Performance of indigenous yeasts in the processing of Chinese strong‐flavoured liquor during spontaneous mixed solid‐state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong‐flavoured liquor, a comparison was conducted between solid‐state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong‐flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong‐flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong‐flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling     

固定化酵母在清香型白酒发酵的应用研究

采用固定化酵母发酵技术对传统白酒生产工艺进行部分改造，对其发酵工艺的研究结果显示，利用固定化酵母的效果均比对照组的产量和出酒率高3％，且不影响原酒的风味和其他理化指标。     

Effect of cellulase‐producing bacteria on the metabolic activities and functional diversity of bacterial communities in Chinese liquor fermenting grains

To study the roles of cellulase‐producing bacteria in the fermentation of Chinese liquor fermenting grains, a strain of cellulase‐producing bacteria was added into the fermenting grains at different levels, and the substrate utilization was profiled to evaluate the bacterial communities’ functional diversity in fermenting grains during fermentation. The results showed that, when the addition level of the cellulase‐producing bacteria was at or below 10² cfu/g, the average well colour development (AWCD_590nm) of the fermenting grains was not affected significantly. When the addition level was more than 10⁴ cfu/g, the average well colour development (AWCD_590nm) of the fermenting grains increased significantly, and the maximum AWCD_590nm was obtained when the addition was 10⁶ cfu/g. The diversity index analysis showed that the addition of the cellulase‐producing bacteria helped to increase the uniformity of bacterial communities in fermenting grains significantly, while the richness and dominance of the microbial communities were not affected. Copyright © 2015 The Institute of Brewing & Distilling     

Microbial succession and metabolite changes during the fermentation of Chinese light aroma‐style liquor

A combination of culture‐dependent and culture‐independent methods and SPME–GC–MS were used to monitor changes of bacterial and yeast communities, and flavour compounds during the fermentation process of Chinese light aroma‐style liquor. Bacillus and Lactobacillus were the main bacterial genera. Pichia anomala, Saccharomyces cerevisiae and Issatchenkia orientalis were the dominant yeast species. There was a close relationship between fermentation time and the shift of microbial community. Compared with the microbiota in the fermentation of other style liquors, higher bacterial diversity and different non‐Saccharomyces composition led to a variety of metabolites. Metabolite analysis showed that esters, acids, alcohols, aromatic compounds and phenols were the main flavour components and most of them were synthesised in the latter phase of fermentation. Principal component analysis further demonstrated that Bacillus and yeast were the most influential microorganisms in the first 10 days of fermentation, and lactic acid bacteria predominated in the later phase. Lactic acid bacteria regulated the composition of other bacteria and yeast, and synthesised flavour compounds to affect the organoleptic properties of liquor. S. cerevisiae and P. anomala were two important yeast species responsible for the characteristic aroma of liquor. These results present a comprehensive understanding of microbial interaction and potential starter cultures to produce desirable liquor quality. © 2018 The Institute of Brewing & Distilling     

Discrimination of Luzhou‐flavoured fresh raw liquor distilled from Zaopei fermented in new,trend to‐be aged and aged pit mud based on their aroma and flavour compounds

Fresh raw liquors distilled from Zaopei fermented in new, trend to‐be aged and aged pit mud, labelled as N1 and N2, T1 and T2, and A1 and A2, respectively, were analysed by sensory evaluation, two‐dimensional gas chromatography/time‐of‐flight mass spectrometry with combined principal component analysis, gas chromatography–flame ionization detection, liquid chromatography–mass spectrometry with combined hierarchical cluster analysis, and partial least square–discrimination analysis. Results showed that samples had the same grouping situations by each method: the N‐group containing N1 and N2; the T‐group containing T1 and T2; and the A‐group containing A1 and A2. The specific markers for the N‐group liquors were 2‐butanol and butanoic acid, and those for the A‐group were 1‐hexanol, ethyl pentanoate and 2‐pentanone. The threshold contents of 1‐hexanol, ethyl pentanoate, and 2‐pentanone for the A‐group liquors were 1207.95 ± 252.59, 469.89 ± 73.08 and 1105.12 ± 141.91 mg L⁻¹, while those of 2‐butanol and butanoic acid for the N‐group liquors were 215.66 ± 179.50 and 430.65 ± 60.47 mg L⁻¹, respectively. The results from modern chromatographic techniques and chemometric analyses verify the correctness of the traditional sensory evaluation used in controlling the quality of Chinese liquor, contribute to further studies related to the aroma and flavour of Chinese Luzhou‐ flavour of fresh raw liquors and may provide significantly helpful thresholds in distinguishing Chinese Luzhou‐ flavour fresh raw liquors distilled from Zaopei fermented in new, trend to‐be aged and aged pit muds. Copyright © 2017 The Institute of Brewing & Distilling     

