Bile Salt Hydrolase Activity Screening and Resistance to the Toxicity of Bile Salt by Indigenous Lactobacillus Isolates of Pakistan; A Research Article
N Ahmed, S Ajmal, S Pervez
bile salt hydrolase, deconjugation, hypocholesterolemia, lactobacillus, precipitation, probiotics.
N Ahmed, S Ajmal, S Pervez. Bile Salt Hydrolase Activity Screening and Resistance to the Toxicity of Bile Salt by Indigenous Lactobacillus Isolates of Pakistan; A Research Article. The Internet Journal of Microbiology. 2009 Volume 8 Number 2.
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
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
MRS (Oxoid) broth and agar were used in all the experiments. Sodium salt of Taurocholate (TCA) and Glycocholic acid (GCA) and Oxgall (Ox) were obtained from Sigma.
Qualitative assay for Deconjugation of bile salts.
Qualitative BSH activity of the isolated strains was evaluated using the procedure described by du Toit et al. (du Toit et al., 1998). Bile salt agar plates were prepared by adding 0.5% of sodium salt of taurocholate and glycocholate to MRS agar. After autoclaving and solidifying, the plates were placed in the GasPak anaerobic jar (Oxoid) for at least 48 h before use. Sterile filter disks were impregnated in an overnight culture of the test strain and placed on MRS agar plates supplemented with 0.5% (wt/vol) taurocholic acid sodium salt (TCA; Sigma), Sodium glycocholic acid (GCA; Sigma). The plates were incubated anaerobically at 37°C for 72 h. BSH activity was present when cholic acid precipitated in the agar medium below and around a colony. The diameters of the precipitation zones were measured. MRS agar plates without supplementation were used as controls. Each strain was tested in triplicate. Subsequently, diameters of the strains that displayed the largest precipitation zones were selected for further study.
Toxicity of conjugated bile salt assay
The isolates were tested for the capacity to resist the bactericidal activity of a conjugated bile salt (TCA) with an assay modified from the assay described by De Smet et al. (De Smet et al., 1998). A stationary-phase culture inoculum (1%) was added to MRS broth supplemented with TCA, GCA and oxgall at a concentration of 0%, 0.3%, 0.5% and 0.6%. At zero time and after 24, 48, and 72 h of anaerobic incubation at 37°C, dilutions of the bacterial suspensions were prepared. Aliquots of the dilutions were smeared onto MRS agar plates, which were then incubated anaerobically at 37°C for 48 h and 72h. Population estimates were made from viable counts.
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
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
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.
Bile Salt Hydrolase (BSH) activity in
Bile Salt Hydrolase (BSH) activity in
Disc Diffusion Assay for Indigenous isolates CMGsaM163 and CMGsaM268 at 0.5 % concentration of bile salts.
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.
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
We thank Dr. Erum Shoaib of Department of Genetics for generous support of this research