Preparation Mechanisms Of Alanine-xylose Amadori Compound And Controlled Formation Of Flavor And Color During Thermal Processing

The increasing demand for healthier,nutritious and quickly cooked food products,and the growing aversion of consumers towards non-natural chemicals added to foods reinforce the need of novel flavor enhancers isolated from natural sources.Currently,Maillard reaction products(MRPs)derived from sugars,amino acids and plant/animal proteins are usually used as the natural flavor enhancers.Although the color and aroma of the freshly prepared MRPs are intense and richness,MRPs always present some problems such as unstable color,loss of aroma,and standardization of quality during processing,storage and application.Therefore,the stabilization of Maillard-type flavors and controlled formation of their flavor and color is a key scientific issue that needs to be tackled in the field of food flavor.To address this issue,this paper demonstrated the preparation of a storage-stable,colorless and tasteless non-volatile flavor precursor,Amadori rearrangement product(ARP),which can be added to foods as a flavor-enhancers,and enable the controlled formation of the desired,fresh and pleasant flavor and color characteristics during subsequent thermal processing.Aiming at the problem that the formation conditions in the aqueous phase of ARP are difficult to be traced and determined.Based on the theory of the stepwise increase of temperature of Maillard reaction(MR),EGCG was screened as a possible tracer of ARP formation from natural food ingredients by using the model system of alanine-xylose(ala-xyl).The qualitative and quantitative analysis of the ARP was established by using the standard ARP which was characterized and confirmed by NMR and LC-TOF-MS,and the accuracy and reliability of EGCG tracer ARP were verified.Then the critical formation conditions of ARP were then determined in the first stage of the reaction at 80°C for 80 min.Model reactions of ARP,reactants(xylose and alanine)and EGCG was constructed to preliminarily clarify the possible mechanism of how EGCG indicated the maximum critical formation of ARP through the point of lowest browning intensity under the stepwise increase of temperature.The reaction mixtures were further analyzed by using LC-TOF-MS and derivatization techniques.The results revealed that the browning reduction was due to the formation of adducts of EGCG with ARP and its primary degradation products 1-DX and 3-DX at the very early stages of the Maillard reaction.thereby influencing or interfering with the formation of browning precursors.In other words,the formation pathway of chromophores was blocked by EGCG,resulting in the lower browning intensity of MRPs.Additionally,the tracer performance of EGCG was investigated at different reaction temperatures and pH values in the first phase of the MR.The results suggested that EGCG has a very significant tracer activity at the lower temperature(80°C)and lower pH values(6.5).The EGCG was further proved to be an effective tracer of peptides MRIs in model reactions of small molecule peptides and plant protein hydrolyzed peptides,which demonstrated the broad applicability of EGCG for tracing ARPs in aqueous phase.In order to solve the problem that the yield of ARP formation in aqueous phase is extremely lower usually not exceeding 2%-5% and difficult to prepare efficiently.Based on the critical formation conditions of ARP traced by EGCG,a normal thermal reaction coupled with a vacuum dehydration reaction system(NTR-VDR)was used to prepare ARP,and it was found that the addition of EGCG after NTR followed by VDR could promote the high-efficiency formation of ARP.The reaction conditions were 2:1 molar ratio of xylose to alanine,initial pH of 7.5,a reaction time of 60 min at 90 °C,the addition of 500 μmol EGCG and vacuum dehydration for 10 min at 90 °C.The ARP yield could reach about 95% under these conditions.In a simplified and more practical simultaneous dehydration-reaction system(SDR),it was found that high pH values facilitated the nucleophilicity of amino groups in synergy with vacuum dehydration and resulted in a more efficient conversion of ARP.The yield of ARP reached around 93% by the optimization of the molar ratio of the reactants,the dosage of EGCG and the initial pH values.It was also found that an increase in the concentration of the xylose was more conducive to an increase in ARP yield,and that an increase in the dosage of EGCG within a certain range could also increase ARP yield.In order to further clarify the mechanism of the addition of EGCG to improve the yield of ARP,the ARP samples prepared by the addition of EGCG were analyzed by