R.

printd Itum the Journalolrhe

An.ncan societyof BewitrACh.nnri, In.
rr40 P,roi Knob R@d, sr. Paut.Min'esora55t2r
Prinred,n rhe u s.A

'ft;

Changesin the ChemicalCompositionof BeerDuring

the BrewingProcessas a Resultof Added Enzymesl
S, Gorinstein,S. Xitov, and S, S,lel, Depa ment of pharmaceuticatChenisnr, Schoot of pharmacy The Heble\9
Univrsity of Jerusalem,Istael, a l O. Berman, M. Bertiner,c. popovich, snd yr, Vennus,Nationat Brev)eryLtd'
T?lA\i!, ltruel
AESTRACT

Termanll 60 L Thh d-arnylaseliquifis adjuncl!. It has a

standardized
activityoi60kilo Novo Amylasunirspe.gran (25).
The lemperatureoplimlm is 50'C in the pH ranse5J.
rr'errlzre.1.J.tThislroteinas,which breaksdownlroteinsro
peptidr,conrains1.5 An6on unirs pr gran (26). Ar 55'C the
optimumpH is in tle 6-7 range.
cetefio 2A0 L This.nzyne, composedofr-slucanaseand oamylase,splib &glucan in malt. It hasa slandardizedacrivilv of
. t 5 0 .C ' r so p r i d u n p H i , : 5 .
2 0 0& B l u c a n a *u n i l rp e r s r d m( 2 7 rA
Fungon!|800 L. Thk a-tfttylasehydrolyzes
starchanddex&ins
1o fcrmentablesuga.s.lt contains800 fDngalamylaseunits pcr
gram(28).The lemperatureoptimumjs 5-l0oC al pH 4.5,

The efccl of addinsenzymq to a nixture ot nall and sorgnun durins

1ne brcwin8 proce$ sas studied accordi.s to rhe following crireia:
p r e s e n c eo f s o l u b l en i r r o s e . - c o n t a i n i n sc o m p o u r u r l n o s u 8 a r s l
filterabilily,physicalstabilityofrhc fini$hedDroducr,anour or exrrac!
yieldedbt rar naroials in lhe mahihgproc.$, and iasle, Addilion ot
Neulrasero ftd malt Eavca significantihcrcalein rhenitrosencontentof
lhe pon and the bee!.Theselo* mol.cutarweishrnilrogcncompounds
l a - d m i n on n r o S e na.d ' n o a c i d r . l o u d o . e .a n d p e p d e r s r . d c c i s . \ Ien
d e r q m i n i n g r h c r a . lt o. .a m . r a b r l r JJ, n dc h i t . s e n r n i v i r y q u .dj lor t o c c , .
Theadvantages
of addinBenzynessuchasNeukaseandrbecarbohldrasss
Telmamyland Cerefloin the brc*ins pro.e$ are sccnin the incrcascd
a m o u n so f \ . l u b l en r r o g e n * o n r J n i ncEo m p o u n d \ , n cd a r b o h ) o G r cas .
d . c r . a . c d \ ! c o 5 i rl Je . d . r g r o h i g h ef irl r r a l r o n r a r c a . a n i n ( l e a " e d ) ( . d . a n d
Mash Samples
l h e p c i b i h r ) o r h i n g h r s e rs h o u n t so f < i c a p q u n m a t l c d
r d j u n o sr o
Kc! votdn Adled antlhc!, Brcwing, Cohpalrion

An imponanl index ofbeerqualiry is its physical stabiljry, which

depends on the mashing procss and on th anounr of minerah,
prottn subslances,and polyphenols in the beverasc(5.10-13,22-

