Quality Of Baijiu Research Articles

Content changes of Jiupei tripeptide Tyr-Gly-Asp during simulated distillation process of baijiu and the potential in vivo antioxidant ability investigation

Baijiu is the national liquor of China. Fermented grain (Jiupei) is the main material for baijiu distillation process. A tripeptide Tyr-Gly-Asp (YGD) with in vitro antioxidant ability was isolated from Jiupei. As one of the non-volatile components, existence of Jiupei peptides after baijiu distillation is still unclear. Study aims to investigate the content changes of total peptides and YGD during the high temperature of distillation process, YGD stability in baijiu and its influence on baijiu characters. AAPH induced oxidative stress Sprague Dawley (SD) rat model was used to evaluate YGD antioxidant and anti-inflammatory activities in vivo. Results showed that YGD content changed under the high temperature of simulated distillation process, exist stably in baijiu and show little influence on baijiu quality. YGD was able to attenuate AAPH-induced oxidative stress by elevating the expressions of Nrf2/Keap1-p38MAPK/PI3K-MafK signaling pathway and downstream antioxidant enzymes. In addition, YGD significantly inhibited the overproduction of inflammatory cytokines and mediator caused by AAPH in rat serum. In light of these findings, YGD is a tripeptide with in vivo antioxidant activity isolated from Jiupei, which proves functional peptides in baijiu are traced from Jiupei and lay a foundation for the development of healthy baijiu.

Isolation and Identification of a High-Yield Ethyl Caproate-Producing Yeast From Daqu and Optimization of Its Fermentation.

Baijiu is an important fermented product in China. A yeast named YX3307 that is capable of producing a large amount of ethyl caproate (EC) was isolated from Daqu, a crude fermentation starter for Baijiu. This yeast was identified as Clavispora lusitaniae on the basis of its morphological properties, physiological and biochemical characteristics, and 26S rDNA sequence. Single-factor experiments were conducted to obtain the optimum fermentation conditions for EC production by YX3307. The highest EC yield (62.0 mg/L) from YX3307 was obtained with the following culture conditions: inoculum size 7.5% (v/v), seed cell age 30 h, sorghum hydrolysate medium (SHM) with a sugar content of 10 Brix and an initial pH of 6.0; incubation at 28°C with shaking at 180 rpm for 32 h; addition of 10% (v/v) anhydrous ethanol and 0.04% (v/v) caproic acid at 32 and 40 h, respectively, static culture at 20°C until 72 h. YX3307 synthesized more EC than ethyl acetate, ethyl lactate, ethyl butyrate, and ethyl octanoate. An intracellular enzyme or cell membrane enzyme was responsible for EC synthesis. YX3307 can produce many flavor compounds that are important for high-quality Baijiu. Thus, it has potential applications in improving the flavor and quality of Baijiu.

Profiling the influence of physicochemical parameters on the microbial community and flavor substances of zaopei

Strong-flavor baijiu is a traditional distilled alcoholic beverage with a long history in China. The fermented grains play a pivotal role in the production of baijiu. The purpose of this study was to evaluate and compare the microbiota and flavor substances present in fermented zaopei (ZP) from pits of different ages. High-throughput sequencing, headspace solid-phase microextraction gas chromatography-mass spectrometry, principal component analysis, community composition analysis, and redundancy analysis were used to analyze and evaluate the impact of environmental factors on microbial communities and flavor substances. Six genera of bacteria (e.g., Caproiciproducens, Syntrophaceticus, Sedimentibacter, Hydrogenispora, Pelotomaculum and Bacillus) and seven genera of fungi (Cladosporium, Debaryomyces, Dipodascus, Auxarthron, Cephalotrichum, unclassified Stachybotryaceae, unclassified Microascaceae and Cephalotrichum) notably affected the production of hexanoic acid (an important flavor compound). Moisture and alcohol content also had considerable effects on the production of the flavor compounds such as ethyl lactate, hexanoic acid, and ethyl hexanoate. The profiles of volatile compounds present in ZP were different between the aged and new pits; these profiles were mainly reflected in the concentration and types of alcohols, aldehydes, esters, and aromatic compounds. This paper provides a comprehensive overview of the physicochemical parameters, flavor substances, and microbial population distribution of ZP. Characterization of various ZP samples help to elucidate the fermentation mechanisms and offer a theoretical reference to control and enhance the quality of Baijiu. © 2021 Society of Chemical Industry.

