J. //«/. Brew., My-June, 19, Vol. 82, pp. 149-3 149 ACTOS ESPONSIBLE O THE DECEASE IN ph DUING BEE EMENTATIONS By N. Coote nd B. H. Kirsop (Brewing Industry eserch oundtion. Nutfield, edhill, Surrey, Englnd) eceived 1 September 19 The reltive significnce of fctors which cuse ph to decrese during fermenttion hs been investigted, using eleven yest strins. Orgnic cid excretion nd bsorption of bsic tnino cids both hve substntil effects: solution of crbon dioxide nd bsorption of primry phos phte contribute to smll extent. Buffering cpcity, over the ph rnge 4 to, increses slightly during fermenttion. Approximtely 3% of the increse in hydrogen ion concentrtion cnnot be ttributed to known cuses; direct excretion of hydrogen ions my be responsible. ermenttion with yest propgted in semi-erobic conditions rther thn in fermentors gives beers of lower ph nd incresed orgnic cid content, but the ltter fctor is not in itself sufficient to ccount for the ph difference. Key words: beer, buffering, fermenttion, ph, wort. Introduction In n erlier study of fctors which ffect the excretion of orgnic cids during fermenttion, it ws noted tht the previous history of the yest hd mrked influence'. Thus the use for fermenttion of cells of Scchromyces uvrum (crlsbergensis) N.C.Y.C. 24 grown scmi-erobiclly in oux bottle led to greter content of pyruvte, lctte, succintc, -ketoglutrtc, -hydroxyglutrle, nd mlte in beer thn did cells grown in stirred fermentors, in which the oxygen supply ws restricted to tht initilly dissolved in the wort used. The reverse sitution occurred with cette. Becuse oux bottles nd stirred fermentors give growth conditions brodly similr to those present in brewery propgtors nd brewery fermentors respectively, experi ments were crried out to sec if the effect of growth con ditions on subsequent cid excretion ws generl one. Additionlly, it seemed desirble to investigte the reltion ship between cid excretion nd the fll in ph which occurs during fermenttion. esults nd Discussion A further ten yest strins were exmined, nd the results re shown in Tble I, together with those for N.C.Y.C. 24. It cn be seen tht the phenomenon of incresed excretion of orgnic cids when fermenttion is crried out with yest grown in oux bottle s compred with stirred fermentor is generl one. The differences rc not result of vritions in the extent of fermenttion: whether propgted in oux bottle or stirred fermentor, ech yest strin pro duced beers of similr finl specific grvity. There ws mrked vrition from strin to strin in both species with regrd to the effect on individul cids. Acette levels were reduced by the use of yest from oux bottle, prticulrly in the cse of S. uvrum (Tble I). Beer ph ws lower when yest from the oux bottles ws used (Tble I), nd it seemed likely tht this ws result of the TABLE I. Influence of previous propgtion conditions on orgnic cid content of beer Yest Strin (NCYC) Conditions of Propgtion cetic pyruvic lctic Orgnic cid concentrtions in beer (mg/litrc) succinic -ketoglutric -hydroxyglutric mlic Totl PH 83 82 18 8 2 1 388 3-93 1 24 3 4 3 9 4 3 248 3-91 3-9 * 32 9 3 9 83 4 1 3 9 3 3-3-84 49 99 4 111 3 II 14 391 3-9 49 3 3 8 4 2 288 3-9 1 38 114 82 98 1 1 1 4 3-82 23 48 4 49 4 3 3-92 4-2 24 3 2 J '1 ^ 2 1 2 4 4 1 8 3 1 3 8 3 4 193 99 2 4 31 4 38 3 4 9 1 4 9 3 4 8 489 4 3-8 4 3-81 3-8 3-88 4 3-8 4- = oux bottle. = ermentor.
