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.     

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.     

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.     

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.     

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）。     

Influence of carbon and nitrogen source on production of volatile fragrance and flavour metabolites by the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus produces a range of volatile molecules with applications as fragrances or flavours. The purpose of this study was to establish how nutritional conditions influence the production of these metabolites. Four strains were grown on synthetic media, using a variety of carbon and nitrogen sources and volatile metabolites analysed using gas chromatography–mass spectrometry (GC–MS). The nitrogen source had pronounced effects on metabolite production: levels of the fusel alcohols 2‐phenylethanol and isoamyl alcohol were highest when yeast extract was the nitrogen source, and ammonium had a strong repressing effect on production of 2‐phenylethyl acetate. In contrast, the nitrogen source did not affect production of isoamyl acetate or ethyl acetate, indicating that more than one alcohol acetyl transferase activity is present in K. marxianus. Production of all acetate esters was low when cells were growing on lactose (as opposed to glucose or fructose), with a lower intracellular pool of acetyl CoA being one explanation for this observation. Bioinformatic and phylogenetic analysis of the known yeast alcohol acetyl transferases ATF1 and ATF2 suggests that the ancestral protein Atf2p may not be involved in synthesis of volatile acetate esters in K. marxianus, and raises interesting questions as to what other genes encode this activity in non‐Saccharomyces yeasts. Identification of all the genes involved in ester synthesis will be important for development of the K. marxianus platform for flavour and fragrance production. Copyright © 2014 John Wiley & Sons, Ltd.     

牛栏山二锅头酵母菌多样性研究及其代谢产物分析

从牛栏山清香型白酒发酵酒醅中共获得120株酵母菌,对其26S rDNA D1/D2区测序分析,对分离酵母菌进行鉴定,并分别以 高粱和酒醅作为酵母培养基,分析不同种酵母菌的代谢产物。结果表明,共分离鉴定得到9株酵母菌,在高粱培养基中,库德里阿兹氏 毕赤酵母（Pichia kudriavzevii）和异常威克汉姆酵母（Wickerhamomyces anomalus）乙酸乙酯产量较高,分别为969.0 mg/L和541.4 mg/L, 9种蒸馏液中正丙醇、异丁醇、异戊醇含量均高于对照组,尼泊尔德巴利酵母（Debaryomyces nepalensis）发酵产乙醛能力最高（58 mg/L）; 在酒醅培养基中,9株酵母菌代谢产物中共检测到24种酯、9种醇、5种酸、2种酮、2种醛、2种酚及1种呋喃,其中W. anomalus产乙酸乙 酯能力最强4 494.27 μg/L,P. kudriavzevii具有较强的产乙酸乙酯、苯乙醇、异丁醇及异戊醇能力,分别为546.21 μg/L、724.96 μg/L、 31.56 μg/L及142.52 μg/L,膜醭毕赤酵母（Pichia membranifaciens）产乙酸能力最强（498.56 μg/L）。     

Inoculated fermentation of orange juice (Citrus sinensis L.) for production of a citric fruit spirit

The yeast Saccharomyces cerevisiae UFLA CA1174 was evaluated for its potential to produce an orange spirit, as a possible alternative to reduce waste and increase income to citrus farmers. The sugar concentration of the orange juice was adjusted to 16ºBrix and the pH to 4.5. The orange juice was inoculated at approximately 7 log CFU/mL, and the fermentation was performed at room temperature until ºBrix stabilization. The yeast used showed high values for the conversion factors of the substrates into ethanol (Y_p/s 0.50 g/g), the volumetric productivity of ethanol (Q_p 1.78 g/L/h), the biomass productivity (P_x 58.47 g/g) and the fermentation efficiency (E_f 97.83%). The sugars were converted quickly, and a high ethanol concentration (58.13 g/L) was achieved after 24 h of fermentation. The orange wine was distilled in a copper alembic, and the head, heart and tail fractions were collected. The orange spirit produced (heart fraction) had high concentrations of acetaldehyde, ethyl acetate, isoamyl alcohol and 2‐phenylethanol. The results showed that orange juice could be a good substrate for fermentation and distillation, and the sensory analysis performed revealed that the produced beverage had good acceptance by the tasters. Copyright © 2013 The Institute of Brewing & Distilling     

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.     

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.     

