Cellular Fatty Acid and Ester Formation by Brewers' Yeast

Transcription

1 Agric. Biol. Chem., 47 (10), , Cellular Fatty Acid and Ester Formation by Brewers' Yeast Kazuo Yoshioka and Naoki Hashimoto* Brewing Science Laboratory and *Applied Bioscience Laboratory ofkirin Brewery Co., Ltd., Miyahara, Takasaki , Japan Received March 2, 1983 The activity of alcohol acetyltransferase, bound to the cell membrane and responsible for the formation of acetate esters, was affected by the fatty acid composition of the cell membrane. When saturated fatty acids, which only slightly inhibit alcohol acetyltransferase activity, were incorporated into the cell membrane, the enzyme activity and ester formation were only slightly affected. On. the other hand, when unsaturated fatty acids, which strongly inhibit the enzyme activity, accumulated in the cell membrane, ester formation was suppressed with inhibition of the enzyme activity. The mechanism of formation of acetate esters by brewers' yeast was explained by the alcohol acetyltransferase activity under the influence of the fatty acid composition of the cell membrane. Acetate esters such as ethyl acetate, isoamyl acetate and js-phenetyl acetate are known to be the main flavor esters of beer and other alcoholic beverages. Nordstrom1 ^3) proposed that the biosynthesis of acetate esters was due to esterification of alcohols with acetyl-coa, probably in the presence of an unknown enzyme. Wepreviously purified the alcohol acetyltransferase responsible for the formation of acetate esters by brewers' yeast.4) This enzyme was localized in the cell membrane of brewers' yeast and was most active at 30 C at ph 7~8. It was least active on C3 alcohol among Q ~C6 alcohols and was slightly more active on straight-chain alcohols than on branchedchain alcohols with the same carbon numbers. The enzyme was strongly inhibited by unsaturated fatty acids, ergosterol, heavy metal ions and sulfhydryl reagents. In general, unsaturated fatty acids in the medium greatly reduce the ester level, while saturated fatty acids raise it.5 ~10) In this study we examined the reason for the effects of fatty acids on the formation of acetate esters by brewers' yeast in relation to alcohol acetyltransferase activity and fatty acid composition of the cell membrane. was MATERIALS AND METHODS Yeast. A typical brewers' yeast (Saccharomyces uvarum) used. Medium and chemicals. A brewers' wort of 1 1 P (grams of extract in 100g of wort) or an unhopped synthetic medium11} was used. The composition of the synthetic edium is shown in Table I. Carbohydrates were purchased from Kishida Chemical Co. Casamino acids from Difco Laboratories and peptone from Kyokuto Seikagaku Kogyo Co. were used as nitrogen sources. Palmitic acid and stearic acid from P-L Biochemical Ind., oleic acid from Sigma Chemical Co:, linoleic acid from Tokyo Kasei Kogyo Co. and linolenic acid from Wako Pure Chemical Ind. were added to the synthetic medium with 0.1 % bovine serum albumin from Sigma Chemical Co. as emulsiner.10) The effects of fatty acids in the medium on ester formation were examined by adding each fatty acid at 0.3 mm.in the study on the effect of the level of fatty acids in brewers' wort on ester formation during beer fermentation, a mixture of 40% palmitic acid, 15% stearic acid, 15% oleic acid, 25% linoleic acid and 5% linolenic acid was added to the synthetic medium. The purities of the fatty acids added to the medium are shown in Table II. All other chemicals were of analytical grade. Fermentation. Fermentation was started at 8 C in a 1000-ml flask by inoculating 3 g of centrifuged yeast into 500ml of wort. The wort was aerated by shaking the flask 100 times both before and just after pitching. After fermentation for 18hr at 8 C in the flask, the wort was

