LIPID METABOLISM AND THE REGULATION OF VOLATILE ESTER SYNTHESIS IN SACCHAROMYCES CEREVISIAE

The specific rates at which ethyl acetate and iso-amyl acetate are produced by yeast increase markedly at that point in fermentation where syntheses of lipids (i.e. saturated fatty acids and squalene) stop. An increase in the acetyl-CoA: CoASH ratio, or a reduced availability of substrates (fatty acyl-CoAs) for acyl transferases, are possible reasons for such a stimulation of ester synthesis. Increased rates of ester production are not sustained but contribute significantly (ca. 30% for ethyl acetate) to the total concentration of acetate esters in beer fermented from 1·040 all-malt wort. Addition of linoleic acid (50 mg litre^?1) suppresses the induction of ester synthesis and reduces overall formation of both ethyl and iso-amyl acetates by ca. 80%. Possibly, linoleic acid exerts these effects either by directly inhibiting the activity of an ester-synthesising enzyme, or by allowing alternative use of acetyl-CoA for the synthesis of saturated fatty acids.     

The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis

For beer wort fermentation the addition of unsaturated fatty acids has sometimes been suggested as an alternative to wort oxygenation. This can however negatively affect the synthesis of acetate esters and consequently beer flavour. This work investigates the effect of supplementing a cropped yeast with an unsaturated fatty acid on the fermentation performance of the pitching yeast. Cropped yeast is in a different physiological state to yeast pitched in unfermented wort. Using a synthetic medium for the fermentations, it was found that the incubation of cropped yeast with linoleic acid resulted in two important changes in the yeasts composition: (1) the ratio of unsaturated fatty acids to total fatty acids increased from 0.53 to 0.66 and (2) the ratio of trehalose to glycogen increased from 0.17 to 0.49. The performance of this yeast in subsequent fermentations was compared to unsupplemented yeast under three conditions: medium pre‐aeration, de‐aerated medium and de‐aerated medium with newly added unsaturated fatty acid. It was found that the supplemented pitching yeast showed growth, attenuation and ethanol formation profiles similar to those obtained with unsupplemented yeast in pre‐aerated medium, which simulated the normal brewing practice. Compared to fermentations with unsaturated fatty acids added to the medium, the supplemented cropped yeast did not induce a reduction in acetate ester synthesis. Results indicated that the supplementation of cropped yeast with unsaturated fatty acids could be an interesting alternative to wort oxygenation to restore the optimal membrane fluidity of the yeast.     

LIPID COMPOSITION OF AEROBICALLY AND ANAEROBICALLY PROPAGATED BREWER'S BOTTOM YEAST

Aerobically grown pitching yeast is very rich in unsaturated fatty acids and sterol esters compared to traditional, anaerobic yeast. The principal fatty acids in aerobic yeast cells are unsaturated palmitoleic and oleic acids, whereas in anaerobic cells saturated palmitic acid predominates. The difference in fatty acid distribution between aerobic and anaerobic cells is most marked in the sterol esters. The fatty acids of phospho-lipids are more stable, although remarkable differences are observed. The sterols of aerobic cells are almost entirely in esterified form and zymosterol is the principal sterol. During the first hours of fermentation a rapid synthesis of palmitoleic acid is observed when anaerobic yeast is used for pitching and the wort is aerated. The synthesis of oleic acid requires more oxygen and time than is available under normal brewing conditions. When aerobic pitching yeast is used no more unsaturated fatty acids are synthesised and the lipid stores of pitching yeast are distributed among the daughter cells. The decrease in acetate ester production by aerobic pitching yeast is concluded to be due to a decrease in acetyl CoA synthesis, which may be caused by the high proportion of unsaturated fatty acids in membrane lipids.     

