Role of Tween 80 and Monoolein in a Lipid-Sterol-Protein Complex Which Enhances Ethanol Tolerance of Sake Yeasts

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1983, p. 821-8 99-224/83/1821-5$2./ Copyright 1983, American Society for Microbiology Vol. 46, No.

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1983, p /83/1821-5$2./ Copyright 1983, American Society for Microbiology Vol. 46, No. 4 Role of Tween 8 and Monoolein in a Lipid-Sterol-Protein Complex Which Enhances Ethanol Tolerance of Sake Yeasts KAZUYOSHI OHTA AND SHINSAKU HAYASHIDA* Department ofagricultural Chemistry, Kyushu University, Fukuoka 812, Japan Received 13 April 1983/Accepted 2 July 1983 An exogenous ternary complex composed of Tween 8, ergosterol, and albumin increased the final ethanol concentration of fermentation by sake yeasts from 17.2 to 19.% (vol/vol) and reduced the fermentation time from 3 to days. Likewise, a complex of monoolein, albumin, and either ergosterol or ergosteryl oleate increased the final ethanol concentration of fermentation to 19.7 or 19.8% (vol/vol), respectively, and reduced the fermentation time to days. Both Tween 8 and monoolein promoted the fermentative activity (Qco,) of cells, and the effect was enhanced by the presence of ergosterol. Sake fermentation results in accumulation of >2% (vol/vol) ethanol in the mash. We previously reported that 2% (vol/vol) ethanol was produced by strains of Saccharomyces cerevisiae Hansen and S. uvarum as well as a strain of sake yeasts in batch cultures through both the stepwise feeding of sucrose to a chemically defined medium and supplementation with Aspergillus oryzae-proteolipid at 2 C (9). The action of proteolipid on yeast cells was completed during the yeast growth phase (9). Supplementation of a chemically defined medium with a crude phosphatidylcholine-albumin (1:2, wt/wt) complex similarly resulted in a final ethanol concentration of the static culture of 2% (vol/vol) (4). The stimulatory effects of added crude egg yolk-phosphatidylcholine on yeast growth and ethanol endurability of the cells by exposure in 2% (vol/vol) ethanol were observed in a nitrogen gas-sparged anaerobic culture (6). Purified phosphatidylcholine increased yeast growth and enhanced fermentative activity (Qco2) of cells, whereas the addition of ergosteryl oleate or oleic acid plus ergosterol enhanced ethanol endurability of cells, using a nitrogen gas-sparged anaerobic culture (8). The work reported below is an extension of these earlier studies that led to the characterization of the lipid-protein complex as a high ethanol concentration-producing factor. The data show the effectiveness of Tween 8 or monoolein in the above-described complex on ethanol fermentation by sake yeasts. MATERIALS AND METHODS Organism. The organism used was a strain of sake yeasts, Kyokai no. 7, which was classified as a member of the S. cerevisiae group (12). The yeast cells were maintained on slants containing 1% yeast extract, 2% peptone, 2% glucose, and 2% agar. Fermentation tests. To assess the effectiveness of exogenously supplied lipid-protein complex in enhancing ethanol tolerance of yeasts, fermentation tests were carried out under anaerobic conditions. For the inoculum, yeast cells from a fresh slant culture were suspended in 1 ml of basal synthetic medium (4) in a -ml culture bottle, and the culture was incubated statically at 3 C for 4 h under the same anaerobic conditions to be used for fermentation tests (see below). A portion (1 ml) of this culture was then transferred to 1 ml of fresh basal medium and allowed to grow at 3 C for 4 h under the same conditions. Yeast cells were harvested by centrifugation and inoculated to give 3 x 17 cells per ml in 15 ml of basal medium supplemented with the lipidprotein complex indicated. The surface of the medium was then covered with a 1-cm layer of liquid paraffin for maintaining anaerobic conditions. Each bottle was fitted with a fermentation bung. The bungs were partially filled with concentrated sulfuric acid, permitting only CO2 to evolve from the culture. The medium was deprived of oxygen by sparging with high-purity nitrogen gas for 1 h. Cultures were incubated statically at 2 C. The fermentation was followed by daily CO2 821 evolution, which was monitored by the decrease in weight of the whole culture. Sucrose concentration was maintained at a level of 5 to 1% (wt/vol) in the middle stages, and 2 to 3% (wt/vol) in the final stages, of the fermentation by stepwise feeding of sucrose to the culture (4). Ethanol. The final ethanol concentration (peli;ent, by volume) accumulated in the culture liquid was determined by use of an ethanol hydrometer after distillation (9). Preparation of ergosteryl oleate. Ergosteryl oleate was prepared by a modification of the method of Knapp and Nicholas (11). Esterification of 2 mg of ergosterol with 5 mg of oleoyl chloride in 1 ml of anhydrous benzene containing 3 ml of pyridine was performed under a stream of high-purity nitrogen gas for 1 h. The ester was eluted from an alumina column (aluminum oxide neutral; activity grade, super I; Woelm) with benzene. Infrared spectroscopy and thinlayer chromatography on Silica Gel G (E. Merck AG)

