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Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

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Chinese Baijiu industry is undergoing industrial modernization. However, the key processes including Daqu processing are directly modified based on empirical knowledge, and the lack of engineering mechanisms lead to the inability of rational design. Here, this study quantitatively analyzed the heat generation and flow during Daqu solid-state fermentation, tracked and recorded the parameters during Daqu processing, simplified the biochemical reactions of the fermentation process, and built a Daqu heat balance model. Through the model, the heat generated during the whole fermentation process was transferred outside of the system through three ways: Water evaporation (54. 22% ), conduction of wall and air flow (42. 00% ), and air temperature rising ( < 1% ). The remaining heat (3. 77% ) was the cumulative heat, which was positively correlated with the temperature rise of the Daqu ( R² =0.85). It was verified that the average error of the model for the temperature change of dacquoise was 5. 05% with R² = 0. 94. Based on the model, the effect of regulating moisture parameters on the temperature of Daqu was explored, which showed the importance of moisture control for regulating the temperature during Daqu preparation. Through the preliminary exploration of the feasibility of regulating and optimizing the future production of Daqu from the perspective of mathematical modeling, the results of the study can provide theoretical support for the construction of modern fermentation process. [ABSTRACT FROM AUTHOR]

BACKGROUND: In industrial production of suancai, baijiu is commonly used to inhibit the spoilage bacteria and enhance the flavor. However, the effects of baijiu on the microbial diversity and metabolic pathways of suancai are rarely reported in the literature. This study aimed to explore the microbial community, its predicted functional roles, and the metabolites formed during fermentation of Chinese Dongbei suancai fermented using a mixed starter with Chinese baijiu as supplementary material. RESULTS: Results showed that Lactobacillus, Enterobacter, and Leuconostoc were the major bacterial genera in the Dongbei suancai fermented by adding baijiu. Linear discriminant analysis effect size indicated that Leuconostoc was the major biomarker in the early stage of fermentation, whereas Lactococcus, Weissella, and Lactobacillus plantarum were biomarkers in the middle and later stages of fermentation. A total of 638 metabolites were detected in suancai fermented by adding baijiu. However, the principal component analysis showed that baijiu significantly affected the metabolites of suancai in the early and later stages of fermentation. Furthermore, 58, 22, and 26 significantly differential metabolites (P < 0.01) were found on day 0, day 2, and day 30 of fermentation respectively. Moreover, Lactobacillus, Lactococcus, and Enterobacter had positive correlations with amino acids, nucleotides, organic acids, alcohols, and esters. Functional analysis implied that carbohydrate, amino acid, energy, and nucleotide metabolism were the major determinants of the characteristics of suancai fermented with baijiu as supplementary material. CONCLUSION: Baijiu changed the metabolites of inoculated fermented Dongbei suancai. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Saccharifying activity of starter culture plays a key role on the simultaneous saccharification and fermentation of Chinese baijiu fermentation. However, the regulation of alcoholic fermentation by Daqu starter or saccharifying enzymes still remains unclear in the baijiu fermentation. In this study, we identified 13 proteins, affiliated to 4 saccharifying enzymes (including alpha-amylase, beta-amylase, glucoamylase and beta-glucosidase) as the key saccharifying enzymes in Daqu by metaproteomic and culture-dependent analysis. These enzymes were metabolized from plant substrate (13.4%) and microbial metabolism (86.6%), and especially alpha-amylase and glucoamylase from Aspergillus and Rhizopus were dominant in Daqu bio-system. Furthermore, we observed the dynamic of saccharifying activity of Daqu (from 241 to 395 U) was driven by the combinations of isolated saccharifying microbes responsible for key enzymes releasing, indicating AR7 group (with a ration of Rhizopus oryzae : Aspergillus oryzae in 10:1) presented the highest saccharifying activity (395 U) in a simulative Daqu production. However, the highest ethanol production (80.5 mg/g) was obtained from the fermentation driven by AR8 group (with a ration of Rhizopus oryzae : Aspergillus oryzae in 50:1) with the saccharifying activity of 389 U. These results indicated that a proper combination of saccharifying microbes could enhance the saccharifying activity of Daqu and then promote ethanol production in baijiu fermentation, as it did not always exhibit a positive correlativity between saccharifying activity and alcoholic fermentation. Our findings should provide a potential approach to regulate the alcoholic fermentation by optimizing the saccharifying microbes or enzymes of starter cultures for baijiu and other fermented food. [Display omitted] • We identified four key saccharifying enzymes in Daqu. • Optimization of microbial proportions improved saccharifying activity. • A proper saccharifying activity promoted alcoholic fermentation. [ABSTRACT FROM AUTHOR]

