Establishment And Application Of Quantitative Determination Method For Milk And Egg Allergens In Infant Cereals Based On Targeted Proteomics

Food allergic reactions have emerged as a growing challenge to the food industry and scientists believe that avoidance is the only line of defense.On January 1,2006,US Congress passed the Food Allergen Labeling and Consumer Protection Act which mandated the labeling of allergenic foods to avoid specific consumers being exposed to food allergens.To meet the requirement of risk management using food allergen labeling,allergen detection methods with a great accuracy and precision need to be established and applied.At present,both polymerase chain reaction assay and enzyme-linked immunosorbent assay are used in Chinese National Standards and are available from several companies.Considering several defects of Chinese National Standards,targeted proteomics is recently regarded as a novel technology applied to accurate quantitation of food allergens.In this paper,an accurate quantitation method of cow's milk and hen's egg allergens in infant cereals was established using targeted peoteomics technology and ultra performance liquid chromatography tandem mass spectrometry,and was applied to validation on food allergen labeling of merchant infant cereals and cross-contact of hand samples in specific food industry.First,a peptide release kinetics model was established for evaluation of peptide enzymolysis property in complex food matrix.Combined with this peptide release kinetics model,a peptide selection workflow was established using high resolution mass spectrometry and dimethylation labeling technology for identification and quantification of tryptic peptides.In this workflow,four properties of tryptic peptides were considered to determine specific quantitative proteotypic peptides for accurate quantitation of targeted protein including enzymolysis property,physical property,specificity and stability.To simplify the description,bovine serum albumin in raw bovine milk was used as an example.After analysis of peptide selection workflow,the quantitative proteotypic peptides of bovine serum albumin were selected as LVNELTEFAK,AEFVEVTK and LWSTQTALA.Method validation results of these quantitative proteotypic peptides showed that the quantification method gave a linear range of 1-100 ppm with the coefficient greater than 0.9995,and the detection limit of bovine serum albumin in milk was from 0.78 mg/kg to 1.38 mg/kg.Second,the quantitative proteotypic peptides of seven cow's milk allergens and four hen's egg allergens were determined using the above peptide selection workflow,including ?-lactalbumin,?-lactoglobulin,?-Sl-casein,?-S2-casein,?-casein,?-casein,bovine serum albumin,ovomucoid,ovalbumin,ovotransferrin and lysozyme C.Peptide selection results showed that the quantitative proteotypic peptides of each allergens were LDQWLCEK,GLDIQK,YLGYLEQLLR,FALPQYLK,VLPVPQK,YIPIQYVLSR,LVNELTEFAK,DVLVCNK,HIATNAVLFFGR,YFGYTGALR and HGLDNYR,respectively.All of these mentioned quantitative proteotypic peptides showed a great performance in quantitative method validation with the coefficient greater than 0,9995,the detection limit less than 1.00 mg/kg,the recovery from 90%to 110%and the precision less than 10%.Next,the quantitative proteotypic peptides of each allergen were used in establishment on the quantitation method of cow's milk and hen's egg allergens in infant cereals.Pretreatment conditions of infant cereal samples were optimized as the extraction solvent of 100 mM NaHCO3-Na2CO3 buffer(pH = 10.4),the extraction temperature of 30 ?,the extraction time of 59 min and the purification method of 10 kDa Centrifugal Filter Units treatment.Results showed that targeted allergens had a great performance in method validation with the coefficient greater than 0.995,the recovery from 85%to 115%,the precision less than 15%and the detection limit from 0.69 to 16.67 ppm which was lower enough than the allergenic threshold.However,lysozyme C was perfomed as an exception,of which the recovery was 64.83%.At last,the quantitation method of cow's milk and hen's egg allergens in infant cereals was applied to validation on food allergen labeling of merchant infant cereals and cross-contact of hand samples in specific food industry.Among the infant cereals from three companies,only one company's products had a consistency between food allergen labels and real contents.In several samples from other two companies,two problems was recognized that(?)what was not labeled on the package was detected in the products and(?)what was listed on the package was not detected in the practical products.According to the investigation results,hazard analysis and risk based preventive control of food allergens was regarded as an important risk management system for food industry.In this paper,an illustration and some suggestions were presented on hazard analysis and risk based preventive controls of food allergens in food industry.