2 2288 K. Yoshioka and N. Hashimoto aerated again by shaking the flask 100 times, and then the wort was poured into a 500-ml cylinder (120cmx 2.5cm). Fermentation was continued for 7 days in the cylinder without aeration. Preparation of cell membrane-bound alcohol acetyltransferase and assay of the enzyme activity. The yeast was collected by centrifugation of fermenting wort at 5500 x g for 10 min, and was washed first with a mixture of petroleum ether-ethyl ether (2 : 1) and then with distilled andeionized water. The washed yeast was treated with Table I. Composition of Synthetic Medium Zymolyase 60,000 for preparation of cell membranebound alcohol acetyltransferase as described previously.4) Alcohol acetyltransferase activity was assayed with 0.8 mm acetyl-coa and 15 mm isoamyl alcohol as substrates, and expressed in milliunits (mu).4) Analysis of acetate esters and higher alcohols. The contents of acetate esters and higher alcohols in the fermenting or fermented wort were determined by head space gas chromatography, after heating 50ml of the sample for 1hr at 40 C in a 300-ml flask with a silicone rubber stopper. The conditions of gas chromatography were the same as those for the assay of alcohol acetyltransferase activity.4) Analysis of the fatty acid composition of the cell membrane. A mixture of 50mg of the cell membrane fraction, 5ml of acetone and 0.2mg of arachidic acid (C2Oo) as internal standard was stirred for 5 min at room temperature in a 10-ml reaction flask with a stopper. The mixture was decanted and filtered through a Toyo No. 2 filter paper. The residue after decantation was further treated successively with 5ml of ethyl ether and 5ml of chloroform-methanol (2 : 1) under the same conditions as those used in the treatment with acetone. The lipid fraction was obtained by evaporating the solvents from the series of filtrates, and was mixed with 0.5ml of 5% HC1-methanol in a 15-ml reaction tube with a stopper and heated for 5hr in boiling water to methylate fatty acids. Then 0.5ml of 2% potassium bicarbonate solution and 2ml ofhexane were added to the sample, and the tube was shaken for 2 min. The methylated fatty acids in the hexane layer were analyzed by gas chromatography by injecting 2[A of the hexane layer on to a Shimadzu GC-5A gas chromatograph with dual flame ionization detectors. The glass column (2mx 3mm) was packed with 15% DEGS on Celite 545 (60~80 mesh). The column oven and the injection port were kept at 190 C and 250 C, respectively. Nitrogen was used as carrier gas at a flow rate of 50ml/ min. The flow rates of hydrogen and air were 35ml/min and 500 ml/min, respectively. Measurement of the ergosterol content of the cell membrane fraction. Fats in the cell membrane were saponified by heating a mixture of 50mg of the cell membrane Table II. Purity of Supplemented Fatty Acids

3 S Cellular Fatty Acid and Ester Formation 2289 fraction and 3 ml of 12% potassium hydroxide in ethanol for 2 hr in boiling water, extracted twice with 5 ml ofethyl ether after adding 3 ml of distilled and deionized water, washed with 3 ml of distilled and deionized water, and dehydrated with anhydrous sodium sulfate. The extract was dissolved in a small amount of chloroform after evaporating ethyl ether, and separated by thin-layer chromatography on silica gel 60 plates (Merck) with chloroform as developing solvent. Ergosterol on the thin layer was detected under UV light, extracted with chloroform and determined from its absorbance at 283 nm. RESULTS uppression of ester formation in brewers' wort by accumulation of unsaturatedfatty acids in the cell membrane When brewers' wort was fermented with aeration by stirring it in a 1000-ml flask, larger amounts of unsaturated fatty acids such as palmitoleic acid and oleic acid accumulated in the cell membrane than during standard anaerobic fermentation, as seen in Table HI. Accumulation of ergosterol was also increased by aeration. On the other hand, when oleic acid was added to the wort, it was incorporated into the yeast cells and its concentration ncreased in the cell membrane. Formation of acetate esters during fermentation was reduced by aeration or addition of oleic acid with a decrease in alcohol acetyltransferase activity. ncorporation offatty acids from the medium into the cell membrane Palmitic acid, stearic acid, oleic acid, linoleic acid or linolenic acid was added at 0.3him to the synthetic medium. Results on their in- Brewers'wort was fermented. Table III. Effect of Cellular Fatty Acids on Formation of Acetate Esters Percentages in the membrane of yeast cells after fermentation. Calculated with the extract in 100g of wort (OE) and the apparent extract in 100g of fermented wort (AE) by the following equation: Apparent attenuation (%) =-^- x100 mu/lo g of centrifuged yeast after fermentation.

4 corporation into the cell membrane are summ K.Yoshioka andn.hashimoto å 2 å ~4 t 8 ioå /^ ^s,å s2" / 'Iki ^ à" / å o12à" / o6- / -«9å / Q) Q) I c 4 / o b / fi / c / o.^--o ---o o. o o o o o o Fig. 1. Incorporation of Fatty Acids from Medium into Cell Membrane of Brewers' Yeast. Amounts of fatty acids are expressed as percentages of the cell membrane fraction. O, no addition; A, palmitic acid; å, stearic acid; #, oleic acid; A, linoleic acid; å, linolenic acid. arized in Fig. 1. These fatty acids were incorporated into the cell membrane at an earlier stage of the fermentation. From the content of fatty acids added to the medium and increases in their levels in the membrane of yeast cells, the percentages of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid incorporated into the cell membrane were calculated tobe22.6, 19.5, 50.5, 47.5 and 58.8%, respectively. When unsaturated fatty acids such as oleic acid, linoleic acid and linolenic acid in the

