Isolation,Purification And Antibacterial Mechanism Of Antifungal Peptides Derived From Lactobacillus,and Its Application In Biological Preservative Film

Antimicrobial peptides derived from lactic acid bacteria are a kind of peptides synthesized and secreted by lactic acid bacteria with inhibitory activity against spoilage bacteria or pathogenic bacteria.Because of its good bactericidal activity against common spoilage bacteria and stable physical properties,it is widely used in food processing and preservation,feed production and medicine.In this study,the antifungal peptides produced by fermentation of lactic acid bacteria were firstly isolated and purified,their physicochemical properties were predicted,and their structures were identified.Then,the antibacterial mechanism of antifungal peptide extracts against C.albicans was analyzed.Finally,the antibacterial film prepared by adding antifungal peptides was characterized,and its fresh-keeping effect in processed cheese was studied.The main results are as follows:1.The antifungal peptides produced by Lactobacillus were isolated and purified.The fractions with higher activity were obtained by the step-by-step separation and purification method,and two active peptides,APTs and APTft were identified by liquid chromatography-mass spectrometry(LC-MS/MS)with molecular weights of 423.18 Da and 710.36 Da,respectively.It can be seen that the peptide APTs with better activity have a molecular weight of 423.18 Da.Through comparison,it is found that both of them are new antibacterial peptides that have not been reported.Select APTs with better activity for further study.Furthmore,the theoretical isoelectric point of APTs was predicted to be 5.24by bioinformatics tools,indicating that APTs have better solubility under neutral conditions.Its aliphatic index is 90.23,indicating that its thermal stability is good.The overall average hydrophobicity index value(GRAVY)was 0.160,indicating it have a good hydrophobicity.There was no transmembrane region of antifungal peptide APTs.In addition,the secondary structure examination showed that it was anα-helical structure.2.To study the antibacterial mechanism of antifungal peptide extracts.By measuring the effect of antifungal peptides on the growth curve of C.albicans,and it was proved that the antifungal peptides had a significant inhibitory effect on C.albicans(P<0.05).The scanning electron microscope showed that antifungal peptides could cause wrinkles,depressions on the surface of cell cells and even some cells appeared holes after 8 h of treatment;The results of Zeta potential showed that the changes of electronegativity of yeast cell membrane surface were significantly reduced after treatment with antifungal peptide(6×MIC),and the potential decreased from-21.4±0.70 m V to-7.47±0.34 m V.The results of the K~+leak test showed that with the increase of the action time of antifungal peptides,the level of K~+released outside the cells increased continuously.The results of cell surface hydrophobicity showed that the adsorption rate of C.albicans cells treated with antifungal peptides(6×MIC)decreased from 86.58±0.91%to 34.43±1.27%.The transmission electron microscopy displayed the cell wall did not change significantly after8 h treatment with the antifungal peptides,the interior of the cell was vacuolated,and the cell membrane was damaged.3.Preparation of biopreservative films and their use in the preservation of processed cheese.The formula of chitosan composite film was determined by single factor and orthogonal experiment:chitosan concentration 1.25%,gelatin concentration 1.75%,glycerol concentration 0.3%.The tensile strength of the composite film was determined to be(70.69±0.50)MPa,and the elongation at break was(12.69±0.12)%;adding different concentrations of antifungal peptides to prepare antibacterial films,it was found that when the concentration of antifungal peptides(6×MIC),the tensile strength and elongation at break were increased by 25.6%and 49.8%,respectively.Moreover,the mechanical properties of the antibacterial film were the best;and the antibacterial film showed a strong inhibitory effect on C.albicans(P<0.05);in addition,the antibacterial film added with antifungal peptides had a broad-spectrum antifungal activity.The results of scanning electron microscopy showed that antifungal peptides had good compatibility with chitosan and gelatin.The results of infrared spectroscopy indicated that there may be covalent bonds or hydrogen between antifungal peptides,chitosan and gelatin molecules.By measuring the preservation effect of the antibacterial film on the reformed cheese coating,the results showed that the moisture content of the cheese packaged in the antibacterial film decreased from the initial(55.25±2.59)%to(32.89±1.64)%after 15 days of storage.The L~*value of the two film treatment groups decreased with the prolongation of time;with the prolongation of the storage time,the hardness of the cheeses in both groups increased significantly(P<0.05).During storage,there was no significant difference in elasticity,but the elasticity of the antibacterial film group increased significantly on the 7th day.On the 7th day of storage,the total number of colonies in the chitosan composite film was(5.11±0.05)lg(CFU/m L),while that in the antibacterial film group was(3.91±0.03)lg(CFU/m L),with a significant difference(P<0.05),the total number of molds and yeasts in the antibacterial film group was(3.83±0.02)lg(CFU/m L),which was significantly lower than that in the chitosan composite film treatment group(5.13±0.10)lg(CFU/m L).The results showed the processed cheese packaged with antibacterial film has a significant antibacterial effect.