不同酵母酒精发酵中乙醛产量特征及影响因素的研究

不同酵母酒精发酵中乙醛产量特征及影响因素的研究

STUDY ON ACETALDEHYDE KINETICS DURING ALCOHOLIC FERMENTATION BY WINE YEAST AND IT’S AFFECTING FACTORS

作者：李二虎　　导师：刘延琳

西北农林科技大学　　葡萄与葡萄酒学2011届博士

摘　要

二氧化硫（SO₂）因存在诸多健康问题日益引起人们的忧虑，减少SO₂的使用量已成为现代葡萄酒酿造学的一个重要目标。然而，葡萄酒中的乙醛可与游离SO₂稳定结合，降低SO₂抗微生物、抗氧化的效果，导致需要添加更多的SO₂以维持葡萄酒的稳定性。论文从酵母菌株的选择和应用出发，在模式体系和葡萄汁发酵中研究不同酵母菌株酒精发酵过程中生成、代谢乙醛的能力，研究发酵速率和SO₂终止发酵等因素对葡萄酒相关酵母乙醛生成的影响，旨在降低葡萄酒中乙醛的含量，为葡萄酒生产中减少SO₂的添加量提供依据。主要研究结果如下：

1.　以酵母静息细胞的悬浮液作为模式体系，研究26株葡萄酒相关酵母生成、代谢乙醛的能力。结果表明：所有酵母菌株在发酵过程中乙醛含量均呈现出先增加后降低的变化趋势，但不同菌株乙醛生成的动态特征及发酵中乙醛含量的最大值（峰值）不同。其中，各酵母菌株在发酵初期乙醛的产量系数（代谢单位还原糖而产生乙醛的量，单位mg/g）与发酵中乙醛的峰值显著相关（r²=0.92），26株酵母在模式体系下乙醛的产量系数在0.4～42 mg/g之间，乙醛峰值的范围为2.2～189.4 mg/L。在该模式体系下，粟酒裂殖酵母（Schizosaccharomyces pombe）的乙醛产量系数和峰值最高，其他非酿酒酵母的乙醛产量系数和峰值均显著低于酿酒酵母（Saccharomyces cerevisiae）。发酵初期添加SO₂增加所有酵母菌株的乙醛产量系数和峰值，其中非酿酒酵母的增加值小于酿酒酵母。所有酵母静息细胞在不添加葡萄糖时，均可代谢外源添加的乙醛，其中粟酒裂殖酵母的代谢速率最小，但各菌株静息细胞代谢外源乙醛的速率和发酵中乙醛的产量系数不相关。

2.　在霞多丽葡萄酒酿造过程中研究26株葡萄酒相关酵母乙醛生成量的动态变化。结果表明：各酵母菌株在发酵初期乙醛生成量迅速增加，当酵母生长至对数期末期时乙醛生成量达最大值，随后发酵葡萄汁中乙醛含量迅速降低。各菌株在发酵初期乙醛的生成速率与峰值过后乙醛的代谢速率显著正相关（r=0.83）。26株酵母乙醛产量系数的范围为0.13～4.4 mg/g，乙醛峰值的范围为9～129 mg/L，发酵结束时不同酵母菌株酿造葡萄酒中乙醛的残留量在2～64 mg/L之间。粟酒裂殖酵母的乙醛产量系数、峰值和残留量均最高，酿酒酵母居中，其他非酿酒酵母最低。发酵前添加SO₂增加了葡萄酒中乙醛的含量，各酵母菌株乙醛含量的增加范围为217～531 µg/mg SO2，平均增加值为325±17 µg/mg SO₂。将葡萄汁发酵和模式体系发酵对比发现，两种发酵体系中各酵母菌株乙醛产量系数和峰值均线性相关。

3.　迟缓发酵在葡萄酒生产中很难预测，试验研究了6株酿酒酵母在霞多丽葡萄汁中快速和迟缓发酵时乙醛生成量的动态变化。结果表明：迟缓发酵时各酵母菌株乙醛产量系数和葡萄酒中乙醛含量均高于快速发酵。其中，迟缓发酵时各菌株乙醛产量系数的平均值为1.59±0.21 mg/g，快速发酵时各菌株乙醛产量系数的平均值为0.95±0.10 mg/g。发酵前不添加SO₂时，2株酵母（EC1118和E1219）快速发酵和迟缓发酵时乙醛产量系数的差异达显著性，添加50 mg/L SO₂时，5株酵母（CY3079、DV10、EC1118、A709和E1219）的差异达显著性（p<0.05）。测量不同酵母菌株发酵初期乙醛的产量系数有可能用于预测酒精发酵是否迟滞。

4.　发酵中添加SO₂抑制酒精发酵常用于生产甜葡萄酒，试验在霞多丽葡萄汁发酵中添加不同浓度的SO₂并结合低温处理，研究中途抑制发酵对2株酿酒酵母（CY3079和EC1118）代谢还原糖并生成乙醛的影响。结果表明：添加大于150 mg/L SO₂可显著抑制酵母糖代谢，不同浓度SO₂处理均提高酵母乙醛的生成量，但提高幅度与SO₂的添加量成反比。本试验中使用不同浓度的SO₂抑制发酵15 d后，各处理葡萄酒中乙醛含量低于79 mg/L，小于葡萄酒中乙醛的嗅觉阈值。