COOTE AND KISOP: ph AND BEE EMENTATION [J. Inst. Brew. greter content of orgnic cids. To check this, cid con centrtions of pir of beers fermented by N.C.Y.C. 24, grown under the two bove-mentioned conditions, were mde identicl by ddition of the pproprite orgnic cids to the beer contining the lower cid concentrtions. It ws found tht this ddition reduced, but did not eliminte, the ph difference between the pirs of beers (Tble II). Therefore, fctors dditionl to the incresed orgnic cid content must be responsible for the incresed fll in ph in fermenttions TABLE II. The effect of orgnic cids on beer ph Beer fermented using yest from oux bottle Beer fermented using yest from stirred fermentor Beer fermented using yest from stirred fermentor with dded orgnic cids NCYC 24 ws used. BeerpH 3-8 4-39 using yest grown in oux bottles. In other experiments, the orgnic cids tht hd ccumulted in beer during the fermenttions giving the results shown in Tble I were dded to wort, nd, s expected, the ph of the wort ws reduced (Tble III). Agin, the decrese ws not s gret s tht which occurs during fermenttion, showing tht, in this sitution lso, dditionl fctors re involved. This is prticulrly pprent when the results re expressed in terms of chnging hydrogen ion concentrtion (Tble III). The literture regrding the cuses of ph decrese during fermenttion is diffuse nd llows no generl conclusion to be drwn. In view of the significnce of ph s fctor ffecting beer qulity, it seemed desirble to consider the sitution fresh, nd obtin quntittive informtion wherever this ws possible. It is well known tht the ph of wort flls most rpidly erly in fermenttion nd chnges only very slowly in the lter stges4-*-1* (ig. I); when yest remins in contct with the beer fter sugr utiliztion is complete, s in the cse shown, the ph my rise to slight extent. The decresing rte of ph chnge during ctive fermenttion reflects the logrithmic nture of the scle, rising from SorensenV conversion of hydrogen ion concentrtion to its negtive logrithm, ph. In consequence, progressively greter qun tities of hydrogen ions re required to decrese ph by, for exmple, 1 unit s the ph vlue of the fermenting wort flls (Tble IV). A reltively smll chnge in ph towrds the end of fermenttion is thus the result of reltively greter chnge in hydrogen ion concentrtion (ig. I). The differences between fermenttions re more redily pprent if they rc expressed in terms of hydrogen ion concentrtion rther thn ph; this cn be seen in the results quoted in Tble III. 4 ph 3-2 4 Durtion of fermenttion (h) - SO 4-4- ig. 1. Chnges in ph nd hydrogen ion concentrtion fermenttion using N.C.Y.C. 24; solid line, ph; line, hydrogen ion concentrtion; open circles, grvity. during broken specific TABLE HI. Chnges in ph & H' concentrtion on ddition to wort of the orgnic cids ccumulting during fermenttions Yest strin (NCYC) BeerpH Increse in hydrogen ion concentrtion during fermenttion (^g ions/litre) Wort ph fter the ddition of orgnic cids * Chnge in hydrogen ion concentrtion due to cid ddition % Contribution of cids to totl hydrogen ion excretion 24 1 32 3-93 4 11 3-91 3-9 3-3-84 9 1 4 192 4-4-48 4-4-2 4-1 4-2 11 23 2 3 14 11 3-9 49 3 4-4- 2 1 3 3-9 1 4-4- 19 38 3-82. 143 4-33 4-4 39 32 2 23 3-92 4 1 92 4-4 4-2 3 2 24 24 3-8 4 1 4-4- 38 2 i. 2 j,. 3-81 3-8 3-88 4 14 4 1 4-4-4 4-3 4-49 28 2 2 3 3-8 4 3! 4-92 4-2 24 ph of wort -11 (hydrogen ion concentrtion = 8/ig ions/litre).
