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.     

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.     

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.     

啤酒酵母酿造葡萄酒及香气成分GC-MS分析研究

以宁夏贺兰山东麓产区酿酒葡萄为原料酿造葡萄酒,测定其感官指标、理化指标,并通过顶空固相微萃取-气质联用（HS- SPME-GC-MS）技术分析啤酒酵母单独发酵（SY）、葡萄酒酵母单独发酵（AY）及二者混菌（1∶1）发酵（MY）酿造葡萄酒的香气物质,并计算气味活力值（OAV）。结果表明,3个样品的感官评分顺序依次为MY＞SY＞AY,理化指标符合相关国标;MY、SY、AY样品GC-MS分析分别共检测出22种、20种、15种香气成分,其中含量最高的均为异戊醇,MY、SY样品中的酯类成分较AY样品种类多;OAV分析结果进一步显示,对3个样品香气贡献最大的物质均为酯类,MY样品OAV＞1的香气成分为4种。啤酒酵母酿造的葡萄酒风味独特,并且混菌发酵可以提升葡萄酒品质。     

HS-SPME-GC-MS法分析发酵过程中荞麦和小麦面团香气成分变化

采用顶空固相微萃取技术和气相色谱-质谱联用技术对比分析荞麦和小麦在不同发酵剂作用下发酵过程中产生香气成分的差异。并基于保留指数和质谱鉴定对挥发性化合物进行鉴定。研究发现未发酵荞麦面团检出38?种香气成分,在酵母和乳酸菌发酵过程中共分别检测出80?种和63?种香气成分;未发酵小麦面团检出11?种香气成分,在酵母和乳酸菌发酵过程中共分别产生42?种和17?种。发酵过程中,荞麦和小麦面团香气种类增加,其中荞麦面团呈现更多香气成分。烃类和酯类是荞麦面团酵母发酵前后主要香气成分,烃类主要为单萜烯和倍半萜烯类,酯类是荞麦面团经乳酸菌发酵前后主要香气成分。小麦经过酵母发酵产生香气成分包括烃类、酯类、醇类、酸类、醛类及醚类,经过乳酸菌发酵产生香气成分种类相对较少,有烃类、酯类、酮类及醛类。荞麦发酵后产生更多活性香气成分。酵母和乳酸菌发酵荞麦和小麦后,香气成分有明显差异。在发酵过程中,香气成分随着发酵时间延长而发生转化。     

一次敲除BAT2同时过表达Lg-ATF1对啤酒酵母产醇酯的影响

乙酸乙酯和高级醇是啤酒中的重要风味物质,为了探究BAT2基因和Lg-ATF1基因对啤酒酵母产醇酯能力的影响,进而解决啤酒中存在的醇高酯低的问题。本研究通过酶切连接法构建重组质粒p UC-PLABBK,采用醋酸锂转化法和胞内同源重组技术,以多倍体啤酒酵母菌株S5为出发菌株,Kan MX基因作为筛选标记,最终获得过量表达Lg-ATF1基因同时敲除BAT2基因的重组菌株S5-Lg。通过啤酒发酵实验和数字PCR探究重组酵母菌株S5-Lg与出发菌株S5醇酯含量与相关基因表达量的变化。结果显示,与出发菌株相比,S5-Lg的总高级醇生成量降低9.12%,其中异丁醇和异戊醇的生成量分别降低了10.63%和9.55%,乙酸乙酯生成量提升了26.81%,BAT2基因表达量降低45.72%,Lg-ATF1基因表达量大幅提升。BAT2基因和Lg-ATF1基因可以影响啤酒酵母产生高级醇和乙酸乙酯的含量,对改善啤酒风味有重要参考意义。     

Sourdough derived strains of Saccharomyces cerevisiae and their potential for farmhouse ale brewing