5 ' Cellular Fatty Acid and Ester Formation 2291 medium were incorporated into the cell membrane, accumulation of palmitic acid increased and that of palmitoleic acid decreased. ffect of incorporated fatty acids on alcohol acetyltransferase activity and ester formation When saturated fatty acids such as palmitic acid hibit and alcohol stearic acetyltransferase acid, which only activity,4) slightly were in- incorporated acetyltransferase into activity the cell membrane, was not affected alcohol greatly acetate (Fig. and 2), isoamyl and the acetate formation was not of ethyl pressed (Fig. 3). On the other hand, when sup- linoleic unsaturated acid and fatty linolenic acids such acid, which as oleic strongly acid, were inhibit incorporated alcohol acetyltransferase into the cell membrane, activity,4) enzyme activity and ester formation were the greatly suppressed. medium ation, but did the not addition affect the of unsaturated apparent attenu- acids such as oleic acid, linoleic acid fatty linolenic acid slightly suppressed the and mation of higher alcohols (Table IV). for- Effect of addition ofafatty acid mixture to the dominant of fatty acids, fatty and acid.12) palmitic The practical acid is the effect pre- fatty acids in brewers' wort on ester formation of during fermentation was examined by supple- T he addition of fatty acids to the synthetic - o ;_> Hw o g O1 à" à" ' ' ' < 0 2 k 6 8 Fig. 2. Effect offatty Acids in Medium on Activity of Alcohol Acetyltransferase Bound to Cell Membrane. O, no addition; A, palmitic acid; å > stearic acid; à", oleic acid; A, linoleic acid; å, linolenic acid. synthetic medium B rewers' wort usually contains 5~ 15 ppm 9à" Ethyl å 1.2å Isoamyl acetate /& acetate ^"- ( ppm) /#r ( ppm) SA 0 2 ^ Fig. 3. Effect of Fatty Acids in Medium on Formation of Acetate Esters. O, no addition; A, palmitic acid; å» stearic acid; #, oleic acid; A, Hnoleic acid; å, linolenic acid.

6 2292 K. Yoshioka and N. Hashimoto Table IV. Effect of Fatty Acids in Medium on Apparent Attenuation, Yeast Growth and Formation of Higher Alcohols Fatty acids were added to the synthetic medium at 0.3 mm. Table III. C alculated with the extract in loog of wort and the apparent extract in loog of fermented wort as shown in Expressed as the weight of centrifuged yeast. mixture reduced the ester formation with a decrease in alcohol acetyltransferase activity. Linoleic acid and linolenic acid, which strongly inhibit alcohol acetyltransferase activity,40 markedly accumulated in the membrane of yeast cells on addition of 35~50 ppm of the fatty acid mixture to the medium. Fermentation, growth of yeast and formation of higher alcohols were slightly accelerated with an increase in the level of the fatty acid mixture added (Table V) ko 50 Fatty acid mixture(pptn) Fig. 4. Effect of Fatty Acid Mixture in Medium on Formation of Acetate Esters. Total amounts of linoleic acid and linolenic acid are expressed as percentages of the cell membrane fraction. O, alcohol acetyltransferase activity; A, total amounts of linoleic acid and linolenic acid; å, isoamyl acetate. meriting with a mixture of palmitic acid, stearicacid, oleic acid, linoleic acid and linolenic acid. As shown in Fig. 4, addition of up to 20 ppm of the fatty acid mixture did not significantly affect the formation of acetate esters. But addition of 35~50 ppm of the fatty acid DISCUSSION The effects of fatty acids on ester formation have been investigated by many workers,5 ~10) and results have shown that unsaturated fatty acids in the medium reduce the ester level. However, it has been unknown why fatty acids in the medium affect ester formation. Ayrapaa and Lindstrom9) reported that oleic acid added to wort was incorporated into brewers' yeast. Ishikawa and Yoshizawa10) observed that fatty acids added to the medium were incorporated intact into cellular lipids such as triglycerides and phosphatidylcholine of sake yeast, even though some of them were ot normally present in the cells. In our experiment, fatty acids added to the synthetic medium were incorporated into the cell membrane of brewers' yeast, unsaturated fatty acids being incorporated preferentially.

