Abstract:Foodborne diseases caused by foodborne pathogens have become the primary issue of food safety in China, posing serious threats to public health. During food processing, osmotic pressure, temperature, pH, and other unfavorable conditions can induce the protective responses of bacteria, leading to abnormal division and filamentous growth. The filamentation of foodborne pathogens enhanced the stress tolerance, enabling the pathogens to rapidly resume division under favorable conditions. It results in a significant underestimation of bacterial count and thus has an adverse impact on food safety. This article introduces the mechanism of inducing bacterial filamentation, aiming to provide theoretical guidance for controlling filamentous foodborne pathogens.

Abstract:The starting point and ultimate goal of the international community’s discussion on pathogenic microbial genetic resources are the prevention, preparedness, and response of public health risks. Timely sharing of pathogens, their nucleotide sequence data and relevant metadata is of paramount importance in enabling early identification, risk assessment, diagnosis, vaccine development, and selection of therapies. Establishing the mechanisms for fair and equitable sharing of the benefits arising from the utilization of the concerned resources has become a critical element of ensuring expedited pathogen sharing. The proposals and discussions on the access and benefit-sharing of pathogenic microbial genetic resources or their data information reflect that the parties pay more attention to the protection of rights and interests of their nations and domestic stakeholders. The regulatory legislation of pathogenic microbial genetic resources in China highlights the prevention, preparedness, and response of public health and biosafety risks, which is in line with the purpose of the current international rules and its discussion process. The establishment of the management system and regime of scientific data as a new factor of production is in the stage of rapid development and necessitates continuous exploration. The industrial practice of domestic microbial genetic resources sharing and utilization gradually matures. This paper reviews the current situation of the sharing and utilization system of pathogenic microbial genetic resources in the international community and China. Based on the development trend of the international system of pathogenic microbial genetic resources and the practical needs of the sharing and utilization of pathogenic microbial genetic resources in China, this paper presents the prospect of the sharing and utilization system of pathogenic microbial genetic resources. Furthermore, this paper puts forward proposals to improve the sharing and utilization system of pathogenic microbial genetic resources in China. First, we should coordinate the consideration of future implementation of the convention with major issues such as overall national security, ethics, politics, and diplomacy, take into account the protection of national sovereignty rights and interests of stakeholders, and actively participate in the building of an international system for the sharing and utilization of pathogenic microbial genetic resources. Second, in view of the weak aspects of the development of the pathogenic microorganism sharing and utilization system in China, we should step up the establishment of the pathogenic microorganism genetic resources access and benefit-sharing system. Third, we should carry out pilot demonstration and take the lead in forming a pattern of sharing, utilization, and benefit-sharing based on industry self-discipline before the improvement of system. Fourth, we should strengthen the infrastructure construction and personnel training for genetic resources of pathogenic microorganisms. Furthermore, we should ensure the security of national resources, improve the prevention, preparedness, and response of public health risks, and support the development strategy of national resources protection, public health, and public safety with high-quality talents.

Abstract:Salinomycin, an important antibiotic widely used in animal husbandry, is a potential drug with anti-tumor stem cell effect. Its biosynthesis and metabolic regulation mechanism have received increasing attention. In this paper, we reviewed the biosynthesis mechanism of salinomycin in Streptomyces albus and the recent progress in the study of its secondary metabolism regulation, aiming to propose some strategies to improve salinomycin production through secondary metabolism regulation technology by analyzing the key proteins of salinomycin biosynthesis gene cluster, key enzymes and regulatory factors of polyketone skeleton chain synthesis. It provides reference for further development of high-yield salinomycin industrial strains.

Abstract:Under extreme water quality conditions such as high salt, high or low temperatures, high ammonia nitrogen, and heavy metals, conventional biological denitrification suffer from reduced biological activity, slowed reaction, and unsatisfactory outcomes. Therefore, the development of efficient functional bacteria capable of withstanding extreme water quality environments is crucial for enhancing the stability of biological denitrification processes. Heterotrophic nitrifying-aerobic denitrifying bacteria can remove nitrogen under completely aerobic conditions. Some of these bacteria can grow under extreme water conditions and demonstrate efficient nitrogen removal capability, offering suitable strain resources for conventional biological nitrogen removal. A variety of heterotrophic nitrifying-aerobic denitrifying bacteria such as Pseudomonas and Acinetobacter capable of tolerating extreme water quality conditions have been screened out. Under extreme water quality conditions, heterotrophic nitrifying-aerobic denitrifying bacteria increase the activities of the enzymes involved in the denitrification pathway to maintain the stability of their denitrification performance. Moreover, they increase the activities of the antioxidant enzymes to adapt to the extreme water environments. We summarize the species, denitrification efficiency, metabolic pathways, and mechanisms of heterotrophic nitrifying-aerobic denitrifying bacteria in extreme water environments characterized by high concentrations of ammonia nitrogen, elevated salinity, extreme high or low temperatures, heavy metal contamination, and extreme pH. Additionally, this paper assesses the current application status of these bacteria in engineering, identifies the technical challenges, and proposes novel research ideas for future investigations.

