Background Solid-state vinegar fermentation, predominantly practiced in China, is characterized by high microbial diversity, multi-substrate co-decomposition, abundant metabolite production, pronounced environmental heterogeneity, and complex interaction networks that collectively shape vinegar quality. However, this process remains largely experience-driven, with limited digitalization and an incomplete understanding of microbial interaction mechanisms. Key challenges include unclear interaction mechanisms, insufficient application of modeling tools such as genome-scale metabolic models, and limited capacity for dynamic process control. Addressing these gaps is essential for improving fermentation efficiency, stabilizing flavor quality, and advancing vinegar modernization. Scope and approach This review is centered on microbial interactions and outlines the metabolic division of labor of key microorganisms in solid-state fermentation and their roles in flavor formation. It examines microbial ecological relationships and interaction mechanisms, and analyzes how environmental heterogeneity regulates microbial interactions. The review further introduces synthetic microbial communities as tools for mechanism validation and functional reconstruction. Finally, based on clarified mechanisms, it discusses how key microbial, metabolic, and environmental information can be translated into model inputs to construct digital twin systems for directional control. Key findings and conclusions Environmental heterogeneity plays a role in shaping microbial interaction patterns, thereby influencing metabolic division of labor, fermentation efficiency, and flavor formation. Microbial interactions, rather than individual species, are drivers of community stability, functional metabolite production, and flavor complexity. The integrated application of synthetic microbial communities, metabolic flux models, and digital twin technologies constitutes a predictive framework for dissecting microbial interactions, optimizing key consortia, and achieving targeted regulation of cereal vinegar fermentation.

Scleroglucan is the extracellular polysaccharide (EPS) produced by Sclerotium rolfsii (S. rolfsii). The low EPS titer and limited substrate utilization of S. rolfsii present significant challenges in the fermentation process, restricting industrial applications of scleroglucan. In this study, we performed a transcriptomic analysis on the mycelium of S. rolfsii fermented with different carbon sources. The key genes involved in polysaccharide biosynthesis (6-phosphofructokinase 1 (PFK1), pyruvate decarboxylase (PDC), aldehyde dehydrogenase (NAD (P)+) (ALDH3), and acetyl-CoA synthase (ACS)) were identified and their roles in the process were investigated. The supplementation of specific precursors—fructose-6-phosphate, pyruvate, aldehydes, and acetate—was shown to enhance both the polysaccharide titer and substrate utilization. By adding precursors, the titer of SEPS produced in a 5 L fermentation tank reached 48.69 ± 3.8 g/L. Notably, the addition of these precursors increased the titer of EPS fermented with sucrose (SEPS) by 65.63% and substrate utilization by 119.3%, while the titer of EPS fermented with lactose (LEPS) rose by 80.29% and substrate utilization rose by 47.08%. These findings suggest that precursor supplementation can effectively improve polysaccharide production and substrate efficiency, thereby minimizing resource waste and environmental impact.

Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1). BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.

Phenols are important functional compounds present in vineagr, however, their composition and formation pathways remain uncertain. Herein, non-targeted metabolomics and macrotranscriptomics methods were applied to identify phenols and analyze their formation network during the brewing process of Shanxi aged vinegar. A total of 82 phenols were detected from the raw material and the brewing process. Results indicated that phenolic acids were the major phenols and were mainly formed during acetic acid fermentation stages. Water, reducing sugars, lactic acid, and 7 amino acids influenced the formation and transformation of phenols, as shown through Spearman analysis. Furthermore, 16 genera and 38 enzymes were involved in substrates decomposition and phenols formation according to the metabolic pathway analysis, with Xenobiotics biodegradation and metabolism identified as the main pathway for phenols formation. Lactobacillus and Acetobacter were the key genera responsible for the phenols transformation. This study provides new insights into the phenols formation mechanisms in cereal vinegars and it is helpful for isolating the functional strains to reinforce the phenols formation.

