Introduction:Huangshui (HS), the liquid fraction that accumulates at the bottom of fermentation pits, serves as a key ecological interface linking microbial diversity and metabolism between fermented grains and pit mud during fermentation. Although HS is increasingly recognized as a reservoir of microorganisms and flavor precursors, how regional heterogeneity shapes its microbiome-metabolome coupling remains unclear. Methods:HS samples derived from nine representative Nongxiangxing Baijiu (NXB) production regions were compared using physicochemical characterization, amplicon sequencing, volatile metabolomics, and ecological association analysis. Results:The results showed marked regional differences in acidity, nitrogen availability, mineral nutrient content, and organic acid composition, indicating distinct fermentation microenvironments. Lactic acid dominated the acid pool in all samples, whereas short- and medium-chain fatty acids varied substantially among regions, suggesting differences in carbon flux allocation and chain elongation activity. Bacterial communities were dominated by Lactobacillus, methanogenic archaea, and Caproiciproducens, whereas fungal communities were enriched with fermentative yeasts (Pichia, Saccharomyces, and Kazachstania). A total of 162 volatile organic compounds were identified, with esters as the predominant aroma class, showing clear regional differentiation. Furthermore, integrated correlation and network analyses indicated that regional physicochemical factors acted as ecological filters, shaping microbial guild assembly and volatile metabolite patterns. Discussion:Lactobacillus emerged as a central taxon associated with acid-ester balance, whereas methanogenic and chain-elongating taxa were linked to carbon redistribution and medium-chain fatty acid formation. Further, these findings support the revised view of HS as an active metabolic interface, highlighting its potential for origin discrimination and precise fermentation control in NXB production.

Pichia kudriavzevii is a dominant yeast species in Chinese baijiu fermentation, yet its intraspecific diversity remains underexplored. This study used metabolomics and metagenomics analysis to investigate the impact of four distinct P. kudriavzevii strains (PK12, PK25, PK97, and PK360) on the metabolite profiles and microbial community structure in a controlled baijiu solid-state fermentation. Metabolomics analysis identified 49 key volatile compounds and 2792 non-volatile metabolites. Strain PK97 exhibited exceptional capacity for butanoic acid metabolism, inducing a 55.27-fold increase in butanoic acid and a 30.54-fold enhancement in ethyl butanoate production. Strain PK25 specialized in acetoin biosynthesis, while PK360 maximized 2-phenylethanol production. Metagenomic analysis uncovered that strains PK12, PK25, and PK360 promoted Lactobacillus acetotolerans population, increasing its relative abundance to 67.39%, 58.57%, and 71.79%, respectively. In contrast, strain PK97 orchestrated a dramatic ecological shift, elevating Enterobacter mori abundance from 0.56% to 17.60%, transforming the community from Lactobacillus-dominated to Enterobacteriaceae-enriched. Integration of metabolomic and metagenomic data revealed that strain PK97's promotion of Enterobacter mori correlated with significant upregulation of key enzymes including α-amylase (EC 3.2.1.1), enoyl-CoA hydratase (EC 4.2.1.17), and succinyl-CoA synthetase (EC 6.2.1.5), creating a metabolic environment favoring enhanced starch hydrolysis, altered TCA cycle flux, and butanoic acid accumulation. Strain PK25 specifically upregulated acetyl-CoA hydrolase (EC 3.1.2.1), facilitating acetic acid and acetoin formation. Strain PK360 enhanced glucose pyrophosphorylase (EC 2.7.7.9) and asparagine synthetase (EC 6.3.1.1) activities, accelerating galactose metabolism and amino acid transformations. These findings illustrate the impact of P. kudriavzevii intraspecific diversity on reshaping microbial ecology and flavor chemistry in Chinese baijiu, offering novel insights for targeted fermentation control and quality enhancement strategies in baijiu production.

