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.     

Microbial modulation of aromatic esters in wine: Current knowledge and future prospects

This review focuses on the considerable amount of research directed at defining the accumulation of esters during fermentation and their contribution to aromas in foods and beverages. From this research it is clear that esters are extremely important for the aroma profile of fermented beverages and various dairy products. A large amount of this research is focused on wine and has yielded the genes involved in ester synthesis and hydrolysis in organisms such as Saccharomyces sp. It is also clear from recent research in both the fermented beverage and dairy context that lactic acid bacteria possess an extensive collection of ester synthesising and hydrolysing activities. This review describes the major esters reported in wine and the enzymes responsible for their hydrolysis and synthesis. Ester impact on wine aroma and formation during primary and malolactic fermentation is also evaluated. Finally the potential applications of current knowledge are outlined.     

不同商品酵母对葡萄酒香气成分的影响

以5种不同商品酵母发酵的赤霞珠干红葡萄酒为原料,采用顶空固相微萃取（HS-SPME）和气质（GC-MS）联用技术对其香气成分进行分析,共检测出66种挥发性化合物,定量分析了各葡萄酒中共有的15种主要香气物质之间的差异。结果表明,不同商品酵母发酵葡萄酒中香气种类差别很小,但含量差别明显。酵母BDX具有较强的高级醇生成能力,其中苯乙醇的含量明显高于其他酵母;酵母BM4×4生成的酯类、高级醇和有机酸的含量均处于中间水平;酵母D254具有较强的酯类生成能力,其中辛酸乙酯和己酸乙酯的贡献最为突出;酵母L2323的乙酸异戊酯生成能力较强;酵母RC212的酯类和高级醇生成量最低。     

Application of Plackett–Burman experimental design for investigating the effect of wort amino acids on flavour‐active compounds production during lager yeast fermentation

Aroma‐active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer's wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus , we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

河源南高丛蓝莓酒精发酵前后挥发性成分变化

目的明确广东河源地区南高丛蓝莓特征风味成分以及蓝莓发酵后香挥发性成分变化规律。方法采用顶空固相微萃取-气质联用法分析了河源南高丛蓝莓的风味成分,以及发酵成蓝莓酒后的风味成分变化。结果河源南高丛蓝莓主要风味成分为柠檬烯以及其他烯烃、醛类等物质,发酵成蓝莓酒后主要特征风味成分为乙酸乙酯、辛酸乙酯等酯类和醇类物质。结论河源南高丛蓝莓在挥发性成分上与以往报道的温带地区蓝莓有一定的区别,蓝莓发酵成蓝莓酒后烯烃、醛类物质显著减少或消失,而酯类和醇类显著增加,同时生成了长链烷烃和脂肪酸酯类物质。     

Free amino acids identification and process optimization in greengage wine fermentation and flavor formation

Greengage wine with low alcohol content is increasing in popularity owing to its fruity taste and rich nutrition. The key to wine aroma and taste is flavor substances like free amino acids (FAAs), volatile fatty acids, higher alcohols, and esters. Amino acid (AA) metabolisms in yeast are an important source of these secondary compounds, which vary with the fermentation conditions. This study explored and optimized the impact of different parameters (carbon source, inoculum, pH, temperature) on FAA contents and dynamics in greengage wine. The results demonstrated that total and essential amino acid (EAA) content rose with a higher proportion of glucose, less yeast inoculation, higher temperature, and higher initial pH. With the results obtained it was concluded that the condition of 22.4°C, pH 4.5, and 3% inoculation was optimum for a 14.9-fold increase of EAAs in fermented greengage wine. In the long run, the research will aid in the development of the greengage brewing industry.     

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 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.     

