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Abstract Butyric acid is a volatile saturated monocarboxylic acid, which is widely used in the chemical, food, pharmaceutical, energy, and animal feed industries. This study focuses on producing butyric acid from pre-treated rape straw using simultaneous enzymatic hydrolysis semi-solid fermentation (SEHSF). Clostridium beijerinckii BRM001 screened from pit mud of Chinese nongxiangxing baijiu was used. The genome of C. beijerinckii BRM001 was sequenced and annotated. Using rape straw as the sole carbon source, fermentation optimization was carried out based on the genomic analysis of BRM001. The optimized butyric acid yield was as high as 13.86 ± 0.77 g/L, which was 2.1 times higher than that of the initial screening. Furthermore, under optimal conditions, non-sterile SEHSF was carried out, and the yield of butyric acid was 13.42 ± 0.83 g/L in a 2.5-L fermentor. This study provides a new approach for butyric acid production which eliminates the need for detoxification of straw hydrolysate and makes full use of the value of fermentation waste residue without secondary pollution, making the whole process greener and more economical, which has a certain industrial potential. Graphical Abstract

Ferulic acid (FA) and ferulic acid methyl ester (FAM) are important phenolic compounds in Baijiu. In this study, the interaction of FA and FAM with human serum albumin (HSA) and lysozyme (LZM) was investigated using multispectral methods and molecular dynamics simulation. FA and FAM could interact with HSA and LZM, changing the conformation and hydrophilicity of the protein. The quenching mechanisms of FA-HSA, FA-LZM, FAM-HSA, and FAM-LZM were all static-quenching. In the FA-HSA, FAM-HSA, and FA-LZM systems, the interaction forces were mainly hydrophobic interactions and hydrogen bonding. In the FAM-LZM system, the interaction forces were mainly hydrophobic interactions, hydrogen bonding, and van der Waals force. Common metal ions such as K+, Ca2+, Cu2+, Mg2+, and Mn2+ could affect the binding ability of FA and FAM to HSA and LZM. Moreover, FA and FAM could increase the stability of HSA and LZM, and the protein bound to FA/FAM was more stable than the free protein. FA and FAM had varying degrees of impact on the physiological activities of HSA and LZM. This study provides relevant information on the interactions and metabolic mechanisms of FA and its derivatives with endogenous proteins.

The thermophilic phase of Daqu fermentation is considered the key period for aroma production in Daqu, but little is known about the changes in substances during this phase. In this study, we combined a metabolomics approach with high-throughput sequencing to analyze the metabolic profiles and identify metabolism-associated microbes during the thermophilic phase of Daqu fermentation. The results revealed that the metabolic sets after 5 and 9 days of fermentation in the thermophilic phase were similar, and several amino acid and biosynthesis-related metabolic pathways were significantly enriched. In addition, pyrazines and alkanes increased and esters decreased significantly after the thermophilic phase. The metabolism of substances during the thermophilic phase involved 38 genera, and the main metabolic pathways involved were glycolysis, TCA cycle, butyric acid metabolism, and five amino acid metabolic pathways. In summary, this study points in the direction for unravelling the mechanism of aroma production in Daqu.

Abstract Understanding how Baijiu facilitates blood circulation and prevents blood stasis is crucial for revealing the mechanism of Baijiu for cardiovascular disease (CVD) risk reduction. Here we established a zebrafish thrombosis model induced using arachidonic acid (AA) to quantitatively evaluate the antithrombotic effect of Wuliangye Baijiu. The prevention and reduction effects of aspirin, Wuliangye, and ethanol on thrombosis were compared using imaging and molecular characterization. Wuliangye Baijiu reduces thrombotic risks and oxidative stress in the AA-treated zebrafish, while ethanol with the same concentration has no similar effect. The prevention and reduction effects of Wuliangye on thrombosis are attributed to the change in the metabolic and signaling pathways related to platelet aggregation and adhesion, oxidative stress and inflammatory response.

Daqu is an important saccharifying starter in the fermentation of Nongxiangxing Baijiu in China. Vanillin is a health and flavor factor in Baijiu. However, only a few research studies on the vanillin content of Daqu are currently not systematic. In order to investigate the metabolic mechanism of vanillin in the fermentation process of Daqu, we analyzed the changes in microorganisms, influencing factors, and enzymes related to vanillin in Daqu. This research found that there were differences between bacterial and fungal genera in each sample, and the abundance of bacteria was greater than that of fungi. Among the microbial genera, Klebsiella, Escherichia, Acinetobacter, Saccharopolyspora, Aerococcus, and Puccinia were positively correlated with vanillin. Meanwhile, we also found that moisture and reducing sugar were the main physicochemical factors affecting the formation of vanillin. The functional annotation results indicate that carbohydrate metabolism and energy metabolism were important microbial metabolic pathways that impacted vanillin production in solid-state fermentation. The feruloyl-CoA hydratase/lyase (EC 4.1.2.61) and acylamidase (EC 3.5.1.4) were positively correlated with vanillin content (p ≤ 0.05) and promote the increase in vanillin content. These findings contribute to furthering our understanding of the functional microorganisms, physicochemical factors, and enzymes related to the change in vanillin content during the fermentation of Daqu and can help to further explore the flavor substances in Baijiu fermentation in the future.

