| Because of its regulatory ability of intestinal microflora balance and other beneficial functions, probiotics are being accepted by more and more consumers. These microorganisms face challenges in harsh gastrointestinal environments when taken orally. To exert health promoting functions, probiotics must be able to survive and reach the human colonization sites. Bile is involved in fat emulsification and absorption in human digestive tract, while the lipid membrane structure of the microorganisms is also damaged. Therefore, bile resistance is a precondition for probiotics screening. The explanations on bacterial bile resistance showed specie variations in Lactobacillus and Bifidobacterium targeted researches, and no applicable or effectively improved bacterial bile resistance policy was raised for food industry applications. In probiotics screening work, survival in 0.3% of bile salts is a commonly used criteria. However, potential probiotic functions are ignored in the eliminated strains under this standard, which is a microbial resource waste. Therefore, the study of the mechanisms of probiotics bile resistance, as well as a based improvement policy, are with theoretical significance and application value. Lactobacillus plantarum is commonly found in fermented foods. Because of its ‘generally recognized as safe’(GRAS) source, it is widely used in probiotic screening and applied in the food industry. In this thesis, thirty five strains of Lactobacillus plantarum isolated from natural fermented foods were involved. Statistical methods were employed to analyze the correlation between physiological indicators and bile resistance. Further, a policy of enhancing bile resistance was put forward to apply in yogurt processing.Most tested strains from natural foods showed poor bile resistance. Reported evaluation methods of bile resistance could not effectively distinguish bile salt sensitive strains, and were unable to quantify the resistant ability. Therefore, a new method based on specific growth rate was established to evaluate bile resistance. By measuring bacterial growth curve of logarithmic phase in the absence of bile salts and 0.1% bile salt concentration, the maximum specific growth rate constant was fitted and the correlation coefficient ranges from 0.9-0.99. Compared with reported methods, the evaluation results of new method for bile tolerant strains are consistent. The new method was able to effectively distinguish and quantify the resistance ability for bile salt sensitive strains.Surface hydrophobicity, total antioxidant capacity, bile salt hydrolase activity, cellular integrity and intracellular p H were commonly involved in studies of bile resistance. Pearson correlation analysis showed that surface hydrophobicity(r =-0.037, P = 0.829), total antioxidant capacity(r = 0.164, P = 0.333), and bile salt hydrolase activity(not detected) were no significant correlation with bacterial bile salt resistance from natural fermented food sources. Compared with the initial strains, the indicators mentioned above showed an inconsistent trend as bile resistance increasing in bile adapted strains. Gram staining and fluorescence microscopy camera technology were used to validate the damaging effects of bile salts on cell morphology, rather than cell lysis. Effects on β-galactosidase activity caused by cell leakage were assayed after challenge in 0.3% bile to evaluate cell integrity. Pearson correlation analysis showed that cell leakage and bile resistance was in significantly moderate negative correlation(r =-0.665, P = 0.000). In another word, the lower ratio of leaking activity, the higher cell integrity, and the stronger bile salt resistance for strains. Compared with the original strain, cellular integrity showed an increasing trend in bile adapted strains. After adapted in bile, intracellular p H in strains showed increases or decreases in different strains. Thus, maintaining cell integrity was the key strategy for strains to survive in bile, and other physiological indicators shifting was a response to bile under the situation of cell integrity being maintained.Cell integrity was maintained by cell outer structure. Cell surface proteins, extracellular polysaccharide production and membrane fatty acid composition were main components of cell outer structure in lactic acid bacteria. Study on the referenced strain Lactobacillus acidophilus NCFM showed that cell surface protein could reduce damage to cell integrity caused by bile salts. However, experimental results and literature search illustrated that surface layer protein(SLP) does not exist in Lactobacillus plantarum. Pearson correlation analysis showed that EPS production was not correlated with bile resistance(r =-0.104, P = 0.541). However, the specific fatty acids showed different degrees of correlation with bile resistance. Palmitic acid(r = 0.766, P = 0.007) and 19 carbon cyclopropane fatty acid(r = 0.659, P = 0.008) contents showed significantly positive correlation with bile resistance. Myristic acid(r =-0.494, P = 0.002) and oleic acid(r =-0.795, P = 0.003) contents showed significantly negative correlations with bile resistance. The high value of saturated/unsaturated fatty acid ratio(r = 0.696, P = 0.001) was beneficial to maintain cell integrity and to improve bile tolerance.Considering the significant impact of membrane fatty acid composition on cell bile resistance, the strategy of adding phospholipids in the cell culture system to improve bile resistance is proposed herein. Experimental results showed that adding soybean lecithin or egg yolk phospholipids in MRS broth could raise C16:0 fatty acid content in Lactobacillus plantarum cells, which improved the cell integrity and bile resistance in bile existed environment. Yolk phospholipids(optimized concentration: 0.6%) showed better protective effect than soy lecithin. By adding soy lecithin into reconstituted milk to ferment yogurt, not only the fermented strain showed better bile resistance, but the stability and processing characteristics of yogurt were also improved. Considering the sensory evaluation, adding the concentration of 0.4% soy lecithin was shown as optimal for yogurt fermentation. |
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