LC-TOF-MS.The results showed that EGCG was very stable during vacuum dehydration,without undergoing isomerization and oxidative degradation.Meanwhile,two types of ARP-EGCG adducts and a variety of mono-DX-EGCG and di-DX-EGCG adducts were detected.The ARP-EGCG adduct was isolated and purified,and its structure was then characterized using UV spectroscopy,highresolution mass spectrometry,IR spectroscopy and 1D/2D NMR techniques.As a result,the C8/C6-ARP-EGCG adduct was formed by aromatic electrophilic addition of the C8/C6 active site on the A-ring of EGCG to the carbonyl group of ARP.The mechanism of interaction of EGCG to promote ARP yield was revealed by the establishment of accurate qualitative and quantitative analytical methods,the thermal reaction models,the comparative experiments and the reaction kinetic analysis,which were carried out by using C8-ARP-EGCG as a standard substance.The mechanism of interaction between EGCG and ARP was summarized as follows:the capture of ARP and its degradation product DXs by the C8/C6 active site of the A-ring of EGCG,which significantly altered the classical degradation pathway of ARP and DXs.The downstream reaction pathway of MR was delayed or blocked by this trapping effect.At the same time,nucleophilic addition of amino acids to the free carbonyl group in DXs-EGCG resulted in the re-release of DXs from the EGCG molecule and the regeneration of ARP with amino acids via a reaction cycle.The effects of various reaction temperatures,times,pHs,reaction concentrations,precursors(xylose,alanine,GO,MGO,DA)and the EGCG tracer on the flavor and color of ARP were investigated in aqueous thermal reaction models.The results showed that ARP was more inclined to form more types of furans and pyrroles than its precursor mixtures(alanine +xylose,AX);ARP was absent of pyrazines at reaction temperatures lower than 120°C;pyrazines were able to be generated in ARP at reaction temperatures greater than 120°C and pyridines were able to be formed in ARP at temperatures above 160°C.Increasing the pH promoted the formation of pyrazines in ARP and mitigated ARP browning,while lowering the pH resulted in greater browning of ARP and the generation of more Furans,but the lack of pyrazines.Increasing the reaction concentrations of ARP promoted the formation of pyrazines,but also enhanced the browning of ARP;the addition of exogenous amino acids facilitated the formation of large numbers of pyrazines and significantly improved the types and numbers of ARP aromas;the addition of exogenous sugars contributed to the production of more furans in ARP,but without an additional increase in the types and numbers of ARP aromas;the addition of dicarbonyl precursors reduced the types and numbers of ARP aromas,while GO and MGO facilitated the ARP to generate pyrazines.However,DA suppressed the formation of pyrroles in ARP;the addition of the browning inhibitor EGCG decreased the flavor intensity of ARP,but with no significant effect on the type and number of ARP aromas.The critical control parameters of ARP flavor and color were established by PLSR analysis,and the ARP flavor enhancement strategies were clarified by PCA analysis.ARP was applied to the biscuit and sausages matrices.The thermal processing suitability of ARP and its contribution on food sensory qualities were examined by applying ARP to crackers and chicken sausages matrices,respectively.The thermal processing adaptability of ARP demonstrated that there was no absence of flavor types in the real food system.The volatile aroma content and numbers of ARP were significantly greater than those of both the control and blank groups,which indicated the significant flavor enhancement capability of ARP.Furthermore,it was found that the higher thermal processing methods,the lower water activity and the rapid heat-up rate were more favorable to the formation of large numbers and richness of the volatile compounds derived from ARP.The results showed that the addition of 0.2% and 1.0% ARP to the crackers resulted in 10 and 25 times higher aroma content than control,respectively;the addition of ARP to the crackers also significantly improved the texture,baking aroma,taste,flavor and color of the crackers samples.The addition of ARP to the sausages imparted and enhanced the characteristic aromas of caramel(furans),roasted sweet(pyrroles)and nutty(pyrazines)aromas in the meat food system,as well as significantly enhanced the color,taste,flavor and overall acceptability of the sausages.The storage stability measurements showed that ARP was more stable stored as a solid and dilute solution,while the rate of ARP decomposition was accelerated at high concentrations,temperatures and moisture levels.