A Study On Mechanism Of Carboxymethyllysine Formation In Lipids Food System

In recent years, advanced glycation end products (AGEs) are found to exist in commonlyconsumed foods, especially in fat diet. Many animal experiments and clinical experimentsshowed that food-derived AGEs were potentially hazardous to human health. In order to studythe formation mechanism of AGEs in fat diet, Nε-carboxymethyllysine (CML) was chose as arepresentative of AGEs. Its formation was associated with food production process. CMLresearch system was extended from simplest model system to real lipid food system in thispaper. CML formation mechanism was investigated from the aspects of reaction mechanism,intermediates, free radicals and carbon source to offer theory for controlling potentialhazardous materials in food production process. The main results are shown as follow.(1) Chemical reaction kinetics of CML in non-lipid model system.The formation rule of CML in lysine (Lys)+glucose (Glu) model system wasinvestigated so as to offer basis for mechanism of lipid effect on CML formation.Firstly, CML and its key intermediates, fructoselysine (FL) and glyoxal (GO), weredetermined with high performance liquid chromatography-mass spectrum (HPLC-MS) inLys+Glu model system heated at100°C,80°C,60°C and40°C. Secondly, The contributionrates of FL and GO to CML were determined by inhibiting GO with o-phenylenediamine(OPD) method. Finally, according to reversible reaction, consecutive reaction and parallelreactions mechanism, CML, FL, GO, Lys and Glu formation kinetics equations weretheoretically deduced. Combining with non-linear fit function of Origin Software,experimental data were fitted to obtain apparent rate constants and apparent kinetics equations.It indicated that CML content could accumulate in food for its thermal stability. There wereboth FL and GO contents maximum points in their formation process. CML derived from FLplayed a major role with contribution rate of84.7%for CML formation, while CML derivedfrom GO played a minor role with contribution rate of15.3%for CML formation. CMLapparent kinetics equations through FL pathway and GO pathway were obtained respectively.Therefore, it is possible to control food-derived CML through adjusting processing variablesand adding inhibitors.(2) Formation mechanism of CML in lipid model system.The formation mechanism of CML was discussed in lipid model system from aspects offree radials and carbon resource. In free radicals aspect, it was proved that hydroxyl radical (OH·) induced by Fentonreagent could promote CML formation in Lys+Glu+Fenton model system andLys+GO+Fenton model system. On the base of that, the effect of Lin, Ole, Tri and Gly onOH·, CML, FL, GO and Glu content in Lys+Glu+Lipid model system, and the effect of Lin,Ole, Tri and Gly on CML and glyoxalic acid (GOA) content in Lys+GO+Lipid model systemwere determined. It was shown that OH· induced by Fenton reagent could promote threepathways for CML formation in Maillard reaction. Lin, Ole and Tri could induce more OH· inMaillard reaction model system which promoted FL and GO conversion to CML. However,Gly could reduce CML content in model system for its scavenging OH· ability. Thepromoting CML formation abilities of lipids on modeling system were in the order of Ole﹥Lin﹥Tri﹥Glu﹥Gly.In carbon resource aspect, Glu and Gly in model system were labeled with13C. Newpathway for CML formation from lipid was proved through tracing13C flow direction.Contribution rates of lipid oxidation and saccharide oxidation for CML formation werecompared through calculating CML content proportion formed from Lin, Ole, Tri and Gly. Itwas found that GO derived from Lin, Ole, Tri and Gly oxidation participated in CMLformation. In100°C heating condition, compared with Glu contribution rate to GO, thecontribution rates of Lin, Ole and Tri to GO reached their maximum value at4.0h with3.53%、3.08%and1.81%respectively. The contribution rate of Gly to GO increased withtime increasing and reached maximum value99.29%at8.0h. Compared with Glucontribution rate to CML, the contribution rates of Lin, Ole and Tri to CML formationincreased with time. They increased to reach their maximum value at8.0h with7.45%,5.31%and4.10%, respectively. The contribution rate of Gly to CML increased with time.They reached maximum value10.33%at6.0h. The promoting effect of Lin, Ole, Tri and Glyon CML formation through oxidation to GO were in the order of FL pathway﹥Gly﹥GOpathway﹥Lin﹥Ole﹥Tri. Therefore, the promoting effect of lipid on CML formationderived from two aspects: Inducing more OH· to promote CML formation; Offering carbonsource for CML formation through oxidation to GO.(3) CML formation in vegetable oils model system.Based on above mechanism of lipid effect on CML formation in model system, the lipidmodel system was extened from lipid model system to vegetable oils model system. Theeffect of five oils on OH·, CML, FL and GO content in Lys+Glu+Oil model system wasstudied. It was shown that vegetable oils including Soybean oil (Soybean), Corn oil (Corn), Olive oil (Olive), Palm oil (Palm) and Rape oil (Rape)) could induce more OH· in Maillardreaction model system, which promoted FL and GO conversion to CML. The promotioneffect of five oils on CML formation was in the order of Soybean﹥Corn﹥Olive﹥Palm﹥Rape. Fatty acid analysis for five oils showed that the more unsaturated fatty acid the oils had,the more OH· they induced in model system. Therefore, oils with more unsaturated fatty acidin food production process were easier to increase CML formation.(4) CML formation in real food system.This part extended vegetable oils model system to real lipid food system. An amino acidbeverage was chose as non-lipid food system, and milk was chose as lipid food system. Theamino acid beverage was storaged at60°C for48h to determine CML formation. It indicatedthat CML content in beverage increased with time. Natural cream of3%,6%and9%wasadded to skim milk (SM) and low lactose milk (LLM) to compared CML formation. Itindicated that under the condition of100°C heating for30min, CML increased with time.LLM generated more CML than SM. Therefore, CML could accumulate in food storageprocess and CML content in lipid system increased with oil content increasing in foodproduction process.