Changes in the content and morphology of residual starch in Zaopei during strong‐flavor Baijiu fermentation are investigated. The results show that the residual starch content increases through the Baijiu production and that the residual starch content of distiller's grains (DGS) is the highest (76.35%). The crystalline structure of starch changes from A‐type (raw grain starch) to B + V‐type (Zaopei, Hongzao, Mianzao, DGS). The upper Zaopei starch has the typical diffraction peaks of B + V‐type (7.4°, 12.8°, 14.9°, 19.7°, 19.6°, 26.6°) and retains the typical diffraction peaks of A‐type (17.0°, 22.2°). The intermediate and bottom Zaopei starch has the typical diffraction peaks of B + V‐type (7.4°, 12.8°, 16.9°, 19.7°, 20.7°, 26.6°) and loses the typical diffraction peaks of A‐type. With the progression of Baijiu production, the gelatinization temperature and enthalpy of starch increase, while the water‐holding capacity, freeze‐thaw stability, solubility, and swelling power decrease. The above results show that the starch complex is the primary form of residual starch in strong‐flavor Baijiu. [ABSTRACT FROM AUTHOR]

Chinese Baijiu is one of the famous distilled liquor series with unique flavors in the world. Under the open environment, Chinese Baijiu was produced by two solid-state fermentation processes: jiuqu making and baijiu making. Chinese Baijiu can be divided into different types according to the production area, production process, starter type, and product flavor. Chinese Baijiu contains rich flavor components, such as esters and organic acids. The formation of these flavor substances is inseparable from the metabolism and interaction of different microorganisms, and thus, microorganisms play a leading role in the fermentation process of Chinese Baijiu. Bacteria, yeasts, and molds are the microorganisms involved in the brewing process of Chinese Baijiu, and they originate from various sources, such as the production environment, production workers, and jiuqu. This article reviews the typical flavor substances of different types of Chinese Baijiu, the types of microorganisms involved in the brewing process, and their functions. Methods that use microbial technology to enhance the flavor of baijiu, and for detecting flavor substances in baijiu were also introduced. This review systematically summarizes the role and application of Chinese Baijiu flavor components and microorganisms in baijiu brewing and provides data support for understanding Chinese Baijiu and further improving its quality. [ABSTRACT FROM AUTHOR]

Chinese Baijiu is one of the famous distilled liquor series with unique flavors in the world. Under the open environment, Chinese Baijiu was produced by two solid-state fermentation processes: jiuqu making and baijiu making. Chinese Baijiu can be divided into different types according to the production area, production process, starter type, and product flavor. Chinese Baijiu contains rich flavor components, such as esters and organic acids. The formation of these flavor substances is inseparable from the metabolism and interaction of different microorganisms, and thus, microorganisms play a leading role in the fermentation process of Chinese Baijiu. Bacteria, yeasts, and molds are the microorganisms involved in the brewing process of Chinese Baijiu, and they originate from various sources, such as the production environment, production workers, and jiuqu. This article reviews the typical flavor substances of different types of Chinese Baijiu, the types of microorganisms involved in the brewing process, and their functions. Methods that use microbial technology to enhance the flavor of baijiu, and for detecting flavor substances in baijiu were also introduced. This review systematically summarizes the role and application of Chinese Baijiu flavor components and microorganisms in baijiu brewing and provides data support for understanding Chinese Baijiu and further improving its quality.