On the basis of 12 sesame-flavor Baijiu spanning multiple vintages, this study systematically revealed aging patterns through chemometrics, sensory, physicochemical, and molecular docking analyses. Sensory profiling showed that aging enhanced sesame and aged aromas while weakening fruity and alcoholic notes. Physically, prolonged aging increased the particle size, regularized the morphology, and intensified yellowing. Gas chromatography-olfactometry-mass spectrometry and omission tests confirmed 21 key aroma contributors to aging traits. Molecular docking demonstrated their strong binding to olfactory receptors, with longifolene exhibiting the highest affinity, suggesting that receptor binding enhances perceived aging characteristics. Crucially, statistical change-point analysis identified the third year of aging as the volatile kinetic transition point, while multidimensional sensory, physical, and chemical cross-validation highlighted 5 years as a key empirical maturation reference. The subsequent 5-12-year period constitutes a continuous maturation phase, where sensory complexity dynamically evolves. These findings elucidate flavor evolution mechanisms, offering practical guidance for optimizing production planning, minimizing overaging losses, and authenticating aged sesame-flavor Baijiu.

Pit age is a key factor shaping microbial succession and flavor formation during Nongxiangxing Baijiu (NXB) fermentation, the ecological coupling among pit mud (PM), Huangshui (HS), and Jiupei (JP) remains unclear. Here, we analyzed the physicochemical properties, microbial communities, and volatile organic compounds (VOCs) of PM, HS, and JP collected from fermentation pits used for 5 and 30 years. Further, mantel tests and co-occurrence network analyses were performed to examine associations among environmental factors, microbiota, and flavor metabolism. The results showed that PM from the 30-year fermentation pits exhibited a more mature physicochemical profile and a more favorable niche for microbial colonization. Lactobacillus was the predominant bacterial genus in JP and HS, whereas Methanosarcina and Fastidiosipila were enriched in PM; Pichia was more abundant in HS, while Monascus was characteristic of PM. Esters were the dominant VOCs in all samples, and HS and JP displayed highly similar volatile compositions. Notably, Lactobacillus remained a core taxon across pit ages, whereas fungal interactions shifted from competitive to cooperative patterns in older fermentation pits. The above findings clarify microecological contributions to flavor formation, and further to offer a theoretical basis for process optimization and quality regulation in industrial Baijiu production.

Phthalate esters (PAEs), as major endocrine disruptors, pose severe risks to human health and environment due to their toxicity. Enzymatic hydrolysis offers a promising detoxification route, but the incomplete enzymatic cleavage of di-ester bonds remains a bottleneck for PAEs hydrolysis. This is largely attributed to a limited mechanistic understanding of how type III hydrolases achieve di-ester bond hydrolysis of PAEs. Integrated molecular docking, molecular dynamics simulations, and site-directed mutagenesis revealed a novel electrostatic guidance mechanism of a representative type III hydrolase PAEIIIase in this work. Specifically, residues E188 and H399 mediated the spatial flip of the mono-ester intermediate via electrostatic interactions, while Y118 stabilized substrates through hydrogen bond and π-π stacking to facilitate sequential ester-bond cleavage by PAEIIIase. Guided by the catalytic mechanism, the homologous type I hydrolase CarB152 was successfully converted into a type III hydrolase CarB152-F118Y capable of complete di-ester bond hydrolysis through targeted engineering. Furthermore, homology screening identified a superior enzyme Est909, which exhibited 116.8% higher hydrolytic activity toward di(2-ethylhexyl) phthalate compared to PAEIIIase. To enhance practical applicability, a robust sodium alginate-based whole-cell immobilization system was constructed. The system demonstrated robust performance, maintaining >90% substrate degradation across 10 consecutive cycles, coupled with a hydrolysis efficiency exceeding 4.0 g/(L·d). Collectively, this study significantly advanced the enzymatic mechanistic understanding of complete di-ester bond cleavage, provided a promising engineering strategy for functionally upgrading closely related homologs, expanded the enzymatic toolbox for PAEs degradation, and established a technically feasible biocatalytic platform with potential applicability in sustainable PAEs remediation.

The blending of Jiangxiangxing Baijiu is achieved through the specific proportional blending of base liquors from distinct distillation rounds, characterized by significant flavor variations attributed to differing volatile and non-volatile flavor compounds. To explore digital-assisted blending methods, sensory profiles of each round were first established to identify characteristic descriptors. Flavoromics analysis was then systematically applied to determine the types, concentrations, and variation patterns of flavor compounds. An 8-sensor electronic nose was built for multi-round Jiangxiangxing Baijiu. Based on flavoromics data, two intelligent models were innovatively developed: a machine learning-based recognition model utilizing electronic nose and chromatographic data for accurate round identification, and a genetic algorithm-based optimization model incorporating a multidimensional flavor compound dataset for intelligent blending. The optimized genetic algorithm model, validated by sensory evaluation and chromatographic analysis, was selected as the superior model for Jiangxiangxing Baijiu blending, providing a scientific basis for the digital transformation of traditional blending techniques.

