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.     

Characterisation of volatile compounds in an alcoholic beverage produced by whey fermentation

An alcoholic beverage (35.4% v/v ethanol) was produced by distillation of the fermented broth obtained by continuous whey fermentation with a lactose-fermenting yeast Kluyveromyces marxianus. Forty volatile compounds were identified in this drink by gas chromatography. Higher alcohols were the most abundant group of volatile compounds present, with isoamyl, isobutyl, 1-propanol, and isopentyl alcohols being found in highest quantities (887, 542, 266, and 176 mg/l, respectively). Ethyl acetate had the highest concentration (138 mg/l) among the esters. Besides higher alcohols and esters, other components, including aldehydes, acids and terpenes were also identified in the whey spirit. Considering that the quality of an alcoholic beverage can be evaluated by the relation between isoamyl alcohol/2-methyl-1-propanol and 2-methyl-1-propanol/1-propanol, which have to be higher than unity, it was concluded that a novel spirit of acceptable organoleptic characteristics can be produced by cheese whey continuous fermentation with K. marxianus.     

Manipulation of volatile compound transformation in durian wine by nitrogen supplementation

This study evaluated the impact of added diammonium phosphate (DAP) and a L‐leucine‐L‐phenylalanine mixture (Leu+Phe) on durian wine fermentation by Saccharomyces cerevisiae var. bayanus EC‐1118. Changes in yeast cell population, ^oBrix, sugars, organic acids and pH were similar in all fermentations, regardless of nitrogen supplementation at a concentration of 150 mg N L^?1. The supplementation of Leu+Phe accelerated the utilisation and reduced the formation of volatile sulphur compounds such as diethyl disulphide, ethyl thioacetate and 2‐(ethylthio)ethanol, which were 64.3%, 26.0% and 48.4% lower than the control, respectively. Nevertheless, the supplementation of Leu+Phe heightened the production of isoamyl alcohol, 2‐phenylethyl alcohol and their corresponding esters, especially isoamyl acetate was 1.6 times and 2‐phenylethyl acetate was 26.5 times higher than the control. The supplementation with DAP exerted negligible effects on the volatile composition. The results of this study suggest that the addition of specific amino acids may be a novel approach to manipulating durian wine flavour by suppressing or accentuating the formation of certain aroma compounds.     

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.     

The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates

The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1- and ATF2-encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1-encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1-encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester-synthesizing and ester-degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2-phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester-producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles.     

Gene expression in wheat beer yeast strains and the synthesis of acetate esters

The synthesis of aroma compounds represents one of the most important parameters in beer production. Although it has been a historical topic of research, exactly how aroma components are formed has yet to be fully explained. Moreover, all of the research that has been published on yeast strains is focused on lagers and ales. Wheat beer yeast strains have not been the focus of aroma and flavour research. In this study, five different wheat beer yeasts were analysed to determine their capacity for producing acetate esters. In this study, the most commonly used wheat beer yeast strains for the production of German‐style wheat beer were analysed. This involved measuring the level of expression of the alcohol acetyl transferase genes ATF1, ATF2 and IAH1 over a period of 4 days (during primary fermentation) and plotting the data to observe the development of expression of the genes over time. Results confirmed their capacity to form acetate esters and showed a distinct correlation with increasing expression of the gene ATF1. However, the findings also indicated that gene expression in different yeast strains can vary considerably during fermentation. Copyright © 2016 The Institute of Brewing & Distilling     

The Saccharomyces cerevisiae alcohol acetyl transferase Atf1p is localized in lipid particles

