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Coffee Enzymes and Coffee Quality

Chapter · June 1977

DOI: 10.1021/bk-1977-0047.ch003

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CoBee Enzymes and CoBee ality

IENRIQUE V. AMORIM and VERA L. AMORIM

Department of t2hemistry, Sector of Biochemistry, Esc. Sup. Agric., “Luiz de Queiroz,”
University o1 Sao Paulo, 15400 Péacicaba, Sp, Brazil

Reprinted from ACS SYMPOS IUM SERIES, No. 47

ENZYMES IN FOOD AND BEVERAGE PROCESSING
Robert i. Ory and Allen J. St. Angelo, Editors
Copyright 1977 by the American Chemical Society
Reprinted by permission of the copyright owner
Cofiee Enzymes and Coflee Quality

HENRIQUE V. AMORIM and VERA L. AM ORIM

Department of Chemistry, Sector of Biochemistry, Esc. Sup. Agrie., ”Luiz de Queiroz,”
University of Sao Paulo, 15400 Piraeieaba, Sp, Brazil

Ooffee beverage is the infusion of tbe roasted

coffee seed. It is the most consumed stimulant
beverage in tbe world and is second in ioternational
trade. The coffee tree is a perennial plant and
grows in tropical and sub-tropical regions. The
price of
ooffee beans (bean, ie a worldwide uaed name , bu$ tbe
correct born should be co I:fee seed ) depends on the
quality. It is almost impossible to define
Quality becauee what is a good coffee £or one,
could be a bad coffee for others. However, it is
important to take into cons id erat ion tire acne ptance
of the product by
the majority oI tbe consumers. It will be
considered in this paper that the best coffee
quality is the one that is accepted as being
high quality by the majority of people involved
in the oof#ee industry.
Goffea arabica L. for example, is considered
the most Ilavorful aad is also the most expensive.
It accounts for J/4 of the world consumption.
Following this comes 0 G e ho , Pierre
ex Froehner, known in lhe iDtemoatio
e by Robusta, and aucouots for l/4. A
small percentage comes from 0. Liberica, Hiern.
Within each specie, coffee is also classified by
its quality, which includes physical aspecta of
raw bean (color, size, shape and impurities) and
the flavor Quality of tbe beverage (acidity, body
and
aroma) a#ter roasting. The quality of the co5fee
within a given specie is dictated by the region
where the plant is grown, by the cultural practices
and by harvesting, processing and storage
conditions.
In spite of the importance of coffee Quality to
tbe price of the commercial bean, little has been
done on the chemical Bnd enzymological aspects ao
coffee seed. Tea is probably $he most studied
stimulant beverage. The flavor precursors of
black tea and several of the chemical components
in the

27
28 ''NZYM€9 IN FOOD AND BEVERAC'E PnOC ''SSINC.

£inisbed infueion are well known and tAeir

effect o quali are fairly well established (l,
2). Gocoa fermeDtation studies have shown that
ohooolate flav precursors develop during this
process (Q, 4).
However , the zhol e me chant em and reactions are not
completlg understood. The activity o£ polyphenol
oxidase and hYdrolytic enzymes are recognized to
pl an important role on flavor precursors
formation of these two commodities.
Go£lee ia perhaps the least studied with
OUTER SKIN reger to the green bean and flavor precursors. A
PULP {ew pape indicate that trigonellin, free sugars
MUCILAGE and peptides are responsible for the coffee
PARCHMENT
aroma (Q, 6). On the other hand, co5fee aroma
after roasting and infusio has be en extensively
— SILVER SKIN
investigated , and more than DC volatiles have been
identified (Q). lot much is kno about co£fe e I as be
and t;he compounds Chi oh able ct' i
(6) . In one aspeet , oo£ ate e ia oompl e t e ly different
from tea and cocoa . For tire formation of flavor
precursors , tea anfi cocoa have to be subjecI ed to s
kind of fermentation, which affects the
chemical composition of the whole leaf (tea)
or cotyledon (noeoa) , while or co//ee, witb tha
except1ort of
auc11age zeaova1 , i b seeae that any k1nd of
Figure I. Gross Section o( a ripe coffee fruit fermentation or change in chemical compositioñ
of t seed after maturity depreciates the flavor
quality. It is not intended to make a complete
review on coffee processing. Instead, full
attention will be given to enzymatic activities
and the posgibi1i*y c these enzymes affecting
the quality of coffee.

Goffee Fruit, flarvestinF and Processing

A cross section of a coffee cber is shown
iz Figure 1. Thie is a schematic drawing of a
ripened fruit. Tbe outer skin can be red or
yellow-orange depending on the variety. To
attain tbe best coffe‹ quality the fruit should
be harvested in tbe fully ripe stage regardless
of speoie or variety. There z two main types of
co5fee processing: Tbe wet ("washed") process
and tbe dry ("natural") prooess. In the wet
process the coffee cber is harvested
‹ transported to a processing plant where the
chem sgueesed in a pulping machine and.the
outer skin ai
pulp are removed . the muo ilagenous and at ipery layi
is then removed f»om the bean by a
natural fermentation (bacteria, yeast and
mold), alkali or added pect inolytic ensyete s
(8) 'the beans are
washi and died to 10-12 per cent moisture.
In the "natural" process the chem is
haveeted aod dried
30 81

