【作者】 陈晓华；

【导师】 张灏；

【作者基本信息】 江南大学 ， 食品科学， 2011， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori)感染能引起胃部疾病如胃炎、胃溃疡甚至是胃癌。H. pylori感染率高,在胃中定植会导致胃炎,但是仅有小部分被感染的人会出现临床病状,而大部分无报警症状的患者常被忽视。H. pylori引发的疾病目前主要采用三联法即质子泵抑制剂加两种抗生素的方法治疗,但此疗法存在副作用强烈、易导致抗药性、愈后复发率高等问题。有研究表明益生菌能够辅助治疗H. pylori感染引起的疾病,并能减轻不良反应。因此,本文为了研究利用食疗的方法干预无症状的H. pylori感染或未感染的人群,预防H. pylori的感染或是降低H. pylori的定植量并减轻炎症,筛选具有拮抗H. pylori作用的益生菌,并尝试阐述益生菌干预H. pylori感染的机制,最后研究筛选到的益生菌在乳品中的应用。利用乳酸菌的耐酸性、疏水性、自凝集和交互凝集作用以及抑制H. pylori生长和尿素酶活性试验,筛选到2株具有潜在拮抗H. pylori作用的乳酸菌,分别为Lactobacillus plantarum 18 (LP18)和L. gasseri Chen (LGChen)。LP18具有耐酸性,能抑制H. pylori生长并降低尿素酶活性。LGChen具有抑菌能力及较强的疏水性、自凝集和与H. pylori交互凝集作用。这两株菌除了可以抑制H. pylori SS1生长,还能抑制其他H. pylori菌株的生长。采用C57BL/6小鼠,建立H. pylori感染的小鼠模型。H. pylori感染的小鼠模型:先用抗生素预处理后,再以H. pylori SS1 (300μL 1×109CFU/mL)结合碳酸氢钠(250μL 0.2mol/L)处理每只C57BL/6小鼠,感染5次,6个月造模结束的方式,可以得到感染率比较高(80%),定植量稳定(106-107CFU/g)的H. pylori感染的小鼠模型。利用小鼠模型评价乳酸菌干预H. pylori感染小鼠的效果。乳酸菌分别为商业菌株L. rhamnosus GG(LGG,作为阳性对照菌)、L. bulgaricus 360(LB)和Streptococcus thermophilus TA460(ST)以及本文筛选出乳酸菌LP18和LGChen,并以药物治疗作为阳性对照。动物实验结果表明药物治疗后H. pylori的定植量及胃组织的尿素酶活性降低;乳酸菌(LGG、LP18和LGChen)干预组也能减轻H. pylori引起的炎症,其中LP18干预组可以显著降低H. pylori的定植量,而LGChen干预组显著降低血液中的抗H. pylori- IgG抗体水平,因此综合比较LP18和LGChen干预效果比LGG好;普通商业菌株LB和ST的作用不明显。同时发现H. pylori感染引起血清、肝脏和胃组织中活性氧自由基(ROS)的升高,谷胱甘肽(GSH)的降低以及丙二醛(MDA)的升高。而乳酸菌干预以及药物治疗后,则能够提高胃和肝脏组织的GSH含量同时降低MDA含量。因此,推测乳酸菌可能具有抗氧化作用而保护机体免受H. pylori感染所引起的氧化损伤。测定了LP18和LGChen的体外抗氧化能力,并用ICR小鼠模型再次评价LP18和LGChen干预H. pylori感染的效果。结果表明这两株乳酸菌都具有抗氧化的能力,LP18和LGChen的DPPH清除率分别为43%和62%,羟自由基的清除率分别为48%和18%,还原力分别相当于62μmol/L和82μmol/L的半胱氨酸的还原力。动物实验表明LGChen和LP18可以降低胃中H. pylori的定植量、尿素酶活性并减轻H. pylori引起的炎症,同时可以提高胃和肝组织中的GSH含量并降低MDA的含量,但没有降低血液中的抗H. pylori-IgG抗体水平。通过琼脂扩散法、共培养法研究乳酸菌对H. pylori的生长和尿素酶活性的影响,以及用细胞实验研究乳酸菌对H. pylori粘附人胃上皮细胞以及诱导细胞分泌IL-8的影响,来探讨乳酸菌干预H. pylori感染的体外机制。结果表明乳酸菌活菌和发酵上清液可以抑制H. pylori生长;上清液的抗菌能力依赖于上清液中的代谢产物(如有机酸和蛋白质类似物)并受pH值影响;共培养时,乳酸菌的活菌和发酵上清液都能抑制H. pylori的生长和尿素酶活性。细胞实验表明乳酸菌发酵上清液、活菌和死菌均能够通过排阻作用抑制H. pylori粘附于SGC7901细胞(抑制率达到50%),但是竞争和替代作用只能通过上清液和活菌起作用(抑制率达30-40%),死菌无作用或者作用非常小。同时,乳酸菌可以抑制由H. pylori诱导的SGC7901细胞分泌IL-8。通过抑菌试验和细胞实验以及两种品系的小鼠体内实验,LP18和LGChen干预H. pylori感染的可能机制:当乳酸菌与H. pylori直接接触时,一方面可以利用乳酸菌的代谢物抑制H. pylori的生长和尿素酶活性,从而让H. pylori失去在酸性环境中赖以生存的条件,另一方面可以通过乳酸菌的空间位阻作用阻碍H. pylori的定植,同时通过交互凝集作用把H. pylori共同沉淀排出体外;当乳酸菌与H. pylori没有接触时,乳酸菌可以通过其抗氧化作用和抑制H. pylori诱导上皮细胞分泌IL-8,保护机体避免因H. pylori感染引起的氧化损伤而造成的病变。研究LP18和LGChen在乳品中的应用,结果表明LP18和LGChen不能利用乳糖发酵,需添加葡萄糖方可在牛乳中生长,但LGChen在此条件下比LP18生长好;两株菌的发酵乳在4℃条件下可保存21d,不发生显著性变化;LP18或LGChen分别与商业菌株(LB和ST)复合发酵,对发酵乳不产生不良影响,表明LP18或LGChen可以应用在乳品中,开发新产品。更多还原

【Abstract】 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.更多还原