822 OHTA AND HAYASHIDA with hexane-diethyl etheracetic acid (9:1:1, by volume) established the identity and purity of the synthetic ester. Preparation of albumin.

2 822 OHTA AND HAYASHIDA with hexane-diethyl etheracetic acid (9:1:1, by volume) established the identity and purity of the synthetic ester. Preparation of albumin. Albumin was prepared in a crystalline form from fresh egg white by Kekwick and Cannan's method (1). The final product was dialyzed against tap water continuously for 3 days and then against deionized water for 2 days at 4 C. The preparation was treated with charcoal (Norit A) at ph 3. by the method of Chen (3) for the removal of fatty acid contaminants, followed by readjustment of the ph to 7.. It was then freeze-dried and stored at -2 C until used. Preparation of lipid-protein complex. Ergosteryl oleate in solution in small amounts of benzene was added to dry, sterile, empty culture bottles in the stated amounts, after which the solution was evaporated to dryness with a stream of high-purity nitrogen gas and the basal medium and defatted albumin were added. Tween 8, oleic acid, ergosterol, monoolein, and triolein were added directly together with defatted albumin to the basal medium. Each bottle was shaken vigorously to form a lipid-protein complex. Nitrogen gas-sparged anaerobic culture of yeasts. The yeast cells were grown anaerobically on a synthetic medium (1 ml) supplemented with lipids with constant nitrogen gas sparging at 3 C for 24 h as previously described (6, 8). Fermentative activities and ethanol endurabilities of yeast cells. The Warburg method was used to determine Qco, under nitrogen gas at 15 C (5). The ethanol endurability of yeasts was defined as the degree of difference in fermentative activities of yeasts before and after treatment of the cells by exposure in buffered (ph 6.) 2% (vol/vol) ethanol for 48 h at 15 C (5). Fatty acid analysis. Freeze-dried cells were subjected to direct methanolysis with methanolic HCI, and the resulting methyl esters of the fatty acids were analyzed by gas-liquid chromatography, using methods described earlier (7). Extraction and estimation of sterols. Sterols in whole cells were extracted after alkaline hydrolysis and estimated as ergosterol from the extinction at 282 nm by the method of Shaw and Jefferies (13). Chemicals. Oleic acid (99% pure), oleoyl chloride (99% pure), monoolein (99% pure), triolein (99% pure), Tween 8, and ergosterol (supplied by Sigma Chemical Co.) were of the highest purity available. They were used without further purification. The fatty acid composition of the batch of Tween 8 used was 75.5% oleic acid, 7.% palmitoleic acid,.9% myristoleic acid, and 16.6% saturated fatty acids. RESULTS Effect of Tween 8 plus ergosterol on ethanol fermentation. Andreasen and Stier (1, 2) provided oleic acid in the form of Tween 8 and ergosterol in an anaerobic shake culture of S. cerevisiae on chemically defined medium. In our first experiments, Tween 8 and ergosterol were added at a level of 1 mg/ml each to a static culture of sake yeasts without added albumin. Figure 1 shows that no significant differences in the rate and extent of CO2 production were seen whether or not Tween 8 and ergosterol were = 2 E U, o 15 1 o 5 APPL. ENVIRON. MICROBIOL Time (doys) FIG. 1. Effect of Tween 8 plus ergosterol on ethanol fermentation. Symbols:, none (17.1%);, 1 mg of Tween 8 per ml (17.9%); A, 1 mg of ergosterol per ml (17.4%); A, 1 mg of Tween 8 plus 1 mg of ergosterol per ml (17.9%). Percentages (by volume) in parentheses indicate the final concentration of ethanol produced. The cell inoculum was 3 x 17 per ml, and fermentation by sake yeast Kyokai no. 7 was carried out statically at 2 C in synthetic medium containing the lipid(s) indicated. added to the culture, and the final ethanol concentration of the culture containing exogenous Tween 8 and ergosterol was 17.9% (vol/vol) in 35 days. Effect of Tween 8 plus ergosterol as albumin complex on ethanol fermentation. In subsequent experiments, Tween 8 or ergosterol or both were added to the basal medium together with albumin. There was a remarkable increase in CO2 formation in the presence of Tween 8 plus ergosterol as albumin complex when compared with either Tween 8 or ergosterol alone as albumin complex (Fig. 2). The final ethanol concentration of the culture was 19.% (vol/vol) in the presence of Tween 8 plus ergosterol as albumin complex in days and 17.8% (vol/vol) in the presence of either Tween 8 or ergosterol alone as albumin complex in 3 days. Effect of oleic acid plus ergosterol as albumin complex on ethanol fermentation. In a previous paper (8), we reported that yeast cells grown in a nitrogen gas-sparged anaerobic culture supplemented with oleic acid and ergosterol at 3C acquired ethanol endurability. Under the static culture conditions, however, the addition of oleic acid and ergosterol as albumin complex resulted in a relatively small increase in CO2 production and a final ethanol concentration of 17.5% (vol/vol) in 3 days (Fig. 3). Necessity of monoolein for the action of the lipid-sterol-protein complex. The superiority of

