Abstract Efficient xylose bioconversion is limited by the catalytic performance of xylulokinase (XylB), which phosphorylates d-xylulose to xylulose-5-phosphate. This study aimed to improve XylB from Lactococcus lactis N8 using a multi-dimensional semi-rational design strategy integrating sequence conservation, structure-based screening, mutagenesis, molecular docking, and molecular dynamics simulations. Asn301 was identified as a key functional hotspot, and Met259 was found to modulate the active-site hydrophobic environment. Combinatorial mutagenesis generated two superior variants, XylBM259H/N301G and XylBM259H/N301H, with more than 200% higher specific activity than the wild-type XylB (XylBWT) while retaining stable expression and environmental adaptability. Mechanistic analyses showed strengthened ATP binding, improved d-xylulose positioning, enhanced ligand affinity, and balanced local flexibility with structural compactness. These findings provide efficient XylB variants and a transferable strategy for engineering lignocellulose-related enzymes.

The flavor formation and quality stability of solid-state fermented foods are closely linked to their storage process, and Chinese baijiu Daqu serves as an ideal model for studying such microecological succession. Analysis of Daqu produced in spring, autumn, and winter showed that despite seasonal differences in initial communities, all exhibited highly convergent succession during storage. Bacillaceae increased steadily, while Lactobacillaceae and Saccharomycetaceae declined. This restructuring corresponded with directional metabolic shifts: metabolites that decreased were enriched in amino acid pathways linked to the TCA cycle. Although amino acid levels declined, the metabolic potential for their utilization increased, while the abundances of core carbon metabolites and several TCA-related intermediates were relatively maintained during storage. Comparative genomic analysis further demonstrated that Bacillaceae MAGs possess a complete TCA cycle and abundant secondary metabolite clusters, whereas Lactobacillaceae lack a full TCA cycle, reflecting contrasting ecological strategies—Bacillaceae as stress-tolerant/homeostasis-maintaining and Lactobacillaceae as rapid-growth/resource-acquiring. Random forest analysis using three core families explained variation in key fermentation functions (cross-validated R2 = 0.504-0.787), supporting an association between microbial succession and Daqu performance. This work suggests that Daqu storage represents a directionally structured ecological succession associated with environmental variation, providing new insight into storage-stage ecological dynamics in solid-state fermentation.

Daqu serves as the saccharifying and fermenting starter in Baijiu production, yet the effects of raw material variation on community succession and metabolite formation during fermentation remain unclear. In this study, amplicon sequencing and metabolomic profiling were applied to compare mung bean Daqu (MBQ), produced by substituting pea with mung bean, with traditional medium-temperature Daqu. Microbial succession patterns were largely conserved after mung bean substitution, whereas stage-specific differences were observed in community composition, environmental associations, and microbial co-occurrence patterns. MBQ was enriched in thermotolerant spore-forming bacteria, particularly Kroppenstedtia, and functional fungi, including Thermomyces, Saccharomycopsis, and Aspergillus. These microbial differences were associated with higher saccharification, liquefaction, and fermentation powers at different stages. Spearman correlation analysis revealed distinct microbe-metabolite associations in MBQ involving pyrazines and plant-derived secondary metabolites. MBQ showed higher accumulation of higher alcohols during the middle stage, particularly phenylethyl alcohol (1.05-23.02 mg/kg), exclusive detection of 2,5-dimethylpyrazine and 2-ethylpyrazine, and flavonoid-related non-volatile metabolite profiles. These findings provide new insights into how raw material variation is associated with microbial succession and metabolic features during Daqu fermentation.

