Preparation,Cytotoxicity And Determination Of Five Kinds Of Amino Acids-Acrylamide Adduct In Food

Acrylamide（AA）is a recognized neurotoxin and a potential carcinogen.High content of acrylamide is often detected in carbohydrate-rich high temperature processed food,such as coffee,potato chips,biscuits and instant noodles.In 10 years,research of acrylamide in food mainly focuses on the formation of acrylamide control,analysis of the development of detection methods,risk assessment,research,and little attention to its whereabouts in the food.However,acrylamide in food is easy to react with free amino acids to form adducts,which may produce new food safety risks while decreasing acrylamide.This thesis studied the mechanism of acrylaimide（AA）elimination with five kinds of amino acid（lysine（Lys）,tryptophan（Trp）,threonine（Thr）,cysteine（Cys）and glycine（Gly））,assessed the cytotoxicity of amino acid-acrylamide adduct,determinded thecontent of adducts in some high temperature processed food,and evaluated the effects of amino acids on the color and texture in the cookies and bisciuts.The main conclusions are as follows:1.The five amino acids showed different elimination effects on acrylamideLysine,tryptophan,threonine,cysteine and glycine reacted with acrylamide for 5h at 80℃,and showed varying degrees of elimination.The cysteine and lysine could eliminate more than 90%of acrylamide,and the other three amino acids could eliminate 15%to 35%of acrylamide.And the elimination of amino acid was related to the formation of the amino acid-acrylamide adduct.The retention time of the adduct formed by lysine,threonine,cysteine and glycine with acrylamide was about 3-5 min,and the maximum absorption wavelength in ultraviolet spectrum（UV）was 192-198nm.The retention time of the tryptophan-acrylamide adduct was about 29.080 min,and the maximum absorption wavelength in UV is 217.90 nm.2.Preparation,isolation and purification,structure identification and cytotoxicity of amino acids-acrylamide adductsThe optimized reaction conditions of the five amino acids and acrylamide were as follows:The ratio of Lys:AA=1:1 was adopted in the Lys-AA reaction system,and the reaction time was 5 h at 80℃.In the Trp-AA reaction system,the ratio of Trp:AA=1:4 was used to react at 95℃for 5 h.Thr-AA reaction system used Thr:AA=1:1 reaction at 95℃for 5 h.The Cys-AA reaction system adopts the ratio of Cys:AA=1:4 and reacts at 80℃for 3 h.Gly-aa reaction system adopts the ratio of Gly:AA=1:2 and reacts at 80℃for 5 h.After semi-preparation by column chromatography and high pressure chromatography,purified adduct was obtained.Combined with the use of UPLC-MS/MS,HRMS and NMR,the structures of five kinds of amino acid-acrylamide were charactered.Their molecular weight and molecular formula were as follows:Lys-AA(217.1490,C₉H₁₉N₃O₃)、Trp-AA(275.1337,C₁₄H₁₇N₃O₃),Thr-AA(190.1021,C₇H₁₄N₂O₄),Cys-AA（192.0645,C₆H₉NO₃S）,Gly-AA 1(146.0760,C₅H₁₀N₂O₃)and Gly-AA2(217.1132,C₈H₁₅N₃O₄)。The above information confirmed that acrylamide reacts with the nucleophilic amino groups on amino acid side chains,which following the mechanism as the Michael addition.The cytotoxicity of Lys-AA,Trp-AA,Thr-AA and Cys-AA adducts was investigated.The cytotoxicity of Lys-AA,Trp-AA,Thr-AA and Cys-AA adducts was significantly lower than that of AA,showing a beneficial effect on the overall survival rate of cells.The cytotoxicity of Trp-AA was higher than AA.In conclusion,The formation of Cys-AA,Lys-AA and Thr-AA adducts significantly reduced the cytotoxicity of acrylamide in Caco-2 cells and Ges-1 cells.3.Determination of amino acid-acrylamide adducts in foodThe contents of acrylamide and amino acids-acrylamide adducts in 12 kinds of high temperature processed foods were determined by UPLC/MS/MS wtih multi-reaction monitoring mode（MRM）.In 10 commercially available foods,the levels of acrylamide in potato crisps and chips were significantly higher than foods made from grains（wheat,millet,brown rice,corn,etc.）.The addition of Cys,Lys,Thr and Gly can reduce the content of AA,and increase the amount of adductsin biscuits and cookies,indicating thatthese four amino acids could promote the transformation of AA to the corresponding adducts in biscuits and cookies.The addition of Trp can mitage the content of acrylamide,but maybe generate new risk caused by the adduct.4.Effects of amino acid addition on moisture content,color and texture of cookies and cookiesAfter the addition of four kinds of amino acids,the pH value of cookies and cookies show a certain change,but the overall range remains within the weak alkaline range meet the national standard;Compared with the blank group,the moisture content of the products increased,but was lower than 4%,which was in line with the product standards of cookies and biscuits.The addition of Cys,Cysteine-L-lysine hydrochloride and Thr could brighten the color of biscuits,while the addition of L-lysine hydrochloride and Trp could make biscuits darker.The addition of L-lysine hydrochloride,cysteine-L-lysine hydrochloride combination,Thr and Trp also made the cookies dark brown,and the addition of Cys had little effect on the color of cookies.The addition of L-lysine hydrochloride and Cys increased the elasticity and chewiness of biscuits and cookies,and the combination of cysteine-L-lysine hydrochloride had better effect than the addition of these two amino acids alone.The addition of Thr and Trp can improve the texture of cookies,but the addition of Thr and Trp has a certain negative effect on the texture of cookies.