Study On Potential Allergenicity And Allergenic Mechanism Of Moringa Oleifera Leaf Proteins

Food allergy has become one of the food safety issues of widespread concern worldwide.With the improvement of global logistics,the introduction of new foods in various countries,and the rapid development of agricultural technology and food processing technology,new proteins are constantly introduced into food.Assessing the potential sensitization of new foods is of great significance for ensuring food safety.Moringa oleifera leaf is a new resource food in China,gradually becoming an important raw material for food processing in China.However,its potential sensitization has rarely been reported and needs further study.This paper takes moringa oleifera leaf as the research object,prepares moringa oleifera leaf proteins(MLP)and optimizes the extraction method and process.The structural characteristics of MLP are studied through amino acid analysis,infrared,circular dichroism,mass spectrometry,SDS-PAGE,etc;Secondly,a BALB/c mouse allergy model was used to detect a series of active substances related to allergies and evaluate the allergenicity of MLP;Then,the changes in different immune cells and cytokines in primary splenic cells were measured to further evaluate the sensitization of MLP,including its impact on immune cells such as dendritic cells,Th cells,and subpopulations;Finally,based on gut microbiota and metabolomics,the sensitization mechanism of moringa oleifera leaf was studied.The main methods,results,and conclusions of the study are as follows:1.With the preparation of MLP as the goal,a specific method for extracting horseradish protein by salting out was optimized and determined.Response surface methodology was used to optimize the extraction conditions.The Na Cl concentration was determined to be 0.177 mol/L,the liquid-solid ratio was 12:1,and the salting out time was 1.5 hours,providing a stable MLP for subsequent experiments.Using infrared,circular dichroism,mass spectrometry,SDS-PAGE to characterize MLP and perform amino acid analysis,it was found that the molecular weight of MLP is mainly distributed in the range of 30-120 k Da,consisting of 22 amino acids(including 8essential amino acids for the human body),a class of proteins with a complete triple helix structure.Through acute oral toxicity testing,MLP was determined to be nontoxic,ruling out the possibility of toxic reactions in sensitization assessment.2.Taking MLP as an object,the food sensitization and stimulation model of BALB/c mice was constructed,and the potential sensitization of MLP was evaluated through humoral immunity,cellular immunity and Effector cell expression.The results showed that compared with the control group,the body temperature of mice in the MLP high-dose group and low-dose group significantly decreased by 0.93 ℃(p<0.0001)and0.21 ℃(p<0.05),the score of systemic allergic symptoms increased by 98.43% and85.71% respectively,the score of stool diarrhea increased to 2.0 and 2.5(the score of the normal group was 0.4),and the average number of mast cell in the intestinal mucosa increased from 4.5/20 fields of the normal group to 13.5-18/20 fields,The serum histamine content increased from normal 443.5 ng/m L to 609.3 ng/m L and 528.1ng/m L,and the serum specific antibody s Ig E significantly increased(p<0.05),while the specific s Ig G1 showed an extremely significant increase(p<0.0001);IFN in mouse serum-γ、 IL-4,IL-13,IL-17 A,and IL-23 were significantly increased compared to the control group.In addition,MLP sensitized and stimulated mice to produce typical allergic clinical manifestations of hypothermia,allergic symptoms and increased diarrhea,which may be closely related to MLP’s ability to strongly activate the expression of allergic Effector cell mast cell in the intestinal mucosa and enhance the release of histamine,the allergic mediator;At the same time,MLP increased the expression of specific antibodies(s Ig E and s Ig G1)related to Th2 in serum,and promoted the secretion of corresponding cytokines in Th2 cells and Th17 cells in mouse serum.Based on various allergenic indicators,it can be concluded that MLP can induce systemic immune responses in mice,inducing allergic reactions to shift towards the Th2 direction,indicating the potential of MLP for sensitization.In addition,through the analysis of intestinal histopathology,it was found that the villi of jejunum and ileum in MLP group were destroyed and disappeared,the glands were arranged irregularly,accompanied by inflammatory cell infiltration,but the colon tissue did not show the above differences,indicating that MLP may play a sensitization potential by destroying the integrity of jejunum and ileum tissue,initiating immune stress response.3.By detecting the changes in different immune cells and cytokines secreted by primary spleen cells stimulated by MLP in vitro,the effect and mechanism of MLP sensitization on cellular immunity were explored.The results showed that the ratio of CD11C+MCH II+(dendritic cells),CD3+CD4+(helper T cells),and CD3+CD4+IL-17A+(Th17)cells in spleen cells stimulated by MLP was significantly increased,while the ratio of CD4+CD25+Foxp3+(regulatory T cells)cells was significantly decreased,and the ratio of Treg/Th17 cells was significantly reduced.The expression levels of cytokines IL-4,IL-9,and IL-17 A in the supernatant after MLP stimulation were significantly increased(p<0.01)using ELISA method.The results indicate that MLP may increase the expression of dendritic cells and helper T cells,inhibit the expression of Tregs cells,promote