Antioxidant-related and kinetic studies on the reduction effect of catechins and esterified catechins on acrylamide formation in a microwave heating model system

Abstract

The reduction effect of catechins and esterified catechins on the kinetic behavior of acrylamide formation and its correlation with the change in antioxidant properties of Maillard reaction products in an equimolar asparagine–glucose microwave heating model system was investigated. Results indicated that both catechins and esterified catechins could effectively reduce the formation of acrylamide and could achieve a maximum inhibitory rate when their addition levels were 10⁻⁹ mol L⁻¹. Furthermore, 5′-phenolic hydroxyl and/or 3-gallate groups in the chemical structure of catechins and/or their derivatives played an important role in the reduction of acrylamide formation. Also, the reduction effect of esterified catechins was better than that of catechins. Meanwhile, inhibitory rates of acrylamide affected by either catechins or esterified catechins correlated well (R²: 0.757–0.940) with the Trolox equivalent antioxidant capacity (ΔTEAC) of Maillard reaction products measured by three different antioxidant evaluation methods, i.e. 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), or the ferric reducing antioxidant power (FRAP) assay. The kinetic study revealed that the processes of the conversion from glucose into fructose, the asparagine–fructose reaction and the generation of acrylamide in Maillard reaction systems were all significantly suppressed, which may help to elucidate the mode of action of catechins and esterified catechins in reducing the formation of acrylamide during the Maillard reaction.

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Introduction

Acrylamide has been classified as Group 2A, “probably carcinogen to humans,” by the International Agency for Research on Cancer.¹ Meanwhile, it is also regarded as a type of neural, genetic and reproductive toxin. In 2002, Swedish scientists found considerable levels of acrylamide as a contaminant in heat-processed, carbohydrate-rich foods.² Soon after its discovery in foods, mechanistic studies revealed that acrylamide can be generated from the Maillard reaction that occurs between the free amino acid, asparagine, and reducing sugars.^3,4 Furthermore, some critical direct precursors contributing to the formation of acrylamide have also been identified, such as 3-aminopropionamide, decarboxylated Schiff base, decarboxylated Amadori product, acrylic acid and acrolein.⁵

Investigations on the reduction of acrylamide in different heat-processed foods have been highlighted in recent years. Various methods have been used to minimize acrylamide levels. The inhibitory measures can be categorized into agronomic and processing strategies.⁶ One representative reduction method is the control of the content of participating substrates, i.e. reducing sugars and asparagine. In addition, other inhibition approaches include pH modification and the use of cations, hydrogen carbonates and specific amino acids during food processing.⁷ Some advanced ways to reduce acrylamide, such as the addition of asparaginase, irradiation and genetic modification techniques have also been used.⁸ Recently, natural antioxidants, especially mono- and poly-hydroxylated, phenolic-rich extracts, were used for the reduction of acrylamide. Zhu et al.⁹ demonstrated that higher addition levels of phenolic compounds were related to lower levels of acrylamide (R = −0.692). In addition, the use of o-diphenolic-rich virgin olive oils may be regarded as a reliable strategy to reduce the formation of acrylamide in fried potatoes.¹⁰ However, the application of phenolic-rich virgin olive oils may either reduce or enhance the generation of acrylamide, depending on their usage levels.¹¹ Therefore, precautions and control of the usage levels of phenolic-rich olive oils should be especially considered. Alternatively, flavonoid-rich herbal extracts were also demonstrated as phenolic-rich antioxidant additives for the reduction of acrylamide in foods. Our previous study found that reductions in acrylamide levels of 74.1% and 76.1% were achieved by the use of antioxidant preparation of bamboo leaves, a flavonoid-rich extract, in potato crisps and French fries, respectively.¹² Mechanistic work showed that naringenin, a compound representative of flavanones, could effectively reduce the formation of acrylamide in the Maillard reaction by directly reacting with its precursors.¹³ In another mechanistic analysis, antioxidants could inhibit acrylamide formation by preventing lipid oxidation, thus limiting the accumulation of carbonyls.¹⁴ Besides, antioxidants may also act as active agents in preventing the generation of a “reactive carbonyl pool,” trapping key Maillard reaction intermediates (e.g. Schiff base), participating in the precipitation of asparagine, reacting with acrylamide via Michael addition reactions and eliminating the formation of acrylamide via the reaction with their oxidized forms.¹⁵