Scleroglucan is obtained from Sclerotium rolfsii and is widely used in many fields. In this study, transcriptomics combined with metabolomics were used to study the global metabolites and gene changes. The results of the joint analysis showed that the DEGs (differentially expressed genes) and DEMs (differentially expressed metabolites) of SEPS_48 (fermented with sucrose as a carbon source for 48 h) and GEPS_48 (fermented with glucose as a carbon source for 48 h) comparison groups were mainly related to cell metabolism, focusing on carbohydrate metabolism, amino acid metabolism, and amino sugar and nucleoside sugar metabolism. We therefore hypothesized that the significant differences in these metabolic processes were responsible for the differences in properties. Moreover, the joint analysis provides a scientific theoretical basis for fungal polysaccharides biosynthesis and provides new insights into the effects of carbon sources on the production. As an excellent bioenergy and biological product, scleroglucan can be better applied in different fields, such as the food industry.

The microbial community plays an important role on the solid-state fermentation (SSF) of Chinese cereal vinegar, where acetic acid bacteria (AAB) and lactic acid bacteria (LAB) are the dominant bacteria. In this study, the top-down (in situ) and bottom-up (in vitro) approaches were employed to reveal the interaction of AAB and LAB in SSF of Shanxi aged vinegar (SAV). The results of high-throughput sequencing indicates that Acetobacter pasteurianus and Lactobacillus helveticus are the predominant species of AAB and LAB, respectively, and they showed negative interrelationship during the fermentation. A. pasteurianus CGMCC 3089 and L. helveticus CGMCC 12062, both of which were isolated from fermentation of SAV, showed no nutritional competition when they were co-cultured in vitro. However, the growth and metabolism of L. helveticus CGMCC 12062 were inhibited during SSF due to the presence of A. pasteurianus CGMCC 3089, indicating an amensalism phenomenon between these two species. The transcriptomic results shows that there are 831 differentially expressed genes (|log2 (Fold Change)| > 1 and, p ≤ 0.05) in L. helveticus CGMCC 12062 under co-culture condition comparing to its mono-culture, which are mainly classified into Gene Ontology classification of molecular function, biological process, and cell composition. Of those 831 differentially expressed genes, 202 genes are up-regulated and 629 genes are down-regulated. The down-regulated genes were enriched in KEGG pathways of sugar, amino acid, purine, and pyrimidine metabolism. The transcriptomic results for A. pasteurianus CGMCC 3089 under co-culture condition reveals 529 differentially expressed genes with 393 up-regulated and 136 down-regulated, and the genes within KEGG pathways of sugar, amino acid, purine, and pyrimidine metabolism are up-regulated. Results indicate an amensalism relationship in co-culture of A. pasteurianus and L. helveticus. Therefore, this work gives a whole insight on the interaction between the predominant species in SSF of cereal vinegar from nutrient utilization, endogenous factors inhibition and the regulation of gene transcription.

Newly synthesized glycosylphosphatidylinositol-anchored proteins (GPI-APs) undergo extensive remodeling prior to transport to the plasma membrane. GPI-AP remodeling events serve as quality assurance signatures, and complete remodeling of the anchor functions as a transport warrant. Using a genetic approach in yeast cells, we establish that one remodeling event, the removal of ethanolamine-phosphate from mannose 2 via Ted1p (yPGAP5), is essential for cell viability in the absence of the Golgi-localized putative phosphodiesterase Dcr2p. While GPI-APs in which mannose 2 has not been remodeled in dcr2 ted1-deficient cells can still be delivered to the plasma membrane, their presence elicits a unique stress response. Stress is sensed by Mid2p, a constituent of the cell wall integrity pathway, whereupon signal promulgation culminates in activation of the spindle assembly checkpoint. Our results are consistent with a model in which cellular stress response and chromosome segregation checkpoint pathways are functionally interconnected.

Steroid C-17-carbonyl reduction is one of the important transformations for the production of valuable steroidal medicines or intermediates for the further synthesis of steroidal medicines, but it remains a challenge through either chemical or biological synthesis. Phytosterol can be obtained from low-cost residues of waste natural materials, and it is preferred as the economical and applicable substrate for steroid medicine production by Mycobacterium .