Abstract:[Background] The exchange rate of substances inside and outside cells has a significant effect on the transformation activity of whole-cells. [Objective] To increase the yield of γ-aminobutyric acid (GABA) synthesized by whole-cell transformation, we investigated the improving effect of ethanol on the apparent activity of whole-cell glutamate decarboxylase (GAD) in Enterococcus faecium. [Methods] We explored the mechanism of ethanol in improving the apparent activity of whole-cell GAD by comparing the effects of ethanol on the enzymatic properties of pure GAD and whole-cell GAD, cell structure, and membrane permeability. [Results] Low-concentration ethanol significantly promoted the activity of whole-cell GAD with the optimal concentration of 7.5%. At this concentration, the apparent activity of whole-cell GAD was increased by 41.63%, and the optimal reaction pH of whole-cell GAD did not change. However, the sensitivity of whole-cell GAD to the changes of extracellular pH and the Michaelis constant (K_m) of whole-cell GAD were both reduced. This concentration of ethanol had no significant effect on pure GAD (P>0.05), and did not cause GAD leakage or cell damage. In addition, 7.5% ethanol increased the activity of GAD only when it coexisted with the cells in reaction system. The results indicated that 7.5% ethanol altered the cell permeability and enhanced the mass transfer rate of cells, thereby improving the apparent activity of whole-cell GAD. [Conclusion] Due to the low price and high safety, ethanol demonstrates great potential of application in the improvement of whole-cell GAD activity for the industrial production of GABA.

Abstract:[Background] The catalase produced by Bacillus sonorensis s262 has the ability to degrade aflatoxin B₁ (AFB₁).[Objective] To express Bacillus sonorensis s262 peroxidase in Escherichia coli, optimize the expression conditions, and determine the enzymatic properties. [Methods] The catalase gene katA was ligated into the pET28a(+) vector to construct the recombinant expression plasmid pET28a-katA, which was then transformed into E. coli BL21(DE3) competent cells. Furthermore, the expression conditions including IPTG concentration, the induction temperature, and the induction time of the recombinant enzyme were optimized. Ni-NTA Sefinose^TM resin was used to purify the recombinant enzyme. The optimal reaction temperature and thermal stability, the optimal pH and pH stability, and the effects of metal ions on the enzyme activity were determined, and the kinetics of the recombinant enzyme was analyzed by double-reciprocal plotting. The degradation rate of AFB₁ by the recombinant catalase was measured by high performance liquid chromatography. [Results] The recombinant expression plasmid pET28a-katA proved to be successfully constructed by double digestion (BamH Ⅰ and Hind Ⅲ) and sequencing. The molecular weight of the purified recombinant enzyme ranged from 55 kDa. The expression conditions in E. coli were optimized as follows: induction with 0.8 mmol/L IPTG at 25 ℃ for 10 h. The kinetic parameters V_max and K_m were (760.17±19.61) mmol/(min·L) and (63.73±3.87) mmol/L, respectively, at the optimal catalytic temperature of 50 °C and optimal pH 7.0. In addition, 0.1 mmol/L Fe²⁺, Zn²⁺, and Cu²⁺ increased the enzyme activity by 40%, 8%, and 12%, respectively, while 0.1 mmol/L K⁺ decreased the enzyme activity by 39%. The degradation rate of AFB₁ by the recombinant enzyme was 61.44%. [Conclusion] We successfully expressed and purified the catalase of B. sonorensis in E. coli, laying a foundation for the industrial production and application of catalase.

Abstract:[Background] Vibrio harveyi is a Gram-negative marine bacterium that has been recognized as an opportunistic pathogen for marine animals. The type III secretion system (T3SS) is a highly-conserved virulence apparatus used by Gram-negative bacterial pathogens to cause infections. [Objective] To decipher the mechanism of T3SS of V. harveyi in inducing the death of fish cells. [Methods] The T3SS disfunctional mutant was established by the in-frame deletion of vcrD in V. harveyi strain 345, and then its complemented mutant was constructed. The wild type and mutant strains were used to infect FHM cells, and then DAPI staining, in situ detection of fragmented DNA (TUNEL assay), Caspase activity assay, and lactate dehydrogenase (LDH) release assay were carried out to reveal the features of cell death. [Results] Dying cells exhibited the features such as cell rounding, nuclear condensation, and DNA fragmentation of apoptotic cells. Caspase-3 was activated by the T3SS, which confirmed that the infection with V. harveyi rapidly induced T3SS-dependent apoptosis in fish cells. Furthermore, the infection with V. harveyi strain 345 led to release of cellular contents including LDH from infected fish cells. Importantly, the inhibition of apoptosis did not prevent cells from releasing cellular contents or rounding. [Conclusion] V. harveyi uses T3SS to induce the activation of apoptosis pathways and the rounding of fish cells, ultimately leading to cell death.