To explore microbial resources for ester production in sub-high-temperature Daqu, this study first established that the esterifying enzyme activity in Daqu predominantly originated from fungi, with Rhizopus being the dominant fungal genus. Six Rhizopus strains capable of decomposing esters were isolated and purified from Daqu. Following secondary screening, strain M1 exhibited the highest esterification activity (40.26 U/mL) and was identified as Rhizopus oryzae based on morphological characteristics and molecular biological analyses. This strain was subsequently designated as Rhizopus oryzae M1 (R. oryzae M1). Using mycelial powder of strain M1 as the inoculum and sterilized wheat bran as the substrate, a pure-culture Fuqu was prepared. Orthogonal array design experiments were conducted to optimize the preparation process of this Fuqu, using esterifying enzyme activity as the evaluation index. Under the optimal conditions, the spore count and esterification activity of the pure-culture Fuqu reached 1.73 × 109 CFU/g and 80.13 U/g, respectively. This pure-culture Fuqu was subsequently used as an inoculum to produce fortified Daqu. Following orthogonal optimization of the Daqu preparation process, the esterification activity of the fortified Daqu reached 103.22 U/g, and its key physicochemical indices met the requirements for high-quality sub-high-temperature Daqu. Analysis of the microbial community structure revealed that Rhizopus was the dominant fungal genus in the fortified Daqu, with its relative abundance increased by 35% compared to the non-fortified Daqu. Consistent with this, the esterifying enzyme activity of the fortified Daqu was 51.79% higher, suggesting that Rhizopus may have been largely responsible for the increase in esterification capacity. In laboratory-scale Baijiu brewing trials, this fortified Daqu produced a base Baijiu with a total ester content of 2.74 g/L, representing a 40.5% increase over the non-fortified Daqu and further confirming the pivotal role of Rhizopus in driving the esterifying enzyme activity. This study successfully screened a high esterifying enzyme-producing strain, R. oryzae M1, systematically optimized its enzyme production and Qu-making processes, and provides an excellent microbial strain and process reference for the preparation of fortified Daqu and the enhancement of Baijiu flavor.

ObjectiveTo solve the problems of single strain, limited fermentation capability, and weak environmental tolerance of yeast Fuqu.MethodsThe yeast community of Jiangxiang Daqu is utilized as the strain source, and bran is used as the raw material to prepare a multi-yeast mixed Fuqu with fortified function. The preparation conditions of yeast Fuqu are optimized by single factor tests combined with orthogonal tests. Then, the acid, ethanol, and high-temperature resistance are systematically evaluated, while the microbial community structure is analyzed via internal transcribed spacer (ITS) rDNA high-throughput sequencing.ResultsThe optimal process parameters are a moisture content of 45%, an incubation time of 48 h, and an inoculum size of 5%. Under these conditions, the yeast count in the fortified Fuqu reaches 7.98×109 CFU/g, which is two orders of magnitude higher than that of the traditional Jiangxiang Daqu (P<0.05). Maintaining robust growth activity in high-acid, high-ethanol, and high-temperature environments, the Fuqu, with high microbial community purity, successfully enriches Saccharomycopsis and Saccharomyces.ConclusionThe multi-yeast mixed Fuqu with high yeast biomass, strong fermentation function, and potent environmental adaptability can be prepared using the yeast community of Jiangxiang Daqu.