Aspergillus niger mycelium residues, a citric acid fermentation by-product, is a raw material for fungal chitosan and glucosamine (GlcN). However, traditional chemical extraction of fungal chitosan and GlcN uses strong acid at high temperatures for long periods and has several drawbacks, including high cost and environmental pollution. In this work, the residues were pretreated by the combination of steam explosion (SE) and ionic liquid (IL) to produce fungal chitosan and GlcN hydrochloride. Following chemical extraction, GlcN hydrochloride yield increased by 65.49 +/- 8.14%, and soluble sugar release reached 0.36-0.46 g/g via SE pretreatment at 2.5 MPa for 1 min. Fungal chitosan was produced via enzymatic hydrolysis. Combined SE and IL pretreatment increased deacetylation degree (1.29 +/- 0.06-fold) of fungal chitosan by chitin deacetylase hydrolysis using Rhodococcus equi CGMCC14861 (ReCDA) at 37 degrees C for 6 h. Besides, fungal chitosan was produced using a by-product recycling strategy, promoting ReCDA fermentation, using supernatant with soluble sugars and proteins. After mixing 0.2 g by-product supernatant with 100 mL medium, ReCDA production was 1.5-fold higher than that in the control. This study demonstrates a green technology for efficient utilization of fungal chitin from A. niger mycelium residues of citric acid fermentation.

Shanxi aged vinegar (SAV), a Chinese traditional vinegar, is produced by various microorganisms. Ammonium is an important nitrogen source for microorganisms and a key intermediate for the utilization of non-ammonium nitrogen sources. In this work, an ammonium metabolic network during SAV fermentation was constructed through the meta-transcriptomic analysis of in situ samples, and the potential mechanism of acid affecting ammonium metabolism was revealed. The results showed that ammonium was enriched as the acidity increased. Meta-transcriptomic analysis showed that the conversion of glutamine to ammonia is the key pathway of ammonium metabolism in vinegar and that Lactobacillus and Acetobacter are the dominant genera. The construction and analysis of the metabolic network showed that amino acid metabolism, nucleic acid metabolism, pentose phosphate pathway and energy metabolism were enhanced to resist acid damage to the intracellular environment and cell structures. The enhancement of nitrogen assimilation provides nitrogen for metabolic pathways that resist acid cytotoxicity. In addition, the concentration gradient allows ammonium to diffuse outside the cell, which causes ammonium to accumulate during fermentation.

Shanxi aged vinegar (SAV), a traditional Chinese cereal vinegar, is produced using solid-state fermentation (SSF) technology. Organic acids are the key flavor compounds of vinegar. However, the metabolic mechanism of organic acids during SSF process is still unclear. In this study, metatranscriptomics was used to explore the metabolic profile of main organic acids in SSF. The results show that carbon metabolism is the dominant pathway during fermentation, among which pyruvate metabolism, glycolysis and starch and sucrose metabolism associated with organic acids were the most abundant. The metabolic pathways of acetic acid and lactic acid shift from acetyl-P and pyruvate pathways at early and middle-early stages of fermentation to acetaldehyde and L-lactaldehyde pathways at later stages, respectively, and Lactobacillus and Acetobacter are the predominant microorganisms contributed to them. Temperature and acetic acid are proven to be the environmental factors that regulate the metabolic activity during SSF. This study sheds new lights on metabolism of flavor substances in the spontaneous ecosystems of traditional fermented food.

该研究将室温等离子(ARTP)诱变与微生物微滴培养(MMC)技术应用于几丁质脱乙酰基酶(CDA)高产菌株的诱变选育,构建高产CDA菌株的诱变及高通量筛选方法.结果表明,经过4轮的ARTP诱变及MMC筛选,从200个不同的液滴中共筛选出5个发酵产酶明显提升的液滴,并通过进一步的平板筛选、24-深孔板复筛,获得了17株产酶提高300％以上的诱变菌株.通过对比分析17株高产菌株产CDA的能力,确定了1株最佳CDA高产菌株B4,其CDA最大产量比出发菌株提高了3.15倍,发酵产酶总量达到419.11 U/mL,为原始菌种的3.90倍.该研究为CDA高产菌株的诱变选育及高通量筛选提供了借鉴.

