Alcohol Dehydrogenase Activities of Wine Yeasts in Relation

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, Nov. 1976, p Copyright 1976 Americn Society for Microbiology Vol. 32, No. 5 Printed in U.S.A. Alcohol Dehydrogense Activities of Wine Yests in Reltion to Higher Alcohol Formtion RAJNDRA SINGH' AND RALPH. KUNK* Deprtment of Viticulture nd nology, University of Cliforni, Dvis, Cliforni Received for publiction 25 June 1976 Alcohol dehydrogense ctivities were exmined in cell-free extrcts of 1 representtive wine yest strins hving vrious productivities of higher lcohols (fusel oil). The mount of fusel lcohols (n-propnol, isobutnol, ctive pentnol, nd isopentnol) produced by the different yests nd the specific lcohol dehydrogense ctivities with the corresponding lcohols s substrtes were found to be significntly relted. No such reltionship ws found for ethnol. The mounts of higher lcohols formed during vinifiction could be predicted from the specific ctivities of the lcohol dehydrogenses with high ccurcy. The results suggest close reltionship between the control of the ctivities of lcohol dehydrogense nd the formtion of fusel oil lcohols. Also, new procedures for the prediction of higher lcohol formtion during lcoholic beverge fermenttion re suggested. During lcoholic fermenttion, yests produce smll mounts of higher liphtic lco-. hols. This mixture of lcohols, often referred to s fusel oil, is composed principlly of n-propnol (1-propnol), isobutnol (2-methyl-i-propnol), ctive pentnol (2-methyl-1-butnol), nd isopentnol (3-methyl-1-butnol). Although these lcohols my contribute some flvor nd body to wines nd wine distilltes, it is generlly considered tht when present in too lrge of mounts, they impir the flvor of the products. The formtion of higher lcohols rises through decrboxyltion nd reduction of the pproprite -keto cid (17, 18). the - keto cid is produced either by trnsmintion of n mino cid or by crbohydrte ctbolism s ws first shown by Thoukis (16), Senthe- Shnmugnthn nd lsden (1), nd SentheShnmugnthn (13). Using cell-free systems these workers obtined expected ldehydes from rnge of -keto cids. They lso studied the formtion of lcohols from these ldehydes, using both cell-free extrcts nd crystlline yest lcohol dehydrogense (ADH). Kunkee et l. (6) studied the formtion of n-propnol by cell-free extrcts of yests. Dehydrogense ctivities for severl stright nd brnched-chin liphtic lcohols in cellfree extrcts of yests hve been previously demonstrted by us (7). Specificity of decrboxylses hs been suggested to be n importnt fctor in determining the proportions between the vrious higher lcohols formed (3, 15). ' Present ddress: Deprtment of nvironmentl Toxicology, University of Cliforni, Dvis, CA The present study concerns the investigtions on the ADH ctivity in vrious wine yest strins in reference to their fusel oil productivity, under vinifiction conditions. Depending on the conditions of growth, yests contin severl isoenymes of ADH tht re reltively unspecific (1,, 8, 11, 12). In recent studies, we (R. Singh, Ph.D. thesis, University of Cliforni, Dvis, 1975) hve found the sme to be true in wine yests. However, in vinifiction conditions we found only one isoyme (ADH-I, 12) to be present in ll of the yest strins used in this work. In spite of this, the ADH specific ctivities were vrible in vrious strins s were the levels of fusel oil produced. A high degree of correltion between these two kinds of vribles ws observed. 666 MATRIALS AND MTHODS Yest strins. The 1 strins of Scchromyces wine yest used re given in Tble 1. The respirtory-deficient mutnt of the Scchromyces cerevisie Montrchet strin ws obtined by mutgen tretment of the prent strin with ethidium bromide nd selection of petite colony formed. Medium nd culture conditions. The cells of vrious yests for enymtic studies were grown in synthetic medium designed to simulte nturl grpe juice. The synthetic grpe juice medium hd the following composition (Trverso, personl communiction): 2% glucose,.3% L-mlic cid,.5% potssium bitrtrte,.2% citric cid,.2% vitminfree Csmino Acids (Difco),.1% K2HPO,.25% MgSO-7H1,.5% CCl12-2H2,.1% trce slt solution, nd 1.% vitmin stock solution; the finl ph of the medium ws djusted to ph 3.5 with 5% potssium hydroxide. The trce slt solution ws

