Introduction

Serum cholesterol is important from the public health because higher concentrations are often associated with a greater risk to cardiovascular diseases. Elevated level of cholesterol in ruminants could possibly due to their increased synthesis to meet the requirements for fat digestion and absorption in the intestine or slower rate of cholesterol disposal (Scott and Cook, 1975). Bile salts are synthesized mainly from cholesterol, conjugated with taurine or glycine in the liver (Batta et al., 1999).

Deconjugated bile acids are excreted more rapidly from the intestinal tract than are conjugated bile salts. While free bile acids are excreted more rapidly from the body, thus the synthesis of new bile salts from cholesterol can reduce the total cholesterol concentration in the body (Gilliland and Speck, 1977) while the enzyme responsible for the deconjugation activity, is bile salt hydrolase (BSH) (Grill et al. 1995). Some natural occurring microorganisms in human intestine are beneficial in terms of lowering cholesterol (Buck and Gilliland, 1994: Fernande, et al. 1987: Fukushima, et al. 1999; Mann and Spoerry, 1974).

It was also reported that Lactobacillus reduces blood cholesterol by direct breakdown of cholesterol and deconjugation of bile salts (Oh and Lee, 2000). Various attempts have shown that species of Lactobacilli were capable of lowering serum cholesterol and reducing the severity of hypercholesterolemia either by lowering total elevated serum cholesterol or reducing low density lipo-proteins LDL.

Therefore, in vitro strain selection for hypercholesterolemic probiotic bacteria can base on bile tolerance and deconjugation of bile salts. This study also aimed to evaluate the potential hypocholesterolemic abilities of indigenous Lactobacillus species.

Material and methods

Media and Chemicals

Bacterial Strains

Qualitative assay for Deconjugation of bile salts.

Toxicity of conjugated bile salt assay

Results and Discussion

All 20 isolates were tested for hydrolase activity of conjugated bile salts GCA and TCA (Table 1).Five of the isolates exhibited GCA hydrolase activity, while six were able to express TCA hydrolase activity. One indigenous isolate CMGsaM135 was able to express hydrolase activity but unable to express TCA hydrolase activity. Conversely, indigenous isolate CMGsa168 and CMGsa212 were positive to produce opaque halo on TCA, unable to produce opaque halo on GCA plates. Opaque halo was considered as positive for Bile hydrolase activity (Table 1). The indigenous isolates were also determined for their resistance to toxicity of conjugated bile salts. Twelve isolates were able to resist the toxicity of GCA and eight were sensitive to GCA toxicity, while in case of conjugated bile salt TCA, nine have shown resistance to TCA toxicity and eleven were sensitive to TCA toxicity (Table 1). Two of the strains CMGsaM163 and CMGsaM268 are able to resist 0.5% GCA and TCA toxicity level for 48 to 72 hours (Table 2a and 2b ) respectively while none of the strains are able to resist 0.6% toxicity of bile salts. Two of the strains CMGsaM163 and CMGsaM268 have also shown opaque halo of bsh in presence of 0.3% TCA.

There are increase evidence that Lactobacilli are able to provide a number of health benefits, including antimicrobial effects against pathogenic bacteria, anti-tumor effects, and protection against antibiotic-associated diarrhea or food allergy (Saxelin 1997; Orrhage and Nord 2000; Cummings and others 2001). Lactobacilli are reported to be acid and bile tolerant and are able to survive in the gastrointestinal tract. It was hypothesized that high BSH deconjugation activity associated with the stationary phase of culture was a result of reduced pH levels in the medium (Corzo & Gilliland, 1999). Eyssen has demonstrated that the increased excretion of bile salts can increase catabolism of cholesterol to bile acids more rapidly (Eyssen. 1973).

The ability of a specific probiotic strain to survive and reproduce in the hostile environment of the gut is the most relevant feature to be checked during the selection procedures, but production of metabolites resulting from effect of bile salts on bacterial cells is unknown (Mallory, et al. 1973), while in vivo testing is time-consuming and expensive. In vitro selection is therefore the first approach used to select few strains that can be further evaluated in vivo.

Nevertheless, results achieved are positive and promising. The development of new probiotic products has produced new scientific achievements and a strong demand for improved and scientifically-based selection criteria.

Figure 1

TABLE 1: Resistance and Hydrolase activity for conjugative bile salts GCA and TCA.

Figure 2

Figure 1a:

Bile Salt Hydrolase (BSH) activity in Lactobacillus strain CMGsaM163 in presence of 0.3% TCA

Figure 3

Figure 1b:

Figure 4

Figure2:

Bile Salt Hydrolase (BSH) activity in Lactobacillus strain CMGsaM268 in presence of 0.3% TCA

Disc Diffusion Assay for Indigenous isolates CMGsaM163 and CMGsaM268 at 0.5 % concentration of bile salts.

Figure 5

Figure 3:

Toxic effect of various bile salts (0.5%gm of Glycocholic acid, Taurocholic acid, Oxgall, Cholic acid) on indigenous isolates CMGsaM163 and CMGsaM268, after 48h of incubation. Each bar represents average of three.

Conclusion

In general, probiotic strain must colonize in GTI of host and must have acid, bile salt – tolerance (Du Toit, et al. 1998; Gilliland, et al. 1985). And from present result, it was suggested that the finally indigenous isolated Lactobacillus strains had an excellent hypocholesterolemic effects. They will be used as a Probiotics to prevent hypercholesterolemia from human health while the mechanisms of regulating serum cholesterol and the effect on serum cholesterol level in vivo needs further extensive investigations

Acknowledgment

We thank Dr. Erum Shoaib of Department of Genetics for generous support of this research