Study On The Anti-soybean Allergy Effect Of Lactobacillus In Effective Form And Development Of Its Bacterial Agents

Soybean is one of the common allergenic foods,and the wide application of soybean products greatly increased the incidence of soybean allergy to a great extent.However,the research on the prevention and treatment of soy allergies is very limited.Studies have shown that probiotics have important physiological functions such as regulating the immune system,stabilizing the intestinal homeostasis and protecting the intestinal barrier.Therefore,probiotics can play an important role in regulating food allergies.Lactobacillus,an important part of probiotics,has attracted much attention for its anti-allergy effect,its related research mainly focused on the direct use of viable bacteria.Furthermore,studies have shown that the direct use of viable Lactobacillus in people with low immunity,may have serious adverse reactions.Thus,the research on the effective form of anti-allergic Lactobacillus strains and their anti-allergic mechanism,will provide a theoretical basis for the production of safe and efficient anti-allergic Lactobacillus products.Three strains of Lactobacillus have been previously proved to have anti-soybean allergy effect.In this study,one strain with best anti-allergic effect was done selected from the three strains as the model strain by KU812 cell degranulation model.The model strain was divided into three forms,including culture supernatant of bacteria,viable bacteria and inactivated bacteria.Firstly,the effects of different forms of Lactobacillus in intervening cell degranulation were evaluated.The anti-inflammatory ability of different forms of Lactobacillus was indicated by Caco-2 cell inflammation model.Then,Caco-2 cell monolayer model was constructed to test the protective effects of different forms of Lactobacillus on intestinal barrier.Finally,mesenteric lymph nodes(MLN)cells from mice were used to assess the ability of different forms of Lactobacillus to regulate the balance of cytokines related to food allergy,in intestinal mucosal immune tissue.In addition,the media composition and culture conditions of model strains were optimized to developed the preparation of Lactobacillus with superior anti-soybean allergy effect.The main methods,results and conclusions of this study are described as follows:1.The degranulation model of human peripheral blood basophil KU812 was established in vitro,and three strains of Lactobacillus with anti-soybean allergy were selected as the research objects.The effects of different forms of Lactobacillus on the degranulation degree of KU812 cells were analyzed.The results showed that there were significant differences among different strains,and Lactobacillus delbrueckii subsp.bulgaricus(CICC 6103)exhibited a better intervention effect,mainly in inhibiting the release of histamine,IL-6 and TNF-α.Lactobacillus delbrueckii subsp.bulgaricus(CICC 6103)was used as a model strain to preliminary explore the anti-allergic effect.It was found that both viable bacteria and inactivated bacteria could effectively inhibit cell degranulation,and there was no significant difference between the culture supernatant of bacteria and MRS.2.The inflammation model of Caco-2 cells induced by lipopolysaccharide(LPS)was established.The effects of different forms of Lactobacillus delbrueckii subsp.bulgaricus on the inflammation of intestinal epithelial cells were evaluated by the levels of IL-6 and IL-8 secreted by the cells.The results showed that both the viable bacteria and inactivated bacteria could inhibit the increase of IL-6 and IL-8,thus,alleviating the inflammatory response caused by LPS.In addition,the levels of IL-6and IL-8 could be restored to the normal level with the appropriate dose of the viable bacteria or inactivated bacteria,but the culture supernatant of bacteria had no significant effect on the inflammatory response of cells.3.Caco-2 cell monolayer model was used to evaluate the influence of different forms of Lactobacillus delbrueckii subsp.bulgaricus on the integrity and permeability of the intestinal epithelial barrier,in which the transepithelial electric resistance(TEER)of the cell monolayer and the permeability of fluorescein sodium were used as evaluation indicators.The results showed that Lactobacillus delbrueckii subsp.bulgaricus and its components did not cause adverse effect on the intestinal epithelial barrier function.However,in the aspect of protecting the intestinal epithelial barrier function,the culture supernatant of bacteria and viable bacteria of the strain showed better ability to maintain the integrity and permeability of the intestinal epithelial barrier.4.MLN was isolated from mice,and the optimal concentration of soybean protein for immune MLN cells was firstly determined.Then,different forms of Lactobacillus delbrueckii subsp.bulgaricus were co-cultured with soybean protein-immunized MLN cells in vitro.The intervention effect of different forms of Lactobacillus delbrueckii subsp.bulgaricus in MLN on the immune response induced by soybean protein was evaluated,using the level of cytokines as the index.The results showed that 20μg/m L of soybean protein significantly reduced the level of IFN-γand IL-10 in MLN cells,which promoted the development of immune response to Th2.The culture supernatant of bacteria had no significant effect on the immune deviation caused by soybean protein.Low doses of viable bacteria lead to excessive secretion of IFN-γin cells,which fail to improve the balance of IFN-γ/IL-4,and their ability to regulate IL-10 levels is limited.However,inactivated bacteria of the strain could increase the levels of IFN-γand IL-10 to the normal values,and restore the IFN-γ/IL-4 balance,thereby reversing the immune deviation in MLN cells induced by soybean protein.5.To obtain a high density of Lactobacillus delbrueckii subsp.bulgaricus,the carbon sources,nitrogen sources and buffer salt needed for the growth of the strain were optimized on basis of MRS medium.Then,the culture conditions were optimized by response surface methodology.The results showed that the optimized culture medium consisted of glucose as carbon source,yeast extract as nitrogen source and sodium citrate as buffer salt.The optimal culture conditions were 37.6℃,initial p H7.4 and inoculation amount of 5%.Culturing with the optimized medium and culture conditions,the colony number of the strain increased by 23.5%and 23.8%,respectively,and the strain concentration of 2.6×10~9 CFU/m L was obtained.A large number of bacteria obtained from fermentation were collected,and inactivated to make inactivated bacterial powder.Prebiotics were added to complete the development of anti-allergic bacteria.