r mining th e activities of th e r e l ev ant
enzymes in h• flavus myc e lia und e r dif fe r e nt conditions. The
data on the enzyme activities are pres ent ed in the next
Ch apt e r •
 215

REF ERENCES

l. Webb J .L., Enzyme and Meta bolic Inh i bitors , vol II ,

Academic Press , New York (1966 ).
2. Singe r T. P . a nd Kear ney E. B., Arch. Bioch e m., 27 , 348
(1 950 ).

3. Blakely E. R. and Cifer r i o., Can. J. Microbiol ., 1

61, (1961).
4. We il- Ma lherbe H. and Bo ne A. D., Biochem. J ., 49
339 (195 1) •

5. Cran e R.K. and Sols A., J . Bio l. Chem., 203 , 27 3

(1 95 3) •
6. Colowic k S . P. and Ka lc kar H. M., J . Riol . Chem ., 148
117 (1943) .

7. Strecker H.J., in 'Metho ds i n Enzymo logy', Edited by

Colowic k S . P . nnd Kapl an N. O., Vol . I , Academic Press ,
New York (1 g55 ), p .335 •

8. Tiss ieres A., Bi ochem . J ., 2Q , 279 (1051) .

o Moog F . and Sp i ege l ma n s., Proc . Soc . Exptl . Bio l. Med .,
1.2 , 392 (1942) •
10 . Rosenburg A.J., Compt . Re nd. Acad. Sc i., 226 , 1751
(1948) .

11. Hotchkiss R.D., Advances i n Enzvmology ., 1, 153 (1 044 ) .

12 . Case E. M. and Mcilwain H., Biochem . J., 48 , 1 (1951)
13 . Lo omis W. F. and Lipmann F., J . Biol . Che m., 172 , 503
(1949) .
 216

14. Spiegelman S ., Kamen M. D. and Sussma n M., Arch . 8ioch em.

~ , 409 (1 948) .
15 . Hoch F . L. and Vallee R~L ., in ' Trace Elements ', Ed it ed
by Lamb C. A., Bentl ey o.~,. and Beatt ie J. M., Academic
Pr ess , New York (195 8 ), p 337 •
• 16 . Rakestraw J.A. and Roberts E. R., Biochim. Bioohys. 4ct a ,
l1 , 388 (1957 ) •
17. Suelter C. H., Deluca M., Peter J . B. and Boyer P. D.,
Nature ., 192 , 43 (196 1) •
18 . King T.E., in ' Methods in Enzymology' , Edited by
Colowick s.P. and Kapl an N. O., Vo l IX, Academic Pres s ,
New Yo r k (1966),p . 98 •
19 . Borei H., Ark iv. Kemi Mineral. Geo l., b2Q, 215 (1 g45) .
20 . Reiner J . M., Proc . Soc . Exptl . Biol . Med,,£~ , 81
(1 946) .
21. Hackett D. P. in Handbuch de r Pfla nz e nphys iolog ic , . Edited
by Ruhl and W. Vo l 12 , part 2 , Springer , Berlin (1Q60)
p . 23 •
22 . Warburg D. and Christian W., Biochem . z., 310 , 384
(1942) .
23 . Rapkine L., Biochem . J. , 2l, 1329 (1Q38).
24 . DeMoss R.D. , in ' Methods in Enzymology', Edited by
Colowi ck S . P. and Kaplan N. O., Vo l I, Academic Pr es s,
New York (1955 )Jp~ 328 •
 2.1 7

25 . Needham D. M. and Pillai R.K ., Biochem . J ., ~l , 1837

(1 937) .

26 . Sanadi D. R., Gibson D. M. , Ayengar P . a nd nuellet L. ,

Biochim. Biophys . Acta , 13 , 146 (1 q54) .
'Zl . Crane R . K. and Lipman F. , J . Biol . Chem . , 201 , 235
(1953) •

28 . Wadkins C . L ., J . Biol . Chem ., 235 , 3300 (1960) .

29 . Azzone C. F. and Ernster L., J . Biol. Chem. , 236 ,
15 10 (1 96 1 ) .

30 . Ch a n P . C., Lehninger A. L . and Enns T., J . Biol . Chem. ,

23~ , 1790 (1 960 ) .

31 . Garen A. and Levinthal C., Biochim. Bi ophys . Acta, 38 ,

470 ( 1960) .

32 . It o E., Kondo S . and Wata n abe S ., J . Biochem. (Tokyo) ,

4 2 , 793 (1955) .
33 . Mac do nald K., Bioch em . J ., 12Q , 154 (1061) .
34 . Umemura Y. , Nishida J ., Akazawa T. and Uritani I .,
Arch . Biochem . Biophys ., 92 , 392 (1961) .
35 . Zentmyer G. A. , Phy topatholog y ~ 33 , 1121 (1 q43 ).•

36 . Bl oc k S . S ., J . Agr . Food . Chem ., 1 , 229 (1955) •

37. Rich s ., in ' Pl ant Pathology', Edited by Ho r sfall J . G.
and Dimond E, A., Vol II , Ac ademic Press , New York (1960 )

p . 507 •
38 . Bentley R., in ' Methods i n Enz·,mology', Edited by
Co l owick S . P . and Kaplan N. O., Vol r· Academic Press,
New York , (1955 ) p - 340 •
3 '1 . G-, ~~ ~.I<. ) V,'.J.> WCv?-> o.Xt;~ L . o,.;n ol V ~ uJ:v;evwiCvrvl a_,,,
TA·, S. UJf:/Y, . M,'01cd,iaf) fi , :243 c_1q71)
-------
 CHAPTER.- VIII

STUDIES ON SOME REJ..]Vl\NT ENZYMES

),.
 218

CHAPTER - VIII

STUDI ES ON SOME RE LEVA.NT ENZYMES

The biosynthesis of koj i c acid is of interest , bec-

ause a wide V.Jriety of subst r ates may be utilized f or its
production. Koj ic acid is reported to be formed from C- 2
compounds ( e . g . ethano l, g lycol l ic acid ), C-3 compounds (e .g .
glycerol , dihydroxy acetone) , pentoses , hexos es , C-7 compo-
unds (e.g. qu inic acid, shikimic acid ) disacch arides and
more complex polysaccharides (e .g . starch, dextrin etc . )
Further , it s eems like ly that the C-6 precursor of this mold
metabolit e i s a compound s ynthesized by the wel l known r ea-
ction sequences of carbohydrate metabolism ; however be caus e
of fea tures unique to the microo r ganis ms producing kojic acid
this precurs or is no lon9.er metabolized i n the usual way ,
ultimate ly to car bo ndioxide . Instead , special (shunt react-
ion) mechanisms co nv e rt the C- 6 precurs or into a r elat iv ely
undegraded pr oduct, that is , koj ic acid . It was hoped that
a study of biosynthesis of koj i c acid in Asperqil lus f lav us
wou ld therefore pr ovide information r egarding both the gen-
era l and special r ea ct io n sequ ences of c ar bohydrate meta-
bolism occurr ing in this organism. Some information has
already been obtained from previous studi es on kojic acid
. . (l 2 3 4 ) 14
b1osynthes 1s. ' ' ' The r esults obt ained with C-labell ed
c ompou nds showed that i n the cas e of kojic ac id produced f r om
,A
 219