Catechins, which belong to the family of flavanols and their derivatives (mainly esterified catechins), have been extracted from green tea. Catechins and their derivatives may have a great potential to reduce acrylamide and exert their antioxidant properties. The correlation between levels of tea catechins and the reduction of acrylamide during the roasting process of green tea has been demonstrated.¹⁶ Nevertheless, few studies have mechanistically investigated the effect of catechins on the reduction of acrylamide and its correlation with antioxidant properties of the Maillard reaction. Besides, a structure–activity analysis of the ability of flavonoids to reduce the formation of acrylamide in our previous study revealed that both the number and position of phenol hydroxyl functional groups play an important role in the inhibition ability of flavonoids,¹⁷ indicating that the different efficiencies of catechins and esterified catechins may contribute to the reduction of acrylamide.

Several different mathematic models have been adopted to better understand the mechanisms for the formation and elimination of acrylamide.⁷ The multi-reaction response model has been widely used to estimate the kinetic constants, which are derived from the dynamic mass balance principle or the network reaction approach. It is possible that the generation and elimination of acrylamide at different stages during the Maillard reaction can be predicted by the knowledge of mechanisms and kinetic models. The reduction of acrylamide via the use of natural antioxidants may affect the generation and elimination of acrylamide on a kinetic basis. Therefore, investigations on the kinetics of acrylamide affected by natural antioxidants are timely.

The aim of this study was to (i) investigate the optimal addition level of esterified catechins and related mechanisms for reducing the formation of acrylamide compared to the use of catechins; (ii) demonstrate the correlation between antioxidant properties of the Maillard reaction products and the reduction of acrylamide affected by catechins or esterified catechins; and (iii) reveal the mode of action of catechins on the reduction of acrylamide during the Maillard reaction on a kinetic basis.

Experimental

Materials

Potato powder made of the Atlantis variety was purchased from Sanjiang (Group) Potato Products Co., Ltd. (Lintao, China). Epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG) were provided by the Tea Research Institute of Chinese Academy of Agricultural Sciences (Hangzhou, China); their chemical structures are shown in Fig. 1.

Fig. 1 The chemical structures of catechins and esterified catechins in the present study.

Chemicals

Acrylamide, D-(+)-glucose monohydrate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was purchased from Shanghai Genebase Technology Co., Ltd. (Shanghai, China). L-asparagine monohydrate was purchased from Biocity Science and Technology Inc. (Beijing, China). D₃-acrylamide, ¹³C₆-glucose and ¹⁵N₂-asparagine were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Formic acid (purity ≥ 96%) was supplied by Tedia (Fairfield, OH, USA), and methanol was obtained from Merck (Whitehouse Station, NJ, USA). Ultrapure water was passed through a Milli-Q Gradient A10 system (Millipore, Bedford, MA, USA) and used throughout the experiments. The Maillard reaction was performed via a microwave heating system in a potato matrix.

Establishment of microwave heating system (MHS)