Application of Wickerhamomyces anomalus in Simulated Solid-State Fermentation for Baijiu Production: Changes of Microbial Community Structure and Flavor Metabolism.

Wickerhamomyces anomalus is conducive to the synthesis of ester compounds in brewing the Chinese liquor Baijiu; esters are crucial for the quality of Baijiu. In this study, simulated solid-state fermentation for Baijiu production was used to explore whether artificial addition of W. anomalus could improve the flavor substance in Baijiu, and the underlying mechanisms. Two experimental groups were studied, in which W. anomalus Y3604 (Group A) and YF1503 (Group B) were added, respectively; in the control group (Group C), no W. anomalus was added. Adding strain Y3604 increased the content of esters in fermentation samples, especially ethyl acetate and ethyl caproate, and reduced the content of higher alcohols. Adding strain YF1503 had little effect on the ester content but decreased the content of higher alcohols. The diversity and abundance of prokaryotic genera in Group A and B samples were similar, but there were some differences compared with Group C. The correlations of genera in Group A or B samples were simple compared with group C. Although the predominant eukaryotic genera in the three groups were consistent, the abundance of each gene varied among groups. Based on our findings, bioaugmentation of Baijiu fermentation with W. anomalus will change the ethyl acetate content and cause changes in the levels of other flavor substances. We suggest that the changes in flavor substances caused by the addition of W. anomalus are mainly due to changes in the microbial community structure that result from the addition of W. anomalus.

Increasing 2-furfurylthiol content in Chinese sesame-flavored Baijiu via inoculating the producer of precursor l-cysteine in Baijiu fermentation

2-Furfurylthiol was an important contributor to the flavor of traditional fermented foods including Baijiu. It is essential to increase 2-furfurylthiol concentration to improve the quality of Baijiu. This study aimed to enrich the content of 2-furfurylthiol in Chinese sesame-flavored Baijiu via two strains we isolated from Baijiu fermentation, Bacillus subtilis LBM 10019 and Bacillus vallismortis LBM 10020, which could respectively produce 56.31 mg/L and 42.81 mg/L l-cysteine, the precursor of 2-furfurylthiol, in sorghum extract. After inoculation of these two strains, the maximal relative abundance of Bacillus increased from 7.48% to 40.38%, the final content of l-cysteine increased by 101.44% in Baijiu fermentation. Moreover, the concentration of 2-furfurylthiol increased by 89.15% in the production. This work provides a novel strategy to improve the quality of Chinese sesame-flavored Baijiu.

Molecular mechanism of the role of Mare Nectaris in the Feng-Flavor Baijiu aging

A special storage container called Mare Nectaris bears a close relationship with Feng-flavor Baijiu quality during aging. The goal of this work is to unveil molecular mechanism of the role of Mare Nectaris in the Feng-flavor Baijiu aging as well as establishes a classification method of vintage Baijiu. Based on ultra high performance liquid chromatography quadrupole-orbitaltrap high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS), most micromolecules especially nonvolatile molecules in Baijiu can be accurately identified and in total of 45 characteristic molecules were discovered through application of the principal component analysis (PCA), partial least square-discriminant analysis (PLS-DA), variable importance in projection (VIP). With the increase of aging year, in addition to 5-fluoro-2,3,4-three chlorobenzoic acid decreased, the other 44 components showed remarkable up-regulation, concretely comprising 10 kinds of esters, 6 organic acids, 8 amino acids, 11 bioactive components and 9 other components. By utilizing artificial neural network recognition model which used 45 characteristic molecules as inputs and 8 groups various aging years as outputs, the prediction accuracy of Baijiu vintage was up to 100%.

Profiling of Fungal Diversity and Fermentative Yeasts in Traditional Chinese Xiaoqu.

