Study On Adhesion Properties Of Lactobacillus Plantarum And Applications In Probiotic Cheese

With increasing awareness of the relationship between diet and health, researchand development of health and functional foods have become one of the importantresearch trends in food science and technology. Previous studies have shown thatingestion of probiotics enhances several physiological functions, particularly thebody’s natural defense system and intestinal well-being. The characteristics of asuccessful probiotic are acid and bile tolerance, ability to adhere and colonize theintestinal tract. Cheese may offer certain advantages as a carrier of probioticmicroorganisms. The matrix of the cheese and its relatively high fat content may offerprotection to probiotic bacteria during passage through the gastrointestinal tract.Having a higher pH than traditional probiotic foods (e.g., yogurts and fermentedmilks), cheese may provide a more stable milieu to support their long-term survival.In the present study,12Lactobacillus strains were identified and one of them,Lactobacillus plantarum C88, showed good adhesion ability and other probioticproperties. The gene related to adhesive protein was cloned and expressed inEscherichia coli, and the recombinant protein was obtained; the adhesion mechanismof L. plantarum C88was discussed; the adhesion and colonization of L. plantarumC88in rat intestines were researched; the probiotic cheese was manufactured by usingL. plantarum C88as a secondary starter culture.Acid tolerance, bile tolerance and adhesion capacity are the most importantproperties for selection of potential probiotic strains. In this study, total12strainswere selected and screened from Inner Mongolia dairy tofu, Tibet mushroom andneonate faeces and found to be able to tolerate acidic conditions and the presence of0.3%(w/v) bile conditions. They were identified to be L. plantarum, L. fermentum, L.brevis, and L. curvatus according to phenotype and API50CH test. Also, these strainswere screened further for their probiotic potential by investigating their surfacehydrophobic properties, adhesion to human colorectal adenocarcinoma cell line Caco-2, in vitro cholesterol-lowering and antioxidative effect. Among the12Lactobacillus strains, L. plantarum C88showed stronger hydrophobicity towardsxylene, ethyl acetate and chloroform, the best adhesion ability to intestional Caco-2cells, and the ability to inhibit the attachment of E. coli CMCC44825, Staphylococcusaureus CMCC26071, Salmonella typhimurium CMCC50, and Shigella flexneriCMCC51061to Caco-2cells under the conditions of competition, inhibition anddisplacement. Furthermore, L. plantarum C88and K25exhibited betterhypocholesterolemic effect and antioxidant activity of scavenging hydroxyl,1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide anion.Exopolysaccharide (LPC-1) isolated from L. plantarum C88had potent hydroxyl andDPPH radical scavenging abilities as well as protective effect on H2O2-inducedCaco-2cells oxidative injury.The potential health-improving effects of L. plantarum C88was evaluated in arat model. After14days orally administration, L. plantarum C88was able to adhereand colonize in different regions of murine intestinal tract, the highest in the cecum,followed by colon, and the least in the ileum. L. plantarum C88was able to firmlycolonize the murine intestine for2days and fallen back to the levels before theadministration. The adhesion ability of L. plantarum C88on the murine intestine wasinvestigated by Laser Scanning Confocal Microscope. The results showed that L.plantarum C88could adhere to intestinal mucosa and form biologic barrier. Inaddition, the effect of L. plantarum C88on the intestinal microbiota was assessed.The results indicated that L. plantarum C88could increase the viable counts oflactobacilli in faeces of rats and decrease the viable counts of enterococci, but hadlittle effect on bifidobacteria, Clostridium perfringens and enterobacilli. Therefore, L.plantarum C88might contribute to the improvement of intestinal health.To study the adhesion mechanism of L. plantarum, the total DNA of L.plantarum C88was extracted and amplified by PCR. The purified PCR products andpMD-18T vector were ligated and transformed into host E. coli strain DH5a. TheGAPDH gene was confirmed by two restriction endonucleases and sequencing. Then,the GAPDH gene was cloned into the prokaryotic expression vector pET28a, resultingin GAPDH-pET28a. The recombinant plasmid was transformed into the host E. coli strain BL21and the inclusion body of recombinant protein was expressed. A strongband at about40kDa was shown by SDS-PAGE. The recombinant protein waswashed and dissolved by urea buffer. The dissolved protein was purified by bothanion exchange chromatography and size exclusion chromatography. When thepurified GAPDH recombinant protein was co-incubated with the bacterial cells, therewas significant decrease (P<0.05) in adhesion of L. plantarum C88, E. coliCMCC44825, Sta. aureus CMCC26071, Sal. typhimurium CMCC50, and Shi. flexneriCMCC51061to Caco-2cells.Among the fermented milk products, cheese is the best carrier for survival ofprobiotics. Therefore the probiotic adjunct L. plantarum K25, L. plantarum C88and L.casei LC6117were inoculated into milk to produce probiotic cheeses. The adjunctcultures and starter cultures could be added in milk at the same time without changingthe traditonal process of Cheddar cheese. Also, the use of probiotic adjuncts inCheddar cheese did not affect the cheese compositions including moisture, protein, fat,salt content and pH value of cheese. During the whole ripening period, the probioticadjuncts maintained their viability (>8.0log cfu/g) that met the least viable countrequirement for probiotic foods. Results demonstrated that Cheddar cheeses can be aneffective vehicle for delivery of probiotic organisms. Assessment of proteolysis duringripening showed no significant differences (P>0.05) in the level of water-solublenitrogen,70%ethanol-soluble nitrogen,5%phosphotungstic acid-soluble nitrogenand urea-PAGE patterns, though the concentration of free amino acids weresignificantly higher in LC6117cheese (P<0.05). Proteolytic activity, however,remained relatively low for all cheeses due to the low temperature of ripening (4℃).The adjunct cultures of L. plantarum C88, K25and L. casei6117could be used inCheddar cheese without negatively affecting texture, sensory properties andacceptance.Assessment of the in vivo cholesterol-lowering property of cheese with L.plantarum K25showed that the levels of serum total cholesterol (TC), low-densitylipoprotein cholesterol (LDL-C), and triglycerides (TG) were significantly lower thanHFC (P<0.05); the LDL-C/HDL-C and TC/HDL-C ratios were also significantlydecreased (P<0.05), suggesting the potential function of the probiotic cheese with L. plantarum K25to reduce the risk of cardiovascular desease.