MUTANT STRAINS OF SACCHAROMYCES YEDO PRODUCING EXCESSES OF FUSEL ALCOHOLS

The possibility of increasing the production of fusel alcohols in fermentation with Saccharomyces yedo by means of induced mutations has been investigated. Mutations were induced by UV-irradiation and mutants that could be expected to produce excesses of isobutyl alcohol, isoamyl alcohol and optically active amyl alcohol were selected in a medium containing norvaline—a partial leucine antagonist. Thus, in the selection of mutants, the relationship between the biosynthesis of amino acids and the formation of higher alcohols was exploited. Using this technique, two different mutants were isolated. In the fermentation of a synthetic medium which was initially lacking in amino acids, one of the mutants produced excess of isoamyl alcohol while the other mutant produced excesses of both isobutyl and isoamyl alcohol.     

WORT COMPOSITION AND BEER FLAVOUR. I. THE INFLUENCE OF SOME AMINO ACIDS ON THE FORMATION OF HIGHER ALIPHATIC ALCOHOLS AND ESTERS

Wort, to which was added various amounts of glucose solution containing either (NH₄)₂SO₄ or one of the amino acids alanine, leucine, isoleucine, valine or proline, was fermented, and in the resulting beers the concentrations of ethyl acetate, isoamyl acetate, n-propanol, isobutanol, and amyl alcohols were determined by gas chromatography. The nitrogen source had some effect on the formation of higher alcohols, in addition to the effect due to conversion of some of the amino acids to the corresponding alcohols via the Ehrlich pathway. Ethyl acetate formation was not significantly affected by the nitrogen source, and iso-amyl acetate formation increased when amyl alcohol formation increased.     

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium‐chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched‐chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO₂/O₂ balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α‐keto‐acids and/or NADH pools. High‐availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2‐phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon‐exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2‐phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase. Copyright © 2014 John Wiley & Sons, Ltd.     

REDUCTION OF HIGHER ALCOHOLS BY FERMENTATION WITH A LEUCINE-AUXOTROPHIC MUTANT OF WINE YEAST

Several auxotrophic mutants requiring branched chain amino acids (valine, leucine, or isoleucine) were isolated in a strain of Montrachet wine yeast. They were tested for their ability to produce lowered amounts of higher alcohols (‘fusel oil’: isobutyl, active amyl, and isoamyl alcohols) in grape juice fermentations. One strain which required leucine was especially good in this respect. This mutation is recessive and is the result of a deficiency for the enzyme α-isopropylamate dehydratase. In trial fermentations with this mutant, the resulting wines contained up to 20% less total fusel oil and 50% less isoamyl alcohol compared to the parent Montrachet strain. An experienced taste panel did not discern any gross degradation of taste quality in wine made with the mutant strain compared to that made with the parent strain. The mutant strain could be of commercial importance in preparation of distilling material for alcoholic beverages since the reduced fusel oil content would not require any special distillation procedures which are normally used to avoid the unpleasant flavour associated with concentrated higher alcohols. Reduction of the isoamyl alcohol content is particularly significant since this fusel oil component is usually present in the highest amount.     

Major constituents of fusel oils distilled from Australian grape wines

Fusel oils from Australian grape wines (Vine sp. Vitis vinifera) have been found to consist substantially of ethanol, n-propanol, isobutanol (2-methyl-1-propanol), isoamyl alcohol (3-methyl-1-butanol) and active amyl alcohol (2-methyl-1-butanol). In addition there are present large numbers of aliphatic esters, believed to be of considerable importance to grape brandy and spirit flavour and quality. The quantities and occurrence of these esters have been found to follow a systematic pattern. The major esterified alcohols are related to the free alcohols present and the major esterified acids occur as a series of acids of even carbon number with a maximum concentration at the n-decanoic acid. These relationships provide a basis for calculating the approximate ester composition of fusel oil.     