Vol. 82, 19] COOTE AND KISOP: ph AND BEE EMENTATION 1 TABLE IV. eltionship between Concentrtion ph ph nd Hydrogen Ion Hydrogen ion concentrtion (fig ions/litre) mesuring the buffering cpcity t intervls between ph 4 nd reveled tht the buffering cpcity of wort nd beer were the sme t ph 4-, but tht beer becme generlly better buffer thn wort t ph vlues bove this (ig. 2). 4-9 4-8 4-4- 4-4-4 4-3 4-2 4 - -8 2 2-1 31-39-8 1 31 9-4 The ph fll must be reflection of yest ctivity, for stored sterile wort does not lter in ph to n pprecible extent. Yest my be envisged s cting to reduce ph by () ltering the buffering cpcity of the wort, (b) bsorbing bses.nd (c) excreting cids. The possible role of ech of these fctors in bringing bout the fll in ph which occurs during fer menttion will be considered seprtely. Buffering Cpcity It hs been suggested tht the removl of buffering sub stnces is prtly responsible for the chnge in ph during fermenttion4-, the inference being tht the cidic substnces of wort then give rise to higher hydrogen ion concentrtion in the less well buffered solution. Worts nd derived beers were therefore exmined to mesure their buffering cpcity, tht is their bility to resist chnge in ph on the ddition of cid or lkli. The numericl vlue for buffering cpcity vries with the ph t which the mesurement is mde. This gin reflects the fct tht the ph scle is logrithmic: the quntity of hydrogen ions necessry to bring bout given chnge in ph diminishes s the vlues pproch neutrlity. Also, in the present cse, it is necessry to crry out mesurements t ph vlues found in wort nd beer: mesurements outside this rnge will be ffected by substnces which my be ineffective s buffers t wort nd beer ph, nd therefore irrelevnt in the present context. Investigtions were ccordingly crried out in the wort nd beer ph rnge of 4 to. When buffering cpcity ws mesured s the quntity of cid or lkli required to chnge the ph of given volume of wort from to 4, nd of the derived beer from 4 to, it ws found tht the buffering cpcities of the wort nd beer were similr (Tble V), the beer being slightly better buffer thn the wort. The buffering cpcity of the ll-mlt wort (sp. gr. 1-4) ws such tht, to increse the hydrogen ion concentrtion by given mount, it ws necessry to dd bout 2 times this quntity. Comprison of beers of different ph vlues, prepred from the sme wort, but using yest propgted with nd without ccess to oxygen, showed tht they were of similr but not identicl buffering cpcity. The beer of the lower ph gve slightly higher vlues nd both beers hd somewht higher buffering cpcity thn the ori ginl wort (Tble V). A more detiled investigtion mde by ig. 2. The vrition of buffering cpcity with ph; closed circles, buffering cpcity of wort (ph -2); open circles, buffering cpcity of beer (ph 49) fermented by N.C.Y.C. 24. Clerly, the fll in ph which occurs during fermenttion is not the result of net loss of substnces which buffer wort in the ph rnge to 4. It cn be inferred tht substnces such s phosphte nd mino cids, which re ssimilted during fermenttion nd which hve been sid to be res ponsible for some of the buffering cpcity of wort1-2, re not effective buffers t the ph of wort. This ws to be ex pected from their titrtion curves", nd pk vlues18, hving regrd to the fct tht substnce buffers over ph rnge of bout one unit on ech side of its pk. The dicrboxylie mino cids, glutmic nd sprtic cids, hve pk2 vlues which re in the pproprite rnge, but their totl concen trtion is too low (bout Iitim)31 for them to contribute substntilly to buffering cpcity. ig. 3 shows tht the 2-4 g 4-. 4 TABLE V'. Buffering cpcity of wort nd beer Smple Wort Beer A BecrB Actul PH -2 49 3- Buffering Cpcity (ml cid or lkli (-IN) required to chnge the ph from - to 4- or vice vers.) 2 NCYC 24 ws used Beer A, fermented by fermentor-grown yest Beer B, fermented by oux-grown yest 3- ml -1N sulphuric cid (ml) ig. 3. Titrtion curves of -% csein hydrolyste (open circles), -% csein hydrolyste nd 1 mm citrte, dded s tri-sodium slt (closed circles), ll mlt wort, sp. gr. 4, (open tringles), nd mm glutmic cid (dded s L-mono sodium slt) (closed tringles). -ml volumes of the bove smples were used.