24.37\.
Berbrewinghasal$,aysinvolv.d rhc acrionofcnzvmsin rhe
mash'ngprocssto breakdo$n high molecularweightproteins,
d c x t r i n sn. o n s r a r c hpyo l y s a c c h a r i d(e1s4 . 3 2 ,g. t u c a n tsl ? ) , a n d
p e n t o s a D1s1 0 , 1 5 a) n d r o i n c r c a s f. i h c r a b j l i r y( 3 t . J 9 , 4 0 )a n d
p.otein stabilily(15,19,36)
of ber.Barleymalr wasorigina y the
mor! rmportantraw materialin beerproccssinS
and vas the only
sourceof esscntialenzymes.Lessxpensive
raw materiaksuchas
ric, maize8.isl, barley,and sorghumhave little or no enzyme
content.excprfor rhe ,-amyla:e or baieyr thetrltarch. proiern.
and glucanhaveto bedigesledby olherenzyrnes
inthegrisr. Thus
theamountofthecheapefadjunctsrha!canbeadded!o themashk
reslrictedby the limiled supplyof enzymesf.om the mah irself.
This rerearchsludiedthe possibililyofinoeasingihe amoDntof
adjuncl(in this cas,sorshDm)in the mixtu.e ofraN malerislsin
the brewingprocessby usingaddedenzyme!.Thefollowingfactors
were examinedto delerminethe orrimum conditionsfor such
treatmenl: l) comparisonof the degreeof fermentationand the
degreeof p.oteolysisin the wo.l as a function ofthe addirionof
enzynes,and 2)quanlitarivedlertrinationof rhevariousniirogencontairingcompoundsin rheraw materialsand in all stagesofrhe
MATERTALS AND METHODS
All the expe.imentswereconductedon lagerbor( 10. B) under
procesing conditions at the National Brewery Ltd., Netanya,
hrael. Six thousandkilogramsof rav malelials,co]nprisedof 65%
nalt and 357,sor8humin additionto 240hi ofwate..vere usedin
themash.Enzynes(NovoIndusbi.s, Denmark)we.euseoroass!
in rh utilization of adjuncts(Termanyl and Neulrase)and to
supploment
the malt qDalitr(Cerefloand Fungamyl).
?re$nrcd d rhe44!hAnnuat M*rins, Toronro,canada,Mry t973.

0361-0470/30/01002304/$03
00/0
o1930Am.icanSoci6lyor Brwingch6misls,Inc.

23

Thc NarionalBrevryLtd. useda mashdecoclionsystem.One

rnashvas 10070ma and conrainedihe enzynesNeulraseand
Cerflo;th othrwascomprisedof rhe sorghumand I07ooi rhe
total wcightof the malrandconraincdthTcrmamylenzyme.Bo!h
w.emashedat 50oC, and the all-mahmashwa! rerainedai thh
temperalurduring th liqucfactionof lhe sorghum.The 1wo
mashcswererhnmixedand heatedto 76oC aftcr saccharificalion
slepsat 60-62oC and at 7l'C. Conkol I wasrhis regularproduct
ot National BreweryLtd,, but wi!hout theenzymesadded.
Con!rol
ll was thc all-mak nash, usedto obrain comparativedara on

Two lestsampleswereured.TestI conrained6570malt and 35Eo

s o r S h u m p l u s 0 . l T o N e u t rI a. 5sS
eb a s d o n r h e w e i g h ti h
oe
f mali.
0 . l T o T r m a m y l 6L0b a s e do n r h w e i s h o
! frhesorghum
a n, d0 . 3e
ofFungamyl600 l, pcr hcctoliterof wo.t. TesrII wasTsl l pll]s
0.025E0
Ce.eflo200 L basedon the weiShtofrhe mah.
Fof all samples,lhe wod was boiled for 1.5 hr wirh tbe hoD
extractaddedin threeponions(t l0 g/hl). After beingboiled.rhe
*ort was pumpedinto a settlingtank and cooledby meansof a
llale heat exchange.to a pircling rmperatxreof l0oc. Il was
pirchedNith Sa..haramrces Corlsbetgensisyeast,with a 50Ea0| v\
solid contnlof yeastsiu|ry in a proportior of 0.5 L/hl. Ar rhis
point, the foudh enzyme(Fungamyl)wasadded!o the resl wort.
T h m a x i m u m l e n p e r a t u r e r e a c h e dd u . i n g ? - 8 d a y s o f
fernentarionwas 12'C.
Theyoungbeerwasl.nnsle(edatauniformapparentdegreeof
frmentationof737.,exceptlorControl I, whichwaslrarsfered at
69Eo.Lagetinglastedfive weeks.Beforefilkalio!. Prolesal(a
.billproof enzymeof SchwarzSe.viceslnternalionatLtd., New
York) wasadded(2 s/hl).
The beerfrom eachbrtch wasclarified by dialonile filt.ation and
polishedthrougha sheetfilter.
The analyses
ofthemall, woit, and beerwercaried out by EBC
methods(9) and by the methodsofBausch(2), KrDgerand Bitig
{18),and Moll el al (20).
The nitlo8enconlenlofsanpleswasde|eIIniDed
by ihe Kieldrhl
or Dumas methods(l), (Buchi nitrogendeterminationsysten:
digertionapparatus,Buchi 425jdisrillationDnit. Euchi320)_Foi
the delerninarion of coagulabland albxmore nirrogen, rhe.
methodsofKolbachandwilharn (4)and De-clerck{7)wereused.
For the actualnikogendeteroinalionafteriheseprocedures,
the
precipilatesand fitrratesofthe$elestsweredigesledand distilled

