| Maillard Reaction has been applicated in the food processing by providing attractive colour, pleasant aroma and taste. However, during the food processing, the chemical changes in food components caused by high heat treatment have contributed to reducing nutritive value and formation of some toxic chemicals, such as advanced glycation end products(AGEs). AGEs have been reported to be toxicants, and a causative factor for various kinds of diseases. Based on animal and clinical studies, AGEs can be accumulated and lead to some impairment in the body if they are absorbed from the diet. Pyrraline, an AGE, has been used as an indicator to evaluate heat damage from the manufacture and storage of food systems. It can be absorbed from the diet and lead to some impairment in the body. In the present study, pyrraline was chosen as a representative marker of AGEs to explore the relationship between formation mechanism of pyrraline and conditions in food processing. The temperatures in the present study covered from pasteurization to frying procedures in food processing. In order to better understanding the formation mechanism of pyrrline, the pyrraline mechanism was investigated from simple model systems(amino acid + saccharides) to complex model systems(dipeptides/tripeptides + saccharides). The formation mechanism was obtained from five aspects(formation pathway, active intermediates, structure of saccharides, structure of peptides and reaction kinetics) to provide a theory for monitoring and controlling potential hazardous chemicals in food processing.The main results are shown as follow:(1) Formation of free-form pyrraline in amino acid+saccharides model systemsPyrraline formation and loss of reactants were monitored to explore the formation mechanism of pyrraline in various amino acid+saccharides model systems(Glc+Lys, Fru+Lys, Lac+Lys and Suc+Lys). The concentration of saccharides was carefully designed so that initial molar ratios of saccharide to lysine were 0.125:1, 0.25:1, 0.5:1, 1:1, 2:1, 4:1 and 8:1, respectively. The mixtures was heated from 60 to 220oC with various times. Pyrraline formation and loss of reactants were obtained by LC-MS/MS. In addition, the stability of pyrraline was investaged in Glc+pyrraline, Lys+pyrraline and net pyrraline model systems. Increase in initial molar ratio of saccharide to lysine could significantly promote the formation of pyrraline. Specifically, the pyrraline formation rate was influenced by the structure of saccharides involved in the reaction, and decreased in the following order: lactose>fructose>glucose>sucrose; the highest pyrraline was generated in lactose–lysine models. The maximum pyrraline was formed at 140oC. Moreover, saccharides and lysine had different effects on the stability of pyrraline. Among the eactants, lysine was themajor factor for the instability of pyrraline; a dipyrraline and a crosslink by pyrraline reacting with lysine could be formed. Pyrraline formation by the saccharide–lysine model system was a dynamic reaction, consisting not only of the pyrraline formation, but also pyrraline elimination with some formation of crosslinks.(2) Formation of petide bound pyrraline in peptide+saccharide model systemsSeven different dipeptides and their corresponding tripeptides with lysine at the N-terminus(Lys-X and Lys-X-Gly, X = Ala, Gly, Ser, Ile, Leu, Thr, Val) were chosen to react with glucose to determine the factors on impact of peptide bound pyrraline formation. Model systems were prepared by heating peptides with Glc from 60oC to 220oC for up to 65 min, the amounts of peptide bound pyrraline were monitored to evaluate the effect of the neighboring amino acid on the peptide bound pyrraline formation. The physico-chemical properties were introduced to explore the quantitative structure-reactivity relationship between physico-chemical properties and peptide bound pyrraline formation. The peptide bound pyrraline and 3-DG production were influenced by side chain of amino acids adjacent to Lys in the following order: Lys-Leu+Glc > Lys-Ile+Glc > Lys-Val+Glc > Lys-Thr+Glc > Lys-Ser+Glc > Lys-Ala+Glc > Lys-Gly+Glc; Lys-Leu-Gly+Glc > Lys-Ile-Gly+Glc > Lys-Val-Gly+Glc > Lys-Thr-Gly+Glc > Lys-Ser-Gly+Glc > Lys-Ala-Gly+Glc > Lys-Gly-Gly+Glc. Dipeptides produced higher amounts of peptide bound pyrraline and 3-DG than the corresponding tripeptides. This fact was caused by the physico-chemical properties of the side chain of amino acid adjacent to Lys. For side chain of amino acid adjacent to Lys in dipeptides, residue volume, polarizability, molecular volume and localized electrical effect were positively related to the yield of peptide bound pyrraline, while hydrophobicity and p Kb were negatively related to the yield of peptide bound pyrraline. In terms of side chain of amino acid adjacent to Lys in tripeptides, a similar result was observed except hydrophobicity was positively related to the yield of peptide bound pyrraline(3) Kinetic study of peptide bound pyrraline formation and elimination inLys-Gly+Glc model systemsThe impact of changing the reactant concentration and ratio on the kinetic parameters describing peptide bound pyrraline(pep-pyr) formation and elimination was evaluated in the peptide+Glc model systems, with microwave heating treatment ranging from 120 oC to 200 oC. Based on the approach of single response modelling, the initial rate of pep-pyr formation decreased with increasing temperature, except at 120 oC. Both the formation rate constant(k F) and the corresponding activation energy(Ea F) of the pep-pyr were independent of the initial concentration of the reactants and ratio. The elimination rate constant of pep-pyr(k E) increased with increasing reactant concentration ranging from 0.01 M to 0.125 M. In addition, by using multiresponse modelling on three model systems with different initial reactant ratios, isomerization should be taken into consideration in the formation mechanism of peptide bound pyrraline. The caramelization occurred obviously during the Maillard reaction in the system with an excess of Glc. Based on some modifications, the mechanistic models were proposed which was able to model all responses simultaneously. 3-DG was an effective intermediate in monitoring pep-pyr and melanoidins. High intercorrelations(correlation coefficient: 0.832 in k F, 0.733 in Ea F) in pep-pyr formation were observed between in the equimolar system and the system with an excess of Glc, as well as that in pep-pyr elimination(0.795 in k E, 0.854 in Ea E).(4) Formation mechnisms and pathways of pyrraline in Lys+Glc model systemsThe formation mechnisms and pathway of pyrraline were investaged in various model systems with 13C-labeled Glc and 15N-Lys involved. The concentration of pyrraline, GO, MGO and GLA were monitored by LC-MS/MS. The weight factors in isotope labeled and regular pyrraline, intermediates were calculated to investage the transition pathways of carbon and nitrogen atoms during pyrraline formation. Particularly, the change of carbon chain was monitored to explore the formation mechnism and pathway of pyrraline derived from 3-DG. The carbon chain of Glc was not always intact during the 3-DG formation: the fragmentation and recombination of carbon chain of Glc may occurred. The fragmentation and recombination in C1-C2 of Glc during the 3-DG formation was comfirmed. The nitrogen atom in the pyrrole ring was from ε-NH2 of Lys. 91% of total pyrraline content was produced via the pathway of Paal-Knorr Pyrrole synthesis reaction, while 9% of total pyrraline was formed via other unknown pathway. |
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