Abstract:[Background] 2,3-dihydroxybenzoic acid decarboxylase (2,3-DHBD) can catalyze the carboxylation of CO₂ with phenolic compounds to generate aromatic carboxylic acids, providing a new strategy for the synthesis of value-added chemicals from fixed CO₂. However, its application is limited by the low conversion efficiency. [Objective] To improve the catalytic efficiency of 2,3-DHBD, we established a high-throughput screening method based on the Biomek i7 automated workstation to achieve efficient screening of benzoic acid decarboxylases with high carboxylation activity, providing a method for enzyme engineering. [Methods] First, 508 clones were obtained by targeted hemi-saturation mutagenesis of the 2,3-DHBD from Aspergillus oryzae (2,3-DHBD_Ao). Then, mutant library construction, clone selection, culture, and enzyme activity assay were performed based on Biomek i7 automated workstation to achieve two rounds of screening for the mutants. The accuracy of the high-throughput screening method was evaluated by high performance liquid chromatography (HPLC). [Results] The decarboxylation and carboxylation reaction system catalyzed by the crude enzyme and the Biomek i7 automated workstation screening process were determined. The two rounds of high-throughput screening from 508 clones yielded 13 positive mutants, and the HPLC confirmed 3 positive mutants. The positive mutation rate of the constructed library was 0.6%, and the mutant with the highest carboxylation activity showed the activity 120% of the wild type. [Conclusion] A high-throughput screening method for the decarboxylases with high carboxylation activity was successfully established based on the Biomek i7 automated workstation. For the first time, the automated high-throughput equipment was combined with mutant screening, which provided the idea of determining the evaluation indexes during the screening. It took 175 h to conduct two rounds of screening for 508 clones, which shortened the screening cycle compared with manual screening.

Abstract:[Background] The vast majority of soil microorganisms exist in the form of biofilms. The available studies mainly focus on the impacts of different agronomic measures on the structures of soil bacterial communities, while the mechanisms of different agronomic measures in regulating the formation of multispecies biofilms remain unclear. [Objective] To explore the effects of different agronomic measures on the formation of multispecies biofilms in paddy fields. [Methods] Fresh rice rhizosphere soil samples were collected from the fields with long-term application of lime (SH), organic fertilizer (SY), and no fertilizer (CK). The microbial communities were extracted as the initial inocula to cultivate multispecies biofilms. The in-situ detection techniques such as attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and confocal laser scanning microscopy (CLSM), were combined with crystal violet staining to reveal the effects of changes in soil physiochemical properties and environmental factors (pH and temperature) on the formation of multispecies biofilms. [Results] The soil multispecies biofilms under both agronomic measures had two growth cycles. The optimal growth temperature for the development and maturity of multispecies biofilms was 25 °C. As the pH value increased, the soil multispecies biofilm biomass showed a trend of first increasing and then decreasing. The biofilm-forming ability in the lime application system was significantly higher than that in the organic fertilizer application system, and the multispecies biofilm biomass followed the trend of SH>SY>CK. [Conclusion] Both agronomic measures promote the formation of multispecies biofilms in paddy fields.

Abstract:[Background] Myxobacteria are a group of prokaryotes producing abundant metabolites with novel structures, which endow myxobacteria with antimicrobial and antitumor activities.Phytophthora infestans is the pathogen causing potato late blight and has a destructive effect on potato production. The distribution of myxobacteria in Ordos Plateau as a special habitat and the antagonistic activity of these myxobacteria against P. infestans remain unclear. [Objective] To investigate the distribution of culturable myxobacteria and their antagonistic activity against P. infestans in the Ordos Plateau area, so as to lay a foundation for the use of myxobacteria in the prevention and control of potato late blight. [Methods] In this study, 46 soil samples were collected from Ordos Plateau of Inner Mongolia, from which myxobacteria were isolated and identified. The correlations of the distribution of myxobacteria with soil types, vegetation types, and environmental factors were determined, and the antagonistic activities of myxobacterial strains against P. infestans were examined. [Results] The soil in this area was dry and had poor fertility. Among the 191 strains cultured, 102 strains were purified, of which 65 strains were identified, belonging to 9 species of 5 genera, including Myxococcus fulvus, M. virescens, Corallococcus exiguus, C. interemptor, C. coralloides, Pyxidicoccus fallax, Archangium minus, Ar. gephyra, and Cystobacter fuscus. The dominant genus was Myxococcus, and the dominant species were M. fulvus and M. virescens. Myxobacteria were mainly distributed in fluvo-aquic soils and aeolian soils, while abundant rare species of myxobacteria were mainly distributed in sierozems and castano-cinnamon soils. Myxobacteria were the most common in woodland, which was followed by cultivated land, grassland, and wasteland. Among the isolated myxobacterial strains, 89.40% showed antagonistic effects on P. infestans. All the identified species had antagonistic strains, and M. fulvus was the dominant species with high resistance to P. infestans. [Conclusion] There are abundant myxobacterium resources in Ordos Plateau, and most myxobacterial strains in this area have antagonistic activity against P. infestans. This study lays a foundation for the development of novel agents from myxobacteria for the biocontrol of potato late blight.

