Pangenome analyses of Clostridium butyricum provide insights into its genetic characteristics and industrial application.

Clostridium butyricum is a Gram-positive anaerobic bacterium known for its ability to produce butyate. In this study, we conducted whole-genome sequencing and assembly of 14C. butyricum industrial strains collected from various parts of China. We performed a pan-genome comparative analysis of the 14 assembled strains and 139 strains downloaded from NCBI. We found that the genes related to critical industrial production pathways were primarily present in the core and soft-core gene categories. The phylogenetic analysis revealed that strains from the same clade of the phylogenetic tree possessed similar antibiotic resistance and virulence factors, with most of these genes present in the shell and cloud gene categories. Finally, we predicted the genes producing bacteriocins and botulinum toxins as well as CRISPR systems responsible for host defense. In conclusion, our research provides a desirable pan-genome database for the industrial production, food application, and genetic research of C. butyricum.

Ameliorative effect of an acidic polysaccharide from Phellinus linteus on ulcerative colitis in a DSS-induced mouse model.

Phellinus linteus, a rare medicinal fungus, displays strong antitumor and anti-inflammatory activities because of its active metabolites, particularly polysaccharides. We investigated effects of P. linteus acidic polysaccharide (PLAP) on amelioration of dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in a mouse model, and associated mechanisms. PLAP treatment alleviated major UC symptoms (weight loss, reduced food intake, increased disease activity index), and ameliorated histopathological colon tissue damage, reduced levels of pro-inflammatory factors (TNF-α, IL-6, IL-1ß), enhanced anti-inflammatory factor IL-10 level, reduced levels of oxidative stress-related enzymes iNOS and MPO, and enhanced expression of tight junction proteins (ZO-1, occludin, claudin-1). qPCR analysis revealed that PLAP downregulated phosphorylation levels of p65 and p38 and transcriptional level of TLR-4. High-throughput sequencing showed that PLAP restored gut microbiota diversity and species abundances in the UC model, and gas chromatographic analysis showed that it increased levels of beneficial short-chain fatty acids. Our findings indicate that PLAP has strong potential for development as an anti-UC agent based on its reduction of inflammation and oxidative stress levels, modulation of gut microbiota composition, and promotion of normal intestinal barrier function.

Loop-mediated isothermal amplification (LAMP)/Cas12a assay for detection of Ralstonia solanacearum in tomato.

Introduction: Bacterial wilt (BW) caused by the aerobic, Gram-negative pathogenic species Ralstonia solanacearum (RS) is a major disease impacting commercial agriculture worldwide. Asian phylotype I of RS is the cause of tomato bacterial wilt, which has caused severe economic losses in southern China for many years. An urgent priority in control of bacterial wilt is development of rapid, sensitive, effective methods for detection of RS. Methods: We describe here a novel RS detection assay based on combination of loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a. crRNA1, with high trans-cleavage activity targeting hrpB gene, was selected out of four candidate crRNAs. Two visual detection techniques, involving naked-eye observation of fluorescence and lateral flow strips, were tested and displayed high sensitivity and strong specificity. Results and Discussion: The LAMP/Cas12a assay accurately detected RS phylotype Ⅰ in 14 test strains, and showed low detection limit (2.0 × 100 copies). RS in tomato stem tissue and soil samples from two field sites with suspected BW infection was identified accurately, suggesting potential application of LAMP/Cas12a assay as point-of-care test (POCT). The overall detection process took less than 2 h and did not require professional lab equipment. Our findings, taken together, indicate that LAMP/Cas12a assay can be developed as an effective, inexpensive technique for field detection and monitoring of RS.

Optimizing the scale-up production of fermented astragalus and its benefits to the performance and egg quality of laying hens.