To increase the safety and quality of baijiu and rice wine in China, controlling the use of traditional Xiaoqu by studying the beneficial yeasts present has recently been considered. The fungal diversity of six Chinese Xiaoqu including five traditional and one commercial samples was investigated to screen fermentative yeasts with low yields of higher alcohols. A high throughput sequencing approach detected fifteen fungal species with relative abundance higher than 1%, and displayed dissimilarities of fungal diversity among Xiaoqu samples. The 15 fungal species were composed of 11 filamentous fungi with Rhizopus arrhizus as the most common specie and four yeast species, containing Hyphopichia burtonii, Saccharomyces cerevisiae, Saccharomycopsis fibuligera, and Saccharomycopsis malanga. Classic culture-dependent approaches, including 5.8S-ITS-RFLP analysis and D1/D2 sequencing of the 26S rRNA gene, identified nine yeast species in the five traditional Chinese Xiaoqu. In addition to the four yeast species also detected by high throughput sequencing approach, the other five yeast species isolated were Clavispora lusitaniae, Cryptococcus neoformans, Komagataella pastoris, Trichosporon asahii, and Wickerhamomyces anomalus. Further micro-fermentations of rice wine were performed using 19 single yeast isolates, and after the fermentation of rice wine, higher alcohols and ethanol were analyzed by gas chromatography. Two yeast strains, Saccharomyces cerevisiae FBKL2.8022 and Wickerhamomyces anomalus FBKL2.8023, were found to have low yields of higher alcohols and could produce 11.70%vol and 7.10%vol ethanol separately. This study for the first time, to the best of our knowledge, explored the fungal resources in traditional Xiaoqu from different regions of Guizhou, China. The screened S. cerevisiae and W. anomalus strains could be used to establish specific starters to promote the standardization of the production of baijiu and rice wine.

Characterization of Microbial Diversity and Community Structure in Fermentation Pit Mud of Different Ages for Production of Strong-Aroma Baijiu.

In the traditional fermentation process of strong-aroma Baijiu, a fermentation pit mud (FPM) provides many genera of microorganisms for fermentation. However, the functional microorganisms that have an important effect on the quality of Baijiu and their changes with the age of fermentation pit (FP) are poorly understood. Herein, the Roche 454 pyrosequencing technique and a phospholipid fatty-acid analysis were employed to reveal the structure and diversity of prokaryotic communities in FPM samples that have been aged for 5, 30, and 100 years. The results revealed an increase in total prokaryotic biomass with an FP age; however, Shannon’s diversity index decreased significantly (p < 0.01). These results suggested that a unique microbial community structure evolved with uninterrupted use of the FP. The number of functional microorganisms, which could produce the flavor compounds of strong-aroma Baijiu, increased with the FP age. Among them, Clostridium and Ruminococcaceae are microorganisms that directly produce caproic acid. The increase of their relative abundance in the FPM might have improved the quality of strong-aroma Baijiu. Syntrophomonas, Methanobacterium, and Methanocorpusculum might also be beneficial to caproic acid production. They are not directly involved but provide possible environmental factors for caproic acid production. Overall, our study results indicated that an uninterrupted use of the FP shapes the particular microbial community structure in the FPM. This research provides scientific support for the concept that the aged FP yields a high-quality Baijiu.

Directional design of a starter to assemble the initial microbial fermentation community of baijiu

Tetramethylpyrazine (TTMP) is an important aroma compound in the sesame-flavored Chinese liquor, baijiu. Similar to other traditional spontaneously fermented foods, it is difficult to directionally increase the key aroma compound in situ fermentation system of baijiu without changing its sensory profile. The assembly of the starting microbial community for fermentation by using a functional starter provides a promising solution which needs careful manipulations. This study aimed to increase TTMP concentration in baijiu using the functional starter inoculated with the indigenous strain B. amyloliquefaciens XJB-104 with high TTMP production ability. After inoculation, the relative abundance of Bacillus in the initial stage of fermentation increased from 82.14% to 88.47%. The TTMP concentration increased by about 26 and 24-fold in the fermented grains (zaopei) and baijiu respectively compared with controls. Moreover, the quality of baijiu was improved according to sensory analyses. In addition, correlation analysis confirmed that the increased TTMP content in baijiu was due to the successful assembly of the initial fermentation microbiota after the inoculation of B. amyloliquefaciens.

