Ergosterol Concentration of Several Different Saccharomyces

Ergosterol Concentration of Several Different Saccharomyces cerevisiae Yeast Strains MARYKE STRYDOM, ADRIENNE F. KIRSCHBAUM and A. TROMP Oenological and Viticltral Research Institte, Private Bag X526, Stellenbosch, 76 Differences in the formation of ergosterol in yeast cells have been fond amongst several ine yeast strains sed in Soth Africa. A higher ergosterol content is generally accompanied by a faster fermentation rate and reslts in a shortening of fermentation time. A high ergosterol concentration in the cell is not a prereqisite for maximal cell formation, bt probably affects the metabolic activity of the cell. Aeration of the mst dring the yeast proliferation stage, broght abot marked increases in ergosterol concentrations. Ergosterol is the major sterol of Saccharomyces cerevisiae and of a nmber of other yeast species (Rattray, Schibeci & Kidby, 1975). Of several hndred fngal strains tested, Sacch. cerevisiae as fond to prodce the largest qantities of sterols (Dlaney, Stapley & Simpf, 1954). Under conditions of aerobic groth, it is able to synthesize ergosterol amonting to 2-1% of its dry mass (Dlaney et al., 1954). The synthesis of sterols depends largely pon the oxygen reqirement of the strain, and different strains behave differently in respect of sterol synthesis in the presence of specific concentrations of oxygen in the groth medim (Kirsop, 1974). Dring anaerobic groth, sterols cannot be synthesized, and nless small qantities of sterol are spplied exogenosly, the cells ill stop groing after a fe generations (David & Kirsop, 1973). The presence of moleclar oxygen is necessary for the biosynthesis of ergosterol and all other yeast sterols. It is reqired for several biochemical reactions in the synthesis process, sch as the indction of the enzyme 3-hydroxy-3-methy lgl tary l-coa redctase, hich catalyses the formation of mevalonic acid from 3- hydroxy-3-methylgltaryl-coa (Berndt et al., 1973); the cyclisation of sqalene to form the first sterol lanosterol (Klein, 1955), as ell as several demethylation and desatration reactions (Aries & Kirsop, 1978). In the beer indstry, aeration of yeast dring the stage of proliferation is condcted ith the specific aim to boost ergosterol prodction (Harding & Kirsop, 1979). Althogh the specific fnction of the sterols in yeast is not clear (Boll et al., 1975), some evidence has been presented sggesting that sterol synthesis and development of respiratory capacity in the cell are closely related (Parks & Starr, 1963). Prodlock, Haslam & Linnane (1969) stated that the main fnction of sterols in yeast is an effect on the strctre and dynamic state of the -cell membrane and ths on its permeability to groth constitents. In respect of ine yeast, Lafon (1978) fond that the addition to mst of groth factors sch as thiamine or diammonim phosphate, cases a more rapid onset of fermentation and a higher fermentation rate, hile the presence of yeast sterols affects only the final stages of fermentation. The present paper reports, firstly, on differences observed beteen locally sed ine yeast strains in respect of their ability to synthesize ergosterol and its possible effect on fermentation behavior; secondly, on the effect of aeration dring the stage of yeast proliferation on the sbseqent fermentation process. MATERIALS AND METHODS Cltral conditions: In the first experiment steam sterilized, clearly settled Chenin blanc mst from the 1979 vintage as sed; the second and third experiments ere done ith sterile filtered Chenin blanc mst from the 1979 vintage, and Chenin blanc and Colombar msts from the 198 vintages respectively. The msts had sgar concentrations of