| Bacteriocins are a class of antibacterial polypeptides produced by ribosomes.Most of them are from lactic acid bacteria(LAB)which is generally recognized as safe bacteria.With the advantages of natural,safe and non-toxic,bacteriocins are suitable for using in food.Although various of bacteriocins have been isolated and identified,few are commercially available.In addition,the mode of action of bacteriocins has not been fully elucidated.With the increasing of drug-resistant bacteria,the detection of methicillin-resistant Staphylococcus aureus(MRSA)in food has been reported in many countries.MRSA is resistant to most of antibiotics.They can produce toxins and form biofilms that are difficult to remove.MRSA is a great threat to food safety.The development of bacteriocins as biological preservatives has great application potential in controlling the spread of MRSA in food and inhibiting food spoilage.In this study,a Lactobacillus plantarum strain with broad-spectrum antibacterial activity was isolated from Guizhou pickled fish,and the bacteriocin plantaricin GZ1-27 was purified and identified from the fermentation broth.Using cell biology,transcriptomics,and proteomics technology,antibacterial mechanism of plantaricin GZ1-27 against MRSA was researched at cell level,gene level and protein level.Finally,effect of plantaricin GZ1-27 in pork storage was evaluated.This study contributed to the use of food-grade lactic acid bacteria resources and the development of new natural biological preservatives.The main results are summarized as follows:1.Screening and identification of lactic acid bacteria with broad-spectrum antibacterial activityIn total,459 lactic acid bacteria strains were isolated from traditional fermented samples.Using agar diffusion method,80 strains with obvious antibacterial activity were selected using Gram-positive bacteria Bacillus cereus AS1.1846 and Gram-negative bacteria Escherichia coli ATCC25922 as indicators.After adjusting pH,7 strains with excellent antibacterial effects was obtained using double-layer agar method.Among them,strain GZ1-27 showed the best activity.Based on morphological observation,physiological and biochemical identification,as well as molecular biological identification,strain GZ1-27 was identified as L.plantarum,and named as L.planta rum GZ1-27.2.Purification and structural identification of plantaricin GZ1-27L.plantarum GZ1-27 was fermented in MRS medium.Antibacterial activity reached the maximum at 36 h.The antibacterial substances in the fermentation broth of L.plantarum GZ1-27 was separated by ammonium sulfate precipitation,Sephadex G-25 and Sephadex G-50 gel column chromatography.Then the single peak with bacteriocin activity was obtained using RP-HPLC.The molecular weight of purified bacteriocin was 975 Da as determined by MALDI-TOF/MS.The antibacterial substance was tested for secondary mass spectrometry analysis using Q-TOF-MS/MS.As analyzed from the mass spectrometry,amino acid sequence of the bacteriocin was Val-Ser-Gly-Pro-Ala-Gly-Pro-Pro-Gly-Thr-His(VSGPAGPPGTH).This bacteriocin was named as plantaricin GZ1-27.Plantaricin GZ1-27 had good thermostability.After treatment at 121℃ for 20min,84.1%of the antibacterial activity still maintained.It’s stable in pH 2.0-6.0 and not easy to be degraded by protease.Plantaricin GZ1-27 is suitable for application in food industry.3.Study on the antibacterial mechanism of plantaricin GZ1-27 against MRS AThe minimum inhibitory concentration against MRSA ATCC43300 was 32μg/mL.MRSA cells could be completely killed within 150 min by 2× MIC plantaricin GZ1-27(64 μg/mL).The K+ ion was used to detect the membrane permeability.It was found that when treated with 1×MIC plantaricin GZ1-27 for 60 min,the content of K+in supernatant increased to 0.6 mg/mL.The results showed that plantaricin GZ1-27 could increase membrane permeability of MRSA cells.The effect of plantaricin GZ1-27 on membrane integrity of MRSA was detected by flow cytometry and CLSM.The results showed that after GZ1-27 treatment,compared with the control group,fluorescence intensity was significantly increased and the proportion of dead cells was significantly increased.The effect of plantaricin GZ1-27 on MRSA cells and internal structure was observed by SEM and STM.The results showed that plantaricin GZ1-27 could induce surface shrinkage,cell membrane dissolution,cytoplasmic leakage and intracellular tissue destruction of MRSA bacteria,and the change was time-dependent.EB combined experiment was used to study the effect of plantaricin GZ1-27 on MRSA genome.After the addition of plantaricin GZ1-27,fluorescence intensity gradually decreased,indicating that plantaricin GZ1-27 could combine with MRSA bacterial DNA in vitro,thus inhibiting the physiological function of bacteria.Moreover,through crystal violet staining and scanning electron microscope observation,low concentrations of plantaricin GZ1-27(1/2 × MIC,1/4×MIC)can inhibit the formation of MRSA biofilms,and high concentrations of plantaricin GZ1-27(2× MIC,4× MIC,8×MIC)can penetrate the biofilm matrix and kill the biofilm cells.It was speculated that plantaricin GZ1-27 could exert its antibacterial effect by changing the membrane permeability of MRSA cells,interfering with the synthesis of genetic materials,and inhibiting the formation of biofilm.4.Antibacterial mechanism of plantaricin GZ1-27 against MRSA based on transcriptome and proteomicsAfter treatment with sub-MIC plantaricin GZ1-27(16 ug/mL),228 proteins showed significant changes both in transcription and translation levels based on RNA-seq sequencing and iTRAQ proteome data.Through further analysis and combined with experiment results in Chapter threes,it was found that membrane permeability increasing and destruction of the synthesis and repair of DNA were the main mechanism of action of plantaricin GZ1-27 on MRSA.Plantaricin GZ1-27 increased the expressing of membrane osmotic protein OpuC which could increase the permeability of cell membrane to small molecule ions.Meanwhile,the expression of protein output protein PrsA,lipid output protein MsbA,and amino acid transport protein EhuA were induced to exclude the macromolecules.After entering into MRSA cell,plantaricin GZ1-27 not only disturbed the synthesis of DNA through the inhibition of urine metabolism,but also blocked the repair of DNA by inhibiting RecA,AddA,XerD and XseB,which lead to the unstable of MRSA genome.In addition,various of MRSA heat shock proteins(GrpE,McsB,HrcA,Asp23)were induced by plantaricin GZ1-27,and the synthesis of amino acid metabolism and secondary product were disturbed.Omics has also revealed the mechanism by which plantaricin GZ1-27 inhibits MRSA biofilm formation.It was inhibition of production of MRSA extracellular matrix(FnBPB.SdrC.Spa.and IsdB)and inhibition of expression of interfering regulators(IcaR,SarA,SigB,LytR,and ArlR).5.Application of plantaricin GZ1-27 in storage of pork during 4℃Plantaricin GZ1-27 could effectively inhibit the growth of artificially contaminated MRS A in pork.MRSA wasn’t detected in the plantaricin GZ1-27+0.5%chitosan and plantaricin GZ1-27+1.0%chitosan treatment group during storage at 4℃.Meanwhile,using sensory evaluation,microbiological indicators analysis,physical and chemical analysis,it was found that after treatment with preservatives,the total plate count was reduced effectively,the increase of TVB-N value and increased pH value was delayed during the process of pork spoilage.In addition,the generation of spoiled gas were inhibited,the sensory quality of pork was improved,and the shelf-life was extended.Plantaricin GZ1-27+0.5%chitosan treatment group have the best preservation effect,extending the storage time of pork at 4℃ to 12 days.This results indicates that plantaricin GZ1-27 is a potential food preservative and anti-MRSA agents.Plantaricin GZ1-27 has a good application prospect in food preservation. |
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