Effects of spontaneous and inoculated fermentation on the volatile profile of lychee (Litchi chinensis Sonn) fermented beverages

The aim of this study was to evaluate the contribution of yeast to the volatile profile of beverages obtained by the fermentation of lychee must through inoculated (Saccharomyces cerevisiae strains UFLA CA116 , UFLA CA1183 and UFLA CA1174 ) and spontaneous fermentation. Higher alcohols and esters were the primary volatiles detected by SPME/GC–MS. A Principal Component Analysis indicated similarities between UFLA CA116 and UFLA CA1183 and between UFLA CA1174 and spontaneous fermentation. Changing the yeast strain used in the fermentation process has the potential to modulate the volatile profile of fermented beverages. The beverage produced by the inoculation of yeast CA1183 showed the most complex aroma profile compared to the other beverages.     

黄酒固定化发酵过程及香气物质形成的研究

将黄酒酵母用海藻酸钙包埋制成固定化酵母,研究游离酵母与固定化酵母的黄酒发酵过程理化指标和香气物质生成量的差异,结果表明:经过主发酵5 d(28℃),后发酵15 d(15℃),固定化酵母发酵过程的酒精度、总糖、总酸变化曲线与游离发酵过程曲线差异不大,固定化酵母发酵性能稳定,杂菌污染可控。用气相色谱(GC)分析乙酸乙酯等9种黄酒特征性香气物质,发现游离发酵黄酒正丙醇、异丁醇、异戊醇、β-苯乙醇的总高级醇含量为680.91 mg/L,固定化发酵第1,2,3,4,5批黄酒的总高级醇含量分别为633.63,649.79,658.17,637.63,614.68 mg/L,说明固定化发酵有利于控制黄酒中的高级醇含量。固定化发酵酯类总量高于游离酵母,且随着固定化酵母发酵批次的增加而递增,即:固定化第5批(115.90 mg/L)>第4批(90.81 mg/L)>第1~3批(80.10~81.81 mg/L)>游离酵母(55.66 mg/L),表明固定化发酵使黄酒的酯类香气愈加浓郁。根据香气阈值计算的固定化发酵酿得的黄酒的香气不比游离酵母酿得的黄酒差,固定化酵母发酵生产黄酒有商业化应用的前景。     

ATF1过表达和BAT2敲除酿酒酵母发酵性能的研究

以啤酒酵母工业菌株S5为对照,对过表达醇乙酰基转移酶编码基因ATF1同时敲除氨基酸转氨酶编码基因BAT2酿酒酵母工程菌株S5-1进行发酵性能的研究。结果表明,与出发菌株相比,突变株生长状况、发酵速度、酒精度等基本发酵性能没有明显变化,而突变株发酵后的乙酸酯总量有较大程度的提升,为85.44mg/L,提高了6.96倍,高级醇总量有较大程度的下降,为79.25mg/L,降低了27.28%。研究结果为啤酒风味的改善奠定了良好的基础。     

杨梅酒酿造工艺优化及香气成分研究

以杨梅为原料,酵母菌RV171为发酵菌种制备杨梅酒。以酒精度为评价指标,以酵母菌添加量、发酵温度、发酵时间为考察因素,通过单因素试验和响应面试验优化杨梅酒发酵工艺,并通过顶空固相微萃取（HS-SPME）结合气相色谱-质谱（GC-MS）联用分析杨梅汁和杨梅酒的挥发性香气成分。结果表明,杨梅酒最优发酵工艺条件为酵母添加量0.3%、发酵温度26℃、发酵时间9 d。在此条件下,制得的杨梅酒酒精度为12.63%vol。香气成分测定结果表明,杨梅酒中共检出挥发性香气成分35种,醛类、醇类、酯类是杨梅酒最主要的挥发性成分,且杨梅酒中醛类、醇类和萜烯类成分相对含量分别显著提高24.37%、14.86%、70.82%(P<0.05),酯类显著降低51.04%(P<0.05)。由此可知,发酵所得杨梅酒香气丰富,可以解决杨梅资源浪费问题,具有重要的生产指导意义。     