Natural exogenous additives (EA) suitable for the tobacco fermentation need to be developed to enhance the fermentation quality and economic value of low-grade cigar tobacco leaves (CTLs). This study analyzed the impacts of three compound Chinese herbal medicine (CHM) on metabolites and microorganisms during CTLs fermentation. The results manifested that EA facilitated the degradation of total sugar, starch and protein, while enhancing the accumulation of reducing sugar in CTLs. Furthermore, EA raised contents of free amino acids (FAAs), while Asp, Glu, Ser and His were found to be key differential FAAs of CTLs. During fermentation, the total contents of volatile flavor components (VFCs) initially increased and then declined. Furthermore, EA contributed to more harmonious compositions of VFCs by promoting the formation of neophytadiene, ketones, esters and aldehydes, as well as facilitating nicotine degradation. According to variable importance in the projection (VIP) > 1 and odor activity value (OAV) > 1, 7 key differential VFCs were identified. EA enhanced positive microbial interactions and led to a more stable and coordinated symbiotic network. Linear discriminant analysis effect size (LEfSe) identified 9 genera as differentially dominant microorganisms in CTLs, which were closely associated with chemical compositions and key differential flavor metabolites. In addition, EA promoted cigar tobacco characteristics (CTCs) by altering bacterial alpha diversity and influencing the assembly of dominant microbial communities. Overall, this study offered theoretical insights into the innovative applications of CHM in CTLs fermentation, and presented new perspectives for enhancing CTLs quality and customizing flavor profiles.

Quorum sensing (QS) is a central system reflecting microbial collective behavior; however, its role in shaping functional microbial communities within complex solid-state fermentation matrices such as Daqu remains insufficiently understood. Here, we integrated amplicon sequencing, metagenomics, proteomics, and metabolomics to investigate autoinducer-2 (AI-2)-mediated quorum sensing dynamics during Daqu storage. Storage induced a directional succession of the microbial community, revealing two distinct ecological stages. The rapid adjustment stage (0-2 months) was characterized by strong homogeneous selection and rapid species turnover, whereas the slow stabilization stage (3-9 months) was dominated by gradual shifts in microbial relative abundances. Notably, the LuxS/AI-2 pathway, the only QS system detected during Daqu storage, declined rapidly and then stabilized, coinciding with the transition between the two ecological stages. During the early stage, the core QS protein LuxS was tightly associated with the dominant taxon Lactobacillaceae and the methyl donor S-adenosylmethionine, forming a synergistic functional module. In contrast, during the late stage, LuxS became decoupled from stress-tolerant taxa and showed weakened associations with resistance-related metabolic networks. This shift was accompanied by a metabolic transition, with carbon flux gradually redirected from active glycolysis toward the pentose phosphate pathway and amino acid biosynthesis during later stages. Collectively, these findings demonstrate that temporal modulation of the LuxS/AI-2 quorum sensing system represents a critical regulatory node reflecting the transition of the Daqu microbial community from cooperative growth to stress-resilient survival, ultimately shaping metabolic phenotypes and ecosystem functions during storage.

This review comprehensively examines the structural architecture, catalytic mechanisms, and targeted molecular engineering of α-amylase (primarily the GH13 family), a pivotal biocatalyst in the food industry. We highlight diverse microbial sources of α-amylases and their cost-effective heterologous expression in well-characterized hosts like Bacillus subtilis and Escherichia coli. To overcome extreme operational bottlenecks—such as elevated temperatures and acidic environments—recent advances in protein engineering are critically evaluated. These strategies, including directed evolution, semi-rational design, and advanced immobilization on nanomaterials, synergistically enhance the enzyme’s thermostability, catalytic efficiency, and reusability. Furthermore, this paper synthesizes the state-of-the-art applications of engineered α-amylases across key food processing sectors, including baking, sugar refining, and brewing. By integrating structural biology with advanced material science, this review provides a targeted roadmap for developing next-generation, high-performance α-amylases to address current and future challenges in sustainable food processing.

Mung bean-flavor Baijiu (MBF) incorporates mung bean to develop a distinctive mung bean aroma (MBA), yet its key aroma compounds and formation mechanisms remain unclear. In this study, through aroma extract dilution analysis (AEDA), quantitative measurements, and aroma addition and omission, 22 key aroma compounds were identified that define the flavor profile of MBF. Among these, 2-isopropyl-3-methoxypyrazine, 1,1-diethoxy-3-methylbutane, 1-hexanol, furfural, furfuryl ethyl ether, and dimethyl trisulfide collectively shape the characteristic MBA. Furthermore, the integration of metabolic pathway analysis with amino acid and flavor profiling of the raw materials revealed that mung bean addition elevates the levels of linoleic acid, valine, leucine, and methionine, thereby promoting lipid oxidation, amino acid catabolism, and Maillard reactions that ultimately drive the formation of MBA compounds. These findings establish a molecular basis for MBA and advance the understanding of raw material driven regulation of characteristic aroma formation.