14
D-glucose-1- C, 70 to 90% of the radioact ivity was located
in C-1 and 6 to 16% i n C- 6 . In the ca se of ~ojic acid pro d-
uced from D- g lucose- 3 , 4- 14 c2 , 90% of the r ad io ac tivity was
located in C- 3 and C- 4 ; and in the ca se of ko jic acid prod-
uced from 1-3 dihyd r oxy-2- propa none- 2- 14 c, 60 to 70% of
(2 3)
the radioactivity was found in C- 2 and C- 5 of kojic acid . '
These radio activity incorporation studies indi cate very
strongly that kojic acid is formed from D-glucose l arg e l y
by direct c onver sion with out spl itting of the carbo n ch ai n.
Similar r es u lts were obtained by Kitada and Fukirnbara! 5 )
fvb reover, if f ree dihydroxyacetone or any other C- 3 compou nd
were an important int ermediate , the convers ion of D- glucose -
14
1- C wou l d h ave lead to a more extens ive incorpor atio n of
14
c into C- 2 to C- 6 of kojic acid . The presence of 6 to 16%
14
of C in C-6 , never th e l ess , point ed towards the occu-
rrence of a minor pathway th r ough 3- C compou nds for kojic
ac id biosynth esis . Desp i te of abov e studies , the exact bio-
s ynt het ic pathway and the enzymes involved are not yet known.
In view of th e above s t ated facts , certa in enzy-
mes which may possibly be involved in kojic acid biosyn-
thesis h ave bee n assay ed in c ell free extrac ts and th e ir
r e l at ionship to kojic acid biosynthesis ha s bee n investi-
gat ed . By studying the enzymes under diff ere nt co nditio ns
us ing gr owing and r esusp ended myc e lia , it should be pos s ible
to distinguish between en zy mes inv olv ed in kojic acid
 2~

synth esis and thos e which only play a r ole i n the g rowth of
th e fungus .

lv1ATERIAIS AND METI-IODS

All the studi es we r e performed on a nontoxigenic
strain of _6sp e rqillus f l avus isolated in this l abo r atory
and termed as Culture Bas ment ioned in Chapter III. Th e
methods pertaining to org an ism and cultural cond itio ns have
already been described in Chapter II . 5 ml of th e unif orm
6
s por e suspension containing 10 spores/ml was us ed to inocu-
late 50 ml of th e growth (YES) medium contained in 250 ml
Erlenmeye r flasks . The flasks wer e incub ated at 3o 0 c as usual
as st at ic cultures . For r es usp ension experime nts, the mycelia
wer e first gr own in YES med ium f or 6 days . They were th e n
r esus pe nd ed i n 50 ml st eri le 0 . 2M phosnhat e buff er, pH 6 .5
with or without 20% g lucos e . For s ome experiments 3 weeks old
myc elia grown on YES medium we r e us ed . In some oth er experi-
me nts t he washed myc eli a we re pre inc ub ated in th e buff er alo-
ne for s even days and wer e then r es us ne nded in 50 ml st erile
buffer supplement ed with 20% g lucose.
The e nzymes we re assayed in mycelial homoge nates
prepared as fo l lows . Th e growth or r esusp ension med ium was
decant ed, the myc elial pads were wash ed wit h the r esp ectiv e
buffe r and ground well with cold buffer and acid washed sand
 221

in a chill ed mortar . Th e de tail ed proc edure has b ee n des -

crib ed in Chapt er I I . The homog e nat e was centrifug ed in the
cold at 20 , 000 r . p . m. f or 30 minut es and the cl ear sup e r -
natant was stored in a deep fr eeze till furth er us e for de t-
er mining t he pr ot ei n conte nt and enzyme activity within 24
hours . Dep ending on th e e nzyme to be assayed diffe r e nt buff-
ers we re used to prepare the homog e nate as already be en
mentioned in Chapter II . The details of th e different e nzyme
assays have also b een described i n Chapt er II .

Pap er Chromatography
0 . 5 ml of th e crude homog enate of~. flavus myce-
lium in O. l M phosphate buff e r , pH 6 . 0 and in O.lM glycyl
glycine buffer , pH 8 . 0 was incubat ed r esp ectiv e ly with 0 . 2 ml
of 0 .6M glucose for 15 minutes and with 0 .4 ml of 0 . 2M glu cose
and O. O~ml of O. lM ATP for 15 and 30 minut es at 25°C . The
r eaction mixtures were conce ntrat ed in vaccum and we r e chro-
matograph ed (desc ~nding) on Whatman no 1 . filt er pap er in
three diff er e nt solvent syst ems in each case. Th e solv e nt
syst ems us ed we r e butanol : acetic acid: wat e r , upper phase
from a mixture of water: s econdary butanol : tertiary but-
anol and isopropanol : cone HCl : water . Furth er details h av e
bee n describ ed in Chapt e r II . Th e chromatograms wer e air
dried and sprayed with hydroxylamin e r e agent in cas e of th e
crude homog enat e incubat ed with g l ucose and with Hans and
 222

Isherwood r eagent.in case of the crude homoge nate incubated

with glu cose and ATP . The Rf va l ues of the spots obta i ned
were compared with t hose given by authentic samples of glu-
conic acid- ~ - lactone , glucose- 6- phosphate , 6 phosphoglu-
co nic acid , ADP and ATP .

RESULTS AND DISCUSSION

I n the comp l ete absence of any infor mat io n o n the
enzymes i nvolved i n the pathway of kojic acid biosynthesis ,
the enzymes to be studied had to be selected on th e basis of
sever al i ndi r ect lines of evide nce .
(1 , 2 , 3)
The studies of Arnste in and coworkers have
established the fact that the g lucose molecu l e is convert ed
dir ectly into kojic acid without a cleavage of th e carbo n
chain into smaller fragments . Theoretically , starting from
the py r anose form of glucose , such a conversion should in-
vo lve a minimum of 3 r eactions , name l y oxidat io n of a
secondary hydroxyl group a nd the i ntroductio n of 2 double
bonds by re moving th e e l ements of water as shown below .
 223

However, sinc e the follo wing enzymes of glucose

metabolism have been r eported in mi croorgani~ms and espe-
cially in fung i , their pos s ible i nvolv ement i n kojic acid
biosynthesis had to be kept i n mi nd. Furth er , the effect of
d i ffe r e nt inhibi tors on kojic acid production under d iff-
erent experimental conditions as described in the previous
Chapter also p r ov ided indirect ev i dence for their pos sible
participation in the synthesis of kojic acid . The r e levant
enzymes are hexokinase , glucose-6 - phosphate dehydrogenase ,
6-phosphogluconate dehydrogenase glucose ox i dase , g lucose
dehydrogenase and gluco nate dehydrogenas e .A lso, preliminary
experiments indicate th e presence of the above enzyme acti-
vities i n crude homog enates of~ . f l filUd.§. mycelia . Paper
chromatog r aphic studies to be described below provided evi-
dence for the formation of 9luconi c acid - ~ - lact one , g lu-
cose- 6- phosphate and 6- phosphogluconic acid from glucose in
such homogen ates .
It may be worthwhile t o discuss here brief:y the
information availab l e i n th e liter ature on some r e lated enzymes
in microorganisms .
Glucose dehydrog enase and gluconate dehvdrogen-
ase have b een reported in a number of bacteria and animal
tissues .( 6 to lO)Glucose dehydrogenase catalyzes the dehy-
drog e nation of glucos e , ge nerally using the coenzymes NAD
or NA.DP . The presenc e of this enzyme has bee n report ed i n
hfil:2erqill~ Oryzae ~ll )The Aspergillus oryza~ enzyme is a
 224

glycof l avoprot e i n cont ai ning one mol ecul e of FAD per ~ol e-
cu l e of enzyme . One of th e majo r charact eristics of ,h. oryzae
g lucose dehydroge nase , distingu ishing it from fungal g lucos e
oxidase , galactose oxidase and algal carbohydr ate oxidase
has been its inability to r eact directly with mol eculer oxy-
gen. 1he failu r e of pyridine nuc l eotides to act as acceptors
fo r h. oryzae enzyme is also i n marked contrast to liv er
g luc ose dehydrogenas e , Bacillus gluc ose dehydrog enase a nd
(1 2)
Pseudo monas aldos e dehydr ogenas e .
Hexokinas e , gluc ose- 6-phosph at e dehydroq e nas e and
6-phosphogluconat e dehydrogena~e are gene rally found through-
ou t the liv i ng ki ngdom i. e . p lants , animals and bacteria .
Hexoki nase c atalyz es the t r ansfer of phosphat e group from
ATP to hexoses t o form correspo nding phosphoryl at ed int er-
mediates . Gluc ose- 6-phosph ate dehydrogena se is generally
NADP- dep e ndent , bu t in Leuconostoc mese nt e!:QiQ§, an e nzyme
3
is pr es ent tha t can utilize eith e r NAD or ~ADP. (l ) 1his
. e d f rom y east and anima
enz yme h as bee n crysta 11 iz (14,15)
. 1 s ourc es.

