Molecular cloning, characterization and heterologous expression of bile salt hydrolase (Bsh) from Lactobacillus fermentum NCDO394.

Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.

Probiotic Lactobacillus rhamnosus GG and Aloe vera gel improve lipid profiles in hypercholesterolemic rats.

OBJECTIVE: The effects of Lactobacillus rhamnosus GG (LGG) and Aloe vera (AV) gel on lipid profiles in rats with induced hypercholesterolemia were studied. METHODS: Five treatment groups of rats (n = 7) were the fed experimental diets: a normal control diet, a hypercholesterolemic diet (HD), HD + LGG, HD + AV gel, and HD + LGG + AV gel. RESULTS: Supplementation with LGG decreased serum total cholesterol by 32%; however, in combination with AV, the decrease was 43%. The decreases in triacylglycerol levels in the HD + LGG, HD + AV, and HD + LGG + AV groups were 41%, 23% and 45%, respectively. High-density lipoprotein increased by 12% in the HD + LGG + AV group, whereas very low-density and low-density lipoprotein values decreased by 45% and 30%, respectively. The atherogenic index in the HD + LGG + AV group decreased to 2.45 from 4.77 in the HD + LGG group. Furthermore, fecal Lactobacillus species counts increased significantly when LGG was fed in combination with the AV gel. The oral administration of LGG fermented milk alone or in combination with the AV gel increased cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase expression) and absorption (low-density lipoprotein receptor expression), whereas cholesterol 7α-hydroxylase mRNA expression levels were lower in the HD + LGG and HD + LGG + AV groups compared with the control HD group. CONCLUSION: The combination of LGG and AV gel may have a therapeutic potential to decrease cholesterol levels and the risk of cardiovascular diseases.