The Production of Isoamyl Acetate from Amyl Alcohol by Saccharomyces cerevisiae

Isoamyl acetate is a natural flavour ester, widely used as a source of banana flavour by the food industry. Fusel alcohols such as amyl alcohol are produced in significant quantities as a waste product, sometimes referred to as “lees oil” or “fusel oil”, of the alcohol distilling industry. By manipulation of brewing yeast fermentation conditions, a significant portion of added amyl alcohol was shown to be converted to isoamyl acetate. This was achieved by the addition of L‐leucine and amyl alcohol in fermentations carried out by a high ester‐producing brewing yeast strain of Saccharomyces cerevisiae and by the use of alkaline fermentation conditions coupled with high gravity media. Mutant strains selected on 5,5,5 trifluoro‐DL‐leucine produced substantially high levels of isoamyl acetate. The adjustment of fermentation conditions outlined in this paper may act as a stepping stone for the potential use of Saccharomyces cerevisiae and other yeasts to produce high levels of natural flavour esters.     

Formation of higher alcohols by wine yeasts,and relationship to taste thresholds

The amounts of n-propanol, iso-butanol and iso-amyl plus active amyl alcohol produced during fermentation of grape juice were found to vary considerably according to the yeast used. The yeasts studied included 11 wine yeasts and one brewery yeast, all belonging to the genus Saccharomyces, and 4 yeasts belonging to genera which sometimes cause wine spoilage. Experiments were carried out in the laboratory and confirmed on pilot-plant scale. The average production of n-propanol in juices from 4 grape varieties varied from 13 to 106 ppm depending on the wine yeast used. The corresponding variations in the production of iso-butanol and isoamyl plus active amyl alcohol were 9 to 37 ppm and 115 to 262 ppm respectively,. Juices from different varieties' of grapes (Vitis vinifera) differed somewhat in the amounts of higher alcohols formed, irrespective of the yeast strain used. An increase in temperature of fermentation from 15° to 25° with four yeasts produced an average of 24% more iso-amyl plus active alcohol and 39% more iso-butanol, and 17% less n-propanol. A yeast/temperature interaction occurred. An increase in pH from 3.0 to 4.2 with four yeasts produced 28% more iso-amyl plus active amyl alcohol, 85% more iso-butanol, and 11% more n-propanol. A yeast /pH interaction occurred. Spoilage yeasts examined also formed higher alcohols and the amounts were not related to sugar consumed during the fermentation. Taste thresholds of the higher alcohols, with the exception of active amyl alcohol, were measured in dry white wine with 7 tasters. Iso-amyl alcohol thresholds ranged from 100 to 900 ppm (mean 300 ppm) and iso-butanol and n-propanol both exceeded 500 ppm. The average threshold of iso-amyl alcohol in distilled water was 4 ppm. For certain tasters strains of yeast can produce sufficient differences in iso-amyl alcohol to be detectable organoleptically, but differences in iso-butanol and n-propanol are not large enough to be detectable.     

Effects of branched‐chain amino acid addition on chemical constituents in lychee wine fermented with Saccharomyces cerevisiae

This study investigated the impact of single branched‐chain amino acid addition (L‐valine, L‐leucine, L‐isoleucine) on nonvolatile and volatile constituents in lychee wine fermented with Saccharomyces cerevisiae MERIT.ferm. The individual addition of branched‐chain amino acids decreased the consumption of proline with significantly different rates. The lychee wine with added L‐leucine and L‐isoleucine had higher odour activity values (OAVs) of isoamyl alcohol, active amyl alcohol and isoamyl acetate. The lychee wine with added L‐valine had higher OAVs of isobutyric acid and isobutyl alcohol (5.55 and 2.98, respectively), which were lower in the wine with added L‐leucine (2.91 and 1.02, respectively) or L‐isoleucine (3.43 and 1.73, respectively) relative to the control wine (4.18 and 1.77, respectively), suggesting interactive effects among the amino acids. These findings demonstrated that lychee wine aroma can potentially be manipulated via the addition of selected branched‐chain amino acids.     