The y east enzyme shows a high spec ificity fo r ~ADP . Us ua lly

6 -phosphogluconat e dehydrogenase is a l s o NADP- specific,
although th e e nzyme obtained fro m ];.eucQ.D~t oc mesent e r oids
. (16 )
r educ es NAD at 25 times th e r ate of NADP I n As pe rgillus
flavus - ory~ , apparently two dehy drogen ases are elaborat ed ,
one uti lizing NAD and th e oth er NADP . (l?) Purification stud-
A
i es hav e b een r eport ed for th e dehydroge nase f rom yea st ,
 225

( 1 8 , 19, 20 )
and animal tissues. Glucose oxi-
dase is found in algae, fungi a nd bact e ria~ 21 to 24) This
enzyme utiliz es oxyg en directly through FAD co e nzyme and pro-
duces H202 • Pseudomonas fluorescens pos es s es an unusual g lu-
cose oxidase, in wh ich oxidation by atmospheric oxygen is
(25)
mediat ed through a cytochrome system.
The r e lev ant enzymes were assayed at dif fe r e nt per-
iods of times during the growth of ,h. flavus and in r esus-
p e nded mycelia under differe nt conditions . An attempt has b een
made to correlat e the data obtained with th e extent of kojic
acid production under these differe nt conditions .
The production of some of th e metabolic int ermed-
iat e s from glucose by crude homogenates was test ed by pap er
chromatography .
Wh en a crude myce lial homogenat e was incubat ed with
g lucos e_for 15 minutes at 25°C and submit ted to chromato-
rgraphy , a single spot giving Rf v alues of 0 . 54, 0 .63 and
0 .77 in th e 3 di ffe r ent solv ent syst ems was obtained 0n spray-
ing the chromatogram with hydroxylamine r eag ent . ( Fig. 16).
These Rf v alues correspond ed to thos e of the standard sample
of g luconic ac id- b -lactone in th ese solv e nts , Glu conic
acid - &-lactone has a l so been identified as a r eaction
product during the dehydrogenat ion of glucose ca talyz ed by
glucose dehydrogenase in As~ergill.!d.§. orY~ill)
FIG . 16 PAPER CHROMATCGRAPHY OF A HOM~ENATE OF S,PERGILLUS

£LAWS I NCUBATED WITH O . 6M GLUCOSE FOR 15 MINUTES

AT 25°C .

~OLV8'JT SYSTEMS :

A) Butanol : acetic acid : water

B) Uppe r phase f r o m a mixtur e of H2 o secondary
butanol : tertiary but ano l •
C) Isopropanol : cone HCl : water .

SPOTS :

l . Glucose s t andard •

2. Gluco nic acid - S- la ct one standard

3. Homogenate+ glucose a t z e ro ti ~e •

4. Homog e n at e + glucose after 15 mi nut es incubati.on

 226

A homogenat e incub at ed with qlucose and ATP fo r

15 minutes at 25 °C yielded t wo spots in case of butanol:
acetic acid: wat e r s olv e nt and three spots in ca se of th e
other t wo solvents , on s pr ay ing th e chromatogram with Hans
a nd Isherwood reagent . One of these h ad th e Rf valu es of
0 . 18 , 0 . 23 and d .74 in th e thr ee solv e nts r espective ly corr-
esponding to an authentic sample of qlucose- 6-phosphate
(Fig . 17 ) . The other spots that gave Rf va1u es of 0 . 17 and
0 .096 , a nd 0 . 40 and 0.45 in two of th e solvents , we re shown
to be ATP and ~DP respect ive ly f r om th e correspond ence in Rf
values . ATP and ADP had th e same Rf value of 0 .065 in butanol-
acetic acid : water solvent system . ATP was already pres e nt i n
the reactio n mixture and ADP should hav e been formed as a
r eactio n product.
Whe n the inc ubation with glucose a nd 4TP was ext -
end ed for 30 minut es , an add itio nal s pot wi th Rf valu es of
0 . 23 , 0 .16 and 0 . 87 i n th e above so lvents was obtained on
spr ayi ng th e chromatogram with Hans and I sh erwo od r eagent .
Th ese Rf valu es correspond ed to th e Rf values of an auth e-
nt ic sample of 6- phosphogluconic acid (Fig . 17) • The spot
due to glucose- 6- phosphat e was comparitiv e l y faint e r in samp-
l es i ncubat ed for 30 minutes . The above data further c onfirm
the pr esence of hexokinas e , glucos e- 6- phosphate dehydrogen-
ase and eith e r glucose oxidas e or glucose dehydroge nas e in
the mycelium .
To confirm that kojic acid was the only ferric
ch l oride positiv e substanc e formed during th e f ermentation
 PAPER CHROMATOGRAPHY OF HOMOGENATES OF ASPERGILLUS

FLh\/tJS I NCUBATED WITH 0 . 2M GUJCOSE ANO O . l M ATP AT

25 o C FOR 15 MINUTES Ci.ND 30 MI NUTES •

SOL VENT SYSTEMS

A) Buta nol ac et ic acid : water

B) I soprop ano l : cone HCI : wa ter
C) Upper phase fr om a mixture of H20 secondary but anol
t ertiary butanol •

SPOTS :

1. ATP s tandard .
2. Glucose - 6- phosphate standard .
3. 6- Phosphogluconic acid standard .
4. ADP s t a ndard.
5. Reactio n mixtur e at ze ro time.
6. Reaction mix ture after 15 minut es incubat ion .
7. Reaction mixtur e aft er 30 minutes incubation .
 227

process , it was desired to chromatograph the f er me nted medium

and mycelial extracts on Whatman no l. filter paper in three
di ffe r ent s olvent systems. The solvents used were butanol:
acetic acid : water , upper phase from a mixture of ethyla-
cetate : water : formic acid and isopropanol : cone HCl
wat e r . Other details h av e been described i n Chapter II •
The chromatograms were air dried and sprayed with ferric
chloride reagent. The Rf values of th e spots were compared
with an au t hentic s amp l e of koji c acid . A single spot having
the Rf values of 0 .74 , 0 . 51 and 0 . 89 was obtained in these
three solvent systems (Fig . 18 ) . These Rf val ues co rr espond-
ed to th e Rf values of a n authentic samDle of kojic acid in
these so l vents . Thus it is conf i rmed that kojic acid is the
only ferric c hloride pos itive substance formed during the
~ flavus f ermentation understudy .
The following enzymes were d et e rmi ned in the myce-
lia of h • flavus at different periods during its growth on
yeast extract sucrose med ium: Gl ucose oxidase , aluc ose de-
hydrogenase , gluconate dehydrogenase , hexoki nase , glucose-
6- ph osphate dehydrogenase and 6- phosphog luconate dehydrooen-
ase . The r esults are expr es sed both as the amount of enzyme
units per ml of th e homogenate and as specific activity or
units per mg protein in Tab l e 61 . The enzyme units us ed
r epres ent the r eaction of l micromole of substrat e p er min-
ute in th e cas e of all th e enzymes . It may b e noted th at l ml
 PAPER CHROMATC'GRAPHY OF THE GROWID MEDIUM AND