abot 2 g/, total S2 concentrations of 75 mg/ and ph vales ranging from 3,3 to 3,4. Pre cltres ere prepared by rehydrating 1 g dried preparations of the organisms in 1 me sterile distilled ater for 3 min. at 42 C, and inoclating sterile filtered grape mst to a concentration of,2 g pre cltre yeast per litre. Inoclations from the pre cltres into the msts to be fermented, ere done to a concentration of 5% Cl,). The organisms ere gron and fermented at l5 C in 1 5 me qantities in 2 glass containers spplied ith stopcocks from hich samples cold be dran. All fermentations ere done in dplicate. Fermentation treatments: In the first experiment Sacch. cerevisiae strains WE 14, WE 353 (Epernay), WE 372 and WE 392 (Montrachet) ere sed. The mst of the second experiment as spplemented ith 1 g/ (NH4)2HP4 to eliminate the possible effect of nitrogen deficiency. Strains WE 14, WE 372 and WE 392 (Montrachet) ere sed. In the third experiment aeration as effected by pmping filtered air throgh the pre cltres for for days at a rate of 25 m /min. Strains WE 14 and WE 432 ere sed. All organisms came from the cltre collection of the Oenological and Viticltral Research Institte, Stellenbosch. Live cell conts: The nmber of live cells present at the inoclation stage and dring fermentation as determined according to the plate cont method on YM agar (Difeo) plates. Colonies ere conted after incbation at 25 C for three days, and the final colony cont as taken as the average of three plates per diltion containing 3 to 3 colonies per plate. Fermentation rate: The mass of the flasks as determined at inoclation - daily for the first for days, and then every second or third day. The mass loss as a reslt of carbon dioxide development as calclated, and a fermentation rate crve dran by plotting mass loss/1 mf mst medim VS time. Yeast dry mass: Fifty me samples of the mst ere tapped after shaking each flask, centrifged at 4 8 g S. Afr. J. Enol. Vitic., Vol. 3. No. I. 1982 23

24 Ergosterol concentration of several different yeast strains for 2 mm., and ashed once ith 5 mc distilled ater. The spernatant as discarded, the cells sspended in 1 mc 5 mm phosphate bffer of ph 7,, the sspension filtered throgh Millipore filters (type HA WP 46) of knon mass, the cells ashed ith 3 mc distilled ater and their mass again determined according to the method of Mas & Pina (198) after 24h at 85 C. Ergosterol analysis: Fifty mc samples of mst ere centrifged at 4 8 g for 2 min., and the cells ashed once ith an eqal volme of distilled ater. The ashed cells ere sspended in 2 mc distilled ater, and their ergosterol contents determined by the method of Breivik & Oades (1957). Abs9rbances ere determined on a Beckman Model 25 spectrophotometer. RESULTS AND DISCUSSION Ergosterol contents of strains: In the first experiment the effect of ergosterol on the rate of fermentation as stdied. Marked differences amongst strains ere observed. Fermentation rates, dry yeast mass prodction, ergosterol content of the cells and in the medim are shon in Figres 1, 2, 3 and 4 respectively. After 35 days the mst inoclated ith strain WE 353, contained 1,5 g residal sgar per litre, hile strain WE 392 contained 4,7 g/c, strain WE 14 16,4 g!c and strain WE 372 27,5 g residal sgar per litre. Only strain WE 353, therefore, as able to complete fermentation at l5 C ithin a reasonable time. Strain WE 353 also had the highest ergosterol concentration per volme (,1 mg/5 mc) and prodced the highest level of dry cell mass (119 mg/5 mc). Hoever, ergosterol content expres- 1 12 16 2 24 28 32 36 F1G.l Fermentation rates of for different ine yeast strains in Chenin blanc mst.,12 > 4 12 16 2 24 3 F1G.2 Dry mass prodction by for different ine yeast strains in Chenin blanc mst. E "' Ol E c.1 -- - --. ---- oo.os / ---------- - - -~-o--o----o ~ -------- -------- ---~~----- cs o '-o..o~----- -------- UJ ~ a UJ 4 12 16 2 24 3 2. s g11.s~ -Ol 1 gos~ ~~ ~o~----o====o=====~-=-=e a ------------ -----=---= ---- - UJ 4 8 12 16 2 24 3 F1G.3 Ergosterol concentration (mg/5 mc) in a Chenin blanc mst fermented ith for different ine yeast strains. F1G.4 Ergosterol concentration (dry mass basis) of for different ine yeast strains in Chenin blanc mst. S. Afr. J. Eno!. Vitic., Vol. 3. No. 1. 1982