Astragalus is a homologous medicine and food that benefits human beings and poultry rearing. Fermented astragalus (FA) is a valuable product obtained by fermentation, but its scale-up production requires optimization and expansion of solid-state fermentation (SSF). In this study, Lactobacillus pentosus Stm was screened as the most suitable LAB strain for fermenting astragalus due to its excellent capacity. After optimization and expansion of SSF, LAB count and lactic acid content reached 206 × 108 cfu/g and 15.0%, respectively. Meanwhile, the content of bioactive compounds in FA was significantly enhanced. Feeding experiments with laying hens indicated that supplementing FA in the diet significantly improved the performance and egg quality, as evidenced by reduced feed-to-egg ratio and egg cholesterol. This was due to the promotion of intestinal health by shifting intestinal microbiota. Therefore, this is a systematical endeavor of producing scaled-up FA with promising potential as a feed additive in the poultry breeding industry.

The Effect of the Microalgae Chlorella vulgaris on the Gut Microbiota of Juvenile Nile Tilapia (Oreochromis niloticus) Is Feeding-Time Dependent.

Chlorella vulgaris is one of the most commonly used microalgae in aquaculture feeds. It contains high concentrations of various kinds of nutritional elements that are involved in the physiological regulation of aquaculture animals. However, few studies have been conducted to illustrate their influence on the gut microbiota in fish. In this work, the gut microbiota of Nile tilapia (Oreochromis niloticus) (average weight is 6.64 g) was analyzed by high-throughput sequencing of the 16S rRNA gene after feeding with 0.5% and 2% C. vulgaris additives in diets for 15 and 30 days (average water temperature was 26 °C). We found that the impact of C. vulgaris on the gut microbiota of Nile tilapia was feeding-time dependent. Only by feeding for 30 days (not 15 days) did the addition of 2% C. vulgaris to diets significantly elevate the alpha diversity (Chao1, Faith pd, Shannon, Simpson, and the number of observed species) of the gut microbiota. Similarly, C. vulgaris exerted a significant effect on the beta diversity (Bray-Curtis similarity) of the gut microbiota after feeding for 30 days (not 15 days). During the 15-day feeding trial, LEfSe analysis showed that Paracoccus, Thiobacillus, Dechloromonas, and Desulfococcus were enriched under 2% C. vulgaris treatment. During the 30-day feeding trial, Afipia, Ochrobactrum, Polymorphum, Albidovulum, Pseudacidovorax, and Thiolamprovum were more abundant in 2% C. vulgaris-treated fish. C. vulgaris promoted the interaction of gut microbiota in juvenile Nile tilapia by increasing the abundance of Reyranella. Moreover, during the feeding time of 15 days, the gut microbes interacted more closely than those during the feeding time of 30 days. This work will be valuable for understanding how C. vulgaris in diets impacts the gut microbiota in fish.

Clostridium butyricum and Chitooligosaccharides in Synbiotic Combination Ameliorate Symptoms in a DSS-Induced Ulcerative Colitis Mouse Model by Modulating Gut Microbiota and Enhancing Intestinal Barrier Function.

Effects of Clostridium butyricum and chitooligosaccharides (COS), singly and in synbiotic combination, were evaluated in a C57BL/6 mouse model of dextran sulfate (DSS)-induced acute ulcerative colitis (UC). Treatment with C. butyricum and/or COS ameliorated UC symptoms in vivo, and the strongest effects were observed for the combination in terms of reduced mortality rates and disease activity indices, increased body weight and colon length, and improved histological features. The C. butyricum and COS combination achieved the following: (i) regulated levels of inflammation-related cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1ß [IL-1ß], IL-6, IL-10) and had a stronger anti-inflammatory effect than either component alone, based on inhibition of Toll-like receptor 4 (TLR-4)/NF-κB/MAPK signaling pathway activation; (ii) enhanced intestinal barrier function by restoring levels of tight junction proteins (occludin, claudin-1, ZO-1) and MUC2; (iii) increased abundance and diversity of beneficial bacteria (gut microbiota) and reduced levels of pathogenic bacteria; and (iv) enhanced production of short-chain fatty acids. Our findings indicate that the synbiotic C. butyricum and COS combination has strong potential as a therapeutic adjuvant for UC. IMPORTANCE Ulcerative colitis (UC), an idiopathic intestinal disease characterized by continuous remission/relapse inflammatory cycles in the colonic mucosal layer, has strong adverse effects on patients' quality of life and considerable costs for health care systems. Probiotics, prebiotics, and synbiotics are regarded as potential therapeutic agents for UC, in terms of safety and efficacy. In this study, we present detailed evaluation of effects in a DSS-induced UC mouse model of a synbiotic composed of Clostridium butyricum and COS (molecular weight [MW], 2,500 Da). We found that synergistic (synbiotic) action of the C. butyricum and COS combination is more effective than either factor alone for prevention and/or therapy of UC by regulating gut microbiota and intestinal barrier function. Our findings indicate that C. butyricum and COS in combination has strong potential for development as anti-UC therapeutic drugs or adjuvant agents in pharmaceutical, food, and livestock industries. Highlights include the following. (i) The C. butyricum and COS combination ameliorated clinical UC symptoms and improved colonic morphology. (ii) The C. butyricum and COS combination displayed strong anti-inflammatory and antioxidant effects. (iii) The C. butyricum and COS combination enhanced expression of tight junction proteins. (iv) The C. butyricum and COS combination inhibited the TRL-4/NF-κB/MAPK signaling pathway. (v) The C. butyricum and COS combination modulated gut microbiota abundance and composition.