A(l.JE(JJS EX TRACTS OF THE .~ . FL AWS MYCEtIUM ,

A FTER 10 DAYS GROWTH ON YEA.S T EX TRACT SUCROSE

MEDIUM •

SOL VENT SYSTEMS :

.4. ) Buta nol : aceti c acid : water

B) I sopropanol : cone HCl : wat er
C) Upper phas e from a mixture of wat er seconda r y
butanol : t ertiary butanol •

SPOTS :

1. Koji c acid standard .

2. Growth mediu m.
3. Mycelial extract .
 .... .. ~
.._ •
..

TABLE - 61
ENZYME ACTIVITI ES IN MYCELIA GROWN ON YEAS T EXTRACT SUCROSE (YES ) MEDIUM
- - -·
Period Glucos e Ox idase Glucos e dehydrogenase Gl uconat e dehydrogenas e
of in-
cub at i on
(days ) Units/ Spec i f ic Units/ Spec if ic Units/ SDec if ic
ml of activity ml of ac tiv i ty ml of activity
homog- ho mog - homo-
enate e nat e genate
- -------
3 0 .093 OiJ 206 0 . 250 0 . 050 0 . 387 0 .088

7 0 .066 1 O. ()77 9 0 . 240 o . 2ss 0 . 312 0 . 36 8

10 0 .0360 o . 0 833 0 . 270 0 . 610 0 .438 0 . 995

15 0 .0205 O .0219 0 . 360 0 . 383 0 . 410 0 . 438

---

rv
S3
 " ... :r ~ I i
=-

..
T2b le 61 contd .

Period Hexokin ase Glucos e- 6- phosph at e 6- p hosphoglucon ate

of in- ~ dehY,s;!f.Qgena~-e ~~ -~g ehyd r og enase
cubation Uni ts 1 Spec if ic Units/ Speciric Un its/ Specific
(days) ml of activity ml of ac tivity ml of activity
homo- homo- homo-
gen ate genate genate

3 43 . 43 9 . 89 1.09 0 . 248 0 . 920 0 . 205

7 16 . 57 19 . 54 0 .137 0 . 16 1 0 .120 0 . 134

10 8 . 54 19 . 8 3 0 .090 0 . 208 0 . 109 0 . 252

15 8 . 34 8. 90 0 .086 0 . 098 0.090 0 .096

~
'

230

of th e homogen ate c orres ponds to 100 mg wet weight of the

myc elium . Th e pattern pr ovided by units of activity and by
s pecific activity are oft e n different in this and i n the
subsequent Tab l es , sinc e the p rot ein cont ent of the homo-
genate v aried with time and usually decre as ed with a n incr-
e ase in the incubation p er iod . Th er efore it is bette r to dr aw
any i nf e rence from th e units per ml of th e homoge nate . The
specific activ i ties of the e nzymes s t udied showed an appa re nt
maximum on th e 10th day wit h the ex cep tion of g luoose-6 - phos-
phate dehydrog enase . Sinc e kojic acid l ev e ls are also maxi-
mum around th e 10th day during g rowth on YES mediu m, this
s eems to sugges t th at all of these en zymes ma y be inv o lv ed
i n kojic ac id biosynthes is . But a l ook at the activity per
ml of the homog ena t e g iv es a diffe r e nt pict ure . Th e activiti es
of glucos e oxidase , hexokinas e , g lucos e- 6- phosphat e dehydro-
genase and 6- phos phogluconate dehydrog e nas e decreased consi-
derably during th e growth on YES medi um and were qu it e low
on the 10th day . On th e oth er ha nd th e activit i es of glucos e
dehydrogenas e and gluco nat e dehydroa e nas e o n the 10th day
wer e comp arab l e to t hos e o n the 3rd day . Th is would s eem to
implicate th es e two en zymes in t he koji c acid production.
Whe n the myc eli a of f: . fl aY.b!.§ grown for 10 days
on YES medium were r esuspended in phos Dhat e buffe r supple-
me nt ed with 20% glucos e , th e specific activiti es of glucose
'
231

oxidase , hexokinase , g lucose- 6- phosphate dehyd rogena se and

6- phosphogluconate dehydrogenas e exh i bited a maximum on the
!st day and those of glucose dehydrooenase and g luc o nate
dehydrogenas e on the 7th day (Ta bl e 62) . Th e activities per
ml of homogenat e of g lucos e ox ida se and 6- phos ohog l uco na te
dehydrogena se did not vary mu ch for the fi r st 3 days and
th en dropped to low values o n 7th and Oth day . 1he act ivity
of hexo Y.inas e increased up to 3 days and it d roped to low
va l ues by the 7th day . Glucos e- 6- phosphate dehyd r ogenas e
activity r each ed a maxi mum o n Ist day and decreased ther e-
after considerably . On the oth er h and the activities of glu-
cose dehydrogenase and gluco nat e dehyd r ogenase increased
r eaching a maximum on the 3rd day and r ema ined high on the
9th day al so . It may be noted that kojic acid l ev e ls r each ed
a maximum by about th e 8th day in such a r esuspended myc el i al
system (Chapter VI , Fig . 10) . Thus the patt e rns of both t he
activities and specific activities of these e nzymes indicate
that of th e six e nzymes , glucose dehydroaenase and g luc onate
dehydrogenase ar e mo st like ly to be involved in koj ic acid
synthes is .
1Nhen ,b. fl~!:!§ mycelia g r own for 3 weeks o n YES
med i um wer e res usp e nded in phosph ate buffe r supplemented
with glucose , th e ac tivity and specific ac tivity of glucose
oxidase dec reased co nsiderab ly with t he period of incubation
(Table 63) . 1he activiti es as we ll as specific activit i es of
 ....
...-
L.! t
;.i
"
-
-- 6-~
TARLE-
ENZYME ACTIVITIES IN 10 D4YS OLD MYCELIA DURI NG R~SUSPENSION ON 0 . 2M PHOSPHATE
BUFFER , PH 6 . 5 , SUPP LEMENTED WITH 20% GLUCOSE

Glucose Glucose Gluco'Flate

Period oxid a se ___ dehydrooe nas_e_,,....,,..,..- __ deh cirooenas e -------
of in-
cub ation
Units/ml
of homo-
Sp~e-c_i_f _i_c
activity
Unit s7ml
of homo-
Specific
activtty
7
Units ml
of homo-
Spec i f i c
activity
( days ) genate gen ate aena t e

0 0 . 101 0 . 086 0 . 283 0 .190 0 .275 0 . 190

l 0 . 087 0 .087 0 . 316 0 . 300 0 .625 0 .600

3 0 . 101 0 .060 0 . 870 0 . 560 1 . 252 0 . 820

7 o ·.0'10 0 .0 10 0 .770 0 .780 1 .070 1 . 10 0

9 0 .OC!? 0 . 004 0. 780 d.460 0 .630 0 . 370

- - ---------- - - ----------- -----

f\)
w
j\.)
 4
'~

'-!11.
I,.-
~ ' t
-
Tab l e 6 2 contd.