Abstract:[Background] The gray mold disease caused by Cladosporium tenuissimum is a common fungal disease infecting protected bottle gourd, often resulting in serious economic losses. [Objective] To identify strain hjt6 with antagonistic activity against the gray mold disease of bottle gourd and evaluate the growth characteristics and antifungal activity of the strain, so as to provide microbial resources for the biocontrol of the gray mold disease of bottle gourd. [Methods] Strain hjt6 was identified based on morphological, physiological, and biochemical characteristics and 16S rRNA gene and gyrB gene sequences. The growth curve of the strain was established, and the temperature, pH, and salt tolerance was examined. The broad-spectrum antagonistic effect of this strain was evaluated by the mycelial inhibition assay. Plate confrontation, co-culture, and detached fruit re-inoculation were conducted to assess the disease control efficacy of strain hjt6 against C. tenuissimum. [Results] Strain hjt6 was identified as Bacillus velezensis. It grew within a temperature range of 10 ℃ to 50 ℃, with an optimal growth temperature of 30 ℃. It could grow within the range of pH 5.0–9.0, with pH 7.0 being the most suitable. The strain tolerated 0% to 10% NaCl. Strain hjt6 exhibited inhibitory activities against eight crop pathogens, with the inhibition rates ranging from 57.38% to 82.18%. It showed an inhibitory ability of “++++” and an inhibition rate of 71.53% against C. tenuissimum on plates. The co-culture results showed that the strain hjt6 reduced the mycelial mass of C. tenuissimum by 77.31%, and achieved the control efficacy of 92.63% on the detached fruits inoculated with the pathogen. [Conclusion] Strain hjt6 demonstrates promising antagonistic effects against the gray mold disease of bottle gourd and could serve as a valuable microbial resource for the biocontrol of this disease.

Abstract:[Background] Three strains of Bacillus subtilis were isolated from camel milk and feces by the conventional culture method, and genome sequencing was performed for strain F10431 with strong probiotic effects in vitro. [Objective] To explore the probiotic potential, functional genes, and genetic basis of the isolates, so as to lay a theoretical foundation for the application and research of B. subtilis. [Methods] The enzyme production, stress tolerance, antimicrobial effect, and feeding safety of B. subtilis F10431, 2N2N, and 2N13N were evaluated in vitro. The second- and third-generation sequencing was performed for the whole genome and functional gene annotation of the isolates. [Results] The three strains of B. subtilis had tolerance to acids and bile salt, and strain F10431 had the strongest tolerance to intestinal and gastric fluids. All of the strains were capable of producing protease, amylase, cellulase, and antioxidant enzymes. Particularly, strain F10431 showed the protease activity of (3.17±0.26) nmol/(min·mL), the amylase activity of (0.48±0.04) nmol/(min·mL), the cellulase activity of (151.02±8.05) μg/(min·mL), and the superoxide dismutase activity of (1.81±0.31) μmol/(min·mL). The strains exerted strong inhibitory effects on Staphylococcus aureus and Escherichia coli and weak inhibitory effects on Salmonella. None of the strains produced hemolysis ring, thus being safe for feeding. [Conclusion] In this study, three camel-derived B. subtilis strains with excellent probiotic properties were isolated. F10431 with outstanding probiotic effects in vitro was sequenced, and the sequencing data were submitted to NCBI to obtain the GenBank accession number of MW721260. This study elucidated the functional mechanism and genomic characterization of F10431, providing theoretical support for the application of relevant probiotic preparations.

Abstract:[Background] Saline-alkali tolerant and plant growth-promoting bacteria are praised for the effects on improving the growth and enhancing the saline-alkali tolerance of crops under saline-alkali stress. [Objective] To acquire the microbial resources that possess both saline-alkali tolerance and growth-promoting effects and decipher the mechanism underlying saline-alkali tolerance, we aimed to isolate a bacterial strain from the saline soil in Yinchuan City, Ningxia Hui Autonomous Region, China. [Methods] The strain was identified by morphological and microscopic observation, Biolog Gen Ⅲ test, and 16S rRNA gene sequencing. The culture method was used to examine the saline-alkali tolerance and growth-promoting effect of the strain. The genome information of the strain was analyzed by functional gene annotation. [Results] Strain YS-AT2 could grow in the LB media with pH 8.0–12.0 and 0%–9% (W/V) NaCl, and showed the alkali reduction rate reaching 13% and above in the media at pH 8.5–9.5. Strain YS-AT2 could use 54 carbon sources and was sensitive to 20 chemical sensitivity test substances. According to 16S rRNA gene sequencing results, YS-AT2 belonged to the genus Citrobacter. strain YS-AT2 could promote the seed growth of Arabidopsis thaliana in a saline environment with NaHCO₃ (pH 9.5) and the germination of soybean seeds under 50–200 mmol/L NaCl or 50–200 mmol/L NaHCO₃ stress. Furthermore, it increased soybean plant height, fresh weight, dry weight, and root length by 19.84%, 18.75%, 10.31%, and 32.58%, respectively, in the soil with pH 8.2 and EC 450 μs/cm. Strain YS-AT2 carried multiple genes associated with saline-alkali tolerance. The saline-alkali tolerance of this strain was probably realized through the transport of saline-alkali substances, the transport and regulation of sodium and potassium ions, and the synthesis of high-concentration cell solutes. [Conclusion] We analyzed the growth-promoting effect of the saline-alkali tolerant strain Citrobacter YS-AT2 on A. thaliana and soybean and mined the genes associated with its saline-alkali tolerance. The findings not only provided a theoretical basis and strain resources for the development of biofertilizers with saline-alkali growth promoting function but also laid a theoretical foundation for deciphering the mechanism underlying the saline-alkali tolerance.

