EFFECTS OF SUPPLEMENTING LACTIC ACID BACTERIA ON FECAL MICROBIOTA, TOTAL CHOLESTEROL,TRIGLYCERIDES AND BILE ACIDS IN RATS.

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Introduction
Cholesterol is the archetypical steroid in mammals.Hyper cholesterol associated with cardiovascular disease is one of the most important causes of death (Yıldız, Öztürk and Aslım 2011, Zhang et al. 2012).Elevated serum cholesterol level is a risk factor for ischemic heart disease and other lethal disease (Watanabe et al. 2013).Reduction in the serum cholesterol has been claimed for lactic acid bacteria (LAB) which are ingredient of several traditional fermented foods and dairy products.LAB have a long history of use in human foods and are naturally present among human gut flora (Szabo et al. 2011).Live microbial feed supplements beneficially affect the host by improving its intestinal microbial balance (Yıldız et al. 2011, Lim 2009).Probiotics (LAB) are normal components of the intestinal micro flora in both humans and animals.
Bile salt is the major route of eliminating cholesterol from the body.Lactobacillus spp were found to excrete bile salt hydrolase (BSH) , the enzyme that catalyzes the hydrolysis of glycine-and taurine-conjugated bile salts into amino acid residues and free bile salts (bile acids) (Liong and Shah 2005) splits glycine or taurine from the steroid moiety resulting in free (de-conjugated) bile salts.Bile-salt hydrolase activity is observed in some strains associated with the gastrointestinal tract (Hyeong-Jun Lim 2004, Zhang et al. 2008).
In a previous study (unpublished), we investigated the cholesterol-lowering effect of lactic acid bacteria screened from Inner Mongolia native meat sausages in China.We defined the cholesterol-lowering effect of Lactobacillus plant arum X 3-2B (L.p X 3-2B )isolated from Inner Mongolia native meat sausages in vitro, and it was considered that the mechanism was incorporation of cholesterol into the L. p X 3-2B .
In this study, to verify our in vitro results and investigate the effectiveness of L. p X 3-2B ingestion in decreasing the risk of atherosclerosis, we investigated the cholesterol-lowering effect of fecal micro biota, triglycerides (TG), total cholesterol (TC) and total bile acid, in rats to further characterize factors affecting and correlation of its ability to hydrolyze cholesterol.

Fecal pH
Fecal samples were collected at 1, 3, 5, 9, and 14 days after treatment.Each 0.1g sample was homogenized in 10ml sterile Saline.The pH of the samples was measured with a pH meter (Wang et al. 2009)

Fecal TC, TG, TBA Concentrations
Fecal samples of 1,3,5,9,14 days respectively were weighed 0.1g and ground to a fine powder, and extracted with a 10-fold weight to volume ratio of saline homogenize at room temperature.The supernatant were then used for the analysis of fecal lipids by TC, TG and TBA commercial assay kits (Huang et al. 2013).

Statistical Analysis
Data analysis was carried out with SPSS Inc. software (version 17.0).A one-way ANOVA was used to check for any significant differences between means with a significance level of P < 0.05 and Pearson's correlation coefficient Test.Critical difference values were used to perform multiple comparisons between means.All data are presented as means with standard deviations.

Fecal Micro Biota
The microbial population compositions of fecal samples in 1, 3, 5, 9, and 14 days respectively after gastric perfusion in different dose, as determined by counting on selective media, are presented in Table 1.There was a trend towards higher counts of lactic acid bacteria in each treated group, compared with the counts in the control, without reaching statistical significance.The counts of Lactobacillus in groups H increased compared with the control group, while the counts of coliform, Enterococcus, and Escherichia coli were reduced.The group of H and M were significantly higher than the control group after intake strain of L. p X 3-2B for 5 days.The micro flora is a complex ecosystem that may vary with intake (Saxami et al. 2012).There was general agreement on the importance of probiotic micro flora in the intestine.
The intestinal micro biota of mammals is complex, numerous and strongly associated with the host's health (Frizzo et al. 2010).The micro biota, by virtue of its metabolic, trophic and protective functions, maintains the integrity of the intestinal barrier for which LAB are critical.LAB as beneficial bacteria normally associated with a balanced normal in the gut flora.Increase in the numbers of lactobacilli can show a normal occurrence in the development of intestinal flora of calves (Bayatkouhsar et al. 2013).In the present study, consumption of lactobacillus by rats not only resulted in increases in fecal numbers of Lactobacillus but also reciprocally suppressed the growth of coliform bacteria, Enterococcus and Escherichia coli.

