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     

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     

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     

固态法发酵白酒中正丙醇产生的机理研究

采用酿酒车间现场取样实测与实验室同步模拟发酵试验方法,测定并分析了"白云边"大曲酒醅中酵母菌和细菌总数与正丙醇含量的关系。结果表明,酒醅发酵过程中酵母菌总数与正丙醇含量成正相关,而与细菌总数成负相关;酒醅的酒精度与正丙醇含量基本呈正相关。小曲酒醅中正丙醇含量在装瓶发酵后24h内激增,4d时正丙醇含量达到最大值。与接种酿酒酵母和根霉的纯种接种剂相比,两者发酵的酒精度和正丙醇含量均接近,表明酵母菌是白酒中正丙醇的主要产生菌。因此推断,固态法发酵白酒中正丙醇主要是在酵母菌的糖厌氧发酵过程中作为酒精的副产物产生的,而不是通过细菌对蛋白质分解导致的。     

Characterisation of volatile compounds in Maotai flavour liquor during fermentation and distillation

Volatile constituents in raw liquor and zaopei during fermentation and distillation were investigated by gas chromatography. Zaopei (steamed grains mixed with Daqu [FG1], stacking fermented grains [FG2], anaerobic fermented grains [FG3] and steamed grains [FG4]) presented changes in physicochemical properties. In all, 72 volatile compounds were identified in zaopei and raw liquor with similarity across 12 zaopei preparations. Principal component analysis and partial least squares discriminant analysis differentiated FG3 from other samples based on content of aroma compounds. FG3 had the highest level of aroma compounds, which increased from the fourth to sixth batches, and was characterised by the presence of 1, 2‐propanediol, acetic acid and linoleic acid, while the others were portrayed by phenylpropionate, ethyl laurate and 4‐ethyl guaiacol. The volatile compounds of raw liquor were dominated by ethyl acetate and ethyl lactate. Correlation analysis illustrated a lack of correlation of volatile compound content between the raw liquors and the zaopei distillation variants. © 2019 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     

基于酒醅乙醇含量和含湿量的白酒蒸馏工艺条件优化研究

针对不同乙醇含量和含湿量的酒醅,以酒产率为指标,研究了酒醅填装高度和加热蒸汽量对蒸馏效果的联合影响。结果表明,白酒蒸馏存在最佳加热蒸汽量和最佳酒醅填装高度。对于水分含量一定但乙醇含量不同的酒醅,其最佳蒸汽量都接近相同,而最佳填装高度随乙醇含量的增加逐渐降低;给定乙醇含量的酒醅,较高的含湿量对应的较高的最佳加热蒸汽量和较低的最佳填装高度。加热蒸汽量与酒醅填装高度联合影响蒸馏效果,较高的填装高度对应的最佳加热蒸汽量较高。     

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.     

中国白酒固态发酵及蒸馏过程中糠嗅味物质变化规律的研究

以三大香型白酒为研究对象,采用旋转蒸发的方法,将酒醅中糠嗅味物质进行浓缩、富集,然后采用气相色谱-质谱联用（GC-MS）技术进行检测。结果表明,清香型白酒酒醅中糠嗅味物质含量最高,其次是浓香型白酒、酱香型白酒;对清香型白酒蒸馏过程进行跟踪检测,初步揭示蒸馏过程中糠嗅味物质的变化规律,并且发现清香型白酒二茬酒醅中糠嗅味物质的含量高于大茬酒醅。     

酱香白酒高温堆积酒醅挥发性风味物质的检测分析

该文采用HS-SPME结合GC-MS对酱香型白酒第三轮次堆积不同时间点、不同位置点酒醅挥发性风味物质进行检测分析。共计检测出挥发性风味物质32种,其中,醇类物质10种,醛类物质3种,酸类物质4种,酯类物质9种,酚类物质1种,其他类物质5种。其醇、醛、酸、酯类物质平均相对含量分别为32.88%、4.37%、9.18%、30.93%。包括酒体中常见的乙酸乙酯、乳酸乙酯、异戊醇、乙酸、丁酸等。分析结果表明,堆积过程在富集微生物的同时,产生了大量酒体有益风味物质,对酱香白酒的品质具有一定的影响。     

Isolation and identification of a black Aspergillus strain and the effect of its novel protease on the aroma of Moutai‐flavoured liquor

Among Chinese traditional distilled liquors, Moutai‐flavoured liquor is the most famous, owing to its complicated process as it is derived from fermented sorghum coupled with the use of a Moutai‐flavoured Daqu. In this study, a novel isolate, belonging to a black Aspergillus, was obtained and identified as Aspergillus hennebergii by ITS‐5.8S rRNA sequencing analysis and conventional morphologic identification. The influences of initial pH, carbon source, nitrogen source and metal ions on the production of an A. hennebergii protease were studied. The results revealed that metal ions exerted a significant effect on enzyme production and activity. Additionally, the potential application of the protease from A. hennebergii was investigated. The enzymatic hydrolysis resulted in the identification and quantification of 42 compounds, including alcohols, aldehydes, pyrazines and esters. These volatile compounds exhibited special flavour properties. Significant differences were observed between the enzyme treatments and the control sample. Samples from the enzyme treatments led to the highest amounts of alcohols, pyrazines and aromatics. These results suggest that A. hennebergii, or its protease, may have some application values for the enhancement of the quality of Chinese liquor and for the improvement of the liquor production process. Copyright © 2014 The Institute of Brewing & Distilling     