Lactobacillus rhamnosus and L. plantarum Combination Treatment Ameliorated Colitis Symptoms in a Mouse Model by Altering Intestinal Microbial Composition and Suppressing Inflammatory Response.

SCOPE: Changes in composition of intestinal microbes may disrupt the balance of their interaction with a susceptible host, resulting in development of inflammatory bowel disease (IBD). METHODS AND RESULTS: The study applied in combination two Lactobacillus strains (L. rhamnosus BY-02, L. plantarum BY-05) ("LS treatment"), previously isolates from feces of healthy human infants, in a mouse model of dextran sodium sulfate (DSS)-induced colitis, and evaluates their ameliorative effect and its possible mechanism. LS treatment suppresses weight loss and colon shortening, and reduces disease activity index in the mice. It also has several additional beneficial effects: i) maintains goblet cell numbers and ameliorates intestinal barrier damage in colonic tissue; ii) alters intestinal microbial composition close to normal by increasing abundances of Muribaculaceae, Akkermansia, Clostridia, Oscillospiraceae, and Lachnospiraceae, and decreasing abundance of Escherichia-Shigella; iii) increases content of short-chain fatty acids; iv) reduces content of pro-inflammatory lipopolysaccharides; v) suppresses overactivation of TLR4/NF-κB inflammatory signaling pathway. CONCLUSION: Combination treatment with two Lactobacillus strains strongly ameliorates colitis symptoms in the mouse model by favorably altering intestinal microbial composition and suppressing inflammatory response.

Enrofloxacin-induced transfer of multiple-antibiotic resistance genes and emergence of novel resistant bacteria in red swamp crayfish guts and pond sediments.

Antibiotic resistance genes (ARGs) can be transferred from environmental microbes to human pathogens, thus leading to bacterial infection treatment failures. The aquaculture polluted by over-used antibiotics is considered as a notorious reservoir of ARGs. However, the origin, diachronic changes, and mobility of ARGs under antibiotic exposure in aquaculture systems remain elusive. Our findings showed that enrofloxacin application also increased the relative abundance of various ARGs in addition to quinolone-resistance genes and induced ARG dissemination in crayfish gut and sediment bacteria. Further investigation indicated that the transposase-mediated recombination was the major driver of horizontal gene transfer (HGT) of ARGs under antibiotic stress. Notably, enrofloxacin application also induced the generation of some metagenome-assembled genomes (MAGs) carrying multiple ARGs, which were identified as novel species. Additionally, Enterobacteriaceae constituted a mobile ARG pool in aquaculture. Therefore, aquaculture provides potential wide environmental pathways for generation and spread of antibiotic resistance. Our findings of ARG temporal variations and dissemination pattern in aquaculture with artificial use of antibiotics are critical to the management of antibiotic resistance, which is of great ecosystem and health implications.

Improving NH3 and H2S removal efficiency with pilot-scale biotrickling filter by co-immobilizing Kosakonia oryzae FB2-3 and Acinetobacter baumannii L5-4.