A portion of potato powder (1 g) was added into a centrifuge tube containing ultrapure water (20 mL). After ultrasonic vibration for 20 min, the sample solution was centrifuged at 15000 r min⁻¹ for 15 min. The supernatant was then passed through a 0.22 μm filter and prepared for injection. The HPLC conditions were optimised according to a previous analytical method.¹⁸ Asparagine was quantified using a diode array detector, and glucose, fructose and sucrose were monitored using a differential refractive detector. HPLC analyses were performed on a 2695 high-performance liquid chromatograph system (Waters, Milford, MA, USA), which includes a 2996 diode array detector and a 2414 differential refractive detector. Chromatographic separation was achieved using a Capcell Pak C₁₈ A.Q. column (150 mm × 2.0 mm i.d., 5 μm) at a flow rate of 1 mL min⁻¹. The mobile phase using an isocratic gradient elution programme consisted of 75% acetonitrile (v/v). The injection volume was 10 μL. Finally, the concentrations of asparagine and glucose in the potato matrix were measured. Based on the determination of these two Maillard reaction substrates in the original matrix, the addition levels of asparagine and glucose were calculated in order to ensure the occurrence of an equimolar asparagine–glucose Maillard reaction system. As a result, 699.3 mL of asparagine (0.2 mol L⁻¹) and 279.9 mL of glucose (0.5 mol L⁻¹) were mixed with a portion of potato powder (50 g) and compensated with 20 mL of phosphate buffer solution 0.1 mol L⁻¹, (pH 6.80) to prepare the Maillard reaction solution (1 L) with an equimolar level of asparagine and glucose (0.14 mol L⁻¹). The microwave heating reaction between asparagine and glucose was performed via an Ethos D microwave digestion labstation from Milestone Inc. (Shelton, CT, USA). The labstation was set up and conducted according to our previous work.¹⁹ In detail, the mixed reaction solutions in sealed digestion vessels were all microwave-heated for 5 min at 180 °C under a working power of 500 W according to a prepared temperature programme of the microwave digestion labstation as follows: room temperature → 120 °C (200 W, 5 min); 120 °C → 180 °C (500 W, 5 min). The fluctuation range of heating temperature in the microwave system was less than ± 1 °C. The present potato-based, equimolar asparagine–glucose Maillard reaction system has many advantages, including high baseline levels of acrylamide, effective generation of acrylamide via microwave heating with high reaction temperature and short reaction time, appropriate mimicking of acrylamide production in characteristic food matrices such as potatoes, etc.^17,20 At the end of heating, the microwave digestion vessels filled with the final reaction products were taken out from the labstation and immediately cooled in prepared ice water to stop any further reaction. The entire cooling procedure was performed in a room with stable air temperature (20 °C) adjusted by air-conditioning. The cooled reaction products were ready for sampling during the pre-treatment of final reaction products.

Reduction effect of catechins and esterified catechins on the formation of acrylamide

Two esterified catechins (i.e. ECG and EGCG) and two non-esterified catechins (i.e. EC and EGC) were investigated for their reduction effects. After dissolving in a small amount of dimethyl sulfoxide (DMSO), they were all diluted to eight different levels, i.e. 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ mol L⁻¹ with 0.1 mol L⁻¹ phosphate buffer (pH 6.80). Then, different levels of catechins and esterified catechins (100 μL) were added into the prepared potato-based Maillard reaction solution (10 mL). A control group was prepared using the same matrix without any addition of catechin compound. When all the groups had been well prepared, the prepared solutions were reacted at 180 °C for 5 min to mimic the Maillard reaction in the microwave heating system.

Pre-treatment of final products from the Maillard reaction

The internal standard solution of D₃-acrylamide (2 μg mL⁻¹) was prepared in advance. An aliquot (200 μL) of Maillard reaction products was diluted to 10 mL with phosphate buffer in a colorimetric cylinder. Then, an aliquot of the dilution (2 mL) was mixed with the internal standard solution (500 μL). After the solution had been well mixed using a vortex mixer, liquid-liquid extraction with ethyl acetate and solid-phase extraction (SPE) purification were successively conducted according to our previous work.²¹ The elution was finally transferred to an auto-sampler vial for ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis.

Determination of acrylamide in Maillard reaction products

Acrylamide levels in Maillard reaction products in each group were measured by UHPLC-MS/MS with the multiple reaction monitoring (MRM) method. The UHPLC was performed on an Acquity ultra-high performance liquid chromatograph system equipped with the micro vacuum degasser, auto-sampler and column compartment (Waters, Milford, MA, USA). Chromatographic separation was performed via using a Waters UPLC BEH C₁₈ (50 mm × 2.1 mm i.d., 1.7 μm) column, guarded with a Hypercarb column (10 mm × 2.1 mm i.d.). Details on the UHPLC-MS/MS conditions were as conducted in our previous study.²²