The Finnish farmhouse ale sahti is unique in that it is fermented with baking, rather than brewing strains of Saccharomyces cerevisae. The custom of maintaining farmhouse yeast cultures is however no longer practiced in Finland, and much yeast derived diversity in sahti beers has presumably been lost as a consequence. Here, the brewing potential of a number of sourdough derived strains was tested with respect to a number of different fermentation traits. Seven strains originally isolated from Finnish or Italian sourdough cultures were used to ferment high gravity sahti wort (20°P), and fermentation performance together with production of volatile compounds were assessed and compared with a reference baking yeast. Strains differed in terms of fermentation rate, yield, yeast viability and beer flavour profile. All were maltotriose positive, but utilisation varied so that alcohol yield could be greater or lower than that of the reference strain, with values ranging from 6.6 to 7.9% (v/v). Production of aroma compounds was also variable so that it was possible to identify strains producing high levels of esters and those with lower production, which could be used to emphasise flavours originating from raw materials. All strains generated 4-vinyl guaiacol and so would be suitable for other beers where this is a part of the normal flavour profile. Results suggest that sourdough isolates of S. cerevisiae are suitable for sahti production, but could also be applied to other beer styles as a way to differentiate products. © 2020 The Authors. Journal of the Institute of Brewing published byJohn Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

The Horace Brown Medal Lecture: Forty Years of Brewing Research

Horace Brown spent fifty years conducting brewing research in Burton‐on‐Trent, Dublin and London. His contributions were remarkable and his focus was to solve practical brewing problems by employing and developing fundamental scientific principles. He studied all aspects of the brewing process including raw materials, wort preparation, fermentation, yeast and beer stability. As a number of previous presenters of the Horace Brown Lecture have discussed Brown's achievements in detail, the focus of this paper is a review of the brewing research that has been conducted by the author and his colleagues during the past forty years. Similar to Horace Brown, fundamental research has been employed to solve brewing problems. Research studies that are discussed in this review paper include reasons for premature flocculation of ale strains resulting in wort underattenuation including mechanisms of co‐flocculation and pure strain flocculation, storage procedures for yeast cultures prior to propagation, studies on the genetic manipulation of brewer's yeast strains with an emphasis on the FLO1 gene, spheroplast fusion and the respiratory deficient (petite) mutation, the uptake and metabolism of wort sugars and amino acids, the influence of wort density on fermentation characteristics and beer flavour and stability, and finally, the contribution that high gravity brewing has on brewing capacity, fermentation efficiency and beer quality and stability.     

啤酒酵母高级醇的代谢与调控研究进展

高级醇是啤酒酵母在啤酒酿造过程中代谢产生的,是啤酒风味物质的重要组成部分。适量的高级醇能赋予啤酒独特的香味,但其含量过高或者过低都会影响啤酒的质量。该文重点综述啤酒酵母中高级醇的生成途径、关键基因、代谢调控机理及选育低产高级醇优良菌株的主要方法,为适当降低啤酒中高级醇含量,进而推动啤酒行业的健康发展提供理论基础。     

影响啤酒总酯水平的单因素分析及验证

啤酒中的酯类物质的组成与含量对啤酒的风味影响很大.本文简单介绍了酯类在啤酒中的作用及形成机理.通过对众多的影响因素逐项进行数据分析,得出了影响总酯的主要因素,并制定了提高啤酒酯香的措施,再运用于大生产实践进行验证.试验表明,使用大米比使用淀粉作辅料更能提高啤酒的总酯水平,通过调整工艺参数,保证酵母旺盛发酵也是提高啤酒总酯水平的有效措施.     

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。     

浓香型大曲中功能性酵母菌的筛选、鉴定及发酵特性研究

以浓香型大曲为研究对象,采用稀释涂布平板法从中分离酵母菌,对其形态特征进行观察;采用2,3,5-氯化三苯基四氮唑（TTC）显色法初筛,通过发酵力、产酒能力、耐高温、耐酒精能力实验进行复筛,筛选一株优良的功能性酵母菌,利用Biolog微生物自动分析系统对其进行鉴定,并利用顶空固相微萃取气质联用（HS-SPME-GC-MS）法对其液态发酵产物进行分析。结果表明,从浓香型大曲中共分离出9株酵母菌,其中菌株NDY-06性能最突出,可耐受42 ℃的高温,16%vol的酒精度,且具有一定的发酵产酒能力;该菌株被鉴定为产香酵母Zygosaccharomyces cidri,从其液态发酵产物中共检测到35种挥发性风味物质,包括醇类物质7种,酯类物质13种,酸类物质7种,吡嗪类物质3种,对白酒香气成分有一定的贡献。     