GC-MS对非酿酒酵母菌发酵赤霞珠葡萄酒香气成分的检测

以新疆石河子地区葡萄自然发酵过程中分离鉴定的7株非酿酒酵母菌为主要发酵菌种,以工业菌株为对照,研究了混合发酵赤霞珠葡萄酒香气成分的气相色谱-质谱联用仪分析结果。结果表明,共检测到香气成分16种,其中酯类物质7种,醇类6种,其他香气物质3种。以Y10（Hanseniaspora sp.）孢汉逊酵母属发酵的7#赤霞珠葡萄酒的主要优势特征香气为：苯乙醇、1,3-丁二醇、2-甲基-丙醇、1-己醇、辛酸乙酯、丁酸乙酯、己酸乙酯、琥珀酸二乙酯、乙酸己酯,且相对含量高于其他菌株和对照工业菌株。     

Production of sensory compounds by means of the yeast Dekkera bruxellensis in different nitrogen sources with the prospect of producing cachaça

The distilled spirit made from sugar cane juice, also known as cachaça, is a traditional Brazilian beverage that in recent years has increased its market share among international distilled beverages. Several volatile compounds produced by yeast cells during the fermentation process are responsible for the unique taste and aroma of this drink. The yeast Dekkera bruxellensis has acquired increasing importance in the fermented beverage production, as the different metabolites produced by this yeast may be either beneficial or harmful to the end‐product. Since D. bruxellensis is often found in the fermentation processes carried out in ethanol fuel distillation in Brazil, we employed this yeast to analyse the physiological profile and production of aromatic compounds and to examine whether it is feasible to regard it as a cachaça‐producing microorganism. The assays were performed on a small scale and simulated the conditions for the production of handmade cachaça. The results showed that the presence of aromatic and branched‐chain amino acids in the medium has a strong influence on the metabolism and production of flavours by D. bruxellensis. The assimilation of these alternative nitrogen sources led to different fermentation yields and the production of flavouring compounds. The influence of the nitrogen source on the metabolism of fusel alcohols and esters in D. bruxellensis highlights the need for further studies of the nitrogen requirements to obtain the desired level of sensory compounds in the fermentation. Our results suggest that D. bruxellensis has the potential to play a role in the production of cachaça. Copyright © 2014 John Wiley & Sons, Ltd.     

产香酵母发酵面团挥发性风味物质分析

采用分离自山东酵子具有优良发酵和产香特性的酿酒酵母（Saccharomyces cerevisiae）CGMCC No.24005,制成干粉发酵剂,并分析其生长发酵特性及发酵面团的香气成分。结果表明,该酵母的最适生长温度、pH分别为28℃、6;发酵16 h时面团体积最大。利用此酵母发酵面团整体香气较强,酯类与醇类的种类及含量显著高于未发酵组（P<0.05）,果香、花香、酒香、甜香等特征明显,其中乙酸异戊酯、异戊醇、辛酸乙酯、苯乙醇等是不同发酵时间产物呈现不同香气特征的关键物质。说明该发酵剂能显著增加面团挥发性物质种类及含量,可提升发酵面食的品质,为传统发酵面食的标准化生产提供理论参考。     

Evaluation of different yeast strains on the quality of beer produced from malted proso millet (Panicum miliaceum L.)