Hexokinase Glucose- 6 - phosphate 6 - Phosphogluconat e

Peri od __ _ _ ~~d ehy_Qrogenase cle.hvdrooenase
of in- Units/ml Spec ific Unitsl ml Specific Unit s'7 rn 1 - S~
r,--e_c_i_f i c
cubat- of homo- activity of homo- activity of homo- ar: tivity
i on q enate ge nate g enate
_(g§Y~ - - -- -- - - - - -

0 11. 40 9 . 90 0 . 600 0 . 520 0 . 290 0 . 260

l 16 .oo 15 .64 0 .720 0 . 703 0 . 270 0 . 270

3 19 .oo 12.30 0 . 418 0 . 270 0 . 290 0 . 200

7 5 .70 5 . 80 0 . 230 0 . 230 0 .120 0 .120

9 5 .70 3.40 0 . 20Q 0 . 120 0 .070 0 .041

---------- -----------------------------------------------------------------

rv
w
w
 ~ ~
IL t
"" "'
-
IABLE=._63
ENZYME \CTIVITI ES IN 3 WEEKS OLD MYC ELIA DtJq I NG RESUS PENSION ON O . 2M PHOS?HA.TE BUFFER,
p H 6 . 5 , SUPPLEMENTED WITH 20% SLUCOSE .

Glucos e Glu cose Gluconat e

Period ox i das e _ ___2ehyd£2~n as L _ _ _ de!}ydroq e n~- - -
of i n- Unit s/ml Spe c if ic Uni t s/ml Spec i f ic Un it s/ ml Specif ic
cub at- of homo- activ ity of homo- ac tiv ity of homo- act iv ity
I onill a en at e g enat e 0e nat e
----------------
J

0 0 . 042 0 . 052 0 .0 20 0 . 025 0 . 385 0 .470

l 0 . 023 0 . 026 0 ·330 0 . 3 70 0 . 590 0 .660

3 o·.0 11 0 . 017 0 . 330 0 . 520 0 .605 0 . 960

7 0 .009 0 . 0 15 0 .40 0 0 . 6 90 0 .700 1. 20 0

9 o·.o07 0 .008 0 . 550 0 . 670 0 .67 5 0 . 820

--
!\)
w
~
 . ~
L t
4
"

Table 63 c ontt: .
---- - ----- - ----- - --------------------------
Period Glucose-6- phosphate 6-phos phool uconat e
of in_. Hexokinase __ deh drgg e n~------ aeh?d rQ.g~fil§_g__ _ _ _
cuba t -
ion
Dn~i~t-s/
--m~l
of homo-
S_p_e_c_i_f-i-c
activity
7
Units ml
of homo-
Specific
activity
Units ml
of homo-
Specif ic
activity
(day s ) ge n ate genate genate
- --------- ------------------------------------------------ ------------
0 8 .oo 10 .00 0 .100 0 . 120 0 . 090 0 . 110

l 11 . 40 13 . 00 0 . 200 0 . 220 0 . 150 0 . 170

3 6 . 20 o.so 0.082 0 . 130 0 .ooo 0 . 140

7 4 . 00 6 . 90 0 .080 0 . 130 0 .070 0 . 120

9 3 .00 3.70 0 . 050 0 .060 0 .055 0 . 067

I\)
w
(J1
 236

hexoki nase , g lucos e- 6-phosphat e dehydrogenase and 6-phospho-

g luconat e dehydrog enas e showed a maximum o n th e Ist day and
gradually declined aft er that . On t he other h a nd the s peci-
fic activiti es of gluc ose dehydroge nase and gluconate de hy-
drogenase increased r ea ching a maximum on 7th day a nd r ema-
ined high on th e 9th day also . Th e activity of g lucos e de-
hydrogenase r o s e fromQ.02 to0.33 units p er ml on Ist day
and then incr eased gradually to0. 55 uni t s per ml on th e 9th
day. That of g luconat e dehydroge na se also increas ed r eaching
a maximum on 7th day and r e mained high on the 9th day also .
It may be po int ed out h er e that 3 we e ks old myc e lia r esus-
pended in phosphate buffer p lus glucos e synthesiz ed kojic
ac id whose lev el r each ed a maximum o n th e 12th day (Chapter VI ,
Fig . 10) . Thus , aga i n g lucose dehydrogenase and gluconate
dehydrogenase are po int ed out as pr obable enzymes i nvolve d
in kojic acid biosynth esis .
Another s ys t e m th a t has been tested , utilized myce-
lium that h ad been grown o n vEs med ium for 10 days a nd then
pre incub at ed in phosph ~t e buffer alo ne befor e r esuspe nsion in
phos ph ate buff er plus glucose . During pr e incubati on in buf fe r
a lone , th e activities as well as ·specific activiti es of the
e nzymes oth er than glucos e ox idase decreased with time a nd
r eached about 1/3 or 1/4 of th e origin a l v a lue by the 7th
day (Table 64 ). The activity and s pecific activity of glu-
cose oxidase sh owed an initi a l de creas e and th en increas ed
 ·~ ~

~
.w .... '-I

-
TABLE- 64
IN -----
WRING
ENZYME ACTI VITIES/10 DAYS OLD MVCELIA / J:U:SUSPENS I ON ON O .2 M PHOSPHA.TE BUFFER , PH 6 . 5
---------
Period of Glucose oxj.d as,g_ _ ~lUf.OSe 9.ehyd.r.2g_gnase Gluco nat e de hydrooenase
incubat-
io n l 1nits/ml Spec i fic Units/ml Specifi c Unit s/ml Specif ic
( days ) of homo- a ctivity of homo- activity of homo - activity
~·e n a t e g e n at e gen a te
- - - - - - - --·- - -----
0 0 . 098 O . 1 27 0 . 266 0 .133 0 . 400 o . 20 0

l 0 . 06 1 0 . 876 0.160 0 . 123 0 . 257 0 .197

3 0 . 140 0 . 091 0 .1 90 0 .118 0 . 200 0 .120

7 0 . 1 20 0 .120 0 . 050 o . 053 0 .1 12 0 .118

f'v
~
 :.- ......
)I ~
r
"
-
Tab le 64 co ntd .

Period Hexokinase r,lucose- 6- phosph at e 6-p hosphog l uco nate

of in- _ dehyjroqenase -~-- -~gehydroo enase
cub ation Init sTrnI-- specif ic Un i ts/ml Specific Vnitslml Spec ific
(days) of homo- activity of homo- activit v of homo- activity
genate gen ate genate
--------
0 17 . 10 22 . 20 0 . 400 0 ~5 10 0 . 260 0 . 359

l 13 . 70 18 . 40 0 . 360 0 . 480 0 . 270 0 . 360

3 14 .00 9 . 40 0 . 360 0 . 230 0 . 1 80 0 .110

7 6 . 20 6 . 50 0. 160 0 . 170 0 .Q70 0 .070

---------------- --------- -----------------

l'0
w
(X)
•
239

considerabiy. Wh en th e preincubated myc e lium was r esuspend-

ed in buffer and g lu cose , t h e patt ern of changes in enzyme
activities r esembl ed that obtained during r esusp ension of
3 weeks old myc elium. 1h e activity and sp ecific activity of
gluc ose oxidase decrea s ed continuously (Table 65). 1hose of
hexokinase. glucose-6-phosphate dehydrog enase and 6- phos-
phogluconate dehydrogenase roJe to a maximum on the Ist day
and thereafter decreased eons iderably. On th e other hand the
Specific activities of glucose dehydrog enase and gluconate
dehydrogenase exhibited a ma~imum on th e 7th e day. 1he acti-
vity of glucose deh ydrogenase rose from 0. 05 to 0 .23 uni ts/ml
of homogenate on Ist day and then increased gradually to
0.40 units on the 9th day . 1hat of gluc onate dehydrogenase
increased from 0 .11 to 0 .60 units on th e Ist day and then
incre ased to0,69 units on th e 3rd day and remained high
(0.615 units) on the 9th day also . 1his ag ain implicates g lu-
cose dehydrogenase and gluconate dehydrog enase in kojic acid
biosynthesis since th e kojic acid l ev e ls r eached a maximum on
the 10th day in this case-{Chapt er vi 1 Fig. io ).
The activ iti es exhibited in vitro by the dif f er-
ent enzymes were compared with the rate of kojic acid prod-
uction to asc ertain if there is any correlation b etween t hes e
two parameters. It has been calculat ed from th e steep nort-
ions of the cuIVes of kojic acid l ev els against period of
incubation from the Flg, 1 of Chapter I V and Fig. 10 of
 ..
; r I~
¥
"'
-
} AB LE- 65