In this study, two NH4+-N and S2- removal strains, namely, Kosakonia oryzae (FB2-3) and Acinetobacter baumannii (L5-4), were isolated from the packing materials in a long-running biotrickling filter (BTF). The removal capacities of combined FB2-3 and L5-4 (FB2-3 + L5-4) toward 100 mg L-1 of NH4+-N and 200 mg L-1 of S2- reached 97.31 ± 1.62% and 98.57 ± 1.12% under the optimal conditions (32.0 °C and initial pH = 7.0), which were higher than those of single strain. Then, FB2-3 and L5-4 liquid inoculums were prepared, and their concentrations respectively reached 1.56 × 109 CFU mL-1 and 1.05 × 109 CFU mL-1 by adding different resuspension solutions and protective agents after 12-week storage at 25 °C. Finally, pilot-scale BTF test showed that NH3 and H2S in the real exhaust gases from a pharmaceutical factory were effectively removed with removal rates > 87% and maximum elimination capacities were reached 136 g (NH3) m-3 h-1 and 176 g (H2S) m-3 h-1 at 18 °C-34 °C and pH 4.0-7.0 in the BTF loaded with bamboo charcoal packing materials co-immobilized with FB2-3 and L5-4. After co-immobilization of FB2-3 and L5-4, in the bamboo charcoal packing materials, the new microbial diversity composition contained the dominant genera of Acinetobacter, Mycobacterium, Kosakonia, and Sulfobacillus was formed, and the diversity of entire bacterial community was decreased, compared to the control. These results indicate that FB2-3 and L5-4 have potential to be developed into liquid ready-to-use inoculums for effectively removing NH3 and H2S from exhaust gases in BTF.

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.

BACKGROUND: Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. RESULTS: To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

Unraveling the microbial community and succession during zha-chili fermentation and their relationships with flavor formation.

Zha-chili is a popular traditional fermented food among people in central and southwest China for its nice flavor. It is produced from grain and fresh chili undergoing spontaneously anaerobic fermentation. In this study, the high-throughput sequencing (HTS), high-performance liquid chromatography (HPLC), and head-space solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) were applied to systematically study the dynamic of microbial community and flavor substances during zha-chili fermentation. Simultaneously, the physicochemical factors were investigated to decode the core factors affecting the microbial succession. Results showed that Lactobacillus and Kazachstania were the most dominant bacterium and fungus, respectively, and lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts dominated the whole fermentation. Besides, acidity, ethanol, and temperature were the core factors that shifted the microbial succession. Herein, the acidity and ethanol drove the bacterial succession from Weissella to Lactobacillus, then temperature affected the succession that Lactobacillus was partially replaced by Acetobacter. Meanwhile, acidity and ethanol actuated the fungal succession that Gibberella was superseded by Kazachstania. Moreover, Lactobacillus, Acetobacter, Kazachstania, Wickerhamomyces, Kluyveromyces, and Cyberlindnera were the core microbes associated with the formation of flavor substances including lactic acid, acetic acid, ethanol, ethyl lactate, ethyl acetate, and umami free amino acids. These findings will be helpful to unravel the formation of flavor substances based on microbial community and succession during zha-chili fermentation, providing a solid foundation for the process optimization and quality control of zha-chili.

Sinomicrobium weinanense sp. nov., a halophilic bacterium isolated from saline-alkali soil.

A Gram-stain-negative, facultative anaerobic, non-motile, rod-shaped strain was isolated from saline-alkali soil collected in PR China, and it was designated as strain FJxsT. Its optimal growth was observed at 37-40 °C in the presence of 0-3 % (w/v) NaCl (pH 7.0). The major fatty acids of strain FJxsT were iso-C15 : 0, iso-C17 : 0 3OH, summed feature 3, C16 : 0 and iso-C15 : 1 G. The predominant respiratory quinone was menaquinone 6. The DNA G+C content of the strain was 45.18 mol%. Whole genome and 16S rRNA gene sequence analyses indicated that strain FJxsT exhibited 94.78 % sequence identity (the maximum) with Sinomicrobium soli N-1-3-6T, 94.36 % with Sinomicrobium pectinilyticum 5DNS001T, and 93.52 % with Sinomicrobium oceani SCSIO 03483T. Analyses of genotypic, phenotypic, phylogenetic and chemotaxonomic characteristics indicated that strain FJxsT represented a novel species of the genus Sinomicrobium. This novel species was named Sinomicrobium weinanense sp. nov. with its type strain as FJxsT (=CCTCC AB 2019251T=KCTC 72740T).