24

V o l . 3 8N o . I

and thir nitrogencontentsdelerminedby Kjeldahl.

The totat protein was precipitatedby 1070trichloroaceticacid
(TCA)j rhe nitrogencontnt,doterminedbytheKjldahlmthod,
wasmutripliedby a factor of6.25 and.eportedascrudprotein.
Total protein and albunin lfere determinedby rpectrolhotofrom a lyophilizedramlle. usinga concenhalionof
melricmahods
100mg/ml for eachdetermination.
Total proreinwasalsodete.minedby the Biu.el reaction,based
on the irleraction belvencopprand pepridebondsofprotcins.
This savea puQle color *ith an absorptionmaximumat 5zl0n.n
{16).
Tolal albuminwasfoundfrom .heractionbelweenalbuminand
Bromcresolgreenin a tuitablbuffer to form an Albumin-Bcc
coBllex with a bluecolorand an absorptionDaximumof 610nm
(8).
The lyophilizedsamplesof Control I and T.sts I and II bee.
containddifferenl amoun$ of unfermentedsolids:ll00 ml of
Control I contained4.25
LTest I3.40 L aDdTertII3.00g). These

$eightsweiethereforusedto calculatetheresul$andrelatethem
10tle originalsadlles.
Total globulinvas calcuiatedasthe differencebel*eenthe total
prorein (froD TCA) and th albumin.
Peptideandfreeaminonitrogenweredeleimined
spectrolhotometrically on a UV-YIS SpeckophotometerVarian Techtron,
model635,in lhe 321 570 no.egion (9,29).ElementalC, H, N, Cl
aDdS weredeterminedby microanalysis.
with an Ostwaldviscorioeterand
The viscositywas'measured
calculatedaccordingto Krugerand Bieli8( l8) on the basisofone
pa.ticular concent.ationof wort and beer.Chill haze(physical
afier one day at
srability)wasmeasuredwith the EBC hazemeter
40oC, folowed by chillirg to 0oCfor 24 br.
The ioam stabilitywas detrminedby the modifiedCarlsberS
method as the Sigma value of beer (33,34).The exponential
equation,which representra unimolecular.eaction,is alplied to
the settlins of a static beerfoam and usedto obtain Si8ma,the
av.raselifetimeof a bubblein the foam.
RTSULIS AND DISCUSSION

TAII,EI
An.lytfu oaM.lt .nd of SorSlumCrlt!
M.lt
l t,9
t,80
N. Kjcld.hl(70,
db)
1.38
N, Dumss(7d,db)
4t,9
c (%,db)
6.59
H (%,db)

cr (%,db)
s (%,db)

10.6
t,54
t.53
40,0
6.E2
0.39
0.30

0,31

Extioci,rsdb(%)

80.3

solubkpror.ii (%db)
solublcN(m8/100nl)

t.0
5.5
98,0

(m8/100ml)
Apparcnl
alicnuElon,
limil oi icmcnbtion(%)