Influence of nitrogen on the biological aging of sherry wine

Nitrogen compounds are essential to the growth and metabolism of yeasts. The uptake and metabolism of nitrogen compounds by Saccharomyces cerevisiae depend not only on the strain and its physiological condition, but also on the chemical and physical properties of its environment. The effect of the addition of different amino acids (L ‐proline, L ‐threonine, L ‐arginine, L ‐glutamic acid, L ‐leucine and L ‐valine) to nitrogen‐depleted natural or nitrogen‐free synthetic wine on the cell growth, flor velum formation and sherry wine compound production was investigated under controlled biological aging by S. cerevisiae var. capensis strain G1 a typical flor yeast. The formation of flor velum was dependent on particular amino acid, oxygen availability and the composition of wine. Consumption of glycerol was related with the cell growth; in contrast, acetaldehyde tended to be released. Amino acid supplementation resulted in the release to wine of amino acids, esters and higher alcohols. The amino acid which was released in nearly all cases was L ‐leucine. Addition of L ‐glutamic acid resulted in the release mainly of ethyl acetate, in the case of L ‐leucine isoamyl alcohols were released, and for L ‐valine isobutanol. In the three cases, 1,1‐diethoxyethane was released in large quantities. The findings might indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential. Copyright © 2006 Society of Chemical Industry     

INFLUENCE OF THE CONCENTRATION OF BRANCHED CHAIN AMINO ACIDS ON THE FORMATION OF FUSEL ALCOHOLS

The influence of concentrations of branched-chain amino acids on the total, anabolic and catabolic production of isobutyl, active amyl, and isoamyl alcohol by Saccharomyces cerevisiae X 2180-1A has been examined in media with high nitrogen levels. It has been found that the total and catabolic production of higher alcohols increased with increasing concentrations of the corresponding amino acids. The anabolic pathway can be suppressed by high levels of the corresponding amino acid. Lower concentrations up to 1 mm for isoleucine and 10mm for leucine stimulate the anabolic formation of active amyl alcohol and isoamyl alcohol respectively. From the data obtained, it is shown that, under the conditions used, 50% or more of the fusel alcohols may arise in a beer wort fermentation from carbohydrates.     

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.     

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.     

Fermentative capacity of Kluyveromyces marxianus and Saccharomyces cerevisiae after oxidative stress

Volatile compound production during alcoholic fermentation has been studied in the production of many beverages. Temperature, yeast strain, nutrients and pH have been identified as important factors in the production of volatile compounds. In addition, other factors could influence this production during the fermentation process as well. Oxidative stress could occur during yeast biomass production because oxygen is an essential nutrient that is added to the growth medium. The fermentation parameters and the volatile compound production of one Saccharomyces cerevisiae strain (MC4) and two Kluyveromyces marxianus strains (OFF1 and SLP1) were evaluated in relation to fermentation parameters after oxidative stress induced by hydrogen peroxide or menadione. These yeasts were compared with S. cerevisiae W303–1A and showed significant differences in ethanol production, ethanol yield and maximum ethanol production rate. K. marxianus (OFF1) showed better fermentative capacity after oxidative stress. The higher alcohol production decreased after oxidative stress by >35% after 72 h fermentation time, and the amyl alcohol decreased at a higher level (>60%); however, the isobutanol production increased after oxidative stress between 1.5 and 4 times. The yeasts produced significant concentrations of esters however ethyl lactate, ethyl caprylate and the ethyl caproate were not detected in the control fermentation, while in the stress fermentation they accounted for up to 3 mg/L. These results demonstrate that oxidative stress can play an important role in the final aroma profile; but it is necessary to guarantee adequate yeast growth to obtain the volatile compounds desired. Copyright © 2017 The Institute of Brewing & Distilling     

Effects of overexpression of the alcohol acetyltransferase‐encoding gene ATF1 and disruption of the esterase‐encoding gene IAH1 on the flavour profiles of Chinese yellow rice wine

Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis. On the other hand, isoamyl acetate is degraded into a higher alcohol under the catalysis of IAH1‐encoded esterase. In this study, Chinese Saccharomyces cerevisiae was used as the parent strain to construct an ATF1 overexpression and IAH1 disruption mutant. The results show that after 5 days of pre‐fermentation, the concentrations of ethyl acetate, isoamyl acetate and isobutyl acetate in the yellow rice wines fermented with EY1 (pUC‐PIAK) increased to 468.94 mg L^?1 (which is approximately 22‐fold higher than that of the parent cell RY1), 99.86 and 7.69 mg L^?1 respectively. Meanwhile, isoamyl alcohol production was reduced to 56.37 mg L^?1 (which is approximately 50% of that produced by the parent strain RY1). Therefore, ATF1 overexpression and IAH1 disruption can significantly increase acetate esters contents and reduce isoamyl alcohol content in Chinese yellow rice wine, thereby paving the way for breeding an excellent yeast strain for high‐quality Chinese yellow rice wine production.     

Chemical characteristics of Śliwowica Łącka and other plum brandies

BACKGROUND: ?liwowica ??cka is a strong, distilled, home‐made plum brandy produced in a submontane region of Poland. The aim of the present study was to determine the chemical composition of this alcoholic beverage (samples from the years 2001–2004) and compare it with that of other plum brandies. Gas chromatography and spectrophotometry methods were used to detect major volatile components. RESULTS: Home‐made Polish plum brandies generally contained more ethanol (64.7–72.5% v/v), methanol (5.59–8.74 g L^?1100°) and butanol (32–335 mg L^?1100°) and less isobutanol (406–491 mg L^?1100°), pentanol (4.3–14.9 mg L^?1100°) and 2‐phenylethanol (61–68 mg L^?1100°) than other samples. The amyl alcohols/1‐propanol and isobutanol/1‐propanol ratios might be used as indices to distinguish spontaneously fermented plum brandies from those produced by monoculture. Statistically significant differences (P < 0.01) were found in the plum brandy sensory characteristics examined. Total sensory scores of Polish plum brandies ranged between 12.0 and 14.3, while Slovakian Slivovica received the highest score (16.7). CONCLUSION: The results showed that plum brandies produced in the ??cko area are characterised by a similar and original chemical composition that results mainly from spontaneous fermentation as well as traditional production technology. Copyright © 2007 Society of Chemical Industry     

Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation

The biochemical formation of yeast-derived sensory-active metabolites like higher alcohols and esters determines the different characteristics of aroma and taste in fermented beverages. In yeast fermentation process, a large number of environmental factors affecting the production of volatile aroma compounds are abundant. Factors like substrate composition in fermentation media as well as process parameters influencing these flavor-active metabolites have already been described. These factors can act on the expression of yeast genes involved in aroma metabolism resulting in concentration differences in esters and higher alcohols important for flavor and taste. The understanding of the function of genes involved in biosynthetic pathways of aroma-active substances as well as their regulatory mechanisms is needed to control the production of ester and higher alcohol synthesis to create specific aroma profiles in fermented beverages. This review discusses the known regulation and function of several individual genes (ATF1, ATF2, EEB1, EHT1, BAT1, BAT2 and BAP2) described in fusel alcohol and ester synthesis mainly in S. cerevisiae and S. pastorianus var. carlsbergensis. Also, different factors like oxygen and temperature that allow ester and higher alcohol synthesis to be controlled during yeast fermentation are described.     

Chemical composition and sensory analysis of cheese whey‐based beverages using kefir grains as starter culture

The aim of the present work was to evaluate the use of the kefir grains as a starter culture for tradicional milk kefir beverage and for cheese whey‐based beverages production. Fermentation was performed by inoculating kefir grains in milk (ML), cheese whey (CW) and deproteinised cheese whey (DCW). Erlenmeyers containing kefir grains and different substrates were statically incubated for 72 h at 25 °C. Lactose, ethanol, lactic acid, acetic acid, acetaldehyde, ethyl acetate, isoamyl alcohol, isobutanol, 1‐propanol, isopentyl alcohol and 1‐hexanol were identified and quantified by high‐performance liquid chromatography and GC‐FID. The results showed that kefir grains were able to utilise lactose in 60 h from ML and 72 h from CW and DCW and produce similar amounts of ethanol (～12 g L^?1), lactic acid (～6 g L^?1) and acetic acid (～1.5 g L^?1) to those obtained during milk fermentation. Based on the chemical characteristics and acceptance in the sensory analysis, the kefir grains showed potential to be used for developing cheese whey‐based beverages.