Determination of antioxidant property in Maillard reaction products

The antioxidant property of Maillard reaction products was simultaneously measured by the DPPH, ABTS and ferric reducing antioxidant power (FRAP) assays. (i) DPPH assay. The DPPH radical-scavenging activity assay was based on a modified procedure of a previous work.²³ In this assay, antioxidants presented in the products reduce the DPPH radicals, and thus minimize the visible absorption at 520 nm. An aliquot of the final products (20 μL) was mixed with 5 mL of the DPPH solution (74 mg L⁻¹). The mixture was then measured at 520 nm using a visible spectrophotometer. The Trolox standard aqueous solution (concentration range: 0–0.4 mmol L⁻¹) was analyzed under the same conditions, and was used for calibration. The antiradical activity of the products was expressed as the Trolox equivalent antioxidant capacity (TEAC), i.e. μmol equivalents of Trolox per mL of sample (μmol Trolox mL⁻¹). (ii) ABTS assay. The ABTS assay was conducted according to previous work, with some modifications.²⁴ In detail, an aliquot of the final reaction solutions (5 μL) was dissolved in 5 mL of the ABTS solution (70 μmol L⁻¹). The thoroughly-mixed products were also detected by visible spectrophotometer at the maximal absorption wavelength (730 nm). Calibration was performed with a linear range of 0–0.12 mmol L⁻¹. Results were expressed as TEAC_ABTS values. (iii) FRAP assay. Based on the procedure described previously,²⁵ the FRAP assay was modified and improved. The FRAP reagent consists of TPTZ (10 mmol L⁻¹), FeCl₃ (20 mmol L⁻¹) and acetate buffer (0.3 mol L⁻¹) with a ratio of 1 : 1 : 10 (pH 3.6). Aliquots of the Maillard reaction solutions (6 μL) were allowed to react with 4.5 mL of the above FRAP solution. Then, the absorbance at 595 nm was determined after assay products had been vortex-mixed and reacted for 2 h at room temperature. Calibration was performed under the same condition as described, with a linear range of (0–0.3) mmol L⁻¹. Results are expressed as TEAC_FRAP values. If the TEAC values measured by the above three assays were above the linear ranges of their calibration curves, the reaction solutions were appropriately diluted with water.

Kinetic study on acrylamide reduction by the use of catechins and esterified catechins in the microwave heating system

A kinetic study on the formation and reduction of acrylamide accompanied by the addition of catechins and esterified catechins was also performed in the microwave heating system. The precursor substrate solution contained equimolar concentrations (0.14 mol L⁻¹) of asparagine and glucose. The optimized addition level of catechins and esterified catechins was selected, which presented the maximal reduction effect on acrylamide formation. An aliquot of the optimized level of catechins or esterified catechins (100 μL) was added into the microwave system in the experimental group, while the same volume of phosphate buffer was added into the control group. The mixed-reaction solutions in both control and experimental groups, in sealed digestion vessels, were simultaneously microwave-heated under the selected heating temperature (180 °C) of the labstation temperature programming, with different heating time treatments (0.1, 1, 2, 3, 4, 5, 7.5, 10, 15, 25 and 40 min, 11 treatments in total). The heating treatment of the asparagine–glucose–catechin mixture in each test was repeated in triplicate (n = 3). Based on kinetic considerations, all the intermediates were modelled into Schiff bases as the proposed central product, and the final products could be divided into acrylamide, melanoidins and other final products. The kinetic data analysis and relative rate constant calculation were performed according to previous studies, with some modifications.¹⁸ The overall changes in acrylamide concentrations can be formulated by a reaction schedule of 6 consecutive reactions in which k₁, k₂, k₃, k₄, k₅ and k₆ represent the rate constants of the asparagine–glucose reaction, conversion from glucose into fructose, asparagine–fructose reaction, acrylamide formation, melanoidin formation and acrylamide degradation, respectively (Fig. 2). The kinetic equations could be written as follows:

Fig. 2 Kinetic model of acrylamide formation and elimination for mimicking Maillard reactions. k₁–k₆, kinetic parameters.

In these kinetic equations, C_Asn, C_Glu, C_Fru, C_Schiff, C_AA, C_M and C_D represent the concentrations of asparagine, glucose, fructose, Schiff base, acrylamide, melanoidins and degradation products, respectively, while t represents the reaction time. For initial conditions, when the reaction time t = 0, C_Asn = C_Glu = 0.14 mol L⁻¹ while C_Fru = 0, C_Schiff = 0, C_AA = 0, C_M = 0 and C_D = 0.