Abstract:[Background] Fungal diseases have gradually become a major factor restricting the crop production in China, and thus it is urgent to develop antifungal agents for disease prevention and control. [Objective] To investigate the mechanism of Bacillus subtilis J-15 secondary metabolites (SMs) in inhibiting fungal growth and development at the molecular level, so as to provide a theoretical basis for their application in fungal disease control. [Methods] The cell viability of Saccharomyces cerevisiae S288C treated with SMs was measured, and the cell necrosis was detected by flow cytometry. Transcriptome sequencing was performed to analyze the effects of SMs on the gene expression in S. cerevisiae, and real-time fluorescence quantitative PCR was employed to verify the results. [Results] SMs caused nuclear membrane lysis, DNA diffusion, and cell necrosis of S. cerevisiae S288C in a time-dependent manner. A total of 1 627 differentially expressed genes were screened out after the treatment with SMs for 12 h, including 851 up-regulated genes and 776 down-regulated genes. A total of 512 genes were differentially expressed after the treatment with SMs for 24 h, including 300 up-regulated genes and 212 down-regulated genes. The differentially expressed genes were involved in multiple pathways such as autophagy, sugar, lipid, and amino acid metabolism, cell wall synthesis, and cell cycle. [Conclusion] The findings provide an experimental basis for revealing the mechanism of SMs in inhibiting fungal growth and a theoretical basis for the application of SMs in the prevention and control of crop fungal diseases.

Abstract:[Background] Banana anthracnose is a fungal disease caused by Colletotrichum musae, which seriously restricts the development of the banana industry. [Objective] To screen the biocontrol bacteria against Colletotrichum musae from mangrove soil samples in Guangxi Zhuang Autonomous Region, and evaluate the biocontrol effect of the fermentation broth. [Methods] The biocontrol bacteria were isolated by dilution plate method, and a plate confrontation experiment was conducted to screen out the strains with antagonistic effects on C. musae. The bacterial strain was identified based on morphological characteristics, physiological and biochemical properties, and 16S rRNA sequencing results. The biological characteristics of the strain screened out were studied by single factor experiments. The antagonistic activity was evaluated based on the mycelial growth rate. The biocontrol effect of the fermentation broth was examined with detached banana. [Results] A strain HSL3-29 was isolated from mangrove soil. It had a strong antagonistic effect against C. musae, with the inhibition rate of 81.83%. HSL3-29 had a broad antimicrobial spectrum, with inhibitory effects on other six plant pathogenic fungal species. HSL3-29 was identified as Bacillus amyloliquefaciens. The strain entered the logarithmic growth stage during 4–24 h. It was sensitive to ampicillin and vancomycin, had tolerance to high temperatures, and could survive in high salt environments. The fermentation broth of the strain showed the biocontrol effect up to 39.4% on the banana fruits infected with anthracnose. [Conclusion] A strain of B. amyloliquefaciens with strong antagonism to C. musae was isolated, which provided strain resources for the development of biocontrol agents for banana anthracnose.

Abstract:[Background] The pork safety problem caused by Listeria monocytogenes not only brings great consumption risks to consumers but also causes huge economic losses to the food processing industry. The virulence of Listeria monocytogenes in pork products should be controlled to curb the occurrence of public health events.[Objective] To explore the inhibitory effect of CO₂ combined with the probiotic Lactobacillus plantarum (CO₂-LP) on L. monocytogenes in pork by the method of predictive microbiology and determine the optimal combination of CO₂-LP. [Methods] The Jameson-effect model was employed to establish the growth curves of L. monocytogenes in pork samples treated with CO₂-LP, and the kinetic parameters including lag phase (λ), maximal growth rate (μ_max), and maximum population density (N_max) were determined. [Results] The inhibitory effect of CO₂-LP was well fitted by the Jameson-effect model. CO₂-LP treatment prolonged the λ and reduced the μ_max and N_max of L. monocytogenes. Moreover, the inhibitory effect of CO₂-LP enhanced as the CO₂ concentration increased. Compared with the control group, the 80% CO₂-LP treatment increased the λ by 0.87 fold and decreased the μ_maxand N_max by 47% and 2.05 lg (CFU/g), respectively. Although 80% CO₂-LP had the best inhibitory effect, the 60% CO₂-LP was selected as the optimal treatment, because the presence of N₂ could prevent pack collapse in modified atmosphere packaging. Compared with the control group, 60% CO₂-LP treatment increased the λ by 0.81 fold and decreased the μ_maxand N_maxby 33% and 1.83 lg (CFU/g), respectively.[Conclusion] CO₂-LP treatment inhibited the growth of L. monocytogenes in pork from different aspects, which reflected the advantages of hurdle technology.