The yeast alcohol acetyl transferase I, Atf1p, is responsible for the major part of volatile acetate ester production in fermenting Saccharomyces cerevisiae cells. Some of these esters, such as ethyl acetate and isoamyl acetate, are important for the fruity flavours of wine, beer and other fermented beverages. In order to reveal the subcellular localization of Atf1p and further unravel the possible physiological role of this protein, ATF1::GFP fusion constructs were overexpressed in brewer's yeast. The transformant strain showed a significant increase in acetate ester formation, similar to that of an ATF1 overexpression strain, indicating that the Atf1p-GFP fusion protein was active. UV fluorescence microscopy revealed that the fusion protein was localized in small, sphere-like organelles. These organelles could be selectively stained by the fluorescent dye Nile red, indicating that they contained high amounts of neutral lipids and/or sterols, a specific characteristic of yeast lipid particles. Purification of lipid particles from wild type and ATF1 deletion cells confirmed that the Atf1p-GFP fusion protein was located in these organelles. Furthermore, a clear alcohol acetyl transferase activity could be measured in the purified lipid particles of both wild type and transformed cells. The localization of Atf1p in lipid particles may indicate that Atf1p has a specific role in the lipid and/or sterol metabolism that takes place in these particles.     

Genetic and enological analysis of selected Saccharomyces cerevisiae strains for wine production

The non‐wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2‐phenethyl acetate and 3‐methyl‐1‐butanol acetate. This study also shows that there is a difference in the ester‐formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.     

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Screening of drug‐resistant mutants of sake yeast strains has been a major method for creation of superior strains. We attempted to create a valproic acid (VPA)‐resistant mutant strain from sake yeast Kyokai No. 7 (K7). VPA is a branched‐chain fatty acid and is an inositol synthesis inhibitor in mammals and yeast. We succeeded in isolating a mutant of strain K7 that can survive long‐term in a VPA‐containing medium. This strain, K7‐VPA^LS, is significantly more resistant to not only VPA‐induced cell death but also ethanol in comparison with the parent strain. Further experiments showed that the new strain is likely to have a deficiency in inositol and/or phosphatidylinositol synthesis. The major characteristics of sake brewed by strain K7‐VPA^LS (compared with K7) were lower amino acidity, higher isoamyl acetate content without an increase in the isoamyl alcohol level and changes in constituent organic acids, particularly higher malate and succinate but lower acetate concentrations. In addition, taste sensor analysis revealed that K7‐VPA^LS‐brewed sake has milder sourness and higher saltiness or richness than K7‐brewed sake does. High isoamyl acetate production may be related to a deficiency in phosphatidylinositol because this compound directly inhibits alcohol acetyltransferase, an enzyme responsible for isoamyl acetate synthesis. Strain K7‐VPA^LS grew more rapidly than the parental strain did in a medium containing acetate as a sole carbon source, indicating that K7‐VPA^LS effectively assimilates acetate and converts it to malate and succinate through the glyoxylate cycle. Thus, strain K7‐VPA^LS shows improved characteristics for brewing of high‐quality sake. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

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.     

ESTER FORMATION IN BREWERY FERMENTATIONS

Esters are secondary products produced by brewing yeasts during the anaerobic metabolism of sugars and constitute one of the largest and most important groups of compounds affecting beer flavour. Many esters can be formed, the most important being ethyl acetate, isoamyl acetate, isobutyl acetate, 2-phenylethyl acetate and ethyl hexanoate. The odour threshold levels for ester detection are very low. Esters are especially important in high gravity brewing, where over production occurs causing unwanted solvent-like flavours. They are also important in low alcohol beer production due to the low levels produced, which can result in beers with little flavour. The factors influencing ester production are reviewed, together with the ways in which they can be used to control ester synthesis. It is believed that acetate esters are synthesized by an enzyme called alcohol acetyl transferase (AAT) which uses as substrates an alcohol and acetyl co-enzyme A; the latter plays a central role in many intracellular reactions. However, esters can also be synthesized by esterase enzymes working in reverse. Several attempts have been made to locate the AAT enzyme and recent work suggests that it is located in either the yeast cell plasma membrane or in intracellular organelles called “vacuomes”.     

