Supplementary Tables

Supplementary Table 1 Physiological properties of the isolate

Growth temperature / pH / % NaCl
15 °C / 37 °C / 45 °C / 65 °C
15 min / 65 °C
30 min / 70 °C
15 min / 3.5 / 4.0 / 4.5 / 8.6 / 6.5 / 10
+ / ++ / ++ / ++ / - / + / ++ / ++ / ++ / ++ / + / vw

+: Growth (> 4 OD), ++: Luxurious growth (> 8 OD), - : No growth, vw: Very weak growth (< 4 OD), Optical Density measured at 600 nm. Maximum growth of the organism was observed at 37°C.

In addition to the above, strain was characterized by conventional methods like Gram staining, catalase production and different carbohydrate utilization test like Lactose, Xylose, Maltose, Fructose, Dextrose, Galactose, Raffinose, Trehalose, Mellibiose, Sucrose, L-Arabinose, Mannose, Inulin, Sodium gluconate, Glycerol, Salicin, Glucosamine, Dulcitol, Inositol, Sorbitol, Mannitol, Adonitol, α-methyl D-glucoside, Ribose, Rhamnose, Cellobiose, Melezitose, α methyl D- mannoside, Xylitol, D-Arabinose, Citrate, Malonate, Sorbose

The organism was Gram positive catalase negative and it was found to utilize all the carbohydrates except citrate and melonate which are characteristics of lactic acid bacteria. 16 S rRNA sequencing with BLAST (National Centre for Biotechnology Information; http://www.ncbi.nih.gov/) data base confirmed the organism as L. plantarum.

Supplementary Table 2 Probiotic properties of the isolate

Acid tolerance a / Bile tolerance a / Cholesterol assimilation in % b / β-galactosidase activity c
pH 2.0 / % survival / pH 2.5 / % survival / 0.3% bile / % survival / cholesterol / cholesterol + 0.3% bile / +
+ / 80.1 / + / 85.4 / + / 82 / 72.4 / 63.1
Antimicrobial activity d
E.coli ATCC (31705) / E.coli ETEC / Yersinia enterocolitica / Listeria monocytogenes / Salmonella typhi / Bacillus cereus
++ / +++ / +++ / ++ / +++ / +++

a Cells with an optical density of 0.280 (50- fold dilution) at 600 nm were inoculated to sterile de Mann Rogosa Sharpe (MRS) broth of pH 2 , 2.5 for acid tolerance test and 0.3 % bile concentration for bile tolerance test, according to Park et al. (2002). Samples were incubated at 37oC for 4 h for acid tolerance and 12 h for bile tolerance. One ml of sample was serially diluted (6 folds) with sterile saline solution and incubated for 24 h at 37oC. The viable cell population was determined by viable cell count.

b Cholestrol lowering test were carried out according to the method described by Searchy and Bergquist (1960). LAB cultures were cultivated in MRS broth supplemented either with cholesterol or cholesterol with 0.3% (w/v) bile salt. About 500 μl of ethanol containing 10 mg of cholesterol was added to 100 ml of MRS broth with or without bile salt. The cultures were grown for 12–24 h at 37°C. Cells were harvested by centrifugation at 9000 × g for 10 min at 4°C. Spent broth was collected and used for the cholesterol assay.

c The test for the presence of β-galactosidase activity was performed according to Karasova et al. (2002). The isolate was streaked on MRS medium containing 0.01% X-gal and 0.1 mM IPTG as an inducer. Plates were incubated at 40oC for 48 h. Colonies producing β-galactosidase appear blue in color.

d For the detection of antimicrobial activity, an agar spot method was used according to Jacobsen et al. (1999). One µL of inoculum with an optical density of 0.280 (50 fold dilution) at 600 nm was spotted on the surface of the MRS agar. Cell population of the isolate in one µL was in the range of log 10-12. The spotted culture plates were incubated at 37°C for 24 h. The inhibitory effect of MRS agar was tested as negative control. One ml of 12 h culture of indicator pathogens was mixed with 7 mL soft agar (0.7%) and poured on the spotted agar plates. The plates were further incubated at 37 °C for 24 h and zone of inhibition was measured in mm. Zone of inhibition in mm: +, between 0 to 9 mm; ++, between 10-19 mm; +++, >20 mm.

Supplementary Table 3 Antibiotic susceptibility test

Antibiotics / Disc conc. (µg) / Interpretive zone dia
(mm)a
Penicillin / 10 / 22 (I)
Kanamycin / 30 / 0 (R)
Metranidazole / 05 / 0 (R)
Vancomycin / 30 / 0 (R)
Ampicillin / 10 / 17 (S)
Chloramphenicol / 30 / 25 (S)
Streptomycine / 10 / 9 (R)
Co- Trimaxazole / 25 / 0 (R)
Tetracyclin / 30 / 12 (R)
Erythromycine / 15 / 15 (R)
Rifampicin / 05 / 16 (R)
Polymixin B / 300 / 0 (R)

a(R)- Resistance; (I)- Intermediate; (S)- Susceptible.

Antibiotic susceptibility was determined semi quantitatively in accordance to Charteris et al. 2000. 100 µl of active L. plantarum culture was spread over MRS agar plates. Plates were incubated for 2 h at 37°C. Antibiotic discs (Himedia, Mumbai, India) were placed on the above plates and incubated for further 24 h at 37°C. Zone of inhibition was measured and reported as susceptible, intermediate and resistant according to the zone size interpretative chart provided with the kit.

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