Investigation of the main parameters during the fermentation of Chinese Luzhou‐f lavour liquor

The aim of this study was to investigate the evolution of some physicochemical properties of fermented Zaopei throughout the fermentation. For each layer of Zaopei, the temperature gradually increased to a maximum after about 2 weeks, and then remained stable for about 15 days before it began to decrease; acidity tended to increase during the entire fermentation, while the starch content decreased throughout the fermentation. The reducing sugar content for all layers reached a maximum after one week and decreased afterwards. Ethanol accumulated with time and reached a peak after 5 weeks for each layer, then tended to decrease but rose again slightly after 7 weeks. The moisture content in the top and middle layers increased gradually and reached saturation after 5 weeks, then remained almost unchanged, while the moisture of the bottom layer increased throughout the fermentation. The ethyl caproate, ethyl acetate and ethyl butyrate content of all layers remained almost constant for the first 2 weeks, then increased gradually and reached a maximum after 6 weeks, and subsequently declined slightly. The ethyl lactate of each layer increased throughout the fermentation. The caproic acid, acetic acid, butyric acid and lactic acid in all of the layers reached the highest level on the sixth week, followed by a continuous decrease until week 8, thereafter followed by a slight increase in trend. Additionally, the qualities of the raw liquor distilled from the different layers of Zaopei were investigated and the results demonstrated that the bottom layer possessed the best quality. Copyright © 2015 The Institute of Brewing & Distilling     

Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Overexpression of vacuolar H+‐ATPase‐related genes in bottom‐fermenting yeast enhances ethanol tolerance and fermentation rates during high‐gravity fermentation

Vacuolar H⁺‐ATPase (V‐ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom‐fermenting yeast strains, in which the V‐ATPase‐related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high‐sugar medium simulating high‐gravity fermentation. Among the strains examined, the DBF2‐overexpressing bottom‐fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high‐gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V‐ATPase is a stress target in high‐gravity fermentation and suggests that enhancing the V‐ATPase activity increases the ethanol tolerance of bottom‐fermenting yeast, thereby improving the fermentation rate and cell viability under high‐gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling     

Effects of cellulase‐producing bacteria on bacterial community structure and diversity during fermentation of Chinese liquor grains

To study role of cellulase‐producing bacteria on bacterial community structure during fermentation of Chinese liquor grains, a cellulase‐producing strain called DM‐4 was added to the grains at different levels. The bacterial community structure and diversity were then studied using a high‐throughput sequencing method. Results showed that the bacterial community structure exhibited varied characteristics at different levels of grain fermentation, which amply illustrated that adding cellulase‐producing bacteria to fermenting grains had significant effect on the bacterial community structure. Diversity analysis indicated that abundance and diversity of bacterial community increased significantly by adding 10⁴–10⁶ cfu/g DM‐4 cellulase‐producing bacteria. Also when 0–10⁶ cfu/g of DM‐4 cellulase‐producing bacteria was added, there was significant correlation between the dose of cellulase‐producing bacteria added and bacterial community diversity. The study thus concluded that bacterial community diversity and uniformity increased by adding cellulase‐producing bacteria during fermentation. Copyright © 2017 The Institute of Brewing & Distilling     

浓香型白酒大曲在发酵和成熟过程中主要功能酶活力分析

为了对浓香型白酒大曲中功能酶系进行深入研究,实验根据大曲制作、发酵与成熟工艺流程,选取10个时间节点的大曲样品,分析曲皮和曲心α-淀粉酶、单宁酶、木聚糖酶和脂肪酶活力及变化规律。结果显示,在进入发酵期后,曲皮中α-淀粉酶活力基本在523.28~765.78 U范围内变化波动;曲心α-淀粉酶活力在曲块发酵期升降变化波动较大,而在成熟期后期基本维持在350 U左右。在整个研究期内,曲皮和曲心单宁酶活力变化规律较为相似,均先迅速升高,后火期达到最大,然后缓慢降低;木聚糖酶活力在曲皮和曲心样品中,都呈现出持续上升至养曲阶段达到最大值(119.72 U和113.70 U)后再下降的趋势;与其它酶相比较,脂肪酶活力在曲皮和曲心中的变化趋势差异较大,其中曲皮脂肪酶活力呈现先升高再降低,再次升高后降低的变化,而曲心中脂肪酶活力则是持续升高至贮藏一个月达到最大(597.50 U)后急剧下降,然后再次缓慢升高的变化规律。针对白酒大曲发酵和成熟过程中主要功能酶活力变化规律的考察,将为高产酶微生物的分离和筛选提供表征性指导作用,同时也将为制曲工艺的改良和加强型特种大曲的研制提供基础数据。     