HS-SPME-GC-MS分析高产酯低产高级醇酿酒酵母发酵酒的风味物质

采用顶空固相微萃取-气相色谱质谱联用(HS-SPME-GC-MS),分析添加不同高产酯低产高级醇酿酒酵母发酵得到酒样中的挥发性成分,并采用气相色谱对其中的主要风味物质进行定量分析。气相色谱质谱联用在酿酒酵母AY15、AY15-BAT2、AY15-BAT2+ATF1、AY15-IAH1+ATF1的酒样中分别分离鉴定出62、56、63、59种挥发性成分,主要包括酯类、醇类、醛类、酸类、烷烃、芳香烃和酚类等。三株具有不同高级醇和酯生成能力的酿酒酵母发酵得到的酒样中酯类与高级醇的比例相比野生菌株AY15均有不同程度的提高,其中,AY15-BAT2+ATF1酒样中新检出乙酸正丁酯、乙酸庚酯、乙酸辛酯、乙酸苯乙酯、乙酸-甲氧基-2-苯乙酯五种乙酸酯。定量结果表明,AY15-BAT2+ATF1与AY15-IAH1+ATF1显著提高了乙酸乙酯和乙酸异戊酯的含量,AY15-BAT2则不影响主要酯类物质的生成;同时,这三株酿酒酵母不同程度的降低了酒中正丙醇、异丁醇和异戊醇的含量。     

Increased esters and decreased higher alcohols production by engineered brewer’s yeast strains

Esters and higher alcohols produced by yeast during the fermentation of wort have the greatest impact on the smell and taste of beer. Alcohol acetyltransferase, which is mainly encoded by the ATF1 gene, is one of the most important enzymes for acetate ester synthesis. Cytosolic branched-chain amino acid aminotransferase, on the other hand, which is encoded by the BAT2 gene, plays an important role in the production of branched-chain alcohols. The objective of this study is to construct engineered brewer’s yeast strains that produce more acetate esters and less higher alcohols. Industrial brewer’s yeast strain S5 was used as the parental strain to construct ATF1 overexpression and BAT2 deletion mutants. The engineered strains S5-2 and S5-4, which feature partial BAT2 allelic genes replaced by the constructed ATF1 overexpression cassette, were obtained. The ester production of the engineered strains was observed to increase significantly compared with that of the parental cells. The concentrations of ethyl acetate produced by the engineered strains S5-2 and S5-4 increased to 78.88 and 117.40 mg L^?1, respectively, or about 7.7-fold and 11.5-fold higher than that produced by parental S5 cells. The isoamyl acetate produced by S5-2 and S5-4 also increased to 5.14 and 9.25 mg L^?1, respectively; by contrast, no isoamyl acetate was detected in the fermentation sample of the parental strain S5. Moreover, S5-2 and S5-4, respectively, produced about 65 and 51 % of higher alcohols produced by the parental strain. The increase in acetate ester content and decrease in higher alcohol concentration shown by the engineered brewer’s yeast strains at the end of fermentation process indicate that the new strains are useful in future developments in the wheat beer industry.     

Cell factory applications of the yeast Kluyveromyces marxianus for the biotechnological production of natural flavour and fragrance molecules

Kluyveromyces marxianus is emerging as a new platform organism for the production of flavour and fragrance (F&F) compounds. This food‐grade yeast has advantageous traits, such as thermotolerance and rapid growth, that make it attractive for cell factory applications. The major impediment to its development has been limited fundamental knowledge of its genetics and physiology, but this is rapidly changing. K. marxianus produces a wide array of volatile molecules and contributes to the flavour of a range of different fermented beverages. Advantage is now being taken of this to develop strains for the production of metabolites such as 2‐phenylethanol and ethyl acetate. Strains that were selected from initial screens were used to optimize processes for production of these F&F molecules. Most developments have focused on optimizing growth conditions and the fermentation process, including product removal, with future advancement likely to involve development of new strains through the application of evolutionary or rational engineering strategies. This is being facilitated by new genomic and molecular tools. Furthermore, synthetic biology offers a route to introduce new biosynthetic pathways into this yeast for F&F production. Consumer demand for biologically‐synthesized molecules for use in foods and other products creates an opportunity to exploit the unique potential of K. marxianus for this cell factory application. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of nutrient supplementation on fermentation kinetics, H2S evolution, and aroma profile in Verdicchio DOC wine production