the differentiation of T cells into Th2 and Th17 cell subpopulations,and secretion of cytokines,thereby inducing the cellular immune system to shift towards Th2 and Th17 directions,resulting in sensitization.4.By sequencing and analyzing the intestinal microflora of allergic mice,we compared the characteristic microflora changes of MLP allergy,and explored the impact of mlp sensitization on intestinal microflora.The results showed that MLP allergic mice had only 22 unique OTUs(approximately 0.19%)compared to control mice.The diversity was analyzed using the Chao1 index,ACE index,Simpson index,and Shannon index,and there was no significant difference.However,through Po CA and OPLS-DA model analysis,there was a significant separation between the CON group and the MLP group.At the phylum level of the community composition,MLP sensitized mouse Proteobacteria and Actinobacteria significantly increased,Desulfobacteria and Spirochetes significantly decreased,and there was a significant difference in the abundance of 15 genera at the genus level,including Bacteroides,Staphylococcus,Eight genera of bacteria,including Corynebacterium,Sporosarcina,Atopostipes,Pseudogramicibacillus,Facklamia,and Yaniella,increased significantly in the MLP group,norank＿f＿＿Desulfovibrionaceae,Desulfovibrio,Lachnospiraceae＿UCG-006,Treponema,Lachnoclostridium,Eubacterium＿xylanophilum＿Group,GCA-900066575 and other seven bacterial genera decreased significantly.The above results indicate that although MLP does not change the population and diversity of intestinal microorganisms,it significantly changes the microbial structure and community composition.Using LEf Se analysis,it was found that a total of 65 species taxons with LDA score >2 and p<0.05 were selected that had a major impact on the species differences between the two groups of flora.According to LDA≥3.50,the taxons significantly enriched in the MLP sensitized mouse flora and produced key distinguishing effects were Actinobacteriota(LDA=3.98,p<0.05)and Corynebacterium(LDA=3.96,p<0.05),The bacteria of Desulfobacterota(LDA=4.07,p<0.01),Spirochaeta(LDA=3.56,p<0.01),Desulfovibrio(LDA=3.58,p<0.05),and Norankin normal mice＿f＿＿Desulfovibrionacea(LDA=3.90,p<0.01),Treponema(LDA=3.56,p<0.05),and other potential biomarkers can be used to distinguish MLP allergy from normal mice.Prediction of intestinal microflora function using PICRSt method,Fc γ R-mediated phagocytosis,Gn RH signaling pathway,endocrine cell proliferation,biosynthesis of type II polyketone products,caffeine metabolism,and steroid biosynthesis are significantly affected.Using Bug Base phenotype prediction,it was found that the biofilm formation of intestinal microflora in MLP sensitized mice was significantly inhibited.Through Spearman correlation analysis with Th2 cytokine and bacterial abundance,it was found that the abundance of dysfunctional bacterial species such as Facklamia,Staphylococcus,Atopostipes,Pseudomonas was significantly correlated with the level of Th2 cytokine in mouse serum(p<0.05),indicating that the above four bacterial species were all thick-walled bacteria,suggesting that the key bacteria associated with Th2 cytokine allergy to MLP might be bacterial thick-walled bacteria.5.High throughput sequencing was used to compare the differences in intestinal metabolites between mice sensitized with MLP and normal mice,explore the differences in metabolic profiles,screen out potential biomarkers related to allergic reactions,and then analyze their metabolic pathways to explore the metabolic mechanism of allergic reactions.The results showed that the metabolites of MLP mice were different from those of normal mice,and 18 different fecal metabolites were selected,mainly steroids and steroid derivatives,fatty acyls,organic sulfuric acids and their derivatives,as well as fat-soluble vitamins,hydroxyacids and their derivatives.The differential metabolites in feces are mainly concentrated in the biosynthesis of unsaturated fatty acids,fatty acid synthesis,longevity regulation pathways,and worm metabolism pathways,which are closely related to indicators related to allergic reactions.Six candidate biomarkers were selected,including DL-2-Aminooctanoic acid,3-Hydroxymethylpyridine,5-Methylthioribose,Pregnan-20-one17-acetyloxy-3-hydroxy-6-methyl-3b5b6a-,Hippuric acid,Goshuyic acid.Through the study of their association with different bacteria and allergy related parameters,it was further shown that the candidate biomarkers Hippuric acid were associated with different bacteria,allergy symptom scores,specific antibodies(s Ig E and s Ig G1),and Th2 Several important allergy indices such as Th17 related cytokines are positively correlated,while the remaining five candidate biomarkers are negatively correlated,indicating that the occurrence and development of MLP allergy is related to changes in differential metabolites,as well as closely related to intestinal microflora in feces.The six potential biomarkers can distinguish sensitized mice from normal mice,with high specificity,and may be potential non-invasive biomarkers for predicting MLP food allergy.Hippuric acid may be the best potential biomarker for positive correlation.These results illustrated that intestinal microflora may affect the pathogenesis of food allergy by regulating host metabolism.In summary,this study successfully constructed a BALB/c mouse sensitization model of MLP,scientifically evaluated its potential sensitization,preliminarily revealed its sensitization mechanism,and provided partial safety assessment data for the safe consumption of MLP.