葡萄酒中酯类物质的生物合成及其影响因素

葡萄酒中的酯类物质主要是酒精发酵过程中,在酵母细胞内由酰基辅酶A及脂肪酸和醇类在相关酶的催化作用下生成的,具有水果或者花的香味,对葡萄酒的香气起着十分重要的作用。酯类物质主要分为乙酸酯和乙基酯两类,乙酸酯是由乙醇/高级醇和乙酰辅酶A在醇乙酰基转移酶的催化作用下合成,乙基酯则是由乙醇和短链/中链脂肪酸在酰基转移酶的催化作用下合成。这些酯类物质是脂溶性的,可以直接穿过酵母的细胞膜释放到发酵液中。葡萄酒中酯类物质的含量受多种因素的影响,主要包括品种、栽培模式、发酵液成分、酵母、发酵温度、苹乳酸发酵和陈酿等。综述了葡萄酒中酯类物质的合成及其影响因素,以期为葡萄酒香气的改善及深入研究提供理论指导。     

异常维克汉姆酵母的乙酸苯乙酯合成途径

以福建红曲黄酒酿造用曲中分离得到的异常维克汉姆酵母为试验菌株,采用发酵过程跟踪法和诱变法相结合,研究该酵母菌的乙酸苯乙酯合成途径。结果表明:该菌在发酵过程中同时存在醇酰基转移酶合成途径和酯酶合成途径,发酵过程跟踪法显示底物乙酸与乙酸苯乙酯的相关性大,说明酯酶合成途径是合成乙酸苯乙酯的主要途径。通过诱变法得到发酵力相同、醇酰基转移酶酶活不变、酯酶合成酶活力和水解酶活力下降的异常维克汉姆酵母突变菌株。该突变菌株发酵结果显示产乙酸苯乙酯水平显著下降,表明酯酶合成途径是合成乙酸苯乙酯的主要途径。通过确定异常维克汉姆酵母对黄酒风味有显著影响的乙酸苯乙酯合成途径,为以分子生物学技术为手段改良和优化酿酒酵母提供酶学基础,也为定向改进黄酒风味和滋味提供科学导向。     

低压贮藏对甜米酒高级醇和酯的影响

低压CO2贮藏海南甜米酒,是一种非热加工贮藏的方法。将进入酵母主发酵后期的甜米酒清液装入耐压容器中,利用酵母自产CO2所形成的压力对甜米酒进行保鲜贮藏。在带压贮藏期中,酵母生长受限于压力和CO2浓度,因而酒中大部分残糖在12个月贮藏后得以保留,使甜米酒依然具备甜的自然属性。压力贮藏令主要风味物质高级醇和酯类的生成受到抑制。高级醇在压力贮藏期前期生成量大,后期由于发生转化作用而有所减少,对风味影响较小。酯类在压力贮藏后期持续增加,主要是因为高级醇的酯化反应生成。主要酯成分乳酸乙酯在压力贮藏期间增加2.45倍,强化了其主要香味特征。     

醋酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响

将白酒生产中常见的1株醋酸菌(巴氏醋杆菌Acetobacter pasteurianus)与高产酯酿酒酵母(Saccharomyces cerevisiae)进行混合发酵,研究醋酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响。保持高产酯酿酒酵母的接种量不变,向高粱汁培养基内添加不同浓度的醋酸菌菌悬液、醋酸菌发酵上清液,结果发现:醋酸菌菌悬液的添加量对高产酯酿酒酵母酒精发酵及酯醇代谢的影响不大。随着培养基内醋酸质量浓度的增加(0～6 g/L),高产酯酿酒酵母乙酸酯和高级醇产量逐渐降低,乙酸酯最多下降60%,高级醇最多下降50%,酒精发酵没有受到影响;当醋酸质量浓度为8 g/L时,高产酯酿酒酵母酒精发酵及酯醇代谢受到严重抑制,乙醇产量下降85%。在培养基初始醋酸质量浓度为6 g/L时,高产酯酿酒酵母共有394个基因转录水平上调,282个下调,这些差异基因主要富集在细胞膜的组成,其中高产酯酿酒酵母的次生代谢产物合成、碳代谢、三羧酸循环(tricarboxylic acid cycle, TCA)、氨基酸生物合成、糖酵解、氧化磷酸化、丙酮酸代谢等多条代谢途径受到影响。     