2 COOTE AND KIKSOP: ph AND BEEK EMENTATION [J. Inst. Brew. concentrtion of glutmiccid necessry to buffer s effectively s wort is the order of mm. Phosphtes become effective buffers t ph vlues bove bout -, nd so will contribute to buffering cpcity if the mesurements mde extend over the rnge - to, s hs sometimes been the cse2. Phosphte will lso ffect the results of titrtble cidity, which is commonly mesured by titrting beer from its ctul ph to vlue in the region of neutrlity. However, chnges in phosphte concentrtion will not influence the buffering cpcity of wort in the prcticlly importnt ph rnge of 4 to. With regrd to the nture of the substnces which ct s buffers in wort nd beer, the orgnic cids, of which citric cid is the predominnt one in wort, hve pk vlues in the correct rnge to buffer t wort nd beer ph19, but re present t too low concentrtions to ccount for the observed buffer ing cpcity (ig. 4). or exmple, wort which possessed buffering cpcity equivlent to tht of mm citrte buffer ws less thn lmm with respect to totl orgnic cid content. > x 2 2 3-1N sulphuric cid (ml) -insodium hydroxide (ml) ig. 4. Titrtion curves of beer from pilot brewery fermen ttion using N.C.Y.C. 24 (solid line), nd solution of orgnic cids contining the sme concentrtions s those in the beer (broken line): cette, mg/litre; pyruvte, 1 mg/litre; lctte, mg/litre; succinte, mg/litre; - kctoglutrte, mg/litre; pyroglutmte, 8 mg/litre; mltc, 4 mg/litre; citrte, 3 mg/litre. -ml smple volumes were used. rom the quntittive point of view, the nitrogenous constituents of wort nd beer merit considertion. Becuse their pk, vlues re low, most mino cids re not importnt with regrd to buffering cpcity in the relevnt rnge. However, it is known tht the pk, vlues of simple peptides re significntly higher thn those of the constituent mino cids". It seems tht peptides my be effective buffers t wort nd beer ph vlues, s suggested by erlier workers1'2. Exmintion of solution of csein hydrolyste (-%; totl nitrogen, 84-3 mg/ ml; non-mino nitrogen, 39-3 mg/ ml) showed tht the buffering cpcity ws similr to tht of wort, prticulrly if the solution ws supplemented with sodium citrte (1 mm citrte) (ig. 3). Even though no de tiled informtion s to the nture of the peptides present in wort or csein hydrolyste is vilble, the results support the view tht the non-ssimilble protein degrdtion products ply substntil role s buffers in wort nd beer. Although buffering cpcity does not chnge pprecibly during fermenttion, it is nevertheless n importnt fctor in determining beer ph. or exmple, consider wort t ph - which, on fermenttion, produces beer t ph 4-; it cn be clculted tht the hydrogen ion concentrtion hs incresed by 9 ms ions/litre. If the buffering cpcity of the wort is now hlved, then ssuming other fctors leding to incresed hydrogen ion concentrtion during fermenttion to be unffected by the dilution, the increse in hydrogen ion concentrtion would be 18 fig ions/litre, which would give rise to beer of ph 32. Absorption of Bses It hs been suggested1 tht the ssimiltion of the dibsic phosphte ion HPO4 leds to incresed cidity during fer menttion, nd experiment showed tht the ddition to beer of the dibsic potssium slt (phosphorus concentrtion mg/litre) led to rise in ph from 3-92 to 4-2. How ever, it hs been estblished88 tht yest cells bsorb phosphte s the monobsic ion H2PO,,-, nd nlysis of yest showed clerly tht potssium nd phosphte re bsorbed in equimolr proportions. The net chnge expected in wort fer menttions is thus the removl by yest of KH:PO4, lbeit s seprte ions. Anlyses of worts nd derived beers showed tht the rtio of K removl: P