VOL. 46, 1983 TWEEN 8 OR MONOOLEIN AND ETHANOL FERMENTATION 823 - E2 LA &I115. 1 2 3 Time (days) FIG. 2. Effect of Tween 8 plus ergosterol as albumin complex on ethanol fermentation.

3 VOL. 46, 1983 TWEEN 8 OR MONOOLEIN AND ETHANOL FERMENTATION E2 LA &I Time (days) FIG. 2. Effect of Tween 8 plus ergosterol as albumin complex on ethanol fermentation. Symbols:, none (17.2%);, 1 mg of albumin per ml (17.3%); A, 1 mg of Tween 8 plus 1 mg of albumin per ml (17.8%); A, 1 mg of ergosterol plus 1 mg of albumin per ml (17.8%); O, 1 mg of Tween 8 plus 1 mg of ergosterol plus 1 mg of albumin per ml (19.%). Percentages (by volume) in parentheses indicate the final concentration of ethanol produced. The experimental Tween 8 to oleic acid in the ternary complex (Fig. 2 and 3) seemed to lie in its surface-active properties. For this reason, monoolein and trio-?ni o1 15 N Time (days) FIG. 3. Effect of oleic acid plus ergosterol as albumin complex on ethanol fermentation. Symbols:, none (16.9%Yo);, 1 mg of albumin per ml (17.3%); A, 1 mg of oleic acid plus 1 mg of albumin per ml (17.1%); A, 1 mg of ergosterol plus 1 mg of albumin per ml (17.9%o); O, 1 mg of oleic acid plus 1 mg of ergosterol plus 1 mg of albumin per ml (17.5%). Percentages (by volume) in parenthesis indicate the final concentration of ethanol produced. The experimental TABLE 1. Effect of various combinations of lipids as albumin complex on ethanol fermentationa Lipid(s) (1 mg/ml) added as Fermentation Ethanol formed albumin complex time (days) (%, vol/vol) None Albumin Albumin + ergosterol Albumin + ergosteryl oleate Albumin + monoolein Albumin + triolein Albumin + ergosterol monoolein Albumin + ergosteryl oleate + monoolein Albumin + ergosterol triolein a Albumin was added at a level of 1 mg/ml. Experimental lein were used for comparison in place of Tween 8. A level of 19.7 or 19.8% (vol/vol) ethanol was obtained with the culture supplemented with monoolein, albumin, and either ergosterol or ergosteryl oleate, respectively (Table 1). However, final ethanol concentrations in the culture supplemented with a combination of triolein, ergosterol, and albumin were almost the same as those in the unsupplemented culture. Action of Tween 8 or monoolein on growth and fermentative activity of yeast cells. Yeast growth was significantly promoted in the nitrogen gas-sparged anaerobic culture supplemented with ergosterol and either Tween 8 or monoolein compared with that supplemented with ergosterol and triolein, and the population was 1 x 18 to 2 x 18 cells per ml at 24 h (Table 2). The fermentative activity per 2 x 18 cells in the presence of 18% (vol/vol) ethanol was remarkably enhanced when the medium was supple- TABLE 2. Effect of lipids on yeast growth No. of Lipid(s) addeda cells per mlb None x 17 Ergosterol x 17 Triolein x 17 Triolein + ergosterol x 17 Monoolein X 17 Monoolein + ergosterol x 18 Tween X 17 Tween 8 + ergosterol x 18 a Triolein, monoolein, and Tween 8 were added at a level of.5 mg/ml and ergosterol was added at a level of 1 mg/ml. b The inoculum size was 16 per ml. The cell number was measured by a hemacytometer after 24 h of nitrogen gas-sparged anaerobic culture at 3 C.