~ Ergosterol concentration of several different yeast strains 25 sed on c dry mass basis for strain WE 353 (,27 g/1 g) Table 1. Final live cell conts 4 days after inoclation, as somehat loer than that of strain WE 392 (,31 ere in accordance ith the ergosterol content and ferg/1 g). It is of interest to note that strain WE 353, mentation rate. This is illstrated by strain WE 392, hich prodced the highest level of total ergosterol in hich at that stage had the highest cell cont (12,8 x the medim, shoed a relatively long lag phase of 1 /mc), shoed the highest ergosterol content abot 2 days before the onset of fermentation (Fig. 1). throghot the fermentation period (minimm,2 Hoever, the fermentation rate of this strain as g/1 g), and as the first to complete fermentation (47 higher than that of any of the other strains. days). Fermentation behavior in this case cold not be In the second experiment the same higher ergosterol ascribed to the size of the inoclm, as strain WE 372, content and faster fermentation rate ere observed. the one that fermented most sloly, had the highest in- Reslts are smmarized in Figres 5, 6, 7 and 8, and the oclm concentration (5,3 x 1 6 cells/ml') compared live cell conts of the three strains are compared in ith 4,8 x 1 cells/me for WE 14 and WE 392). 1 9 E ~~-5 1 _J <{ ::::; 12 16 2 24 28 32 36 4 12 16 2 24 28 32 36 4 Tl ME, days F1G.5 Fermentation rates of three ine yeast strains in sterile filtered Chenin blanc mst. o WE 392; WE 14; D WE 372 F1G.6 Dry mass prodction by three ine yeast strains in sterile filtered Chenin blanc mst. o WE 392; WE 14; D WE 372,12 : ;-.----- - '--------- () Qa.4 ~ 1-----------,,---------- (f) <!) er: 12 16 2 28 32 36 4 TIME. days FrG.7 Ergosterol concentration (mg/5 mc) in a sterile filtered Chenin blanc mst fermented ith three different ine yeast strains. o WE 392; WE 14; D WE 372 ~---:-~ 1 a 12 16 2o 24 FrG.8 Ergosterol concentration (dry mass basis) of three ine yeast strains in sterile filtered Chenin blanc mst. o WE 392; WE 14; D WE 372 36. g 4 S. Afr. J. Enol. Vitic., Vol. 3. No. 1. 1982

26 Ergosterol concentration of several different yeast strains 12 16 2 28 l2 36 4 Tl ME, days FIG.9 Fermentation rates of aerated and naerated pre cltres of to ine yeast strains in sterile filtered Chenin blanc mst. D WE 14 aerated; WE 14 naerated; l:,. WE 432 aerated;.&. WE 432 naerated F1G. l Live cell conts of aerated and naerated pre cltres of to ine yeast strains in sterile filtered Chenin blanc mst. D WE 14 aerated; Ill WE 14 naerated; l:,. WE 432 aerated;.&. WE 432 naerated.17r o~~ ~ 2 ~ 28 32 36 ~ F1G. ll Dry mass prodction by aerated and naerated pre cltres of to ine yeast strains in sterile filtered Chenin blanc mst. D WE 14 aerated; WE 14 naerated; l:,. WE 432 aerated;.&. WE 432 naerated if ~o 15: /;"_,,-"-."~---------. ---------:_ '" 'E :::_ ;-- -- -------- E "' I ' ~5fl_ 1)" ------- f- U') CJ./ _ -------. ------ 4 B 16 2 24 32 4 F1G.12 Ergosterol concentration (mg/5 mt) in a sterile filtered Chenin blanc mst fermented ith aerated and naerated pre cltres of to ine yeast strains. D WE 14 aerated; WE 14 naerated; l:,. WE 432 aerated;.