打瓜啤酒生产工艺的优化

在啤酒生产过程中添加打瓜,通过单因素和正交试验对打瓜啤酒酿造工艺进行优化,利用电子舌、气相色谱（GC）和高效液相色谱（HPLC）测定打瓜啤酒味觉特征、香味物质和有机酸。结果表明,打瓜啤酒最佳生产工艺条件为打瓜汁添加量18%,酵母添加量为1.0 g/L、初始pH值5.2、主发酵温度10.5 ℃。在此优化条件下,打瓜啤酒的感官评分为93.3分。打瓜啤酒丰富度饱满,整体滋味偏咸味和鲜味;通过GC在打瓜啤酒中检测出10种香气成分,其中醛类物质1种、醇类物质4种、酯类物质5种;HPLC检测分析的9种有机酸均有检出,其中柠檬酸含量最高,为（9.30±0.038）g/L,是打瓜啤酒有机酸组成的主体。     

混合微生物发酵生产啤酒醋及饮料开发

以大麦芽为主要原料,进行啤酒醋发酵,调配制备麦香啤酒醋饮料。优化后的啤酒醋发酵工艺条件为：麦芽糖化后接种啤酒酵母进行酒精发酵,24 h后接种生香酵母,继续发酵4 d,啤酒酵母和生香酵母的添加比例为4∶1;醋酸发酵阶段醋酸菌接种量为10%,30 ℃条件下发酵,发酵结束时啤酒醋酸度为（3.21±0.09） g/100 mL,乙醇含量＜0.05%vol。开发麦香啤酒醋饮料,最佳配方为啤酒醋12%,麦芽汁70%,白砂糖3%,橙色素0.04‰,最终产品的酸度为（0.38±0.07） g/100 mL,糖度为（84.32±0.05） g/L,酸糖比为1∶22。感官评定表明,该方法制备的麦香啤酒醋饮料色泽金黄、澄清透明、酸甜爽口,醋香和麦芽香气浓郁。     

岩藻多糖啤酒生产工艺的研究

以55%大麦麦芽、30%小麦麦芽和15%焦香麦芽为原料进行糖化、过滤和煮沸,随后采用二次添加工艺分别添加70%苦型颗粒90型和30%香型颗粒45型酒花,麦汁经过沉淀和冷却,选用德国上面发酵酵母NO.354作为主发酵的生产菌株、下面发酵酵母NO.308作为后发酵的生产菌株进行发酵,过滤后添加岩藻多糖,得到的岩藻多糖啤酒口味更加纯正、香味独特、泡沫洁白细腻、杀口力强.     

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     

葡萄籽百香果啤酒发酵工艺优化

该研究以大麦芽、小麦为主要原料,葡萄籽、百香果为主要辅料酿制啤酒,利用单因素试验考察混合主料（大麦芽、小麦与水）料液比、萨兹香型酒花添加量、混合辅料（葡萄籽、百香果与麦汁）料液比、初始pH值、酵母添加量和主发酵温度等因素对葡萄籽百香果啤酒感官评分的影响,并通过响应面优化葡萄籽百香果啤酒发酵工艺条件。结果表明,最优葡萄籽百香果啤酒发酵工艺条件为：大麦芽、小麦与水料液比1.0∶4.0（kg∶L）,初始pH值5.0,萨兹香型酒花添加量0.6 g/L,百香果汁添加量80 g/L,葡萄籽、百香果与麦汁料液比2.0∶1.0（kg∶L）,酵母添加量0.10%,主发酵温度21 ℃。在此优化发酵工艺条件下,葡萄籽百香果啤酒感官评分为88.44分,酿制的啤酒香气丰富有层次感,麦香突出,是一款理想的烈性啤酒。     

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