Daqu is a saccharifying agent, starter culture agent and aroma generating agent in the process of liquor brewing. The quality of Daqu directly affects the quality of liquor. In order to find the Daqu’s optimal molding process parameters during the pressing process of Daqu’s multi position press, this paper deployed area ratio of surface pulping and the uniform factor of internal moisture content of Daqu as evaluation indexes. Surface response experiment studied the influence of three factors of different pressing speed, stepping times and forming times on Daqu，s pulp extraction effect and moisture content uniformity. The research results showed that the impact of each process parameter on the pulping effect of Daqu ranked as follows: Stepping times>forming times>pressing speed. The influence on the uniformity of Daqu water content was pressing speed>stamping times>forming times. The best process parameters were: Pressing speed (A)=20 mm/s, times of trampling (B)=20, and times of molding (C)=7. Under the conditions of the process parameters, the Daqu obtained by pressing Daqu showed that the effective area ratio of pulping was 85.66%, and the uniformity factor of water content inside Daqu was 6.37. The thesis provided productive and practical technical support for the standardization and industrialization of liquor Daqu pressing production.

Abstract Background Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown. Results In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region. Conclusions This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.

Traditionally, Ya’an Tibetan tea is routinely consumed by local people in the Tibet region. It is believed to possess promising anti-inflammatory benefits. This study was conducted to elucidate the protective impact of Tibetan tea extract (TTE) on dextran sodium sulfate (DSS)-induced colitis in mice. Mice were split into four groups: control (C) group, Tibetan tea (T) group, DSS-induced model (CD) group, and Tibetan tea + DSS (TD) group. The intake of TTE significantly reduced the clinical symptoms of ulcerative colitis (UC) by alleviating the impact of cellular damage and reducing glandular hypertrophy and the infiltration of inflammatory cells. UC led to a prominent shift of the microbial communities in the gut. Interestingly, the beneficial microbes, such as Lactobacillus reuteri, Bifidobacterium choerinum, and Lactobacillus intestinalis, were significantly increased in TTE-treated mice when compared to any other experimental group. The transcriptome analysis revealed that the positive effect of TTE on UC could be attributed to changes in the G alpha (i) signaling pathway and the innate immune system. The genes related to inflammation and immune system pathways were differentially expressed in the TTE-treated group. Moreover, the relative expression of genes linked to the inflammatory TLR4/MyD88/NF-κB signaling pathway was significantly downregulated toward the level of normal control samples in the TD group. Overall, this study revealed the modulatory effect by which TTE reversed the development and severity of chronic colon damage.

Strong aromatic liquors, also known as strong aromatic Baijiu (SAB) in China, are manufactured by solid fermentation, with a multi-microbe mixing and cooperative fermentation process that uses Daqu as a brewing starter. Bacillaceae have a specific action in food fermentation, such as soybean and wine, and more recent studies have found Bacillaceae play important roles in the SAB making industry. This review describes the diversity, functionality, and influence of Bacillaceae in Daqu, pit mud, Zaopei, Huangshui within making processes of SAB. Furthermore, aromatic flavor components from the Bacillaceae metabolism of SAB are discussed in this review. Ultimately, the resulting improvements and deeper understanding will benefit practical efforts to apply representatives of Bacillaceae in improving the quality of SAB as well as biological control of the micro-ecological environment of brewing.

Actinomycetes (a group of filamentous bacteria) are the dominant microbial order in the Daqu (DQ) fermentation starter and in the pit mud (PM) of the Baijiu fermentation microbiome. Actinomycetes produce many of the key enzymes and flavor components, and supply important precursors, which have a major influence on its characteristic aroma components, to other microorganisms during fermentation. This paper reviews the current progress on actinomycete research related to Baijiu fermentation, including the isolation and identification, distribution, interspecies interactions, systems biology, and main metabolites. The main metabolites and applications of the actinomycetes during Baijiu fermentation are also discussed.

Daqu is an important saccharifying and fermenting agent. It provides various microorganisms and enzymes for the fermentation of Baijiu and plays a vital role in the formation of Baijiu flavor. However, it is difficult to obtain information on microbial growth and metabolism in time for Daqu production. Therefore, the “Qu Xiang” obtained by smelling is an important index in the traditional production process to evaluate the microbial fermentation in the process of Daqu-making, “Qu Xiang” mainly represents the volatile flavor compounds in Daqu. The microbial diversity and volatile metabolites on 0, 6, 16, and 29 days of the fermentation process were measured using high-throughput sequencing and gas chromatography–mass spectrometry. Significant differences were found in the composition of the microbial community. Pseudomonas, Weissella, Bacillus, and Pelomonas were the main bacterial genera. Alternaria, Rhizopus, and Pichia are the main fungal genera. A total of 32 differential volatile metabolites were detected in samples at four time points using differential metabolic analysis. The correspondence of prevailing microorganisms with differential metabolites distinguished by Spearman correlation and two-way orthogonal partial least square analysis show that Saccharopolyspora exhibited a significant connection for the 12 differential metabolites. A significant positive correlation was observed between Rhizomucor and 13 different metabolites. These findings further understanding of the metabolism of microorganisms in Daqu fermentation and also help to control the microorganisms in the Daqu-making process, to obtain more stable Baijiu products.