about l2 per cent moisture coffee, in relation to Arabica species found by

in the whole fruit. WILBAUX (IO), was confirmed in l9}2 by OLI7EIRA (
outer layers are then
removed The )
by hulling.
A detailed of each processing step aaâ YA OTWA ( 12)
description
oan be seen in SIVnT5 and £O0TE'8 DOOR ( H ) • i Among tbebifferen0 varieties o# C. arabica in
gure
shows the steps of the
Beasil , no di I ferenoe was found in the qua i y of the
and natural beverage if tbe coffee is processed in tbe same way.
main wet
processes The activity of polyphenol oxidase is also very
WASHED COFFEE NA'P URAL COFFEE
similar.
Table I sbows the activity of polypbenol
Harvesting oxidase in four different species and three
Traoaportation Harvesting different varieties.
Transportation
Storage Storage
Sorting Sorting TABLf I. Relative activity of polyphenol oxidase
Pulping in coffee seeds ( DOPA as subafirat e) of
lying different species and varieties
Mueilage Removal (Storage)
Washing Mulling reca1cu1ated z'oa OLIy5IRA 11
Drying Storage e ative
(Storage) PPO aotivit7
dulling lTemoval o£ Impurities COFFEE
Roasting . ewevrei e i et Durant
storage C minding
Removal of Izpuri ti es var• ñ°exevrei IOO
Roasting Indusi on ( cothe e beverage ) a0ge ho a Pierre ex Froehn er
Grinding u o e sHiern
G. liberica is 298 •2
5.J
Infusion (coffee beverage)
ii Linoeu
Figure 2. Main steps of coffee-han‹lling proce mo on smarelo 10.
var Nundo novo 15.
Enz e var. Oatuai emarelo 12.2
Activities in different Coffee S ecies
arieties ond Tbe quality of tbe beverage of tbree G. arabica
The report ooffeee is the seme, it is cbaracteristio of the
first green of ensue detection in
coffee beans was made by HZRNDLHOFER in Brazil specie and indistinguishable smong them. However, it
in
*9)2 ). Lipase, protease, o lase, catalase and is very different £rou 0. canephora, C. liberica
( and 0. dewevrei which have their characteristic
"trace aof peroxidase were the enzymes detected. flavor,
Polyphenol oxidase could not but is not mucR appreciated.
confirmed in the Recently, PSYNE et al. ( ) utilised malate
be tO t to
dried seeda. However, the correlate
de drogenase and general protein banding patterns
first of coffee was to
enzyme
quality activity with the
VIEBAUX in l9}8 (IO). Ce was in measuring differentiate coffee species, varieties and cultivars
sucessfull
the activity of polyphenol and peroxidase with some success.
oxidase in
the dried seeds using substrates. In Although variations in different protein
various his
extensive work, WILBAUX compared tbe activity patterns end en& e activities may contribute to
of
lipase, oatalase, oxidase and di fferent Ilavors aft er the beans have been roach ed,
polyphenol peroxidase
in seven d ifJerent cofeee species aztd. cozze1ated. thea eoSZ eee oZ d ifferent speci es al so have different
witb tbe properties. Although be did not contents of caffeine, chlorogenio acid, trigonelline,
orgazoleptic
try to these differences iD fats, and possibly other components (). For this
correlate enzymatic
activities with the quality of the beverage reason, it seemg reasonable to speculate that the
(because
other components such a6 caffeine and oil difference in flavors ruAoag species is given by their
also
d1 If ered among the species), this pioneering work different contents of several chemical components.
was
not followed bg other ensYmatic Rec eat;1y , However, the effect of ens atic activities on flavor
studies.
the higher activity of polyphenol oI can not be excluded.
oxidase Robusta
32 ENZYMES iN FOOD AND BEVERAGE PnO CESSlriG
3. xvoniu on xcores H offee end Coffee Quality