The followingindiceswcredeterninedby EBC Methods(9) but

li.nitsofConroll, arenol
bccausctheywcrelrithin the prescribd
presenred
in thc rablsrfor malt and sorghrm-moisture, 1,000kerncl{,eight,and gcrminationtcsqfor laboratory\rort-extract,
llltration time,
color beforcandaficrboiling,pH, saccharificstion,
HartongVZ 45, and ITT; for lcchnological$ort-xtroct, color,
and aroma;for beer-apparenl and
olarity,pH, saccharification,
alcohol,
real xtract,o.iginal gravily,color, pH, sacchar'ficalion,
disc.ryl, iron, coppcr, oxyg.n, ITT, SO,, COr, air, Asbach,
Hartong,bilterness,.ndturbidity. In addition,individualanino
ftodl 120/3
acidswcreevaluatedin the bccr!, usiDsa BeckmanD
a mino acidanalyzerand th two-columnprocedure(2I ,38).Here,
too. ro diffrenccsouisidethe limils of exprimentalrror wre
found,and the rcsultsare not rcportedin thc tables.
Analyticaldaiaobtain.d on the malt and sorghumSritsusedin
th expefimentsarc sivcn in Table L Th sorshum srits had
s o m c w h a tl o \ r e r p r o t e i n c o n t n t a n d s u b s l a n i i a l l yh i g h . r
laborato.yextrad valuethandid th malt.
Analytical and procesrdata othr than fof th nitrogenous
areshownin Tablcll for both th wortsand rcsutant
constiluents

85.1

20
7t , 4

TAILEII
Atr.ly36 of Comm.rcil Worr! .nd 8..4'

c (%, dh)
H (%, db)

cr (%,db)
s (%,db)

II

4t.01

37,85
6.t0
0.35

38,39

0.54
0.10
1.97

o:1.'

0.69
0.15
1.90

It
38.6E
6_4t
0.69
0.12
|,71

Apparent atlennario. linit

Exlnd oI sp&ge waier (%)

69.0
0,97

12.0

5.0
t56
69,1

8,4
l6l
70,3

12.5
0.3?

,o.0
3.9

tt,

39.10
6.80
1.20
0.60
t,t6

t9.66
6,28
0,93
0,44

39,91
6.63
1.0?
0.45
1.43

II
39.40
6.51
0.9?
0,56
t.34

740.

tn:1

85..'8_

'-9

,.u

0
28.8

l
24.2

0.8
t22

0.8
lI5

t.t
165
10.5

Tasr. test (.udber oi voics

(nB/L)
Anthocyanoecns

Ir

l6l

5
l0-6
3,8
3.9

3.1

'.1.
Ll

'A11brqs ar. fion a nal/sorshun

nash, except for Control II, from an au-nalt mah,

5,3

t.r_

'.1

125

ASBCJournal

Microanatyticalresultsindicatedthat the carbonand hydrogen

valueswerecomparableliom worr to beerand sidilar to those
slown to! the brwingmalerials(Table I). However,direrences
sere observedfor chlorine and lulfur. which may have some

25

fractionswereincreasedby th enzymtreatmenl;in the caseof

peptideN, the increasewas to valuesabovthar of Control U.
TcA-precipit.ble prote;n,prolein found by th Biuret Dethod.
albunin, and globulinvalueswere.ll infieasedmarkedlyoverlhe
Conlrol I beer lalues by the enzyne treatmnt. This was
larticularly apparenttor the TCA protein,the Biu.el protein,and
albumir Because
th Tstland Tesi lI beeisand $orts are quite
similar in nirrogen ard prolein patterns, the hajor erect is
obviouslythat ofth.'mash treatment presumablyrhe aclivity of
the nroteinaseNeut'lse 1.5S.