In order to investigate the influence of temperature on pH, alcohol production, carbohydrate profile and aroma compounds, a variety of top-fermenting yeast strains were used to ferment wort obtained from proso millet malt. Champagne (Saccharomyces bayanus), altbier, kölsch and ale (Saccharomyces cerevisiae), as well as Brettanomyces bruxellensis were used in two isothermal (12 and 18 °C) fermentation trials. During the fermentation, changes in pH, extract conversion and the monosaccharide profile were monitored. After 2 weeks of storage, the levels of higher alcohols and esters were also measured in the finished beers. In many cases, a strong resemblance in the behaviour of the selected strains to Saccharomyces was observed during the fermentation. All other influencing fermenting parameters like pitching rate, yeast generation, fermenting vessels geometry, amount of assimilable nitrogen, yeast nutrients, oxygen and CO₂ content and the sugar content were the same for all trials regardless of the yeast applied. However, at higher temperatures Brettanomyces showed more rapid rates of sugar conversion. Concerning the development of aroma components, no uniform picture was evident in these trials, neither with respect to temperature dependence nor to what extent the incurred values are comparable to those already known for beers brewed using barley malt wort. The values for aliphatic alcohols obtained during experimentation, ranged from 23 to 267 μg/L, and the sum of aromatic alcohols, from 2,437 to 11,844 μg/L. Values for ester content were recorded between 188 and 2,543 μg/L. Overall, wort produced using proso millet malt was found to be a substrate that lends itself to alcoholic fermentation, exhibiting a sufficient decrease in pH over the course of fermentation, as well as reasonable alcohol yield and a broad range of aroma components.     

Effects of mediums on fermentation behaviour and aroma composition in pure and mixed culture of Saccharomyces cerevisiae with Torulaspora delbrueckii

The nutrient status and composition in mediums have a significant effect on yeast metabolism and phenotypic characteristics in wine fermentation. In this study, the effects of three frequently used mediums, including synthetic grape juice (SGJ), grape juice without grape pericarp and seeds (GJ) and grape must with grape pericarp and seeds (GMPS), on yeast fermentation behaviour and aroma compounds produced by pure and mixed culture of Saccharomyces cerevisiae T73 with Torulaspora delbrueckii TD20 were investigated after alcoholic fermentation. The results showed that high fermentation activities and cell population were always found in GJ medium irrespective of inoculated approach. More esters and higher alcohols were produced in GMPS medium fermented by pure S. cerevisiae, while SGJ medium had increased levels of fatty acids. Consistent with previous literatures, the mixed fermentation of T. delbrueckii and S. cerevisiae produced more acetate esters and fatty acids than the pure culture of S. cerevisiae, while this enological trait was only found in SGJ and GJ, not in GMPS. Our results highlighted that more attention should be paid to the fermentation medium when evaluating the enological and aromatic properties of selected yeasts used in industrial winemaking. In this regard, the combined use of GJ and GMPS medium might be a suitable choice.     

A novel approach for the production of a non‐alcohol beer (≤0.5% abv) by a combination of limited fermentation and vacuum distillation

Despite the increasing demand, the production of non‐alcohol beers is still limited by unsatisfactory or artificial flavour and taste. In this study, a novel approach to producing non‐alcohol beer is presented, in which the alcohol‐reducing techniques, limited fermentation and vacuum distillation were combined. Starting from barley and wheat malts, wort with a low level of fermentable sugars was prepared by infusion mashing and lautering. Limited fermentation was carried out by Saccharomycodes ludwigii at 18°C. When the level of fermentable sugar was reduced by 25%, the fermented wort was quickly cooled from 18 to 0°C and held at that temperature for two days. The young beer was obtained after degassing and removal of yeast and was then subjected to vacuum distillation at 0.06 MPa to remove the alcohol. The concentrated extract is suitable for storage and transportation. The final product of non‐alcohol beer was obtained by dilution with deoxygenated water and carbonation with 6.0 g/L CO₂, followed by addition of 8–12% of regular beer and equilibration for 2–3 days to develop normal beer aroma. The results showed that the non‐alcohol beer had several favourable properties, including the alcohol level of <0.5% (v /v), colour 7.0 (EBC), thiobarbituric acid value of 1.05 and ratio of alcohols to esters of 1.08. Compared with other methods for the production of non‐alcohol beer, this novel approach produced a favourable alternative to regular beers with similar flavour characteristics and satisfactory stability. Copyright © 2017 The Institute of Brewing & Distilling