浓香型白酒酿造相关酵母与发酵糟醅中乙酸乙酯和乙酸的相关性

为探讨浓香型白酒酿造过程中酵母与乙酸乙酯及乙酸生成的关系,将分离自浓香型酿造诸环节的122株酵母分别接种至半固体培养基（入窖粮糟+黄水）中发酵,并设立空白对照和窖内对照,75d后测定糟醅中乙酸乙酯和乙酸的含量。结果显示,122株酵母中能够使糟醅中乙酸乙酯含量高于2个对照的菌株分别有84株和54株,其中乙酸乙酯含量最高可达3.74g/L,为窖内对照的3.94倍;使乙酸含量低于2个对照的分别有120株和15株,其中乙酸含量最低仅为2.03×10-4g/L,仅为窖内对照的0.13%;122株酵母所对应的糟醅中乙酸乙酯、乙酸的浓度间不存在明显的对应关系。      

Microbial community structure of pit mud in a Chinese strong aromatic liquor fermentation pit

Pits are necessary solid bioreactors for the production of Chinese strong aromatic liquor, and the microbial community in these pits significantly affects the quality and yield of the liquor. In this study, the microbial community structures of different aged pit muds were evaluated by phospholipid fatty acid analysis. The results indicated that the microbial community was composed of bacteria, actinomycetes and fungi, with Gram‐positive (G⁺) bacteria and anaerobic bacteria being dominant. The biomass of the pit mud increased and the microbial community shifted to G⁺ bacteria as the pit age increased. Principal component analysis showed that PC1 consisted mainly of fatty acids of saturated (15:0, 16:0 and 18:0), methyl‐branched (a15:0, i16:0, a16:0, i17:0 and 8Me18:0), polyunsaturated (16:1ω7c, 16:1ω9) and cyclopropane (cy17:0 and cy19:0), and PC2 mainly of 18:1ω9, 18:1ω10 and a14:0, The characteristics of the microbial community structure in the pit mud resulted from the stress effects of low oxygen concentration, low pH and a high alcohol concentration. Copyright © 2013 The Institute of Brewing & Distilling     

白云边酒优势酵母菌的分离鉴定及其发酵特征分析

从白云边大曲酒发酵堆积料和出池酒醅中共分离获得7株占优势的酵母菌,依次编号为Z1、Z2、Z3、Z4、BYB-2、BYB-3和FJ5。应用26S rDNA D1/D2区域序列分析,结果显示,Z1、Z2、Z3和FJ5属于东方伊萨酵母;Z4、BYB-2和BYB-3分别为热带假丝酵母、酿酒酵母和毕赤酵母。表明白云边大曲酒酿造过程中存在酵母菌的多样性,而优势酵母菌为东方伊萨酵母。对7株酵母菌进行耐高温、耐酒精能力以及酒精发酵产物的测定和分析,结果表明,东方伊萨酵母除具有一定的产酒精能力外,还具有耐热性好、耐酒精能力强和乙酸乙酯产生量高的特点。采用高粱为原料进行液态酒精发酵结果表明,Z1和FJ5菌株乙酸乙酯平均产量达到204.19mg/L。     

Bacterial and fungal diversity in the traditional Chinese strong flavour liquor Daqu

In this study, the diversity of bacterial and fungal communities in the strong flavour Daqu production process was investigated using polymerase chain reaction denatured gradient gel electrophoresis (PCR‐DGGE). The band number, dominance, diversity and similarity of the 16S rDNA differed in the DGGE patterns, reflecting the wide diversity of the microbial communities present in the different phases. The band abundance and dominance of the eukaryotic microbial community varied in the different phases. To provide insight into factors contributing to this diversity, the physicochemical properties of Daqu were explored. The core temperature of Daqu gradually increased to a maximum during the Dinghuo phase followed by a decline. The moisture and starch content decreased throughout fermentation. The total acid, reducing sugar, amino nitrogen, α‐amylase activity, glucoamylase activity, protease activity, bacterial and mould number increased gradually to a maximum value at the end of the Peijun phase before decreasing in the Dinghuo phase increasing slightly in the Huanluo phase and then remaining almost unchanged. The esterase activity exhibited a similar change to that of other enzymes and then slightly increased after the Peijun stage. For yeasts, the highest number was obtained at the Peijun stage and then decreased. Correlation analysis revealed that the diversity of both bacteria and fungi was negatively correlated with moisture and starch and positively correlated with other indices. The reducing sugar, amino nitrogen, α‐amylase and protease activities showed significant positive correlation with bacteria, and glucoamylase and esterase showed significant correlation with fungi. © 2019 The Institute of Brewing & Distilling     