Quantitation of pyrazines in Baijiu and during production process by a rapid and sensitive direct injection UPLC-MS/MS approach

Pyrazines are one type of the most important aroma compounds in Baijiu, and a novel method of pyrazines detection was developed in this work. Baijiu samples were analyzed by direct injection ultra–performance liquid chromatography coupled with tandem mass spectrometry operating in positive electrospray ionization mode. The approach achieved good extraction recoveries (78.24–102.58%), good repeatability with intra-day, inter-day, and inter-week relative standard deviations of 0.84–4.17%, 0.78–6.71%, and 4.50–8.61% in respective, and short analysis time. Moreover, the optimized method was applied to the analysis of different typical aroma-type Baijiu. The unsupervised principal component analysis clearly differentiated four typical aroma-type Baijiu samples based on the pyrazines concentrations. The pyrazines concentrations showed a significant increase during fermentation steps and distillation time. This work provides an efficient strategy for determining the pyrazines levels in Baijiu and is beneficial for improving Baijiu quality.

Multiple Batches of Fermentation Promote the Formation of Functional Microbiota in Chinese Miscellaneous-Flavor Baijiu Fermentation.

Baiyunbian baijiu, the most popular miscellaneous-flavor baijiu in China, has been widely consumed for decades. Similar to many Chinese baijiu, Baiyunbian baijiu is fermented in five successive batches every year. Sensory analysis demonstrated that the raw baijiu obtained from the last two fermentation batches always has better quality than that produced from the former three batches. In this study, the microbial compositions of fungi and bacteria in each fermentation batch were investigated via high-throughput sequencing. The results showed that Bacillus, Virgibacillus, and Lactobacillus dominated the bacterial community in the last two batches, and the most prevalent fungi were Paecilomyces, Saccharomyces, and Zygosaccharomyces. In contrast, large percentages of fungi belonging to Thermomyces, Thermoascus, Monascus, and Issatchenkia and prokaryotes belonging to Acetobacter, Lactobacillus, and Thermoactinomyces were observed in the former three fermentation batches. GC-MS analysis revealed that the fermented grains sampled from the latter two batches contained high concentrations of ethyl lactate, 2,3-butanediol and ethyl caproate, which were mainly generated by co-fermentation of Lactobacillus and yeast. The high acidity of the fermented grains in the fourth and fifth fermentation batches as well as the large contents of ethanol and moisture promoted the formation of the functional microbial community. This study provides insight into factors that influenced the baijiu fermentation and is helpful for developing new fermentation techniques with higher baijiu quality.

Schizosaccharomyces pombe can Reduce Acetic Acid Produced by Baijiu Spontaneous Fermentation Microbiota.

The spontaneous fermentation of alcoholic beverage is a bioprocess donated by microbiota with complex stress environments. Among various microbes, non-Saccharomyces yeasts have high stress tolerance and significantly affect the taste and quality of products in process. Although many researchers have focused on the influence of acid stress, the mechanism of non-Saccharomyces yeasts to tolerant stress remains unclear in microbiota. To bridge the gap, we constructed in situ and in vitro studies to explore the reduction pathway of acetic acid in non-Saccharomyces yeasts. In this study, we found Schizosaccharomyces pombe has special capacities to resist 10 g/L acetic acid in laboratory cultures and decrease the average concentration of acetic acid from 9.62 to 6.55 g/kg fermented grains in Chinese Maotai-flavor liquor (Baijiu) production. Moreover, Schi. pombe promoted metabolic level of mevalonate pathway (high expressions of gene ACCAT1, HMGCS1, and HMGCR1) to degrade a high concentration of acetic acid. Meanwhile, Schi. pombe also improved the concentration of mevalonic acid that is the precursor of terpenes to enhance the taste and quality of Baijiu. Overall, the synchronicity of reduction and generation in Schi. pombe advances the current knowledge to guide more suitable strategies for mechanism studies of non-Saccharomyces yeasts in fermented industries of alcoholic beverages.

