| Listeria monocytogenes is one of the four food-borne pathogens.Infection with L.monocytogenes will cause great harm to human health,mainly manifested as sepsis,meningitis,and increased mononuclear cells.As a probiotic,lactic acid bacteria can produce a variety of bacteriostatic substances,including bacteriocin,of which type Ⅱ bacteriocin can specifically inhibit L.monocytogenes,and has high safety and stability.In order to obtain highly effective bacteriocins for L.monocytogenes and clarify its characteristics,this study screened and identified bacteriocin-producing lactic acid bacteria,isolated,purified,and evaluated the bacteriocins produced by it Mass spectrometry analysis and whole gene sequencing performed genomic analysis on the amino acid sequence of bacteriocin,which provided theoretical support for the practical production and application of high-producing bacteriocin lactic acid bacteria.The specific research results are as follows:(1)Seven lactic acid bacteria that had antibacterial effect on L.monocytogenes were selected from 481 lactic acid bacteria deposited in the laboratory by the Oxford Cup double-layer agar method.After excluding the interference of hydrogen peroxide and organic acids,6 lactic acid bacteria were identified as bacteriocin-producing strains by protease sensitivity test.The strain No.270 with the largest bacteriostatic diameter was selected as the research object.The strain was identified as L.plantarum by combining its morphological characteristics,physiological and biochemical characteristics,and 16S rRNA sequence,and named it LP270.(2)Through the measurement of the growth curve of lactic acid bacteria LP270,it was found that there was a corresponding relationship between bacteriocin LP270 synthesis and bacterial growth.Bacteriocin was separated from 1,500mL of LP270 fermentation broth by cell adsorption-desorption method.The total protein mass of the obtained crude bacteriocin extract was 126.6mg,and the specific activity was 6066.35AU/mg.The crude bacteriocin extract was evaporated under reduced pressure and purified by acidic buffer dialysis to obtain a total protein of 3.33 mg and a specific activity of 76,876 AU/mg.After purification by high performance liquid chromatography,a total of 2.28mg of bacteriocin was obtained,with a specific activity of 89825.56AU/mg.After Tricine-SDS-PAGE electrophoresis,bacteriocin was a single band with a molecular weight between 3.4-6.9KDa.It was named bacteriocin LP270.(3)The bacteriocin LP270 has good stability through thermal stability test,and it still has 85%antibacterial activity after being treated at 100℃ for 20min.Acid-base tolerance test shows that bacteriocin L270 has strong acid tolerance and can maintain its antibacterial activity in the pH range of 2-7.Proteolytic hydrolysis results showed that in addition to its resistance to pepsin,it was sensitive to trypsin,papain,and proteinase K.The bacteriostatic spectrum showed that bacteriocin LP270 had antibacterial activity against Listeria,but did not show antibacterial activity against other test strains.Among them,the minimum inhibitory concentration(MIC)of L.monocytogenes reached 1.78 μg/mL,and the bactericidal kinetics measurement also showed that bacteriocin LP270 had a good inhibitory effect on L.monocytogenes.(4)The genome of L.plantarum LP270 was found to contain 1 chromosome and 2 plasmids(P1,P2),which were 2,952,197bp,11,899bp,12,458bp in length,and the chromosome GC content was 44.93%.After analyzing the entire genome sequence on antiSMASH bacteriocin prediction website,it was found that a synthetic gene cluster of bacteriocin exists on plasmid P1.The gene cluster includes a bacteriocin immune gene,a bacteriocin-encoding gene,an ABC transporter gene,and a bacteriocin transport helper protein.The mature bacteriocin encoded by this gene synthesis cluster contains 44 amino acids,and the sequence contains the conserved sequence of class Ⅱ bacteriocin,YGNGV-.Its molecular weight is 4.6 KDa,which is consistent with the results of Tricine-SDS-PAGE and mass spectrometry.This indicates that the lactobacillus produced by this strain and the pathway-related protein factors are encoded by plasmid 1.Compared with the eggNOG annotations of the other 8 bacteriocin-producing lactic acid bacteria in Genebank,we found that LP270 has a strong carbohydrate utilization capacity.Among the strain’s genome,204 genes are related to carbohydrate transport and metabolic functions,and 120 genes are related to energy production and transformation.At the same time,48 genes related to the PTS transport system that can transport a variety of carbohydrates were identified.The above analysis shows that the LP270 strain has strong energy production and transformation capabilities,which is helpful for its use as an industrial strain producing bacteriocin.At the same time,genomic analysis revealed that LP270 has strong intestinal adaptability,which contains 2 bile hydrolases and 3 chloroacyl glycine hydrolases.In addition,lactic acid bacteria LP270 is also highly adaptable to the environment,containing 12 pH tolerance genes,12 temperature tolerance genes,5 osmotic pressure regulating genes,and 1 phage stress protein PspC coding gene.This indicates that this strain is expected to become a good intestinal probiotic. |