BACKGROUND:Research on shelf-stacking fermentation is growing, with the aim of reducing labor and improving Daqu stability, but it still falls short compared to traditional stacking fermentation, especially for high-temperature Daqu (HTD). The heterogenetic mechanisms underlying+ these two Daqu remain unclear. RESULTS:The improved micro-oxygen environment in shelf-stacking fermentation significantly reduced acidity and increased liquefaction and esterification activities compared to traditionally stacked HTD (HTD-T). These changes substantially impacted the bacterial community and its succession, though fungi were minimally affected. Specifically, shelf-stacked HTD (HTD-S) showed higher relative abundances of Weissella and Thermoactinomyces in later stages than HTD-T, whereas Bacillus and Kroppenstedtia prevailed under traditional conditions. Redundancy analysis indicated that moisture and acidity mainly drove the differentiation of the bacterial community. Shelf-stacking fermentation also reduced the contents of furfuryl alcohol, furfural, acetic acid and isovaleric acid. Thermoactinomyces, Aspergillus and Thermoascus significantly contributed to these changes in characteristic flavor compounds. Functional prediction further revealed that microbial communities in HTD-T demonstrated enhanced abilities in glycolysis, 2,3-butanediol synthesis and phenylalanine metabolism. CONCLUSION:The findings provide potential control targets for precision regulation to improve the quality of shelf-stacked HTD and will contribute to mechanized HTD production. © 2025 Society of Chemical Industry.

Strong-flavor Baijiu (SFB) is produced using a solid-state fermentation system, but the impact of stratified fermented grains on fermentation remains unclear. Therefore, in a typical distillery plant, we evaluated the physical and chemical composition, volatile compound profile, and microbial community of fermented grains in SFB both above (FG-A) and below (FG-B) the Huangshui line. Significant differences in fermentation parameters between FG-A and FG-B were observed after 30 days of fermentation (P < 0.05). Additionally, the partial least squares projection to latent structure discriminant analysis (PLS-DA) revealed distinct differences in volatile compounds between FG-B and FG-A, identifying 26 discriminant markers. The diversity of short-chain fatty acids (SCFAs) and their esters were higher in FG-B compared to FG-A. Furthermore, microbial diversity and abundance have differed significantly between the two layers of fermented grains (P < 0.05), included 17 differential genera. Correlation and pathway enrichment analyses indicated that the higher SCFA content in FG-B could be attributed to the greater abundance of acid-producing microorganisms compared to FG-A. This study highlights the differences between the two layers of fermented grains in SFB fermentation, offering new insights into solid-state fermentation and expanding the current understanding of the traditional SFB fermentation process.

BACKGROUND:Norovirus (NoV) is the leading cause of foodborne disease outbreaks worldwide, typically spreading via contaminated food and water. Rapid, sensitive, and portable detection of NoV is crucial. RESULTS:Here, we presented a magnetic CRISPR/Cas12a-SERS nanobiosensor capable of detecting NoV with high sensitivity, accuracy, speed, and portability. In this nanobiosensor, SERS nanoprobes linked to magnetic nanoprobes via linker single-stranded DNAs (ssDNAs). The presence of NoV nucleic acid triggered Cas12a's trans-cleavage activity, degrading the linker ssDNA. After magnetic separation, the dissociated SERS nanoprobes were efficiently separated from the magnetic nanoprobes. This enhanced the SERS signal in the supernatant, detectable using a portable Raman spectrometer. The detection limit for NoV is 100 copies/mL within 60 min. The nanobiosensor was further assessed in real-world settings, demonstrating excellent sensitivity and selectivity for detecting trace NoV in complex food samples. SIGNIFICANCE:This approach not only broadens CRISPR-based pathogen detection but also provides a reliable tool for monitoring foodborne viruses. Its potential extends beyond NoV, promising enhanced surveillance of various pathogens in food safety, environmental monitoring, and public health sectors.

Ethyl caproate, a significant aromatic component in strong-flavor baijiu, is synthesized requiring caproic acid as an essential precursor. In this study, a novel caproic acid-producing bacteria Rummeliibacillus suwonensis J-1 was isolated from pit mud. Subsequently, a dual bacterial co-culture system (DBCS) was successfully established by combining J-1 with the acid-producing bacterium Enterococcus sp. D-1, resulting in a 21-fold increase in yield, which reached 4.41 g/L. A new immobilization strategy was developed, utilizing luffa sponge as a carrier for DBCS to facilitate pit mud-free strong-flavor baijiu production. The findings indicated a substantial increase in the ethyl caproate concentrations, with a 218 % increase, reaching 0.625 g/L. Transcriptomic analysis showed that in the two-bacterial system, crucial genes implicated in the biosynthesis pathway of caproic acid in J-1, including Crt, Scad, Ptb, pdxK, L-cysteine dehydrogenase, and l-serine decarboxylase were significantly upregulated, which enhanced the synthesis of caproic acid. These findings suggest that DBCS may have potential applications in fermentation without pit mud and could potentially enhance quality of strong-flavor baijiu.