&. WE 432 naerated " Days WE14 WE372 WE392 TABLE 1 Live cell conts of strains WE 14, WE 372 and WE 392 dring fermentation. Cell cont (x 1'/mf) 4,8 5,3 4,8 5 13,4 16,5 1,7 12 11,6 12,4 11,4 4 9,8 8, 12,8 Effect of aeration: Using Chenin blanc mst, the reslts of aeration of pre cltres ith regard to fermentation rates, live cell conts, dry cell mass prodction, ergosterol content in cells and in the media, are given in Figres 9, 1, 11, 12 and 13 respectively. Similar reslts ere obtained in the case of the Colombar mst. (Reslts not shon.) As can be seen from Figre 9, aeration of both strains led to a higher fermentation rate and earlier completion of fermentation. In the case of WE 14, fermentation ith the aerated pre cltre as completed ithin 34 days after inoclation, hile the naerated cltre reqired 41 days. With strain WE 432 this difference as of the same order, ith 38 days for the aerated and 46 days for the naerated cltre. In Table 2 the inoclation cell concentrations, final ergosterol contents per cell and per volme, as ell as the dration of fermentation are compared for both strains and both cltivars. It can be seen that althogh the inoclm sizes differed largely, the final ergosterol contents on a dry mass basis ere mch higher in the case of the aerated cltres (13 times higher than the naerated cltre for strain WE 14, and abot 3 times higher than the naerated cltre for strain WE 432). S. Afr. J. Enol. Vitic., Vol. 3. No. I. 1982

1, Ergosterol concentration of several different yeast strains 27 OJ z OJ - (.) c (.),5 _J ~ (f) CD 4. D... 8 12 16 2 24 28 32 36 4 44 48 F1G. l3 Ergosterol concentration (dry mass basis) of aerated and naerated pre cltres of to ine yeast strains of sterile filtered Chenin blanc mst. D WE 14 aerated; II WE 14 naerated; L,. WE 432 aerated;.a. WE 432 naerated TABLE 2 Comparison of aerated and naerated cltres in respect of inocla, days fermented and final ergosterol concentrations. Ergosterol Strain Inoclm Days (x 1 6 fermented g/1 g mg/5 mt cells/me) yeast medim Chenin blanc WE 14 U 1,25 41 OJJ4,82 WE14A 28,6 34,52,144 WE432 U,635 48,2,45 WE432A 8,935 38.59,133 Colom bar WE14U 1,245 46,2,86 WE14A 23,11 3,26,172 WE432 U,55 46,2,54 WE432A 8,25 38,63,144 'U = naerated, A = aerated CONCLUSIONS In the fermentation of grape mst ergosterol plays an important part in the yeast cell, since the more rapid fermentation in either nfiltered or filtered mst as accompanied by a relatively high ergosterol content. Similar observations have been reported by Lare, Lafon-Laforcade & Riberea-Gayon (1979) ho fond, in addition, that sterols and other so-called "srvivial factors" permit the yeast to complete fermentation in the presence of higher sgar levels in the mst. The yeast strain prodcing most ergosterol in its cells does not necessarily prodce the highest qantity of dry mass (for example strain WE 392 in the first experiment ith clear settled jice). Ths ergosterol is probably not directly responsible for the formation of ne cells, bt may only be concerned ith the physiological state of the cell, permitting it to maintain a higher level of activity and enabling it to complete fermentation more rapidly. Aeration of a yeast cltre greatly enhances ergosterol prodction. This treatment does not exert its effect only hile being applied, bt probably permits the yeast to maintain a more active metabolism leading to more rapid fermentation. Dring fermentation aerated cltres maintain a higher ergosterol concentration per dry mass, and their total ergosterol concentration per volme is also mch higher. According to Jakobson & Thorne (198) yeasts groing in the presence of dissolved oxygen (aerobiosis) synthesize more sterols and nsatrated fatty acids than the. minimm concentration necessary