Example 5
The Lactobacillus ingested through the oral cavity passes through the stomach with the lower acidity and the intestines with high digestive enzymes and are exposed to low pH of gastric acid, pepsin, intestinal bile salts and digestive enzymes. Therefore, in order to utilize microorganisms as probiotics, gastric juice resistance is essential to survive in low pH and enzymes, and bile juice resistance is essential to survive in extreme intestinal environment. In accordance with the present disclosure, experiments were conducted to identify resistance to artificial gastric juice and bile juice of the above two strains with superior inhibitory effects against Gardnerella vaginalis and Candida albicans. The pH of the gastric juice in the body is maintained at about 3.0, and the food passes through the stomach for about 3 hours. In general, when maintaining viable cell count for 3 hours or more at pH 3, the cells has the high resistance to acidity. In order to identify the intestinal viability of Lactobacillus, survival experiments for artificial gastric juice and artificial bile juice were conducted with reference to Maragkoudakis' method. MG4272 and MG4288 strains were streaked on MRS plate medium and incubated at 37° C. for 24 hours, and the resulting colonies were inoculated in MRS liquid medium and incubated (37° C., 24 hours). Then, 2% passage was incubated for 24 hours in fresh MRS medium. The culture medium was then centrifuged (4,000×g, 4° C., 5 minutes) and washed twice with phosphate-buffer saline (PBS, pH 7.4). The washed cells were adjusted to OD600 1.0 (108 to 109 CFU/mL) and used for resistance experiments to the artificial gastric juice and artificial bile solution, respectively. As a control, 900 μL of pH 7 PBS was added to 100 μL of diluted Lactobacillus and the mixture was shaken and the number of viable cells was measured immediately. In order to identify the resistance to gastric juice, pepsin (Sigma-Aldrich, Saint Louise, USA) was dissolved in 3 g/L of pH 3 to pH 4 PBS to prepare an artificial gastric juice. 100 μL of lactobacillus diluent was added to 900 μL of artificial gastric juice, shaken, and cultured at 37° C. In 3 hours, the viable cell count was measured. To identify resistance to the artificial bile juice, pancreatin (Sigma-Aldrich, Saint Louise, USA) was dissolved in 1 g/L at pH 7 to pH 8 to prepare artificial bile juice. 100 μL of lactobacillus diluent was added to 900 μL of artificial bile juice, shaken and incubated at 37° C. In 4 hours, the viable cell count was measured. The measured results are shown in Table 1 in terms of log CFU/ml.
As shown in Table 1 both strains of MG4272 and MG4288 were identified to maintain the viable cell count of 108 CFU/mL or more after 3 hours at pH 3, thereby identifying excellent acid resistance. In the artificial bile resistance test, both strains of MG4272 and MG4288 were identified to maintain the viable cell count of 108 CFU/mL or more, thereby identifying excellent bile resistance.
