Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Efficient production and characterization of a newly identified trehalase for inhibiting the formation of bacterial biofilms.

Trehalase has attracted widespread attention in medicine, agriculture, food, and ethanol industry due to its ability to specifically degrade trehalose. Efficient expression of trehalase remains a challenge. In this study, a putative trehalase-encoding gene (Tre-zm) from Zunongwangia mangrovi was explored using gene-mining strategy and heterologously expressed in E. coli. Trehalase activity reached 3374 U·mL-1 after fermentation optimization. The scale-up fermentation in a 15 L fermenter was achieved with a trehalase production of 15,068 U·mL-1. The recombinant trehalase TreZM was purified and characterized. It displayed optimal activity at 35 °C and pH 8.5, with Mn2+, Sn2+, Na+, and Fe2+ promoting the activity. Notably, TreZM showed significant inhibition effect on biofilm forming of Staphylococcus epidermidis. The combination of TreZM with a low concentration of antibiotics could inhibit 70 % biofilm formation of Staphylococcus epidermidis and 28 % of Pseudomonas aeruginosa. Hence, this study provides a promising candidate for industrial production of trehalase and highlights its potential application to control harmful biofilms.

High-Level Extracellular Expression of Hyaluronate Lyase HylP in Bacillus subtilis for Hyaluronan Degradation.

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20 L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40 °C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40 °C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.

Rapid detection of six Oceanobacillus species in Daqu starter using single-cell Raman spectroscopy combined with machine learning.

Many traditional fermented foods and beverages industries around the world request the addition of multi-species starter cultures. However, the microbial community in starter cultures is subject to fluctuations due to their exposure to an open environment during fermentation. A rapid detection approach to identify the microbial composition of starter culture is essential to ensure the quality of the final products. Here, we applied single-cell Raman spectroscopy (SCRS) combined with machine learning to monitor Oceanobacillus species in Daqu starter, which plays crucial roles in the process of Chinese baijiu. First, a total of six Oceanobacillus species (O. caeni, O. kimchii, O. iheyensis, O. sojae, O. oncorhynchi subsp. Oncorhynchi and O. profundus) were detected in 44 Daqu samples by amplicon sequencing and isolated by pure culture. Then, we created a reference database of these Oceanobacillus strains which correlated their taxonomic data and single-cell Raman spectra (SCRS). Based on the SCRS dataset, five machine-learning algorithms were used to classify Oceanobacillus strains, among which support vector machine (SVM) showed the highest rate of accuracy. For validation of SVM-based model, we employed a synthetic microbial community composed of varying proportions of Oceanobacillus species and demonstrated a remarkable accuracy, with a mean error was less than 1% between the predicted result and the expected value. The relative abundance of six different Oceanobacillus species during Daqu fermentation was predicted within 60 min using this method, and the reliability of the method was proved by correlating the Raman spectrum with the amplicon sequencing profiles by partial least squares regression. Our study provides a rapid, non-destructive and label-free approach for rapid identification of Oceanobacillus species in Daqu starter culture, contributing to real-time monitoring of fermentation process and ensuring high-quality products.

Lactic acid fermentation of goji berries (Lycium barbarum) prevents acute alcohol liver injury and modulates gut microbiota and metabolites in mice.

