| In recent years,food allergy has become one of the public food safety issues that threaten human health,as the incidence of food allergy is increasing worldwide.Food allergy imposes a heavy physical and psychological burden on food allergy sufferers.Besides,sustainable development issues have put demands on new protein resources with clean and high productivity,such as insect proteins,microbial-derived proteins,genetically engineered expressed proteins and other new protein resources or production technologies.But these proteins have allergenic risks,which bring new challenges to food allergy risk management,and allergenicity evaluation will become the basis for ensuring food safety in the future.The current gold standard method for evaluating food allergenicity is the double-blind controlled dietary challenge test with human subjects,which is difficult to operate,and risky.Animal models have been widely used for food allergenicity evaluation,but the existing evaluation index system cannot reflect the mechanistic changes of food allergy,and is difficult to predict the potential allergenic capacity of samples.Improving allergenic risk evaluation models is necessary to introduce novel protein resources with low allergenic risk into people’s diet and to reasonably control safety risks.In this study,a food allergy BALB/c mouse model was constructed using OVA as the positive control.The intestinal flora,serum metabolomic and immunobiochemical indicators were jointly analyzed to improve the allergenicity evaluation system.The established allergenicity evaluation model was applied to evaluate the allergenicity of porcine myoglobin expressed by genetically engineered Komagataella phaffii,providing directions for the optimization of its production purification process and the control of safety risks.The main research contents and results are as follows:(1)A food allergy BALB/c mouse model was successfully constructed by multiple gavage and stimulated by intraperitoneal injection.OVA was given by gavage five times at an interval of 7 days,and BALB/c mice were treated with OVA by intraperitoneal injection 7days after the last gavage.The mice showed allergic symptoms such as scratching nose,erect hair and reduced activity within 60 min after intraperitoneal injection of OVA,and the immune-related biochemical indexes of OVA-specific Ig G,Ig E,HIS and m MCP-1 in serum were significantly increased.Through isolation of active splenic lymphocytes,immunofluorescence staining,flow cytometry sorting and in vitro allergen restimulation,it was found that the splenic lymphocytes of OVA-treated mice showed a balanced differentiation of Th1/Th2 to Th2 type.A Th2 type immune response induced by OVA specifically occurred in OVA-treated mice,indicating that the OVA allergic mouse model was successfully constructed.(2)Through serum metabolomics and intestinal flora detection and analysis and multi-omics combined immunobiochemical index data mining,it was found that the occurrence of OVA allergy was closely associated with changes in intestinal flora and serum metabolome in BALB/c mice.By analyzing the α-diversity and β-diversity between OVA-treated and control mice,it was found that the intestinal flora species abundance was significantly decreased and the community composition characteristics were changed in OVA-allergic mice,and a total of 12 significantly different flora(family level)were identified.Serum metabolome was significantly changed in OVA-allergic mice,and a total of 18 different metabolites were identified(p <0.05,VIP <1,FC >1.5).Correlation analysis between differential intestinal flora,metabolites and immunobiochemical indicators,combined with intestinal function prediction and serum metabolite pathway enrichment analysis,revealed that species of Muribaculaceae,Erysipelotrichaceae,Rikenobacteriaceae,Lactobacillus and Clostridium in the intestine,indole metabolite derivatives and aminoacyl t RNA biosynthetic pathway abundance was associated with the development of food allergy.(3)The developed allergenicity evaluation model was applied to the allergenicity evaluation of porcine myoglobin expressed by Komagataella phaffii.The yeast-expressed porcine myoglobin extract was allergenic,and the allergenicity might be generated by the impurities.The BALB/c mice were given multiple gavage and single intraperitoneal injections of 60% and 88% purity fermented porcine myoglobin,respectively.Both showed allergic symptoms such as scratching of the nose,reduced activity or erect hair,and a significant increase in immune parameters in serum and T cell analysis.But the 60% purity fermented porcine myoglobin-treated mice showed a higher degree of alteration in most of the immunobiochemical parameters than the 88% purity fermented porcine myoglobin.The 60%purity group also showed similar allergy-induced changes in intestinal flora as the OVA-allergic mice,including multiple differences in flora abundance and changes in the abundance of intestinal aminoacyl t RNA synthesis pathway.The 88% purity group only showed a similar trend of changes,and the degree of changes was lower than that of the 60%purity group.Natural porcine hemoglobin in its pure form,which is similar in structure and function to porcine myoglobin,did not exhibit sensitization.(4)Allergen sequence similarity searches and in vitro digestion simulations showed that porcine myoglobin was not allergenic and the allergenic components of the fermented protein extract were impurities.The similarity search of porcine myoglobin sequences in AOL and NCBI databases did not reveal the possibility of cross-reactivity between porcine myoglobin and existing allergens or toxic proteins.The major protein bands in the 60% and 88% pure fermented porcine myoglobin extracts were degraded after in vitro digestion,indicating that porcine myoglobin does not have anti-digestive properties.Combined with the results of mouse model evaluation,it was inferred that the porcine myoglobin produced by yeast expression was not allergenic,and that the allergenicity was produced by impurities,which provided a direction for the optimization of the purification process and safety risk control of microbially expressed porcine myoglobin production. |
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