| In recent years,the abuse of antibiotics has led to the emergence of a variety of drug-resistant bacteria.Moreover,the use of broad spectrum of antibiotics to kill pathogenic bacteria can cause damage to the probiotics that are symbiotic with the human and animal intestinal tract,which leads to flora imbalance affecting the health ofthe body.Bacteriocin has attracted attention from researchers as a potential substitute for antibiotic because of its naturally occurring protein-based antibacterial substances,non-toxic,non-residue and non-resistance bacteria.At present,probiotics capable of producing bacteriocins are widely used in food,aquaculture and human health to inhibit pathogenic and spoilage bacteria.Bacillus subtilis is widely distributed in the soil,water and mammalian gut,and it was included in the Catalogue of Feed Additives by the Ministry of Agriculture of China.It can be used as a probiotic for direct feeding to animals.B.subtilis is usually produced in the form of spores and is widely used in many fields such as food,aquaculture,livestock,poultry feed and plant disease control.In addition,the application fields of strains can be further developed through the mining and analysis of genomic information.In this paper,two bacteriocin-producing strains,LF-9 and LF-11,were screened by bacteriostatic activity assay.After 16S rRNA sequence analysis,they were identified as Bacillus subtilis.Firstly,the biological characteristics of the two strains were analyzed and found to have many probiotic characteristics.Subsequently,the whole genome of the two strains were sequenced and analyzed for information,and the functional genome related to probiotic characteristics was explored.Finally,the bacteriocin produced by B.subtilis LF-11 was isolated,purified,structurally characterized and analyzed,and the bactericidal mechanism of Bac-LFll was preliminarily explored.The specific contents and results are as follow:1.Study on probiotic characteristics of bacteriocin-producing strainsThe pathogens L.monocytogene.STCC19114 and M.luteus 2016 were used as indicator bacteria,and the well-diffusion method was used to determine the antibacterial activity of41 strains of Streptococcus thermophilus and 20 strains of lactic acid bacteria and Bacillus strains screened from different sources.The results showed that LF-9 and LF-11 had strong antibacterial activity,and they,still had antibacterial activity,after excluding the interference of organic acid and H2O2,.indicating that the antibacterial substance produced was bacteriocin,and the sequence analysis of'the 16S rRNA of these two strains showed that they were both Bacillus subtilis.The probiotic characteristics of the strains were studied.It was found that both strains were tolerant to acid and bile salts at certain extent.The survival rate of B.subtilis LF-9 and S.subtilis LF-11 after treatment for 2 h at pH=2 reached 59.77%and 58.74%,respectively.B.subtilis LF-9 can tolerate 0.15%bile salt,while B.subtilis LF-11 can tolerate bile salt with concentration up to 0.3%and the survival rate was 39.07%after treatment for 3 h.In addition.both strains have high surface hydrophobicity and autoaggregation.which are beneficial to the adhesion of bacteria in the intestine.Moreovern they have strong coaggregation ability with the pathogens L.monocytogenes ATCC19114 and M.luteus 2016,which help them to effectively combine pathogens and exert bactericidal or bacteriostatic action.2.Analysis of genomic information of B.subtilis LF-9 and B.subtilis LF-11With tlhe rapid development of high-throughput sequencing technology and bioinformatics,it has become more and more time and labor-savng to,mine genomic information.In this chapter.the whole genome of bacteriocin-producing B.subtilis is sequenced.and the basic infornation of the genome and the genetic information of enzymes and antibacterial gene clusters related to animal feed in the genome are summarized.among which the full length of B.subilis LF-9 genome is 4.343.962 bp.the GC content is 43.03%?and there are 4691 ORE.accounting for 87.57%of the total genomic length.the full length of B.subtilis,LF-11 genome is 4.341.047 bp.the GC content is 43.02%,and there are 4792 ORF,accounting for 88.52%ofthe total genomic length.The results showed that both strains contained genes encoding related enzymes which can degrade cellulose,hemicellulose,pectin,starch,chitin and anti-nutritional factor phytic acid.Moreover,B.subtilis LF-11 contairns a gene encoding ?-mannanase.In addition,6 and 5 antibacterial gene clusters were predicted in the genomes of B.subtilis LF-9 and B.subtilis LF-11,respectively.The above theoretical analysis is excepted to provide theoretical basis for application of the strain in the field of animal feed.3.Purification and characterization of bacteriocinThe bacteriocin is mostly a small molecule oligopeptide,the yield is low,and purification of bacteriocin is very difficult.The bacteriocin is separated and purified by a series of operations such as ammonium sulfate salting out,ion exchange chromatography,gel exclusion chromatography and HPLC,and the purified bacteriocin is identified by mass spectrometry,it is preliminarily judged to be a novel bacteriocin.The molecular weight is around 5 kDa.Then,the results of the characteristic study showed that bacteriocin Bac-LF9 and Bac-LF11 had strong antibacterial activity at pH=l-9,and the antibacterial activity completely disappeared at pH=10-12.The results of temperature sensitivity test showed that Bac-LF9 and Bac-LF11 retained partial antibacterial activity after treatment at 90? for 15 min.Therefore,the bacteriocin has good thermal stability and is resistant to acid but not to alkali conditions.The above characteristics make bacteriocins more stable and advantageous in practical applications.In order to preliminarily explore the probiotic mode of Bac-LF11,M.luteus 2016 and L.monocytogenes ATCC19114 were treated with a certain amount of purified bacteriocin Bac-LF11,and the number of viable bacteria was determined after treatment for different time,compared with the control group,the number of viable cells of L.monocytogenes ATCC19114 showed a signi:ficant downward trend in the experimental group.After 24 hours of treatment,the number of viable cells decreased by nearly 5 orders of magnitude,and the electron microscope showed that the surface of L.monocytogenes ATCC19114 is severely damaged,and,most of the cells are in fragments.so the mode of action of Bac-LF11 on L.monocylogeens ATCC19114 is bactericidal.While the number of viable cells of M.Luters 2016 was basically the same as that at the beginning.but it was lower than the control group without bacteriocin,and the electron microscope showed that the surface of the bacteria was only slightly damaged,so the mode of action of Bac-LF11 on M.luteus 2016 is bacteriostatic.In summary,through the study of biolofgical characteristics and genomic information,the two strains of B.subtilis selected in this paper have a variety of probiotic characteristics and have a lethal effect on pathogens,so they have potential application in the fields of feed and food. |
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