R.printd Itum the Journalolrhe An.ncan societyof BewitrACh.nnri, In. rr40 P,roi Knob R@d, sr. Paut.

Min'esora55t2r Prinred rhe u s.A ,n

'ft;

Changes the Chemical in Composition BeerDuring of the BrewingProcess a Resultof Added Enzymesl as
S, Gorinstein,S. Xitov, and S, S,lel, Depa ment of pharmaceuticat Chenisnr, 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

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 e f s o l u b l en i r r o s e . - c o n t a i n i n s o m p o u r u r l n o s u 8 a r s l o c filterabilily,physical stabilityofrhc fini$hed anour or exrrac! Droducr, yieldedbt rar naroials in lhe mahihgproc.$, and iasle, Addilion ot Neulrase ftd malt Eavca significant ro ihcrcalein rhenitrosencontentof lhe pon and the bee!.Theselo* mol.cutarweishrnilrogcncompounds l a - d m i n o n r o S e n .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 . \ I n n a e d e r q m i n i n g r h c r a . l 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 . j lo t o c c , . t. , d r Theadvantages addinB of enzynes suchasNeukase rbecarbohldrasss and 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 nc o m p o u n d \ , n c a r b o h ) o G r ca . E d s d . c r . a . c d \ ! c o 5 i rl J . d . r g r o h i g h eirl 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 e f 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 dd j u n o sr o r Kc! votdn Adled antlhc!, Brcwing, Cohpalrion

Termanll 60 L Thh d-arnylase liquifis adjuncl!. It has a standardized activityoi60kilo Novo Amylas unirspe.gran (25). The lemperature oplimlm is 50'C in the pH ranse5J. rr'errlzre.1.J.tThislroteinas, which breaks downlroteinsro peptidr,conrains1.5 An6on unirs pr gran (26). Ar 55'C the optimumpH is in tle 6-7 range. cetefio 2A0 L This.nzyne, composed ofr-slucanaseand oamylase, splib &glucan in malt. It hasa slandardized acrivilv of ( . 2 0 0 B l u c a n a * n i l rp e r s r d m2 7 rA t 5 0 .C ' r so p r i d u n p H i , : 5 . u & Fungon!|800 L. Thk a-tfttylase hydrolyzes starchand dex&ins 1o fcrmentable suga.s.lt contains800 fDngalamylase units pcr gram(28).The lemperature optimumjs 5-l0oC al pH 4.5,

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$,ays involv.d rhc acrionofcnzvmsin rhe mash'ngprocss breakdo$n high molecularweightproteins, to 14 . d c x t r i n sn o n s r a r c hp o l y s a c c h a r i d(e s . 3 2 ,g t u c a n t l ? ) , a n d . y s p e n t o s a D s1 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 1 ) . ( p.otein stabilily(15,19,36) ber.Barleymalr wasorigina y the of mor! rmportantraw materialin beerproccssinS vas the only and of source esscntial enzymes. Less xpensive materiaksuchas raw ric, maize8.isl, barley,and sorghumhave little or no enzyme content.excprfor rhe ,-amyla:e or baieyr thetrltarch. proiern. and glucanhaveto bedigesled olherenzyrnes by inthegrisr. Thus theamountofthecheapef adjuncts rha!canbeadded themash !o k reslricted the limiled supplyof enzymes by f.om the mah irself. This rerearch sludiedthe possibilily ofinoeasingihe amoDnt of adjuncl(in this cas, sorshDm) the mixtu.e ofraN malerisls in in process usingadded the brewing by enzyme!. followingfactors The were examinedto delerminethe orrimum conditionsfor such treatmenl: l) comparisonof the degree fermentation of and the degree p.oteolysis the wo.l as a function ofthe addirionof of in enzynes, 2)quanlitarive and dlertrination rhevarious of niirogencontairingcompounds rheraw materials in all stages in and ofrhe MATERTALS AND METHODS All the expe.iments wereconducted lagerbor( 10. B) under on procesing conditions at the National Brewery Ltd., Netanya, hrael. Six thousand kilograms rav malelials, of co]nprised 65% of nalt and 357,sor8hum additionto 240hi ofwate..vere usedin in themash. Enzynes(Novo Indusbi.s, Denmark) we.euseoro ass! in rh utilization of adjuncts(Termanyl and Neulrase) and to supploment malt qDalitr(Cerefloand Fungamyl). the
?re$nrcd d rhe44!hAnnuat M*rins, Toronro,canada,Mry t973.

