| China is the largest egg producer and has abundant egg resources.But at present,the degree of deep processing of egg products is low and the degree of high-value product development is insufficient,which seriously restricts the development of high quality and high efficiency of China’s egg processing industry.The application of innovative means to develop egg-derived bioactive peptides is a key initiative to broaden the application prospects of egg derived-products and enhance the economic value of egg-derived functional foods.Probiotic fermentation could change the molecular structure of food-borne proteins,and it has become a new method to prepare food-borne bioactive peptides,but egg white cannot provide the growth environment and energy requirements for probiotics.Therefore,the development of "probioticassistant" heterogeneous protein co-fermentation technology can effectively solve the above bottleneck problems.Meanwhile,the fermented egg source products prepared by heterogeneous protein co-fermentation can combine the functional and nutritional properties of antioxidant,anti-inflammatory and amino acid supplementation,which is suitable for the adjuvant treatment of ulcerative colitis,a global disease.To confirm and deeply analyze the intestinal stabilizing effect of fermented egg source products and investigate its mechanism of action,this study takes the technology of heterogeneous protein co-fermentation for the preparation of functional foods of fermented egg source as the core of the research.Firstly,we carry out the exploration of the process parameters and evaluation of the physicochemical characteristics of fermented custard prepared with the key technology of heterogeneous protein cofermentation,as well as separates and purifies the peptides of fermented custard and identifies the sequences.By constructing an animal model of ulcerative colitis,we preliminarily evaluated the intestinal stabilizing ability of fermented egg milk peptides from the perspectives of clinical symptom recovery,intestinal barrier repair and intestinal bacterial flora regulation,etc.Subsequently,we analyzed the molecular mechanism of the fermented egg milk peptides in intervening in ulcerative colitis from the three dimensions of "active ingredient-disease target-functional network" based on the theory of bioinformatics and the approach of network pharmacology and screened the most potential for intestinal stabilizing.Finally,based on the animal model of ulcerative colitis and proteomics analysis,we evaluated and analyzed the clinical efficacy and molecular mechanism of the fermented egg white peptides FGY and LFR in assisting in the alleviation of ulcerative colitis from the perspectives of clinical symptom restoration,intestinal barrier repair,inflammatory response modulation,and disease target intervention.This study lays a theoretical foundation for the effective development and popularization of egg white-derived bioactive peptides,and provides new ideas for the symptomatic relief and adjuvant treatment of ulcerative colitis.The main research content and key conclusions of this paper are as follows:(1)Using fresh egg white and milk as the main raw materials,and ABY-8 probiotic bacteria as the fermentation strain,we applied the key technology of heterogeneous protein co-fermentation of ‘probiotic-assistant’ technology to prepare fermented egg milk and fermented egg milk peptides,and identified and analyzed the sequence of the fermented egg milk peptide.The composition and content of free amino acids were based on mass spectrometry and high-performance liquid chromatography(HPLC).Then we detected the functional and nutritional properties of fermented egg milk peptides.Functional and nutritional properties of the peptides were confirmed.The fermented egg milk peptides prepared by the key technology of heterogeneous protein co-fermentation had antioxidant activity and amino acid supplementation properties.After in vitro digestion,the total amino acid,free essential amino acid,free Lysine and free Arginine levels of the fermented egg milk peptide increased by 182.10%,91.85%,228.25% and 30.71%,respectively.(2)A mouse model of ulcerative colitis was constructed to evaluate the intestinal stabilizing ability of fermented egg milk from the dimensions of clinical symptom recovery,physical barrier repair,and intestinal flora regulation.Compared with the ulcerative colitis group,the length of the colon was significantly restored after fermented egg milk intervention,the symptoms of inflammatory factor infiltration disappeared,the crypts were structurally intact.The expression of key proteins ZO-1and MUC-2 was uniformly distributed and restored,the secretion level of proinflammatory cytokines(TNF-α,IL-1β,IL-6)was suppressed,and the oxidative damage protection enzymes(CAT,SOD)were restored.Fermented egg milk intervention was also able to improve the structure of intestinal flora in mice with ulcerative colitis,with a decrease in the abundance of Akkermansia and an increase in the abundance of Lactobacillus in the cecum contents of mice after 14 days of intervention.In addition,the levels of short-chain fatty acids such as isobutyric acid,butyric acid,isovaleric acid,valeric acid,isocaproic acid,and caproic acid were significantly restored after the fermented egg milk intervention.(3)Based on bioinformatics theory,we analyzed the functional network and molecular mechanism of the fermented egg milk peptides sequence in regulating the disease targets of ulcerative colitis from the three dimensions of ‘active ingredientdisease target-functional network’.The fermented egg milk peptides can intervene in69 targets related to ulcerative colitis,including ALB,SRC,MPO and GRB.Besides,fermented egg milk peptides intervene in molecular functions such as inflammatory response,response to peptide process,antioxidant biological process,and signaling pathways closely related to inflammation such as MAPK signaling pathway,TNF signaling pathway and Jak-STAT signaling pathway.Meanwhile,the fermented egg milk peptides FGY and LFR have higher potential for intestinal stabilization in the‘fermented egg milk peptides-disease target’ functional network.(4)The intestinal stabilizing effects of the fermented egg milk peptides FGY and LFR were demonstrated with the help of a mouse model of ulcerative colitis.The Results showed that the fermented egg milk peptides FGY and LFR were able to improve the clinical symptoms of ulcerative colitis mice,such as loose feces,anal redness and swelling,and restore their weight loss.Both peptides were able to repair damaged intestinal tissues,restore and improve crypt and cup cell structures,and repair mucin expression and distribution.At the same time,they alleviate the inflammatory response and oxidative damage response process,improve the symptoms of increased intestinal permeability,and reduce the secretion level of tissue growth factors(VEGF,FGF).(5)Based on proteomics analysis technology,the intestinal stabilizing molecular mechanism and functional network of fermented egg milk peptides FGY and LFR were deeply analyzed.The Results showed that a total of 45664 peptides and 4893402 amino acid residues were identified in the sample tissues,among which,7110 peptides matched the data with high protein confidence.Differential protein analysis showed that the expression of phosphatidylinositol 5-phosphate 4-kinase was elevated by 12.75%in the colonic tissues of group M mice,and the expression content of this protein decreased by 3.48% and 76.37% after the intervention of FGY and LFR,respectively.The differential proteins were mainly enriched in protein metabolism function,calcium ion binding,acute inflammatory response,TORC1 signaling pathway regulation of endocytic organelles,membrane-bound organelles,cellular regulatory functions,and protein annotation functions,as well as signaling pathways such as phosphatidylinositol metabolism(map00562),degradation of valine,leucine and isoleucine(map00280),arginine and proline metabolism(map00330),and tyrosine metabolism(map00350). |
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