Unraveling the composition and succession of microbial community and its relationship to flavor substances during Xin-flavor baijiu brewing.

A novel baijiu type, Xin-flavor, has been developed via combining Qu and craftsmanship from three famous baijiu types including light-flavor, sauce-flavor, and strong-flavor. However, the microbial community and its relationship to physicochemical factors and flavor substances were little known during the three-stage brewing processes including saccharification, stacking, and fermentation. In this study, 21 phyla, 407 genera, and 615 species distributed in bacteria and fungi were identified via high-throughput sequencing (HTS). For bacterial community, the dominant bacteria at saccharification stage were Weissella and Leuconostoc, which were superseded by Acetobacter and Gluconobacter at stacking stage, then quickly replaced by Lactobacillus at fermentation stage. For fungal community, Rhizopus, dominant at saccharification stage, was partially replaced by Thermomyces at stacking stage, then was completely superseded by Thermoascus and three yeasts (Saccharomyces, Kazachstania and Apiotrichum) at fermentation stage. The moisture, ethanol, and acidity, as key factors affecting the microbial community, explained this microbial succession. Interestingly, Apiotrichum was firstly detected in fermented grains. Furthermore, the rapid accumulation of ethanol mainly occurred in the first 10 days of fermentation stage, and highly correlated with Saccharomyces, while Kazachstania and Apiotrichum were related to the formation of flavor substances (acetic acid, lactic acid, caproic acid, ethyl lactate and ethyl caproate). Therefore, this study provides fundamental basis for the rational control of baijiu production and improving the craft and quality of Xin-flavor baijiu.

Chitin derivatives ameliorate DSS-induced ulcerative colitis by changing gut microbiota and restoring intestinal barrier function.

Chitin derivatives (CDs), including chitosan (CS), chitooligosaccharides (COS), and glucosamine (GlcN), were administrated in dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mice. UC symptoms such as body weight loss, reduced food intake, and increased disease activity index were relieved (except GlcNL group). CDs (except GlcNL) exerted a strong protective effect on colon length and colonic structure. Treatment with CDs (except GlcNL) increased IL-10 level, reduced levels of IL-1ß, IL-6, TNF-α, myeloperoxidase, and inducible nitric oxide synthase, and enhanced expression of tight junction proteins significantly. CDs (except GlcNL) significantly upregulated IκB-α level, and downregulated p65 and p38 phosphory lation and TLR-4 mRNA transcription level, indicating inhibition of TRL-4/NF-κB/MAPK signaling pathway activity. CD treatments increased relative abundance of gut microbiota, modulated its composition, and increased the concentrations of SCFAs. Our findings indicate that CDs exert an ameliorative effect on UC by change of gut microbiota composition and restoration of intestinal barrier function.

Comprehensive utilization of palm kernel cake for producing mannose and manno-oligosaccharide mixture and yeast culture.

Palm kernel cake (PKC) is an agricultural waste derived from palm kernel oil manufacturing, and its production is increasing year by year. It is very urgent to process this agricultural waste in an environmentally friendly way. Here, PKC was used to produce mannose and manno-oligosaccharides mixture (MMOM) and yeast culture (YC) through enzymolysis and solid-state fermentation (SSF). In enzymolysis, five factors were optimized separately and a response surface methodology analysis was performed. Then, enzymolysis of PKC was carried out at the optimal condition, and the extraction efficiency of mannose and manno-oligosaccharides reached 68.90% with mannose concentration achieving 60.27 g/L. After enzymolysis, the enzymatic hydrolysate was dried by spray drying, and the contents of MMOM reached 42.9%. In SSF, the enzymolysis residues were utilized with inoculating Saccharomyces cerevisiae for yielding YC. After optimization, the cells number of S. cerevisiae reached 2.08 × 109 cells/g and the crude protein content was increased to 27.31%. Therefore, a novel approach to produce feed additives, including MMOM and YC, with high value by comprehensive utilization of PKC was proposed, which has good application prospects in the breeding industry. KEY POINTS: • New idea for the comprehensive utilization of PKC is proposed. • PKC was used to produce mannose and mannan-oligosaccharides mixture (MMOM) by enzymolysis and spray drying. • The enzymolysis residues were reused via SSF for producing yeast culture (YC).