Enhanced production of ethyl acetate using co-culture of Wickerhamomyces anomalus and Saccharomyces cerevisiae

The style and quality of Baijiu is greatly influenced by ethyl acetate. Therefore, improving and controlling the ethyl acetate levels in Baijiu is important. This study investigated ethyl acetate production using a co-culture of Saccharomyces cerevisiae Y3401 and Wickerhamomyces anomalus Y3604. More ethyl acetate was produced in mixed fermentations using both yeasts than in single fermentations. The highest ethyl acetate yield was 6.41g/L using a Y3401/Y3604 ratio of 3:1. Synergistic fermentation using both yeasts not only improved ethyl acetate production, but also increased the contents of other flavor compounds, such as β-phenethyl alcohol and phenethyl acetate. Therefore, the co-culture of S.cerevisiae and W.anomalus had a positive effect on ethyl acetate production and provides opportunities for altering the aroma and flavor perception of Baijiu.

Improving Ethyl Acetate Production in Baijiu Manufacture by Wickerhamomyces anomalus and Saccharomyces cerevisiae Mixed Culture Fermentations.

Ethyl acetate content has strong influence on the style and quality of Baijiu. Therefore, this study investigated the effect of Saccharomyces cerevisiae Y3401 on the production of ethyl acetate by Wickerhamomyces anomalus Y3604. Analysis of cell growth showed that Y3401 influences Y3604 by nutrient competition and inhibition by metabolites, while the effect of Y3604 on Y3401 was mainly competition for nutrients. Mixed fermentation with two yeasts was found to produce more ethyl acetate than a single fermentation. The highest yield of ethyl acetate was 2.99 g/L when the inoculation ratio of Y3401:Y3604 was 1:2. Synergistic fermentation of both yeasts improved ethyl acetate production and increased the content of other flavor compounds in liquid and simulated solid-state fermentation for Baijiu. Saccharomyces cerevisiae had a positive effect on ethyl acetate production in mixed culture and provides opportunities to alter the aroma and flavor perception of Baijiu.

Characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method

Chinese Nong-flavor (NF) daqu has been enriched with plenty of active enzymes by man-made environment for thousand years. Based on our previous metatranscriptomics, an endo-β-glucanase gene (NFEg16A), which showed high expression level in NF daqu, was directly obtained and expressed in Escherichia coli BL21 (DE3). NFEg16A shared the highest sequence identity of 87% with endo-1,3-1,4-β-glucanase from Paecilomyces thermophile. It was optimally active at pH 6.5 and 60 °C and highly stable (>75% residual activity) at pH 3–8 and temperature 30–90 °C. The activity of NFEg16A was strongly inhibited by 10 mM Fe3+ and Hg2+. Compared with endoglucanases with high similarities, NFEg16A was more stable at 70 °C and had higher half-lives of 3.4 h and 1.4 h at 80 °C and 90 °C, respectively. Its specific activity was 85.3 U/mg on barley β-glucan. Moreover, NFEg16A could efficiently hydrolyze substrate at high concentration of 15 mg/mL, and released glucose and cellobiose as its main end-products. Therefore, this work to some extent verified the important role of NFEg16A in NF daqu, and it would stimulate the acquisition of more enzymes from NF daqu to improve the baijiu quality in future. High thermostability of NFEg16A could also strengthen its potential applications in feed industry.

Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Wickerhamomyces anomalus for Chinese Baijiu making

Yeasts are the most important microorganisms in the fermentation of Chinese Baijiu and the interaction of these yeasts could impact the quality of Baijiu. In this study, we initially characterized the Baijiu yeasts and evaluated their fermentation potential. Wickerhamomyces anomalus GZ3 and Saccharomyces cerevisiae G20 were found to generate high yields of ethyl acetate (2.76g/L) and 2-phenylethanol, respectively. Results also indicated that the use of W.anomalus along with S.cerevisiae increased volatile compounds production, the maximum ethyl acetate production was observed in S.cerevisiae and W.anomalus at 106:106 ratio and have increased by 33 % compared with single culture of W.anomalus. Besides, there was a significant increase of 2-phenylethanol (3.29g/L) production in single culture of S.cerevisiae with the addition of l-phenylalanine. However, the conversion of l-phenylalanine in mixed culture had significant impact on the yeasts interaction and end flavor of Baijiu. Thus, the present study provided new insights into yeasts interactions in Baijiu fermentation and the effect of some primary metabolites on the end flavor and Baijiu quality.