2 VOL. 32, 1976 TABL 1. Wine yest strins UCD enology no. Species Strin nme 51 S. cerevisie Burgundy 55 S. bynus Cliforni-Chmpgne 513 S. cerevisie Distillers' 519 S. fermentti Flor 522 S. cerevisie Montrchet 522(RD)b S. cerevisie Montrchet respirtory deficient 53 S. cerevisie Toky 585 S. cerevisie Austrlin Wine Reserch Institute no S. cerevisie Austrlin Wine Reserch Institute no S. bynus Chmpgne (Psteur Institute) University of Cliforni enology culture collection number. b RD, Respirtory-deficient mutnt. prepred by dissolving g of MnSO H2O, g of ZnSO, 1 g of CuSO-5H2O, 1 g of KI, 1 g of H3BO3, 1 g of MoO3, nd.5 g of Co(NO3)2 6H2O in 1 liter of distilled wter. The vitmin stock solution ws prepred by dissolving 2.5 mg of clcium-pntothente, 1,g of biotin,.25 mg of thimine,.25 mg of pyridoxine HCl, nd 2 mg of inositol in 1 liter of distilled wter. The medium ws sterilied by being utoclved. A totl of 3 ml of yest suspension of the -dy-old cultures grown in the bove medium ws used to inoculte 15 ml of medium in 25-ml rlenmeyer flsks. Growth ws llowed to proceed in stnding cultures t 25 C mbient temperture until the medi were fermented to less thn.2% residul reducing sugrs (12 to 16 dys). The fermenttions were followed by dily mesurements of reducing sugr with Dextrocheck tblets (Ames Co., lkhrt, Ind.). In the initil stges the flsks were occsionlly shken to provide some ertion nd uniform cell distribution. Preprtion of cell-free extrcts. The yest biomss ws hrvested by centrifugtion t 1, x g for 1 min, wshed twice with potssium phosphte buffer (.2M phosphte), ph 7., nd ground with glss beds to mke cell-free extrcts s described previously (6). Protein concentrtions, in the cellfree extrcts, were determined by the biuret method (2) using bovine serum lbumin s stndrd. Mesurements of enyme ctivity. ADH ctivity in cell-free extrcts (with ny of the lcohols under study s substrtes) ws ssyed by the mesurement of nicotinmide denine dinucleotide reduction t 3 nm in Zeiss spectrophotometer, model PMQ II (9). The optiml concentrtions of the substrte lcohols used were: ethnol,.5 M; n-propnol,.3 M; isobutnol,.6 M; isopentnol,. M; nd ctive pentnol,. M (7). nyme concentrtions were djusted so s to be in the liner rnge of the ssy. One unit of ctivity ws defined s the mount of enyme cusing chnge in bsorbnce of.1/min, nd the specific ctivity ws units of ctivity per milligrm of protein. Anlysis of higher lcohols. The concentrtions of the fusel lcohols were mesured in distilltes of fermented broths. The modified method of Khn nd HIGHR ALCOHOL DHYDROGNASS IN WIN YASTS 667 Blessinger (5), using Hewlett-Pckrd model 5711A gs chromtogrph equipped with hydrogen flme ionition detector, ws used. The column consisted of hevy-wll, stinless steel tube (6 feet by.125 inch [outer dimeter] [c m by.32 cm]) pcked with 1- to 12-mesh Gs-Chrom R (firebrick) coted with 2% ech of glycerol nd 1,2,6- hexnetriol. Column, injector, nd detector tempertures were mintined t 65, 1, nd 125 C, respectively. Four different stndrd solutions contining vrying mounts of lcohols were prepred; 3-pentnol ws used s n internl stndrd. Chemicls nd substrtes. The source of chemicls, unless given bove, nd techniques for purifiction of substrte lcohols re described in previous publiction (7). Sttisticl nlysis. The sttisticl nlysis for correltion between ADH ctivity nd the production of fusel lcohols by different yest strins ws done on Hewlett-Pckrd model 981A computer coupled to Hewlett-Pckrd model 9862A clcultor plotter. RSULTS AND DISCUSSION As hs been previously indicted (1, 18), we lso found the mount ofhigher lcohol production to be dependent upon the strin of wine yest. In ddition, we found the ADH ctivity for the vrious lcohols to vry from strin to strin. To show whether reltionship existed between the ADH ctivities nd higher lcohol productivities of vrious wine yests, we compred these two spects under vinifiction conditions. Figure 1 shows the ADH specific ctivities observed in the cells of vrious strins hrvested from vinous fermenttions nd higher lcohols produced by the strins. The curves representing these two prmeters followed the sme pttern, suggesting tht if strin possessed higher ADH ctivity for certin fusel lcohol it lso produced the higher quntities of the ltter. This ws prticulrly true when the strins producing the highest mounts of fusel oil were considered. For exmple, strin 586, which hd the highest ADH ctivity for n-propnol, produced the highest mounts of this lcohol, s compred with other strins. The sme ws true in the cse of strin 595 for isobutnol production nd in the cse of strin 51 for the production of ctive pentnol nd isopentnol. These observtions suggested definite reltionship between the ADH ctivities of yests nd their fusel oil productivity. This reltionship between the ADH ctivity nd the fusel oil productivity ws further confirmed by doing the sttisticl nlysis for coefficients of correltion nd liner regressions between the two prmeters. The regression nlysis results (Fig. 2) showed liner reltionship between the ADH specific ctivities