N-propanol is a vital flavor compound of Chinese baijiu, and the proper n-propanol contents contribute to the rich flavor of Chinese baijiu. However, the excessive content of n-propanol in liquor will reduce the drinking comfort. Based on the Box–Behnken design principle, the response surface test was used to optimize the factors affecting the production of n-propanol in a simulated liquid state fermentation of Chinese baijiu, and the best combination of factors to reduce n-propanol content was determined. Results showed that the content ratio of additional glucose to threonine and temperature had a significant effect on the production of n-propanol (p = 0.0009 < 0.01 and p = 0.0389 < 0.05, respectively). The best combination of fermentation parameters obtained was: the ratio of additional glucose to threonine content was 6:4, the temperature was 32 °C, and the initial pH was 4.40. Under these conditions, the production of n-propanol was 53.84 ± 0.12 mg/L, which was close to the theoretical value. Thus, the fermentation parameter model obtained through response surface optimization is reliable and can provide technical guidance for regulating the production of n-propanol and realizing high-quality baijiu brewing. [ABSTRACT FROM AUTHOR]

d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W−N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W−N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS–tNDPS1 fusion protein, and the best strain yielded 9.8 mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation. [ABSTRACT FROM AUTHOR]

Appearance features serve as pivotal metrics for regulating the Daqu fermentation process and assessing its quality. Nevertheless, uncertainties persist regarding the variances in microbial community structure and potential functions during Daqu fermentation with distinct appearance features. Employing amplicon sequencing technology, this study scrutinized low-temperature Daqu sample groups (Y and C) exhibiting discernible differences in appearance features. The results elucidated that microbial α-diversity within the Y group aligned with fluctuations in temperature and moisture during LT-Daqu fermentation, a contrast observed in the C group. Concomitant β-diversity and dominant microbial succession analyses revealed disparate microbial community structures between these groups. Co-occurrence network analysis indicated heightened microbial interconnectivity within the Y group. Compared with the C group, the Y group showcased a greater abundance of relevant genes in the Glycolysis/Gluconeogenesis pathway. T test analysis delineated markedly distinct microorganisms at various fermentation stages, with Streptomyces , Staphylococcus , and Saccharomycopsis enriched in the Y group, and Lactobacillus , Wickerhamomyces , and Aspergillus in the C group. This comprehensive investigations revealed the difference of microbial community structure and function of LT-Daqu with different appearance features, providing insights for refining fermentation procedures and quality evaluation. Moreover, it contributes to an enhanced understanding of fermentation mechanisms in analogous domains. [Display omitted] • The microbial community exhibited significant differences between two types of LT-Daqu with distinct appearance features. • Two types of Daqu with different appearance features had the same dominant microorganisms. • Daqu with different appearance features can provide distinct functionality and flavor contributions. [ABSTRACT FROM AUTHOR]

Chinese starter Jiuqu , traditionally produced by spontaneous fermentation and always squeezed into bricks, serves as a vital saccharifying agent for simultaneous saccharification and fermentation of Chinese Baijiu. It is important to reveal the key saccharifying microbiota and the driving force to improve the quality of Jiuqu. Here we studied the compositions of the microbiota by high-throughput amplicons sequencing analysis in Jiuqu , and revealed eight bacterial and seven fungal genera as the dominant community members. Among them, Lactobacillus, Aspergillus, Pichia, Saccharomyces, Rhizopus were the main contributors of proteins by metaproteomics analysis. Whereas, only Lactobacillus, Pichia, Rhizopus appeared as key actors for saccharification by secreting three glycosidases and two glycosyltransferases, and it indicated they were the key saccharifying microbiota in Jiuqu. Especially, Rhizopus secreted the most abundant glucoamylase. Interestingly, these three active genera significantly decreased and the key saccharifying enzymes were down-expressed, when Jiuqu was produced in diffused shape with a low volumetric weight. Rhizopus microsporus , the main producer of glucoamylase, was positively correlated with volumetric weight of Jiuqu. It indicated volumetric weight was the major driving force of the key saccharifying microbiota in Jiuqu. This work provides deep insights of key saccharifying microbiota, and indicates the main driving force for the key microbe. Furthermore, this finding can contribute to the improvement of saccharifying agent for food fermentation. [ABSTRACT FROM AUTHOR]

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded β-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages. [ABSTRACT FROM AUTHOR]