Agaricus bisporus is the most widely consumed edible mushroom and an important source of dietary protein and bioactive compounds. The casing layer plays a critical role in its cultivation, where microbial communities and metabolic activities strongly affect yield and quality, however, the stage-resolved research gap remains that it is still unclear which microbial and metabolic shifts in the casing layer are associated with primordia initiation and subsequent quality formation. In this study, the ecological and metabolic basis of mushroom quality formation was investigated by the combination of microbiome sequencing, non-targeted metabolomics, and functional genomics. Microbial communities exhibited stage-specific dynamics, with significant restructuring during primordia formation, when community cohesion and niche breadth reached their highest levels (0.68 +/- 0.06 and 4.48 +/- 1.05, respectively). A total of 1108 non-volatile metabolites were identified from metabolomic profiling. The adenosine and tryptophan exhibited significant changes and were enriched in energy and amino acid metabolism pathways. A representative strain, Pseudomonas putida AT130, was isolated from the genus Pseudomonas. The gene clusters related to phosphate solubilization, potassium mobilization, lignin degradation, and indole-3-acetic acid biosynthesis were revealed by genome analysis with the multifunctional activities being confirmed through in vitro assays. Pot experiments further showed that AT130 inoculation improved mushroom performance, increasing fruiting body yield by 123.55% and enhancing nutritional traits (protein and soluble sugars increased, whereas ash decreased) relative to the control. These findings linked casing-layer microbiota with mushroom quality and identified AT130 as a promising food-grade bioinoculant to enhance A. bisporus nutritional value and productivity.

Daqu, a solid-state microbial starter for Baijiu, undergoes pronounced storage-driven ecological and functional shifts, yet how these changes functionally translate into downstream fermentation outcomes remains insufficiently understood. Here, we systematically tracked Daqu stored for 3, 6, and 9 months and evaluated its effects during controlled Zaopei fermentation. With increasing storage duration, the Daqu microbiota shifted from nutrient-sensitive taxa, including Saccharomycetaceae and Thermoascaceae, toward stress-tolerant groups such as Bacillaceae and Trichocomaceae. Concomitantly, functional potential transitioned from hexose metabolism and glycan biosynthesis toward amino acid metabolism and nucleotide-sugar biosynthesis, accompanied by a significant decline in the levels of 14 free amino acids. Fermentation experiments revealed that short-term stored Daqu promoted the migration of Mucoraceae and Enterobacteriaceae into Zaopei, enhanced saccharification and fermentation performance, and increased ester production, including ethyl E-2-hexenoate and ethyl 3-methylthiopropanoate. Conversely, nine-month stored Daqu favored the dominance of Bacillaceae and Trichocomaceae, intensified arginine and proline metabolism, accelerated amino acid turnover, and increased the formation of nitrogen-containing flavor compounds, notably pyrazines (2-ethyl-3,5-dimethylpyrazine; 2,3-diethylpyrazine). Collectively, these results demonstrate that Daqu storage functionally regulates fermentation performance and flavor formation through microbial and metabolic reprogramming, providing a basis for targeted flavor regulation in Baijiu and other solid-state fermented foods.

Honey is a nutrient-rich natural functional food, with its color being considered as a key indicator of quality and consumer acceptance. Although various secondary metabolites are closely related to the color of honey, the specific substances and their exact effects on honey color remain unclear. In this study, the chemical composition, such as organic acids, polyphenols and vitamins, in different colored honey (Acacia, buckwheat, linden, vitex, lychee and jujube honey) were quantitatively analyzed using liquid chromatography-high resolution mass spectrometry (LCHRMS). The correlations between the chemical composition and the honey color were established by the mantel test. The results showed that quinic acid, ascorbic acid, and other composition were identified as significantly related with honey color. The validation experiments indicated that adding these significantly related substances to honey samples could multiplicatively deepen the honey color, and the value of the deepening color was positively correlated with that of the initial color. The study developed a practical and systematic LCHRMS method for analyzing chemical composition in honey. The results provided valuable scientific insights into the relationship between chemical composition and honey color. Additionally, it introduced a novel and feasible approach for investigating the effects of substances on the color of complex mixture systems.

