Establishment And Application Of An Efficient Identification Method Of Glutelin And Amino Acids In Food For Special Medical Purposes

In recent years,the market scale has been expanding and the types of products have increased of Food for Special Medical Purposes(FSMP)in China.However,it is difficult to ensure the safety,effectiveness and controllability of FSMP,as a result of the imperfect laws and regulations.The raw materials and food additives used in Infant formulas for Special Medical Purposes(IFSMP)must not contain glutelin according to GB 25596-2010.The amino acids should be limited in types and contents for formula foods with amino acid metabolism disorders according to GB 29922-2013.Therefore,it is necessary to establish an efficient and accurate detection standard system for glutelin and amino acids in FSMP in order to promote the healthy and sustainable development of FSMP in China.In this study,firstly,the glutelin in rice was extracted by alkali method,and then was heated at different temperatures to determine its heat stable subunit.Cooked rice was prepared referring to the processing technology of infant rice flour,then the glutelin in cooked rice and high-quality protein(milk protein concentrate(MPC),soy protein isolate(SPI),MPC+SPI)in FSMP were prepared according to different proportions to establish an efficient and accurate detection method of glutenin in the presence and interference of protein matrix.The high-efficiency hydrolysis,derivative method and gradient elution procedure were explored,and an efficient detection method of amino acids in FSMP was established.Finally,it will be applied to the milk powder samples on the market and form a technical standard(Draft).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)results showed that subunit II of glutelin was the most stable subunit for heat treatment.The retention time(RT)of glutelin in infant rice flour(10.462 min)was in keeping with the glutelin in cooked rice(10.445 min).The results of methodological validation show that:the value of LOD was 9.7μg/m L for pure glutelin solution,R~2=0.9909,and it was 10-50mg/g(glutelin/other protein)at the presence and interference of other protein matrix,R~2=0.9517-0.9983.Commercial milk powder samples were found to have no glutelin by this method,and to be in line with the requirements of GB 25596-2010.The high-pressure 120℃and 2 h hydrolysis conditions of amino acids could not only greatly shorten the hydrolysis time,but also increase the amount of amino acid hydrolysis.The phenyl isothiocyanate(PITC)derivative method determined in this study was simple and easy to operate without heating.Eighteen amino acids were all detected within 35 minutes by the gradient elution procedure.The method validation of the amino acid detection method established in this study showed that the linear relationship range of 18 amino acids was 1.465-93.75μg/m L,R~2=0.9993-0.9999,R~2were all greater than 0.999.The LOD was 0.09-0.32μg/m L,the precision,repeatability and stability were 0.07%-2.44%,0.32%-2.22%and 0.19%-2.77%respectively,and the spiked recovery was 85.92%-107.55%.The content of single amino acid in milk powder samples on the market was≥8 mg/g,which did not meet the requirements of GB29922-2013.Compared with GB 5009.124-2016《Determination of amino acids in foods》,the method established in this study could detect more three amino acids:tryptophan(Trp),asparagine(Asn)and glutamine(Gln).