Chitin deacetylase (CDA) can hydrolyze the acetamido group of chitin polymers and its deacetylated derivatives to produce chitosan, an industrially important biopolymer. Compared with traditional chemical methods, biocatalysis by CDA is more environment-friendly and easy to control. However, most reported CDA-producing microbial strains show low CDA producing capabilities. Thus, the enhancement of CDA production has always been a challenge. In this study, we report co-culture fermentation to significantly promote the CDA production of Rhodococcus equi CGMCC14861 chitin deacetylase (ReCDA). Due to co-culture fermentation with Staphylococcus sp. MC7, ReCDA yield increased to 21.74 times that of pure culture of R. equi. Additionally, the enhancement was demonstrated to be cell-independent by adding cell-free extracts and the filtrate obtained by 10 kDa ultrafiltration of Staphylococcus sp. MC7. By preliminary characterization, we found extracellular, thermosensitive signal substances produced by Staphylococcus that were less than 10 kDa. We investigated the mechanism of promotion of ReCDA production by transcriptomic analysis. The data showed that 328 genes were upregulated and 1,258 genes were downregulated. The transcription level of the gene encoding ReCDA increased 2.3-fold. These findings provide new insights into the research of co-culture fermentation for the production of CDA and quorum sensing regulation.

Polyphenols extracted from Shanxi-aged vinegar (SAVEP) can alleviate oxidative stress and inflammatory stress caused by high-fat diets, improving intestinal microbial disorders. SAVEP may be a novel treatment of the metabolic syndrome.

Scleroglucan is widely used in the food and chemical industries because of its good rheological property, stability, and emulsification activity. To investigate the influence of different carbon sources on the properties and synthesis of exopolysaccharides (EPS), the three EPSs (GEPS, glucose was used as the carbon source; LEPS, lactose was used as the carbon source; and SEPS, sucrose was used as the carbon source) were determined, respectively. It was found that the yield and viscosity of exopolysaccharides were different. When sucrose and glucose were used as the carbon sources, the viscosity and yield of EPS were both higher than lactose. The scanning electron microscopy (SEM) images showed that the three EPSs had different morphologies, but the monosaccharide analysis showed that they were all composed of glucose units. Fourier transform infrared spectroscopy (FT-IR) proved that there were no additional substituents for the three EPSs. Furthermore, the high performance liquid chromatography (HPLC) results showed that SEPS and LEPS had two fractions. Through the analysis of proteomics data, there were few differences in the metabolic pathways between GEPS and SEPS, but a significant difference between LEPS and SEPS. Our study provides a theoretical basis and reference for understanding the biosynthesis of exopolysaccharides and the development of different types of EPS products.

Chitin deacetylase (CDA) can hydrolyse the acetamido group of chitin polymers to produce chitosans, which are used in various fields including the biomedical and pharmaceutical industries, food production, agriculture, and water treatment. CDA represents a more environmentally-friendly and easier to control alternative to the chemical methods currently utilised to produce chitosans from chitin; however, the majority of identified CDAs display activity toward low-molecular-weight oligomers and are essentially inactive toward polymeric chitin or chitosans. Therefore, it is important to identify novel CDAs with activity toward polymeric chitin and chitosans. In this study, we isolated the bacterium Rhodococcus equi F6 from a soil sample and showed that it expresses a novel CDA (ReCDA), whose activity toward 4-nitroacetanilide reached 19.20 U/mL/h during fermentation and was able to deacetylate polymeric chitin, colloidal chitin, glycol-chitin, and chitosan. Whole genome sequencing revealed that ReCDA is unique to the R. equi F6 genome, while phylogenetic analysis indicated that ReCDA is evolutionarily distant from other CDAs. In conclusion, ReCDA isolated from the R. equi F6 strain expands the known repertoire of CDAs and could be used to deacetylate polymeric chitosans and chitin in industrial applications.