Juice fermented with lactic acid bacteria (LAB) has received attention due to its health benefits, such as antioxidant and anti-inflammatory. Previous research on LAB-fermented goji juice mainly focused on exploring the changes in the metabolite profile and antioxidant activity in vitro, whereas the liver protection properties of LAB-fermented goji juice in vivo are still unknown. This study aimed to investigate the effects of Lacticaseibacillus paracasei E10-fermented goji juice (E10F), Lactiplantibacillus plantarum M-fermented goji juice (MF), Lacticaseibacillus rhamnosus LGG-fermented goji juice (LGGF) on preventing acute alcoholic liver injury with physiology, gut microbial, and metabolic profiles in mice. Compared with goji juice, E10F, MF, and LGGF enhanced the protective effect against liver injury by reducing serum alanine transaminase (ALT) levels, improving the hepatic glutathione (GSH) antioxidant system, and attenuating inflammation by decreasing the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß. Furthermore, E10F, MF, and LGGF increased intestinal integrity, restructured the gut microbiota including Bacteroides and Lactobacillus, and altered gut microbial metabolites including kyotorphin, indolelactic acid, and N-methylserotonin. Pretreatment of different LAB-fermented goji juice in mice showed significant differences in gut microbiota and metabolism. The correlation analysis demonstrated that the increase of Lactobacillus, indolelactic acid, and N-methylserotonin by E10F, MF, and LGGF was positively correlated with reduced inflammation and improved liver and gut function. Taken together, E10F, MF, and LGGF all have the potential to be converted into dietary interventions to combat acute alcoholic liver injury. It provided a reference for the study of the hepatoprotective effect of LAB-fermented goji juice.

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris.

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum-related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. KEY POINTS: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation.

Microbial biogeography of pit mud from an artificial brewing ecosystem on a large time scale: all roads lead to Rome.

IMPORTANCE: Baijiu is a typical example of how humans employ microorganisms to convert grains into new flavors. Mud cellars are used as the fermentation vessel for strong-flavor Baijiu (SFB) to complete the decomposition process of grains. The typical flavor of SFB is mainly attributed to the metabolites of the pit mud microbiome. China has a large number of SFB-producing regions. Previous research revealed the temporal profiles of the pit mud microbiome in different geographical regions. However, each single independent study rarely yields a thorough understanding of the pit mud ecosystem. Will the pit mud microbial communities in different production regions exhibit similar succession patterns and structures under the impact of the brewing environment? Hence, we conducted research in pit mud microbial biogeography to uncover the impact of specific environment on the microbial community over a long time scale.

Technology and functional insights into the nicotinamide mononucleotide for human health.

Nicotinamide mononucleotide (NMN), a naturally occurring biologically active nucleotide, mainly functions via mediating the biosynthesis of NAD+. In recent years, its excellent pharmacological activities including anti-aging, treating neurodegenerative diseases, and protecting the heart have attracted increasing attention from scholars and entrepreneurs for production of a wide range of formulations, including functional food ingredients, health care products, active pharmaceuticals, and pharmaceutical intermediates. Presently, the synthesis methods of NMN mainly include two categories: chemical synthesis and biosynthesis. With the development of biocatalyst engineering and synthetic biology strategies, bio-preparation has proven to be efficient, economical, and sustainable methods. This review summarizes the chemical synthesis and biosynthetic pathways of NMN and provides an in-depth investigation on the mining and modification of enzyme resources during NMN biosynthesis, as well as the screening of hosts and optimization of chassis cells via metabolic engineering, which provide effective strategies for efficient production of NMN. In addition, an overview of the significant physiological functions and activities of NMN is elaborated. Finally, future research on technical approaches to further enhance NMN synthesis and strengthen clinical studies of NMN are prospected, which would lay the foundation for further promoting the application of NMN in nutrition, healthy food, and medicine in the future. KEY POINTS: • NMN supplementation effectively increases the level of NAD+. • The chemical and biological synthesis of NMN are comprehensively reviewed. • The impact of NMN on the treatment of various diseases is summarized.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.

Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins. Accounting for the accumulated knowledge of the pivotal factors in the solubility and folding stability of proteins, this review focuses on advanced technologies and tools of protein engineering, protein quality control systems, and the redesign of expression platforms in prokaryotic expression systems, as well as advances of the cell-free expression technologies for membrane proteins production.

Genetic variation reveals the enhanced microbial hyaluronan biosynthesis via atmospheric and room temperature plasma.

