Screening Of Probiotics With Antagonistic Activities Against Helicobacter Pylori And The Study Of Intervention Mechanisms

Helicobacter pylori may cause stomach diseases such as chronic gastritis, peptic ulcer, and gastric cancer. At present, traditional therapies in the treatment of H. pylori infection have some disadvantages, such as side effects, antibiotic resistance, and high recurrence rate. Several studies reported that lactobacilli have inhibitory effects on H. pylori. Therefore, the purpose of the study was to use the method of food therapy to intervene with the infection of H. pylori, maintain the low level of colonization of H. pylori, and alleviate inflammation caused by H. pylori for the asymptomatic infected or uninfected people. The contents of this research were to screen the probiotics with antagonistic activities against H. pylori, explain the mechanisms of probiotics intervening with the infection of H. pylori, and explore the potential application of the strains in the fermented milk.Thirty-eight Lactobacillus strains were screened for anti- H. pylori activity using in vitro methods, including survivability under the simulated gastric conditions, agar plate diffusion, urease activity, coaggregation, autoaggregation, and hydrocarbon analysis. The results indicated that two Lactobacillus strains showed potential anti- H. pylori activity in vitro, Lactobacillus plantarum 18 (LP18) and L. gasseri Chen (LGChen), respectively. LP18 had the largest zone of inhibition and significantly reduced the urease activity of H. pylori. LGChen had higher coaggregation rate and hydrophobicity than the other strains. The two lactobacilli not only can inhibit the indicator strain- H. pylori SS1, but also can inhibit other strains of H. pylori.The mouse model of H. pylori infection was established, and every C57BL/6 mouse pretreatment with antibiotics was gavaged with H. pylori (300μL at 1×109CFU/mL) and sodium bicarbonate (250μL at 0.2mol/L) for five times. This mouse model was able to obtain high infection rate (80%) and colonization counts of H. pylori (106-107CFU/g tissue).LGG (positive control), LP18, LGChen, LB (common commercial strain) and ST (common commercial strain) were evaluated for intervening with the infection of H. pylori in the C57BL/6 mouse model. The results showed that pretreatment with LGG, LGChen or LP18 and PPI treatment reduced the counts and urease activities of H. pylori in the stomach, and alleviated the inflammation of mice caused by H. pylori infection. Among the tested strains, the group of LP18 showed significant decreasing of H. pylori bacterial density and the group of LGChen showed significant reducing the level of anti- H. pylori - IgG in the serum. Therefore, the effects of LP18 and LGChen on H. pylori infection were better than LGG. However, common commercial strains had no obvious effects. At the same time, infection of H. pylori could increase the levels of ROS and MDA in tissues of liver and stomach as well as ROS in the serum, and reduce the levels of GSH in the tissues of liver and stomach. Pretreatment with LGG, LGChen or LP18 and PPI treatment could reduce the levels of ROS and MDA in tissues of liver and stomach as well as ROS in the serum, increase the levels of GSH in the tissues of liver and stomach. Therefore, the results suggested that inhibition of H. pylori infection with these two strains of lactobacilli in the mice may be related to anti-oxidative activities. Tests of the scavenging rates of DPPH and hydroxyl radical and the reducing activities of LGChen and LP18 were evaluated. The results showed that the scavenging rates of DPPH of LP18 and LGChen were 43% and 62%, scavenging rates of hydroxyl radical were 48% and 18%, and reducing activities were equivalent 62μmol/L and 82μmol/L cysteine, separately. The ICR mouse model was used to evaluate antagonistic abilities of the strains of LP18 and LGChen against H. pylori. The results showed that LGChen and LP18 could reduce the density and urease activities of H. pylori in the stomach, alleviate the inflammation of mice caused by H. pylori infection, increase the levels of GSH and reduce the levels of MDA in the tissues of liver and stomach. However, the strains could not reduce the levels of anti- H. pylori -IgG in the serum.In this study, the antagonistic activities of LGChen and LP18 were assessed by agar plate diffusion assay, and tests that determined the growth and urease activity of H. pylori cocultured with lactobacilli, and the adherence of H. pylori to human gastric epithelial cells in the presence of lactobacilli. The results showed that the Lactobacillus strains had significant anti- H. pylori activity, and this activity may be contributed by the cell-free supernatants (CFS) of lactobacilli and live Lactobacillus strains in vitro. The antagonistic activity of the CFS against H. pylori depended on the pH and the presence of metabolites, such as organic acids and proteases. In the coculture conditions, the cells of lactobacilli and the CFS both could reduce the number and the urease activity of H. pylori. In the adherent assays, the Lactobacillus strains containing CFS, live cells and dead cells could inhibit H. pylori adherence human gastric epithelial cells via the function of exclusion; CFS and live lactobacilli cells could inhibit H. pylori adherence human gastric epithelial cells via the function of competence and replacement, but dead cells had no these functions. In the cell assays also showed that lactobacilli were able to reduce the levels of IL-8 in the human gastric epithelial cells induced by H. pylori.According to the above assays, the mechanisms of LP18 and LGChen intervening with the infection of H. pylori were summarized as follows: when the lactobacilli came in contact with H. pylori, the metabolites of lactobacilli could inhibit the growth and urease activity of H. pylori, and live lactobacilli cells could inhibit the adherence of H. pylori and colonization in the host via steric hindrance, and/or remove H. pylori from the host stomach via co-aggregation; when the lactobacilli did not came in contact with H. pylori, lactobacilli with anti-oxidative activities and reducing the levels of IL-8 induced by H. pylori protected host against oxidative damage caused by H. pylori infection.LP18 and LGChen could not utilize lactose, and their growths in the milk need glucose. LP18 or LGChen could cocluture with commercial starters in the milk with glucose. The complex fermented milks and commercial starters fermented milk had no differences in titration, pH, viscosity, bacterial counts, construct and sensory. Therefore, LP18 and LGChen could be used as potential probiotics with antagonistic activities against H. pylori application in the fermented milk.