Coffee Enzymes on Harvesting, Transport and Rio flavor also develops wAen tbe beans is
drying in the sun and gets wet by rain. After the
otoraKe Natural toffees. In Brozil, coffee is pulp is removed, no Rio flavor develops. Working
with coffee harvested in different regions, AMORIM
classified and SILVA ( ) fouod a positive
with respect to quality oI the beverage, bv the correlation between the activity
following grades: (from the best to the poor) Soft of polyphenol oxidaae (DOPA as eubstrate) of the
(mild), Almost Soft, Hard (astringent), Slightly green bean and quality of the beverage of C.
Rio and Rio (medicinal). arabica.
When the coffee chem is harvested in the Several papers confirmed their result l, , ,
fully
ripened stage, the moisture content is about 65 per , ). Polyphenol oxidase, or o-diphenol odase,
:ent and this kind of Fruit can produce the ks a copper eozYme (EO, 1.10. .1.) which oxidizes
bes#
beverage. Immature beans and black beans if not o-diphenols to quinones.
separated from ripe beans, depreciate the final Figure ) shows the results obtained by OLIVEIRA
liquor. Chese defective beans will not be (11) who uaed 7 oof ate e sampl es o I each kind of
considered in this parer. beverage aod compared them with the activity
In regions where all the fruits reach maturity of pol henol oxidaae. The correlation was
at almost the some time, the harvesting of onlv significant at the 1 per cent level. The
ripe cherries becomes impossible. The early mature activity of peroxidase aod catalase were also
beans maybe over-ripe in the tree or £a11 dow to studied by OLIVEIRA (11).
the ground. If the weather is dry, the quality oI However, only peroxidase gave a significant
the beve rat;e of the se beans may be fair t o good result. Tbe Rio co££ee bad lower activity in
HoSever relation to the others.
, i5 the climate is humid, hgdrolytic and oxidative Localization of polyphenol oxidase in
reactions caused by the fruit enzymes and coffee eeed has been unsucessf.ul up to now
microorganisms deteriorate the Quality of the However +
,
resu3.ting beverage. peroxidase was local i sed ( p-phertyl enediamine i
One experiment utilizing fruits collected in (TO) chiefly in the outer layers of the seed
the same tree, but harvesting in different stages the embryo (Figure 4). The intensity of oxidation
oi maturity, and dried in different ways showed of p-pheoylenediamine in Soft cof£ees is much
that the quality o£ the beverage and the activity greater than in Rio colfeee. In geoeral, Rio
o£ polyphenol oxidase di5fer, depending upon the coffee is light green or yellow-brown, even when
process used (Table III. The poorer the liguor it is o€ the current year's crop. Tbe localisation
quality, the lower was the activity of polyphenol of more intense peroxidase activity in the outer
oxidase. layers of the endosperm increases the chance of it
being associated with the discoloration generally
TABLE II. Relative activity o5 polyohenol oxidase found i0 Rio coffees
oI green coffee and quality of the Bg using alkaline gel electrophoresis (21,
beverage of cherries harvested and 22) of crude extracts without dialysis, several
prepared in polyphenol oxidases and one peroxidase were found
in
set i e e t a Dried in the ground li a e co tio
Relative Beverage PRO act. Quality
Are aLment
Pipe cherry, 100
pulped and sun
dried
Over ripe, dried 46.9 Slightly Rio
in the tree /§.1 Slightly Rio
Sample (1)
Sample (2)
green co£fee ( Figure 9 ) . In was In Drasil, where most of the commercial beans are
beans. However, not possible to "natural coffee", these different qualities result
the number of di£ferentiate £rom different methods of harvesting, handling and
PRO bands depends Soft from Rio dying. If one harvests only ripened Irui€ or
on the coffees by the collects green, ripe and over-ride fruits, but
intensity of the classifies them before dying, the ripe fruit alw s
substrate used ( ) bands, because of give a good beverage ( Soft ) . The classi Hication of
great variations.
lb days 16.8 Rio the Hard coffee in Brazil is a puzzle. The
QUAJGO, T1IZEIBA, LIORIM, 1971 (unpublished). characteristic of Hard co£fee is its astringency.
However, some cof£ees have no astringency, no Rio
flavor and also lack the full taste and aroma of the
 ENZYMF',S IN FOOD AND DEYr,RAC '• PIIOCEOSINC•
3. xvoniv wD rooms Coffee and G• Bee Quality

Soft cofiee. Dhese coffees are then classified by

the professional Masters as Hard, considering that
tbey ocoupy an intermediate position in the
Quality scale (A.A.TKIXEI8A, personal
communication). The literature shows that may
kinds of defective
PPO ACTIVITY f t reenn›
beaoa, dying temperatures and attack by fungi may
give rise to this peculiar flavor (24).
Although the activity of polyphenol oxidase
seems *o be correlated witb the Quality of
Brazilian "natural" cof£ees, other estimates of
chemical components o[ green coffee were made.
Soluble carbohydrates, polysaccharides ( ),
chlorogenic acids, total soluble phenolics,
hydrolysable phenols
T - RI0 (26), soluble proteins (2 ), eletrophoretic patteros
of soluble proteins a multiple linear
regression analysis of all dala were calculated (
QUALITY OF BEVERAGE ( infueioe)
). The most significant results showed
that Rio coffee had less
Figure 8. Activity o/ polyphenol ozidore in rant codec drolyzable phenols and less soluble
end the qua.litiJ of the beoeroge after rowin g end infu- proteins (DGA precipitable). Tbe dif£erent
mpu. Eoch point represents on average o/ seven different patterns of soluble proteine in agar gel
eletrophoresie were explained by tbe fact that, in
the extraction procedure, chlorogenic acid was
more oxidised by polyphenol oxidase (higher in the
Soft coffees) and it binds with proteins changing
their electrical charge ( 24t
).
One teotative explanation for the lower
activity of polyphenol oxidase fouod in Rio
coffees is this: in ay step
Buring harvesting and processing the enzvme has a
chance to come into contact with the substrates.
Tbe oxidized phenolics are yet restive
and bind covalenlly witb the enzyme, inhibiting
its activity ( ).
Concerning the lesser amount of TCA-
precipitable protein reported earlier in Rio
coiiee ( ), recent results
confirmed this £act and sbed more light on the
protein differences between Soft and Rio coffees
(21). It was found tbat, although tbe total
soluble nitrogen is apparently the s&me between
these two coffees, 3DS (sodium dodecyl sulfate)
gel eletrophoresis of tbe water-soluble proteins
(treated witb 2-mercaptoethanol) shows a different
pattern between tbe proteins extracted from 5
different samples of Rio coffee and A samples of
Soft coffee (Figure 6). SDS gel eletropAoresis
fi’igure 4. Coca(ination (btucE oreo in th
yreer oo|jee seed using ate separates most of the proteinc according to
0 produced by cato[ase orfirity. t;he ir molecular we i ght s
(1). I£ this holds true £or coffee proteins, the
gel patterns show that Soft coffee has some
proteins o£ high molecular weight (-
64,000 Daltons) that are absent in
Rio coffee. On the other hand, Rio
coffee shows some bands of low
molecular weight that are
36 ENZYMES IN FOOD AND BEVEIIAGE PTIO LESSING
3. xMOHJM AND xMOTlIM to ffee and Co$ee Quality 37

