Preparation And In Vitro Evaluation Of Anti-Food Allergic Fermented Soymilk Beverage

Food allergy has become a food safety issue of worldwide concern.In recent years,dietary interventions have begun to be favored by patients with food allergy.Many studies have shown that the imbalance of intestinal flora in the body is one of the important causes of food allergy.Therefore,the main strategy of dietary intervention on food allergy is to regulate the balance of intestinal flora.Prebiotics and probiotics can regulate the balance of intestinal flora and have an important effect on food allergies.Lactobacillus,a class of important probiotics,has attracted much attention for its role in intervening in food allergy.Soybean is rich in nutrients.In addition to protein and fat,soybean also contains oligosaccharides,lecithin and soybean isoflavones,and it is the most nutritious material of plant-based food.Compared with other soy products,soymilk is mainly consumed for breakfast in China,and its market demand is wide and there is a lot of latitude for improvement.Studies have shown that soybean protein can be degraded into active peptides with immunomodulatory functions after fermentation of soymilk by Lactobacillus.Furthermore,oligosaccharides in soybean can promote the metabolism of Lactobacillus to produce short-chain fatty acids（SCFAs）,which can activate relevant immune cells and promote their differentiation,thereby maintaining the body’s immune tolerance to food allergens.In addition,studies also found that the amount of free soy isoflavones in soymilk increased after fermentation,and isoflavones have also been confirmed to have the effect of intervening in food allergies.Based on the anti-allergic effect of Lactobacillus and some anti-allergic active ingredients produced during the fermentation of soymilk,adding prebiotics and other ingredients,through their synergistic effect,it is expected to produce a significant anti-allergic effect.Therefore,in this paper,soymilk was used as the main raw material,fructooligosaccharide（FOS）was added,and Lactobacillus with anti-allergic effect was selected to ferment soymilk.The anti-allergic fermented soymilk beverage was prepared by adjusting and optimizing the process parameters of fermentation.Then the physicochemical indexes,microbial indexes and anti-allergic active ingredients of fermented soymilk beverage were analyzed.Simultaneously,the anti-allergic properties of the digested product of fermented soymilk beverage were evaluated by the inhibition rate of hyaluronidase and the degranulation model of KU812 cells.Through the work of this thesis,it is expected to provide a theoretical basis for the research and development of nutritious and healthy anti-allergy fermented soymilk beverages.The main research methods,results and conclusions are as follows:1.Soymilk was used as the main raw material,and a certain amount of FOS was added,and Lactobacillus acidophilus（CICC 6081）and Lactobacillus plantarum subsp.Plantarum（CICC 20988）were selected and mixed to ferment soymilk.The content of SCFAs,the number of Lactobacillus and p H value were used as evaluation indicators,and the single factor and orthogonal tests were used to optimize the preparation process of fermented soymilk.The results showed that the SCFAs produced by fermented soymilk supplemented with FOS were mainly acetate and butyrate.According to the single factor test results,the optimal soybean-water ratio was 1:6,and the inoculation amount was 4%.In the orthogonal test,the information entropy weighting method was used to calculate the comprehensive score.Finally,the optimized fermentation conditions were obtained as follows:fermentation time was 24 h,fermentation temperature was 34°C,the addition amount of FOS was 4%,and the inoculation ratio was 1:1.The verification results of the orthogonal test showed that in the fermented soymilk,the acetate concentration was 6.69 m M,the butyrate concentration was 0.27m M,the number of Lactobacillus was 1.99×10⁹ CFU/m L,the p H was 4.30,and the comprehensive score was 88.38.To enhance the functionality of the product and adjust the flavor of the product,the optimized fermented soymilk was added with 0.5%GSE,and finally an anti-allergic fermented soymilk beverage was made.2.According to the results of process optimization,the prepared fermented soymilk beverage was taken as the research object.Firstly,the physicochemical indexes and microbial indexes stipulated in GB/T 30885-2014"Vegetable Protein Beverage-Soymilk and Soymilk Beverage"were measured.Then the p H value and acidity of the fermented soymilk beverage,as well as the number of Lactobacillus and p H changes during storage at 4°C were determined.Finally,the content of anti-allergic active ingredients such as total phenols,total flavonoids,acetate,and butyrate were determined.The results showed that the fermented soymilk beverage met the requirements of physicochemical indicators and microbial indicators in the national standard,which means it had good quality,and maintained a relatively stable number of Lactobacillus and p H value within 21 days of cold storage.In the fermented soymilk beverage,the total phenolic and total flavonoid contents were 0.91 mg/m L and 10.37mg/m L,respectively,and the acetate and butyrate contents were 6.69 m M and 0.27m M,respectively.These contents were significantly higher than those in soymilk and fermented soymilk without added anti-allergic active ingredients.3.In vitro simulation of gastrointestinal digestion of fermented soymilk beverage,SDS-PAGE and Tricine-SDS-PAGE were used to analyze the changes of protein molecular weight in fermented soymilk before and after digestion.Then,the anti-allergic effect of fermented soymilk beverage digested product was evaluated in vitro by using the inhibition rate of hyaluronidase and the degranulation model of KU812cells induced by OVA.The results showed that in the fermented soymilk,except for the acidic polypeptide chain of 11S glycinin,other protein peptide chains were degraded to a large extent.After simulated digestion,the protein was further degraded to a molecular weight lower than 15 k Da,soybean peptides with molecular weight less than 5 k Da appeared in the digested products.The inhibition rate of hyaluronidase in the digested product of fermented soymilk beverage was 87.35%,which showed strong anti-allergic effect.The digested product inhibited the degranulation of KU812 cells,mainly manifested as a significant decrease in intracellular Ca²⁺content and a decrease in the release of bioactive mediators including histamine,β-HEX,TNF-αand IL-6.Therefore,the prepared fermented soymilk beverage has an effective anti-food allergy effect.