Biosynthesis Regulation Mechanism Of Plantaricin Em F And Its Application In Beef Preservation

Bacteriocins of lactic acid bacteria,a kind of ribosome-synthesized low molecular weight antibacterial polypeptides or proteins,with the advantages of biodegradation,no drug resistance,and low hemolytic activity,especially produced by Lactiplantibacillus plantarum(L.plantarum)were generally recognized as safe food preservatives.Due to the complex regulatory mechanism of plantaricin biosynthesis,the yield is relatively low in the wild strain,and it has not been able to be applied on a large-scale in commerce.Hence,it is of great significance to study the regulatory mechanism of plantaricin biosynthesis and improve the yield via molecular modification at the gene level to promote the large-scale production and commercial application of plantaricin.In this study,plantaricin EmF,a novel bacteriocin,was isolated,purified,and identified from the fermentation broth of L.plantarum 163 which produce broad-spectrum antibacterial active substances.The potential regulatory genes of plantaricin EmF biosynthesis were selected by combined analysis of multiple omics,and the regulatory mechanism was further elucidated.Lastly,the potential application of plantaricin EmF as a natural food preservative was studied in beef preservation experiments.The main results are as follows:1.Isolation,purification and identification of plantaricin EmFThe L.plantarum 163 seeds were cultured in MRS medium for 48 h to harvest the fermentation supernatant.The bacteriostatic active substances A and B were obtained from the concentrated supernatant by ammonium sulfate precipitation,Sephadex G25 and Sephadex LH-20 column chromatography,and HPLC.Their molecular masses were identified by MALDI-TOF-MS as 1638 and 3702 Da,respectively,and their amino acid sequenceswerepredictedtobeFNRGGYNFGKSVRHand VFHAYSARGVRNNYKSAVGPADWVISAVRGFIHG,respectively by MALDI-TOF-MS/MS.Based on the published articles and the blast result in NCBI,antimicrobial active substance A is a natural mutant of plantaricin E and B is the plantaricin F.Therefore,substances A and B are a natural mutant of the two-peptide bacteriocin plantaricin EF,named plantaricin EmF.The isoelectric points of plantaricin Em and F are 11.16 and 10.71,respectively,and both of their net charges is 3.Their hydrophobicity values are 0.117 and0.408,respectively,and their hydrophobic moment values are 0.473 and 0.264,respectively.As a two-peptide bacteriocin,the antibacterial activity of plantaricin EmF remained above80%after treatment at 100°C for 30 min and its antibacterial activity was higher than 60%in the range of p H 2-10.These results indicated that plantaricin EmF exhibited broad-p H adaptability and thermostablility,and possessed the potential to be used as a preservative.2.Whole genome sequencing of L.plantarum 163The whole genome sequencing of L.plantarum 163 was performed by Illumina Hi Seq4000 and Illumina Hi Seq 2000 sequencing platforms.The genome contains one circular chromosome and four circular plasmids.The total length of the genome was 3317196 bp,and the GC content was 44.46%.The chromosome size of the genome was 3131367 bp,and the GC content was 44.71%.The genome of L.plantarum163 was similar to that of other five L.plantarum in terms of nucleic acid level,amino acid level,core genes and pan-genes,but they also had their own characteristics.Among them,the difference between L.plantarum163 and L.plantarum GBLP1 was found to be minor,while the difference between L.plantarum 163 and the other four strains was substantial.By analysis of the genome of L.plantarum 163,a pln locus that synthesizes plantaricin was found.The structure of pln locus and the possible regulatory mechanism of plantaricin EmF were analysed,laying a foundation for the study of the regulatory mechanism of plantaricin EmF biosynthesis in L.plantarum 163.3.The effects of maltose on the biosynthesis of plantaricin EmFThe effects of different carbohydrates on the yield of plantaricin EmF were studied,which showed that maltose significantly promoted the biosynthesis of plantaricin EmF.The fed-batch fermentation of maltose showed that feeding maltose at 36 h can obtain the highest yield of plantaricin EmF.The maximum yields of plantaricin Em and F were 10.547 and22.938 mg/L,respectively,and the maximum yields of plantaricin EmF was 33.49 mg/L,which was 2.508-fold higher than that of the medium without fed-batch of maltose.The effects of maltose on the