-ORIGIN absent in the So£t cof£ee. Furthermore, the bands

I-1
(-|-)
Figure S. Alkaline polyacnj lamide gel efectrop6o-
sis (7 —— 79•) of seed neater-soluble proteins:
protein trending pattern (1); polyphenol oxidase,
DOPA (2), and caffeei acid coupled u:ith m-phen-
yfenedfnmine (3) as substrates; and peroxidase, o-
dienizidine -|- HCOi in 10W• acetic acid (4)
oi small polypeptides (-8,000 Daltons) are much
more intense in the Bio cof£ee than in So£t
coffees. This euggests that the cof£ee beana which
give Rio [lavor undergo drolysig during
processing and/or modifioatioñ o5 the tertiary
structure of their proteins. However, at the
present time oQe caonot be sure that *hese
polypeptides are the cause of the Rio flavor,
because other hydrolytic reactions also take
place, probably a* the same time: for example, the
total extractable lipids (chloroform/methanol)
were lower in Rio coffee in relation to Uo£t
coflee as shown in Table III.
TABLE III. Extracrable lipids (chloroform/methanol) of
Arabica coffees which di5[er in the ualitof the bevera e and a
ii id li
14.84 16.28
1) 51 1.98
i5
ld .12
mean1).7 mean level.
’Statistical significant at 1°

The density, thickness of cell wall and

volume of cell walls are lower in Rio co£fee in
comparison with Soft cof£ee ( ), as shown in
Toble IV.
TARIE IV. Physical measurements of the whole bean and the ha
sJ,0Oo

39,60 0

2S,9O 0 CofieeWt. )0O ume of mean)

Gell wall thickness cell wall
Samplebeans density 6.2 72 gâ
l6,7OO 1.085 (751)
1J6OO Soft71.0é §0.7
Rio58.21 0.967 J7
.24 61 .6

EEEEEEEzz Although all samples were £rom the same

Figure 6. SDS polyacrylamide gef electrophoresis (T —— 10 &) of
green coffee, teeter-co[uble proteins ((rent ed ttnth 2-mercaptoethonol) oJ
Soft and ftio samples
coffee variety (G. arabica L., var. h.mdo Novo),
the 8ge of the beans were different. Soft-1, So£t-
2 and Rio-1 were kept in sealed cans for three
years and hio-2 was 7 years old. (In a later
section we will discuss
38 ENZYMES IN FOOD AND BEVEHAC'E PRO CEOSIVG series o£ different conditions and using multiple linear
regression analysis, these same
the effect of storage conditions on physical and
chenical aapect s of raw coffee) .
Coffee beans contain several glycosidases (
) and three d-galactosidaseg were
recently purified by affinity chromatography ( ).
BARHAM and coworkers
( ) found only one #-galactosidase (they did
not mention the coffee specie) and found a
molecular weight of 26,)00. SHADAKSHARABWANY
and RAMACHANDRA
( , ) observed tbat the activity of
a-galactosidase and #-iructofuranosidase incresed
when the seed was soaked in water. This might
Partlg explain the thinner cell wall in Rio
cof£ees.
However, differences in soluble carbohydrates
between Rio aod Soft coffee ( )
were not [ound, probably because the simple sugars
were metabolized, giving rise to CO2. This
accounts for part of the d weight
loss in Rio coffees.
It may be inferred from these chemical and
physical analyses, that the Rio flavor is
associated with drolytic and oxidative
reactions that take place during harvestiog and
subeeguent processing, while the bean is still wet
and covered with the pulp.
Washed toffees. The physiological stage o5
fruit developmen* upon harvesting which gives the
best beverage for "natural" coffees, also applies
to washed coffees. The ripe chem, if well
processed, gives the best beverage.
A£ter barves$ing, tbe cof£ee chem should be
"pulped" as soon as possible. Tbe time reQuired to
initiate the reactions which lead to "off" flavors
depends on the temperature of the environment and
the storage conditions. If the whole beans are kept
under a stream of water, the time from harvesting
to pulping may take 48 hours without ailecting cup
Qua1i*g.
AhGILA and VALENCIA ( ), in Colombia, studied
several factors which affect tbe quality o£ the
beverage of washed cof£ees and tried to correlate
them with activity of polyphenol oxidase of the
dried bean. The enzvme activity is higher in
coffee pulped immediately; that is within a l2-
hour interval from harvesting to pulping. It drops
in 24 to 56 hours and again in 48 to 72 hours.
Statistical analysis oi the data showed Whet ens
atic activities were
different and inversely correlated with acidity,
the higher the activity, the lower the acidity. No
difference was found in the body of the beverage,
although the 72-hour treatment yielded a ver
poor aroma. By considering a
3. AMORIM AND AMORIM Go ffee and Co B• • 0•• '‹r 39 oI D-galactose. they also isolated a highly
branched Zalactoaraban in which
authors found that the activity of PPO L-arabinofuranose residues linked (1-§) and (1-b)
was negatively correlated with acidity apparently occur in the exterior chain, with a
and positively correlated with the box core of a branched¿galactan composed of (1-6) and
of the beverage ( ) (1-5)
, linked D-galaétopyranose residues.
It is believed that upon Reducing sugars, sucrose, ca£feine,
harvesting, hydrolytic and oxidative chlorogenic acids ( ) and amino acids are also
reactions in the pulp and mucilage found in the mucilaye 8)a The water-
begin. If the cberry ig injured, attack soluble protein patterns are shown in *igdre 7.
bg microorganisms speeds up the se react Although the mucilage bas no
ions. be have been able to detect
activity of pectin esterase,
polygalac turonas e, p-gala ctosidase, peroxidase
and
polyphenol oxidase in the mucilage of
uninjured ripe fruits (uopublished
results). Polgac lamide gel
electrophoresis ( ) o£ mucilage water-
soluble proteins indicated that there
are 2 polyphenol oxidases and four
protein bands (Figure }). The
contribution to "o£f" flavors
developed before pulping by
microorganisms and by degradative and
oxidative reactions catalysed by the
enzymes of the pulp/mucilage have not
yet been stablished. However, ROOTON (O)
states that brown pig;ment s formefi in
the pulp and mucilage layers
depreciate the Quality of the
beverage, as well physical aspects oi
the bean, if it di
rhus es through the parchment and reache s the
bean .