Monascus vinegar (MV) exerts beneficial effects on cardiovasular diseases. The present work explored the lipid-lowering mechanism of MV and the correlation among gut microbiota, lipid metabolism and inflammation. The results illustrated that the administration of MV ameliorated hyperlipidemia by regulating Peroxisome proliferator-activated α (PPARα)-, Nuclear factor-E2-related factor 2 (Nrf2)- and Nuclear factor κB (NF-κB)-mediated signals and modulating the gut microbiota composition in hyperlipidemia rats. Moreover, Lactobacillus, Roseburia and Lachnostridium showed positive correlation with antioxidative parameters and negative correlation with lipid metabolism and inflammation parameters. Whereas Clostridium, Desulfovibrio and Streptococcus exhibited the opposite effect. Importantly, the differences in lipid-lowering effect between high-dose of MV (HD) group and low acetic acid and lovastatin (LLA) group were evaluated, which indicated that the other components in MV could improve the lipid-lowering effect of acetic acid and lovastatin. Our findings provided a novel insight into the lipid-lowering effect of MV.

The structure and antidepressant like activity of MEPS2 extracted from Marasmius androsaceus subjected to submerged fermentation was systematically studied. MEPS2 is a pyranoid polysaccharide composed of glucose and arabinose, which have a molar ratio of 0.56:0.08. The molecular weight was 85,944 Da. The NMR spectrum suggested the extracted MEPS2 contained uronic acid, and the glucosyl linkage was in α form, in accordance with the analysis of FT-IR spectrum. MEPS2 can considerably enhance the levels of noradrenalin (NE) and dopamine (DA) by ELISA. In addition, western blotting results indicated that MEPS2 can enhance the expression levels of TH, D2DR, and CAMKII. Furthermore, we found that AMPT, raclopride, and prazosin blocked the immobility and time-reducing effect of MEPS2. Overall, the antidepressant-like effect of MEPS2 may be involved in catecholamine synthesis and release, and TH, D2DR and CAMKII play an important role in this process.

Chitin is the second most abundant renewable polymer on earth, and its deacetylated derivative, chitosan, is a highly useful biopolymer. This work studied for the first time the application of ionic liquid (IL) tetrabutylammonium hydroxide ([TBA] [OH]) and chitin deacetylase from Rhodococcus equi CGMCC14861 (ReCDA) for the efficient conversion of chitin into chitosan at room temperature. Results confirmed that chitin had good solubility in 18 wt% aqueous [TBA] [OH] solution at 80 degrees C. In addition, efficient chitin deactylation was observed with high concentrations of [TBA] [OH] exceeding 12 wt% and showing potential application in chitin conversion. ReCDA activity on chitin was activated by [TBA] [OH] pretreatment. Sequential and simultaneous strategies were also compared, and the results showed that the simultaneous one-pot deacetylation provided the highest acetic acid yield of 3.78 mg/g chitin powder after 24 h. This study serves as a guide for the dissolution and deacetylation of chitin to produce high value-added chitosan products.

SummarySediments will spontaneously form in traditional Chinese vinegars (TCVs), which negatively affect the product acceptability. Here, sediments in Shanxi aged vinegar, a representative TCV, were investigated to unravel the microstructure and composition. Morphologically, crystals, amorphous substances and thalli were three common forms. Composition analysis by confocal laser scanning microscopy manifested starch/polysaccharides and proteins were present. Proteins (~30%), carbohydrates (>10%), crude fats (~17.00%) and metallic elements (~3%) were four main components determined, and polyphenols and flavonoids accounted for about 1% in the sediments. The crystals were recognised as calcium oxalate crystals by micro‐area energy‐dispersive X‐ray and micro‐Raman spectrometry, and they were mostly in dihydrate form. This is the first report on the discovery of calcium oxalate crystals in the vinegar. Crystallisation into crystals, self‐assembly and polymerisation of polymers, aggregation of particles, as well as interactions between polymers and particles form a basic model of sediments formation in TCV.