Viscosilydrta for lhe worts ofthe mall/sorghu!, brewsreflect

the influenceotenzymeadditionduringprocesing.F.omT.st II,
vhich had Cereflo200Laddedto the mash.a low beerviscosityof
l.14 was obtained.This loweredwort viscositycoincidedwith a
subslantial.eductionin mashfitration time to lossrhan4 h..
SUMMARY
The aplarenl!trenuationlimit otfrmntarionwasi.np.ovedby
thc useofcnzymesin the mash.This can be seenin TesrsI and II
The influenceof lhe enzymeson mashing,on worl fermentation
worts and mosl dramaticaliyin the Test I and II beers,where
andberstorageprocsses.
and on beerqualityitselfwasexamired
Fungamyl600L waspresentduringfrmentationand slora8e.The
in the NationalBreweryLtd. Addition ofth proteolyticenzyme,
valueof8770isresardedassome$hathighforlasrber-Howeler,
Neulrase,10the oash, in .ddition to oihe. enzymesupplemenls,
the 74% fot the Cont.ol I beer is lower than desi!d.A more
.esultedintheinc.easednitrogenconlenlofthevonandtheber.
.estrictedlvolofenzymadditionshouldDrovidthe desiredlelel
Th increase
in lh los molecularweight
nitrogenous
compounds,
ot 80 82ED.
suchasaminoacids(freanino N), albumoses,
and peptidcsmay
Thequantityof anthocyanogens
andpolyphnols
wassomevhal
havea decisiverole in detrminingthe tasle,foam, stability,and
lowerin theTestI and ll be6thaninthe ControlIIber, no doubl
chiu sensitiviiycharacteristics
of beer.
becauseof the useof sorghumgrih and hop extraci in rhe tsr
Animbalancein the nitrognouscompoundsmayalsoleadto a
brws.Valuesin all samples*ere Iowrthan would beanticipated
darkercoloredbeerandto physical,chmical,and lasteirstability;
in regular ber production, about 220 in8/L (18). The
tfe p.oductionof thesecompoundsmus! therforbe carefully
polymcrizationindex for all samplesh hiSher rhan would be
resulated(23,24).
expcredrrom olher repo.tson all-lnall beers(3,6).
W i t h t h e u s e o f e n z y m e s ,t h e a m o u n t o f f e r m n l a b l e
Froman econoInics
standpoinl,theenzymearcared
gave
Inashs
ca.bohyd.ales(estimatd
bytheapparentattenuationlinit) in the
a 17, highe. brewhouseyicld than did Control L With regardro
\t/ortand beeralsoincreascdg.early.
beerqualily,the enzyme-proccssd
br$sgav.subsrantially
bencr
Tastc differenccs1fere noticcable in th producr, but no
beerfoam stabilitythaD.lid the Control I brcw. Howcver,when
stathticallysignificantprefcrnccwasobtaincd.
ighttactrscompared
the beersfromthe enzyftebrcwswithbeer
The advaniagcsofenzymeaddition in thc brcwingproccssare
from Control I for arorda. tastc, and intensityand qualiry of
lecn in th increasedalnounts of solublc nitfogcn-containing
bittcrncss,
threeof thetsstcrsprfcrrcdrhcleslproducrandf ivrh
c o m p o u n d ss n d f e r m n t a b l ec a r b o h y d r a t e st,h e d e c r e a s d
control.This singletcst cannotbe considercdsignificantbul docs
v i s c o s i t y l a d i n 8 t o h i g h e r f i l l r a l i o n r a r el T
s ,otihncc r a s e i n y i e l d ,
mphasizethe need for conrinuedettenrion to this import&nr
and in ihe possibilityof usin8 larSeramounts of ihe cheapr
quality factor.
unmalted&djunctsto obtain a similarproducl.
Thc nitrognou!content,bytyp,in thcwortsandbe..sisshown
This has beenthe firsr siagein the srudyof enzymusin ihe
in Tabl. III, with referencedata given for Conrrol II. Usc of
b.ewingprocess.
Fulu.estudicsareplannedto considcriheffccts
nzymesin bre$,ingsubstantiallyincrased
both thc won and Deer
of an increasein the .elativeamountsof unmaltedadjunch and
t o l a l n i l r o g e n v a l u c s o v e r t h s e f r o m C o n r r o l l b u t w i r h l e v e l s svarialion
till
in theproportionsofenzymcsobtsindfrom a numberof
substantiallylower than thosefound for Cont.ol IL Freamino
nilroSenwas also increasedand, in this instance,reachedlvels
ACXNOWLEDGMENT
similar to rhat of th all-malt conrrol. On the olher hand,
wca.csraicfulro
thc
Minbr.yof
lndusrry!
Trad.lndToudsm(Offhcof
coagulablenitrogen was not significandyincreasedovef rhe
ih. Chhf Sci.nris0and 10 NsdonalBrcweryLtd., Nstanaya,who
Control I wort or beeraDd.emainedrelativelylow comparedwirh
lupportedrhisvork b, conrlact,Manythantsro Dr. R. viclor (The
Conrol IL
H.brewU.iv.rsity)ior he!nelph 1neorgadzation
oltht nanu*ripl,and
Furtherexaminationofthe nitrogenolrconstituenrs
of rhfour
ro S, Fcldman,
G, K.upnit,S, Melrr (NationalBleeoryLld.),andA.
beeB showed rhat albumose dtroSen and peptide nitrogen
Convay(TheHeb!.wUnir*iry) lor assisL.cc
in oblaining
rcsulk,