ENZYME ACTIVITIES I N 10 DAYS OLD MYCELI A. PREINCUBATED ONO . 2M PHOSPHATE BUFFER ;

pH 6 . 5 FJR 7 DAYS DURI NG RESUS PENSION ON O . 2M PHOSPHATE BUFFER, pH 6 . 5 , SUPPLE-
MENTED WITH 20% GLUCOSE
- ·- ---- - -
Glucose oxida1.,e Glu cose dehydroge n- Gluconate dehydro-
Period __ sTmI ______
ase oenase - - -
of in- ----s7rr-
Units ml - --Specific-
--- Unit s ml Spe cif ic ----7rr-
Units ml - - - Specific
cu bat- of .homo- activ i ty of homo- ac tivity of homo- act i vity
i on genate genate ge nat e
_{gro:~

0 0 . 120 0 . 120 0 . 050 0 . 053 0 . 112 0 . 118

l 0 . 080 0 . 081 0 . 233 0 . 233 0 . 600 0 . 600

3 0 .026 0 . 042 0 .330 0 . 390 0 .690 0 . 810

7 0 . 0 11 0 .024 0 . 370 0 .soo 0 . 640 1.400

9 0 .012 0 . 0 10 0 . 4 00 0 . 330 0 . 6 15 0 . 5 10
--

f\.)

~
 -{
:'ii
r ~
¥
•
-
Table 65 contd .

----------------------·
Hexoki nase Glucose- 6- phosphate 6-phospho?luconate
__ dehldrog_gna_s_e____,._,,__ dehydrogen,2,2.§_ _
Period Unitslml Specific Units ml Specific Unifs?ml Spe cific
of in-· of homo- activity of homo- activi ty of homo- ac tivity
cubat- g e'12te ge n ate gen ate
ion
(days)

0 6 . 20 6 . 50 0 .16 0 .170 0 . 070 0 . 070

l 9 .10 9 .10 0 . 260 0 . 260 0 .109 0 .109

3 6 .. 80 8 . 01 0 .180 0 . 210 0 . 090 0 .106

7 3 . 40 ·7 . 40 o.664 0.140 0 . 055 0 .100

9 6 . 80 5 .70 0 .045 0 . 037 0 . 046 0 .038

------------- -----------------------

I\)
~
1--
•
242

Chapter VI that the maximum r ate of p r oduct ion of koj ic ac id

was 0 .0470 , 0 .0580 , 0 .0230 and 0 .0309;tt- moles per mi nut e per
10 0 mg of wet we i ght of mycelium during g r owth on YES med-
ium and during r esuspensio n of 10 days old mycelium , 3 wePks
old myc e lium and 10 days old but preincubated myc elium res -
pectively . One ml of the homoge nat e correspo nd s to 10 0 mg
wet weight of myc e lium . Th e5e rates may be compar ed with th e
activit i es of the r e lev ant enzymes at the time at which kojic
acid lev e ls r eached a maximum . (Tabl es 66) . Of the six enzymes
stud i ed glucose oxidase had the activities of the order O .OC!?
to 0 .036)-l moles of subst r ate per minut e per ml of homog e nate .
This is lower than the observed rat es of kojic acid synth esis .
Thus g l ucose ox id as e i s p r obably not involved i n th e biosynthe-
sis of koj ic acid . Hexokinas e showed th e hioh est activity at
the r e lavant peri od ranging from 3 .00 to 8 .54}1 moles of sub-
str ate per mi nute per ml of homoge nat e . Glucose- 6 - phosphate
dehy dr ogenase a nd 6- phosphogluconate dehydrogenase exh ibit ed
activities r a nging from 0 .05 to 0 . 13 and 0 .046 to 0 . 12,11
mol es of substrate per minut e per ml of homogenat e resp ec t iv e ly .
The act ivities of these two enzymes i n most of the resus -
pension systems studied are suffic i e nt to account fo r th e
rat es of kojic acid sy nth es is ment ioned above . Thus hexo -
ki nas e , glucose- 6 - phos phat e dehydroaenase and 6 - phospho-
gluco nate dehydrogenase ma y be involved i n kojic acid bio-
synthe! is . Data on i nhibition studies also sugo ested th e
 f- ~ (
~
.... "'
-

TA] LE- 66
ACTIVITI ES CF DI FFERENT ENZ YMES ~T A TIME CLOSE TO THE PERIOD OF M~XI MIJM KOJ IC ACID LEVELS
----- - ----------
Enzyme On 10 th day On 7th day of On 9th day of On 9t h day of r es -
du r i ng g r owth resus pens i on r esuspe nsio n US De ns i o n of 3 wee ks
on YES medium of 10 days old of pr e i ncubat- ol d myce l i um
myc elium ed myc elium
--------------- ,--~-~-·----~--~- - -----
Glucose 0. 0 360 0 .0 10 0 . 012 0 .o0'7
oxidase
Glucose 0 . 270 0 .770 0 , 400 0 . 550
dehy d.:,:- ')o e nas e
Gl ucon ate 0 . 438 1.070 0 .6 15 0 . 675
de hydro1cnase
Hexokj nase 8 . 54 5 .70 6 . 80 3 · 00
Gl ucose- 6- 0 . 090 0 . 230 0 "045 0 .050
phosph at e dehy-
dr ogen2s e
6-phosphog lu cona t e 0 . 109 0 . 120 0 . 046 0 .055
dehydrogenase
_________________
,

~
w
 244

roles of phos phory Lit ed int er mediates in koj ic ac id biosvn-

th esis . However , the ac tiviti es of g l ucos e dehydrogenase
and glu conate dehydrogenase which were in th e range of 0 . 27
to O. 77 and O. 438 to 1 .07 ),\, mo l es of substrate per minu t e p er
ml of homogenat e ar e v e ry much high e r than those r equired
to acc ount for the observed rates of kojic acid synthesis .
It is v ery lik ely that glucose dehy dr ogenas e and glucon ~t e
dehydrog enase are compo ne nts of th e biosynthetic pathway •
Also patter n of changes in enzyme activities during r esuspe.-
ns ion str ongly support ed the involv ement of above two enzy-
mes in kojic acid biosynth es is .

1he above enzymes h ave bee n assayed by standard

>
assay procedur es . 1he assay h as dep e nded on the production of
acid in the ca se of h exok in ase , formation of dye in the c ase
of g lucose ox idase , r eduction of nucleotide co e nzymes in th e
ca se of h exose mo nophosph ate dehydrogenase and r eduction
of dyes in the case of q lu cose dehydroge nase ~nd gluconate
deh yd rogenase . 1hough the for mat io n of g luconolact:oJ,,~) cj,tA.,.-
cos e- 6- phosphate and 6- phosphog luconic ac id by the homogen-
ates has been shown qu alitativ e ly ne ith e r th e ide ntity of
the products or the stoichiometery of the prod ucts ha s b ee n
established comp l etely . Thus, the identification of the
e nzymes can only be co ns id ered to be provisional •
•
245