Kouzijiao (KZJ) is a traditional Jian flavor Baijiu (JFB) that exhibits flavor characteristics typical of both soy sauce flavor Baijiu (SSFB) and strong flavor Baijiu (SFB). However, the key aroma compounds and compositional changes during aging have not been clearly characterized, and manufacturers lack objective criteria to determine the optimal aging period for peak KZJ flavor. A total of 58 aroma compounds were identified in the KZJ from different years using a combined approach of headspace solid-phase microextraction (HS-SPME) and liquid--liquid extraction (LLE) coupled with gas chromatography-olfactometry (GC-O). There were 19 important aroma compounds (FD >= 9) with odor activity values (OAVs) >= 1 revealed by aroma extract dilution analysis (AEDA) and odor threshold calculations. Among them, ethyl 3-methylbutanoate and ethyl furfuryl ether were first identified as key aroma compounds in JFB. Recombination and omission experiments further confirmed that ethyl acetate, ethyl hexanoate, hexanoic acid, and dimethyl trisulfide made significant contributions to the characteristic aroma profile of KZJ. Moreover, one-way analysis of variance (ANOVA) showed that the levels of key aroma compounds remained relatively stable in KZJ3. This study provides critical insights into the quality control parameters and dynamic trends of key flavor compounds during the aging of KZJ.

Minglv-flavor (MLF) Baijiu, brewed with mung beans, exhibits a characteristic aroma profile. However, key aroma compounds and dynamic evolution during aging have not been systematically investigated. Through recombination and omission experiments, 21 key aroma compounds were confirmed, including ethyl hexanoate, ethyl butyrate, ethyl pentanoate, ethyl octanoate, ethyl 3-methylbutanoate, ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, hexanoic acid and pentanoic acid, which synergistically constituted the aroma profile of MLF Baijiu, characterized by a prominent mung bean aroma accompanied by harmonious fruity and floral notes. Additionally, 1-hexanol, 1,1-diethoxy-3-methylbutane, dimethyl trisulfide, furfural, and furfuryl ethyl ether were recognized as the major contributors to mung bean aroma. Approximately 66.67 % of key compounds peaked during 3-5-year aging and subsequently declined. Ethyl 3-methylbutanoate, furfuryl ethyl ether, 1-butanol, 1,1-diethoxy-3-methylbutane and dimethyl trisulfide with VIP > 1 were identified as aging markers. These findings provide a theoretical basis for the sensory characterization and aging evaluation of Baijiu.

Chinese Baijiu brewing relies on the vertical layering of Zaopei, yet its effect on microbial-flavor interactions remain unclear. This study employs result-oriented backward analysis of distinct flavor patterns to decipher how spatial differences govern metabolic networks and flavor distribution. GC-IMS/GC-MS revealed spatial divergence: the surface layer concentrated 62.89 % of total volatile flavors, significantly higher than the bottom layer (18.88 %) and the middle/upper layers (8.67-9.55 %). Fungal communities played a key role in layer differentiation (p = 0.002) through temperature and chemical changes. Deterministic selection dominated in hot and acidic (3.39 g/mol) surface layer (40.74 % |βNTI| > 2), while random drift prevailed in cool and high-ethanol (3.49 % v/v) bottom layer (NST = 78.14 %), with limited dispersal linking the layers. Five key flavor-related microbes were identified, highlighting the role of microbial metabolism in flavor development. Overall, the vertical structure of Zaopei guides the evolution of Baijiu's complex, multi-layered flavor profile.

The pyruvate dehydrogenase complex (PDHc) in Lactococcus lactis, composed of pdh E1α, E1β, and E2, links glycolysis to the tricarboxylic acid cycle (TCA cycle). This study investigated the effect of the pdh E2 knockout on metabolism. The knockout strain showed 76% biomass, 48% lower PDHc activity, and 13.4% reduced ATP versus wild type but a 1.5-fold higher NAD+/NADH ratio. Lactate production increased by 5.6% in the knockout and 45.3% in overexpressing strains. Next to this, the lactate dehydrogenase activity was induced, achieving the highest enzyme amount with the addition of 5 ng/mL nisin after 48 h of induction (28.7% higher than that of wild-type L. lactis). In addition, the transcript levels of genes related to the pyruvate metabolic pathway also changed: pdh E2 decreased by 67.62%, while pdh E1β was upregulated 15-fold. Our study provides insights for enhancing lactate production and understanding pdh E2 function.