Enzymes in Coffee Fermentation

Mucilage removal after the bean is
pulped is a very important step in
waehed coffee processing. I[
*he mucilage is not removed, the dying
process becomes difficult. On the other
hand, the mucilage is easily attacked by
microorganisms which add "o£f" flavor
into the bean and taint the beverage.
The chemical composition oI the
mucilage has been partialy resolved.
GOLEMAN and coworkers (41) determined that
pectic acid is one the polysaccharides of
the mucilage; it is a polymer of
polygalacturonic acid (77.6° d. wt.
basis), with a small amount of arabinose,
galactose, close and rhemnose. Further
work carried out by C0RR#A and his group
(42, , 44) showed
polymer containing (1-4) linked residues
40
ozidnie, DOPA, alkaline PAGf, Z - 79•
(2); gnd SDK, no 2-mezcaptoethanol treat-
ment, T - 10'» (3).
3. xvoniv on svo un H offee end Coffee Quolit y 41
celluar structure (46) it has several hydrolytic
and oxidative ensues, such as pectinesterase ( ),
galacturonase, J-galactosidase, peroxidase and
polyphenol oxidase (catechol and DOPA as substrate)
(AMOEIM, TEIXEIRA, OLIVEIRA, O0STA - in preparation).
However, these drolytic enzymes of the mucilage
seem to be unable to remove the mucilage when the
seed is kept in microorganism £ree environment
(4R). GARBONNEL and VILANOVA (46) explain this
fact bg the equilibrium between tbe substrate and
product of an enzymatic reaction. Apparently, the
fruits do not have enough enzyme to completely
catalyze solubilizatioo of *he mucilage;
conclusive proof for this is still lacking.
The fermentation process whioh includes the
removal of the mucilagenous layer adhering to the
parchment, is achieved first by the endogenous
a e ine i
and Aspergillus sp from Brasilian
all mold strains produced in the culture
medium enz es able to break down pectic
acid and galactoaraban from coffee pulp and
mucilage. A
decrease in pH is caused by exposure of the
carboxyl groups of pectin ( ), and
by the production of acetic and lactic acids
(40). There is also a decrease in carbohydrates
during I erme nd ati on ( ) and a
concurrent product ion of ethanol (J) For
low-groom co the e , the fermentation is last and
ethanol production is vet high in the
begining, but tben it disappears. For high-grown
coffees, ethanol increases steadlly but does not
reach lB/ of that by low-grown coffee production.
Acetic acid production is steady and reaches 15°
o£ all volatiles. Propionic and butyric acid are
also produced, but they increase only after 20
bours fermentation ( ). Propionic and
butyric acids give a bad flavor to tAe coffee
beverage. For this reason, fermentation time
should be kept as sbort as possible. On the
other hand, if the co£fee is exposed to the
liQuor (degradation and oxidized products of
mucilaqe) for too long, these products may pas s
through the pa rohment , cause a poor appearance of the raw
coffee (brown silver skin and center cut) and
possibly taint the beverage characteristics
(40). *hese oxidized products are possibly
cblorogenic acids which are oxidised by
pol henol oxidase and peroxidase and link
covalently with amino acids and polypeptides ( ).
o avoid tbese browning reactions, WOOTON
(4T// added a
 ENZYMES IN FOOD AND BEVERAGE PROCESSI1 fC• S. rooms xuoniu • Bee and Coffee Quality 43

