It has been demonstrated that normal and so-called  SO2-forming yeasts differ considerably in sulphate absorption, i. e.  SO2 forming strains consume on an average twice as much sulphate. The addition of increasing concentrations of methionine and/or cysteine to the grape must reduces the uptake of sulphate and the formation of sulphite with all strains during fermentation. Since cysteine c auses changes similar to methionine it is assumed that it also regulates the uptake and activation of sulphate.Sulfataufnahme und Sulfitbildung in Beziehung zum Methionin- und/oder Cystein-Gehaltvon Traubenmost während der Vergärung durch einige Stämme von SaccharomycescerevisiaeEs konnte gezeigt werden, daß sich normale und sog. SO2-bildende Hefestämme hinsichtlich der Sulfatabsorption betrlichtlich unterscheiden, d. h.  SO2-bildende Stämme verbrauchen durchschnittlich die doppelte Sulfatmenge. Die Zugabe steigender Konzentrationen von Methionin und/oder Cystein zu Traubenmost reduziert bei allen Stämmen die Aufnahme von Sulfat und die Bildung von Sulfit während der Gärung. Da Cystein ähnliche Veränderungen verursacht wie Methionin, wird angenommen, daß es ebenfalls die Aufnahme und Aktivierung von Sulfat regelt

Eschenbruch, R.

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Vitis 11, 222-227 (1972) Oenological and Viticultural Research Institute, Stellenbosch, South Africa Sulphate uptake and sulphite formation related to the methionine and/or cysteine content of grape must during the fermentation by strains of Saccharomyces cerevisiae by R. EscHENBRUCH Sulfataufnahme und Sulfitbildung in Beziehung zum Methionin- und/oder Cystein­Gehalt von Traubenmost wahrend der Vergarung durch einige Stamme von Sac­charomyces cerevisiae Z u s  a m  m e n  f a s  s u n g .  - Es konnte gezeigt werden, da13 sich normale und sog. S02-bildende Hefestamme hinsichtlich der Sulfatabsorption betrlichtlich unterscheiden, d. h. SO,-bildende Stlimme verbrauchen durchschnittlich die doppelte Sulfatmenge. DieZugabe steigender Konzentrationen van Methionin und/oder Cystein zu Traubenmost re­duziert bei allen Stammen die Aufnahme van Sulfat und die Bildung van Sulfit wlih­rend der Glirung. Da Cystein lihnliche Verlinderungen verursacht wie Methionin, wirdangenommen, da13 es ebenfalls die Aufnahme und Aktivierung van Sulfat regelt.Introduction Recent investigations showed that the so-called S02-forming yeast strains are able to produce H2S during the fermentation of grape juice in quantities similar to those of normal strains (EscHENBRUCH 1972 a). These findings were contradictory to those of DITTRICH and STAUDENMAYER (1968) who postulated that S02-forming yeasts 240 200 160 120 80 so= uptake 4 mg/l /J.-/J. M 0-0 R x-x 0 o-o WEI·-· WE 14 ------· 40. ,--.:g =-::0A 500 1000 2000 Methionine addition mg/ l SO format ion 60" 2 mg/ l 50 40 30 20 10 • �- . 500 1000 2000 Methionine addition mg/l Fig. 1: Sulphate uptake (A) and sulphite formation (B) related to increasing concentra­tions of methionine added to Steen must, vintage 1971. r Sulphate uptake and sulphite formation by S. cerevisiae 223 were defective mutants, unable, on account of a genetical blockage, to reduce the sulphite, deriving from the sulphate of the must, to H2S. Consequently S02 would accumulate. On the other hand we demonstrated, that the concentration of both methionine and cysteine in the must can decisively participate in the regulation of S02 forma­tion (EscHENBRUCH 1972 b). Sufficiently high levels of these two sulphur containing amino acids decreased the sulphite synthesis to less than 10 mg/1, not only with normal but also with S02-forming strains. Hence the question arose whether differences existed in the absorption of sul­phate from the must by normal and S02-forming strains and to what extent this uptake could be influenced by methionine and cysteine. The influence of these two amino acids was already demonstrated by KLEINZELLER et al. (1959) and MAW (1963, 1965) and could possibly explain our observation in that S02-forming strains produce only 20 mg S0/1 in some musts and 80 mg/1 in others. WORDIG and SCHLOTTER (1971) also reported that considerable variations in the S02 synthesis occur when different fruit juices were fermented with one and the same strain. Therefore sulphate uptake and sulphite synthesis had to be investigated with regard to increasing concentrations of methionine and/or cysteine not only with normal but also with S02-forming yeasts, without addition of sulphite to the