Fecal pH
There was no significant effect of treatment on fecal pH (Table 2).That was in agreement with previous reports (Bayatkouhsar et al. 2013).
Such a shift of the micro-ecological balance in favor of overall improved population ratios of micro flora may lead to healthier gastrointestinal tract conditions.These results may have been due to the production of high amounts of lactic acid or other organic acids during fermentation of carbohydrate, keeping the fecal pH low.

Fecal Lipid Analysis
Fecal cholesterol and triglyceride concentration of rats fed strain of L. Plant arum X3-2B increased after 5 days compared with the control group (P < 0.05) (Tables 3 and 4).Groups H and M showed higher cholesterol and triglyceride concentrations than did group L, and group D.

Fecal TBA Concentrations
The fecal chloric acid levels differed significantly among the experimental groups (P < 0.05) (Tables 3 and 4).Bile acids are amphipathic molecules synthesized from cholesterol in the liver.Bile acid synthesis is a major pathway for endogenous cholesterol metabolism in humans and other animals.Enhancement of faecal bile acid excretion is important for reducing serum cholesterol levels.We found that the H, M, L groups showed considerable increases in the excretion of cholic acid in their feces.After 14 days of feeding, the maximum fecal cholic acid excretion (87 mmol/L) was recorded in the H group, whereas in the D group, the corresponding increases in fecal cholic acid occurred in male rats.However, after 14 days of feeding, the maximum fecal cholic acid excretion (93 mmol/L) was recorded in the H group in female rats.In another study, both the low and high-dose lactobacilli fed rats showed significantly more increased levels of daily faecal TBA excretion similar to our findings (Guo and Li 2013).

Females showed higher cholesterol
and triglyceride concentrations than males.LAB treatment leads to cholesterol reduction.These findings are in agreement with previous reports (Guo and Li 2013, de Almeida Jackix et al. 2013, Chiu et al. 2005).
a, b, c: different superscripts in the same column differ significantly (p < 0.05).A,B,C: different superscripts in the same horizontal line differ significantly (p < 0.05).C1,C2,C3,C4,C5:Viable counts (log cfu/mL) of Lactic acid bacteria, Bifidobacterium, Enterobacteriaceae, Enterococcus, Escherichia coli.H,M,L,D: High doses group, medium dose group, low-dose group, control group Correlation Net Works among Fecal Micro Biota and Fecal TC, TG, TBA Concentration Correlations among fecal micro biota and fecal TC, TG, TBA concentrations are shown in Fig 1.. LAB indicated positive correlation (r=0.942,p=0.017) with fecal TBA and negative correlation (r=-0.901,p=0.037)with fecal Enterococcus while Bifidobacterium indicated positive correlation (r=0.962,p=0.009)with fecal TG in the H1 group.LAB and Bifidobacterium indicated positive correlation with fecal TC, TG and TBA while indicated negative correlation with fecal Enterobacteriaceae and Enterococcus.Fecal triglyceride, total cholesterol and total bile acid indicated positive correlation within the four groups.

Figure 1 :
Figure 1: Correlation networks among fecal microflora, total cholesterol (TC), total triglyceride (TG) and Total bile acid (TBA) in rat of different lactic acid bacteria.Solid lines indicate C2 correlations and dotted lines indicate C1 correlations.H1: High doses group of Male rat.H2: High dose group of female rat.M1: medium dose group of Male rat.M2: medium dose group of Female rat.L1: low-dose group of Male rat.L2:

Table 1 :
Fecal microbiota in rats fed different doses of L. plantarum X3-2B.Viable counts (log cfu/mL) of Lactic acid bacteria, Bifidobacterium, Coliform, Enterococcus, Escherichia coli.H,M,L,D: High doses group, medium dose group, low-dose group, control group a, b, c: different superscripts in the same column differ significantly (p < 0.05).

Table 2 :
Fecal pH of rats fed different doses of L. plantarum X3-2B on different days.
H,M,L,D: High doses group, medium dose group, low-dose group, control group

Table 4 :
Fecal TC, TG, TBA excretion levels of female rats fed different dose of L. plantarum X3-2B