| Food is susceptible to microbial contamination during production,transportation and storage,leading to frequent occurrence of various safety incidents.These food-borne pathogens can reduce food quality and even endanger human health.The common method to solve this problem is to use chemical preservatives,but its side effects are obvious.Therefore,it is urgent and necessary to find safer and more reliable alternatives to chemical preservatives from biological sources.Bacteriocins are a type of polypeptide synthesized by ribosomes,which have attracted much attention because of their inhibitory effects on harmful microorganisms.Bacteriocins can reduce the risk of spreading foodborne diseases and extend the shelf life of products.The use of bacteriocins instead of chemical preservatives has less adverse effects on the receptors because they are easily hydrolyzed by proteases,and the decomposition products are non-toxic and harmless.The most important thing is that bacteriocins can kill bacteria through a variety of methods,making the target bacteria less likely to develop drug resistance.With the development of society and the advancement of technology,more new bacteriocins will be discovered.However,traditional bacteriocin separation and purification methods are time-consuming and highly uncertain.In addition,due to the wide variety of bacteriocins and obvious differences in properties,there is no fixed purification method,which makes the cost of obtaining bacteriocins higher.Therefore,the discovery of new bacteriocins has changed from traditional activity screening method to genomic data analysis method to eliminate hidden dangers of omissions,so that researchers can more effectively and conveniently mine bacteriocins on a large scale.Sichuan pickle is a traditional Chinese food,especially in Sichuan and Chongqing.The Lactobacillus rhamnosus LS-8 used in this study was isolated from traditional Chinese Sichuan pickle.Previous studies have determined that its extracellular secreted protein had good antibacterial ability,so this strain is the focus of research.In this paper,by integrating genomic and peptide group information,potential bacteriocins were discovered at the genetic level,and heterologous expression was performed in the E.coli expression system.Subsequently,the stability and antibacterial mechanism of the selected bacteriocins were studied.The specific researches and main results are as follows:（1）The growth of Lactobacillus rhamnosus LS-8 was inhibited in an environment that simulates oral digestion and gastric digestion,and was not affected in the digestive environment of the small intestine,indicating that this strain was sensitive toα-amylase,acidic environment and pepsin,but not sensitive to trypsin and bile salts.Through the optimization of ammonium sulfate saturation and adsorption and desorption p H,the optimal extraction conditions for antibacterial proteins（90%saturated ammonium sulfate,adsorption p H5.85 and desorption p H1.95）were obtained,and crude protein samples were prepared for mass spectrometry analysis.After comparison with four databases,a total of 215non-repetitive peptide sequences were matched.Among them,25 peptide sequences were identified using APD3（Antimicrobial Peptide Database）,21 of which were derived from the ammonium sulfate precipitation method,and 4 were derived from p H adsorption and desorption method.（2）The genomic DNA of Lactobacillus rhamnosus LS-8 was extracted and sequenced on the Illumina Novaseq platform.Some conclusions could be known through the functional annotation of the open reading frame,the analysis of protein,lipid and carbohydrate hydrolysis and utilization,and the study of species evolution.There are 2835 protein-coding genes in the genome of Lactobacillus rhamnosus LS-8.GO（Gene Ontology）annotation showed that 50.04%of the genes were classified as biological processes in the three major classifications.KEGG（Kyoto Encyclopedia of Genes and Genomes）annotation showed that96.86%of genes were involved in metabolic pathways,but 37.21%of protein-coding genes have not been annotated with COG（Cluster of Orthologous Groups of proteins）function.In addition,Lactobacillus rhamnosus LS-8 had a wide range of available nutrients,active cell metabolism and strong environmental adaptability.Through the analysis of the species evolution of Lactobacillus rhamnosus,it could be seen that LS-8 genome was genetically stable,with few mutations and recombination genes,and no large-scale insertions,deletions,and inversions have occurred.