Abstract:[Background] Although being a major producer of edible fungi, China remains to improve the breeding of new varieties and strengthen the excavation and utilization of wild edible fungal resources. [Objective] To cultivate the fruiting bodies of Neolentinus lepideus and evaluate its development potential based on the biological activity analyses. [Methods] The wild fruiting bodies of N. lepideus were collected from Jiaocheng County, Shanxi Province, and the cultures were isolated and induced to produce fruiting bodies in the laboratory. The biological activities and nutrient composition of the artificially cultivated fruiting bodies were determined. [Results] From the wild fruiting bodies of N. lepideus, the strain ZYJ0862 was obtained and identified as N. lepideus by the phylogenetic analysis. On the medium with pine sawdust as the main material and corn meal as the complementary material, the strain was successfully induced to generate fruiting bodies. The artificially cultivated fruiting bodies had consistent morphological characteristics with the wild fruiting bodies. Compared with five other commercially cultivated mushrooms, the artificially cultivated fruiting bodies of N. lepideus showed strong antioxidant and cellulase activities, as well as certain lignin-degrading enzyme activities. In addition, the artificially cultivated fruiting bodies of N. lepideus had high protein, low calorie, and low fat. [Conclusion] The distribution of N. lepideus in Shanxi was reported for the first time in this study. The strain ZYJ0862 capable of generating fruiting bodies under artificial cultivation conditions demonstrated high development potential. This study provided a theoretical basis for the large-scale cultivation of N. lepideus.

Abstract:[Background] Food-borne pathogens are easy to grow and reproduce in food and have high pathogenicity, posing critical threats to food safety and public health.[Objective] To analyze the antimicrobial stability of Bacillus velezensis PJP10 from multiple perspectives, discover the genes encoding antimicrobial enzymes and involved in the biosynthesis of antimicrobial substances by genome sequencing, and measure the in vitro activities of enzymes and active metabolites, so as to provide data for the application of this strain in industry and agriculture. [Methods] The microbial inhibition tests were carried out to examine the effects of pH, temperature, protease, metal ions, ultraviolet (UV), and salt concentration on the antimicrobial stability of the strain. The potential genes encoding antimicrobial enzymes of PJP10 were investigated by whole genome sequencing and the enzyme-producing abilities assessed by enzyme activity assays. The secondary metabolites of PJP10 were predicted by antiSMASH. HPLC and microbial inhibition tests were employed to analyze the components of the crude extract obtained by the acid precipitation and ethyl acetate extraction. [Results] The cell-free fermentation supernatant exhibited excellent stability at high temperatures, strong acid, strong alkali, high salt and UV, and was sensitive to trypsin and metal ions, but not proteinase K or pepsin. The genome of PJP10 carried abundant genes encoding amylase, aminopeptidase, protease, chitinase, cellulase, esterase, pectinase, glucanase, and amidase, and the strain could produce protease, amylase, cellulase, and esterase. The antiSMASH predicted that strain PJP10 had a variety of gene clusters for the synthesis of secondary metabolites, including bacillaene, difficidin, macrolactin H, surfactin, bacillomycin D, fengycin, bacilysin, bacillibactin, amylocyclicin, and plantazolicin. In addition, the extract obtained by acidification exhibited strong inhibitory activities against Salmonella enteritidis and Escherichia coli but no activity against Ralstonia solanacearum. The ethyl acetate extract showed strong inhibitory effects on Staphylococcus aureus and R. solanacearum and weak inhibitory effects on S. enteritidis and E. coli. According to HPLC results, we hypothesized that the antimicrobial substances in the cell-free supernatant of PJP10 were iturin, fengycin, and surfactin. [Conclusion] The strain PJP10 shows high antimicrobial stability and carries rich genes encoding active enzyme and abundant gene clusters for the synthesis of antimicrobial substances, demonstrating a promising prospect of application in industry and agriculture.