酿酒酵母与球拟酵母混合发酵对白酒风味物质的影响

为研究球拟酵母在白酒发酵过程中对风味物质生成量的影响,将工业酿酒酵母AY15、高产酯酿酒酵母MY15分别以不同接种体积比、不同接种顺序与球拟酵母WY2进行混合发酵,以3种酵母单独接种发酵作为对照,检测其发酵过程中发酵性能、乙酸酯及高级醇生成量的变化。在不同接种体积比试验中,高产酯酿酒酵母和球拟酵母接种体积比为1∶2时,乙酸乙酯生成量达到351.65 mg/L,比MY15菌株纯种发酵提高27.78%,且发酵周期缩短至4 d。在不同顺序接种试验中,先接种酿酒酵母AY15和MY15,再接种球拟酵母,对应的乙酸乙酯生成量分别提高了24.55%和12.78%,而异戊醇生成量分别降低了10.58%和24.24%。结果表明,酿酒酵母与球拟酵母混合发酵时,球拟酵母对酿酒酵母的产酯能力具有强化作用,且不影响酿酒酵母的产酒精能力。     

CO2背压啤酒发酵中乙酰辅酶A与酿酒酵母生长和酯类生成的相关性分析

该研究探讨CO2背压发酵中乙酰辅酶A（acetyl-CoA）与酿酒酵母生长和酯类生成的相关性。结果表明：常压和CO2背压发酵条件下,acetyl-CoA含量在发酵初期（0～20 h）急速增长,并在20 h时达到峰值,在100 h之后均趋于稳定一致;酵母细胞数量分别在100 h、140 h时达到峰值,在180 h之后趋于一致;总酯含量分别在100 h、180 h时达到峰值,在180 h之后趋于稳定,常压发酵条件下总酯含量始终高于CO2背压发酵。常压条件下,啤酒发酵中acetyl-CoA含量与酵母数量呈极显著正相关（P＜0.01）,与总酯含量呈极显著负相关（P＜0.01）,相关系数R分别为0.937 17、-0.833 96;CO2背压条件下,啤酒发酵中acetyl-CoA含量与酵母数量呈极显著正相关（P＜0.01）,与总酯含量呈极显著负相关（P＜0.01）,相关系数R分别为0.905 66、-0.750 42。     

可同化氮对葡萄酒发酵香气物质积累及代谢调控的影响

葡萄汁中酵母可同化氮的含量和组成,不仅影响酵母生长和酒精发酵,还对葡萄酒中高级醇、支链酸、酯类等发酵香气物质的积累起决定性影响。支链氨基酸、芳香族氨基酸等由酿酒酵母通过Ehrlich路径代谢生成相应的高级醇及酸,高级醇和酸与糖代谢及脂肪酸代谢产物结合生成酯类,构成发酵香气的主要成分。合理的葡萄汁氮源组成和外源添加氮源对葡萄酒发酵过程中优良香气物质的积累,减少不良香气物质,提高酒的感官品质有重要意义。本文总结了酵母可同化氮量与发酵香气积累相关的研究成果,综述酿酒酵母氮代谢产生香气物质的路径及其基因表达调控的研究进展。     

啤酒生产过程中高级醇形成因素及控制

高级醇是啤酒生产发酵过程形成的,目前可检出的高级醇有30多种.啤酒中高级醇的生成途径主要有氨基酸、α-酮酸途径和糖类物质合成高级醇途径.高级醇的生成与麦汁发酵过程的pH值、α-氨基氮含量、麦汁充氧量、麦汁浓度、发酵强度、酵母菌种及其接种量等因素有关,控制麦汁α-氨基氮含量、可发酵性糖、麦汁充氧量、发酵工艺条件、乙醛含量、酵母菌种及其接种量可有效控制啤酒中的高级醇含量.     

己酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响

将己酸菌与高产酯酿酒酵母在高粱汁培养基中进行混合发酵,研究己酸菌对高产酯酿酒酵母酒精发酵及酯醇代谢的影响。结果表明,己酸菌菌悬液对高产酯酿酒酵母的生长及代谢没有直接影响;己酸菌的代谢产物对高产酯酿酒酵母的发酵速度及酒精产量有所促进,在培养基己酸浓度为220mg/L时,酒精产量提高了6.63%;在己酸浓度为45~220mg/L时,高产酯酿酒酵母的乙酸酯类和主要高级醇受到的抑制作用逐渐增强,乙酸乙酯最多降低65.32%,乙酸异丁酯最多降低91.32%,乙酸异戊酯最多降低82.14%,高级醇最多降低71.14%;在己酸浓度为220mg/L时,与己酸浓度为0mg/L时对比,高产酯酿酒酵母共有1032个基因转录水平上调,有1037个基因转录水平下调,这些转录水平变化的基因主要涉及到高产酯酿酒酵母的氮代谢、糖酵解、苯丙氨酸代谢、碳代谢等多条代谢途径。     