In order to explore the effect of Wickerhamomyces on the production of flavor compounds in Xiaoqu Baijiu (XQBJ), this study examined the correlation between the fungal communities in Xiaoqu and ester compounds. It was hypothesized that Wickerhamomyces contributes to the aroma of Xiaoqu. Intensified fermentation methods were used to validate the role of Wickerhamomyces anomalus in XQBJ brewing. Compared to traditional Xiaoqu, intensified fermentation significantly increased the acidity of fermented grains and starch utilization rate (p < 0.05). The fungal communities in fermented grains were analyzed by high-throughput sequencing technology. The dominant fungi in both control and test groups were Saccharomyces, Cyberlindnera, Rhizopus, and Meyerozyma, with Wickerhamomyces replacing Saccharomymycopsis in the enhanced group. Flavor compounds in the fermentation experiments were analyzed, revealing that ethyl acetate content in XQBJ increased by 949.94 mg/L in the test group, while isoamyl alcohol decreased by 23.62 mg/L and isobutanol decreased by 34.81 mg/L. Functional prediction analysis using PICRUSt2 confirmed a higher relative abundance of enzymes involved in ethyl acetate metabolism and a lower relative abundance of enzymes involved in higher alcohol metabolism in the test group. These findings demonstrate that Wickerhamomyces enhances ethyl acetate production and reduces higher alcohols during XQBJ brewing, offering a theoretical foundation for enhancing the quality of XQBJ.

A systematic study was undertaken of the flavor disparities of Jiangxiang-flavor Baijiu produced across southern and northern China. Five non-volatile organic acids, seven amino acids, and twenty-one volatile compounds (OAV > 1) were identified, with 14 key aroma-active compounds determined through aroma omission and triangle tests. Quantitative targeted flavoromics and partition coefficient method were undertaken through descriptive sensory analysis. Sensory evaluation shows the intensities of floral, fruity, acidic, and grain aromas of the fourteen key aroma-active compounds were enhanced in the presence of non-volatiles. Partition coefficient analysis demonstrates that non-volatiles enhance the aroma release of most key aroma-active compounds, which aligns with sensory evaluation results showing significantly intensified fruity and floral notes following non-volatile supplementation. Co-adding organic acids and amino acids had a lower promoting effect on some volatiles than adding organic acids alone. The findings guide Baijiu production by optimizing fermentation to control non-volatile accumulation and modulate volatile release.

Objective:The production process of esterified Monascus bran koji was optimized,and its effect on Luzhou-flavor liquor was explored.Methods:Five strains of Monascus were screened from Daqu,and the esterifying enzyme production ability of the strains was detected by the ester decomposition method.The strain with the most prominent enzyme production ability was selected to prepare Monascus bran koji.The koji-making process was optimized by single factor and orthogonal experiments and applied to liquor production.Results:Among the five strains,the J-3 strain had the strongest ability to produce esterification enzyme.Under the conditions of 50％moisture,4％inoculation amount,pH 4.0 and 32 ℃ culture temperature,the esterification enzyme activity was as high as 77.78 mg/g bran koji.The bran koji was added to the fermented grains according to different inoculation amounts.Compared with the fermented grains without bran koji,when the inoculation amount of bran koji was 1％,the content of ethyl acetate in the fermented grains increased by 4.84 times,and the content of ethyl caproate increased by 3.55 times.When the inoculation amount was 1.5％,the content of ethyl butyrate increased by 4.72 times.The addition of esterified Monascus bran koji can improve the utilization rate of alcohol and starch,and significantly increase the total ester content in fermented grains.Conclusion:Adding Monascus bran koji to fermented grains according to the appropriate inoculation amount can improve the quality of Luzhou-flavor liquor.

