Study On Biosynthesis Of Exopolysaccharide Produced By Lactobacillus Plantarum

Fermented milk has shown beneficial physiology properties on human body.Those properties are partially attributed to the constituents of lactic acid bacterial's cell wall, specifically the exopolysaccharide(EPS).The EPS-producing strains are utilized in numerous kinds of fermented milk containing kefir,the traditional fermented milk which has its origin in the Caucasian mountains.Kefir is a natural probiotic dairy product produced by the actions of bacteria and yeasts contained in kefir grains which have been described as a symbiotic community.In vitro and animal trials have shown kefir and its constituents to have various kinds of bioactivitiy.Exopolysaccharide(EPS),one kind of carbohydrates,is secreted by lactic acid bacteria during its growth metabolism.Many researchers reported EPS have many health benefits such as antitumor effects, immunostimulatory activity and cholesterol lowering ability.This study was aimed to develop a novel yoghurt starter of EPS producing strain.The exopolysaccharide production ability was tested on 10 strains of lactic acid bacteria isolated from Tibet kefir in different medium by the phenol-sulphate acid method.Strain KF5, whose yield was determined as 49.32 mg/L,was screened out for its better sticky characteristics and WHEY media 1 was selected as basic medium.KF5 was identified as Lactobacillus plantarum by morphologic observation and physiological-biochemical identification.Effect of different kinds of fermentation conditions,incubation time,inoculated amount, and initial pH of culture medium on EPS production was studied in order to enhance EPS production.Fermentation conditions was optimized by cultivation time,inoculated amount and initial pH of culture medium through an orthogonal experiment L₉(3⁴).The results indicated that under aerobic cultivation at 37℃,the optimization of fermentation condition was as follows:culture time of 30 h,inoculation percentage of 3%(v/v) and initial pH6.3 of culture medium.Under this condition,a corresponding EPS yield of 95.58 mg/L was obtained.Comparative study of different isolation method indicated heat treatment of the samples as a first step in the EPS isolation procedure is critical for complete recovery of the EPS. Three times volume was the proper amount of ethanol and precipitated for 36 h at 4℃when ethanol was used to precipitate EPS.As compared to Sevag method,trichloroacetic acid method was more suitable to purify EPS roughly.The optimal trichloroacetic acid was 8%.The characteristic of FT-IR released EPS was indeed pyrylium polysaccharide that chiefly constituted byα-glycosidic bonds.The monosaccharide compositions of EPS were mannose,glucose and galactose in the molar ratio of 1:1.46:1.43 by hydrolyzing EPS with trifluoroacetic acid(TFC) and then analyses with GC.The molecular structure of EPS was observed with atomic force microscopy(AFM).The results of AFM showed a spherical lump.By testing the characteristics of milk fermented by KFS,it showed that KF5 has intermediate coagulation speed and gave desirable product when fermented in milk. Postacidification of the fermented milk can be inhibited and the extracting rate of whey was lower than that of contral during the period of storage at 4℃.Besides,it exhibited a better stability when stored under 4℃.These results indicated KF5 has the potential to be applied in dairy industry.In vitro,the exopolysaccharides could not inhibit the growth of E.coli,Staphylococcus aureus,Bacillus subtilis and streptococcus mutans.