must before or during the fermentation. Materials and Methods A must of the grape variety Steen, vintage 1971, with a sugar content of 21.3° Brix, 6.8 g total acid/1, 260 mg sulphate/I (as K2S04) and with a pH 3.6 was used for the tests where either methionine or cysteine was added. For the simultaneous addition of these two amino acids a Steen must, vintage 1972, with 20° Brix, 6.7 g total acid/1, 230 mg sulphate/I (as K2S04) and with a pH 3.3 was used. No sulphite was added to the must either during or after fermentation. Aliquots of 150 ml of must were measured into 250 ml ERLENMEYER flasks sealed with fermentation caps. Methionine and cysteine were always added after sterilisation at 0.5 atm for 5 min. An inoculum was chosen in order to obtain 1500 cells per mm3 aliquot and the fermentation temperature was 25° C. The yeast strains employed were as previously described (EscHENBHUCH 1972 a). After completion of fermentation, sulphite and sul­phate were determined according to PAUL (1958) and REBELEIN (1969) respectively. The fermentation rate was determined in terms of daily loss in weight, and yeast growth by cell count checks. In the sulphate determination all samples were sterile filtered, allowed to stand for 10 min after the addition of BaC12, centrifuged for 10 min at 3000 rpm and weighed after cooling for 30 min in a dessicator. Instead of platinum, porcelain crucibles were used. Sulphate was determined in triplicate, S02 singly and mean values for three parallel fermentations were calculated. Results Figs. IA and B demonstrate the sulphate uptake and the S02 formation of the two normal strains WE 1 and WE 14 (two pure cultured yeasts which are supplied to wineries) as well as the three socalled S02-forming strains R, 0 and M. With increasing concentrations of methionine the uptake of sulphate decreases with ail five yeasts; with strain M, for instance, from 197 mg/1 to 98 mg/1 and with WE 14 from 76 mg/1 to 30 mg/1 after 2000 mg methionine per litre had been added. The un­fermented must contained 260 mg sulphate per litre. 224 ATP­Sulphurylase APS­Kinase R. EscHENBRUCHPAPS- Sulphite-Reductase Reductase so" APS p APS so" H ,s 4 3 Scheme 1: Sulphate reduction sequence. 7' Methionine '"' Cysteine The formation of S02 is reduced simultaneously. With strain M, for instance, S02 decreases from 56 to 22 mg/1. The yeasts R and O show the same tendency. However, the S02 synthesis of WE 1 and WE 14 is not influenced. Two facts become evident. Firstly, yeast strains which form anomalously high amounts of sulphite (R, 0, M) absorb much more sulphate than normal straim: (WE 1, WE 14). Secondly, methionine decreases the uptake of sulphate by the yeast cell. This automatically causes less S02 formation, since sulphite is an intermediate of the reduction sequence of sulphate to sulphide (see Scheme 1). The addition of cysteine instead of methionine yields data shown in Figs. 2A and B. Again, with all five strains, increasing concentrations of cysteine decrease the uptake of sulphate considerably. Similarly the formation of sulphite by the strains R, 0 and M is clearly reduced, to as low as 10 mg/1. Again, WE 1 and WE 14 are not influenced. It should however be noted that the effect of cysteine on the uptake of sulphate and on the formation of S02 seems to be more pronounced than that of methionine. Figs. 3A and 3B show the results obtained after the simultaneous addition of both methionine and cysteine to the must which contained 230 mg sulphate per litre. In contrast to the results shown in Figs. 1 and 2, a distinct influence on the S02 formation of the strains WE 1 and WE 14 is evident. Under these conditions they produce 50% less sulphite, i. e. the S02 synthesis is reduced from 8 to 4 mg/1. The so= uptake 240 I- 4 mg/l 200� \""-160 II- b\ 80 500 /J.-/J.M D-o RX-X Q o-o WE1•-• WE 14�-==----==­v • • -------0 ' 6 1000 2000 Cysteine addition mg/l 60 50 40 30 20 "6 ' ,or O e � �=-=, 500 1000 2000 Cysteine addition mg/l Fig. 2: Sulphate uptake (A) and sulphite formation (B) related to increasing concentra­tions of cysteine added to Steen must, vintage 1971. 1 ~ r \\ 120 \ --- X 0 F .. , .~. 200 160 120 80 40 Sulphate uptake and sulphite formation by S. cerevisiae 225 6-t:i. Mo--o Rx--x 0 0--0 WE1 - ·--· WE 14 ....... O'l E Cl) ::it:. 