CHEMICAL CHARACTERIZATION AND STEREOSPECIFIC ANALYSIS OF LIPIDS SYNTHESIZED BY CERTAIN YEAST STRAINS

Fatty acid analysis of lipids from yeast strains, Apiotrichum curvatum ATCC 10567, Cryptococcus albidus ATCC 56297, Lipomyces starkeyi ATCC 12659, and Rhodosporidium toruloides ATCC 10788, grown on whey permeate revealed palmitic, stearic, oleic and linoleic acids as the predominant fatty acids in the triacylglycerol fractions. The phospholipid fractions were dominated by oleic and linoleic acids. The stereospecific distribution of fatty acids in the triacylglycerol fractions of lipids from two yeast strains, Apiotrichum curvatum ATCC 10567 and Lipomyces starkeyi ATCC 12659, was determined and revealed the presence of almost entirely unsaturated fatty acids at the sn-2 position (91.9% and 82.8%, respectively). The dominance of unsaturated fatty acids (in the range of 61.5 to 72.3%) at the sn-1 position was also observed. Oleic acid was the predominant fatty acid at positions sn-1 and sn-2 for both yeast strains. Position sn-3 had the greatest concentrations of saturated fatty acids, with palmitic acid as the predominant fatty acid.     

Influence of triglycerides and free fatty acids in milk replacers on calf performance, blood plasma, and adipose lipids

In a 4-wk study of 48 3-day-old calves we compared effects of feeding various fats or their free fatty acids in skim milk-powder based milk replacer, on calf performance, feed utilization, and blood plasma and adipose lipids. When fat was fed, calf performance and feed utilization were equivalent for tallow and coconut oil diets but markedly poorer for corn oil. Complete replacement (tallow) or one-half replacement (coconut and corn oils) of the fats with their free fatty acids reduced calf gains and feed efficiency. Tallow free fatty acids gave lower digestibilities of palmitic and stearic acid and reduced calcium absorption. Free fatty acids from both coconut and corn oils reduced diet palatability and intake; those from tallow and coconut oil markedly interfered (in vitro) with rennet clotting of milk replacers. The main lipid classes in blood plasma for all treatments were cholesteryl esters and phosphatidylcholine. High concentrations (56 to 87%) of linoleic acid occurred in cholesteryl esters for all diets despite low concentrations of linoleic acid in the tallow and coconut oil diets.     

Linoleic Acid Supplementation of a Cropped Brewing Lager Strain: Effects on Subsequent Fermentation Performance with Serial Repitching

Most breweries collect yeast from a previous fermentation cycle for further use in a subsequent cycle. However, the cropped cells are deficient in membrane sterols and unsaturated fatty acids (UFAs) which are required for good fermentation performance in the next cycle. Consequently, the cellular levels of these compounds must be restored to obtain an optimal fermentation performance. There are currently three possibilities to satisfy this requirement. The common practice is aeration of the wort before pitching, thus providing oxygen needed for lipid synthesis during the first stages of fermentation. Oxygenation (aeration) of cropped yeast slurries is a second alternative. Finally, the addition of the required lipids to wort is sometimes suggested as an alternative to aeration. We examined a fourth possibility, namely the supplementation with UFA of cropped cells. Previously, we reported that the supplementation of stationary phase cropped brewer's yeast with linoleic acid is a good alternative to wort aeration. This conclusion resulted from results obtained with a well‐defined stirred synthetic fermentation medium. We also showed that cells cropped from non‐stirred tall‐tube fermented malt wort incorporated linoleic acid into different cellular lipid fractions, when suspended and supplemented in fermented wort. Now, we report that such yeast, pitched in malt wort in non‐stirred tall‐tubes, showed growth and attenuation profiles comparable to unsupplemented yeast in pre‐aerated wort. Moreover, the synthesis of acetate esters, which is known to be affected when UFAs are added directly to the wort, was not significantly affected. We hypothesize that the active uptake of linoleic acid during fermentation and its activation by coenzyme A (CoA) and phospholipid synthesis are responsible for the effects on ester synthesis, through repression of the alcohol acetyltransferase‐encoding gene, ATF1. In supplemented cropped yeast, these reactions occur prior to fermentation, thus avoiding interferences with acetate ester synthesis. In serial repitching experiments with repeated linoleic acid supplementation of the cropped yeast, the fermentation performances of the yeast remained comparable to those of non‐supplemented yeast in pre‐aerated wort. However, due to a progressive increase of cellular UFA, negative effects on acetate ester synthesis appeared. Nevertheless, the supplementation of cropped yeast with UFAs can be considered as an interesting alternative to wort oxygenation to restore optimal membrane functions.     