Abstract:[Background] Traditional handmade dairy products are the favorite food in Tajik agricultural and pastoral areas. They contain abundant lactic acid bacterial resources which need to be exploited and utilized. [Objective] By analyzing the diversity and functions of lactic acid bacteria in traditional homemade dairy products from Tajik agricultural and pastoral areas, we aim to aid in the exploration and protection of microbial resources and provide a theoretical basis for evaluating the safety of traditional dairy products. [Methods] The V3–V4 variable region of bacterial 16S rRNA gene was subjected to high-throughput sequencing. The culturable suspected lactic acid bacteria were screened by the conventional culture methods in the MRS, Lee’s, and M17 media and identified by physiological and biochemical tests and 16S rRNA gene sequence homology. Furthermore, the microbial species and gene function information in the dairy products in Tajik autonomous county of Taxkorgan were analyzed. [Results] There were 37 bacterial genera and 65 bacterial species in the 4 dairy samples, among which Lactobacillus and Streptococcus were the dominant bacterial genera in cheese and yogurt, and Lactobacillus helveticus was the dominant species. A small amount of pathogenic bacteria existed in the cheese samples. Moreover, 38 culturable suspected lactic acid bacterial strains were identified from MRS, M17, and Lee’s media, including 3 strains of Bacillus subtilis, 1 strain of B. luti, 1 strain of B. paranthracis, 9 strains of Lactobacillus paracasei, 3 strains of Enterococcus hormeachei, 1 strain of Staphylococcus heamolyticus, 1 strain of Klebsiella pneumoniae, 10 strains of Leuconostoc pseudomesenteroides, 2 strains of Leuconostoc mesenteroides, 4 strains of Lactobacillus plantarum, 1 strain of Acetobacter senegalensis, 1 strain of Enterococcus durans, and 1 strain of Enterobacter faecalis. Among them, the dominant genus was Leuconostoc, followed by Lactobacillus. The 38 strains were classified into 7 genera and 13 species. The results indicated that the conventional culture method can accurately identify lactic acid bacteria to the species level. Representative 11 strains of lactic acid bacteria were selected from 38 strains for antibacterial tests, and all 11 the strains had inhibitory effects on Escherichia coli and B. subtilis. The COG (clusters of orthologous groups of proteins) function prediction showed that the microorganisms in cheese carried rich genes involved in amino acid transport and metabolism. Although there were significant differences in the bacterial community composition between samples, their functions were highly similar. [Conclusion] The diversity of lactic acid bacteria in four traditional handmade dairy products in Tajik autonomous county of Taxkorgan, Xinjiang was studied by combining the molecular biological methods and the conventional culture method. The richness and diversity of lactic acid bacteria in the dairy products were low, and there were some pathogenic bacteria. The combination of the two methods in this study comprehensively revealed the microbial composition in the dairy products. The findings provided basic data for the industrial production and protection of the abundant microbial resources in this area and laid a foundation for evaluating the safety of lactic acid bacteria in traditional dairy products.

Abstract:[Background] Ras-GTPase-activating protein SH3 domain binding protein 1 (G3BP1), an important component and iconic protein of stress granules (SGs), plays a role in host resistance to pathogen invasion. [Objective] To construct a PK-15 cell line with stable knockdown of G3BP1 gene, and to explore the effects of knocking down G3BP1 gene on the replication of vesicular stomatitis virus (VSV), seneca virus (SVV), and vaccinia virus (VACV) and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), myxovirus resistance protein 1 (Mx1), interferon-stimulated gene 54 (ISG54), and interferon-β (IFN-β). [Methods] The lentiviral vector was used to construct a PK-15 cell line with stable knockdown of porcine G3BP1 gene. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and quantitative real-time polymerase chain reaction (qPCR) were carried out to analyze the effects of G3BP1 knockdown on the replication of VSV, SVV, and VACV and the expression of TNF-α, IL-6, Mx1, ISG54, and IFN-β after infection with VSV, SVV and VACV. The bioinformatics tools were used to characterize the G3BP1 gene. [Results] A PK-15 cell line with stable knockdown of G3BP1 was successfully constructed. RT-qPCR and qPCR results showed that knockdown of G3BP1 gene promoted the replication of VSV and SVV (P<0.000 1), while it had no significant effect on the replication of VACV (P>0.05). RT-qPCR results showed that VSV and SVV infections up-regulated the mRNA levels of TNF-α, IL-6, Mx1, ISG54, and IFN-β (P<0.05, P<0.000 1), and knocking down G3BP1 gene down-regulated the mRNA levels of these cytokines (P<0.01, P<0.000 1). RT-qPCR results showed that VACV infection inhibited the production of these cytokines (P<0.01, P<0.000 1), and knocking down G3BP1 gene had no significant effect on the expression of the cytokines inhibited by VACV (P>0.05). The deduced porcine G3BP1 protein was an unstable, hydrophilic, and non-secretory protein with 30 phosphorylation sites, with no transmembrane region. The secondary structure of the protein was mainly composed of random coils (52.47%). The protein was mainly located in the cytoplasm (34.38%) and nucleus (31.25%). [Conclusion] In this study, a PK-15 cell line with stable knockdown of porcine G3BP1 gene was obtained. Knocking down this gene promoted the replication of VSV and SVV but had no significant effect on the VACV replication. Furthermore, it significantly inhibited the production of TNF-α, IL-6, Mx1, ISG54, and IFN-β. The protein had 30 phosphorylation sites and was an unstable, hydrophilic, and non-secretory protein with no transmembrane region. The results help to further reveal the role of G3BP1 in viral infection.