High content of ethyl acetate may destroy the typical flavor of Chinese Nong-xiang Baijiu. Formation of ethyl acetate is closely related to its precursor acetic acid from Daqu starter in the fermentation process of Chinese Baijiu. In this study, the potential functional microbiota responsible for production of acetic acid was explored by correlation analysis and function prediction in Nong-xiang Daqu from different geographical regions. Our results revealed that the microbial community of Daqu starter was correlated with its acetic acid content. In addition, the relative abundances of Weissella , Lactobacillus , Bacillus , Kroppenstedtia , and Pichia were higher in the Daqu with high content of acetic acid. Furthermore, Acetobacter , Weissella , Lactobacillus , Pichia , Candida , and Rhizomucor were significantly (p < 0.01) and positively correlated with acetic acid. Meanwhile, higher abundances of most genes that encoding enzymes participated in glycolysis and pentose phosphate pathways for acetic acid synthesis, were observed by PICRUSt2 approach in the Daqu starters with high content of acetic acid. Taken together, Acetobacter , Weissella , Lactobacillus , Pichia , and Candida were determined as the potential functional microbiota for producing acetic acid in Nong-xiang Daqu starters. These results provided evidence of microbiota for revealing the possible causes of high acetic acid and ethyl acetate in Nong-xiang Baijiu brewing process, and laid theoretical foundation for regulation of Baijiu fermentation by functional microbiota. [Display omitted] • Microbiota of Daqu starter was correlated with its acetic acid content. • Acetobacter , Weissella , Lactobacillus , Pichia and Candida were involved in acetic acid formation. • Potential pathways for acetic acid formation were proposed by functional prediction. [ABSTRACT FROM AUTHOR]

Chinese Baijiu (Chinese spirit or Chinese liquor) is one of the six major distilled spirits in the world, and is renowned overseas. Chinese Baijiu has a long history of more than 1000 years when people first begin to brew liquor using distillation. So far, Chinese Baijius have formed 12 types according to their flavour characteristics. The strong flavour Chinese spirit is regarded as one of the typical representatives, and occupies a > 70% market share in China. The liquor brewing process is under an open environment, with grains as the fermentation substrates, as well as Daqu as the saccharifying ferment. The brewing process is complex and impacted by multiple factors. Among the factors, the microorganisms have important influences on the brewing process and product quality of liquors. In the past decades, a series of research achievements have been made with strong flavour Chinese spirits. However, reviews related to this field are relatively few. In this paper, we have reviewed strong flavour Chinese spirits including the introduction, brewing process, microorganism diversity of Daqus, fermented grains and pit muds, and the application of microbes. Copyright © 2017 The Institute of Brewing & Distilling [ABSTRACT FROM AUTHOR]

• Effects of mechanization on microflora and flavor in Baijiu production were studied. • Pichia kudriavzevii was more active than Saccharomyces cerevisiae. • Five core microbes were detected both in metagenomic and metatranscriptomic analyses. • The expression level of 9 genes related to higher alcohol formation were determined. Microorganisms play an important role in the flavor formation of Chinese Baijiu. Mechanization is the way to develop Baijiu. Therefore, it is necessary to study the effects of mechanization on microbial community and flavor in Baijiu production. The microbial communities exhibited differences between two technologies with the fermentation, and the numbers of yeasts and bacteria in new mechanical technology were significantly higher than those in traditional technology at the peak of fermentation. Both metagenomic and metatranscriptomic analyses showed 5 core microorganisms in the fermentation, namely Saccharomyces cerevisiae , Rhizopus delemar , Pichia kudriavzevii , Lactobacillus helveticus , and Rhizopus oryzae. S. cerevisiae was generally regarded to be the most dominant yeast in Baijiu fermentation, but our metatranscriptomic data showed that P. kudriavzevii was more active than S. cerevisiae. These two analyses indicated that higher initiation abundance of S. cerevisiae and P. kudriavzevii and lower initiation abundance of R. delemar and R. oryzae were observed in traditional technology than in new technology, and that Lactobacillus displayed apparent advantages in traditional technology, whereas Lactobacillus and yeast exhibited obvious advantages in new technology at the end of fermentation. In addition to S. cerevisiae, other microorganisms including non-saccharomyces yeasts, molds, and bacteria were involved in higher alcohol formation. This work provides insight into the microbial dynamics and higher alcohol formation, as well as an efficient strategy for process improvement in Baijiu fermentation. [ABSTRACT FROM AUTHOR]

• Two l -cysteine high-producing Bacillus were inoculated in Baijiu fermentation. • l -cysteine content increased after Bacillus inoculation in Baijiu fermentation. • Bacillus inoculation increased 2-furfurylthiol contents in Baijiu. • Bacillus inoculation affected the microbial community structure. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]