| ε-polylysine is a polycationic natural antimicrobial peptide.It can interact with anions on the surface of cell membranes to cause damage to cell membranes and cell death.So it can effectively inhibit most Gram-positive and Gram-negative bacteria.Due to its non-toxic and effective antibacterial properties,more and more researchers have used it for food preservation and achieved many remarkable results.However,there are problems with the application ofε-polylysine in food preservation.Whenε-polylysine is used in some fruit juices or dairy products,its cations will interact electrostatically with the anionic polysaccharides or proteins in the system.Undesired flocculation or precipitation leads to a decrease in the antibacterial efficacy ofε-polylysine.In order to solve the practical problems in the application ofε-polylysine,this paper combinesε-polylysine with polyanionic sodium hexametaphosphate(SHMP)to form a polymer.The physicochemical properties of polymers formed under different conditions and the antibacterial properties of polymers were explored.he antibacterial mechanism at the molecular level was explored through transcriptomics.Finally,it was applied in pasteurized milk to investigate its application value.The main research contents and results are as follows:(1)The ionization properties ofε-polylysine and SHMP at different pH were determined by potentiometric titration.Then the physical and chemical properties of polymer formation under different conditions(proportion,pH,ion concentration,andε-polylysine concentration)were studied,investigating the phase behavior of the polymer,Zeta potential,and particle size.The results showed thatε-polylysine and SHMP had different degrees of electrostatic interaction at different pH,resulting in changes in the phase behavior of the system.As the H~+concentration increased,the salt ions in the solution could electrostatic shielding at low concentrations(10 m M and 20 m M).The salt ions at high concentrations(50 m M and 100 m M)could produce many insoluble polymers due to the high charge density,making the particle size larger and even causing phase separation.In this study,the structural properties of the polymer were analyzed by Fourier transform infrared spectroscopy,and it was found that the formation of the polymer was affected by the electrostatic interaction between the phosphoric acid groups in SHMP and the free amino groups inε-polylysine.(2)Selected two target strains of Staphylococcus aureus(S.aureus)and Bacillus cereus(B.cereus),and revealed the antibacterial properties of polymer by examining the size of the inhibition zone,the minimum inhibitory concentration(MIC)and the dynamic sterilization curve.The research found that the polymers of R20 and R25 had better antibacterial effect on S.aureus and B.cereus,the MIC for S.aureus was 40mg/L,and the MIC for B.cereus was 20 mg/L.It was shown that the polymer could reduce the number of colonies by 5-6 orders of magnitude within 12 h.The antibacterial mechanism of the polymer was preliminarily determined by investigating the cell contents’leakage,the cell membrane’s depolarization,the apoptosis,and the destruction of the bacterial cell membrane.The results showed that the electrostatic interaction between the negatively charged cell membrane and the polymer could lead to the collapse of the damaged cell membrane surface,the generation of holes,and the leakage of the cell contents to destabilize the osmotic pressure.(3)Further research on the antibacterial mechanism of polymers at the molecular level based on transcriptomics.The genes with significantly different expression levels were obtained by transcriptome sequencing.B.cereus had 829 differential genes,of which 390 were up-regulated and 439 were down-regulated;S.aureus had 290differential genes,of which 188 were up-regulated and 102 were down-regulated.Through GO and KEGG enrichment analysis of up-regulated differential genes,it is found that the impact of polymers on bacteria can be attributed to four aspects:the impact on metabolism,gene replication and repair,translation,and membrane transport and effects on group stress responses.Real-time fluorescent quantitative PCR verified the regulation of related genes,and the verification results were consistent with the transcriptome results.(4)Apply the polymer to pasteurized milk and study its antibacterial efficacy in pasteurized milk and its effect on the physical and chemical properties of the milk system.The results showed that the polymer had an excellent inhibitory effect on the total number of colonies in milk within one week,while the total number of colonies in the blank control group exceeded 10~4 CFU/m L on the fourth day.The inhibitory effect had a similar trend under the action of the three polymers(R15,R20,R25),the number of colonies of B.cereus could be kept below 10~6 CFU/m L within 7 days,while the number of colonies of S.aureus was more than 10~6 CFU/m L after the third day.During one-week storage period,the pH of the polymer-treated pasteurized milk did not change significantly,and the relative viscosity increased slightly.Using SDS-PAGE to analyze the protein in milk,it was found that with the increase of storage days,ε-PL combined with related proteins in milk and precipitated,and the protein in the polymer treatment group was more stable.Stability analysis showed that the stability ofε-polylysine treated alone was the worst,and no precipitation was found in the pasteurized milk system added with polymer.Therefore,this study found that adding polymers has an excellent antibacterial effect and will not significantly impact the physicochemical properties of pasteurized milk within one week. |
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