This study reveals the genetic and biochemical changes underlying the enhanced hyaluronan (HA) biosynthesis in Streptococcus zooepidemicus. After multiple rounds of atmospheric and room temperature plasma (ARTP) mutagenesis combined with novel bovine serum albumin/cetyltrimethylammonium bromide coupled high-throughput screening assay, the HA yield of the mutant was increased by 42.9% and reached 0.813 g L-1 with a molecular weight of 0.54 × 106 Da within 18 h by shaking flask culture. HA production was increased to 4.56 g L-1 by batch culture in 5-L fermenter. Transcriptome sequencing exhibits that distinct mutants have similar genetic changes. Regulation in direction of metabolic flow into the HA biosynthesis, by enhancing genes responsible for the biosynthesis of HA including hasB, glmU and glmM, weaking downstream gene (nagA and nagB) of UDP-GlcNAc and significantly down-regulating transcription of wall-synthesizing genes, resulting in the accumulation of precursors (UDP-GlcA and UDP-GlcNAc) increased by 39.74% and 119.22%, respectively. These associated regulatory genes may provide control point for engineering of the efficient HA-producing cell factory.

Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin.

Phospholipase D (PLD) is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification. However, the efficient heterologous expression of PLD is limited by its cell toxicity. In this study, a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL. A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was established, and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation, 191.30-fold higher than that of the control. Under optimum reaction conditions, a 61.61% conversion ratio and 21.07 g/L of phosphatidylserine production were achieved. Finally, the synthesis system of PL derivates was established, which could efficiently synthesis novel PL derivates. The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application, and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates. As far as we know, this work reports the highest level of extracellular PLD expression to date and the enzymatic production of several PL derivates for the first time.

Machine learning based age-authentication assisted by chemo-kinetics: Case study of strong-flavor Chinese Baijiu.

The aged Chinese liquor, Baijiu, is highly valued for its superior organoleptic qualities. However, since age-authentication method and aging-mechanism elucidation of Baijiu is still in the exploratory stage, high-quality aged Baijiu is often replaced by lower-quality, less-aged product with fraudulent mislabeling. Authentic high-quality strong-flavor Baijiu was analyzed by gas chromatography-mass spectrometry. Total esters decreased with aging, while acids, alcohols, aldehydes, ketones, terpenes, pyrazines increased. Although concentrations of partial compounds showed non-monotonic profiling during aging, a close positive linear correlation (R2 = 0.7012) of Baijiu Evenness index (0.55-0.59) with aging time was observed, indicating a more balanced composition in aged Baijiu. The reaction quotient (Qc) of each esterification, calculated by the corresponding reactant and product concentration, approached to the corresponding thermodynamic equilibrium constant Kc. This result demonstrated that the spontaneous transformation driven by thermodynamics explained part of the aging compositional profiling. Furthermore, an aging-related feature selection and an age-authentication method were established based on three models combined with five ranking algorithms. Forty-one key features, including thirty-six compound concentrations, four esterification Qc values and the Evenness index were selected out. The age-authentication based on neural network using forty-one input features accurately predicted the age group of Baijiu samples (F1 = 100 %). These findings have deepened understanding of the Baijiu aging mechanism and provided a novel, effective approach for age-authentication of Baijiu and other liquors.

Protein engineering of NADH pyrophosphatase for efficient biocatalytic production of reduced nicotinamide mononucleotide.

Introduction: NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide, has potential applications in the food, cosmetic and pharmaceutical industry. Methods: Here, we investigated the effects of vector screening, promoter and RBS strategies on NADH pyrophosphatase expression and protein engineering on its enzymatic activity and thermal stability. Results: In this study, we describe a NADH pyrophosphatase derived from Escherichia coli (EcNudc). Strategies focusing on expression regulation including screening vectors, optimizing promoters and ribosome binding sites were utilized to enhance the productivity of EcNudc (1.8 U/mL). Moreover, protein engineering was adopted to further improve the catalytic properties of EcNudc, achieving 3.3-fold higher activity and 3.6-fold greater thermostability at 50°C. Furthermore, fermentation for the combined mutant R148A-H149E (EcNudc-M) production in a 7 L fermenter was implemented and the enzyme activity of EcNudc-M reached 33.0 U/mL. Finally, the EcNudc-M was applied in the catalysis of NADH with the highest NMNH yield of 16.65 g/L. Discussion: In conclusion, we constructed a commercially available genetically engineered strain with high activity and thermal stability of NADH pyrophosphatase, laying a broad foundation for the biocatalytic industrial production of NMNH and expand its application range.