Simultaneous determination of acrylamide, asparagine, glucose and fructose by UHPLC-MS/MS during the kinetic study

In the kinetic study, acrylamide, asparagine, glucose and fructose were simultaneously determined by UHPLC-MS/MS according to our previous work.²⁶ Briefly, chromatographic separation was performed using a Hypercarb column (100 mm × 2.1 mm i.d., 5 μm, Thermo Electron, San Jose, CA, USA). The mobile phase was formic acid (0.2%, v/v) at a flow rate of 0.2 mL min⁻¹. The chromatographic column was maintained at 40 °C with a run time of 5.5 min per sample. Tandem mass spectrometry was performed on a Quattro Ultima triple-quadrupole mass spectrometer (Micromass Company Inc., Manchester, UK) using the electrospray ionization (ESI) source. The detection of asparagine and ¹⁵N₂-asparagine was operated in negative ion mode due to matrix peak interferences in positive ion mode. All other analytes, including isotope compounds, were monitored in positive ion mode. In addition, the melanoidins were measured using a spectrophotometer, at the visible absorption maximum of 470 nm. The concentration of melanoidins was calculated from the Lambert–Beer equation, with an extinction coefficient of 282 L mol⁻¹ cm⁻¹ and a 1 cm cuvette path length.

Statistical analysis and calculation of kinetic parameters

All acrylamide levels were given as mean ± standard deviation (SD) values in triplicate, and corresponding inhibitory rates were subsequently calculated. Compared to the TEAC_DPPH, TEAC_ABTS and TEAC_FRAP values in the control groups, three TEAC values of the final reaction products in experimental groups were calculated. The ΔTEAC values were then obtained via observing the difference between the TEAC of Maillard reaction products in the treatment groups and the control group. The correlation between ΔTEAC determined by three antioxidant evaluation assays and the degree of inhibition of acrylamide formation in the presence of catechins or esterified catechins was then investigated. Kinetic studies on the calculation of six kinetic parameters were statistically evaluated by the SAS software, version 8.2 (SAS Inst. Inc., Beijing, China). The kinetic parameters of both control and experimental groups were estimated by the Marquardt nonlinear least squares regression method. The significance test for kinetic parameters of each experimental group was conducted using the Student's t-test method compared to the control group.

Results and discussion

Comparison study on the reduction effect of catechins and esterified catechins on the formation of acrylamide

To evaluate the thermal stability of catechins/esterified catechins, eight different levels of each were added into currently established MHS (final concentrations: 10⁻¹¹–10⁻⁴ mol L⁻¹) without the presence of asparagine and glucose. Mimicking the microwave heating conditions of dose–response and kinetic studies, the catechin-containing, potato-based phosphate buffer solution was thermally processed at 180 °C. The alteration of catechin/esterified catechin levels was analysed in relation to the increase in microwave heating time by LC-MS/MS, according to previous work.²⁷ Results showed no significant change in all catechins/esterified catechins with the elapse of microwave heating time, indicating their sufficient thermal stability. A nonlinear and bell-shaped dose–response correlation between acrylamide concentrations in Maillard reaction products and addition levels of catechins/esterified catechins is shown in Fig. 3A. Results indicated that with different concentrations of catechin/esterified catechin treatments, the extent of inhibition of acrylamide formation caused by EC, ECG, EGC and EGCG ranged from 24.4%–50.1%, 50.6–71.6%, 32.5–58.4% and 58.7–91.9%, respectively. The optimal addition level of either catechins or esterified catechins for suppressing the formation of acrylamide was 10⁻⁹ mol L⁻¹. Results for all acrylamide concentrations in the final reaction products in experimental groups were significantly different from the acrylamide concentrations in the control group (P < 0.05). In detail, negative and positive dose–response relationships of acrylamide-formation inhibition rates were observed with the catechin/esterified catechin treatment ranges of 10⁻¹¹–10⁻⁹ mol L⁻¹ and 10^-9–10⁻⁴ mol L⁻¹, respectively. Such reverse tendency on the reduction of acrylamide formation may be related to the antioxidant activity of food matrices, the antioxidant property of Maillard reaction products, the inherent property of antioxidants, etc., which is a clear manifestation of the so-called “antioxidative paradox”.²⁸ First, a previous study demonstrated that the increase of cooking time leads to the enhancement of acrylamide levels, antioxidant property and colour in a biscuit product.²⁹ Such observation indicated that acrylamide concentrations may increase significantly with increasing the addition level of catechins/esterified catechins and advance the antioxidant property of reaction products due to the promotion of the Maillard reaction. Second, the acrylamide concentrations may decline due to the quinine–amine interaction between antioxidants and the direct precursor of acrylamide. Quinones are generated via the oxidation of the catechins/esterified catechins, and subsequently react with key intermediates such as 3-aminopropionamide.³⁰ The degree of inhibition of acrylamide formation via the effect of antioxidants may be evaluated by taking both factors into consideration and ensuring which factor plays a predominant role during the Maillard reaction.¹⁷