Short communication: Dietary conjugated linoleic acid down-regulates fatty acid transporters in the mammary glands of lactating rats

Recent studies indicated that reduction of milk triacylglycerol concentrations by dietary conjugated linoleic acid (CLA) involves an impairment of both de novo fatty acid synthesis and uptake of fatty acids from circulating triacylglycerol-rich lipoproteins into the mammary gland. However, nonesterified fatty acids (NEFA) in the plasma released from adipose tissue and taken up into the mammary gland by fatty acid transporters are a further important source of fatty acids available for milk triacylglycerol synthesis. Therefore, the aim of the present study was to investigate the effect of dietary CLA on plasma concentrations of NEFA and the expression of fatty acid transporters in the mammary glands of lactating rats fed either a CLA diet or a control diet. Dams fed diets with CLA had a greater concentration of NEFA in plasma than those fed the control diet. In addition, relative mRNA concentrations of fatty acid transporters (fatty acid translocase/CD36, fatty acid transport protein, and plasma membrane fatty acid binding protein) were about 45, 75, and 70% lower, respectively, in the mammary gland of dams fed diets with CLA compared with those fed the control diet. In conclusion, the present findings indicate that reduced uptake of circulating NEFA released from white adipose tissue into the mammary gland could also contribute to the reduction of milk triacylglycerol concentrations by dietary CLA in rats. The mechanism through which CLA inhibits expression of fatty acid transporters deserves further study.     

Effects of dietary corn oil and fish oil concentrate on lipid composition of calf tissues

Lipid composition of calf blood plasma, liver platelets, muscle, heart, and brain was measured, as affected by high dietary intake of linoleic acid from corn oil or of polyunsaturated fatty acids from fish oil concentrate. Plasma total lipids, phosphatidylcholine, and cholesteryl esters were reduced by corn oil and fish oil concentrate. Dietary fatty acid composition had no influence on percentage distribution of the major phospholipid components of liver, heart, muscle, and brain, but did alter the polyunsaturated fatty acid composition of major phospholipids in plasma, liver, platelets, muscle, and heart. In general, high linoleic acid intake increased linoleic acid and decreased oleic, arachidonic, and linolenic acids in tissue phospholipids, and fish oil concentrate high in eicosapentaenoic and docosahexaenoic acids increased phospholipid concentrations of these fatty acids. The fatty acid composition of brain phosphatidylcholine and phosphatidyl-ethanolamine was relatively resistant to dietary lipid alterations. The fatty acid changes in tissue phospholipids that resulted from dietary lipid alterations may have important implications in eicosanoid metabolism, and in the structure and function of cell membranes.     

THE INFLUENCE OF LIPIDS DERIVED FROM MALT SPENT GRAINS ON YEAST METABOLISM AND FERMENTATION

The addition to wort of lipids derived from malt spent grains had a pronounced effect on yeast metabolism. The lipids allowed the fermentation of de-oxygenated wort and also stimulated yeast growth and the corresponding rate and extent of fermentation of air-saturated wort by yeast strains having a high oxygen requirement. The lipids increased the fusel alcohols content of beer and decreased the content of esters and medium chain-length fatty acids. The yeast incorporated sitosterol and unsaturated fatty acids from the spent grain lipids and the unsaturated fatty acids changed the pattern of fatty acids and sterols synthesized by the yeast. The fatty acids were present in the spent grain lipids mainly as triglycerides, free fatty acids and phospholipids. Using pure lipid compounds it was shown that the triglycerides were inactive and that the spent grain lipids exerted their effect on fermentation through the synergistic action of free unsaturated fatty acids, sitosterol and phospholipid. Phospholipid could be replaced by the detergent, Triton X-100. The effect of the lipids on the synthesis of esters, fusel alcohols and medium chain fatty acids could be explained solely by their content of unsaturated fatty acids.     