3 668 SINGH AND KUNK APPL. NVIRON. MICROBIOL. 1r w ml C (.2 u U- U w. -C UJ O Isopentnol n 21 w IO -J W oi1 I I I I I Jlo o l' J o b (RD) (RD) STRAIN NUMBRS FIG. 1. Reltionship between ADH specific ctivity (A) nd the formtion ofhigher lcohols () by vrious wine yests indicted by strin number (see Tble 1). 8 Uh (5 -J 6 so Active Pentnol go is A 'C I 1U ADH SPCIFIC ACTIVITY (UNITS/mg PROTIN) FIG. 2. Regression nlyses ofadh ctivities nd higher lcohol productivities found in the vrious wine yest stins.

4 VOL. 32, 1976 nd production of fusel oil components: n-propnol, isobutnol, ctive pentnol, nd isopentnol. In Tble 2 re presented the clculted vlues for coefficients of correltion (r), coefficients of determintion (r2), nd levels of significnce, corresponding to the r vlues obtined from Student's t tble. It is evident tht there is significnt correltion between the ADH ctivities nd the fusel oil productivities of vrious wine yest strins. Active pentnol nd isopentnol, the mjor components of fusel oil, showed the best correltions (r =.931 nd r =.899, respectively). The r2 vlues for these two lcohols showed tht 86.6 nd 8.9% of the vribles were ccounted for, respectively. thnol, which ws produced in high quntities by ll of the yest strins, showed no correltion with ADH ctivity when plotted s in Fig. 1. The sttisticl vlues for ethnol (Tble 2) ber this out. In Tble 2 the vlues for liner regression constnts ( nd b) re lso given. Using these constnts, stright-line eqution (y = + bx) could be derived for ech of the four higher lcohols; y represents the mount of higher lcohol produced, nd x is the specific ctivity of TABL 2. Sttisticl nlyses for correltion between ADH ctivities nd higher lcohol formtion Coeffi- Constnts of Higher lco- Coef-lcient of cient of regression Level of determi- signifihol correlntion b (in- cnce tion (r) (r2) (slope) tercept) (%) n-propnol Isobutnol Active pen tnol Isopentnol thnol TABL 3. HIGHR ALCOHOL DHYDROGNASS IN WIN YASTS 669 ADH for tht lcohol. From these equtions, then, the mounts of higher lcohols tht would be expected to be formed by given wine yest strin could be predicted from the specific substrte ctivities of ADH for tht strin. In Tble 3 re presented the predicted vlues of higher lcohols s clculted from these equtions nd the vlues of the lcohols tht were ctully produced. There ws striking correltion between the predicted nd observed vlues, with the exception of the isobutnol vlues for the respirtory mutnt nd for n-propnol, which is formed in low quntity. The coefficient of correltion clculted for the observed nd predicted totl higher lcohols (lst two columns oftble 3) ws.91. This shows highly significnt reltionship between the two. The strong correltion between the predicted formtion of higher lcohols nd those ctully obtined should be helpful tool for enologists. Mesurement of the ADH specific ctivities in yest cells would be useful for predicting, in dvnce, the formtion of the totl nd individul higher lcohols in wine, brndy, nd other distilled spirits productions. Higher lcohol formtion hs been shown to be prtly yest strin dependent (1), but the formtion is lso influenced by the growth medium of the yest (R. Singh, Ph.D. thesis). If subsequent reserch revels tht the ADH specific ctivities re lso dependent upon the growth medium in the sme wy s is the higher lcohol formtion, this could show how importnt ADH is s controlling fctor in the production of these lcohols. ACKNOWLDGMNTS We thnk dwrd Crowell for his technicl ssistnce in the determintion of fusel lcohols, Mrilynn Vils for her help in sttisticl nlysis, Sergio Trverso-Rued for prep- Observed nd predicted vlues of higher lcohol formtion Higher lcohol formtion (mg/1 ml) Strin n-propnol Isobutnol Active pentnol Isopentnol Totl no. Ob- Pre- Ob- Pre- Ob- Pre- Ob- Pre- Ob- Preserved dicted served dicted served dicted served dicted served dicted (RD)b The predicted vlues were clculted from the liner regression equtions for vrious lcohols derived from sttisticl nlysis by pplying ADH specific ctivities in the corresponding equtions (see text). b RD, Respirtory-deficient mutnt.