Thc NarionalBrevryLtd. useda mashdecoclion system. One rnashvas 10070 ma and conrainedihe enzynesNeulraseand Cerflo;th othrwascomprised rhe sorghumand I07ooi rhe of total wcightof the malrandconraincd Tcrmamylenzyme. th Bo!h w.emashed 50oC, and the all-mahmashwa! rerained thh at ai temperalur during th liqucfactionof lhe sorghum.The 1wo mashcs wererhnmixed heated 76oC aftcr saccharificalion and to sleps 60-62o and at 7l'C. Conkol I wasrhis regularproduct at C ot National Brewery Ltd,, but wi!hout theenzymesadded. Con!rol ll was thc all-mak nash, usedto obrain comparative dara on

Two lestsamples wereured.TestI conrained 6570 malt and 35Eo s o r S h u m p l u s 0 . l T o N e u t r a5 S b a s d o n r h e w e i g h t h e m a l i . I. se i of 0 . l T o T r m a m y l 6 0 a s e d n r h w e i s h o f r h e s o r g h u m n d0 . 3e Lb o ! a, ofFungamyl600 l, pcr hcctoliterof wo.t. TesrII wasTsl l pll]s 0.025E0 Ce.eflo200 L based the weiShtofrhe mah. on Fof all samples, wod was boiled for 1.5 hr wirh tbe hoD lhe extractaddedin threeponions(t l0 g/hl). After beingboiled.rhe *ort was pumpedinto a settlingtank and cooledby meansof a to of llale heat exchange. a pircling rmperatxre l0oc. Il was pirchedNith Sa..haramrces Corlsbetgensis yeast,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 frmentation of737.,except lorControl I, whichwaslrarsfered at 69Eo.Lagetinglastedfive weeks.Beforefilkalio!. Prolesal(a .billproof enzymeof SchwarzSe.vices lnternalionatLtd., New York) wasadded(2 s/hl). The beerfrom eachbrtch wasclarified by dialonile filt.ation and polished througha sheet filter. The analyses ofthemall, woit, and beerwer caried out by EBC (9) methods and by the methods ofBausch(2), KrDgerand Bitig {18),and Moll el al (20). The nitlo8enconlenlofsanples wasde|eIIniDed ihe Kieldrhl by or Dumas methods(l), (Buchi nitrogendeterminationsysten: digertionapparatus, Buchi 425jdisrillationDnit. Euchi320)_ Foi the delerninarion of coagulabland albxmore nirrogen, rhe. methods ofKolbachandwilharn (4)and De-clerck{7)wereused. For the actualnikogendeteroinalionafteriheseprocedures, the precipilates and fitrratesofthe$elestsweredigesled and distilled

0361-0470/30/01002304/$03 00/0 o1930Am.icanSoci6ly Brwing or ch6misls, Inc.

23

24

V o l . 3 8N o . I resul$andrelate usedto calculatethe them $eightsweietherefor 10tle originalsadlles. as bel*eenthe total Total globulinvas calcuiated the difference prorein (froD TCA) and th albumin. Peptideand freeaminonitrogenweredeleimined spectrolhotoVarian Techtron, metrically on a UV-YIS Speckophotometer Elemental H, N, Cl C, model635, lhe 321 570 no.egion (9,29). in by aDdS weredetermined microanalysis. was'measured with an Ostwaldviscorioeterand The viscosity ofone according Krugerand Bieli8( l8) on the basis to calculated pa.ticular concent.ation wort and beer.Chill haze(physical of with the EBC hazemeter afier one day at srability)wasmeasured 40oC, folowed by chillirg to 0oCfor 24 br. by The ioam stabilitywas detrmined the modifiedCarlsberS method as the Sigma value of beer (33,34).The exponential .eaction,is alplied to equation,which representr unimolecular a 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) (7d, 1.38 N, Dumss db) 4t,9 c (%,db) 6.59 H (%,db) (9) The followingindices wcredeterninedby EBC Methods but li.nitsofConroll, arenol bccausc theywcrelrithin the prescribd presenred thc rablsr malt and sorghrm-moisture, 1,000for in for \rort-extract, kerncl{,eight,and gcrminationtcsq laboratory llltration time, color beforc andaficrboiling,pH, saccharificstion, $ort-xtroct, color, HartongVZ 45, and ITT; for lcchnological and olarity,pH, saccharification, aroma;for beer-apparenl and 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 in a acidswcreevaluated the bccr!, usiDs BeckmanD proced (2I ,38).Here, ure and th two-column a mino acidanalyzer rror wre ouisidethe limils of exprimental too. ro diffrenccs are found,and the rcsults not rcportedin thc tables. Analyticaldaiaobtain.d on the malt and sorghum Sritsusedin th expefiments arc 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 are constiluents shownin Tablcll for both th wortsand rcsutant

