Screening Of Lactobacillus Plantarum With High Antimicrobial Activity And Purification Of Plantaricin And Its Properties

Now a days,people payed close attention to food safety caused by foodborne pathogens.It was a pressing matter for government,enterprises and people's daily life to improve food safety and extend food safety period.Using large doses of traditional chemical preservatives could cause cancer,so that it was rejected by people as its security risks.The more people were in favor of biological preservatives.Therefore,the development of a broad spectrum,efficient,stable and safe natural biological preservative become a good development trend in today's food manufacturing industry.Lactobacillus plantarum(Lb.Plantarum)widespread in traditional fermented foods ware considered as food grade microorganism.It played a significant effect on the inhibition of foodborne pathogens.Protein inhibitory factor plantaricin metabolism produced by Lb.plantarum and other natural antibacterial substances such as organic acids,hydrogen peroxide and diacetyl not only inhibited the growth of foodborne pathogens,but also could improve food flavor and good sensory.In order to obtain Lb.plantarum with an efficient antibacterial activity.In this study,the traditional fermented foods ware collected in Yunnan.The 19 Lb.plantarum strains were screened by using traditional microbiology gradient dilution method,then identified by gram staining method and 16S rRNA molecular biology and drawing a phylogenetic tree.Physiological and biochemical properties of Lb.Plantarum are determined.It found that catalase test,producing hydrogen sulfide,gelatin liquefaction test,indole test,starch hydrolysis,V.P.test and nitrate reduction test were negative,while the methyl red test was positive for Lb.plantarum.In sugar fermentation experiment,Lb.plantarum could ferment most sugar such as glucose,maltose,lactose,mannose and fructose but not xylose,Arabia sugar and rhamnose.The 19 Lb.plantarum strains were screened twice with respectively G+bacteria(Listeria monocytogenes)and G-bacteria(E.coli 0157)as indicator bacteria.The first screening used double-plate method(spot-on-lawn),the rescreening used Oxford-Cup method for excluding non-inhibitory factor proteins such as organic acids and hydrogen peroxide.At last,Lb.plantarum QB3-1 with highly efficient antibacterial activity was obtained.The output of plantaricin produced by Lb.plantarum conventional fermentation was relatively low.Optimization of plantaricin production conditions was an effective to improve the plantaricin output.In this paper,three strains of Lb.plantarum with various levels of antibacteria were chosen as experimental material,the impacts of fennentation time,temperature,initial pH value of culture solution and medium composition on the production of plantaricin were studied.Research findings that the production of plantaricin was increased by 1.5 times after the optimization of culture conditions.The fermentation supernatant of Lactobacillus plantarum was isolated by 5 kinds of method such as ammonium sulfate precipitation,organic solvent extraction,organic solvent precipitation,pH dependent adsorption method and ultrafiltration.The ultrafiltration method was the best separation method through contrast and analysis,the recovery rate was more than 69%.The initial extract of Lb.plantarum QB3-1 fermentation supernatant was obtained by ultrafiltration method.The initial extract was purified HiTrap CM FF exchange chromatography and Bio-Gel gel filtration chromatography respectively.Plantaricin QB3-1 was obtained after bacteriostatic test and protein test were confirmed.The molecular weight of plantaricin QB3-1 was about 4.4 kDa by Tricine-SDS-PAGE.The biological characteristics of plantaricin QB3-1 also were studied.Plantaricin QB3-1 had high antibacterial activity and wide antimicrobial spectrum,which had different antibacterial activity onindicator bacteria including G+ bacteria,G-bacteria and fungi.Plantaricin QB3-1 had a strong thermal stability because antibacterial activity was still at heating 15 min at 100?.Plantaricin QB3-1 was better stability in weak acidic and neutral pH,low activity even loss at alkaline pH.Plantaricin QB3-1 was very sensitive to protease and had protein properties.Through the above research,Lb.plantarum QB3-1 with high antimicrobial activity was isolated from traditional fermented foods in Yun Nan and plantaricin QB3-1 had a better biological characteristics,which had potential application as a new biological preservatives in food preservation.