Systematic Characterization of the Metabolism of Acetoin and Its Derivative Ligustrazine in Bacillus subtilis under Micro-Oxygen Conditions.

Bacillus subtilis is an important microorganism for brewing of Chinese Baijiu, which contributes to the formation of flavor chemicals including acetoin and its derivative ligustrazine. The first stage of Baijiu brewing process is under micro-oxygen conditions; however, there are few studies about B. subtilis metabolism under these conditions. Effects of various factors on acetoin and ligustrazine metabolism were investigated under these conditions, including key genes and fermentation conditions. Mutation of bdhA (encoding acetoin reductase) or overexpression of glcU (encoding glucose uptake protein) increased acetoin concentration. Addition of Vigna angularis powder to the culture medium also promoted acetoin production. Optimal culture conditions for ligustrazine synthesis were pH 6.0 and 42 °C. Ammonium phosphate was shown to promote ligustrazine synthesis in situ. This is the first report of acetoin and ligustrazine metabolism in B. subtilis under micro-oxygen conditions, which will ultimately promote the application of B. subtilis for maintaining Baijiu quality.

Isolation and Identification of High-Yield Ethyl Acetate-Producing Yeast from Gujinggong Daqu and Its Fermentation Characteristics

ABSTRACTEthyl acetate is used to judge the style and quality of baijiu. Therefore, it is of significance to improve and strengthen the stability of baijiu quality by improving and controlling the levels of ethyl acetate. The aroma-producing yeasts are the main strains that produce ethyl acetate and have been used to better effect in baijiu production. In this article, a high-yield ethyl acetate-producing yeast strain named Y3604 was isolated from Gujinggong Daqu by screening and ethyl acetate analysis. According to the morphological properties, physiological and biochemical characteristics, and 26S rDNA sequence analysis, strain Y3604 was identified as Wickerhamomyces anomalus. The optimum fermentation conditions of Y3604 for producing ethyl acetate were obtained by single-factor experiments. When W. anomalus Y3604 was inoculated into sorghum hydrolysate medium (SHM) with the addition of 4% anhydrous ethanol and 0.1% acetic acid, fermented at 25°C with a shaking speed of 210 rpm for 96 h, and a 8°Brix medium, the total ethyl acetate production was 16.92 g/L. This strain has the potential to increase the content of ethyl acetate in baijiu production and enhance the aroma characteristics of baijiu. Thus, it has potential application value in improving the quality of baijiu.

Quantification of volatile compounds in Chinese soy sauce aroma type liquor by stir bar sorptive extraction and gas chromatography–mass spectrometry

Considering the important of aroma compounds on the quality of baijiu, stir bar sorptive extraction (SBSE) coupled with gas chromatography-mass spectrometry was applied to the aromas analysis of baijiu (Chinese liquor). Based on the results of optimization, a total of 76 volatile compounds were identified from 14 Chinese liquors, including 25 esters, 10 alcohols, 9 aldehydes and ketones, 8 aromatic compounds, 5 furans, 3 nitrogen-containing compounds, 6 acids, 4 phenols, 3 terpenes, 1 sulfide-containing compound, 1 lactone, and 1 acetal. By principal component analysis based on quantification results, 14 Chinese liquor samples could be classified into three groups. SBSE is a fast, simple, effective, and reliable method for extracting volatile compounds from Chinese liquor. The chemometrics approach revealed that LJ liquor with soy sauce aroma could exhibit more prominent sauce flavor through extending the time of storage, and its peculiar manufacturing practice was responsible for the soy sauce flavor.