Characterization of volatile compounds from Daqu‐a traditional Chinese liquor fermentation starter

Daqu is a fermentation starter and substrate complex that is used to initiate the solid fermentations for the production of Chinese liquor. The aroma of Daqu is one of the most important factors that influence the flavour of Chinese liquor. To further study the aroma of Daqu, volatile compounds in ten Daqu samples were extracted by headspace solid‐phase microextraction (HS‐SPME) and analysed by gas chromatography–mass spectrum (GC‐MS). The samples were pre‐equilibrated at 50 °C for 10 min and extracted with stirring at the same temperature for 30 min prior to injection into a GC‐MS. A total of 75 volatile compounds were characterised by GC‐MS, including twelve alcohols, eleven esters, eight acids, nine aromatic compounds, four phenols, two furans, fourteen nitrogen‐containing compounds and fifteen aldehydes and ketones. By a principal component analysis (PCA), the ten Daqu samples could be classified into three groups according to their origins and, in particular, the production technologies.     

The influence of porosity of the grain medium on the flavour formation of Chinese liquor during solid‐state fermentation

Solid‐state fermentation is a unique technology used for Chinese liquor production and the porosity of the grain medium is one of the important characteristics in the process. However, research on the effects of porosity on flavour generation of liquor in fermented grains is rarely reported. Therefore the present paper introduces the concept of porosity from the field of soil studies and establishes a method for determining the porosity of the fermentation grain medium. Based on the practicality in liquor production, the influence of the addition levels of rice husk and the grinding intensity of the raw material on the porosity of the grain medium was studied. On this basis, the process of solid‐state fermentation was simulated and the influence of different porosities on liquor flavour production was examined. The results showed that there were obvious differences in the flavour profiles of the fermented grains with different porosities. The most abundant content and species of volatile compounds appeared when the porosity of the fermented grain medium was ~33%. The development patterns of volatile flavour compounds, in both varieties and quantities, were examined in relationship to the porosity of the fermentation medium. Copyright © 2016 The Institute of Brewing & Distilling     

Characterization of volatile compounds in Fen‐Daqu – a traditional Chinese liquor fermentation starter

Fen‐Daqu is a saccharifying agent and fermentation starter for the production of Chinese liquor Fen (alcoholic spirit) and Fen traditional vinegar. The volatile compounds produced at seven incubation steps were analysed by HS‐SPME‐GC‐MS. A total of 83 major volatile compounds were identified, including 23 esters, 8 acids, 24 alcohols, 18 ketones and aldehydes, 6 pyrazines and 4 acetals. Data obtained by HS‐SPME‐GC‐MS were subjected to principal component analysis. The trajectory plots of volatile compounds in Fen‐Daqu samples obtained during successive steps of incubation were revealed. The major compounds that contributed to discrimination were hexanal, (E)‐2‐octenal, (Z)‐2‐octen‐1‐ol, nonanoic acid, 1‐octanol, 2‐decen‐1‐ol, hexyl acetate, (E)‐2‐octen‐1‐ol, acetic acid, ethyl acetate, phenylethyl alcohol, ethyl alcohol, octanoic acid, 1‐octanol, 3‐methyl‐2‐buten‐1‐ol and pyrazines. Copyright © 2012 The Institute of Brewing & Distilling     

Identification of microorganisms producing lactic acid during solid‐state fermentation of Maotai flavour liquor

Lactic acid is the main acid produced during the Maotai liquor brewing process, influencing the quality of the base liquor and fermentation process. However, the microorganisms responsible for lactic acid production have not been identified. In this work, the dynamic changes in bacterial community structure in the Zaosha round (second sorghum feeding and fermentation) of the brewing process were analysed by 16S rRNA high‐throughput sequencing. Results show that lactic acid bacteria (LAB) and Bacillus spp. are the dominant bacteria in the brewing process, where Bacillus spp. are found in the early stage, whilst LAB are found throughout the brewing process. Furthermore, 10 types of LAB and five Bacillus spp. were isolated from Zaopei (a mixture of fermented grains including sorghum and wheat) by a culture‐dependent method. Lactobacillus panis accounts for 68% of the LAB, and Bacillus amyloliquefaciens for 54% of Bacillus spp. Solid‐state fermentation experiments were performed with L. panis and B. amyloliquefaciens and lactic acid production was consistent with the accumulation of lactic acid in Zaosha. The results showed that L. panis was the main producer of lactic acid in pits, while B. amyloliquefaciens plays an important role in the production of lactic acid in the early stages of fermentation. The approach used in this study may facilitate the identification of key microorganisms with specific functionality involved in other food and beverage fermentation processes. © 2018 The Institute of Brewing & Distilling