Effects of chlorogenic acid on growth, metabolism, antioxidation, immunity, and intestinal flora of crucian carp (Carassius auratus).

In recent years, with the harm caused by the abuse of antibiotics and the increasing demand for green and healthy food, people gradually began to look for antibiotic alternatives for aquaculture. As a Chinese herbal medicine, leaf extract chlorogenic acid (CGA) of Eucommia ulmoides Oliver can improve animal immunity and antioxidant capacity and can improve animal production performance. In this study, crucian carp (Carassius auratus) was fed with complete feed containing 200 mg/kg CGA for 60 days to evaluate the antioxidant, immuno-enhancement, and regulation of intestinal microbial activities of CGA. In comparison to the control, the growth performance indexes of CGA-added fish were significantly increased, including final body weight, weight gain rate, and specific growth rate (P < 0.01), while the feed conversion rate was significantly decreased (P < 0.01). Intestinal digestive enzyme activity significantly increased (P < 0.01); the contents of triglyceride in the liver (P < 0.01) and muscle (P > 0.05) decreased; and the expression of lipid metabolism-related genes in the liver was promoted. Additionally, the non-specific immune enzyme activities of intestinal and liver tissues were increased, but the expression level of the adenylate-activated protein kinase gene involved in energy metabolism was not affected. The antioxidant capacity of intestinal, muscle, and liver tissues was improved. Otherwise, CGA enhanced the relative abundance of intestinal microbes, Fusobacteria and Firmicutes and degraded the relative abundance of Proteobacteria. In general, our data showed that supplementation with CGA in dietary had a positive effect on Carassius auratus growth, immunity, and balance of the bacteria in the intestine. Our findings suggest that it is of great significance to develop and use CGA as a natural non-toxic compound in green and eco-friendly feed additives.

Detection of viable and total fungal community in zaopei of Chinese strong-flavor baijiu using PMA combined with qPCR and HTS based on ITS2 region.

BACKGROUND: Chinese strong-flavor baijiu (CSFB), one of the three major baijiu types, is the most popular baijiu type among consumers in China. A variety of microbes are involved in metabolizing raw materials to produce ethanol and flavor substances during fermentation, which fundamentally determined the quality of baijiu. It is of great importance to study microbial community of fermented grains (zaopei) during baijiu brewing process for improving its quality. In this study, we firstly used propidium monoazide (PMA) to treat zaopei samples from 5-year pit and 20-year pit for removing the interference of non-viable fungi, and analyzed the diversity of total fungi and viable fungi by quantitative PCR (qPCR) and high-throughput sequencing (HTS) based on ITS2 gene. RESULTS: The results showed that total fungi and viable fungi displayed no significant differences at OTU, phylum, or genus levels during fermentation within two kinds of pits. A total of 6 phyla, 19 classes, and 118 genera in fungi were found based on OTUs annotation in zaopei samples from 5-year pit and 20-year pit. Besides, non-viable fungi had little effect on the fungal community diversity during the fermentation cycle. It was found that the most dominant viable fungi belonged to Saccharomyces, Kazachstania, Naumovozyma, and Trichosporon, and Naumovozyma was firstly detected in zaopei samples of CSFB. Moreover, based on the variation of flavor substances in zaopei samples, the quality of CSFB produced from older pit was better than that produced from younger pit. CONCLUSION: The non-viable fungi had little effect on the fungal diversity, structure, and relative abundance in zaopei samples of CSFB, and Naumovozyma was firstly detected in zaopei samples of CSFB. Our findings can be applied as guidance for improving the quality and stability of CSFB.

