Characterization Of Bacillus Subtilis LF-11 And Its Bacteriocin

Bacillus subtilis,an important industrial microorganism as well as the direct-fed microbial additive in ’Varieties Catalogue of Feed Additive’,can antagonize pathogenic bacteria,regulate intestinal flora,enhance immunity and improve the production performance of farm animals.Nowadays,probiotics preparation with B.subtilis and bacteriocins as the main components are widely used as in-feed antibiotic alternatives in husbandry.However,strains homogenization,indistinct function and unclear mechanism make the efficacy and quality of products indistinctive and unstable.Therefore,the theoretical basis for the rational application of probiotics should be provided via mining the characteristics of B.subtilis and analyzing its mode of action deeply.Previous comparative analysis of functional genes was carried out between strain LF-11 and another probiotic B.subtilis screened by our laboratory.More functional genes in strain LF-11 were involved in cell biosynthesis,signal transduction,substance metabolism,post-translational modification and other physiological functions in LF-11 genome,which promising probiotic was proved by previous studies in vitro.The aims of this study are to explore the probiotic mechanisms and identify the antibacterial compounds of B.subtilis LF-11.The functional genes related to probiotics and bacteriocin biosynthesis were analyzed based on the annotation and analysis of its whole genome information.Firstly,the antibacterial substance was identified after isolation,purification and structural analysis,and the properties and bactericidal mechanism of the bacteriocin were analyzed.Besides,the yield of bacteriocin was increased by optimizing the culture conditions.Finally,the mechanism of B.subtilis LF-11 protecting broilers against Salmonella infection was explored through breeding trial and enterocyte cell culture in vitro.All the studies lay foundations for the industrialized application of this probiotic strain.The research content and landmark results are as follows:1.Mining the gene cluster encoding the bacteriocin based on the functional genome analysis in B.subtilis LF-11It is helpful to clarify the probiotic functions and develop new products to analyze and characterize the functional gene in B.subtilis LF-11.Based on the whole genome sequencing of B.subtilis LF-11,the functional genes were classified and analyzed according to the standards in GO and KEGG database.A total of 10 gene clusters in 6 classes encoding secondary metabolites were obtained in the genome of B.subtilis LF-11 using online antiSMASH software.Four potential areas of interest(AOI)were recognized from the gene clusters involved in ribosomal synthetic and post-translational modified polypeptides(RiPPs)using BAGEL 4.Four AOIs encode one quorum sensing competence and three potential bacteriocins,which play the important roles in physiological processes including antibacterial,immune-promoting,anti-stress,metabolic regulation and so on.Besides,lots of genes related to physiological regulation were found in B.subtilis LF-11 genome,suggesting that the strain was tolerant to various environmental pressures such as oxidation,temperature,bile salt and pH.In addition,genes involved in adhesion and aggregation indicated that the strain has advantages in intestinal colonization,pathogen antagonism and provision of special nutritional elements,suggesting that the strain had potential antibacterial activity and other probiotic properties,which lay a foundation for the study of its phenotypic characteristics.2.Purification,identification and property analysis of antibacterial compound produced by B.subtilis LF-11The growth kinetic curve of B.subtilis LF-11 in LB medium showed that inhibitory activity of the fermentation broth appeared in the exponential phase and reached the maximum in stationary phase.The antimicrobial compound was purified from cell-free supernatant by ammonium sulfate precipitation,ion exchange chromatography,gel filtration chromatography and RP-HPLC.The final purity of the antimicrobial compound reached 98.7%.The molecular weight was 3.4 kDa by Tricine-SDS-PAGE and mass spectrometry.The amino acid fragment analyzed by secondary mass spectrometry matched with subtilosin A in the genome,which was named subtilosin A-11.Property studies have shown that the antibacterial activity of subtilosin A-11 treated by pepsin or trypsin for 2 hours remained(95.8±1.4)%and(96.6±1.6)%,respectively;the activity lost indistinctively in the range of pH 4-10 and still maintained(89.5 ± 3.5)%after treatment at 80 ℃ for 30 min,indicating that it can tolerate the proteolysis in the digestive tract and exert antimicrobial effect under some degrees of temperature and acid.In addition,the strong inhibitory activity against pathogens and good biosecurity also suggested that