TABLEIII
Nlroger Conr.rr'of Conn.r.iit Wortsrnd 0..8"

II

III

1.43
1.39
82.4

2.03
1.89
t23.2
24
12.1

1.19
1.65
92.4
23
,,:

,,:

t.72
t.54
92.1
23
':.

t.26
0.65
60.5
0.8
l6.l

.'..
' 4 l l L a l u e s e n . n d . ei n
m g 1 0 0d l . e r c e p r o - r f e K i r , o a hal n d D u m a \\
' { l b r e r sa E l - o m n a l r / s o r s h u n
mrh. ercepL
l o . a o n r o , I l , l r o ma n a l l - m a lm
r dsh.
"

III

28.1
32_4
I l.?

83.3
t2
L8
6,I
18.5
ll5.0
1t8.7
76.8
33.I

II
0.98
0.30
12.3
t2
t,0
20.6
46.1
88.4
21.8

t.02
0.80
67.9
ll
0.8
t.0
20.9
8?.5
89.0
66.0

26

Vol. 3ENo. I

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l. Aslociaiion of Offlcial A.alytical Chemists.Official Methods of
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Lkiebskonholle, p.,1417. (41ned.).PautPakey:Berlin, 1963,
l. Brauw.lt Brevier, vonussctzuns fiir die Qualitairserhalluns
ftrr
Cetr:inte,p. 58-60,9?. Braleelt: Nn.nbers,1978,
4. Bul8ahov,N., Projzvod(vennyyi Labolatohyy Konirol, Solodorashcheriyai Pivovareniya.
Pishcheprorizdat,Moscow,J6:214,264,
304 1959,
5, Crabb,D.. ard HndsonJ. R. J. Ih!- Brew.3l:96, 1915.
6. Daiber,K.H- J, Sci.Food Asric.26(9)1399,1975.
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LehransrahftrBlauelei: Bedin,I, 1964tII, 1965.
8, Doumas,B. T,, watson,W.,atuBiEEs,H c. Clin. Chin. Acta7ta1,
1911.
9. Elropean lre*ery Convehlion.Analytica,EBc(3rd ed.). Schweizer
Braued-Rlndschau,Zuich, SwitTarland,
1975.
10. Gori.srein,S,. J.,4rro., Ol Anal. Chen.85(4)t793,1915.
I | . Gorinstein,S.,L:lsrid F, od Cheh. 21(l)145,1975.
12. Corinstein.S,, J. ,trrrc. Ol Anal. Ch.h.59(6)1380, \976.
13. Gorinstein,S,, J,,.tsrid,Foarl cheh. 261204,1978,
14. Crill, W., End Plspok, J., tai Bev, Conr, Ptoc, Canet, t4th,
Sdlzbetg,1973,p, 11l.
15. Kalashnil(ova,
A, M.,Ezbov,I. S,,Nakhapctyan,
L. A., Menyailova,l.
L, end Cracheva,L M, Chem,Abst. 85:44874x,
l9?5.
l6 Kin!slcy,C. R.In:coopcr, G. R., (ed.).sundard M erhodsor Clinjcal
Chemhrr, (7th cd.),p. 199.AcademicPre$:Ncw Yori, 1972.
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