Identification Of Major Allergens In Almonds And Their Desensitization Methods

In recent years,food allergies have received widespread attention in the public health sector around the world.Among them,allergies caused by almonds in tree nuts account for a high proportion.Almonds are seeds of the genus Rosaceae or apricot,which are rich in various nutrients and are often used as food ingredients,thus increasing the chance of almond allergy.This paper mainly studies the molecular characteristics of almond allergen protein,identifies almond allergen protein,and studies the desensitization method of almond protein,which lays a foundation for the development of less allergenic nut products and oral immunotherapy.The crude protein was extracted with methylene chloride and TBS buffer,and the crude protein was separated by ammonium sulfate with 100% saturation.The almond-sensitized protein was purified by dialysis and ion exchange column,and the almond was induced by SDS-PAGE.The components of sensitive proteins were identified by Western Blotting,and the nano-magnetic beads-double-antibody sandwich ELISA method was established.The qualitative analysis was carried out by means of amino acid quantification and mass spectrometry.Finally,desensitization was studied.The main findings are as follows:(1)The test determined that the optimum saturation of ammonium sulfate was 100%.The protein content and concentration of each stage of separation and purification were determined by BCA protein kit,and the content of almond protein without any treatment was about 36%,and the protein content after degreasing was 58%,the concentration was 3.4 mg/mL;The protein content after precipitation was 46%,the concentration was 4.4 mg/mL;the protein content after dialysis was 30%,the protein concentration was 5.7 mg/mL;the protein content after purification was 25%,and the protein concentration was 7.1 mg/mL.(2)Through SDS-PAGE electrophoresis experiments,multiple and complex protein bands were obtained,and the main enriched protein bands were 20-22 KD and 38-41 KD.On the basis of the crude protein obtained from the almonds after separation,it was carried out.After purification,a clear protein band diagram wasobtained,and the purification effect was better and the purity was higher.(3)Western Blotting identified allergic proteins with 21 KD and 38 KD.A nano-magnetic bead-double antibody sandwich ELISA method was established,and the LOD value was(5.31±1.02)?g/L,and the LOQ value was(15.31±2.16)?g/L.The nano-magnetic beads-double established in this experiment The anti-sandwich ELISA method is significantly more sensitive than the detection methods reported in other literatures.(4)Amino acid quantitative analysis obtained 17 kinds of amino acids of almond protein,of which glutamic acid content was the highest.The polypeptide sequence of the 21 KD protein band was obtained by mass spectrometry: GAYSLSLR,GFFFLVEGGR,GSSIFGLAPGK and LNTLETEEWFFK.Among them,GSSIFGLAPGK is not found in other proteins and is unique in this protein band.The proteins that may be present are available as Rubber elongation factor protein and Glutelin protein.At the same time,the 38 KD protein band polypeptide sequence is identical to the 21 KD protein band polypeptide sequence,and the following proteins may be present: Glyceraldehyde-3-phosphate dehydrogenase3,cytosolic,Glyceraldehyde-3-phosphate dehydrogenase 2,cytosolic,Rubber elongation factor protein,Sucrose synthase 2,Tubulin beta-1 chain,Elongation factor 1-alpha and ADP-ribosylation factor 2-B.(5)Three kinds of juices POM,BB,and CG treated almond protein,and it was found that POM juice showed greater ability to form a complex with almond protein.Nanoparticle size and Zeta potential measurements showed that the almond protein extract particle size increased sharply to about 3800 nm,and less protein was analyzed by electrophoresis after treatment with POM juice.