Barnase, derived from Bacillus amyloliquefaciens, is a key enzyme in biocatalysis with widespread applications in pharmaceutical synthesis. However, its stability under extreme conditions, such as high temperatures and extreme pH, limits its industrial applications. Therefore, enhancing both its catalytic efficiency and stability through genetic engineering has become a critical focus of research. In this study, AlphaFold was employed to predict the structure of Barnase, followed by molecular docking and molecular dynamics simulations using GROMACS to design and construct 24 mutants. The results demonstrated that the enzymatic activity of the S28H and D101 K mutants increased by 75.28% and 71.86%, respectively, while the stability of D101 K declined under high temperatures. To address this, D101 K was immobilized onto a ZIF-8 carrier. Under optimized immobilization conditions (1.5 M 2-methylimidazole, 1.5 mL enzyme solution, 20 °C), ZIF-8@D101 K exhibited significantly enhanced thermal stability and pH adaptability. Recycling experiments showed that 96.21% of its activity was retained after three cycles, and 72.47% after eight cycles, demonstrating superior reusability and stability, making it more suitable for industrial applications.

The summer-autumn green tea is hard for consumers to accept because of its bitter taste and low quality. This study reveals the core mechanism by which inoculation with the single fungal strain Aspergillus cristatus in naturally enhanced fermentation improves the quality of summer-autumn green teas. Multi-omics (including microbiomics through Illumina MiSeq high-throughput sequencing, non-volatile metabolomics through high-performance liquid chromatography-mass spectrometry, and volatile metabolomics through gas chromatography-mass spectrometry) and sensory analysis were used to evaluate the metabolites and quality of summer-autumn green tea before and after fermentation. The results indicated that Aspergillus rapidly dominated the microbial community during the early stages of fermentation, maintaining an absolute dominance (accounting for over 99 % of total fungi). It significantly reduces the content of bitter compounds such as flavonoids, tea polyphenols, and catechins through key metabolic pathways (e.g., flavonoid biosynthesis, flavonoid and flavanol biosynthesis, and arginine biosynthesis), while optimizing the composition of free amino acids (Increasing in fresh and sweet-tasting amino acids and decreasing in salty and bitter-tasting ones). Furthermore, Aspergillus drove a significant increase in volatile organic compounds (Geraniol, Phenylethanol, Methyl salicylate, etc) through isoprenoid synthesis and phenylalanine metabolic pathways. This shift led to a transformation in aroma profile from esters to ketones, while creating a complex fragrance characterized by floral, fruity and sweet scents. Correlation analysis further indicated that Aspergillus metabolism directly regulated the conversion of characteristic non-volatile and volatile components. This research elucidates a complete pathway through which a single fungus reshapes the metabolic profile of summer-autumn green tea via directed metabolism, providing a theoretical foundation for optimizing natural fermentation processes.

Honey is known to promote alcohol metabolism effectively. However, the effects of its individual chemical components system on alcohol metabolism and their mechanisms of action have not yet been fully elucidated. We constructed polyphenols and ascorbic acid systems (PAAS) of six different floral kinds of honey by exogenous substances, to investigate their effects on alcohol metabolism in the liver. PAAS consists of 22 kinds of polyphenols (including arbutin and caffeic acid) and ascorbic acid. The results demonstrated that PAAS improved the activity of alcohol dehydrogenase (ADH) and the expression of adh1, but had no significant effect on acetaldehyde dehydrogenase (ALDH) and expression of adh2, which caused a decrease in blood ethanol concentration but no difference in acetaldehyde concentration. Correlation analysis illustrated that arbutin and trans-4-hydroxycinnamic acid in PAAS were important potential substances for promoting alcohol metabolism. In addition, PAAS could also reduce the deleterious effects of alcohol by modulating unsaturated fatty acid biosynthesis, purine metabolism, and other metabolic pathways in the liver. These findings revealed the mechanisms by which PAAS promoted hepatic alcohol metabolism as well as protected the liver and provided a theoretical basis for exploring the mechanisms in honey synergistically promote alcohol metabolism.