reducing agent, sodium sulfide (1°), with well proc es ed

apparent sucess, bu* tbe fermentation waa
delayed; probably because of inhibition oI Possible Role of Ensymea During the D iag
microorganiems.
OANINT and VALTLGIA ( ) found thot long Process The d irg
fermentation times of more than 24 hours,
depreciate process is an important step in
quality o£ the beverage and lower activity of co#fee preparation. If dying is no1 done properly it
polyphenol oxidage of the dried bean. may change the physical aspect of the bean and
To avoid a loog fermentation time, one can tbe quality of the beverage. FERRAZ and VEIGA
add several enzymes that are commercially in l9§4 ( ), were tbe first to call
available, but attention to tAe Importance of drying temperature
these are not used on a large scale because of the o£ natural co flee
added cost. Denefax (@4) is a material produced and the quality of the beverage. The best tem erature
from
molds and contains lC of a useful crude enzyme was found to be 45 C. Dryiog between 90 to
preparation (8). Recently, Giba-Gei S.A. the worst ct fe eI s on the quali ty of’ the beverap;e
marketed ULTEAZIM lOO, wbich is used by orange temperatures above 6O°C and up to 9O°C gave a fai rly
juice
industries. It hgs been used to remove coffee good beverage for They speculate
mucilage with success in Kega ( ) and natural that activate enzymes
Gua$euala temperatures around wbich
( ), end in Brasil we observed a drastic I in turn produce compounds deleterious to the
reduction in time when 4 mg o£ the commercial coffee flavor. On the other band, other
ULTRAZIM 100 was mixed witb 2 kg of pulped temperatures activate (or inactivate) enzymes
cof£ee, as demonstrated in Table V. which can produce compounds of good or bad
flavors. Although no
DABLE Y. Fe en ion ensvmatic activities were measured, the
e speculation of FERRAZ and VEIGA ( ) should be
d meoo uepeeratu£ l8-2e' takeo iAto
Treatment e considerat ion, be cause t empe rafiure nay a*lect bean
initial enzymatic activities.
end In Colombia, ARCILA and VALENCIA ( ) studied
Natural , dry 16 4. 02 .64 the e ffeot of drying temperature on the activity of
Natural , under water 18 $ .2g 5•7J polyphenol oxidaae and on tlte quadi by o£ the
ULTRAZIM 100 beverage the best I emperaYurea £ oz ac i dity, body and
(O mg - 100 rl/2 kg) 9 l7 fi. 6g aroma were found to be 40 JO and 8O°G . Temperatures
Natural + of 30, 60 and 7O°G gave a poor beverage , with respect
Metabisulfite
OO l0O ml 2 k 26 It to note that , abter fe zme nt ation,
AMORIM, TEIXEIRA, GOS A, OLIVLIEA, the beaos should be wasbed to remove mucilage and
1 polgsaccharide degradation products (free sugars).
preparation) Otherwise, these free sugars bind Co the silver skin
in the region of the center cut and, after the
roasting process, the center cut becomes brown (j8).
Tbis commercial ensyme may be reused for at This brown center cut indicates that the bean was
least ) consecutive fermentations by recycling the
decanted liguid. To avoid browning of tbe ailver not
skin, 2§O ppm of metabisulfite my be added per kg
o£ pulped coffee. No iohibition of the ensue was
noted.
Purbhernore , met;abi cut Hi t e may react with
acetaldehyd e and other carbonyl compounds , avoiding
seed contamination and affecting the quality o£
the beverage
to these three organoleptic characteristics. The
activity of pol heaol oxidase generally decreased
when temperature increased, with one exception at
40°G. The ensYme activity at 40’0 was lower than
thet at 30 and 9O°C . from the data presented , it
vrould appear tbat lhere is still no direct
relationship between Quality, temperature and ens
atic activities.
In the section on roasting the resistance oI
some coffee ensues to high temperatures, probably
because of the dry state o5 the bean, will be
discussed. However, it does seem that both
enzpnatic and nonenzjw ti:• reaction might be
involved in changing lhe chemical composition
during the dying process, but this aspect needs
further investigation.
 46
ENZ•f MES IN FOOD AT BEVEHAC•E PRO YESSING

Role of Enzymes During Ooffee Storage

Ooffee storage in humid and warm regions is
a serious problem , because the cothee bean becomes
white , or ;yellow to brown, aepend ing on tche degree or
moisture in tAe air and the time of storage.
This change in color is accompanied by a
decrease in flaVor QualiLg»
flULTON and his group ( 60, 61, 62) have done a
series oi studies on coffee storage in different
environment conditions, analyzing microorganisms,
water absorption, and ensjnnatic activities, and
comparing tbem witb the Quali of the beverage.
Their major fimdinga may be summarised as follows:
above 79% relative humidity, tbe coffee bean
absorbs a sigoi£icant emount o£ water and lhe
increase in moisture cooteni is positively
correlgted to fungal growth, although the number
of bacteria decreases Tbe quality of the beverage
is completely changed and after 50 days at 9J°
relative humidity, the coffee beaos are completely
rotten. At 95% relative humidity there is also sm
increase of lipase activity, witb a
concurrent increaee in £ree fatty acids. In
addition, ribonucleaae and protease activities seem
to increase
under these conditions. Tbe d weight loss may
reacb 25° in 2OO djs if the relative
humidity of the air ie above 907. In Brazil,
JORDZO et al. ( ) also found an increase in free Time (in moDtha) 0{ StO¥Og8
fatty acids during three years storage of green
coffee. The peroxide value increased only after Figure 8. Polyphenol
2 years storage. In Africa, ESTEYES ( ) studying
acidity in the oil of Robusta and Arabi ca
cofJ e es also found an increase in titratable acioit7
with increasing time of storage in both t;ypes o E eo E
See a.
PEREIRA ( ) in Portugal was the first to
report a decrease in polyphenol oxidase activity
in green coffee witA storage time. His results
were confirmed later b OLIVEIRA ( ), VALENGIA (12)
and UonIM et

Figure 8 9hows the polypbeool oxidaae

activities obtained by OLIVEIRA (llJ with different
co£fee species aiter ditierent times o£ storage.
the value for Arabicas represents the average o£
£our different varieties grown in dif£erent
regions; all gave similar patterns.
3tored green coffees tbat give dif£erenL
Qualities of beverage also show decreases ia
polyphenol oxidase activity and total carbo le
with time of storage (Dable VI).
 47
46 ENZ'YMES IN FOOD AND BEVETtADE PROCESSJNH

Total curbonyls in coffee oil and polyphenol oxidase (PPO) activity of Arabica green coffee beans (each symbol is

SO
A lmost S
beverag
Quali
o £

AlBO
Ooffee Bomple Abs. 10 min/ owder

stored in k i thecent
were made with four

rd
Ha
Ha
ol/oil 1.?2 a
So£t Rio
stored 1 yr) 115.7 a .72 b
stored 1 Yr) 88.5 b

yellowis
yellowis
gre
Soft stored 2 yr)

en
Rio_ (stored 2 .6 b1 d

activity in sealed
iiierent sqols mean s1i icancet b°’,
level.

h
MC02tIM et al.