7 ROH Cellular Fatty Acid and Ester Formation 2293 Table V. Effect of Fatty Acid Mixture in Medium on Apparent Attenuation, Yeast Growth and Formation of Higher Alcohols Fatty acid mixture was added to the synthetic medium. a Calculated with the extract in loog ofwort and the apparent extract in loog of fermented wort as shown in Table III. b Expressed as the weight of centrifuged yeast. CH3COOR (Acetate esters) Alcohol acetyltransferase (Ester synthesizing enzyme) 1 Inhibition Accumulation of unsaturated fatty acids and ergosterol (Alcohols)l Aeration Unsaturated fatty acids Fig. 5. Proposed Mechanism for Formation of Acetate Esters by Alcohol Acetyltransferase Activity under Influence of Fatty Acid Composition of Cell Membrane of Brewers' Yeast. When C18 unsaturated fatty acids were incorporated into the cell membrane, the accumulation of palmitic acid increased and the accumulation of palmitoleic acid was suppressed. This finding suggests that in brewers' yeast, the degree of unsaturation and the chain length of fatty acids in the cell membrane are regulated to maintain normal functions of the ell membrane. Several explanations have been proposed for why the incorporation of unsaturated fatty acids into yeast cells suppresses ester formation, but there is no clear evidence for any f these explanations. Suomalainen and Nurminen13) showed that the movementof various compounds into and out of yeast cells was regulated by the lipid composition of the plasma membrane. During L-glutamic acid fermentation, the permeability to L-glutamic acid was influenced by the diacyl groups of membrane phospholipids.14) Nykanen et al.15) showed that a high pro-

8 p e 2294 K. Yoshioka and N. Hashimoto portion of esters remained in the yeast cells. From these findings, Ishikawa and Yoshizawa10) supposed that ester formation by sake yeast was affected by permeability of the membrane depending on the kind of acyl chains or the fatty acid composition of membrane lipids. Anderson and Kirsop6) proposed that oleic acid stimulated the synthesis of triglycerides and phospholipids, decreased the availability of acetyl-coa for ester formation and sup- ressed the formation of acetate esters. Ayrapaa and Lindstrom5) suggested that the ester synthesizing enzyme was inhibited by unsaturated fatty acids. The results obtained in this and previous studies4) clearly show that unsaturated fatty acids in the medium reduce the ester level through their incorporation into the cell membrane and their strong inhibitory effect on membrane-bound alcohol acetyltransferase. Wort aeration also reduces the ester level by inhibiting alcohol acetyltransferase activity as a result of accumulation of unsaturated fatty acids and ergosterol in the cell membrane. In practical brewing, the fatty acids usually contained in brewers' wort seem to have little ffect on the formation of acetate esters. A proposed mechanism for ester formation by alcohol acetyltransferase is illustrated in Fig. 5. Thus far, the supply and consumption of acetyl-coa have been thought to be dominant factors in ester formation. Now the mechanism of ester formation by brewers' yeast can be explained clearly by the influence of the fatty acid composition of the cell membrane on the alcohol acetyltransferase activity. The activity of this enzyme, bound to the cell membrane and responsible for the formation of acetate esters, is inhibited by unsaturated fatty acids and ergosterol. Therefore, the accumulation of these lipids in the cell membrane, which is caused by wort aeration or unsaturated fatty acids in the medium, results in the reduction of ester formation through the inhibition Acknowledgments. The authors wish to thank the management of Kirin Brewery Co., Ltd. for permission to publish this work, and Miss Atsuko Miyazawa for her technical assistance. 1) K. Nordstrom, /. Inst. Brew., 67, 173 (1961). 2) K. Nordstrom, /. Inst. Brew., 68, 398 (1962). 3) K. Nordstrom, /. Inst. Brew., 69, 142 (1963). 4) K. Yoshioka and N. Hashimoto, Agric. Biol. Chem., 45, 2183 (1981). 5) T. Ayrapaa and I. Lindstrom, 14th Eur. Brew. Conv., Proc. Congr., Salzburg, 1973, p ) R. G. Anderson and B. H. Kirsop, /. Inst. Brew., 80, 48 (1974). 7) A. K. Palmer and H. Rennie, J. Inst. Brew., 80, 447 (1974). 8) K. Yoshizawa, Nippon Nogeikagaku Kaishi, 50, 115 (1976). 9) T. Ayrapaa and I. Lindstrom, 16th Eur. Brew. Conv., Proc. Congr., Amsterdam, 1977, p ) T. Ishikawa and K. Yoshizawa, Agric. Biol. Chem., 43, 45 (1979). ll) F. Weinfurtner, Brauwissenschaft, 14, 109 (1961). 12) J. K. MacPherson and G. K. Buckee, J. Inst. Brew., 80, 540 (1974). 13) H. Suomalainen and T. Nurminen, /. Inst. Brew., 82, 218 (1976). 14) M. Kikuchi and Y. Nakao, Agric. Biol. Chem., 36, 1135 (1972). 15) L. Nykanen, I. Nykanen and H. Suomalainen, J. Inst. Brew., 83, 32 (1977). R