Differences in Acid Stress Response of Lacticaseibacillus paracasei Zhang Cultured from Solid-State Fermentation and Liquid-State Fermentation.

Liquid-state fermentation (LSF) and solid-state fermentation (SSF) are two forms of industrial production of lactic acid bacteria (LAB). The choice of two fermentations for LAB production has drawn wide concern. In this study, the tolerance of bacteria produced by the two fermentation methods to acid stress was compared, and the reasons for the tolerance differences were analyzed at the physiological and transcriptional levels. The survival rate of the bacterial agent obtained from solid-state fermentation was significantly higher than that of bacteria obtained from liquid-state fermentation after spray drying and cold air drying. However, the tolerance of bacterial cells obtained from liquid-state fermentation to acid stress was significantly higher than that from solid-state fermentation. The analysis at physiological level indicated that under acid stress, cells from liquid-state fermentation displayed a more solid and complete membrane structure, higher cell membrane saturated fatty acid, more stable intracellular pH, and more stable activity of ATPase and glutathione reductase, compared with cells from solid-state fermentation, and these physiological differences led to better tolerance to acid stress. In addition, transcriptomic analysis showed that in the cells cultured from liquid-state fermentation, the genes related to glycolysis, inositol phosphate metabolism, and carbohydrate transport were down-regulated, whereas the genes related to fatty acid synthesis and glutamate metabolism were upregulated, compared with those in cells from solid-state fermentation. In addition, some genes related to acid stress response such as cspA, rimP, rbfA, mazF, and nagB were up-regulated. These findings provide a new perspective for the study of acid stress tolerance of L. paracasei Zhang and offer a reference for the selection of fermentation methods of LAB production.

Deciphering Succession and Assembly Patterns of Microbial Communities in a Two-Stage Solid-State Fermentation System.

Although the importance of microbiota in the natural environment and in industrial production has been widely recognized, little is known about the formation and succession patterns of the microbial community, particularly secondary succession after disturbance. Here, we choose the Xiaoqu liquor brewing process as an experimental model in which sorghum grains were first aerobically saccharified and then anaerobically fermented after being stirred and acidified to explore multistage community succession patterns. We analyzed microbial composition, physicochemical factors, and metabolites of brewing grains inoculated with two different starters, pure starter and traditional starter, respectively. Two groups showed similar succession patterns where the saccharification microbiota was mainly derived from starters, while environmental microorganisms, mainly Lactobacillaceae and Saccharomyces, dominated the fermentation microbiota regardless of the original saccharification community composition. Species replacement shaped the bacterial community, while species replacement and loss both contributed to fungal community succession in both groups. Grain acidification and hypoxia led to the succession of bacterial and fungal communities during fermentation, respectively. Despite inoculation with starters containing different microorganisms, similar microbial communities during the fermentation stage of the two groups exhibited similar metabolite composition. However, higher abundance of Rhizopus in the saccharification of the pure starter group led to more alcohols, while higher abundance of Monascus and Saccharomycopsis in the traditional starter group promoted acid and ester metabolism. These results revealed the microbial succession patterns of two-stage liquor brewing and its influence on flavor metabolism, which could be used to regulate the microbial community in food fermentation to further promote the modernization of the fermented food industry. IMPORTANCE Revealing formation and assembly mechanisms of microbiota can help us to understand and further regulate its roles in the ecosystems. The Xiaoqu liquor brewing system is a tractable microbial ecosystem with low complexity. This two-stage microbial ecosystem can be used as an experimental model to analyze the multistage temporal succession pattern of microbial communities. Our results demonstrated the dynamic composition and succession pattern of a microbial community in the two-stage liquor brewing system. The results also revealed the microbial origins determining community composition, the ecological processes dominating microbial community succession patterns, the determinants affecting microbial community successions, and the effect of microbial community changes on metabolite synthesis. Overall, our study not only provides an insight into multistage succession patterns of microbial communities in liquor brewing systems but also provides reference for optimizing the quality of fermented products, which will be helpful to understand the succession patterns of microbial communities in other natural ecosystems.