Fermentation ability and alcohol production ability are important quality indicators of Chinese liquor Daqu, reflecting microbial growth and metabolic capacity and ethanol production capacity of Daqu microbiota, respectively. However, information on the microbial community related to the fermentation ability and alcohol production ability is unclear. In this study, fermentation functional microbiota (FFM) and alcohol functional microbiota (AFM) were obtained by correlating fermentation ability and alcohol production ability with Daqu microbiota. FFM and AFM consisted of 50 and 49 genera, respectively, which were basically the same at the phylum level but differed at the genus level. Correlation analysis showed that FFM and AFM were mainly affected by moisture, acidity, and humidity in the early stage of Daqu fermentation, and oxygen content was a critical factor for microbial succession in the middle stage of fermentation. FFM and AFM had commensal or synergistic interactions with multiple microbes. Function predictions indicated that fermentation functional bacterial microbiota was active in product synthesis and transport-related metabolic functions, and alcohol functional bacterial microbiota was very active in raw material utilization and its own metabolic synthesis. This study reveals the structural characteristics and formation mechanism of FFM and AFM, which is important for control of Daqu quality.

Jiang-flavor (JF) daqu is a liquor starter used for production of JF baijiu, a well-known distilled liquor in China. Although a high temperature stage (70°C) is necessary for qualifying JF daqu, little is known regarding its active microbial community and functional enzymes, along with its role in generating flavor precursors for JF baijiu aroma. In this investigation, based on metatranscriptomics, fungi, such as Aspergillus and Penicillium, were identified as the most active microbial members and 230 carbohydrate-active enzymes were identified as potential saccharifying enzymes at 70°C of JF daqu. Notably, most of enzymes in identified carbohydrate and energy pathways showed lower expression levels at 70°C of JF daqu than those at the high temperature stage (62°C) of Nong-flavor (NF) daqu, indicating lowering capacities of saccharification and fermentation by high temperature stage. Moreover, many enzymes, especially those related to the degradation of aromatic compounds, were only detected with low expression levels at 70°C of JF daqu albeit not at 62°C of NF daqu, indicating enhancing capacities of generating special trace aroma compounds in JF daqu by high temperature stage. Additionally, most of enzymes related to those capacities were highly expressed at 70°C by fungal genus of Aspergillus, Coccidioides, Paracoccidioides, Penicillium, and Rasamsonia. Therefore, this study not only sheds light on the crucial functions of high temperature stage but also paves the way to improve the quality of JF baijiu and provide active community and functional enzymes for other fermentation industries.

Strong flavor baijiu (SFB), also called Luzhou-flavor liquor, is the most popular Chinese baijiu. It is manufactured via solid fermentation, with daqu as the starter. Microbial diversity of the SFB ecosystem and the synergistic effects of the enzymes and compounds produced by them are responsible for the special flavor and mouthfeel of SFB. The present review covers research studies focused on microbial community analysis of the SFB ecosystem, including the culturable microorganisms, their metabolic functions, microbial community diversity and their interactions. The review specifically emphasizes on the most recently conducted culture-independent analysis of SFB microbial community diversity. Furthermore, the possible application of systems biology approaches for elucidating the molecular mechanisms of SFB production were also reviewed and prospected.

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.

This study aimed to investigate the antioxidant activity and release behavior of anthocyanin (ANC) loaded within FA-g-MD wall (ANC-FA-g-MD microcapsule) in vitro. The microencapsulation of ANC was prepared by spray drying and displayed a biphasic release profile. The combination of ANC and FA-g-MD (0.0625–1 mg/mL) showed a higher antioxidant activity than that of both individuals. A possible intermolecular interaction between ANC and FA-g-MD was studied by UV-vis spectra. Intracellular reactive oxygen species (ROS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test, and protein expression of quinone oxidoreductase 1(NQO1), glutathione reductase (GSR) and γ-glutamate cysteine ligase catalytic subunit (γ-GCLC) were measured through human colon cancer cells (HT-29). After a 24-hour incubation of the HT-29, the combinations (0–60 μg/mL) exhibited a high potential to diminish the ROS level. And the distinct upregulated expressions of GCLC and NQO1 of HT-29 were detected after treatment with combinations compared to those of single ones. These results suggested that the ANC-FA-g-MD microcapsules exerts enhanced antioxidant effect with capability of the modulation of GCLC and NQO1.