Fig. 3 The bell-shaped correlations between different treatment levels of non-esterified/esterified catechins and (A) acrylamide concentrations, (B) TEAC_DPPH values, (C) TEAC_ABTS values, or (D) TEAC_FRAP values in Maillard reaction products via microwave heating. Data are expressed as mean ± SD (n = 3).

It is well known that the reduction effect of food additives on the formation of acrylamide is closely related to the chemical structure of additives.³¹ The carbonyl value, a representative indicator for evaluating the state of lipid oxidation, may contribute to the formation of acrylamide. A positive correlation between the carbonyl value and acrylamide formation was observed in a model system.³² Further mechanistic study demonstrated that catechins reduce the formation of acrylamide, presumably by trapping carbohydrates and/or preventing lipid oxidation.³³ On the other hand, EC, ECG, EGC and EGCG belong to the flavanol group and their derivatives on a structural basis. The typical differences of chemical structure among different catechins and their derivatives are the functional groups in the 3- and 5′-positions of the flavanol skeleton. Results indicated that catechins containing a phenolic hydroxyl group in the 5′-position and/or a gallate group in 3-position of the flavanol skeleton showed a higher inhibitory effect on acrylamide formation than the catechins that did not contain these functional groups. Among all of the catechins, EGCG exerted the strongest inhibitory effect on the formation of acrylamide at all of its addition levels, which may be mainly ascribed to the chemical structure of both 5′-phenolic hydroxyl and 3-gallate. Compared to the effect of catechins EC and EGC, results revealed that the inhibitory effects of esterified catechins (ECG and EGCG) seemed much better, indicating that the 3-gallate group predominantly contributed to the inhibitory effects of catechins. Besides, the presence of the 3-gallate group in the chemical structures of ECG and EGCG introduces three additional aromatic phenol hydroxyl functional groups (Fig. 1), which may substantially contribute to the inhibitory effects on the formation of acrylamide.

Correlation between antioxidant properties of Maillard reaction products and inhibition of acrylamide formation