中国白酒窖池真菌的研究进展

窖池是白酒酿造过程中的重要固态生物反应器,其中参与发酵的菌群结构对白酒风格和质量具有重要影响。该文综述了酱香型、浓香型、清香型等不同类型白酒的窖池真菌多样性及其功能与作用,提出了窖池真菌研究方法和研究技术的未来发展方向。     

Yeast community associated with the solid state fermentation of traditional Chinese Maotai-flavor liquor

Yeasts are the most important group of microorganisms contributing to liquor quality in the solid-state fermentation process of Chinese Maotai-flavor liquor. There occurred a complex yeast community structure during this process, including stages of Daqu (the starter) making, stacking fermentation on the ground and liquor fermentation in the pits. In the Daqu making stage, few yeast strains accumulated. However, the stacking fermentation stage accumulated nine yeast species with different physio-biochemical characteristics. But only four species kept dominant until liquor fermentation, which were Zygosaccharomyces bailii, Saccharomyces cerevisiae, Pichia membranifaciens, and Schizosaccharomyces pombe, implying their important functions in liquor making. The four species tended to inhabit in different locations of the stack and pits during stacking and liquor fermentation, due to the condition heterogeneity of the solid-state fermentation, including the different fermentation temperature profiles and oxygen density in different locations. Moreover, yeast population was much larger in the upper layer than that in the middle and bottom layers in liquor fermentation, which was in accordance with the profile of reducing sugar consumption and ethanol production. This was a systematical investigation of yeast community structure dynamics in the Maotai-flavor liquor fermentation process. It would be of help to understand the fermentative mechanism in solid-state fermentation for Maotai-flavor liquor.     

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。     

强化发酵丢糟再生产白酒的研究

通过加入强化发酵菌株、糖化酶、大曲粉、复合酶对白酒丢糟进行强化发酵生产复糟酒,研究结果表明,强化发酵菌株、糖化酶、大曲、复合酶的加入量为6.0‰、60U/g、6‰、5‰,发酵时间为35d时产酒最多且酒质最好.     

浓香型白酒酒醅发酵过程中淀粉和还原糖的变化规律研究

该试验跟踪采取白酒生产整个发酵期的酒醅样品,采用连续流动化学分析仪分析窖池不同空间位置的酒醅中淀粉、还原糖两个指标在发酵过程中的变化规律,结果发现:发酵过程中酒醅的淀粉含量随发酵时间的增加呈下降趋势,而且发酵前期上层酒醅淀粉含量下降最多;发酵中后期三层酒醅淀粉含量缓慢下降;酒醅中的还原糖含量在发酵前期呈上升趋势,下层酒醅中的还原糖含量增加最多;发酵中后期还原糖含量呈下降趋势.     

Improvement of flavour compound synthesis in a mixed culture system at high temperature by solid‐state fermentation

In order to enhance the synthesis of flavour compounds in solid‐state fermentation (SSF) at a high temperature, Bacillus subtilis XJ‐013 and Saccharomyces cerevisiae Z‐06 were used as a mixed culture with Monascus HQ‐3. The culture temperature was enhanced from 37°C to 56°C by the synergetic effect, and the synthesis of esterase was enhanced from 85.43 U/g to 129.65 U/g in the mixed culture system (over 50% higher than that of the culture using a single strain). This resulted in the synthesis of favourable flavour compounds in the solid‐state fermentation. These results signified that a mixed culture of Monascus and S. cerevisiae was favourable for enzyme production. The mixed culture of Monascus and B. subtilis resulted in a high culture temperature that promoted flavour compound synthesis in the solid‐state fermentation system dramatically. These results present a model to explain the synergetic effects between the fungus and the Bacillus in the solid‐state fermentation. Copyright © 2015 The Institute of Brewing & Distilling     

山西老陈醋醋酸发酵阶段醋醅疏松度的研究

通过对山西老陈醋醋酸发酵过程中醋醅质量容积的测定来反应醋醅疏松度的适合程度。经感官试验得知,质量容积在350.0～450.0g/L时醋醅疏松度适宜。通过L9（34）正交试验确定醋酸发酵时醋醅各项指标的最优组合。结果表明,最优组合为含水量65.0%～70.0%、酒精度5.0%vol～5.5%vol、麸皮量110.0%、质量容积350.0 g/L,在此条件下山西老陈醋总酸为4.96 g/100 g醋醅。     

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     

双轮底发酵过程中酒醅温度变化规律的探讨

温度对窖池酒醅发酵过程有着极为重要的影响,酒醅在发酵过程中的温度变化主要是环境温度、酒醅入窖温度及窖池微生物生长代谢三者共同作用的结果。该试验利用窖池温度检测系统对成都某酒厂采用浓香型白酒双轮底工艺的三口窖池酒醅温度进行实时精确检测,并绘制出其温度变化曲线。结果发现：采用双轮底工艺时,酒醅温度变化基本符合“前缓、中挺、后缓落”的传统经验,相比于普通工艺,双轮底酒醅温度变化更为平缓。