| Ovotransferrin is one of the four major allergens of egg white,and it can induce type I hypersensitivity in human,leading to threaten seriously the health of egg allergy patients.At present,the study of ovotransferrin mainly focused on functional properties,while the effect on the digetibility and allergenicity are limited to its structure whitout the human body's impact.Food allergy is mainly sensitized by the intestine,and allergen's resistance to gastrointestinal digestion and formation of larger fragment are the prerequisites for allergenicity of intestinal mucosal immunity.In addition,the current allergenicity assessment by digestion stability is based on whether the intact protein degrades within a certain period of time,but does not take into account the digestive residual fragments and their allergenicity.In this work,we aimed to assess the allergenicity of ovotransferrin by the digetibility of ovotransferrin combined with the allergenicity of digestied products.It will help to further understand the allergenic process of ovotransferrin,resulting in improving our understanding of ovotransferrin allergens.Egg white was pretreated by acid precipitation,followed by refing by column chromatography to prepare ovotransferrin with high-purity in this work.Then,the simulated digestion in stomach,small intestinal and small intestinal brush border membrane enzymes of infants and adults was performed in vitro,respectively,and the fragments of digested ovotransferrin were characterized by Tricine-SDS-PAGE,RP-HPLC and MALDI-TOF-MS,The purpose of this study was to elucidate the effect of simulated digestive fluid on the stability of ovotransferrin digestion.Moreover,the specific IgE and IgG binding ability of digested ovotransferrin were tested by ELISA,followed by evaluating the transport of ovotransferrin and IgE binding of the transported products in the Caco-2 cell monolayer model.Furthermore,allerginicity of the digestied ovotransferrin was evaluated in a KU812 cell degranulation model.The main methods,results and conclusions of this work were as follows.In vitro digestion model of infants,ovotransferrin can be digested by simulated gastric fluids,and the digested protein was mainly at about 45 KDa,while a few fragments existed at 3-12 KDa.After further digestion by simulated intestinal fluid,the ovotransferrin was almost degraded with a very weak band in the electrophoresis patterns in Tricine-SDS-PAGE,while the 3-12 KDa fragment had no obvious change.Afterwards,the digested ovotransferrin was further digested by BBM enzyme.It was shown that ovotransferrin was completely degraded into small molecular peptides,and most of the 3-12KDa fragments were further degraded into smaller fragments,indicating the produced components increased and ovotransferrin was more thoroughly digested.However,fragments of more than 3 KDa still existed.When ovotransferrin was digested by simulated gastric fluids in vitro digestion model of adults,it was completely degraded into fragements of 55 KDa,35 KDa and less than 10 KDa.After further digestion by intestinal fluids,the two macromolecular fragements of 55 KDa and 35 KDa were completely degraded,and the fragments below 10 KDa were also further degraded.Moreover,the digested ovotransferrin was further digested by BBM enzyme.It was shown that there was almost no images tesded by Tricine-SDS-PAGE,and all ovotransferrin was digested into fragments below 3 KDa,suggesting that the simulated digestion fluids of adults have better digestion of ovotransferrin than that of infants.The indirect ELISA method was used to determine the binding capacity of IgG and IgE to digested ovotransferrin.It was shown that the specific IgG binding capacity of digested ovotransferrin by simulated fluids of infant stomach,stomach-small intestine,stomach-small intestine-BBM enzyme decreased by 51.14%,66.80% and 77.0%,respectively,and the crossponding specific IgE binding capacity decreased by 34.21%,62.31% and 68.22%,respectively.Regarding to the simulated gastric fluids of adults,the crossponding specific IgG binding capacity decreased by 75.32%,89.20%,and 93.26%,respectively,and the specific IgE binding capacity decreased by 57.27%,77.81%,and 84.59%,respectively.The results suggested that the potential allergenicity of digested ovotransferrin decreased gradually followed the digestive processing,and the allergenicity of the digeted overtransferrin in stimulated gastrointestinal fluids of adults was weaker than in stimulated gastrointestinal fluids of infants when compared the same digestion stage.The transport of ovotransferrin and the digested ovotransferrin was tested in a Caco-2 cell monolayer model,and the apparent permeability coefficient of the adults digested ovotransferrin was Papp>1×10-5 cm/s,indicating a good absorption.The transfer rate of the digested ovotransferrin by the simulated fluids in infant stomach,stomach-small intestine and stomach-small intestine-BBM enzyme within 2h was 5.31%,6.56% and 8.39%,respectively.The specific IgE binding capacity of the transported product of the digested ovotransferrin decreased by 45.59%,69.31% and 84.06%,respectively when compared with the undigested ovotransferrin.Regarding to the corresponding digesting in adults,the transmembrane transport rates of the digested products were 6.77%,9.32%and 9.78%,respectively,and the specific IgE binding capacity of the transported products decreased by 61.64%,78.43% and 86.62%,respectively.The results suggested that the transport rate of the digested ovotransferrin increasd gradually and the potential allergenicity of digested ovotransferrin decreased gradually followed the digestive processing.When comparing the digested ovotransferrin by stimulated gastrointestinal fluids of infants and adults at the same digestion stage,the latter had a higher transmembrane transport rate and lower allergenicity.The allergenicity of the digested ovotransferrin was evaluated by measuring the degranulation and relevant cytokine contents released from the actived basophil KU812 cells.The release contents of ?-HEX,histamine,TNF-?,IL-4,IL-6 and INF-? from KU812 cells induced by ovotransferrin and digested ovotransferrin by simulated fluids of infant stomach,stomach-small intestine,stomach-small intestine-BBM enzyme were 19.09%,14.06%,10.95%and 8.31%;665.13,446.62,374.24 and359.67 ng/mL;15.07 pg/mL,8.96 pg/mL,2.90 pg/mL and 2.07 pg/mL;5.97 pg/mL,2.04 pg/mL,0.95 pg/mL and 0.82 pg/mL;155.92 pg/mL,155.53 pg/mL 154.43pg/mL and 139.08 pg/mL,respectively.Regarding to the same stages of stimulated digestion of adults,the release contents of?-HEX,histamine,TNF-?,IL-4,IL-6 and INF-?from KU812 cells were 13.95%,7.31% and 5.65%;373.70 ng/mL,230.05ng/mL and 224.62 ng/mL;4.29 pg/mL,0.73 pg/mL and 0.51 pg/mL;1.81pg/mL,1.13 pg/mL and 0.64 pg/mL;140.07 pg/mL,129.88 pg/mL and 127.00 pg/mL;3.58 pg/mL,12.96 pg/mL and 13.17 pg/mL,respectively.The results shown that the degranulation ability of digested ovotransferrin decreased gradually followed the digestive processing,indicating the potenticall allergencity decreased gradually.Moreover,the degranulation ability of the digested overtransferrin in stimulated gastrointestinal fluids of adults was weaker than that in stimulated gastrointestinal fluids of infants when compared the same digestion stage.In conclusion,ovotransferrin has anti-digestive ability to some extent,and it is more stable in the stimulated gastrointestinal fluids of infants than in that of adults.The digested ovotransferrin still has strong potential allergenicity,and the allergenicity of the digested overtransferrin by the stimulated gastrointestinal fluids of infants is stronger than by that of adults,suggesting that ovotransferrin is more likely to cause allergies in infants. |
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