YEAST STRAIN AND KINETIC ASPECTS OF THE FORMATION OF FLAVOUR COMPONENTS IN CIDER

An apple juice was fermented at 8°C using twelve strains of Saccharomyces uvarum. Glucose, fructose, malic and L-lactic acids, isobutanol, 2,3 butanediol, isoamyl alcohol, ethyl acetate and titratable acidity were determined and monitored in the course of fermentation. It was observed that yeast strains differed from one another mainly in fermentation rates. When components were determined in ciders of the same remaining fructose concentration, rather than after the same fermentation time, the only significant effect of the yeast strain was on the amounts of glucose and ethanol in sweeter cider (fructose 34 g/litre), or on the amounts of glucose, acetic acid, isobutanol and amyl alcohols in dryer ciders (fructose 17 g/litre). A sensory analysis showed that standard deviations of concentrations of remaining or formed components in ciders fermented by different yeast strains, was sufficient to be detected by the taste panel. Added glucose in this range of concentration (1.5 g/litre) in cider reduced the perception of sourness while the addition of acetic acid (0.3 g/litre) reduced the perception of the scented flavour. Added Isobutnol (6 mg/litre) reduced the perception of sweetness. No significant effect of added isoamyl alcohol (30 mg/litre) could be detected. Chemical determinations were correlated by means of regression equations derived from earlier work with some organoleptic characteristics. No anticipated effect of the yeast strain could be detected on any studied flavour characteristic. It is concluded that in ciders of the same attenuation, the effect if it does exist, of the S.uvarum strain isolated from ciders, must be low.     

外源添加单一氨基酸对桑葚酒风味品质的影响

为探究外源添加氨基酸在改善桑葚酒风味品质中的影响,在桑葚果汁中分别添加400 mg/L的异亮氨酸（Ile）、亮氨酸（Leu）、苯丙氨酸（Phe）和缬氨酸（Val）制备桑葚酒,并分析桑葚酒理化指标、挥发性风味物质和感官品质。结果表明,与对照相比,外源添加单一氨基酸可显著增加桑葚酒的挥发性风味物质含量（42.38～84.58 mg/L）（P＜0.05）,添加Ile显著增加活性戊醇、乙酸活性戊酯含量,添加Leu显著增加异戊醇、乙酸异戊酯含量,添加Phe显著增加苯乙醇和乙酸苯乙酯的含量,添加Val显著增加异丁醇和乙酸异丁酯含量,添加四种氨基酸都能增加辛酸乙酯的含量（P＞0.05）;添加Phe可提高桑葚酒花香,添加Ile、Leu或Val提高果香,添加四种氨基酸的桑葚酒的口感评分均显著提高（P＜0.05）。     

FORMATION OF HIGHER ALCOHOLS FROM 14C-LABELLED VALINE AND LEUCINE

The formation of isobutanol and 3-methylbutanol during alcoholic fermentation by brewer's yeast has been studied by adding uniformly ¹⁴C-labelled valine and leucine to the complex amino acid mixture which is used as nitrogen source. In the presence of ¹⁴C-leucine, only 3-methylbutanol becomes radioactive whereas, in the presence of ¹⁴C-valine, both isobutanol and 3-methylbutanol acquire label. The specific radioactivity of these compounds is, in all cases, inferior to that of the parent amino acids showing that a part of these higher alcohols is always formed by the synthetic functions of the yeast. The results suggest that the regulating effect of the valine and leucine level of the medium on the synthesis of these amino acids in yeast cells is rather limited. They furthermore indicate that intact assimilation of amino acids is not the only mode of their utilization, even in media with excessive levels of amino acids. An attempt is made to explain the dependence of the anabolic and catabolic modes of higher alcohol formation upon the nitrogenous nutrient level of the medium. The presence of radioactive isoleucine as impurity in the ¹⁴C-leucine used has allowed an approximative calculation of the formation of 2-methylbutanol from isoleucine which is very similar to the formation of isobutanol from valine.     

Sake: Production and flavor

Abstract

Sake, its history in Japan, the unique brewing process, and microbes concerned with the characteristics of flavor are described. The main flavor components derived mainly from the fermentation process are higher alcohols, isoamyl acetate, ethyl caproate, and phenethyl acetate. These are the compounds which give an accent to the flavor of sake. The esters are formed mainly by yeast during mash fermentation. Isoamyl acetate is produced by the reaction of acetyl CoA with isoamyl alcohol catalyzed by alcohol acetyl transferase. The enzyme, bound to the yeast cell membrane, is unstable to heat and unsaturated fatty acids. The ester formation is regulated by the amount of isoamyl alcohol produced. Acyl CoA alcohol acyl transferease catalyzes the formation of ethyl caproate from caproyl CoA and ethanol. In this reaction, the amount of caproyl CoA supplied is important. The mechanism of higher alcohol formation, including the biosynthetic pathway of amino acids and its feedback regulation, is discussed. Finally, breeding and the practical use of sake yeast with high productivity of higher concentrations of higher alcohols and esters are described.     