4 824 OHTA AND HAYASHIDA APPL. ENVIRON. MICROBIOL. 12 r 2 _ o u _~ _ x 75 >. at (N4 Z - a 1 F 8-6 F 4 F lhnhf7i 2 T1 None Ergosterol Triolein Triolein Ergosterol Monoolein Monoolein Tween 8 Tween8O Ergosterol Ergosterod FIG. 4. Effect of lipids on fermentative activity of yeast cells. Fermentative activity is shown in the presence of 18% (vol/vol) ethanol. Cells were grown in a nitrogen gas-sparged anaerobic culture at 3 C for 24 h. Triolein, monoolein, and Tween 8 were added at a level of.5 mg/ml and ergosterol was added at a level of 1 mg/ml. mented with Tween 8 or monoolein alone or in conjunction with ergosterol, but not in the case where the medium was supplemented with triolein or ergosterol or both (Fig. 4). However, fermentative activity of any type of cells was lost after exposure to 2% (vol/vol) ethanol (data not shown). Incorporation of oleic acid and ergosterol into yeasts grown in nitrogen gas-sparged anaerobic culture. Yeast cells grown in nitrogen gassparged anaerobic culture were examined for the incorporation of oleic acid provided in the form of Tween 8 and ergosterol from the growth medium. Cells grown in media containing Tween 8 and ergosterol contained 35.4% oleic acid of the total fatty acid, whereas the oleic acid content of control cells accounted for only 5.% (Table 3). When the medium was supplemented with Tween 8 and ergosterol, the ergosterol content of dry-weight cells increased from.5 to.35%. DISCUSSION Independent studies (7, 14) have shown that the action of ethanol on yeast cells was influenced by the lipid composition of the plasma membrane. The addition of Tween 8 plus ergosterol to the basal medium as albumin complex made it possible to obtain higher concentrations of ethanol than when oleic acid plus ergosterol was used as albumin complex. However, the addition of Tween 8 plus ergosterol to the basal medium without albumin failed to stimulate ethanol fermentation, although Tween 8 not only dispersed ergosterol into the liquid medium but also provided a soluble source of oleic acid. Likewise, the final concentration of ethanol produced was not significantly increased by the sole addition of crude phosphatidylcholine (4). Supplementation of the basal medium with monoolein plus either ergosterol or ergosteryl oleate in the presence of albumin allowed the formation of about 2% (vol/vol) ethanol, whereas triolein plus ergosterol as albumin complex possessed a lower stimulatory effect on ethanol fermentation. Monoolein contains hydrophobic and hydrophilic portions similar to Tween 8 and under- TABLE 3. Fatty acid composition and total ergosterol content of sake yeasts grown anaerobically in medium containing Tween 8 and ergosterola Composition (%) in medium containing: Fatty acid No Tween 8 + addition ergosterol 12: : : : : : : Miscellaneous Total ergosterol content.5.35 (mg/1 mg, dry wt) a Yeast cells were grown in synthetic medium containing Tween 8 (.5 mg/ml) and ergosterol (1 mg/ml) under nitrogen gas-sparged anaerobic culture conditions at 3 C for 24 h. Fatty acids are denoted by the convention, number of carbon atoms:number of unsaturated linkages.