5.　通过研究8株酿酒酵母在霞多丽葡萄汁发酵过程中其他三种主要SO₂结合物丙酮酸、α-酮戊二酸和2-丁醇-3-酮含量的动态变化，并结合各菌株乙醛产量的研究结果，对不同酵母菌株发酵葡萄酒结合游离SO₂的能力进行计算。结果表明：酵母菌株显著影响葡萄酒中SO₂结合物的含量。商业酵母EC1118酿造的霞多丽葡萄酒中，SO₂结合物的总量最低，本土筛选菌株E1219酿造葡萄酒中SO₂结合物的总量最高。以葡萄酒中游离SO₂水平需达到50 mg/L为例，EC1118酿造的葡萄酒中需要加入的SO₂总量不应低于89 mg/L，而E1219酿造的葡萄酒中需要添加的SO₂总量不低于120 mg/L，是前者的1.34倍。因此，在酵母菌株筛选研究及葡萄酒生产中，可将不同菌株产乙醛等SO₂结合物的能力作为重要的参考指标。

关键词　葡萄酒　酵母　乙醛　SO₂结合物

Abstract

The reduction of SO₂ concentrations in wine is a primary objective because of regulatory pressure or consumer concerns with regards to asthma or allergic reactions. Several wine constituents may bind to SO₂,acetaldehyde (ethanal) typically being the most important because of its quantity and binding power. This research evaluated the ability of 26 yeast strains,including Saccharomyces and non-Saccharomyces strains and commercial starters to form and degrade acetaldehyde in a model medium and during grape must vinifications. The effect of fermentation vigor was considered as well as yeast response to SO₂ additions at different fermentation stages. Specifically,the effect of using SO₂ to stop alcoholic fermentation with the aim of achieving wines with residual sugar was compared with cooling. In addition,the ability of production other SO₂ binders by 8 S. cerevisiae was investigated in grape must fermentation with the aim of calculation bound SO₂ concentrations in different wines. The major results were summarized as following.

1.　Acetaldehyde metabolism of 26 yeast strains was evaluated in a reproducible resting cell model system. Acetaldehyde kinetics and peak values were highly genus,species,and strain dependent. Peak acetaldehyde values varied from 2.2 to 189.4 mg/L and correlated well (r² = 0.92) with the acetaldehyde production yield coefficients that ranged from 0.4 to 42 mg acetaldehyde per g of glucose. S. pombe showed the highest acetaldehyde production yield coefficients and peak values. All other non-Saccharomyces species produced significantly less acetaldehyde than the S. cerevisiae strains and were less affected by SO₂ additions. All yeast strains could degrade acetaldehyde as sole substrate,but the acetaldehyde degradation rates did not correlate with acetaldehyde peak values or acetaldehyde production yield coefficients in incubations with glucose as sole substrate.

2.　In addition,acetaldehyde production property of 26 enological yeast strains was investigated in a natural grape must. All yeast strains led to uniform kinetics where acetaldehyde reached an initial peak value at the beginning of fermentation followed by partial reutilization. Peak acetaldehyde values and final concentrations in wine ranged from 9 to 129 mg/L and 2 to 64 mg/L,respectively. Acetaldehyde production yield coefficient at the beginning of alcoholic fermentation was found to be yeast strain specific and can be used as a discriminating factor between yeast strains. Initial addition of SO₂ led to higher acetaldehyde concentrations throughout the fermentation and the extent of increases at final values in wine was highly strain dependent,which ranged from 217-531 μg acetaldehyde per mg of SO₂ addition. Compared with previous work reported in resting cell system,acetaldehyde yield coefficients calculated as acetaldehyde produced per sugar degraded of 26 yeast strains were also correlated with the values in grape must fermentation and could be used as a criteria to discriminate among yeast.

3.　In wine making,slow or sluggish alcoholic fermentations still occur frequently and are difficult to predict. Acetaldehyde kinetics of 6 S. cerevisiae strains at different fermentation speed was studied in Chardonnay wine vinification. By comparison with fast fermentations,sluggish fermentations led to higher acetaldehyde yield coefficient as well as final concentrations in wine. The average value of acetaldehyde yield coefficient was 1.59±0.21 mg acetaldehyde per g of sugar for sluggish fermentations,but 0.95±0.10 mg acetaldehyde per g of sugar for fast fermentations. The significance was evidenced in 2 strains (EC1118 and E1219) in absence of SO₂ and 5 strains (CY3079,DV10,EC1118,A709 and E1219) with 50 mg/L SO₂ addition.

4.　The effect of using SO₂ to stop alcoholic fermentation on acetaldehyde production was also studied. Sugar metabolism was stopped with over 150 mg/L SO₂ addition and acetaldehyde production was increased with any SO₂ addition during fermentations. Acetaldehyde formation rate after the time-point of SO₂ adding was decreased as the increase of SO₂ dosage. However,final acetaldehyde concentration was below 79 mg/L in all wines,which did not over the acetaldehyde threshold in wines.

5.　Concentrations of other SO₂ binders as pyruvate,α-ketoglutaric acid and acetoin were studied throughout Chardonnay wine fermentation. Pyruvate and acetoin had similar kinetics with acetaldehyde during alcoholic fermentation. The amount of bound SO₂ in wines was yeast strain dependent. As calculated by three major binders,wine made with yeast strain EC1118 required at least 89 mg/L total SO₂ addition to reach a free SO₂ concentration of 50 mg/L,while wine made with yeast strain E1219 needed at least 120 mg/L total SO₂ addition,which was 1.34 fold higher.

Key words　Wine　Yeast　Acetaldehyde　SO₂ binding compounds