A ctivi
PEROI
The higher emount of total carbonyls found

8
1
ty
in the best coffees agrees in part with tbe work

O5
i
of GALLE (66) in Colombia and GOPAL pt al. ( ) in

changes of green cotI

India, who found more aldehydes in the best

2
coffees.
In a prelimina experiment with Arabica
coffees, MELO and coworkers (68) ond TEIXEIRA et

2
Activi
al.

i 0O
(J6) oobbsseerrvveedd tI hat green ooffee stored in sealed cans
or plast ic baga for 21 months were still green,

ty
•

o dianisidine
whereas coffee stored for the seme period of time

relativ t
in baga made of paper, cotton or vegetable Siber

p
were wAite-yellow and yielded a poor beverage.
these bleached beans showed concurrent lower

ee
densities because oi an increase in sise (swelling)

}
Solub
(Table VII). The activities of polyphenol oxidase

1.
52
1 .

2
le
% *

O
and peroxidase were also lower in the spoiled

•
i
coffees. It is interesting to note that the beans
kept in cans and plastic had practically a constant
moisture content

O .05
containers during
21 replications).
(-lOQ), but coffee etored in paper, cotton
anB vegetable lib er cont aine rs changed moisture

1.124
Densi

DOPAiPM
content s from 9 to l 3gé, depending upon the season •

0 8
ty

0 .
O•
sical and
It is well known tbat, upoo stgrage green

in
coffee gradually becomes white. The discoloration

9g vt.
starts in the outer hers o2 cells aad goes towards
the center. Dhe color of green coffee oi a new

1000
crop, if well process ed , is Sank gre en or gre eni

( substrate: PPO =
sh-blue F/i th increased time o£ storage it

1
bea

120

8›
l l
becomes light green and white. Depending upon the

ns

.5
environmental conditions (humid icy and temperature ) ,
the bean nay change to yellow or brown.

*Relative
This change in color o# green coffee with

activity
fiber
O ott on
storage was extensively studied by BAGCHI () in
ainer

getable
Brasil. BY using two varieties o5 G. arabica,
type
DABtE

different methods of processing (wet aod

Coot

P ap

Ve
natural), different pulping and hulling
,

processes, and different storage conditions,

BAGGflI concluded that
48
3. AMOBIM AND AMoRIm Coffee and Coffee Qualify 49
the Rios t import ant la ct or
which causes the bean phenolic acids play an important role in the taste of
te
become is mechmnscal injury , from Carves ting tO tiqe beverage (6, )
while
hulling, regardless of the coffee Tbe activities of several enzymes in the green
whether is
processed by W0t o» #he natural The bean muat plag an important role in modifying the
the method.
environmental fectOr which enhances ost the quality o£ tbe final beverage, as haa been discussed
o5
change COlOy i9 tht of the air. in this papér. In several processing steps tbere are
in moisture
The chemical mechanism coffee discoloration
or has not yet been However, from the occasions *o Bodie the chemical composition of the
establhished. da ta bean. It is not probable that ensue activities, per
available the andfruits
by Comparison
in
discoloratio inliterature,
and
vegetables seems ( ), it with se, play a major role in coffee flavor during the
that green co Lie e bean discoloration is roasting process,
and thebecause the coffee would
bean is
to due dehydrated high temperatures inactivate
enzymatic oxidation phenolic compounds all enz es. However, due to the fact that 2%/ o£ the
of by
polyphenol oxidaae peroxidase, a activity of polyphenol oxidase could be retained
end with concurrent
OXld8tiOn of acid ( ). ascorbic after 5 hours exposure of green co£fee beans to 12§’C
ascorbic The acid
oxidation might coupled the reduction (TIO and AMORIM, 1972, unpublished), it was
be with o£
Qu:nones produced by action o£ PPO and PER necessa to look at the activity o5 several
the on
phenolics ( ). a general discoloration drolytic ( #-galactosiñase, #-glucosidase, acid
In manner, in
fruits followed by a in phenolics, phosphatase, ( )) and oxidative (polyphenol oxidase,
is decrease
as co rbi c acid and Polyphenol oxidase activity ( peroxidase) eos es during the roasting process.
) • In coffee, AMO8IM et al. (Z6j [Ound Figure 9 shows the relative activities of
less
bydrolyzable in Rio coffee than in several envies plus the water-soluble proteins (TCA
phenolics Soft
cof£ee, but the chlorogenic acid s (A . 0 .A. c .
soluble procedure ( in the coffee was precipitatable) during roasting. As can be seen, the
)) best degree of inactivation of the enzymes with increasing
content
s
r,i gnificantly lowe in
comparison with the
other t enperalure , di f hers depending on the enzyme At 9
qualities OhloroZenic determination min. ( IJO°C) , the powder has no co thee arona.
acid in
green sbould be reinvestigated Instead, an odor reminiscent of roasted peanut aroma
because
different types of may give results, was observed. At 8 min. (l#7°#), a clearly defined
ooflee different
depending upon the used ( ). There
method possibility that coffee
min. aroma was detected, which increasesof
atproteins
12 azd
phenolics is bOund to cell are 19 of roasting. The insolubilization
wall
responsible for discoloration reactions
the coincides witb the inactivation of enzymes and coffee
coffee. in
It is well
documented in the literature, nroma formation. between the Cth to 8th minute, the
that
injury causes discoloratio n
Or/and browning
reactions free amino acids, peptides, trigonelline and free
in plant tissues. beans seem to be Oo surars should be reacting to produce the coffee
Coffee
exception. However, aroma.
methods
analysisfor
better
should be devised in If the free amino acids and polypeptides
chemical and order to explain maoy of the are important in coffee aroma [ormation (Q,
phys¿qgl det changes whi ch o ccur in cothee ), the
erioration. ratio of these amino acids, as well as their
total amounts should be important to the flavor
of the
Ens e Act ivit i es and Protein Bolubilit final beverage. Also, the amino acid composition
t of the Proteins and perhaps their tertia
Durin structures a I so ply a rol e
Roast ink Process in eo Pte e *favor . Tt i e well know
Coffee aroma and taste develop
only after
the that ba s i c and sul Our amino ac ids are dev troyed by
bean is The temperature is 24O°C the roast ing process ( , 8O , 81) . Furthe more
roasted. roasting or
higher and inrequires 5 to lb min. FOKORNT et al . ( Sl ) obse rvefi that Are e sub rur
depending upon
the Memperature,load, machinery ,
etc. Coffee
aroma basic amino acids in green co£5ee decrease with
begins tO fOrB when the bean rea chea 180-1 O°G ( , storage. In Brazil, DOMONT et al. (92), comparing the
› ). The orincipal precursor
of coffee
aroma behavior oi auiQo acids during the roasting of Soft
seems to be trigonelline, sucrose, «nd lio coffees, observed that Rio coffees had 2b,
fructose, glucose,
free acids and peptides (Q), Carbo lic
more free amino acids than Soil coffees, and the
emino speed of pyrolysis of serine, threouine and chiefly
and
50 ENZYMES IN FOQD AND BEVERAGE PROC,EgPING
3. xuoniu nrro xuonni Coffee end Goffee Qoolity

