Study On The Preparation And Structure Of Low Allergenicity Parvalbumin From Alaska Pollock Based On Glycation Reaction

Fish is rich in high-quality protein,unsaturated fatty acid and fat soluble vitamin,which has became the main source of high-quality protein intake for Chinese residents.Among them,Alaska Pollock is loved by consumers because of its tender meat and delicate taste.However,as one of the "eight allergens",about 6-8%of children and 2-4%of adults around the world had allergic reactions.At present,there is no particularly effective treatment for fish allergy.Strictly avoiding the intake of fish and its products is the only and most effective way to prevent fish allergy,which will inevitably lead to the lack of nutrients in the body.Therefore,the research on the screening and desensitization of fish allergens is the focus and hotspot at home and abroad.In this paper,parvalbumin(PV)was used as the object to study the desensitization of glycated PV.The antigenicity of glycated PV was used indirect competitive ELISA to study and screen a low antigenicity PV;in order to reveal the mechanism of reducing the antigenicity of glycated PV at the molecular level,the effects of glycation on the primary and advanced structures of PV were studied by spectroscopy,chromatography and mass spectrometry;the antigenicity and structural change of low antigenicity PV in the digestive process were analyzed by simulating the digestive system in vitro to reveal its digestive characteristics,and provide a theoretical reference for the new low antigenicity foods.The specific conclusions are as follows:(1)Study on extraction of PV and the antigenicity of glycated PV.PV and its rabbit anti-PV serum were prepared by ammonium sulfate precipitation and intravenous injection respectively,to prepare PV and rabbit antiserum,analysising the purify of PV,and the specificity and titer of the antiserum.Then the IgE/IgG binding ability was used as the evaluation index to study the antigenicity and structural changes of PV modified by different reducing sugars(glucose,fructose,ribose,lactose,galactose).The results showed that the purity of PV and the titer of rabbit antiserum were both higher;the access of ribose,galactose and lactose can effectively reduce the IgE/IgG binding capacity of PV,among them,the IgE/IgG binding capacity of PV had decreased most after the access of ribose,while the access of glucose and fructose can increase the IgE/IgG binding capacity of PV afer glycation;the primary and advanced structures of PV had changed after covalent binding of different reducing sugars to PV,which indicated that the IgE/IgG binding capacity of PV was changed by modifying its linear and conformational epitopes.(2)Study on the best preparation technology of low antigenicity PV.Taking the IgE-binding ability of PV as an index,the preparation process of low antigenicity PV was optimized by single factor and orthogonal test.The changes of amino acid and apparent structure of the glycated PV obtained under the optimum preparation conditions were analyzed.The results showed that when the mass ratio of PV to ribose was 1.5:1,the pH of buffer was 6.5,and the reaction time was 65 min,the IgE binding capacity of glycated PV was the lowest,which was 31.67%.The lysine conten of low allergenicity parvalbumin(PV glycated under the best preparation process,PV-Gly-U)obtained under this preparation condition had decreased significantly and a cross-linked network structure was formed after glycation.(3)Analysis of the glycation sites of low antigenicity PV.High resolution mass spectrometry was used to analysis the glycation sites,number and reaction degree of low antigenicity PV,and to explore the molecular mechanism of low antigenicity PV by glycation.The results showed that there were six glycation sites in the glycated PV,they are K20,K32,K45,K65,K84 and K97,respectively,which indicated that glycation could reduce the antigenicity of PV by destroying the conformational epitope and modifying the linear epitope of PV.(4)Study on the antigenicity and structural changes of low antigenicity PV during the digestion process in vitro.Taking the IgE/IgG binding ability as the index,to explore the antigenicity and structural changes of glycated PV obtained under the best preparation conditions during the simulated digestion in vitro.The results showed that the digestibility of PV-Gly-U had decreased;the IgE/IgG binding capacity of PV and PV-Gly-U had decreased first and then increased in the process of digestion;they were easy to be degraded under the condition of pepsin and had strong resistance to trypsin;the structure of PV and PV-Gly-U were expanded,and the hydrophobicity of Tyr residues of them had increased and new emission peaks were appeared when after digestion;the peptides with molecular weight less than 500 Da produced by pepsin enzymolysis were regrouped under the action of trypsin,and the Trp residue of digestive products were gradually buried.The results showed that the antigenicity of glycated PV was closely related to the changes of its structure in the process of digestion,it can reduce the antigenicity of glycated PV by modifying and destroying the epitope of protein.The combination of glycation modification and simulation of human gastrointestinal tract digestion can effectively reduce the sensitization of protein.