Metabolomic analysis of the effects of a mixed culture of Saccharomyces cerevisiae and Lactiplantibacillus plantarum on the physicochemical and quality characteristics of apple cider vinegar.

Introduction: This study compared differences in physicochemical characteristics of the vinegar made by a mixed culture (MC) of Saccharomyces cerevisiae and Lactiplantibacillus plantarum and a pure culture (PC) of Saccharomyces cerevisiae. Methods: The fermentation process was monitored, and metabolomics analysis by Liquid Chromagraphy-Mass Spectrometry (LC-MS) was applied to the compositional differences between PC and MC vinegars, combined with quantification of organic acids, amino acids and B vitamins. Results: A total of 71 differential metabolites including amino acids, organic acids and carbohydrates, and six possible key metabolic pathways were identified. MC enhanced the malic acid utilization and pyruvate acid metabolism during fermentation, increasing substrate-level phosphorylation, and supplying more energy for cellular metabolism. Higher acidity at the beginning of acetic acid fermentation, resulting from lactic acid production by Lactiplantibacillus plantarum in MC, suppressed the cellular metabolism and growth of Acetobacter pasteurianus, but enhanced its alcohol metabolism and acetic acid production in MC. MC vinegar contained more vitamin B, total flavonoids, total organic acids, amino acids and had a higher antioxidant capacity. MC enhanced the volatile substances, particularly ethyl lactate, ethyl caprate and ethyl caproate, which contributed to a stronger fruity aroma. Discussion: These results indicated the mixed culture in alcoholic fermentation can effectively enhance the flavor and quality of apple cider vinegar.

High-level expression and characterization of a highly active hyaluronate lyase HylC with significant potential in hyaluronan oligosaccharide preparation.

Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.5 M NaCl. The enzyme exhibited the optimal activity under 37 °C and pH 5.5, and was activated by Ca2+, K+, Zn2+, Ni2+ and Li+. Analysis of degradation product proved it cleave HA in endolytic manner, releasing unsaturated disaccharides as final product. Then, through optimization of promoter and construction of dual promoter, expression level of HylC improved from 1.10 × 104 U/mL to 2.64 × 104 U/mL on shake-flask level. Finally, through batch fermentation, a highest activity of 2.65×105 U/mL was achieved in a 5-L fermenter. Taken together, this work demonstrates the potential of HylC and its recombinant strain in industrial applications. To our knowledge, the HA lyase production reported in this study was the highest level in literatures to date.

Metformin alleviates liver fibrosis in mice by enriching Lactobacillus sp. MF-1 in the gut microbiota.

