Isolation A Bacteriocin-produced Lactobacillus Paracasei And Study On The Characterization Of Bacteriocin

Contamination of spoilage bacteria and foodborne pathogens is the main cause of food spoilage and food poisoning.At present,chemical preservatives are often used to prevent and control microbial contamination in food industry,but chemical preservatives are at risk of carcinogenesis,teratogenesis,and allergic reactions,long-term overconsumption of chemical preservatives can affect human health.Therefore,the development of safe and efficient natural food preservatives has attracted increasing attention.Bacteriocin of lactic acid bacteria(LAB)is a type of ribosomally synthesized low-molecular weight proteins or peptides with antimicrobial activity.It exhibits great potential in food preservation because of its high efficiency,safety and green characteristics.In this study,L.paracasei ZFM54 producing broad-spectrum bacteriocin was screened from the feces of newborn infants in Women's hospital school of medicine Zhejiang University.The bacteriocin of L.paracasei ZFM54 was purified by a four-step purification procedure for studing on its biological characteristics.The aim of this research was to provide theoretical basis and reference value for the application of L.paracasei ZFM54 and its bacteriocin in food preservation,pharmaceutical industry and other fields.The main research contents are as follows:1.Screening and identification of bacteriocin producing lactic acid bacteria.LAB was isolated from the feces of newborn infants,after primary screening,a strain ZFM54 with strong broad-spectrum antimicrobial activity was obtained.To avoid antagonism by organic acids and hydrogen peroxide,secondary screening was done.Protease hydrolysis experiment confirmed that the antibacterial substance was protein or peptide.The strain ZFM54 was identified as Lactobacillus paracasei by physiological and biochemical characteristics and 16S rDNA sequence analysis,and named L.paracasei ZFM54.2.Isolation and purification of bacteriocin produced by L.paracasei ZFM54.The bacteriocin of L.paracasei ZFM54 was successfully purified by four steps of XAD-16 macroporous resin adsorption chromatography,cation exchange chromatography,Sephadex G-25 gel filtration chromatography and reverse phase high performance liquid chromatography(HPLC).Tricine-SDS-PAGE analysis showed that the molecular weight of this bacteriocin was approximately 5 kDa.According to the protein standard curve,42.49 mg bacteriocin was obtained from the fermentation supernatant of L.paracasei ZFM54.After purification,the bacteriocin titer was 5.03 fold than the initial culture.3.Antibacterial spectrum and minimum inhibitory concentration of bacteriocin of L.paracasei ZFM54.According to the agar well diffusion test and microtiter plate well method detection,the inhibition spectrum and minimal inhibitory concentration(MIC)were achieved.The result showed that bacteriocin of L.paracasei ZFM54 had a broad antibacterial spectrum,it can inhibit not only Listeria monocytogenes,Staphylococcus Musca,Micrococcus luteus and other Gram-positive bacteria,but also Salmonella,Pseudomonas aeruginosa and other Gramnegative bacteria,which makes up for the inadequacy of Nisin's anti-Gramnegative bacteria activity.But this bacteriocin has no inhibitory effect on molds and yeast.Among them,the bacteriocin of L.paracasei ZFM54 performed the strongest activity against Micrococcus luteus and Salmonella typhimurium,the MICs were 3.00 ug/mL and 3.50 ug/mL,respectively.4.Biological characteristics of the bacteriocin of L.paracasei ZFM54.Thermal stability study showed that this bacteriocin was thermostable and it still retained 72%antibacterial activity after heat treatment at 100 C for 30 min.In the study of pH stability,it was found that the antibacterial activity gradually decreased with the increase of pH value.In the environment of pH 5,the antimicrobial activity decreased significantly,but when pH was above 5,the antimicrobial activity was completely lost.Enzyme hydrolysis experiments found that this bacteriocin was sensitive to common proteases,but not to alpha-amylase,lysozyme,lipase and ribonuclease A.5.Preliminary study on Bacteriostatic Mechanism of the bacteriocin of L.paracasei ZFM54.Through the fluorescence leakage test,it was found that the cell membrane of the indicator bacteria treated with the bacteriocin of L.paracasei ZFM54 was damaged,which increased the permeability of the cell membrane and led to the dissipation of membrane potential difference.According to the results of the inhibition zone test,Lipid II had no effect on the antimicrobial activity of the bacteriocin of L.paracasei ZFM54,suggesting that Lipid II was not the specific target of this bacteriocin on cell membrane.