and thir nitrogencontents delermined Kjeldahl. by The totat protein was precipitated 1070 by trichloroacetic acid (TCA)j rhe nitrogencontnt,doterminedbythe Kjldahlmthod, wasmutriplied a factor of6.25 and.eportedascrudprotein. by by Total protein and albunin lfere determined rpectrolhotofrom a lyophilized of melricmahods ramlle. using concenhalion a 100mg/ml for eachdetermination. Total proreinwasalsodete.mined the Biu.el reaction, by based on the irleraction belvencopprand pepride bondsofprotcins. maximumat 5zl0 n.n This savea puQle color *ith an absorption {16). Tolal albuminwasfoundfrom .heraction belween albuminand greenin a tuitablbuffer to form an Albumin-Bcc Bromcresol Daximumof 610nm coBllex with a bluecolorand an absorption (8). The lyophilizedsamples Control I and T.sts I and II bee. of containddifferenl amoun$ of unfermented solids:ll00 ml of Control I contained4.25 LTest I3.40 L aDdTertII3.00g). These

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

0,31 80.3 t.0 5.5 98,0 20 7t , 4

10.6 t,54 t.53 40,0 6.E2 0.39 0.30 85.1

Extioci, rsdb(%) solubkpror.ii (%db) solublc N(m8/100 nl) (m8/100 ml) Apparcnl alicnuElon, limil oi icmcnbtion(%)

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

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

II

4t.01 0.54 0.10 1.97 69.0 0,97 5.0 t56 69,1

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

Apparent atlennario. linit

37,85 6.t0 0.35

38,39 0.69 0.15 1.90 12.0 8,4 l6l 70,3

o:1.'

It 38.6E 6_4t 0.69 0.12 |,71 12.5 0.3? t.t 165 10.5

I
39.10 6.80 1.20 0.60 t,t6 740.

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

39,91 6.63 1.0? 0.45 1.43 85..'8_

II 39.40 6.51 0.9? 0,56 t.34

tn:1
l6l

'-9

Exlnd oI sp&ge waier (%)

Tasr. test (.udber oi voics (nB/L) Anthocyanoecns

,o.0 3.9

tt,

5 l0-6 3,8 3.9 3.1

'.1.
Ll

,.u

0 28.8

5,3

t.r_

l 24.2

'.1

0.8 t22

0.8 lI5

125

'A11brqs ar. fion a nal/sorshun

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

ASBCJournal

25

Microanatytical results indicated that the carbonand hydrogen valueswerecomparable liom worr to beerand sidilar to those (Table I). However, slown to! the brwingmalerials direrences sere observed for chlorine and lulfur. which may have some