光皮树籽抽出物的成分分析

为了分析光皮树籽抽提物的化学成分,采用无水乙醇、丙酮、正己烷、石油醚和乙酸乙酯分别抽提光皮树籽,抽出物经活性炭脱色后进行GC-MS 分析。结果发现,无水乙醇和丙酮抽出物中主要含有游离的棕榈酸、油酸和亚油酸,其中亚油酸相对含量最高,达到52% 以上,油酸相对含量最低,不超过8.2%。正己烷、石油醚和乙酸乙酯抽出物中甘三酯的脂肪酸组成主要是棕榈酸、硬脂酸、油酸和亚油酸,其中油酸相对含量最高,达到42% 以上,其次是亚油酸,总不饱和脂肪酸相对含量超过70%。抽出物中未发现三不饱和脂肪酸。     

ESTERASES OF BAKER'S YEAST. II. SUBSTRATE SPECIFICITIES TOWARDS ESTERS FORMED DURING SUGAR FERMENTATIONS

Ethyl esters of fatty acids (C₂–C₁₂), isoamyl acetate and 2-phenethyl acetate were studied as substrates for yeast esterases and compared with the synthetic substrates, p-nitrophenyl esters and β-naphthyl esters. Intact yeast, the 55% and 55–75% ammonium sulphate precipitate of centrifuged yeast homogenate, and partly purified esterases were used for the determination of the hydrolysation activity towards the esters. The results showed that the yeast esterases prefer to hydrolyse the ethyl esters with acyl chain length of C₅ to C₁₂. The acetate esters, ethyl acetate, isoamyl acetate and 2-phenethyl acetate are only very slowly hydrolysed or remain unaffected. The substrate specificity of different esterases varies and can be used for their characterisation. Investigating pH optimum curves using intact yeast and a crude esterase preparation and different substrates confirmed the earlier result that there are esterases on both sides of the plasma membrane. The specificity of intracellular and periplasmically located esterases is, however, different.     

气相色谱-质谱联用法分析罗布麻蜂蜜中脂肪酸

目的建立气相色谱-质谱联用法快速测定脂肪酸的方法,分析新疆罗布麻蜂蜜中组成成分。方法样品用正己烷乙醚混合提取,氢氧化钾-甲醇法甲酯化,归一化法计算含量。结果罗布麻蜂蜜中含有28种脂肪酸成分,其主要成分是亚油酸(26.57%)、棕榈酸(17.6%)、十七烷酸(7.34%)等。饱和脂肪酸含量为38.83%,不饱和脂肪酸为61.17%。结论罗布麻蜂蜜中不饱和脂肪酸含量丰富,可为罗布麻产品的开发利用提供依据,该方法简便快速,适合于大批量样品的测定。     

Influence of vacuum filtration of Viura must on the concentration of fatty acids and their utilization in fermentation

Fatty acids such as palmitoleic, oleic, linoleic and linolenic along with sterols constitute growth or survival factors for yeast. The objective of this work was to study the influence of vacuum filtration of Viura must on fatty acid contents and their use during fermentation. The results were compared with unclarified must, the control. Filtration drastically reduced the total fatty acid concentration (81.5%) and especially unsaturated fatty acids (97.1% of linoleic, and 100% of linolenic), as well as the minor saturated acids, arachidic and behenic. In the first half of fermentation, fatty acids were excreted in the filtered must (76.8%) whereas they were consumed in the control (46.8%). In the second half of fermentation, there was greater consumption in the control sample (74.5%) than in the filtered sample (37.4%).     