Inspite of this limitat i on, the evide nce for the

involvement of g l uc os e dehyd r oo enas e and g luconate dehydr o--
g enas e is ouite strong . It therefore becomes nec essary to
modify th e simple pathway , based purely on t heoretical grounds
and presented earlie r in this Chapter (page 7.'ll.) • The above
pathway ha s to be modif i ed to includ e gluconic acid as an
intermed iat e . The produ ct f or med by 9luco nate dehydr ogenasG
has not b ee n ide ntified in this sy stem . Probable products
are 2- ket ogluconate , 5- ketogluconat e 2 , 5-d iket ogluco nate and
3- ketog luconate . 1he first three are known to be produc ed in
different enzyme systems such as gluconate dehydrogenase
(EC 1 . 1 . 99 . 3) , 5- ketogluco nate reducta se (EC 1.1 . l 6Q) and
ketogluconat e dehydroqena se (EC 1 . 1.09 . 4) . In addition to
this , the followin g r el at ed compounds hav e been r eport ed in
dif fer ent biolog ical syst ems .
Preis s and Ashwell ( 26 ) h ave reported that 3- keto-
2 deoxy- D gluconic acid is fo rmed during th e bact erial meta-
bolism of po lygalacturonic ac id . This compound has been shown
to arise from 3- deoxy-D- g lyc ero-2,5 - hexo diu l soni c acid by
s pecific pyridine link ed dehydro0enase . 3- Ket og l uconic acid-
6- phosph ate h as bee n postulat ed as an intermed iat e in the
formation of ribos e 5- phosphate f r om 6- phosphogluconic acid
(27)
by a y ea st en2yme, Furth er Mos es and Hansen h ave r eport-
ed the formati on of 3- ke togluconic acid- 6- phosphate i n algae
 I

246

(28)
and fu ng i during carbon dioxide fix at ion in dar kness In
additio n, a ketogluconic acid wh ich is not the usual 2- or
5- ket o ac id and is pr obab ly 3- ketog lu co nic acid has been
29
isol at ed from hf,etobacter melanoqenum~ ) This seems espe-
ciall y signif i cant since the acet1c ac id bacteria are the
on ly g roup of bact eria known to produc e koj ic ac id . 3 Ke t o-
gluconic acid or a derivativ e of 3 ketogluco nic acid ha s been
includ ed in th e proposed pathwa y . The pathway beyond this is
pur e ly hypoth etic al but the reac ti ons suggested h ave s imi-
la ? i ties to other well known enzymatic r ea ctions . The ten-
tative b iosynthctic pathway is given below . Three al t ernative
pathways are suggest ed .
Th e first stage i n the pathway may be the r emov al
of 2H atoms f r om carbo n atom 3 of D- glucopyranos e ( I ) under
the influ enc e of g luc ose de hydroge nase . The second step may
be the c onve r sion of g lu co nic ac i d- b- l acfone (II) t o 3-
ketogluc onic acid l actone (III) by the actio n of oluconat e
deh ydrogena se . Koji c ac id (V) may be formed from 3 ke t oqlu- ~
) .
conic ac id l actone (III) by either of th e t wo pathways shown .
In one , 3 ketogluco nic acid l ac tone is reduc ed to 3 keto-
glucose (IV) followed by loss of two molecules of water to
form kojic acid . I n th e other 3 ketog luconic acid l ac t one
loses one mol ecule of water to give oxyk oj ic acid (VII )
foll owed by r eduction of the ca rbonyl a r oup and loss of a
 ~
~
' 'ttl • I~ l

-
H H OH 0
HJ H
OH HO H
Dehyd r oge n ~ti on Reduction
~
H - 2H c.ti on + 2H II
0 -2H
" ' C•·2 OH
0
/ CH ,>H
2
II III IV
I
Gluc on ic c.c i d - 3 Kc t ogluc on ic EWid 3 Ket og b c ose
Gluc opyre.noa e
lac t one l oo t one
Dehycl.r -.ti on Dehydrati on -a20 Dehydrati on - ~O

HO &ehydrog enati snH

~
-2H

c:a: 2 o:a ~ .cn2 oo C.u.2

7
OH. GH Ori
0 2
:1

VI V
VII Ifo jic aci d
O:xyko jic a cid

I\)

\
 •
248

~econd molecule of water . ~ third oossib ility may be that

gluconic acid - ~ - lactone (II) is deh yd rat ed first and then
dehydrogenated to oxykojic acid (VI I ) . lhis may then be redu-
ced and lose anoth er molecu l e of wat e r to oive kojic acid . Jne
compound (VII ) oxykojic acid, postu l ated as an intermed iat e
(32)
has been report ed by Yabuta. Jne reactions are r epres-
ented as follows. lh e order i n which th e r eac tions within
each branch of the pathway are shown to occur is pu r e ly
arbitary. Also the ste reochemistry of the postulated int e r-
med iates is uncertain .
lhere is e nough evid ence to suqqest that the post-
ulated reductions CO---t CHOH , wo uld r ea lly occur . Lipmann
pointed out that g lucose-6- phosphate and 6-phosphoglucono-
lactone system had a hiohe r oxidation r eduction ootential
than corresponding ald ehyde carboxylic acid system and sugg-
est ed that th e (phospho) lactone would be more re adily redu-
( 30)
ced to th e glucose level . Strecker and Korkes a lso pointed

, out that in oxliv er preparations , th e r e action .

+ +
Glucose+ DPN ~ Gluconolactone + DPNH + H
should be r eversible and we r e ab l e to sho,.., that this was
(31)
so .
Jnough th e evid enc e of th e rol es of glucose de-
hydrogenase and gluconate dehydroge na se is very strong both
the data on enzyme activiti es in r esuspended myc elia and
on the effect of inhibitors , do not rul e out th e involv e ment
 •
249

of phosphorylated intermediates in kojic acid biosynthesis~

If this is true , the above postulated pathways caA still be
considered provided gluconic acid~ - lactone and later inter-
mediate ; are replaced by 6 phosphoaluconic acid and oth er
phosphorylated derivatives . However , one has to expect the
a
action of/phosphatase at some stage before the formation of
kojic acid .
Considerabl e amount of further wo rk preferably
with cell- free systems and with radioactive precursors will
be needed to check the validity of the proposed biosynthetic
pathway .

>
 I

250

REFERENCES

l. Ar nstei n H. R. V. and Bentley R. , Nat ure , 16 6 , q4s

( 1940 ) •

2. Ar ~stein H. R. V. and Bentl ey R., Bi och em . J ., 21

493 , 508 , 516 (1Q53) .

3. Arnst ei n H. 11. V . Be nt ley R., Ri ochem . J . , 62 , 403

( 1 956 ) •
4. Denison F, W,, Car s on S . F. and Foster J . w., Rac t .
Pr oc ., 9 9 (1 954) .
5. Kit ada M. and Fukimbara T. , Ha kko Kagaku Zasshi ,
i2 , 847 (19 5 1 ) .
6. Br i nk N. G., Acta Chem . Scand . , 1 , 1081 (1953) .
7. Strec ker 1-1 . J . a nd Korkes s ., J . Bio l . Chem ., lq§. ,
769 (1952) .
8. Ramkrishna n T. an d Ca mpbell J .J . R., Ri och i m. Bi o-
phys. Acta , l I , 122 ( 195 5 ) .
9. De Ley J. and Stouth amer A. J ., Biochim. Bionhys.
Act a , J1 , 171 (1Q59 ) .
> 10 . Blakl ey E. R. and Cif err i o., Can . J . Microb i ol ., 1,
6 1 (1% 1 ) .

11 . Gibak T . and Sato R . Bio chi m. Biophys . Acta ., 1 3~ ,

265 (1967) .