5 67 SINGH AND KUNK rtion of the respirtory mutnt, nd Bryce Rnkine of the Austrlin Wine Reserch Institute for the two Austrlin strins of wine yest. LITRATUR CITD 1. bisuki, K., nd. S. G. Brron Yest lcohol dehydrogense II. Arch. Biochem. Biophys. 69: Gornll, A. G., C. J. Brdwill, nd M. M. Dvid Determintion of serum proteins by mens of biuret rection. J. Biol. Chem. 177: Hrrison, G. A. F., nd. Collins Gs-chromtogrphic determintion of individul -keto cids in beer. Proc. Am. Soc. Brew. Chem. 1968: Heick, H. M. C., J. Willomot, nd N. Begin-Heick The subcellulr loclition of lcohol dehydrogense ctivity in Bker's yest. Biochim. Biophys. Act 191: Khn, J. H., nd. T. Blessinger Collbortive study of the quntittive gs-liquid chromtogrphic determintion of fusel oil nd other components in whisky. J. Assoc. Off. Anl. Chem. 55: Kunkee, R.., J. F. Guymon, nd. A. Crowell Formtion of n-propyl lcohol by cell-free extrcts of Scchromyces cerevisie. J. Inst. Brew. 72: Kunkee, R.., nd R. Singh Dehydrogense ctivity for higher lcohols in cell-free extrcts of Scchromyces cerevisie. J. Inst. Brew. 81: Lutstorf, U., nd R. Megnet Multiple forms of lcohol dehydrogense in Scchromyces cerevisie. I. Physiologicl control of ADH-2 nd properties of ADH-2 nd ADH-. Arch. Biochem. Biophys. 126: APPL. NVIRON. MICROBIOL. 9. Rcker, Alcohol dehydrogense from Bker's yest, p In S. P. Colowick nd N.. Kpln (ed.), Methods in enymology, vol. I. Acdemic Press Inc., New York. 1. Rnkine, B. C Formtion of higher lcohols by wine yests, nd reltionship to tste threshold. J. Sci. Food Agric. 18: Roche, B., nd. Aouly Regultion des lcool deshydrogenses che Scchromyces cerevisie. ur. J. Biochem. 8: Schimpfessel, L Presence et regultion de l synthese de deux lcool deshydrogenses che l levure Scchromyces cerevisie. Biochim. Biophys. Act 151: SentheShnmugnthn, S The purifiction nd properties of the tyrosine-2-oxo-glutrte trnsminse ofscchromyces cerevisie. Biochem. J. 77: SentheShnmugnthn, S., nd S. R. lsden The mechnism of the formtion of tyrosol by Scchromyces cerevisie. Biochem. J. 69: Suomlinen, H., nd A. J. A. Kernen Keto cids formed by Bker's yest. J. Inst. Brew 73: Thoukis, G The mechnism of isomyl lcohol formtion using trcer techniques. Am. J. nol. Vitic. 9: Wtson, T. G., nd J. S. Hough Conversion of - keto-iso-cproic cid to isomyl lcohol by yest pyruvte decrboxylse nd lcohol dehydrogense. J. Inst. Brew. 75: Webb, A. D., nd J. L. Ingrhm Fusel oil. Adv. Microbiol. 5: Downloded from on September 11, 218 by guest