0 '-X 'b�U) A _.J 50 100 500 60 50 40 30 20 10 SO formation 2 B mg/I X � 0�'�',I� -�50 100 500 Simultaneous addition of Methionine and Cysteine mg /l Fig. 3: Sulphate uptake (A) and sulphite formation (B) related to increasing concentra­tions of both methionine and cysteine added to Steen must, vintage 1972. Methionine and cysteine were each added at the shown concentrations, e. g. 50 indicates that 50 mg of each was added. effect of the simultaneous addition of 100 mg/1 of each of both amino acids is more pronounced than in the case where they have been added separately. These dif­ferences can not be ascribed to growth or fermentation differences since these were not influenced by the addition of these two amino acids. Discussion 1. The assumption of previous investigations, viz. that the concentration of the sul­phur containing amino acids, methionine and cysteine, present in the must couldpossibly influence the amount of S02 being formed during fermentation via sulphateuptake or sulphate activation could be confirmed (EscHENBRUCH 1972 b). The extentto which increasing concentrations of methionine and/or cysteine reduce the sul­phate uptake and the linked synthesis of sulphite, can be seen in the data of Figs.1-3.DE Vno and DREYFuss (1964) reported, that the ATP-sulphurylase of baker'syeast, which activates the sulphate of the substrate to adenosine -5'-phosphosulphate (APS), is repressed by methionine and that the su1phite-reductase, which reduces sulphite to sulphide, is repressed by cysteine. However, no inhibition of the ATP­sulphurylase either by cysteine or methionine, sulphite or thiosu1phite could be demonstrated. This is elucidated in Scheme 1. The influence of methionine on the absorption of sulphate can therefore be ex­plained as a repression of the ATP-su1phurylase. A suppressed activation of sul­phate consequently reduces the formation of sulphite (Fig. 1). 226 R. ESCHENBRUCHThe fact that the addition of cysteine does not cause an accumulation of sui­phite, as could be expected from the repression of the sulphite-reductase by cysteine, and the fact that the sulphate uptake decreases (Fig. 2), could be an indication that the activation and transport of sulphate is regulated by cysteine. With SalmoneUa typhimurium, for instance, DREYFuss and MONTY (1963) proved that the sulphate­transport-system was in fact repressed by cysteine. The more effective influence of the simultaneous addition of both methionine and cysteine on the decrease in sulphate uptake and sulphite synthesis could re­sult from a marked reduction in the sulphate turn-over rate (Fig. 3, Scheme 1). In effect a decreased rate of methionine and cysteine synthesis would occur. Since they are already present as ready-formed molecules, the necessity of their synthesis by means of the complex and energy-requiring sulphate reduction sequence is not critical. 2. If the extent to which the addition of 100 mg of both methionine and cysteine perlitre influences the sulphite synthesis by sulphite-forming yeasts is considered (Fig.3), one can understand why these yeasts in some musts form only 20 and in others80 mg S0/1. Different concentrations of these amino acids could have caused thesevariations. Only very few references on the free amino acids content in musts are available which support this postulation. RAPP and RuurnER (1971) found that theamount of methionine varied between 17-90 mg/1 with different grape varieties. DITTRICH et al. (1970) analysed four musts with 17-30 mg methionine per litre. Some musts of Spanish grape varieties contained only traces or no methionine (LLAGUNO­MARCHENA 1972). Furthermore, it has been emphasised by BERGNER and HALLER (1969)that increasing pressure during grape pressing raises the free amino acids content of the must. However, data on cysteine concentrations in grape juices are to our knowledge not available. On this basis it also appears unlikely and contrary to published work (DITTRICH and STAUDENMAYER 1970) that sulphite formation by S02-forming strains is independant of the substrate. 