Abstract:[Background] Clostridium perfringens has been a major pathogen affecting the poultry industry, and its infection rate has been on the rise since the use of antibiotics was restricted. Bacteriophages with unique antibacterial effects and safety without residues have become a primary approach to reduce the use of antibiotics. [Objective] To evaluate the synergistic effects of Clostridium perfringens phage, Salmonella phage, and Bacillus subtilis on laying hens by analyzing the histological changes of the intestinal structure, the reduction of C. perfringens, and microbial diversity, so as to provide a scheme for the prevention and control of bacterial diseases in poultry with antibiotic reduction. [Methods] A total of 105 260-day-old Hyline Brown hens were selected and randomized into 5 groups of A: Clostridium perfringens phage SD72, B: SD72+Bacillus subtilis, C: Salmonella phage JDF1-6, D: SD72+JDF1-6+ Bacillus subtilis, and E: blank control. After the hens were treated for 42 days, the cecum contents were collected to determine the number of C. perfringens, and the intestinal structure was observed. The 16S rRNA gene amplicon was sequenced to reveal the intestinal microbial community structure, diversity, and metabolism. [Results] Compared with the blank control group, D group showed decreased number of C. perfringens (by 1.48 log₁₀ (CFU/mL)), decreased depth of jejunal crypts (P<0.05), and increased villus height/crypt depth (P<0.05). Firmicutes and Proteobacteria were dominant in cecum contents of laying hens in all the treatment groups after drinking water, and Lactobacillus(11.26%) had higher relative abundance at the genus level. The serum levels of IgA, IgG, and IgM were significantly increased (P<0.05), and intestinal microbial genes were mainly enriched in the genetic information processing pathways. The abundance of enriched genes in excretory system and immune disease-related pathways in group D was 755.95% higher than that in the blank control group. [Conclusion] The combination of mixed phages and B. subtilis could improve the intestinal health and immunity of laying hens by promoting the expression of microbial genes related to immune disease and improving the immunometabolism of intestinal flora. The findings provide a theoretical basis and biological resources for the prevention and control of bacterial diseases in poultry with reduced antibiotics.

Abstract:[Background] Hyperlipidemia, a key risk factor for cardiovascular diseases, is associated with gut microbiota. Lactic acid bacteria and other probiotics can lower the blood lipid levels by regulating gut microbiota. [Objective] To investigate the effects of Loigolactobacillus coryniformis Lc7 on the serum lipid levels and gut microbiota in hyperlipidemic hamsters. [Methods] The hyperlipidemic hamster model was established with a high-fat diet and then administrated with L. coryniformis Lc7 suspension or phosphate buffered saline by gavage for 6 weeks. The serum levels of lipids and inflammatory cytokines and the pathological changes of the liver and epididymal adipose tissues in hamsters were measured. Furthermore, 16S rRNA gene amplicon sequencing was employed to analyze the gut microbiota in cecal contents. [Results] Oral administration of L. coryniformis Lc7 in hyperlipidemic hamsters lowered the serum levels of total cholesterol (P<0.001), triglyceride, and low-density lipoprotein cholesterol (P<0.05) and elevated the serum level of high-density lipoprotein cholesterol (P<0.001). Moreover, it reduced the serum levels of interleukin-1β (P<0.05), interleukin-6 (P<0.01), and lipopolysaccharide (P<0.05) and attenuated liver tissue injury and epididymal fat accumulation induced by the high-fat diet. Furthermore, the intervention improved the diversity and structure of gut microbiota, as manifested by the decreased ratio of Firmicutes to Bacteroidota (P<0.05), increased relative abundance of beneficial bacteria such as Oscillibacter and Bacteroides, and decreased relative abundance of Helicobacter and Negativibacillus in hyperlipidemic hamsters. The relative abundance of Helicobacter was positively correlated with the concentration of total cholesterol (P<0.01).[Conclusion] L. coryniformis Lc7 can ameliorate lipid metabolism disorder and modulate gut microbiota in hyperlipidemic hamsters, serving as a promising candidate for the prevention and treatment of hyperlipidaemia.

Abstract:[Background] Saccharomyces cerevisiae is an important microorganism in the fermentation of glucose to yield ethanol. However, high concentrations of ethanol and sugar, high temperature, low pH, and other highly stressful conditions often occur during the fermentation, which affect the fermentation efficiency of S. cerevisiae. [Objective] To obtain a S. cerevisiae strain with improved tolerance to high concentrations of ethanol and sugar and high temperatures and evaluate the fermentation performance of the domesticated strain in the case of high sugar levels. [Methods] A laboratory-preserved strain (CICC 33068) of S. cerevisiae was gradually acclimated to an environment of highly concentrated ethanol. The domesticated strain was compared with the original strain in terms of the growth curves and fermentation performance under high concentrations of ethanol and sugar, high temperature, and low pH for the evaluation of its tolerance. [Results] Compared with the original strain, the domesticated strain displayed a complete growth cycle and normal morphology in the broth medium with 13% ethanol. The domesticated strain was capable of growing in the presence of 450 g/L glucose and at 45 ℃ and pH 3.5, with the glucose-to-ethanol conversion rate of 79.22%, which was 7.72% higher than that of the original strain. The gene expression levels indicated that the improved tolerance was associated with the up-regulation of intracellular trehalose synthesis pathway. [Conclusion] A S. cerevisiae strain with improved tolerance to high concentrations of ethanol and sugar and high temperatures and low pH was obtained by environmental acclimation to high levels of ethanol. The domesticated strain outperformed the original strain in the fermentation with high glucose for ethanol production. The results demonstrate that adaptive acclimation could enhance the strain tolerance by upregulating intracellular trehalose synthesis. The findings provide fundamental data for subsequent domestication and screening of other strains.