Formation And Reduction Mechanism Of N~?-(carboxymethyl)lysine And N~?-(carboxyethyl)lysine In Maillard Reaction

Advanced glycation end-products?AGEs?are a group of complex and stable compounds formed mainly by nonenzymatic glycation reaction between free amino groups?proteins,amino acids,lipids and nucleic acids?and carbanyl group of reducing sugars through condensation,rearrangement,cleavage and oxidative modification.Dietary AGEs are important sources of AGEs in the human body.Excessive intake of dietary AGEs may increase inflammation and oxidative stress,induce diabetes mellitus and its complications.Therefore,reducing the exposure of AGEs such as N^?-?carboxymethyl?lysine?CML?and N^?-?carboxyethyl?lysine?CEL?in food is of great importance to ensure food safety.In this study,a new method for the simultaneous determination of CML and CEL in Maillard reaction with high resolution was developed by high performance anion exchange chromatography,which provided important technical means for the study of CML and CEL formation.The effects of pH,types of sugars,metal ions,sulfites and molasses on the formation of CML and CEL in the Maillard reaction system were systematically studied.By monitoring the reaction substrate and intermediate products,the mechanism of CML and CEL regulation was proposed.An efficient method was developed for the simultaneous determination of CML and CEL in Maillard reaction using high performance anion exchange chromatography with pulsed amperometric detector?HPAEC-PAD?.The method had the advantages of low cost,simple operation and high resolution.CML and CEL had a good linearity?R> 0.997?between 3.125-25 mg/L,which was suitable for the routine analysis of CML and CEL in laboratory.The new method laid the technical foundation for the follow-up study.The effects of different pH?4.92,5.91,6.98,8.04 and 9.18?and different heating time?0-4 h?on the formation of CML and CEL were studied in a glucose and lysine model reaction system,respectively.The results showed that pH and heating time had great influence on the formation of CML and CEL.The CML and CEL in the Maillard reaction system were formed largely at the initial stage of heating?0.5 h?,while the contents of CML and CEL had a slight increase with the prolonging of heating time?0.5-4 h?.In addition,the levels of methylglyoxal?MGO?and glyoxal?GO?formed under acidic conditions were lower than those of alkaline conditions,resulting in less CML and CEL formation at low pH.Therefore,short-term,acidic processing technology was conducive for CML and CML inhibition.The changes of substrate sugar and lysine in the reaction system were studied using HPAEC-PAD.GO and MGO were determined by high performance liquid chromatography-diode array detector?HPLC-DAD?.The mechanism of action of acidic sugar?glucuronolactone?and basic sugar?glucosamine?on CML and CEL formation was proposed.The results showed that acidic and basic sugars significantly inhibited the formation of CML and CEL in Maillard reaction,and the browning intensities in acidic,neutral?glucose?and basic sugar system had little difference.In particular,the acidic sugar as raw material could significantly inhibit the formation of CEL in the system.The basic sugar as raw material had the highest inhibition for CML and almost did not generate CML.The possible explanation was that the reaction between glucosamine and lysine could not form Amadori rearrangement products?ARPs?and further oxidized to form CML.In addition,the GO content in the basic sugar system was lower than that in the neutral sugar system,which affected the further reaction with lysine to form CML.The content of CML in the acidic sugar system was also lower than that in the neutral sugar system.This was mainly because that the ARPs formed in acidic sugar system were unstable and easily underwent back-reaction to glucuronic acid/glucuronolactone and lysine,and thus could not be oxidized to form CML through ARPs.The effects of Ca²⁺ and Mg²⁺?0,50,100,150 and 200 mmol/L?on the formation of CML and CEL were studied in glucose and lysine model reaction system.The inhibitory rate of Ca²⁺ on CML and CEL formation was higher than that of Mg²⁺.Especially when the concentration of Ca²⁺ was 200 mmol/L,the CEL was not detected in the system.This may be due to the fact that Ca²⁺ and Mg²⁺ could form complexes with ARPs,thus the amount of ARPs was reduced and the further degradation into MGO and GO was inhibited.As MGO and GO were the precursors of CEL and CML formation,the CEL and CML content was decreased.In addition,Ca²⁺ and Mg²⁺ addition could reduce the browning intensity of the model reaction system.Therefore,the addition of Ca²⁺ and Mg²⁺ could reduce the harmful products formation and the browning intensity in the system.The safety of sulfite in food was studied from the point of view of CML and CEL.In the glucose and lysine model reaction system,different concentrations of SO32-?0,20,40,100 and 200 mmol/L?was added,it was found that the formation of GO through the oxidation reaction was inhibited due to the reducibility of sulfite while the nucleophilicity of sulfite promoted the formation of MGO through the non-oxidative pathway.The addition of sulfite significantly increased the amount of CEL in the model system due to the promotion of MGO formation,which was the precursor of CEL formation.For CML,low concentrations of sulfites promoted the formation of CML,whereas the CML formation was inhibited by high concentrations of sulfites.This was mainly due to the fact that sulfite could form adduct with carbanyl group in GO,thus inhibiting the reaction between GO and lysine to form CML.On the other hand,high concentrations of sulfites could also form adduct with carbonyl in glucose through the nucleophilic addition,although the adduct could be further condensed with lysine,but the condensate could not form Schiff's base and ARPs,thus it was unable to form CML by oxidative cleavage of ARPs..The total phenol content of molasses after extraction was found to be 25.5 mg gallic acid equivalent/g extract.Molasses extract had strong antioxidant activity and could effectively scavenging DPPH free radicals.The addition of ethyl acetate and n-butanol extracts into the glucose and lysine model reaction system showed that these two extracts had significant inhibitory effect on the formation of MGO and GO,which were the precursors of CEL and CML,respectively.In addition,vanillic acid and syringic acid,which were the main phenolic acids in sugarcane molasses,also had an inhibitory effect on the formation of CML and CEL This was mainly due to the fact that the oxidation reaction were involved in the formation of AGEs such as CML and CEL,and these phenolic compounds had strong antioxidant activity,could scavenge the free radicals generated in the Maillard reaction,thus inhibiting the oxidation reaction in the Maillard reaction and reducing the CML and CEL formation.In addition,these phenolic compounds may react with dicarbonyl compounds such as MGO and GO to form adducts,thereby inhibiting their reaction with lysine to form CEL and CML.Therefore,the inhibition of CML and CEL in the Maillard reaction was theoretically feasible by using molasses,a by-product of the sugar industry,as an additive.