Liong, Min Tze (2006) In-Vivo and In-Vitro Cholesterol Removal by Lactobacilli and Bifidobacteria. PhD thesis, Victoria University.

Abstract

Four strains of Lactobacillus acidophilus, eleven strains of L. casei and five strains of Bifidobacterium were studied for their acid and bile tolerance. Possible mechanisms of cholesterol removal were also examined. All strains showed varying levels of tolerance at pH 2.0 for two hours. L. acidophilus ATCC 4962, L. casei ASCC 290, L. casei ASCC 292, B. longum BB536 and B. infantis 17930 were more acid tolerant than the other strains studied. All strains could tolerate the presence of bile, with greater tolerance to cholic acid and oxgall, however the strains showed inhibition by taurocholic acid. All strains were able to assimilate cholesterol at varying levels ranging from 4.17 to 32.25 ìg/mL. Cholesterol assimilation patterns suggested that its removal was associated with growth of the organisms. Cholesterol removed by dead- and resting cells ranged from 0.79 to 3.82 mg/g of dry weight, indicating the possibility of removal of cholesterol via binging to cellular surface. Fatty acid methyl esters, as quantified using gas chromatography, showed changes of hexadecanoic-, octadecanoic-, total saturated-, and unsaturated acids in cells grown in the presence of cholesterol compared with those grown without cholesterol, suggesting that cholesterol from the medium was incorporated into the cellular membrane. Similar strains of Lactobacillus and Bifidobacterium were also evaluated for their bile salt deconjugation ability, bile salt hydrolase activity (BSH) and co-precipitation of cholesterol with deconjugated bile. Results showed that more cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate. BSH activity indicated that substrate specificity was also more towards glycine-conjugated bile compared to taurineconjugated bile. Most strains of lactobacilli and bifidobacteria exhibited highest total BSH activity (1.60-1.99 U/mL for lactobacilli and 0.87-1.39 U/mL for bifidobacteria) in bile salt mixture (2.8 mM sodium glucocholate and 1.2 mM sodium taurocholate) than individual conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both conjugated bile, and increased rapidly with decreasing pH levels below 5.0, with maximum co-precipitation at pH 1.0. Based on these preliminary experiments, we selected the more acid and bile tolerant strains (tolerated pH 2.0 for more than 120 mins and 0.3% bile) with best cholesterol removal ability from each of L. acidophilus, L. casei and Bifidobacterium for subsequent optimization. L. casei ASCC 292, L. acidophilus ATCC 4962 and B. infantis ATCC 17930 were grown in the presence of six prebiotics, namely, sorbitol, mannitol, maltodextrin, highamylose maize, fructooligosaccharide (FOS), and inulin, in order to determine the best combination of inoculum size (probiotic) and best concentration of prebiotics for removing the highest level of cholesterol. First-order models showed that the combination of L. casei ASCC 292, FOS, and maltodextrin was the most efficient for the removal of cholesterol, while L. acidophilus ATCC 4962 in the presence of mannitol, FOS and inulin was best for cholesterol removal. B. infantis ATCC 17930 in combination with sorbitol, inulin and maltodextrin were significant for removal of cholesterol (P < 0.05). Optimum experimental regions were developed using the steepest ascent. Subsequent second-order polynomial regression model estimated that the optimum condition of the factors for cholesterol removal by L. casei ASCC 292 was 1.71% (wt/vol) inoculum size, 4.95% (wt/vol) FOS, and 6.62% (wt/vol) maltodextrin, while that for L. acidophilus ATCC 4962 were 2.64% w/v inoculum size, 4.13% w/v mannitol, 3.29% w/v FOS and 5.81% w/v inulin. Similarly, optimum cholesterol removal (52.18 ìg/mL) was achieved from inoculum size of B. infantis ATCC 17930 at 2.70% (w/v), sorbitol at 6.30% (w/v), maltodextrin at 4.60% (w/v) and inulin at 8.60% (w/v). Validation experiments showed that the response surface method was reliable for developing the model, for optimizing factors, and for analysing interaction effects. Analyses of growth, substrate utilization, growth yield, mean doubling time, and short-chain fatty acid (SCFA) production by the use of quadratic models indicated that cholesterol removal was growth associated and was encouraged by higher growth and substrate utilization rates. The production of organic acids also appeared to be growth associated and highly influenced by the types and concentrations of prebiotics. In addition, the production of lactic and acetic acids was relatively sensitive to the end-product fermentation of maltodextrin, sorbitol, FOS and inulin. Increased production of lactic acid also showed cessation of growth of the organisms, indicating inhibition of growth at high concentration of lactic acid (approximately beyond 109 mmol/L). Increased