for groth, so that they do not have any demand for oxygen hen placed in a ne medim here conditions soon change to anaerobiosis. A frther advantage of aeration is that aerated cltres have higher live cell conts. Its inoclation into another mst therefore cases an earlier onset of fermentation than old be the case ith an naerated cltre. Strain WE 432 obviosly has a disadvantage as regards the formation of ne cells. Althogh efforts ere made to obtain similar inoclation concentrations, strain WE 432 prodced approximately three times less cells ith aeration than did strain WE 14. Strain WE 432 as nevertheless a better ergosterol prodcer and contained a higher ergosterol concentration (,6 g/1 g) than the aerated strain WE 14 (,4 g/1 g) at the end of the aeration period. This reslt also proves that ergosterol is not directly connected ith the formation of ne cells. Frther stdies are at present being ndertaken to determine hether the general tendency of higher ergosterol concentration and faster fermentation rate applies to other ine yeast strains as ell. The effect of different periods of aeration of pre cltres and its inflence on ine qality are also investigated. S. Afr. J. Eno!. Vitic., Vol. 3. No. 1. 1982

28 Ergosterol concentration of several different yeast strains LITERATURE CITED ARIES, V., & KIRSOP, B. H., 1978. Sterol biosynthesis by strains of Saccharomyces cerevisiae in the presence and absense of dissolved oxygen. J. Inst. Bre. 84, 188-122. BERNDT, J., BOLL, M., LOWEL, M., & GAUMERT, R., 1973. Reglation of stern! biosynthesis in yeast: indction of 3-hydroxy-3-methylgltaryl-coA redctase by glcose. Biochem. Biophys. Res. Commn. 51, 843-848. BOLL, M., LOWEL, M., STILL J., & BERNDT, J., 1975. Stern! biosynthesis in yeast. Er. J. Biochem. 54, 435-444. BREIVIK,. N., & OWADES, J. L., 1957. Spectrophotometric semimicrodetermination of ergosterol in yeast. Agricltral and Food Chemistry 5, 36-363. DAVID, M. H., & KIRSOP, B. H., 1973. Yeast groth in relation to the dissolved oxygen and stern! content of ort. J. Inst. Bre. 79, 2-25. DULANEY, E. L., STAPLEY, E.., & SIMPF, K., 1954. Stdies on ergosterol prodction by yeasts. Appl. Microbial. 2, 371-379. HARDING, S. A., & KIRSOP, B. H., 1979. Relative significance of oxygen and other ntrients as fermentation reglators in Saccharomyces cerevisiae. J. Inst. Bre. 85, 171-174. JAKOBSEN, M., & THORNE, R. S. W., 198. Oxygen reqirements of breing strains of Saccharomyces varm (carlsbergensis) - bottom fermentation yeast. J. Inst. Bre. 86, 284-287. KIRSOP, B. H., 1974. Oxygen in breery fermentation. J. Inst. Bre. 8, 252-259. KLEIN, H.P., 1955. Synthesis of lipids in resting cells of Saccharomyces cerevisiae. J. Bacterial. 69, 62-627. LAFON, S., 1978. Les steriodes "facters de srvie" des levres a cors de la vinification. Ann. Technol. Agric. 27(1), 215-217. LARUE, F., LAFON-LAFOURCADE, S., & RIBE REAU-GA YON, P., 1979. Les differents roles fonctionnels des stero"ides Sr Jes Jevres dans le mot de raisin en fermentation: notion de facter de srvie. Ann. Microbial. (Inst. Paster) 13A, 231-243. MAS, J., & PINA, E., 198. Disappearance ofnystatin resistance in Candida mediated by ergosterol. J. Gen. Microb. 117, 249-252. PARKS, L. W., & STARR, P. R., 1963. A relationship beteen ergosterol and respiratory competency in yeast. J. Cell. Comp. Physiol. 61, 61-65. PROUDLOCK, J. W., HASLAM, J.M., & LINNANE, A. W., 1969. Specific effect of nsatrated fatty acid depletion on mitochondrial oxidative phosphorylation in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commn. 37, 847-852. RATTRAY, J.B. M., SCHIBECI, A., & KIDBY, D. K.. 1975. Lipids of yeasts. Bacterial. Rev. 39, 197-231. S. Afr. J. Enol. Vitic., Vol. 3. No. 1. 1982