removl (on n tomic bsis) during fermenttion ws 1:1-. This vlue is clerly more com ptible with the bsorption of KH8PO4 thn of K2HPO4. KH2PO«is cidic, lthough slightly more bsic thn beer, nd its ddition to beer, to replce the phosphte removed during fermenttion, incresed the ph from 3-92 to 3-9. In terms of hydrogen ions, this difference represents />g ions of hydrogen/litre. In reltion to the totl mount of hydrogen ions tht ccumulte during fermenttion, phosphte ssimil tion is therefore of only minor significnce. Other bsic sub stnces considered include the bsic mino cids, nd the purine nd pyrimidinc bses nd nucleosides. With regrd to the mino cids, lysine, rginine nd histidinc re strongly bsic t beer ph, while lnine, thrconine, glycine nd glutminc rc very slightly bsic. In contrst, glutmic nd sprtic cids rc cidic t beer ph. The net contribution of mino cid uptke to the fll in ph ws mesured by dding to beer those mino cids which re ssimilted from wort: the quntities used were those listed by McWillim for British infusion wort. This resulted in n increse in ph from 4- to 4-23, corresponding to n increse in hydrogen ion con centrtion of 3 Mg ion/litre. The literture suggests tht the overll chnges in concentrtion of purine nd pyrimidine bses nd nucleosides during fermenttion is slight3-". It is therefore unlikely tht these compounds would significntly ffect the fll in ph tht occurs during fermenttion. Excretion of Acids. Crbon dioxide, which gives rel tively wek cid in solution, nd the orgnic cids rc the mjor cidic molecules known to be excreted by yest during fermenttion. Crbon dioxide is produced in lrge quntities nd sturted the wort, in the fermenttion shown in ig. 1, when the specific grvity hd fllen from 1-4 to 1-32. Addition of this quntity of crbon dioxide to wort decresed the ph from -14 to 4-3. In terms of the hydrogen ions present this mounts to n increse of 11-4 pg ions/litre. Yest hs been reported to excrete nucleotides, which re cidic, during fermenttion18. However, the mounts present in beer rc low showing tht their excretion is unlikely to contribute significntly to the fll in ph; it ws found by experiment tht cytidinc monophosphte hd little effect on beer ph, s the ddition to beer of mg/litre of this compound only reduced the ph by 2 (from 4 to 43). Although orgnic cids re excreted throughout ferment tion, nd contribute to the ph fll (s shown in Tble II), it is cler tht the whole fll in ph cnnot be ttributed to cid excretion. The results given in Tble I llowed the effect of the cids to be quntified, by their ddition to wort so tht the concentrtions were the sme s those of the beers. When this ws done, it ws found tht the hydrogen ion concentrtion ws incresed from 8 to 32 /*g ions/litre, giving men in crese of 24 f*g ions/litre. This is equivlent to 23 % of the totl increse in hydrogen ion concentrtion occuring during these prticulr fermenttions. Clerly, the mjor prt of the increse in hydrogen ion con centrtion occuring during fermenttion is due to fctors other thn the ccumultion of orgnic cids. If one ssumes tht
Vol. 82, 19] COOTE AND KIKSOP: PH AND DEE EMENTATION 3 the uptke of mino cids nd primry phosphte, nd the solution of crbon dioxide, hd the effect found bove in the sets of fermenttions listed in Tble I, then the totl effect of the known fctors which cuse the hydrogen ion concentr tion to increse is of the order of 9 us ions/litre. In these prticulr fermenttions, this represents % of the men increse in hydrogen ion concentrtion: the reminder must therefore be ssigned to unknown fctors. One such fctor my well be the direct excretion of hydrogen ions. This is known to