乙酰辅酶A含量对酿酒酵母乙酸乙酯合成的影响

通过缺失乙酰辅酶A水解酶（ACH）基因和过表达乙酰辅酶A合成酶（ACS）基因技术提高酿酒酵母合成乙酰辅酶A（acetyl- CoA）能力的同时,过表达醇酰基转移酶（ATF）基因,提高乙酸乙酯合成能力,并考察acetyl-CoA含量对酿酒酵母合成乙酸乙酯能力的影响。结果表明,敲除ACH1基因、且在敲除ACH1基因基础上过表达ACS1、ACS2基因均能提高酿酒酵母Acetyl-CoA含量,进而提高乙酸乙酯含量。较亲本菌株α5,缺失突变株α5ΔACH1、重组菌株α5-A1、α5-A2的Acetyl-CoA的含量均分别提高了52.5%、80.33%、52.79%,乙酸乙酯含量分别提高10.59%、26.12%、23.70%。在敲除ACH1基因、过表达ACS1和ACS2基因的基础上同时过表达ATF1基因,得到工程菌株A1-ATF1和A2-ATF1,较亲本菌株α5,乙酸乙酯含量分别提高226.09%、530.43%、289.57%,工程菌株A1-ATF1乙酸乙酯产量最高,为72.52 mg/L。研究表明,提高乙酰辅酶A含量能够促进乙酸乙酯的合成,为提高乙酸乙酯生成量提供了新思路。     

Top Pressure and Temperature Control the Fusel Alcohol/Ester Ratio through Yeast Growth in Beer Fermentation

Temperature and top pressure are key factors for maintaining a consistent quality of lager beer. Their influence on yeast growth, CO₂ production, final concentrations of fusel alcohol and ester and production kinetics was analysed under industrial conditions. Fermentations of 12°P lager wort were performed at 10°C or 16°C temperature and 1.05 bars or 1.8 bars top pressure, corresponding to dissolved carbon dioxide concentrations of 1.98 g/litre to 3.65 g/litre. Analysis of variance was performed to test the significance of temperature and dissolved C₂. Results show that temperature increases fermentation rate and the production ratio and final concentration of fusel alcohol, independently of the top pressure applied. Conversely, dissolved carbon dioxide controls the production rate and final concentration of ester by limiting yeast growth. Relationships between initial or maximum ester production rates and maximal growth rates were shown. Considering the metabolic pathways occurring during anaerobic growth of yeast, a limited production of acetyl CoA was expected in cultures with high concentrations of dissolved carbon dioxide. Also, final ester concentration and biomass produced are linearly correlated. Furthermore, whatever the ester considered, its synthesis is not influenced by corresponding fusel alcohol availability. It was demonstrated that fermentations performed with a reasoned combination of temperature and top pressure can result in a beer of distinctive aroma without resorting to modification of the initial wort or yeast strain.     

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”.     

THE FORMATION OF ESTERS AND HIGHER ALCOHOLS DURING BREWERY FERMENTATION; THE EFFECT OF CARBON DIOXIDE PRESSURE

The influence of the size and geometry of brewery fermentation vessels on beer flavour and aroma formation is generally attributed to carbon dioxide pressure. In order to study this pressure effect, brewery batch fermentations were carried out on the laboratory scale with Saccharomyces cerevisiae. The formation rates and yields of esters and fusel alcohols were studied in relation to the growth of metabolically active biomass. The results indicate that the observed reduction in the formation of esters and fusel alcohols with increased carbon dioxide pressure is mainly caused by reduced yeast growth. The overall formation of fusel alcohols is less affected than the formation of esters.     