arginine, ia mucb fastly in Rio than in Soft coffee

samples. These results agree in part with the
observation that Rio coffee bas more low molecular
weight polypeptides than Soft coffees, as seen in
Figure 6. Tbus, there is a possibility that these
polypeptides may affect the precursors of aroma
formation.
a o beta goloc tosidose
Conclusion
SOLUBLE PROTEINS
p-a peroxidase From a review of the literature and the data
o - ----a beto glucosldose presented in tbis report, there is only one ensyme
that ie certain to depreciate coffee quality. This is
the polyphenol oxidase of tbe pulp and mucilage (two
ENZYMATIC
ACTIVITIES

enzymes detected), which oxidises chlorogenic acid

aoA other phenolics to quinones. These quinones then
form complexes witA amino acids and polypeptides
having a brown color, that bind to tbe center cut of
the
silver ekin. Dbis brown color of the center cut
and silver skin depreciate the quality of raw
end coffee and it s appearance after roast ing. Yowever , whether
these pigments positively a£fect the Quality of the
23 5 I5 ( min
170
beverage, is still disputed.
25 165 155 187 245 ( °C} Commercial pectinolytic enz es may be used in
P-&aiactosidasee (p-nitzop conjunction with reducing agents to speed up
peroxidase (pyrogallol mucilage removal and avoid browning reactions.
Although polyphenol oxidase 0nd peroxidase
octivitiee in many instances are correlated with
quality of the beverage, proof of the mechanism and
tbe compounds which take part in these changes and
affect cup quality is still lacking. Secauee of the
fact that peroxidase is located chiefly in the outer
1 er6 of the eeed, it seems probable that this
ensyme is associated with coffee discoloration
upon storage.
Considering all hydrolytic reactions catalysed
by coifee seed ensues, the lipases and other ensues
oi lipid metabolism should be important, because oil
is tbe aroma carrier. Also, the protein hydrolases
deserve attention because they affect a known
important coffee aroma precursor: tbe amino acids and
polypeptides. By affecting the ratio oI these aroma
(and probably taste) precursors, it is reasonable to
believe tbat both aroma and taste should be altered.
From the available literature on coffee
chemists it seems that the characteristic flavor of
each coffee specie is given chiellg by the
inherited chemical composition of tbe bean.
However, the variation in quality (p eical and
orgonoleptic) wbithin each apecie, seems to be
associated with
52 ENZYMES IN FOOD AND BEVERAC.E PBOGESSINC•
3. xMOnIM AND AMOnlM G0 0e and Co fee Qualil y 53
variations in chemical
composition, caused by 12. Valencia, G.A., 0enica£e (Colombia) (1972) :
the
action Of dzolytlC and enzymes of )-18.
oxidative the
bean and/or microorgan i sas . The deleterious effects Payne, R.C., Oliveira, A.R., Fairbrothers, D.E.,
of enzymes oO the quality of the resulting beverage Bi ochem Sys lemal . ( 1975 ) 1 : 9Q-61 •
uay be prevented using more suitable methods for 14. Streuli, H., 6eme Colloque International sur la
by
coffee processing and ato rage . Ghemie dev Gates (ASIO) p. 61-72, Bogota,
Acknowled ent Colombia. 197a.
lb. Amorim, H.V., lilva, D.M., Nature (1966) :
Research was
c8rried )8l-ñ82.
ou
t with grants mom the
Insti*uto
Brasileiro do Cake , Oonselho Nasional do Rotemberg, B., lachan, A., Rev. Bras. Tecnol.
Desenvolvimeoto (1971) 2: 67-69.
e Tecnologico e Fundaqâo
de Ampa ro a Pesquisa do £stado hotemberg, B., lachan, 5., Rev. Bras. Tecnol.
grateful to Dr. A. A. de 8âo Paulo. Ue (lH72) /: 1§b-lJ9.
Teixeira are and Dr. O. M. Gopal, N.F., Annual Detailed Technical Report,
£or helpful discussions and dance Dr. Robert L. Oolfee Board (India) (197"-75) B: lOH-lJ9.
to reviewing the manuscript. 0 for Amorim, H.V., Legendre, H.G., Amorim, Vera L.,
St. Angelo, A., Of, R.L., Turrialba (1976)
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