occur in yest nd other micro-orgnisms, both in reltion to trnsport phenomen, nd in order to regulte the internl environment of the cell8--11'"-". However, it is not possible t present to ssess the mgnitude of such excretion (or the indistinguishble uptke of hydroxyl ions) in fermenting wort. Experimentl Yest strins were obtined from the Ntionl Collection of Yest Cultures (The Brewing Industry eserch oundtion, Nutficld, Surrey, Englnd), nd were propgted cither in shken oux bottles for 3 dys t 2CC, so tht ir ws present in the hcdspcc of the bottle8, or in glss fermentors with nitrogen pssing through the hcdspce, s described erlier9. All fermenttions were crried out under stirred conditions3. Both propgtions nd fermenttions were crried out in llmlt hopped wort; both the wort nd the experimentl beers used to determine the effects on ph of the ddition of phos phtes, mino cids, nd nucleotides were prepred in pilot scle brewery by the method of Hudson & Birtwistle18. Orgnic cids were ssyed by prtition chromtogrphy on silicic cid columns s described erlier. Crbon dioxide con centrtions were estimted mnomctriclly by the method of Mrtin"; phosphte ws determined by the method of ogg & Wilkinson11, nd potssium concentrtions were estimted by tomic bsorption spectroscopy. Csein hydrolyste (cid) ws obtined from Oxoid Ltd, London, Englnd. Acknowledgement. We thnk Allied Breweries for crrying out nlyses for potssium in wort nd beer smples. eferences I. Anon., Brewers Digest, 19, 32, (July), 8. 2. Brown, M. L., & Kirsop, B. H., Journl of the Institute of Brewing, 192,8,39. 3. Budy, A. Z., Bcllcu, G. & Vn Ghcluwc, G., Proceedings of the Americn Society of Brewing Chemists, 192,. 4. Bunker, H. J., ood, 19, 19, 8.. Burger, M., Glenislcr, P.., & Becker, K., Proceedings of the Americn Society of Brewing Chemists, 192,.. Conwy, E. J., & O'Mlley, E., BiochemiclJournl, 1, 4,9.. Cootc, N., & Kirsop, B. H., Journl of the Institute of Brewing, 1, 8,. 8. Dvid, M. H., & Kirsop, B. H., Journl of the Institute of Brewing, 193,4,2. 9. Duggn, P.., Biochimic et Biophysic Ad, 19, 88,. Eddy, A. A., in The Chemistry nd Biology of Yests, 19, 8 Edited by A. H. Cook, Acdemic Press, New York & London. 11. Eddy, A. A., & Nowcki, J. A., Biochemicl Journl, 191, 1,1.. Eddy, A. A., Seston, A. & Inkson, C, Biochemicl Journl, 193, 1, 31.. Edsll, J. T., in Proteins, Amino Acids nd Peptides, 13, 84-8. Edited by J. E. Cohn & J. T. Edsll, einhold Publishing Corportion, New York. 14. ogg, D. N., & Wilkinson, N. T., Anlyst, 19, 83. 4.. Hrris, G., & Prsons,., Journl ofthe Institute of Brewing, 19.3,.. Higuchi, M., & Uemur.T., Nture, London, 199,184,81. 1. Hopkins,. H., Journl of the Institute of Brewing, 19, 2,. 18. Hudson, J.., & Birtwistle, S. E., Journl of the Institute of Brewing, 19, 2, 4. 19. KortQm, G., Vogcl, W., & Andrussow, K., Dissocition Constnts of Orgnic Acids in Aqueous Solution, 191, Butterworths, London. 2. McKcnzic, K. G., & Kenny, M. C, Journl ofthe Institute of Brewing, 19, 1,.. McWillim, I. C, Journl of the Institute of Brewing, 198,. Mrtin, P. A., Journl of the Institute of Brewing, 19,, 4. 23. Mulc, D.., Pinnegr, M. A., Portno, A. D., & Whitcr, A. L., Journl of the Institute of Brewing, 19, 2, 488. 24. Pickett, J. A., Journl of the Institute of Brewing, 1, 8,. 2. ink, H., Proceedings of the 4th Interntionl Symposium on Yests, Vienn, 1, Prt I, A33,. 2. othstcin, A., Membrne Trnsport & Metbolism, 191, 2, Edited by A. Kleinzeller & A. Kotyk, Acdemic Press New York & London. 2. Sshr, T., & Miyoshi, T., eport of the eserch Lbo rtories ofkirin Brewing Co. Ltd., 19,, 1. 28. Sorcnscn, S. P. L., Comptes endus Crlsberg, 199, 8, 1.