（3）The genome sequence of Lactobacillus rhamnosus LS-8 was analyzed by anti SMASH and BAGEL4 mining software,and 5 gene clusters related to bacteriocins were found.Subsequently,the uncharacterized protein sequences were compared with the genome,and 16 potential bacteriocin sequences were mined.Using Ex PASy to predict the physical and chemical properties of these sequences,the molecular weights of the sequences ranged from 7,840.41 Da to 49,323.06 Da.Except for p H4 and S75,other sequences had good stability.When using Prot Scale to predict the hydrophilicity and hydrophobicity of the sequences,it was found that S81 and S137 had strong hydrophobicity.The penetration structure predicted by TMpred showed that S68,S81,S109 and S137 had strong membrane penetration function.There were signal peptides in only four of all sequences.The secondary structure and tertiary structure predicted that only p H25 does not containα-helical structure.The ratio of BFP<0 in S81 and S137 was the highest,indicating that these two sequences had better stability（4）The E.coli BL21（DE3）expression system was used to heterologous express 16potential bacteriocin sequences and prepare crude protein samples.E.coli ATCC 25922 and S.aureus ATCC 25923 were used as indicator bacteria to verify that there were 14 expressed proteins possessing antibacterial activity.These sequences were p H4,p H23,p H25,p H35,p H45,S7,S58,S68,S75,S79,S81,S93,S109 and S137,respectively.Four bacteriocins（p H25,S68,S81,and S137）were selected as the focus of later research,and their expression conditions were optimized.The final optimized conditions were 0.6 mg/m L Kana,OD600=0.45,116.81μg/m L IPTG;0.8 mg/m L Kana,OD600=0.40,95.32μg/m L IPTG;0.6 mg/m L Kana,OD600=0.35,71.49μg/m L IPTG和0.6 mg/m L Kana,OD600=0.40,95.32μg/m L IPTG.The antibacterial test on 19 kinds of Gram-negative bacteria（G-）and 10 kinds of Gram-positive bacteria（G+）showed that these four sequences have broad-spectrum bacteriostasis,and the effect on G-is better than G+.（5）The four bacteriocins were purified by dialysis,cation exchange chromatography and reversed-phase high performance liquid chromatography.The single peak in HPLC with antibacterial activity were collected,and then subjected to LC-MS/MS identification after lyophilization and concentration.The fragment coverage showed that the antibacterial proteins have been successfully cloned and expressed,and a relatively complete and pure bacteriocin proteins have been obtained.The MICs of the four purified bacteriocins for E.coli,S.aureus,L.monocytogenes and C.sakazakii ranged from 5.38 to 19.84μg/ml.Fourier transform infrared spectroscopy showed that the four bacteriocins are proteins containing unsaturated bonds.Among them,p H25 contained aromatic rings,S68 had aryl ketones,S81had an unsaturated C=C double bond,and S137 had an alkyne structure.Using 3×MIC bacteriocins to treat pathogenic bacteria,there was no signs of growth within 48 hours,indicating that the four bacteriocins had strong antibacterial and even bactericidal effects.In addition,the four bacteriocins had self-killing ability for expression vectors,of which p H25had the worst ability to inhibit the growth of expression vectors,and S137 was the strongest one.（6）The four bacteriocins（p H25,S68,S81 and S137）were not sensitive to ultraviolet radiation and temperature changes.The antibacterial ability will not be affected under acidic and neutral conditions,but slightly reduced under alkaline conditions.The four bacteriocins are sensitive to proteinase K,pepsin and trypsin.3%Na Cl and Fe2+will affect the antibacterial ability of p H25.S81 was sensitive to Ca2+,while Zn2+,Mn2+,methanol,acetone and SDS would destroy the stability of these four bacteriocins.Tween-20 and Tween-40reduced the ability of p H25 to inhibit S.aureus,but N-butanol could enhance the antibacterial effect of p H25.p H25 was sensitive to VC,and VE would affect the stability of S137.Besides,S137 could show good stability in various low temperature environments.According to the results of the stability test,the overall stability of the four new bacteriocins can be ranked:p H25<S68<S81<S137.（7）The bacteriocins had a bactericidal effect on E.coli,S.aureus,L.monocytogenes and C.sakazakii under 2×MIC,and the bacteriocins had better ability against E.coli.At32×MIC,S68 and S137 had 13.93%and 20.37%hemolysis rates,respectively.Through membrane potential monitoring,AO/EB fluorescent staining and DNA leakage test,it was found that bacteriocin p H25 and S81 had the ability to destroy the cell membranes of E.coli and S.aureus.S68 had no effect on the cell membrane integrity of these two pathogenic bacteria,while S137 had no membrane-breaking effect on E.coli,but it damaged the cell membrane of S.aureus.Scanning electron microscopy and transmission electron microscopy results showed that the bacteriocin p H25 could enhance the permeability of the cell membranes of E.coli and S.aureus,causing the content to flow out and cell death.S68inhibited DNA/cytoplasm production or repair of E.coli and S.aureus,leading to cell death.S81 can directly cause the rupture of the cell membrane of two pathogenic bacteria.S137will cause G+bacteria cell membrane damage,and bactericidal mechanism of G-bacteria may mainly occur in the cell.（8）Add a mixture of E.coli and S.aureus as well as bacteriocins（p H25,S68,S81,S137）with a final concentration of 2×MIC to fresh beef and milk,and observe the growth of pathogenic bacteria in food within 7 days through colony counting method.It was found that p H25 and S168 had no significant inhibitory effect on pathogenic bacteria in beef,but they had a good bactericidal effect on pathogenic bacteria in milk.Subsequently,the four bacteriocins were mixed with Nisin and processed with beef and milk.It was observed that S81+Nisin and S137+Nisin showed significant joint effects on the beef.But after adding bacteriocin p H25 and Nisin into milk,the bacteriocins had the weakest antibacterial ability. |
