MATERIALS AND METHODS

Bacterial Strain and Culture Conditions
Lactobacillus plantarum KCA-1 a previously characterized (Anukam et al., 2005) vaginal isolate from a healthy premenopausal woman was employed in this study. Isolation and molecular characterization was done by using Polymerase Chains Reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) at the Canadian Research and Development Centre for Probiotics (CRDC) Gene sequencing was done in the summer of 2004 at Robart’s sequencing facility, University of Western Ontario. The isolate was prepared from the stock culture preserved in glycerol stored at -80°C and inoculated in MRS broth/agar. The preparation was incubated anaerobically using BBL^™ GasPack system (Becton Dickinson, NY, USA).

Bile Tolerance
The ability of Lactabicllus plantarum KCA-1 to grow in the presence of bile salts was examined at CRDC between 2006/2007. Two types of bile were used, namely; porcine bile extract containing glycine and taurine conjugates of hyodeoxycholic acid and other bile salts (Sodium cholate and sodium deoxycholate) and oxgall (BD Difco^TM, USA). Porcine bile salt tolerance was evaluated by preparing different concentrations of the bile (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0%) wt/vol in MRS broth. Lactobacillus plantarum KCA-1 strain (10⁸ cfu mL^-1) that produces biosurfactants was inoculated and incubated anaerobically at 37°C for 48 h. The control was composed of MRS broth without bile salt. Bacterial survival was monitored by measuring the absorbance with a Multiskan Ascent V1.24 (Ascent software version 2.6) at 600 nm. The absorbance values obtained were plotted against the different bile concentrations (%) weight/volume.

For oxgall bile, concentrations ranging from 0, 0.05, 0.1. 0.5, 1.0, 1.5. 2.0, 2.5, 3.0, 3.5 and 4.0% wt/vol were prepared in MRS broth and 100 μL of (10⁸ cfu mL^-1) Lactobacillus plantarum KCA-1 was inoculated and incubated at 37°C for 48 h and absorbance read with the Multiskan Ascent.

To determine the number of surviving cells in the different bile concentrations after 48 h of incubation, 10-fold serial dilutions were made using MRS broth. Ten micro-liter was taken out from each dilution and plated onto MRS agar in duplicates and the plates were incubated anaerobically by using the BBL^™ GasPack system (Becton Dickinson, NY, USA). After incubation of plates at 37°C for 48 h, colonies were counted and results expressed as percentage inhibition with respect to the cell count in the absence of the bile salts. The growth inhibition was calculated as follows:

Where Nmc = No. of viable count in the control MRS broth after 48 h of incubation and Nmb = No. of viable count in MRS broth containing the bile salts after 48 h of incubation.

Acid Tolerance
Acid tolerance of the Lactobacillus plantarum KCA-1 cultures was studied by incubating the organisms in MRS broth at different pH values (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0) adjusted with hydrochloric acid (HCl) and the value determined with digital pH-meter (Φ240 pH/Temp Meter, Beckman, USA) and cultures were incubated at 37°C for 48 h. Bacterial survival in the different pH values was monitored by measuring the absorbance with a Multiskan Ascent V1.24 (Ascent software version 2.6) at 600 nm. The absorbance values obtained were plotted against the different pH values. To determine the number of surviving cells in the different pH preparations after 48 h of incubation, 10-fold serial dilutions were made using MRS broth. Ten microliter was taken out from each dilution and plated onto MRS agar in duplicates and the plates were incubated anaerobically by using the BBL^™ GasPack system (Becton Dickinson, NY, USA).

Acid tolerance was determined by comparing the colony forming unit (CFU mL^-1) on the plates from the absorbance values from the Multiskan.

RESULTS

The ability of Lactobacillus plantarum KCA-1 to grow and survive in the presence of porcine bile and oxgall was examined. After 48 h of incubation, the optical densities (OD) of the medium containing different concentrations of the bile salts were read. Table 1 shows an increase from 0.25 to 0.53 OD₆₀₀ nm^-1 within the porcine bile concentrations tested (0.1 to 1.0%) and there was a corresponding increase in the viable cell counts with a 3 log cycle increase (Data not shown). To determine whether the Lactobacillus strain can survive in high concentrations of bile, the organism was inoculated in oxgall with varying concentrations ranging from 0 to 4.0%. The result in Table 2 indicates that at high concentrations of oxgall bile, growth of the Lactobacillus strain was inhibited. Table 3 shows the corresponding increase in growth inhibition (%) as the concentration of oxgall increases.

