Study On Mechanisms And Influncing Factors Of Cholesterol-Lowering Function Of Human Origin Probiotics

Cardiovascular and cerebrovascular diseases are presently the leading causes of death in many countries, and hypercholesterolemia is a major reason leading to such diseases. Medications are the main approach for managing hypercholesterolemia. However, there are intractable side effects of the medication. Probiotics are viable microorganisms that have a beneficial effect to human heath when ingested. Probiotics are generally regarded as safe for human consumption, and therefore, use of probiotics to prevent or control hypercholesterolemia has important significance. In this study, 150 lactic acid bacteria (LAB) were isolated from 27 human fecal samples. The probiotics with potential cholesterol-lowering function were screened from the 150 LAB, and subsequently the in vitro cholesterol-lowering mechanisms, influencing factor of functional properties and in vivo cholesterol-lowering effects of the selected strains were investigated.Based on bile salt deconjugation pathway, 4 LAB (Lactobacillus casei F0822, Lactobacillus casei F0422, Enterococcus faecium F0511 and Enterococcus faecium IN7.12) were screened out. Based on deoxycholic acid (DCA) removal pathway, L. casei F0822 was screened out. L. casei F0822 was a potential cholesterol-lowering probiotic that had stronger bile deconjugation ability as well as a stronger DCA removal ability.The 4 LAB as mentioned above were investigated for the bile salt deconjugation property. The following conclusions were drawn: (1) Bile salts markedly affected bile salt hydrolase (BSH) location of the strains. In the absence of bile salts, BSH of the strains was located in intercellular fractions almost entirely, and in the presence of bile salts, a portion of intercellular BSH was transfered to extracellular broth. (2) Cellular membranes blocked the deconjugation action of intercellular BSH against conjugated bile salts, and thus resulted in that the lysed cells of the strains had stronger bile salt deconjugation ability than the whole cells. (3) The viable cells from L. casei F0822 and L. casei F0422 showed stronger deconjugation activity against glyco-conjugated bile salts, and the viable cells from E. faecium F0511 and E. faecium IN7.12 showed stronger deconjugation activity against tauro-conjugated bile salts.The mechanisms on DCA removal for L. casei F0822 were investigated. It was concluded that the DCA removal action for L. casei F0822 should be attributed to binding of the S-layer protein on DCA. The binding action belongs to hydrogen-bond binding that occurs under hydrophobic action of DCA. Carboxyl of DCA was the main group bound to the S-layer protein. The physicochemical properties of the S-layer protein were determined using modern instruments, and results demonstrated that molecular weight, isoelectric point and thermal denaturation temperature of the S-layer protein were 43 kDa, 9.7 and 78℃, respectively. Moreover, the S-layer protein contained 40.8% hydrophobic amino acids and are absent of cysteine.The effects of tween 80 on functional properties of L. casei F0822 and L.casei F0422 were investigated in vitro. It was found that 0.2% (v/v) tween 80 could significantly (P<0.05) enhance bile salt dedonjugation ability and bile salt tolerance ability of the strains. The mechanism on the tween 80 enhanced bile salt deconjugation ability of the strains was related to the tween 80 enhanced activity of extracellular BSH of the strains. The mechanism on the tween 80 enhanced bile salt tolerance ability of the strains was related to the tween 80 reduction in the degree of intercellular material leakage and enhanced the percentage of intercellular 16:0 free fatty acid to total intercellular free fatty acids.The effects of CaCl2 on functional properties of L. casei F0822 and L. casei F0422 were investigated in vitro. It was found that 0.2% (m/v) tween 80 could significantly (P<0.05) enhance adhesive ability of the strains. For L. casei F0422, such phenomenon was related to the Ca ion mediated adhesive action between lipteichoic acid and the epithelial cell. For L. casei F0422, such phenomenon was related to the Ca ion mediated adhesive action between the S-layer protein and the epithelial cell. The in vivo cholesterol-lowering effects of L. casei F0822 and L. casei F0422were investigated in rats. The results demonstrated that L. casei F0822 could significantly (P<0.05) decrease serum TC, LDL-C and liver TC levels of rats at a low dose(1×108 CFU/mL driking water), while L. casei F0422 could significantly (P<0.05) decrease serum TC, LDL-C and liver TC levels of rats only under a high dose(1×109 CFU/mL driking water). It was the main cholesterol-lowering mechanism for the two strains to increase daily total fecal bile acid excresion of rats.The mixture of 0.6% (v/v) tween 80 and 0.1% (m/v) CaCl2 could significantly (P<0.05) enhance the potential capability of L. casei F0822 and L. casei F0422 to decrease serum TC and LDL-C levels of rats. This finding suggested that such a mixture might have potential as a facilitating agent that helps L. casei F0822 and L. casei F0422 to exert a cholesterol-lowering function.