3. The sulphate determinations resulted in another important aspect, viz. yeast strains forming little sulphite take up comparatively little sulphate (WE 1 andWE 14). On the other hand the socalled S02-forming strains have a high rate of S02 formation but also have a correspondingly high sulphate uptake. It therefore seems unlikely that in these yeasts it is the defective sulphite-sulphide reduction which causes S02 accumulation (DITTRICH and STAUDENMAYER 1968). It would rather appear that the cell membrane and sulphate activation to APS and PAPS are more im­portant in this system. When it is furthermore considered that S02-forming strains (R, 0, M) form amounts of sulphide similar to that of normal strains (WE 1, WE 14) it will be apparent that the sulphite-sulphide blockage supposition is less acceptable (EscHENBRUCH 1972 a).Considerable differences in activities of many enzymes occur during the fer­mentation of wort with flocculating and non-flocculating bottom yeast strains of Saccharomyces carlsbergensis (PrnNDL 1971). Similar considerations could apply to normal and sulphite forming strains of Saccharomyces cerevisiae. Summary It has been demonstrated that normal and so-called S02-forming yeasts differ considerably in sulphate absorption, i. e. S02 forming strains consume on an average twice as much sulphate. The addition of increasing concentrations of methionine and/or cysteine to the grape must reduces the uptake of sulphate and the formation t Sulphate uptake and sulphite formation by S. cerevisiae 227 of sulphite with all strains during fermentation. Since cysteine causes changes similar to methionine it is assumed that it also regulates the uptake and activation of sul­phate. Acknowledgement I wish to thank Miss A. VAN WYK, and Miss M. BouwMEESTER for skilful technical assistance, Dr. c. S. ou PLESSIS and Mr. G. HoFMANN for reading the manuscript and suggesting appropriate linguistic corrections. References BERGNER, K. G. und HALLER, H. E., 1969: Das verhalten der freien Aminosauren von Wei/3-weinen im Verlauf der Garung, bei Ausbau, Lagerung und Umgarung. Mitt. Klosterneu­burg 19, 264-288. DE VITo, P. C. and DREYFuss, J., 1964: Metabolic regulation of adenosine triphosphate sulphurylase in yeast. J. Bacterial. 88, 1341-1348. DITTRICH, H. H., LEIDENFROST, E. und TEPE, W., 1970: Ubertriigt sich eine hohe N03-Dtingung der Reben auf den N03-Spiegel im Most? Wein-Wiss. 25, 130-132. - - und STAUDENMAYEn, TH,, 1968: S02-Bildung, Biickserbildung und Biickserbeseitigung. Dt. Weinztg. 104, 707-709. - - und - - , 1970: Uber _die zusammenhange zwischen der Sulfitbildung und der Schwefel­wasserstoff-Bildung bei Saccharomyces cerevisiae. zentralbl. Bakteriol. Parasitenk. rn­fektionskrankh. Hyg., 2. Naturwiss. Abt. 124, 113-118. DREYFuss, J. and MONTY, K. J., 1963: Coincident repression of the reduction of 3'-phosphoadeno­sine-5'-phosphosulfate, sulfite and thiosulfite in the cysteine pathway of SalmoneUa typhi­murium. J_ Biol. Chem. 238, 3781-3783. EscHENBnuctt, R., 1972 a: Zur Substratabhiingigkeit der H2S- und S02-Bildung bei Saccharomyces cerevisiae Stiimmen. Wein-Wiss. 27, 40-44. - - , 1972 b: Der EinfluJl von Methionin und Cystein auf die SOrBildung einiger Stiimme von Saccharomyces cerevisiae bei der Vergarung von Traubenmost. Vitis 11, 53-57. KLEINZELLER, A., KOTYK, A. and KovAc, L., 1959: Utilization of inorganic sulphate by baker's yeast. Nature 183, 1402--1403. LLAGuNo-MARCHENA, c_, 1972: Autoanalysis in oenology laboratories. 3. Internatl. Oenol. Sym­posium, Cape Town 1972, paper No. 18. MAw, G. A., 1963: The uptake of inorganic sulphate by a brewer's yeast. Folia Microbiologia (Prag) 8, 325-332. - - , 1965: The role of sulphur in yeast growth and in brewing. Wallerstein Lab. Commun. 28, 49-70. PAUL, F., 1958: Die alkalimetrische Bestimmung der freien, gebundenen und gesamten schwef­ligen Siiure mittels des Apparates von LIEB und ZACHERL. Mitt. Klosterneuburg 8, 21-27. PIENDL, A., 1971: The enzymatic behavior of bottom flocculating and non flocculating yeast dur­ing fermentation. Amer. Soc. Brewing Chem. Proc., 63-79. RAPP, A. und REUTHER, K. H., 1971: Der Gehalt an freien Aminosiiuren in Traubenmosten von gesunden und edelfaulen Beeren verschiedener Rebsorten. Vitis 10, 51-58. REBELEIN, H., 1969: Beitrag zur Bestimmung der Schwefelsiiure in Traubenmost und Wein. Mitteilungsbl. GDCh-Fachgr. Lebensmittelchem. u. Gerichtl. Chem. 23, 107-109. WOno1c, G. und SCHLOTTER, H. A., 1971: Uber das Vorkommen S02-bildender Hefen im naturli­chen Hefegemisch des Traubenmostes. Dt. Lebensm.-Rundsch. 67, 86-91. Eingegangen am 3. 7. 1972 Dr. R. ESCHENBRUCH Research Institute for Oenology and Viticulture Private Bag X5026 Stellenbosch Rep. of South Africa 

Sulphate uptake and sulphite formation related to the methionine and/or cysteine content of grape must during the fermentation by strains of Saccharomyces cerevisiae

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Sulphate uptake and sulphite formation related to the methionine and/or cysteine content of grape must during the fermentation by strains of Saccharomyces cerevisiae

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