12 . Gibak T. and Sat o R., Bio ch im . Rioph·is . Ac t .a ., 146 ,

3 17 (1967) .
•
251

13 . Demoss R. D. in ' Methods in Enzymo l ogy ', Ed i ted by

Colowick S . P . nnd Kaplan N. O. Vo l I , Academic Press ,
New York (1q55 ) p-331 .
14 . Noltmann E.A ., Gubler C . J . and Kuby S . A., J . Biol .
Chem ., 136 , 1225 (1Q61) .
15 . Julian G. R., Wolfe R. G. ~nd Reithel F .J ., J . Blol .
Chem ., 236 , 754 (1Q61) .
16 . DeMoss R. D. in ' Methods in Enzymoloqy ', Edited by
Colowick s .o. and Kap lan ~.o., Vol I , ~cademi c
Pr ess , New York (1955) 1 p . 334 •
17. Hochster R. M. ,Arch . Riochem . Riophys. , 66 , 499 (1957)
18 . Horecker B. L . and Smyrniot is P. Z., J . Biol . Chem .,
193, 371 (1951).
1q . Scott D. B. M. and Cohen S . S ., Biochem . J ., .21, 33
(l q53).
20 . Dic ke ns F. and Glock G. E., Riochem . J ., 50 , 81
(1951) .
21 . Roberts E. C., Cain C. K., Mu ir R. D., Reithel F. J . and
Gaby w. 1 ., J . Bio l. Chem ., 1 47 , 47 (1943) .
22 . Coulthard C. ~., Michael is R. and Short W. F., Bio-
ch em . J ., ~ , 24 (l Q47).
23 . Keilin D. and Hart ee E. F., Nature , 1 57 , 801 (1946) .
24 . Kelin D. and Hartee E. F., Biochem . J ., 12 , 221
(1 948) .
 •
252

11
25 . 1 ood . 1t'. A. and S chwe rdt R.E ., J . Bio l. Chem ., l Ol ,
501 ( 1953) .

26 . Preiss J . and As hwel l G., J . Bio l. Ch e m., 238

1571 (1 963 ).

27 . Horecker B. L . a nd Smyrniotis o . z ., Arch . Bioch em .,

£2 , 232 (1950) .
28 . Mo9 es M. a nd Hansen O., J. 13act ., 11, 70 (1 950) .
29 . Ri ed l-Tumova E. and Be rnhau e r K., Bioch em . 7 .,
~ 20 , 47 2 (1050) .

30 . Lip mann F., in ' Phosphoru s Me t abol i sm ', Edited by

McElroy W. D. and Glass B., The Johns Hopkins Pr es s,

Baltimore (1951), p . 158 •

31 . Strecker H. J . nnd Korkes S ., Nat ure , 12.S , 193

(1 951) .

32 . Yabuta T., J. Che m. Soc ., 37 , 1185 (19 16) .

>
 •

>
 253

Isolation and Sel ect io n of the Culture :

A numb er of fu nga l cultures were isolaterl from a ir
and s oil and high koj ic acid pr oduci ng cu l tu res· were screened
out . I t was not ed that in the case of all t h e cultures , koji c
acid level in fer me ntation med ium rapidly inc~e ased with the
period of incubation and r eached the maxi mum arou nd 7 t o 11
days , after which it decreased rapidly. The ir r elat iv e vaJ.-
ues varied with the s trains and period of incubation . Consi-
derable variation was also observed in the rate of kojic acid
production . It was observed that culture B produced the maxi-
mu m amount of kojic acid (75.6 mg/ml) on 10th day, corr espo-
nding to 37 . 8% yi eld on the basis of sugar initially pr esent
in the medium . Cultures Hand A als o yie lded somewhat compar-
able r esults producing maximum kojic acid (67.6 ~/ml) on
11th and (6 2 . 3 mg/ml) on 10th day r espect ively. The lowes t
amount of koj ic acid (24 . 2 mg/ml ) was produced by culture K
on the 9th day of incubation . In ge neral the amo unt of kojic
acid produced by different cultures ranged f r om 24 mg to
75 .6 mg/ml .
It was further obs erv ed that the:,:-e was a ve ry goo d
correlation between th e levels of kojic ac i d produced and
my celial growth. The maxi mu m grow 6 . 98 g wr1s obtained in
case of culture B, that produced the maximum amou nt of kojic
 •
254

acid . Similarly cultures Hand A which had al s o produced com-

paratively high amounts of kojic acid also exhibited oood rrr-{-

celial growth . Since among all the cultures tested , culture B

gave good mycelial growth a nd maximum yield of kojic acid ,
subsequent studies were performed on this organism.

Optimization of Fermentation Parameters :

The optimum ferment at ton conditions hav e been
standard i zed for the selected strain of th e organims with
respec t to medium , pH temperat ure ferme ntation pe~iod, spore
inoculum size , carbon source , nitrogen source and other growth
factors . The optimu m conditions for kojic acid production
were the YES medium containing 20% sucrose and 2% ye ast extr-
act , a temperature of 3o 0 c, pH 6 . 5 and an inoculum size of
6
10 spores/ml . The amino acids cystine , lysine , aspartic
acid and glycine and th e vitamins ascorbic acid, fo l ic acid
and pyridoxine were stimulatory . The clos e corr elation bet-
we en kojic acid production and growth was observed in t h ese
experiments also .

Studies on Fermentation of Cane Molasses :

The production of kojic acid from in expe nsive
and abudantly av a ilable carbon sourc es lik e cane molas ses
wa s then studied . It was found that kojic acid p roduction
- on unclarified molasses started from the third day of
incubation and its level increased upto 20 days . Th e yields
 255

of kojic ac i d also i ncreased wi t h an increase in molas ses

concentr ation from 10 to 30° br i x . No myc elial growth was
observed on 40° brix molas ses solution eve n upto 10 days ,
however it appeared on 11th day after which it started for -
ming kojic ac i d upto a maximum of 22 mg/ml by the 22nd day .
In ca s e of 50° brix molasses solution no myc e l i a l g rowth
was noted even upto 20 days. Furth er it was observed that
additio n of 1% yeast ext r act to t he medium not only stimu-
lated kojic acid production i n all the cas es but also r edu-
ced the fe r mentatio n period from 20 to 14 days . It was obser-
ved that simple additio n of 1% yea s t extract to the unclari-
fied mo l as ses of 10 , 20 and 30° brix c oncentrat io n increased
the yie l d of kojic acid by 208 , 217 and 230% resp ectively .
Clarif i cation of molasses by chemical methods decreased the
yi eld obtained on th e molasses med ium except in the case of
clarif i cation by ammonium sulphate or egg albumin . On t he
other ha nd all th e. clar ifi c-at ion proc edur es decreased the
y i eld obt ained on the ch emically clar ifi ed molasses medium
supplemented with y ea st e xtract . Treatme nt of molasses with
i on exch ange resin t oo could not e nhance th e kojic acid
productio n wheth e r t he medium was suppl ement ed wi th yeast
extract or not . Supp l ementation with a salt mixture incre-
ased kojic acid y i elds on a molasses medium from 7 .5 t o 15 .43
mg/ml. The data obta in ed on a yeast extr act supp l ement ed
molasses medi um were j ust opposite • In this case , th e

res in~ slight l y r educed th e amou nts of kojic acid produc ed

by r esuspend ed my ce l i a . Tre atment with both the r es ins r educed
the r ate of kojic acid production but th e maximum yield wa s
not much affected . Whe n th e molas5 es r esuspe nsio n med ium was
_,, __ , ____ ..,.._..J ... ~+i... ... - .......... ..L. ..... ..... ..&..---..&.. -- · -- -- -- -..&. · -- --··--\...-~-- - - - -
•
262

g l uco ni c aci d- ~ - l ac t one , 3 ketog luconic ac i d lac tone a nd

oxy ko j i c ac id has bee n suggested fo r t he biosy nth esis of
kojic ac i d . Some r e l at ed alt ernat iv e pathways ar e also
indi cat ed . I f phosphory lat ed int er mediates ar e taking par t
in th e for mation of koj ic acid , t h es e may be r e l at ed t o t he
i ntermedi ates sugges t i ng the abov e pathways •

.. .
,... ....
'~ ·

' ..