concentration of FOS contributed to the increased production of propionic acid, while mannitol and maltodextrin exhibited a positive correlation on the production of formic acid. We subsequently evaluated the hypocholesterolemic effects of the optimized synbiotics using rats kept on a high-cholesterol diet (1% cholesterol). L. casei ASCC 292, L. acidophilus ATCC 4962 and B. infantis ATCC 17930 were evaluated in the presence of all prebiotics combined as well as with individual prebiotics. Three synbiotics were given to male Wistar rats (n = 6) including L. casei ASCC 292 and fructooligosaccharides (FOS) (LF), L. casei ASCC 292 and maltodextrin (LM) and L. casei ASCC 292, FOS and maltodextrin (LFM). The control group had no probiotic or prebiotics. The effect of the synbiotic on intestinal microflora and concentration of organic acids was also investigated. The spleen, liver and kidney were analysed to determine the presence of lactobacilli which may indicate translocational property of the probiotic. The LFM diet lowered serum total cholesterol and triglycerides levels, while the LM diet increased serum HDL-cholesterol level. The LFM diet decreased the population of staphylococci, bacteroides, E. coli and total coliforms in most bowel segments, possibly contributed by increased concentration of lactic acid in those segments. Similarly, four diets namely L. acidophilus ATCC 4962 and fructooligosaccharide (LF), L. acidophilus ATCC 4962 and mannitol (LM), L. acidophilus ATCC 4962 and inulin (LI) and L. acidophilus ATCC 4962 and all three prebiotics (LFMI) were evaluated in rats. The LFMI diet reduced serum total cholesterol, triglycerides and low-density lipoprotein (LDL) cholesterol levels by 32.40%, 32.51% and 42.95%, respectively. The LI diet decreased the pH values in the intestines resulting in decreased population of total aerobes, staphylococci, Escherichia coli, coliforms and bacteroides. Rats were also given diets containing B. infantis ATCC 17930 namely B. infantis and sorbitol (BS), B. infantis and maltodextrin (BM), B. infantis and inulin (BI), and B. infantis and all three prebiotics (BSMI). Rats on the BM diet decreased total serum cholesterol, triglycerides and low-density lipoprotein (LDL) cholesterol level compared to the control, possibly due to increased production of propionic acid. Diet BS, BM and BI increased the population of Bifidobacterium in the cecum and colon, accompanied by increased concentration of acetic acid. These led to decreased counts of total aerobes, Escherichia coli and bacteroides in those intestinal segments. There was no Lactobacillus and Bifidobacterium detected in the spleen, liver and kidney of all synbiotics studied, indicating no occurrence of translocation. The synbiotic containing L. acidophilus ATCC 4962, mannitol, FOS and inulin showed the most promising hypocholesterolemic effects in rats, and thus was used for further evaluation on plasma lipid profiles and red blood cell (RBC) membrane properties in hypercholesterolemic pigs. Twenty four White male Landrace pigs were randomly allocated to four treatment groups for 8 weeks (n = 6). Treatment factors were the supplementation of synbiotic (with and without) and dietary fat (5% and 15%). Plasma lipoprotein profiles were measured using commercial assay kits while RBC properties were studied using Wright’s stain, commercial kits and fluorescent anisotropy. The supplementation of synbiotic reduced serum total cholesterol (P = 0.001), triacylglycerol (P = 0.002) and LDL-cholesterol (P = 0.045) for both dietary fat. Although pigs given the high-fat diet remained hypercholesterolemic after the experimental period, pigs on the lowfat diet supplemented with the synbiotic achieved normal levels after 8 weeks, while those on the control diet remained hypercholesterolemic. A higher concentration of esterifiedcholesterol in HDL of pigs supplemented with synbiotic than the control regardless of dietary fat (P = 0.036) indicated that cholesterol was reduced in the form of cholesterylesters (CE). Reduced concentration of CE (P < 0.001) and increased concentration of triacylglycerol (P = 0.042) in LDL of pigs on synbiotic suggested that LDL-cholesterol was reduced via the hydrolysis of smaller and denser LDL particles. The deformity in RBC of pigs on the high-fat diet was more prevalent that those given the low-fat diet. Despite this, pigs without synbiotic showed higher occurrence of spur cells than the pigs given synbiotic, as supported by the higher cholesterol:phospholipid ratio in RBC (P = 0.001). Also, fluorescence anisotropy that targeted the apolar, polar and interfacial regions of phospholipids in RBC of pigs was significantly lower (P < 0.001) when pigs were supplemented with synbiotic compared to those without supplementation for both dietary fat content, indicating reduced membrane rigidity and improved fluidity.

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