transcriptional levels of related genes of plantaricin EmF biosynthesis were investgated,which demonstrated that maltose significantly promoted the transcriptional levels of the synthetic genes(pln Em,pln F)and regulatory genes(pln A,pln B,and pln D)of plantaricin EmF.Additionally,maltose also increased the transcriptional levels of processing and secretion genes(pln G1,pln G2,pln H,pln ST,pln U,pln V,pln W)of plantaricin EmF,thereby promoting the processing and secretion of plantaricin EmF and enhancing the production of extracellular plantaricin EmF.4.Analysis of the effects of maltose on plantaricin EmF biosynthesis based on transcriptome and proteomicsThe effects of maltose on plantaricin EmF biosynthesis in L.plantarum 163 was analysed using the transcriptomic and proteomic techniques.A total of 2967 genes and 2042proteins were identified,and there are 1689 genes and 309 proteins were significantly different at the quantitative level.Thereinto,2016 genes and proteins were associated with each other at the identification level,while only 188 differentially expressed genes(DEGs)and differentially expressed proteins(DEPs)were associated with each other at the quantitative level.Maltose promoted glycolytic pathway,amino acid metabolic pathway,tricarboxylic acid cycle and purine metabolic pathway in L.plantarum 163 cells,and provided a lot of energy,while inhibiting pyruvate metabolic pathway and pentose phosphate pathway,changing the metabolic pathways in L.plantarum 163 cells,thereby promoting plantaricin biosynthesis.Through the protein interaction network analysis,the pln locus may be related to eight two-component systems(RRP1/HPK1,RRP3/HPK3,Plt R/Plt K,RRP5/HPK5,RRP6/HPK6,Lam R/Lam K,RRP11/HPK11,and Lam A/Lam C)and two regulators(Lp＿2642 and Lp＿3040),especially the seven regulatory factors Plt R,Lam A,Lam R,RRP6,Lp＿2642,RRP5 and Lp＿3040,which were closely related to the pln locus.5.The regulatory factor Lp＿2642 regulates the biosynthesis of plantaricin EmFSeven regulatory factors Plt R,Lam A,Lam R,RRP6,RRP5,Lp＿2642,and Lp＿3040were overexpressed in L.plantarum 163,respectively.The results showed that the overexpression of the regulatory factor Lp＿2642 significantly increased the production of plantaricin EmF and the highest yield was 17.35 mg/L,which was 1.29-fold higher than that of the wild bacteria.In addition,the overexpression of the regulatory factor Lp＿2642 can up-regulate the transcriptional levels of plantaricin EmF synthetic genes(pln Em,pln F)and regulatory genes(pln A,pln B,pln D).Moreover,it did not affect the growth and cell cycle of L.plantarum 163.Furthermore,the DNase I footprinting results demonstrated that Lp＿2642can specifically bind to three sites of pln A promoter,which enhances its transcription and expression,thereby increasing plantaricin EmF production.Molecular docking results showed that the concave domain of Lp＿2642 protein could bind to the major groove of three DNA sequences,and their specific binding modes were different.As a regulator of the Tet R family,this is the first report to show that Lp＿2642 can promote the biosynthesis of plantaricin EmF and enhance its production in L.plantarum.6.The application of plantaricin EmF in beef preservationPlantaricin EmF damaged cell membrane integrity of L.monocytogenes,resulting in the leakage of cytoplasm,changes in cell structure and morphology,and ultimately cell death.Meanwhile,plantaricin EmF effectively inhibits the growth of L.monocytogenes in beef samples,especially in combination with chitosan.Plantaricin EmF(16μg/m L)+1.0%chitosan can inactivate all the L.monocytogenes in beef samples in 3 d.In addition,plantaricin EmF(16μg/m L)+1.0%chitosan effectively inhibits the growth of microorganisms in beef samples and control the total viable count.During the whole storage period,the total viable count in beef samples was significantly lower than the minimum detection limit(≤10~6 CFU/g)in the national standard.Meanwhile,plantaricin EmF(16μg/m L)+1.0%chitosan can effectively reduce the TVB-N content,TBARS content,p H value and storage loss in beef samples,and the color change also effectively maintained.All of these results indicated that plantaricin EmF(16μg/m L)+1.0%chitosan effectively improved the quality index and sensory quality of beef samples,and the shelf life can be extended to 15 d.It shows that plantaricin EmF has the potential application value to replace chemical preservatives as a natural food preservative in the field of food preservation.