Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

BACKGROUND:Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt. METHODS AND FINDINGS:An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5-2.0 and toxicity of 1.5-2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of approximately 37% and autoaggregation of approximately 31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4+/-1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus. CONCLUSION:The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals

Antibacterial activity of Lp9 isolate against various pathogens.

a<p>Diameter of the well −7 mm.</p>b<p>The values shown are mean of three replicates.</p

Amplification of <i>mub</i> and <i>fbp</i> genes by PCR.

<p>Genomic DNA of respective strain was used as template DNA and primer sets used in the PCR assay are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008099#pone-0008099-t001" target="_blank">Table 1</a>. Lane 1–500 bp ladder, lane 2 – <i>MUB</i> domain of Lp9 (425 bp and 1050 bp), lane 3 – <i>MUB</i> domain of <i>L. plantarum</i> CSCC5276, lane 4 – N-terminal domain of <i>mub</i> in Lp9 (1.6 kbp), lane 5 – N-terminal domain of <i>mub</i> in <i>L. plantarum</i> CSCC5276, lane 6 – <i>fbp</i> in Lp9 (1.5 kbp), lane 7 – <i>fbp</i> in <i>L. plantarum</i> CSCC5276.</p

Antioxidative activities of Lp9 and other probiotics.

<p>The free radical scavenging activity measured by ABTS method in the extracellular (open) and intracellular (solid) extracts of the Lp9 isolate (circle), <i>L. johnsonii</i> LA1 (triangle) and <i>L. acidophilus</i> LA7 (square). Phosphate buffer saline (dash line) and MRS broth (dotted line) were used as controls for intracellular and extracellular extracts, respectively. The solid continuous curves are the non-linear least-square fit of first order kinetic model given by equation 4 to the experimental data.</p

Identification of Lp9 isolate by molecular-typing methods.

<p>Molecular typing of Lp9 isolate by using LbLMA1/R161genus-specific (lane 1) and species-specific primers (lane 2) and <i>L. plantarum</i> specific primers for amplification of full length 975 bp <i>bsh</i> gene (lane 3) and partial 231 bp <i>bsh</i> gene (lane 4) by PCR. Lane 5–100 bp DNA ladder.</p

List of primer pairs used in PCR amplification.

<p>List of primer pairs used in PCR amplification.</p

Cell surface hydrophobicity of Lp9 and other probiotic cultures.

a<p>All the values are shown as mean±sd (standard deviation) of 3–5 replicate experiments.</p

The kinetic parameters for antioxidative potential of Lp9 isolate of <i>L. plantarum</i> and other probiotic <i>Lactobacillus</i> species.

a<p>The values given for extracellular milieu also include contribution from MRS broth also. For MRS as a blank, <i>k</i>_obs =<i>k</i>_mrs, while contribution of cultures as <i>k</i>_cul or reaction halftime (T_1/2 = 0.69/<i>k</i>_cul) has been deascribed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008099#s2" target="_blank">results</a> section.</p>b<p>All the values are shown as mean±sd of three replicate experiments.</p