subtilosin A-11 is potential in application.3.Analysis of the antimicrobial mechanism of subtilosin A-11The clarification of the mode of action of bacteriocin will provide theoretical basis for its application.In present study,the mechanism of subtilosin A-11 inhibiting indicators L.monocytogenes ATCC 19114 and S.agalactiae ATCC 13813 was explored.Firstly,the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of subtilosin A-11 against L.monocytogenes ATCC 19114 were 1.9 mg/L and 3.9 mg/L,respectively,meanwhile the MIC and MBC against S.agalactiae ATCC 13813 were 250 mg/L and 500 mg/L.The growth inhibition curve and bactericidal curve showed that bactericidal activity of subtilosin A-11 was concentration-dependent against L.monocytogenes ATCC 19114 and time-dependent against S.agalactiae ATCC 13813.The scanning electron microscope(SEM)showed that subtilosin A-11 formed irregular holes on the surface of L.monocytogenes ATCC 19114 as well as hemispherical holes on the ends of S.agalactiae ATCC 13813 but no holes in meso-coccus.Further analysis showed that high concentration of subtilosin A-11 caused the destruction of cell membrane,the disintegration of proton motive force and the rapid release of intracellular ATP,while low concentration of subtilosin A-11 caused the increase of cell membrane permeability,the dissipation of proton motive force and the delay of intracellular ATP release of indicators.The fluorescence microscope further proved that the bactericidal action of subtilosin A-11 against L.monocytogenes ATCC 19114 and S.agalactiae ATCC 13813 are concentration-depended and time-depended.4.The overproduction of subtilosin A-11 by B.subtilis LF-11Single factor test was conducted to optimize the production conditions of subtilosin A-11 by B.Subtilis LF-11.The medium was determined to contain molasses 15 g/L,glycerol 10 mL/L,yeast powder 30 g/L and NaC1 5 g/L,and the conditions were 35℃,150 rpm and initial pH 7.5,which were further confirmed via Plackett-Burman test and Box-Behnken design,including yeast powder 41 g/L,glycerol 14.6 mL/L,molasses 15 g/L and NaCl 5 g/L.The yield of subtilosin A-11 reached 328.8 mg/L under the conditions of initial pH 7.5,temperature 35℃ and rotation speed 142 rpm,which was 11 times higher than that under the initial conditions.It was also found that the accumulation of subtilosin A-11 was negatively correlated with sporulation.The synthesis of subtilosin A-11 ended when sporulation was abundant.Therefore,sporulation rate can be used as a fermentation indicator to indicate the synthesis process of subtilosin A-11.Besides,the preparation process of subtilosin A-11 was optimized through the orthogonal test of NaCl precipitation method and acid precipitation method,which simplified the extraction process and laid the foundation for the industrial production of subtilosin A-11.5.B.subtilis LF-11 protects broilers against Salmonella infectionAntimicrobial spectrum showed that B.subtilis LF-11 inhibited gram-positive pathogens such as L.monocytogenes and S.agalactiae,but did not inhibit gram-negative bacteria such as Salmonella spp.and Escherichia coli.The broilers feeding trial showed that B.subtilis LF11 could effectively protect broilers from Salmonella infection,and the diarrhea rate and mortality reduced to 63%and 37%respectively.To verify the protection of this strain on broilers,B.subtilis LF-11 and S.braenderup H9812 were co-incubated with model enterocyte NCM460 cells in exclusion and competition experiments,respectively.The results showed that B.subtilis LF-11 could effectively inhibit the adhesion and infection of S.braenderup H9812 to NCM460 cells.The count of Salmonella adhering to and infecting NCM460 cells was decreased by 47.4%and 81.1%respectively in exclusion experiment and 33.1%and 61.7%in competition experiment.The viability of NCM460 cells increased by(105.5±2.3)%and(57.0±5.7)%.Meanwhile,RT-PCR and western blot analysis confirmed that B.subtilis LF11 up-regulated the transcription and expression levels of tight junction genes CLDN-1,OCLN,JAM-1 and ZO-1 in NCM460 cells.The immunofluorescence intensity observation further showed that B.subtilis LF-11 improved the distribution uniformity of 4 tight junction proteins.In addition,RT-PCR analysis showed that the gene transcription levels of proinflammatory factors IL-6,IL-8 and TNF-α in NCM460 cells were down-regulated respectively by 50.7%,49.7%and 11.7%in exclusion experiment.ELISA analysis also confirmed that the secretion of IL-8 protein was significantly reduced.All results suggested that B.subtilis LF-11 played the probiotic roles by reducing the infection of S.braenderup H9812 on enterocyte,strengthening the epithelial cells barrier,and attenuating the inflammatory response.The clarification of probiotic mechanism provides the theoretical reference for the industrial production and application of this strain.