Viscosilydrta for lhe worts ofthe mall/sorghu!, brewsreflect the influence otenzymeadditionduringprocesing.F.omT.st II, vhich had Cereflo200Laddedto the mash. low beerviscosity a of l.14 was obtained.This loweredwort viscosity coincided with a .eductionin mashfitration time to lossrhan4 h.. subslantial SUMMARY The aplarenl!trenuationlimit otfrmntarion i.np.oved was by thc useofcnzymes the mash.This can be seen TesrsI and II in in The influence lhe enzymes mashing, worl fermentation of on on worts and mosl dramaticaliyin the Test I and II beers,where andberstorageprocsses. on beerqualityitself examired and was Fungamyl L waspresent 600 duringfrmentation slora8e. and The in the NationalBreweryLtd. Addition ofth proteolytic enzyme, valueof8770is resarded some$hat as highforlasrberHoweler, Neulrase, the oash, in .ddition to oihe. enzyme 10 supplemenls, the 74% fot the Cont.ol I beer is lower than desi!d.A more .esultedintheinc.easednitrogenconlenlofthevonandtheber. lvolofenzym .estricted additionshouldDrovid desired the lelel Th increase lh los molecularweight in nitrogenous compounds, ot 80 82ED. suchasaminoacids(freanino N), albumoses, peptidcs and may Thequantityof anthocyanogens polyphnols somevhal and was havea decisive role in detrmining tasle,foam, stability,and the lowerin theTestI and ll be6thaninthe ControlIIber, no doubl chiu sensitiviiy characteristics beer. of because the useof sorghumgrih and hop extraci in rhe tsr of Animbalancein the nitrognous compounds mayalsoleadto a brws. Values all samples in *ere Iowrthan would beanticipated darkercoloredbeerandto physical, chmical, lasteirstability; and in regular ber production, about 220 in8/L (18). The tfe p.oductionof thesecompounds mus! therforbe carefully polymcrizationindex for all samples hiSher rhan would be h (23,24). resulated expcred rrom olher repo.tson all-lnall beers (3,6). W i t h t h e u s e o f e n z y m e s ,t h e a m o u n t o f f e r m n l a b l e Froman econoInics ndpoinl,theenzymearcared sta gave Inashs (estimatd ca.bohyd.ales bytheapparent attenuation linit) in the yicld than did Control L With regardro a 17, highe. brewhouse g.early. \t/ortand beeralsoincreascd beerqualily,the enzyme-proccssd br$sgav.subsrantially bencr Tastc differenccs1fere noticcable in th producr, but no beerfoam stabilitythaD.lid the Control I brcw. Howcver,when prefcrncc obtaincd. stathtically significant was ighttactrscompared beersfromthe enzyftebrcwswithbeer the The advaniagcs ofenzymeaddition in thc brcwingproccss are from Control I for arorda. tastc, and intensityand qualiry of lecn in th increased alnounts of solublc nitfogcn-containing prfcrrcd leslproducr bittcrncss, threeof thetsstcrs rhc andf ivrh c o m p o u n d ss n d f e r m n t a b l e a r b o h y d r a t e st,h e d e c r e a s d c control.This single tcst cannotbe considercd significant docs bul 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 eT , tih c r a s e i n y i e l d , l so nc mphasize the need for conrinuedettenrion to this import&nr and in ihe possibilityof usin8 larSeramounts of ihe cheapr quality factor. unmalted &djuncts obtain a similarproducl. to Thc nitrognou! content, bytyp,in thcwortsandbe..sisshown This has beenthe firsr siagein the srudyof enzym in ihe us in Tabl. III, with reference data given for Conrrol II. Usc of b.ewingprocess. Fulu.estudics planned considcrih are to effccts nzymes bre$,ing in substa ntiallyincrased both thc won and Deer of an increase the .elativeamountsof unmaltedadjunch and in 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 svarialionin theproportions till ofenzymcs obtsind from a numberof substantially lower than thosefound for Cont.ol IL Freamino nilroSenwas also increased and, in this instance, reached lvels ACXNOWLEDGMENT similar to rhat of th all-malt conrrol. On the olher hand, wca.csraicfulro Minbr.yof thc lndusrry! Trad.lndToudsm(Offhcof coagulablenitrogen was not significandyincreasedovef rhe ih. Chhf Sci.nris0 and 10 NsdonalBrcweryLtd., Nstanaya, who Control I wort or beeraDd.emained relatively compared low wirh rhis Manythantsro Dr. R. viclor (The lupported vork b, conrlact, Conrol IL H.brew U.iv.rsity) he!nelp 1ne ior h orgadzation oltht nanu*ripl,and Furtherexamination ofthe nitrogenolrconstituenrs rhfour of ro S, Fcldman, K.upnit,S, Melrr (National G, Bleeory Lld.),andA. beeB showed rhat albumose dtroSen and peptide nitrogen (TheHeb!.w Convay Unir*iry) lor assisL.cc oblaining in rcsulk,

fractionswereincreased th enzym by treatmenl;in the caseof peptideN, the increase was to valuesabovthar of Control U. TcA-precipit.ble prote;n,prolein found by th Biuret Dethod. were.ll infieasedmarkedlyoverlhe albunin, and globulinvalues Conlrol I beer lalues by the enzyne treatmnt. This was tor larticularly apparent the TCA protein,the Biu.el protein,and albumir Because Tstland Tesi lI beeisand $orts are quite th similar in nirrogen ard prolein patterns, the hajor erect is obviouslythat ofth.'mash treatment presumably aclivity of rhe Neut'lse 1.5S. the nroteinase

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

II

III

III

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

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 . en i m g 1 0 0 l . e r c e p r o - r f e K i r , o a ha n d D u m a \\ d l ' { l b r e r sa E l - o m n a l r / s o r s h u n m r h . e r c e p Lo . a o n r o , I l , l r o ma n a l l - m a lm d s h . l r "

28.1 32_4 I l.?