VOL. 46, 1983 goes spontaneous dispersions in liquid media. In contrast, triolein, oleic acid, ergosterol, and ergosteryl oleate themselves are almost water insoluble.

5 VOL. 46, 1983 goes spontaneous dispersions in liquid media. In contrast, triolein, oleic acid, ergosterol, and ergosteryl oleate themselves are almost water insoluble. Although the addition of oleic acid plus ergosterol to the nitrogen gas-sparged anaerobic culture enhanced the ethanol endurability of cells (8), the addition of oleic acid plus ergosterol to the static culture as albumin complex did not affect fermentation significantly. Both Tween 8 and monoolein promoted the fermentative activity of cells grown in a nitrogen gas-sparged anaerobic culture. Phosphatidylcholine is also a surface-active lipid, and similar stimulatory effects on the fermentative activity of cells were observed when purified phosphatidylcholine was added to the nitrogen gassparged anaerobic yeast culture (8). In addition, the balance between hydrophobic and hydrophilic properties of the lipid may determine the capacity to form a lipid-sterol-protein complex in static cultures as in sake mash. LITERATURE CITED 1. Andreasen, A. A., and T. J. B. Stier Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J. Cell. Comp. Physiol. 41: Andreasen, A. A., and T. J. B. Stier Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a defined medium. J. Cell. Comp. Physiol. 43: Chen, R. F Removal of fatty acids from serum TWEEN 8 OR MONOOLEIN AND ETHANOL FERMENTATION 8 albumin by charcoal treatment. J. Biol. Chem. 242: Hayashida, S., D. D. Feng, and M. Hongo Function of the high concentration alcohol-producing factor. Agric. Biol. Chem. 38: Hayashida, S., D. D. Feng, and M. Hongo Physiological properties of yeast cells grown in the proteolipidsupplemented media. Agric. Biol. Chem. 39: Hayashida, S., D. D. Feng, K. Ohta, S. Chaltiumvong, and M. Hongo Compositions and a role of Aspergillus oryzae-proteolipid as a high concentration alcohol-producing factor. Agric. Biol. Chem. 4: Hayashida, S., and K. Ohta Cell structure of yeasts grown anaerobically in Aspergillus oryzae-proteolipidsupplemented media. Agric. Biol. Chem. 42: Hayashida, S., and K. Ohta Effects of phosphatidylcholine or ergosteryl oleate on physiological properties of Saccharomyces sake. Agric. Biol. Chem. 44: Hayashida, S., and K. Ohta Formation of high concentrations of alcohol by various yeasts. J. Inst. Brew. 87: Kekwick, R. A., and R. K. Cannan The hydrogen ion dissociation curve of the crystalline albumin of the hen's egg. Biochem. J. 3: Knapp, F. F., and H. J. Nicholas Liquid crystalline properties of ergosteryl fatty acid esters. Mol. Cryst. Liq. Cryst. 1: Kodama, K Sake yeast, p In A. H. Rose and J. S. Harrison (ed.), The yeasts, vol. 3. Academic Press, Inc., New York. 13. Shaw, W. H. C., and J. P. Jefferies The determination of ergosterol in yeast. II. Determination by saponification and ultra-violet absorption spectroscopy. Analyst 78: Thomas, D. S., J. A. Hossack, and A. H. Rose Plasma-membrane lipid composition and ethanol tolerance in Saccharomyces cerevisiae. Arch. Microbiol. 117:

Organisms used. The routine test organism was a putrefactive anaerobe, Company, and Bacilus stearothermophilus, strain NCA 1518.

Organisms used. The routine test organism was a putrefactive anaerobe, Company, and Bacilus stearothermophilus, strain NCA 1518. THE EFFECT OF OXIDATIVE RANCIDITY IN UNSATURATED FATTY ACIDS ON THE GERMINATION OF BACTERIAL SPORES NORMAN G. ROTH2 AND H. 0. HALVORSON Department of Bacteriology, University of Illinois, Urbana, Illinois