The nonlinear and bell-shaped dose–response relationship between the TEAC_DPPH/TEAC_ABTS/TEAC_FRAP values and the addition levels of catechins/esterified catechins is also shown in Fig. 3B–D. ΔTEAC_DPPH, ΔTEAC_ABTS and ΔTEAC_FRAP values representing the differences between TEAC of Maillard reaction products determined by three assay methods in the catechin treatment groups and the control group were calculated accordingly. A linear regression analysis was performed to examine whether ΔTEAC values showed a close correlation with extent of inhibition of acrylamide formation (Fig. 4A–C). The correlation coefficients (R²) showing the ΔTEAC-inhibitory rate relationship ranged from 0.757 to 0.907, indicating a close linear correlation between ΔTEAC values and acrylamide inhibitory rates among all of the investigated experimental data. It can be inferred from the phenomenon of the reaction that acrylamide formation was effectively suppressed, while the color of the final reaction products became faint. The decrease of melanoidins generated from the Maillard reaction may explain the reduction of antioxidant properties of the reaction system. This also means that catechins and esterified catechins may affect the Maillard reaction process; this needs to be confirmed by further kinetic studies. Summa et al.²⁹ investigated the relationship among the antioxidant activity, acrylamide concentration and colour of self-prepared cookies. However, the direct correlation between the antioxidant property of the reaction products and acrylamide concentration was not addressed. Subsequently, such direct relationship has been systemically investigated in various food matrices using different antioxidant evaluation methods. For example, a linear relationship between acrylamide concentration and total antioxidant capacity (TAC) was found during the frying process (R² = 0.8322).³⁴ Also, acrylamide concentration in biscuits made from two different wheat flour types—wholemeal and white flour type 550—correlated with FRAP and lightness (R²: 0.47–0.85).³⁵ These pioneer studies showed the establishment of a model system for simulating the Maillard reaction and the relationship between acrylamide levels and antioxidant properties of reaction products via changing the heating processing conditions, such as baking time, baking temperature, moisture content, etc. Our present study simultaneously demonstrated the reduction of acrylamide formation by catechins/esterified catechins and the correlation between the extent of inhibition of acrylamide formation and the antioxidant property of the reaction products. Moreover, high linear correlation coefficients were also observed to quantitatively demonstrate the close relationship. Ciesarová et al.³⁶ investigated the effect of other extracts (e.g. pimento, black pepper, marjoram and oregano) on the reduction of acrylamide contents, which was shown to correlate with antioxidant capacities, in particular with their DPPH-scavenging capacities (R = −0.996). The current study provides a novel experimental system comprised of a characteristic food matrix and equimolar asparagine–glucose model system. Such design of the experimental system not only avoids the interference from complex food matrices, but also shows the correlation between the reduction of acrylamide formation and antioxidant properties in mimic food systems.

Fig. 4 Correlation between the effect of catechins/esterified catechins on the reduction of acrylamide and ΔTEAC of Maillard reaction products. (A) ΔTEAC_DPPH-inhibitory rate relationship, (B) ΔTEAC_ABTS-inhibitory rate relationship, (C) ΔTEAC_FRAP-inhibitory rate relationship. Catechins, EC and EGC; Esterified catechins, ECG and EGCG.

Kinetic study of acrylamide formation and reduction by optimized addition of catechins

The optimized addition level of both catechins and esterified catechins (10⁻⁹ mol L⁻¹), which exerted a maximal reduction effect on the acrylamide formation, was used for the kinetic study in the equimolar asparagine–glucose model system. The kinetic model mimicking the Maillard reaction and related kinetic parameters are shown in Fig. 2. A representative UHPLC-MS/MS chromatogram describing the simultaneous determination of asparagine, glucose, fructose and acrylamide in Maillard reaction products using ¹⁵N₂-asparagine, ¹³C₆-glucose and D₃-acrylamide as isotope internal standards is shown in Fig. 5. Eqn (1)–(7) describe the dynamic formation and elimination processes of acrylamide. However, these equations need to be simplified in order to conveniently calculate the kinetic parameters. Based on the kinetic model, calculation results of the kinetic parameters, i.e. rate constants, describing the formation and elimination of acrylamide in the buffered equimolar asparagine–glucose microwave heating system are shown in Table 1. The results indicate that the kinetic parameters k₁, k₅ and k₆, which reflect the rate constants of the asparagine–glucose reaction, melanoidin formation and acrylamide degradation, respectively, were not significantly changed (P > 0.05). However, parameters k₂, k₃ and k₄, which represent the rate constants of conversion from glucose into fructose, asparagine–fructose reaction and the formation of acrylamide were much lower than those of the control group (P < 0.05). Therefore, the current study showed that catechins/esterified catechins selectively affect the kinetic process of the formation and elimination of acrylamide and significantly reduce the asparagine–fructose reaction pathway, not the asparagine–glucose reaction. Besides, the rate constant of acrylamide formation, k₄, calculated from the study in esterified catechin-containing, asparagine–glucose MHS was lower than the k₄ calculated from the study when catechins were added into the equimolar asparagine–glucose MHS; this could also indicate that esterified catechins exert a better reduction effect on acrylamide formation than catechins. Flavanol-related structures, such as catechins and esterified catechins, are known to interfere with the Maillard reaction through scavenging of reactive dicarbonyl compounds such as decarboxylated Amadori compound, which is an important intermediate present in the conversion from Schiff base into acrylamide.^37,38 Results of current kinetic investigations regarding the alternation of k₄ via the effect of catechins and esterified catechins were in accordance with the above mechanism. Combined with previous work regarding the correlation between the reduction of acrylamide and aromatic phenol hydroxyls in the chemical structures of catechins and esterified catechins, the finding that esterified catechins exert a better reduction effect on acrylamide formation than the reduction effect of catechins could be demonstrated in both dose–response and kinetic results. However, future mechanistic work should also focus on the effect of catechins and esterified catechins on the prevention of the asparagine–fructose reaction, though not the original asparagine–glucose reaction.