YEAST STRAIN AND THE FORMATION OF FLAVOUR COMPONENTS IN CIDER

Two apple juices were fermented at 8°C and 18°C using thirteen strains of Saccharomyces uvarum. Glucose, fructose, malic, lactic, isobutanol, 2–3 butanediol, isoamyl alcohol, ethyl acetate and titratable acidity were determined in the two resulting experimental ciders. This data was correlated by means of multiple regression equations derived from earlier work with the following organoleptic characteristics: sweetness, sourness, fruity, scented, sharp and irritating flavours and a “global hedonic score” for overall acceptability. A significant effect of yeast strain could be detected on all component concentrations except ethanol, ethyl acetate, amyl alcohols and titratable acidity. The amounts of malic acid and titratable acidity found in cider depends mainly on the source of the juice. The anticipated effect of the source of the juice on sourness and on sharpness and fruity flavours is therefore significant. Yeast strain is also expected to have a significant effect on fruity and sharp flavours as well as on overall acceptability.     

Online-Based Kinetic Analysis of Higher Alcohol and Ester Synthesis During Winemaking Fermentations

The production of the major volatile carbon compounds corresponding to the predominant higher alcohols and esters synthesized during wine alcoholic fermentation was monitored using an online GC system. The accuracy and frequency of measurements made it possible to calculate kinetic parameters, in particular rates and specific rates of production. Using this original GC system and a model describing the evolution of the gas–liquid ratio during the fermentation process, kinetic gas–liquid balances of the synthesis of volatile compounds could be followed. This showed that, for esters—substantial proportions of which are lost in the off-gas (up to 40 % of the total production at 20 °C)—measurements of the liquid concentration, rather than of the total production (liquid content?+?losses), can lead to misinterpretations of yeast metabolism. The specific production rate of individual higher alcohols reached their maximum values before the exhaustion of the corresponding precursor amino acids. Isobutanol and isoamyl alcohol were formed from carbon metabolites and nitrogen metabolites and consequently were produced continuously throughout the fermentation process. In contrast, propanol synthesis was strongly correlated with the presence of assimilable nitrogen, during both the growth and stationary phases. Therefore, propanol appears to be a highly pertinent marker of nitrogen metabolism. Acetate ester concentrations correlated linearly with the concentrations of the corresponding higher alcohols, indicating that the availability of the precursors is the main limiting factor for the production of these esters. These results open possibilities for innovative approaches based on metabolic flux analysis and taking their dynamics into account.     

Quantitative determination of the odorants of young red wines from different grape varieties

Fifty‐two young monovarietal red wines made with Grenache (17 samples), Tempranillo (11 samples), Cabernet Sauvignon (12 samples) and Merlot (12 samples) grapes have been analysed by HRGC–MS to obtain quantitative data on 47 odorants previously identified as potential aroma contributors by olfactometric techniques. Thirty‐three odorants were present in the wines at concentrations higher than their corresponding odour thresholds. These include ethyl octanoate, β‐damascenone, ethyl hexanoate, isovaleric acid and isoamyl acetate as the most important, which together with isoamyl and β‐phenylethyl alcohols, fatty acids, 2,3‐butanedione and ethyl butyrate are always found at concentrations higher than their odour thresholds. In some cases the ethyl esters of isobutyric and isovaleric acids, β‐ionone, methionol, isobutyric acid, ethyl cinnamate, ethyl dihydrocinnamate, γ‐nonalactone, eugenol, c‐3‐hexanol, geraniol, guaiacol, 3‐isobutyl‐2‐methoxypyrazine, 4‐ethylguaiacol, acetoin and t‐whiskylactone were at a concentration high enough to be odour‐active. There were 30 compounds that were found to differ significantly between varieties. These include 3‐isobutyl‐2‐methoxypyrazine, isoamyl acetate, isovaleric acid, ethyl isobutyrate, ethyl isovalerate, fusel alcohols, c‐3‐hexenol, methionol, eugenol, guaiacol and γ‐nonalactone. © 2000 Society of Chemical Industry     

Ethyl acetate and fusel oil determination in distilled spirits by gas-liquid chromatography and confirmation by mass spectrometry

A gas-liquid chromatography (g.l.c.) method was developed for the determination of ethyl acetate, n-propyl, isobutyl, primary active amyl and isoamyl alcohol using Carbowax 400 as a liquid phase. These compounds were determined with a single injection at isothermal conditions with a minimum of bleed from the substrate. The procedure described, has certain points of advantage over the Official A.O.A.C. method because of improved column efficiency and resolution as shown in the separation of primary active amyl and isoamyl alcohols. Mass spectrometry was used to identify the individual compounds and to show its feasibility in such types of analysis.     