THE CONTROL OF VOLATILE ESTER SYNTHESIS DURING THE FERMENTATION OF WORT OF HIGH SPECIFIC GRAVITY

Some effects have been studied of fermenting wort of unusually high specific gravity, followed by dilution with water to give beer of normal original gravity. This procedure permits increased overall rates of beer production, but matching of flavours requires control of the level of flavour determinants. If not controlled, the concentration of volatile esters may be disproportionately increased so that, after dilution, beer flavour is markedly different. Higher alcohol production is not affected in this way. The concentration of esters can be adjusted to appropriate levels by increasing the production of yeast mass during fermentation. Unsaturated fatty acids, which increase yeast dry matter production without altering the rate of fermentation, are particularly effective in reducing the extent of ester synthesis.     

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     

高浓高温对啤酒酵母发酵性能的影响

以啤酒酵母S-6为实验菌株,研究了主发酵温度和原麦汁浓度对啤酒发酵的残糖、酒精度、风味物质和絮凝性等性能指标的影响。结果表明,原麦汁浓度一定时,主发酵温度对高级醇和乙酸酯的含量影响较大,主发酵温度由10 ℃提高至16 ℃时,高级醇含量提高了10%～20%,乙酸酯含量提高了8%～16%,但CO2累积质量损失、残糖、酒精度和絮凝性基本不受温度的影响;主发酵温度一定时,原麦汁浓度对酵母絮凝性影响较大,原麦汁浓度越高,酵母絮凝性越低,将高浓（18 °Bx）发酵液稀释50%至常浓（12 °Bx）,残糖、酒精度和高级醇的含量与常浓发酵液基本相同。该研究为选育高温高浓发酵低产高级醇同时强絮凝性酵母菌株提供了重要依据。     

降低上面发酵小麦啤酒头痛感的酿造条件优化

啤酒上头的原因主要是较高的醇酯比和乙醛含量。为降低上面发酵小麦啤酒头痛感,对其发酵条件进行优化,并通过气相色谱（GC）检测啤酒中高级醇、酯类和乙醛等风味物质含量。结果表明,最优发酵条件为发酵温度18 ℃（醇酯比2.02、乙醛含量3.51 mg/L）、发酵压力0.12 MPa（醇酯比2.01、乙醛含量2.75 mg/L）和零代酵母（醇酯比2.20、乙醛含量2.76 mg/L）。在此条件下发酵的小麦啤酒醇酯比为2.0～2.5,乙醛含量＜4 mg/L,不易引起上头。     

不同固定化酵母载体对啤酒风味成分影响研究

采用气相色谱分析法,主要考察了山毛榉木片、多孔陶瓷和海藻酸钠3种固定化酵母载体对啤酒风味物质成分的影响。结果表明,不同固定化酵母载体对啤酒风味物质成分的形成有较大影响,其中海藻酸钠载体固定化酵母的牢固程度最好,且载体正丙醇、异丁醇和异戊醇含量分别高于成品啤酒9%、8%和12%,啤酒含醇量较高,适于醇厚型啤酒发酵;山毛榉木载体生成的酯类物质较多,载体乙酸异丁酯、乙酸乙酯、乙酸异戊酯和己酸乙酯含量分别高于成品啤酒20.1%、14.3%、12.5%和17.4%,含酯量较高,适于淡爽型啤酒发酵;与另两种载体相比,多孔陶瓷载体双乙酰含量达到0.14 mg/L,不适合用于啤酒发酵。     

YEAST ESTERASES AND AROMA ESTERS IN ALCOHOLIC BEVERAGES

It has been shown that the proportion of fatty acid ethyl esters retained by the yeast cell increases with increasing acyl chain length as the ester becomes more lipid soluble. The distribution of esters depends on the yeast strain and on the fermentation temperature; larger amounts of esters were found to transfer from the cells into the medium at higher temperature. It was shown that esterase activity is located both inside and outside the yeast cell plasma membrane. Intact yeast was capable of hydrolysing the ethyl esters of caproic, caprylic and capric acid. Acetate esters, were hydrolysed only very slowly or not at all. The hydrolytic activity of baker's yeast was studied with ethyl caprylate as substrate. The hydrolysis was very fast at the beginning. The equilibrium attained depended not only on the concentration of ester and alcohol but also on the pH, a higher amount of ester remaining in solutions of lower pH. It was also shown that the esterases possess appreciable ester synthesizing ability and an equilibrium was attained by incubating yeast with caprylic acid and ethanol. The experiments described show that the ester level in an alcoholic beverage, such as beer, is not dependent solely on the ester concentration formed during fermentation: in the presence of yeast the level can be shifted in either direction by changing temperature, pH or alcohol concentration — or the amount and type of yeast.