Different yeast nutrient additions were studied for the 2008 and 2009 vintages of Verdicchio grape juice fermentation. Addition of yeast derivatives at the beginning of fermentation and/or different amounts of diammonium phosphate at various times within the first half of fermentation were examined, with initial yeast assimilable nitrogen concentrations set at 200 and 250 mg l^?1. Supplementation with glutathione in combination with this nitrogen addition was also evaluated. Fermentation rates were monitored throughout these fermentations carried out under different nutrient conditions. H₂S production during fermentation and synthesis of volatile compounds in the finished wines were quantified; the wines also underwent sensory evaluation. The fermentation kinetics were almost exclusively influenced by the inorganic nitrogen supplementation with diammonium phosphate. H₂S evolution was more affected by assimilable nitrogen than glutathione. Diammonium phosphate significantly reduced H₂S production, with a further reduction in the presence of yeast derivative. This nitrogen supplementation yielded higher concentrations of acetate esters, and in particular of isoamyl acetate (fruity aromas), which positively influences the analytical and aroma profile of wines and results in a general reduction in 2-phenylethanol production (floral aromas). Overall results (two harvesting times and vintages) indicate that the management with diammonium phosphate and yeast derivative supplementation improves the kinetics of fermentation and provides a good tool to reduce H₂S formation and increase the analytical and sensory quality of Verdicchio wine.     

Volatile Bio-ester Production from Orange Pulp-Containing Medium Using Saccharomyces cerevisiae

The dynamics of orange pulp (OP) as a fermentation feedstock for the production of volatile esters of “fruity” aroma by using a commercial wine yeast strain (Saccharomyces cerevisiae var. cerevisiae) was investigated. To achieve this goal, the kinetic behaviour of yeast (cell growth, substrate assimilation and volatile ester formation) was studied in OP complemented with nutritive medium under two different aeration conditions. The results were compared with those obtained from conventional processes in nutritive medium containing glucose as a sole carbon source. The results obtained revealed that the yeast cells were able to grow in the OP-containing medium after a 12-h lag period, probably due to the inhibitory effect of limonene. OP was found to stimulate the de novo synthesis of isoamyl acetate, phenylethyl acetate and ethyl esters (hexanoate, octanoate, decanoate and dodecanoate) by S. cerevisiae. This was strongly evident in the case of limited oxygen supply. Based on the above findings, OP appears to be a promising choice for bioflavour production by yeast.     

SPME-GC-MS分析不同商业葡萄酒酵母香气物质合成的差异性

采用顶空固相微萃取(HS-SPME)和气质联用法(GC-MS)分析了4株常用商业葡萄酒酵母的挥发性香气成分。对酵母代谢的19种香气成分进行定性定量分析,菌株CY3079具有较高的高级醇生成能力,其中异戊醇和2-苯乙醇的合成均高于其它酵母菌株。D254酵母具有较高的酯类和挥发酸类合成能力,其中乙酸乙酯和乙酸对酵母D254总酯和总挥发酸的含量贡献较大。EC1118高级醇生成量最低,但是具有较高的乙酸-2-苯乙酯生成能力。RC212酒样具有较低的酯类和挥发酸。感官品评试验表明,D254酒样的果香比较明显;CY3079酒样的刺激性大于其它菌株;EC1118酒样花香比较突出。     

Evaluation of the potential of commercial non-Saccharomyces yeast strains of Torulaspora delbrueckii and Lachancea thermotolerans in beer fermentation

This work evaluated the potential of three commercial non-Saccharomyces yeast strains Torulaspora delbrueckii (Biodiva and Prelude) and Lachancea thermotolerans (Concerto) for beer fermentation. The fermentation performance, volatile and non-volatile profiles were compared. Growth behaviours of all three yeast strains exhibited similar trends during the initial fermentation phase although a marked population decline was detected in strains Prelude and Concerto, which also showed a rapid utilisation of maltose, while strain Biodiva was unable to consume maltose and consumed lesser amounts of amino acids. Additionally, terpenoids inherently absent in the wort such as β-caryophyllene and geranyl acetone were produced in all beers, significantly higher in beers fermented with strain Prelude. For volatile profiles, Prelude and Concerto produced more ethanol and significantly higher amounts of acetate esters and long-chained ethyl esters. Strain Biodiva, on the other hand, produced higher amounts of isoamyl alcohol and ethyl butanoate.