Enhanced production of isoamyl acetate from beet molasses with addition of fusel oil by Williopsis saturnus var. saturnus

Fusel oil which contains high level of amyl alcohols (approximately 45–55%) is a by-product obtained from the distillation of alcohol made by fermentation of molasses. Williopsis saturnus is a yeast which is able to convert isoamyl alcohol into isoamyl acetate. The aim of this study was to increase the formation of isoamyl acetate by the addition of fusel oil at the ratios of 1%, 2% and 3% (v/v) to molasses based fermentation medium using W. saturnus. It was found out that bioconversion of added fusel oil into isoamyl acetate was possible and an addition of 1% fusel oil led to an increase in isoamyl acetate concentration from 118 to 354 mg/L.     

SYNTHESIS OF HIGHER ALCOHOLS IN THE GENUS ZYMOMONAS

Several strains of bacteria of the genus Zymomonas were examined with respect to their potential for higher alcohol synthesis. All strains studied were able to produce higher alcohols during growth in a simple medium containing glucose and yeast extract. The higher alcohols produced were mainly n-propanol and iso-amyl alcohol. In contrast to Saccharomyces cerevisiae, only trace amounts of higher alcohols were produced from glucose by resting cells. When amino acids or other precursors were added to the fermentation medium, the resting cells formed higher alcohols. The stimulation of n-propanol synthesis by precursors was the most pronounced. The results obtained indicate that, with minor differences, the mechanisms of higher alcohol synthesis are comparable to these used by yeasts.     

贵州迎宾酒绵柔酱香型白酒酒体感官特征与特征风味的相关性分析

以绵柔酱香型白酒为研究对象,采用感官定量描述分析和色谱分析等技术剖析绵柔酱香型白酒具体的感官品质和风味特征,同时利用spearman指数研究二者的相关性。结果表明,绵柔酱香型白酒具有酱香突出,花果香和糊香较好,陈香较舒适,空杯留香较持久,酒体绵甜爽净,柔和协调,酱香风格典型的特征;风味构成特征为总醇类21%～32%,总酯类38%～45%,总酸类19%～22%,总醛类8%～12%,总酮类0.9%～2.0%,总芳香类0.22%～0.28%,具有相对较高的醇类和酮类占比以及较高的乳酸/乙酸值;对酱香型白酒绵柔品质正向影响较大的风味物质有正丙醇、仲丁醇、2,3-丁二醇、3-羟基-2-丁酮、丙酸。酱香、陈香、醇香、焦香、酸味、苦味、陈酒味、回味、柔和感和爽净感等感官特征易受风味物质影响,影响较大的风味物质有异戊醇、异戊酸乙酯、丁酸乙酯、乙缩醛、苯甲醛、活性戊醇和苯乙醇。     

Apple Aminoacid Profile and Yeast Strains in the Formation of Fusel Alcohols and Esters in Cider Production

The amino acid profile in dessert apple must and its effect on the synthesis of fusel alcohols and esters in cider were established by instrumental analysis. The amino acid profile was performed in nine apple musts. Two apple musts with high (>150 mg/L) and low (<75 mg/L) nitrogen content, and four enological yeast strains, were used in cider fermentation. The aspartic acid, asparagine and glutamic acid amino acids were the majority in all the apple juices, representing 57.10% to 81.95%. These three amino acids provided a high consumption (>90%) during fermentation in all the ciders. Principal component analysis (PCA) explained 81.42% of data variability and the separation of three groups for the analyzed samples was verified. The ciders manufactured with low nitrogen content showed sluggish fermentation and around 50% less content of volatile compounds (independent of the yeast strain used), which were mainly 3‐methyl‐1‐butanol (isoamyl alcohol) and esters. However, in the presence of amino acids (asparagine, aspartic acid, glutamic acid and alanine) there was a greater differentiation between the yeasts in the production of fusel alcohols and ethyl esters. High contents of these aminoacids in dessert apple musts are essential for the production of fusel alcohols and most of esters by aromatic yeasts during cider fermentation.