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

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

26

Vol. 3ENo. I

T,ITERAIURE CITED l. Aslociaiion of Offlcial A.alytical Chemists.Official Methods of AnatFt (llrhed.), p.858, The Asociation: warhington,DC, t9?0. 2, Bausch, H. A. Arbeitsvoscnrift.n znr chedisch-bEulecnhisncn 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, Solodorashcheriya Pivovareniya. i 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. 7. De-Clerck,J. Lhrbuch dei Erauerei (2nd cd.). Versuchy ud ftrBlauelei: Bedin,I, 1964t 1965. Lehransrah II, B. 8, Doumas, 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. S., I | . Gorinstein, L:lsrid F, od Cheh. 21(l)145, 5. 197 12. Corinstein. J. ,trrrc. Ol Anal. Ch.h.59(6)1380, \976. S,, 13. Gorinstein, J,,.tsrid,Foarl cheh. 261204,1978, S,, 14. Crill, W., End Plspok, J., tai Bev, Conr, Ptoc, Canet, t4th, p, Sdlzbetg, 1973, 11l. 15. Kalashnil(ova, M.,Ezbov,I. S,,Nakhapctyan, A., Menyailova,l. A, L. L, end Cracheva, M, Chem,Abst. 85:44874x, L l9?5. l6 Kin!slcy,C. R.In:coopcr, G. R., (ed.). sundard M erhods Clinjcal or Chemhrr, (7th cd.),p. 199. Academic Pre$:Ncw Yori, 1972. t7. Ktoppet,w. J. bateett, I l5(8)1205, I975. 18. Kruser, E., and Eielis, H. L Berricb$ und Qoaherskonrlollei. Brauereiund alkobolfreierOctr?in*c,indusrrie. Paul PakcyrBdlin, 1976. 19, Li.bcrfrrn, E. R., taz]rrd Techhal. Die, 4(2):69, 1975, 20. Moll, M., Thir, v., Schmir! A., and Parisoi,M. J, ,4r.. So.. arer. ch.n. 341187, 1976.

21. Mol, M., Winn-Tnal,and Nod, J- P. ,ior (/{a,c, 3(}8):293, l9?2, 22. Naoyuti Kunilate. xrpp,h Jazo Klakoi Zasshi 7119):6A2,1916. 23. Narzi$, L., Kienin8er, and Reicheneder, ,.a!,eL, 116(31)i999, H., E. 1976. 24. Na.zns.L., Reicheneder,8., RnsitzLa, Stipple.,K.,and Hunkel,L. P.. Brau||elt, I 15(21):90 1975. 1. 25. Novo Elzyne Inform ion. Novo Indusllia/s, Novo Alle, DK, 2880 Bagsvaerd. Dc.nark. l2la-GB {1975), AF4/3a-GB ( I976),AF 9/3acB(r976), F?0/2B(r977). A 26. Novo Enzymelnfo.nalior. Novo lndusria/s, Novo Alle, DK,2380 (1976). Bassvacrd, Dennark. F-763754/BSO/HF 27. Novo Enzyne Infomation, Novo lndust a/s, Novo,{llc, DK, 2880 Bassvacrd, Dennark. lo9a-GB(1975). 28. Novo Enzyoe I.formalio.. Nolo Industria/s.Novo Alle, DK,2880 aagsvaeld, Denmark.079b-cB(1975). 29. Petetrhofer, and Frey,y . Mikro.hih. A.to, p. 981, 1969. C., 10. Potrcvskaya, N. V., (ilya(ova, o. v., roakova, R. A., and Nefedola,Ya. v. ched. Abslt. 8l:241264, t974. ll. Pollock, J, R. A., and weir, M. J, Te.h, Q. Malet ore||. Aso.. .ah. \3lt)122,1916. 32. Protsen(o, N, Scmenola,T,L,and Maftsev,P M. Cbcm Abstr. A. 8l:167800v,19?4. 13. QA 9 AnalydcalMethods,p.92, John Labatlt: Londo., Onrlrio, t912. 14. Ross, S.,and Clark,C. L. llalledein tub. Caflhh, 6:46,1939, Jt, Sglnsnovs,L, S,, Zhda.ova.L, A., and Sheprun, S, Chcm.Abstr. L. 85:14570tj,I975. 36, S.riban, R,, Hebcn, J, P,, and Dclillc^, G. ,irr (r'r'dr.l), 7(4)142, 1976. 37, Somme!,G. ,r.r, D/a.47(ll)160,197? 38, Spackman, H,, Stein,W, E,. and Mookc,S. /lral Ch?4.l0rll90, D,

r9t8. 19.wi.B,A.J. F*nearotiat(4))85, l 1915.

40. Wic8,A J, Ptoces Bioth?n. 1t(6)t21,1916,

July lReceived J, 19781