NongXiangXing Baijiu (NBJ) is produced using a solid-state fermentation system, but the impact of stratified fermented grains on fermentation remains unclear. Therefore, in a typical distillery plant, we evaluated the physical and chemical composition, volatile compound profile, and microbial community of fermented grains in NBJ both above (FG-A) and below (FG-B) the Huangshui line. Significant differences in fermentation parameters between FG-A and FG-B were observed after 30 days of fermentation (P < 0.05). Additionally, the partial least squares projection to latent structure discriminant analysis (PLS-DA) revealed distinct differences in volatile compounds between FG-B and FG-A, identifying 26 discriminant markers. The diversity of short-chain fatty acids (SCFAs) and their esters was higher in FG-B compared to FG-A. Furthermore, microbial diversity and abundance differed significantly between the two layers of fermented grains (P < 0.05). Correlation and pathway enrichment analyses indicated that the higher SCFA content in FG-B could be attributed to the greater abundance of acid-producing microorganisms compared to FG-A. This study highlights the differences between the two layers of fermented grains in NBJ fermentation, offering new insights into solid-state fermentation and expanding the current understanding of the traditional NBJ fermentation process.

In order to explore the effect of Wickerhamomyces on the production of flavor compounds in Xiaoqu Baijiu, this study examined the correlation between the fungal communities in Xiaoqu and ester compounds. It was hypothesized that Wickerhamomyces contributes to the aroma of Xiaoqu. Enhanced fermentation methods were used to validate the role of Wickerhamomyces in Xiaoqu Baijiu brewing. Compared to traditional Xiaoqu, enhanced fermentation significantly increased the acidity of fermented grains and starch utilization rate (P<0.05). The fungal communities in fermented grains were analyzed by high-throughput sequencing technology. The dominant fungi in both control and test groups were Saccharomyces, Cyberlindnera, Rhizopus, and Meyerozyma, with Wickerhamomyces replacing Saccharomymycopsis in the enhanced group. Flavor compounds in the fermentation experiments were analyzed, revealing that ethyl acetate content in Xiaoqu Baijiu increased by 949.94 mg/L in the test group, while isoamyl alcohol decreased by 23.62 mg/L and isobutanol decreased by 34.81 mg/L. Functional prediction analysis using PICRUSt2 confirmed a higher relative abundance of enzymes involved in ethyl acetate metabolism and a lower relative abundance of enzymes involved in higher alcohol metabolism in the test group. These findings demonstrate that Wickerhamomyces enhances ethyl acetate production and reduces higher alcohols during Xiaoqu Baijiu brewing, offering a theoretical foundation for enhancing the quality of Xiaoqu Baijiu.

This study investigates innovative approaches to improve the quality and aroma characteristics of Muscat Hamburg wine production by substituting the conventional Saccharomyces cerevisiae yeast with an efficient fermentation strain of Schizosaccharomyces pombe. The typical use of S. cerevisiae in Muscat Hamburg wine often leads to uniformity and prolonged processing times, requiring subsequent malolactic fermentation to degrade excessive malic acid. The study advocates for the replacement of S. cerevisiae with a specific S. pombe strain, Sp-410, isolated from the fermented grains of sauce-flavor Baijiu, a Chinese spirit. Muscat Hamburg wine fermented with the S. pombe strain demonstrates decreased malic acid levels, offering a potential alternative to malolactic fermentation. However, exclusive S. pombe fermentation may result in an overproduction of acetic acid metabolites, leading to a monotonous taste. In response, the study proposes a mixed fermentation approach, combining the S. pombe strain with a Saccharomyces uvarum strain and a non-Saccharomyces yeast, Torulaspora delbrueckii. The optimized mixed fermentation strategies (M:SP+TD and M60SP+TD) involve specific proportions and intervals of inoculation, aiming to enhance the quality and aroma complexity of Muscat Hamburg wine. In conclusion, this research contributes to advancing the production of high-quality Muscat Hamburg wines, utilizing S. pombe as the primary yeast strain and implementing mixed fermentation methodologies.

The study clarified the succession of microbial community structures in fermented grains (FG) and their association with flavor compounds, along with their potential metabolic functions. The community diversity, functional genera and metabolites in FG were investigated by polyphasic detecting approaches. There are 13 dominant microorganisms in FG (relative abundance > 1