Isolation And Identification Of Lactobacillus And In Vitro Evaluation Of Its Probiotic Function

ObjectiveTo screen the candidate strains of probiotics Lactobacillus for further evaluation,we isolated and identified the Lactobacillus strains with acid-resistant and bile-resistant capability from the breast milk and neonatal faeces,then evaluated the properties of cholesterol-lowering,antibacterial,antitoxin and anti-inflammatory in vitro.Methods1.Isolation and identification:The Lactobacillus strains were isolated from serial diluted feces samples from breast milk and neonatal feces basing on their morphology on MRS agar plates after 48h incubation.The strains with good capability of acid-resistant and bile-resistant capability were further identified with the 16S rDNA gene sequence and the API 50 CHL system.2.(1)Evaluation of capability of lowering cholesterol in vitro:We used a commercial Cholesterol E-test Wako kit to measure the cholesterol in the culture supernatant.The effect of strain viability,incubation time and amount of inoculation on capability of lowering cholesterol were determined.By using ethanol to delude the cholesterol from Lactobacillus pellets,we further explored the mechanism of lowering cholesterol of these strains.(2)Antimicrobial activity:The antimicrobial activity of selected strains against E.coli 0157:H7,Salmonella typhimurium and Shigella sonnei was determined by well-diffusion assay method.The factors affecting the antimicrobial activity were also determined by using proteinase K or adjusting the pH value.(3)The cell free culture supernatant of Vibrio vulnificus or E.coli 0157:H7 has been pre-incubated with the Lactobacillus for 3hrs.Then the Caco-2 and Vero cells were treated with those pre-incubated supernatants containing cytotoxins for 12hrs respectively and the cytotoxicity of cells was determined by Lactate dehydrogenase(LDH)releasing assay to evaluate the effect of Lactobacillus on cytotoxicity of toxins secreted by Vibrio vulnificus or E.coli O157:H7.3.The mouse PMs were infected with V.v then incubated with L.plantarum DOMLa with different MOI for 3hrs.Lactate dehydrogenase(LDH)releasing assay was used to determine cytotoxicity and the cytokine productions of IL-6.IL-1? and TNF-a were measured by ELISA.Results1.We isolated 10 strains of Lactobacillus from neonatal faeces,including 6 strains of L.plantarum,2 strains of L.fermentum,one strain of L.acidophilus and one strain of L.delbrueckii.2.(1)L.fermentum H109 reduced 42.15%of cholesterol in the MRS-CHOL broth without bile salt.The L.delbrueckii Y20 reduced 90%cholesterol in the MRS-CHOL broth with 0.1%bile salt and most of the cholesterol in the pellets can be washed out by ethanol.The cholesterol-lowering activity of both L.fermentum H109 and L.delbrueckii Y20 were inhibited obviously by heating treatment.(2)The CFCS of Y7-4?Y20?H109?HI 15-5 and H121-1 all exerted antimicrobial activity against E.coli 0157:H7,S.typhimurium and S.sonnei with Y20 possessing the most antimicrobial activity.Further studies showed that the CFCS of L.delbrueckii Y20 remained the antimicrobial active after proteinase K treatment but attenuated the antimicrobial activity when the CFCS was adjusted to pH 6.0.(3)L.plantarum Y7-4 and L.delbrueckii Y20 reduced the cytotoxicity of Caco-2 cells caused by V.v supernatants from 75%to 20%and 10%respectively.The protective effect of L.plantarum Y7-4 and L.delbrueckii Y20 could not be observed on cytotoxicity of supernatant of E.coli 0157:H7.3.The L.plantarum DOMLa significantly inhibited the cytotoxicity of PMs treated with V.v as MOI of 100 and also reduced the production of IL-6,IL-1? and TNF secreted by V.v-infected PMs.Conclusion(1)We isolated 10 strains of Lactobacillus with acid-resistant and bile-resistant capability from healthy neonatal faces which is a good source of Lactobacillus.(2)L.fermentum H109 and L.delbrueckii Y20 possess the highest cholesterol removal ability as potential probiotics candidate to reduce serum cholesterol levels.L.fermentum H109 reduces the cholesterol in the medium mainly through absorption directly and L.delbrueckii Y20 reduces the cholesterol through secreting BSH which make cholesterol co-precipitation with de-conjugated bile salts.(3)L.delbrueckii Y20 exerts a good antimicrobial activity in vitro mainly due to the production of organic acids.(4)L.plantarum Y7-4 and L.delbrueckii Y20 demonstrat a good antitoxin activity by pre-incubated with the supernatants of V.v.These antitoxin activity is strain specific and toxin specific.(5)L.plantarum DOMLa could inhibit the cytotoxicity of mouse PMs infected with V.v and had an anti-inflammatory effect on the production of proinflammatory cytokines ex vivo,which suggest that L.plantarum DOMLa has potential value for an anti-inflammatory use.