Fig. 5 A representative UHPLC-MS/MS chromatogram describing the simultaneous determination of asparagine, glucose, fructose and acrylamide in Maillard reaction products using ¹⁵N₂-asparagine, ¹³C₆-glucose and D₃-acrylamide as isotope-labelled internal standards.

Table 1 Parameter estimation of kinetic models via the effect of catechins on the reduction of acrylamide (n = 3)

Catechins/esterified catechins	Parameters of kinetic models^a
	k1 (min^−1)	k2 (min^−1)	k3 (min^−1)	k4 (min^−1)	k5 (min^−1)	k6 (min^−1)
a The significant difference was evaluated by Student's t-test. *P < 0.05, **P < 0.01.
Control group	0.143 ± 0.026	0.636 ± 0.009	0.718 ± 0.005	5.093 ± 0.447	4.121 ± 0.397	0.099 ± 0.009
EC	0.135 ± 0.043	0.577 ± 0.065*	0.591 ± 0.069*	3.033 ± 0.344**	4.167 ± 0.398	0.112 ± 0.036
ECG	0.105 ± 0.019	0.524 ± 0.019**	0.548 ± 0.018**	2.780 ± 0.541**	4.308 ± 0.223	0.101 ± 0.029
EGC	0.133 ± 0.037	0.529 ± 0.021**	0.558 ± 0.020**	2.888 ± 0.391**	4.115 ± 0.661	0.099 ± 0.014
EGCG	0.094 ± 0.015	0.523 ± 0.025*	0.537 ± 0.012**	2.134 ± 0.515**	4.050 ± 0.411	0.083 ± 0.012

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Conclusions

In the present work, we investigated the effect of catechins and esterified catechins on the formation of acrylamide in a potato-based, equimolar asparagine–glucose MHS via dose–response, antioxidant correlation and kinetic studies. Both catechins and esterified catechins could effectively reduce the generation of acrylamide in the Maillard reaction during microwave heating, with an inhibitory rate range of 24.4–91.9% in a nonlinear, bell-shaped dose–response manner using optimized addition levels of 10⁻⁹ mol L⁻¹, which was ascribed to the so-called “antioxidant paradox” phenomenon. The 5′-phenolic hydroxyl and 3-gallate groups in the chemical structures of catechins/esterified catechins play an important role in the reduction of acrylamide on a structural basis; thus the reduction effect of esterified catechins on the formation of acrylamide was better than the effect of catechins. Also, the acrylamide formation inhibition rates correlated well with the antioxidant properties of Maillard reaction products as simultaneously evaluated by three antioxidant capacity assays. Finally, the optimized use of both catechins and esterified catechins could significantly reduce the processes of conversion from glucose into fructose (k₂), the asparagine–fructose reaction (k₃) and the formation of acrylamide (k₄) during the Maillard reaction on a kinetic basis. The current study indicates that catechins or catechin-rich extracts (e.g. tea polyphenols) may substantially contribute to the reduction of acrylamide formation during microwave heat processing.

Acknowledgements

The authors gratefully thank the financial support by the National Natural Science Foundation of China (Grant no. 21277123), Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars of China (Grant no. LR12C20001) and Specialised Research Fund for the Doctoral Program of Higher Education of China (Grant no. 20120101120147).

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