| Recently, the findings, analytical methods, formation mechanism and inhibition pathways of advanced glycation end-products (AGEs) in food have attracted wide attention all over the world. Many studies showed that Nε-(carboxymethyl)lysine (CML), as the representative of AGEs, has causal relationship with diabetes and complications and progression of aging, and were potentially hazardous to human health. Dietary AGEs is the important source of pool’s AGEs in human body, therefore, reducing the exposure of AGEs in food is an important measure of preventing the hurt to human. Formation mechanism, impact factors and effective inhibition pathways of CML produced during heat processing of lipid food was studied in this paper. The study was started from real food system, and CML research system was extended from complex real food system to simplest fatty acids-lysine model system. Plant flavonoid extracts were added to reduce CML formation in food systems and the inhibition mechanism was studied, which provided the theory basis for its effectively reducing CML in food production process. Main research content and results were as follows:The analytical method of isotope dilution ultra-performance liquid chromatography electrospray tandem mass spectrometry (UPLC-MS/MS) with derivatization by9-fluorenylmethyl chloroformate (FMOC-C1) for the quantification of CML and Nε-(carboxyethyl)-lysine (CEL) in food was investigated. The method was validated by evaluating its sensitivity, linearity, precision (repeatability and reproducibility), and recovery in food samples. The limit of detection (LOD) and quantification (LOQ) of CML and CEL was2ng/mL and7ng/mL. The recovery of the method ranged from98.9%to99.6%(RSD<7%) for CML and from91.1%to96.2%(RSD<7%) for CEL. The results indicated that this method was stable, rapid, good producibility and high senstivity for determining CML and CEL contents simultaneously in food matrices. Finally, this method was applied to determining CML and CEL contents simultaneously in59commercial products including heat processing food, infant formulas and protein powder. The results showed that the level of CML and CEL in heat processing food was significantly higher than other food. The value of CML and CEL contents ranged from3.9mg/kg to50.3mg/kg and from0.6mg/kg to50.5mg/kg (n=12) in fried food. The value of CML and CEL contents ranged from2.3mg/kg to171.2mg/kg and from0.9mg/kg to57.2mg/kg (n=20) in baked foods. The value of CML and CEL contents ranged from27.8mg/kg to100.0mg/kg and from3.8mg/kg to14.5mg/kg (n=5). The value of CML and CEL contents ranged from8.6mg/kg to304.7mg/kg and from3.8mg/kg to106.5mg/kg (n=11) in meat products. The value of CML and CEL contents ranged from40mg/kg to87.3mg/kg and from0.7mg/kg to2.3mg/kg (n=6) in infant formulas. The value of CML and CEL contents ranged from51mg/kg to479.8mg/kg and from4.1mg/kg to21.8mg/kg (n=5) in protein powder.Cocoa beans were chose as real food system. The effect of five concentrations of0.01%,0.05%,0.1%,0.3%and0.5%(w/w) of three hydrophilic antioxidants water-soluble antioxidant of bamboo leaves (AOB-w), water-soluble tea polyphenols (TP-w), water-soluble rosemary extracts (RE-w) on the formation of CML was investigated in baked cocoa beans (130℃/30min). The result showed that the average level of CML in blank sample without antioxidants was89.1mg/kg. Three kinds of antioxidants can lower the content of CML in baked cocoa beans. The average levels of CML in baked cocoa beans could reduce to68.8mg/kg and67.5mg/kg when they were treated by0.3%and0.1%(w/w) of AOB-w, and the inhibition ratio of CML were22.8%and24.2%. The average levels of CML in baked cocoa beans could reduce to59.4mg/kg when they were treated by0.5%(w/w) of TP-w, and the maximum inhibition effect was33.3%. The average levels of CML in baked cocoa beans could reduce to65.4mg/kg when they were treated by0.1%(w/w) of RE-w, reaching to the maximum inhibition ratio (26.6%).The formation regulation of CML under different heating conditions was studied in model systems of stearic acid/palmitic acid/oleic acid/linoleic acid-lysine. The study showed that the formation of CML was closely related to reacting temperature, time and substrates. In the same conditions, polyunsaturated fatty acids were easier to form CML, and the order of reactivity for the formation of CML was linoleic acid> oleic acid> palmitic acid> stearic acid. The formation of CML was studied in the fatty acid-lysine model systems. And the experimental design was performed by the response surface methodology (RSM) with heating temperature, time and the molar ratio of substrates as the parameters. Results showed that CML was optimally generated in model systems when the equimolar linoleic acid and lysine were heated at180℃for25minutes. And this linoleic acid-lysine model system was set as the bank control for the following study on inhibition effect of CML.This paper investigated the effects of plant polyphenol rich (flavonoid and phenolic acids) extracts of hydrophilic/hydrophobic antioxidants of bamboo leaves, tea polyphenol and rosemary extracts, i.e. water-soluble antioxidant of bamboo leaves (AOB-w), water-soluble tea polyphenols (TP-w), water-soluble rosemary extracts (RE-w), oil-soluble AOB (AOB-o), oil-soluble TP (TP-o) and oil-soluble RE (RE-o) on the formation and elimination kinetics of CML in linoleic acid-lysine model system. The maximum reduction effect (66.0%and69.8%) of CML was observed in the model system when the addition levels of AOB-w and AOB-o were10-5mg/mL, respectively. The maximum reduction effect (68.0%and67.9%) of CML was observed in the model system when the addition levels of TP-w and TP-o were10-5mg/mL, respectively. The maximum reduction effect (45.6%and40.9%) of CML was observed in the model system when the addition levels of RE-w and RE-o were10-4mg/mL and10-3mg/mL, respectively. Meanwhile, the effect of six plant polyphenols on the kinetics of CML in linoleic acid-lysine model system was described by the Logistic-exponential kinetic model. Results indicated that addition of AOB-w, AOB-o, TP-w, TP-o, RE-w and RE-o could significantly reduce the formation kinetics of CML while no effect on its elimination kinetics.The current study completed the establishment of CML and CEL simultaneous detection method in food and carried out the investigation of AGEs content in food in the market, on the basis of this, we concluded that the heat processed food with high content of fat is one of the main AGEs-riched food. The raw cocoa beans of chocolate production material was chosen as the representative of the real food system, plant polyphenols antioxidants were used to study the inhibitory effect of dAGEs and the fatty acid-lysine model systems were used to study the formation of CML in the heating process, and its inhibition kinetics. The research has enriched the knowledge of other associated hazards of maillard reaction in the heat processing food other than the acrylamide, and provided idea and method of the synchronous control of AGEs and acrylamide. |