Table 1:	Survival of Lactobacillus plantarum KCA-1 in porcine bile salt after 48 h of incubation

Table 2:	Survival of Lactobacillus plantarum KCA-1 in oxgall after 48 h of incubation

Table 3:	Acid tolerance of Lactobacillus plantarum KCA-1 after 24 h of incubation

Table 4:	Percentage growth inhibition of Lactobacillus plantarum KCA-1 by Oxgall Bile

At 2% oxgall concentration, there was a 25% growth inhibition while at a concentration of 4%, a corresponding 68% growth inhibition of the strain was observed.

The ability of Lactobacillus plantarum KCA-1 to survive at different pH values is shown in Table 4. At pH of 2 to 3, the strain exhibited resistance for 24 h (data not shown) with a reduction of only 2 log cycles but between 4 and 6.5 there was an increase in growth as shown by increase in the optical density at 600 nm.

DISCUSSION

The selection criteria for probiotcs have been highlighted by various guidelines including the World Health Organization and Food and Agricultural Organization (WHO/FAO, 2001). Previous study by Anukam and Reid (2007) on this strain presents properties that may allow its use as a biotherapeutic agent for infections of the urogenital tract. However, as the strain looks promising, oral administration portends that the organism will have to traverse the acidic and bile environment of the gastrointestinal tract. This is an important probiotic characteristic as the small intestine contain high concentrations of bile acids which is inhibitory to a large proportion of enteric bacteria (Fernandez et al., 2003). However, there is still no consensus about the precise concentration to which the selected strain should be tolerant. The physiological concentrations of bile acids in the small intestine have been shown to hover between 5000 and 20,000 μM by Hofmann (1991). In this study, there was a stimulatory effect of oxgall bile at 0.1% but the trend seems to decrease as the concentration increases above 1%. At a concentration of 0.5% bile, equivalent to 12,255 μM as shown by Kociubinsky et al. (1999), the lactobacillus strain was still resistant, contrary to 0.15-0.3% that have been used by other investigators (Golden and Gorbach, 1992; Fernandez et al., 2003) of bacterial probiotic characteristics. Lactobacillus plantarum KCA-1 exhibited a high level of tolerance against oxgall bile, thus suggesting that the strain could be a potential probiotic candidate for gastrointestinal disorders. However, at higher concentrations of oxgall bile, the strain started decreasing. This is in line with the recent study of Taranto et al. (2006) demonstrating that at higher concentrations, bile salts induces complete permeabilization of cells, abolish glucose uptake and severely distorts the cell envelope, as shown by electron microscopy. Also detailed analytical studies revealed a change in the phospholipid to glycolipid ratio and also in lipid proportions of the cell membrane. In another study Kurdi et al. (2006) reported that free bile acids at the minimum inhibitory concentration, disturbance of the membrane integrity occurs and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion and other cellular components and eventually cell death. This study has revealed that under high concentration of oxgall bile, Lactobacillus plantarum KCA-1 survives well and this findings supports previous work of Liong and Shah (2005) indicating the survival of Lactobacillus acidophilus at higher concentrations of bile salts.

In addition to bile acid resistance, probiotic microbes are mostly delivered in a food matrix that must pass through the stomach with high pH between 1.5 and 3.0. In this study, Lactobacillus plantarum KCA-1 survived pH 2 and 3 and these values have been suggested to be optimal for probiotic strains (Usman, 1999). Hutt et al. (2006), recently observed the screening of Lactobacillus and Bifidobacterium sp. strains according to their activity in various acid conditions which could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections. The present results from Lactobacillus plantarum KCA-1 have demonstrated a high degree of bile and acid tolerance. Further in vivo studies are now planned to establish whether this strain will be used as a probiotic in conferring health benefits on the host.

ACKNOWLEDGMENT

This study was supported in part by Dr. Gregor Reid, the director, Canadian Research and Development Centre for Probiotics, Lawson Health Research institute, London Ontario, Canada.