传统豆酱中脂肪酸酯化方法的选择与组成分析

为更好地研究传统豆酱中脂肪酸的组成,先用索氏提取法提取豆酱中的脂肪酸,分别利用酸酯化法、碱酯化法和酸碱酯化法对脂肪酸进行甲酯化,正己烷萃取后,采用气相色谱-质谱法检测,根据标准谱库NIST11.L,结合有机质谱学规律对脂肪酸甲酯进行定性分析,并用面积归一化法测定其相对含量。结果表明：衍生化后,脂肪酸甲酯在33 min内完全分离,利用酸酯化法可分析出10 种脂肪酸,其中含量较高的物质有棕榈酸甲酯13.62%、油酸甲酯23.95%、亚油酸甲酯48.91%;利用碱酯化法可分析出4 种脂肪酸,其中含量较高的物质有油酸甲酯10.42%、亚油酸甲酯19.63%;利用酸碱酯化法可分析出10 种脂肪酸,其中含量较高的物质有棕榈酸甲酯13.64%、油酸甲酯24.16%、亚油酸甲酯49.07%。本方法无需标准品即可快速定性检测豆酱中的脂肪酸,结果准确、可靠。     

Isolation of Delta12 and omega3-fatty acid desaturase genes from the yeast Kluyveromyces lactis and their heterologous expression to produce linoleic and alpha-linolenic acids in Saccharomyces cerevisiae

Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Delta12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited omega3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced alpha-linolenic acid. Co-expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and alpha-linolenic acids. The yield of alpha-linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; alpha-linolenic acid reached 8.7% when exogenous linoleic acid was available.     

Effect of exogenous long-chain fatty acids on lipid biosynthesis in dispersed ruminant mammary gland epithelial cells: esterification of long-chain exogenous fatty acids

Dispersed epithelial cells from lactating bovine and goat mammary glands incorporated acetate into all fatty acids (C4 to C16) that were incorporated into mainly triacylglycerols. The cells secreted free fatty acids only into the incubation medium, and this secretion was dependent on the concentration of albumin and the type and amount of exogenous fatty acid added to the medium. Addition of palmitic acid to the incubation medium stimulated synthesis and incorporation of fatty acids synthesized de novo into triacylglycerols, whereas stearic and linoleic acid were inhibitory.     

正丁酯对共轭亚油酸异构体分析的影响

研究了正丁酯对共轭亚油酸异构体分析的影响。通过选择合适的分析条件 ,可以将脂肪酸正丁酯与脂肪酸甲酯完全分开。研究结果表明 ,脂肪酸正丁酯与脂肪酸甲酯在氢火焰电离检测器上具有类似的响应值 ,两者的加和可以粗略地代表混合物经皂化、甲酯化后脂肪酸甲酯的相对含量。     

利用气相色谱-质谱联用技术测定燕麦中脂肪酸的组成

以无水乙醚-石油醚为溶剂,用索氏抽提法提取燕麦中粗脂肪,经KOH-甲醇进行甲酯化后以正已烷萃取,用气相色谱-质谱测定脂肪酸甲酯,分析鉴定出15种脂肪酸,并测定出各脂肪酸的相对含量,其中不饱和脂肪酸相对含量约占79.45%,以油酸、亚油酸、亚麻酸为主.     

Improvement of isoamyl acetate productivity in sake yeast by isolating mutants resistant to econazole

Sake yeast strains were improved so as to produce larger amounts of isoamyl acetate than the parental strain by isolating econazole-resistant mutants. Econazole, an imidazole antimycotic, directly interacts with unsaturated fatty acids in the yeast cell membrane, where it also inhibits the synthesis of ergosterol and decreases the ratio of unsaturated to saturated fatty acids. In contrast, alcohol acetyltransferase (AATase), which catalyzes the synthesis of isoamyl acetate, is inhibited by unsaturated fatty acids. Fifty econazole-resistant mutants were isolated from a sake yeast, Kyokai no. 701, several of which produced approximately 1.4 to 2.4 times more isoamyl acetate and an almost equal amount of isoamyl alcohol compared with the parental strain. The AATase activities of the mutants in koji extract were 1.2 to 1.4 times higher, and the unsaturated to saturated fatty acid ratios were lower, than in the parental strain.