BACKGROUND: Liver fibrosis is associated with gut dysbiosis. Metformin administration has emerged as a promising method for the treatment of organ fibrosis. We aimed to investigate whether metformin ameliorates liver fibrosis by enhancing the gut microbiota in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and the underlying mechanism. MATERIALS AND METHODS: A liver fibrosis mouse model was established, and the therapeutic effects of metformin were observed. We administered antibiotic treatment and performed fecal microbiota transplantation (FMT), and 16S rRNA-based microbiome analysis to evaluate the effects of the gut microbiome on metformin-treated liver fibrosis. We isolated the bacterial strain preferably enriched by metformin and assessed its antifibrotic effects. RESULTS: Metformin treatment repaired the gut integrity of the CCl4-treated mice. It reduced the number of bacteria in colon tissues and reduced the portal vein lipopolysaccharide (LPS) levels. The FMT performed on the metformin-treated CCl4 mice alleviated their liver fibrosis and reduced their portal vein LPS levels. The markedly changed gut microbiota was screened out from the feces and named Lactobacillus sp. MF-1 (L. sp. MF-1). In the CCl4-treated mice, daily gavage of L. sp. MF-1 maintained gut integrity, inhibited bacterial translocation, and reduced liver fibrosis. Mechanistically, metformin or L. sp. MF-1 inhibited the apoptosis of intestinal epithelial cells and restored CD3+ intestinal intraepithelial lymphocytes in the ileum and CD4+Foxp3+ lamina propria lymphocytes in the colon. CONCLUSIONS: Metformin and its enriched L. sp. MF-1 can reinforce the intestinal barrier to alleviate liver fibrosis by restoring immune function.

Diversity and Comparison of Intestinal Desulfovibrio in Patients with Liver Cirrhosis and Healthy People.

Desulfovibrio belongs to Sulfate-reducing bacteria (SRB), which are widely present in anaerobic environments, including the human gut. Desulfovibrio has been associated with many human diseases, including chronic liver disease. However, the characteristics and difference of Desulfovibrio from fecal samples of healthy volunteers (HV) and patients with liver cirrhosis (LC) have not been fully elucidated. Here, we isolated Desulfovibrio from the feces of 6 HV and 9 LC, and 88 Desulfovibrio strains were obtained. In the feces of HV, 55% of isolated strains were D. desulfuricans, followed by D. intestinalis (15%), D. simplex (11%), D. piger (9%), D. legallii (4%), Cupidesulfovibrio oxamicus (4%) and D. fairfieldensis (2%). However, only D. desulfuricans (60%) and C. oxamicus (40%) were isolated from fecal samples of patients with LC. Our results suggest that there was a significant difference in the desulfurization ability and the H2S production ability of different Desulfovibrio. Desulfovibrio. Furthermore, we found that Desulfovibrio isolated from the patients with LC generally had a higher hydrogen sulfide production capacity, gastrointestinal tolerance, and levels of antibiotic resistance than the same species isolated from HV. Our findings suggested that Desulfovibrio may be associated with the occurrence and development of liver cirrhosis.

Bacteria and filamentous fungi running a relay race in Daqu fermentation enable macromolecular degradation and flavor substance formation.

As the saccharifying and fermentative agent, medium-temperature Daqu (MT-Daqu) plays an irreplaceable role in the production of strong-flavor Baijiu. Numerous studies have focused on the microbial community structure and potential functional microorganisms, however, little is known about the succession of active microbial community and the formation mechanism of community function during MT-Daqu fermentation. In this study, we presented an integrated analysis of metagenomics, metatranscriptomics, and metabonomics covering the whole fermentation process of MT-Daqu to reveal the active microorganisms and their participations in metabolic networks. The results showed that dynamic of metabolites were time-specific, and the metabolites and co-expressed active unigenes were further classified into four clusters according to their accumulation patterns, with members within each cluster displaying a uniform and clear pattern of abundance across fermentation. Based on KEGG enrichment analysis in co-expression clusters and succession of active microbial community, we revealed that Limosilactobacillus, Staphylococcus, Pichia, Rhizopus, and Lichtheimia were metabolically active members at the early stage, and their metabolic activities were conducive to releasing abundant energy to drive multiple basal metabolisms such as carbohydrates and amino acids. Thereafter, during the high temperature period and at the end of fermentation, multiple heat-resistant filamentous fungi were transcriptionally active populations, and they acted as both the saccharifying agents and flavor compound producers, especially aromatic compounds, suggesting their crucial contribution to enzymatic activity and aroma of mature MT-Daqu. Our findings revealed the succession and metabolic functions of the active microbial community, providing a deeper understanding of their contribution to MT-Daqu ecosystem.