Corn silk, an agricultural by-product rich in bioactive compounds, has long remained underutilized. In this study, we systematically examined how probiotic co-fermentation influences the bioactivity, flavor profile, and metabolic landscape of corn silk. The results demonstrated that fermentation significantly enhanced the total phenolic content by 73.31 % and flavonoid content by 80.20 %, along with a substantial improvement in antioxidant capacity. Volatile compound analysis identified 36 key aroma-related substances. Notably, 4-methylguaiacol and 4-ethylguaiacol contributed to both improved flavor and antioxidant function, highlighting a synergistic relationship between sensory quality and bioactivity during fermentation. Non-targeted metabolomics further revealed substantial changes in the metabolic profile post-fermentation, with 110 differential metabolites identified-75 upregulated and 35 downregulated. These metabolites were primarily enriched in the TCA cycle, phenylpropanoid biosynthesis, and amino acid metabolism pathways. Together, these findings uncover the molecular mechanisms by which probiotic fermentation remodels metabolic networks to enhance both the functional and sensory attributes of corn silk. This work offers a theoretical foundation and technical guidance for the high-value utilization of agricultural by-products and the development of functional foods.

The succession of microbial communities during the fermentation process in sesame-flavored Baijiu cellars profoundly influences the flavor profile of the liquor. However, the key factors driving microbial succession in these cellars remain unclear. This study focuses on the fermentation process of sesame-flavored Baijiu Zaopei in traditional Tongcheng cellars. Samples were collected from the surface, middle, and bottom of the cellar, categorized by fermentation time. Various techniques were employed to analyze the physicochemical properties (including moisture, ethanol, total acid, starch, and reducing sugars), flavor compounds (volatile substances and amino acids), and microbial communities (bacteria and fungi) of the Zaopei during fermentation. A total of 68 flavor compounds were detected, with 16 key flavor compounds and 16 amino acids identified. Microbiologically, the Lactobacillus genus dominated in the later stages of fermentation, while the Issatchenkia species were the predominant fungi. Correlation analysis indicated that environmental factors play a significant role in driving microbial community succession. Acetobacter, Staphylococcus, Pichia, Bacillus, and Kroppenstedtia species may contribute to the synthesis of key flavor compounds. The relative contents of acetic acid, 2-phenylethyl ester, and Benzenepropanoic acid ethyl ester were influenced by multiple microbial groups, suggesting a synergistic fermentation effect. PICRUSt2 predictions revealed significant differences in 41 KEGG pathways at level 2 and 293 pathways at level 3 across different fermentation intervals. These pathways are primarily associated with amino acid, ester, and nucleotide metabolism, as well as bacterial transcription, translation, and signal transduction. This research provides a theoretical foundation for understanding the fermentation mechanisms of sesame-flavored Baijiu.

In this study, wheat bran was used to prepare dietary fiber by Monascus anka in liquid fermentation. The structural and functional characteristics of wheat bran dietary fiber were analyzed. Scanning electron microscopy and X-ray diffraction analysis indicated that the insoluble dietary fiber matrix was disrupted during the liquid fermentation. Infrared spectroscopy and differential scanning calorimetry analysis demonstrated that intramolecular hydrogen bonds were broken and the oligosaccharides increased. The soluble dietary fiber content increased from 10.7 g/100 g to 16.5 g/100 g, which contributed to improvements in the water-holding capacity, oil-holding capacity, and swelling capacity of wheat bran dietary fiber. UV–Vis spectroscopy analysis demonstrated that the M. anka wheat bran fermentation broth (MWFB) mainly contained yellow pigments (236.6 μ mL−1). HPLC-MS spectrometry further showed MWFB contained three known Monascus pigments: monasine (observed. m/z 359.1853 [M+H]+), ankaflavin (observed. m/z 387.2151 [M+H]+), and monascorubrin (observed. m/z 382.2007 [M+H]+). In conclusion, M. anka can make the most use of wheat bran and improve the structure and function of dietary fiber, thereby expanding its application potential in functional food additives, gut microbiota modulation, and low-calorie baked goods.