Separation and characterization of the antioxidant active component from Maillard reaction products in xylose-lysine system

ABSTRACT

Maillard reaction products (MRPs) play a crucial role in preserving food quality due to their antioxidant capacity. In this study, we isolated and characterized the antioxidant components from MRPs using a xylose-lysine system under optimal conditions. Methylene chloride and ethyl acetate were employed for sequential extractions, and the ethyl acetate phase underwent thin-layer chromatography and silica column separation. The identified compound, 3-hydroxy-4-methyl-2(5 H)-furanone (3H4MFN), was analyzed using FT-IR, GC-MS, and NMR, revealing its volatile nature with a chemical formula of C₅H₆O₃. Further activity analysis demonstrated that 3H4MFN exhibited higher or comparable antioxidant activity to vitamin C (V_C) and vitamin B6 (V_B6). This newly discovered compound shows potential as a natural antioxidant in cosmetics, food, and nutraceutical applications.

Highlight

• A novel antioxidant has been identified from MRPs.

• The structure was determined by NMR, FT-IR and GC-MS.

• The newly identified compounds showed higher antioxidant activity than Vc and VB6.

KEYWORDS:

• Antioxidant activity
• Maillard reaction products
• xylose-lysine system
• 3-hydroxy-4-methyl-2(5H)-furanone
• separation and purification

1. Introduction

Non-enzymatic browning reaction, also well known as Maillard reaction (MR), is a very complicated reaction, which can be attributed to a series of different chemical changes during cooking, thermal processing, and food storage. It occurs between amino groups from amino acids, peptides, proteins, or any nitrogenous compounds and carbonyl groups of reducing sugars, aldehydes, and ketenes. Maillard reaction products (MRPs) such as ultraviolet absorbing intermediates, aroma compounds, and dark-brown polymeric compounds (usually have high molecular weight) called melanoidins (Jing & Kitts, 2004; Laroque et al., 2008; Morales & Jiménez-Pérez, 2001; Wijewickreme et al., 1997b), which play an important role in food properties, including stability, flavor, color, and shelf-life of several foods (Yilmaz & Toledo, 2005). Except for the food industry, MR has also been widely applied in other fields (Zhang & Tang, 2018). For instance, MR has been used to accelerate the absorption of acid and toxic dyes in the textile field (Trézl et al., 1995); in the leather field, modified MR is generally utilized to improve the affinity of toxic dyes to crust leather (Haroun & Mansour, 2008); MR has also been applied to colorize polyamide fibers in the fiber field, which increase the anti-bacterial activity of these fibers (Ohe & Yoshimura, 2013). More recently, the effects of MRPs on gut microbiota and their metabolic profile have also been studied (Liang et al., 2023).

The most well-known property of MRPs is their antioxidant activity (Hwang et al., 2011; Kim & Lee, 2009). Many studies have explored the correlation between the antioxidant activity and the browning intensity (A420 nm) of MRPs (Chen & Kitts, 2008, 2011). Non-volatile and high molecular weight compounds involved in MRPs are mainly contributing to their antioxidant activity, yet some volatile compounds isolated from MRPs may also possess antioxidant properties as reported. In recent years, a wide range of research has focused on sugar-amino acid model systems (Mondaca-Navarro et al., 2017), in which the antioxidant activity of MRPs was identified (Osada & Shibamoto, 2006; Yoshimura et al., 1997), sugar-protein model systems (Benjakul et al., 2005; F. Gu et al., 2009; Jing & Kitts, 2002), sugar-peptides model systems (Kim & Lee,2009, 2010; Oh et al.,1991, 1992), as well as in food applications (Manzocco et al., 2000; Morales & Jiménez-Pérez, 2004a). By using these systems, various active new components have been isolated from MRPs. Dai et al. investigated the functional properties and characteristics of MRPs generated from α-lactalbumin and polydextrose systems, resulting in increased radical-scavenging activity and reducing power (Dai et al., 2023). Feng et al. summarized several examples where active components were separated from MRPs, such as fructosyl arginine [N-α-(1-deoxy-D-fructos-1-yl)-L-arginine; Fru-Arg], a low molecular weight MRP that can be isolated from aged garlic extract, exhibits antioxidant activity in vitro (Feng et al., 2022).

In the present study, the 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity of MRPs in xylose-lysine system was first detected. Afterward, to separate and purify the active component from MRPs, a new separation system was developed through a single-factor experiment and uniform experimental design. Moreover, the structure of the newly identified component was confirmed by Nuclear Magnetic Resonance Spectroscopy (NMR), Fourier Transform Infrared Spectroscopy (FT-IR), and Gas Chromatography-Mass Spectroscopy (GC-MS). Finally, the antioxidant activities of the newly identified component were assessed.

2. Materials and methods

2.1. Chemicals and reagents

D-Xylose, L-Lysine and DPPH were ordered from Sigma Chemical Co., Ltd. (now Merck, U.S.A.). 2,2-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) was obtained from Tokyo Chemical Industry Co., Ltd. (Japan). Trichloroacetic acid (>99%), Potassium ferricyanide (>98%) and Ferric chloride (98%) were purchased from Alfa Aesar. K₂S₂O₈ (>99%) was ordered from Acros Organics. The other chemicals in this study were obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China) and analytical grade.

2.2. Preparation of MRPs

The reaction conditions and model systems used in this study were described in our previous study (Zhang & Zhang, 2014; Zhang & Zhou, 2013). DPPH radical-scavenging activity was described as one of the indicators of antioxidant activity. Four factors (temperature, heating time, pH value, and mass ratio) were taken into consideration. The objective of this study is to optimize the antioxidant activity of MRPs produced from a xylose-lysine model system using a single-factor experiment and a uniform experimental design (Zhang & Zhang, 2014). The optimal conditions were temperature 140°C, mass ratio (lysine: xylose) 3:1, pH 7.0 and retention time 60 min. The samples consisted of lysine (37.5 g) and xylose (12.5 g), dissolved in 450 ml of deionized water, and the pH was adjusted to 7.0 with 6 M NaOH or 4 M HCl. Then, we diluted the solution with deionized water to 500 ml as a final volume, and transferred it to 1000 ml round-bottom flasks, covered tightly and heated in a thermostatic oil bath at 140°C for 60 min. After being heated, the round-bottom flasks were immediately cooled down on ice and kept at 4°C until separated and purified.

2.3. Separation of antioxidant from MRPs

A large number of MRPs were prepared using the optimum reaction conditions. Every time, 500 ml MRPs mixture was extracted with methylene chloride and ethyl acetate in turn, each separation being performed three times using a 2000 mL separatory funnel. Finally, the methylene chloride phase and the ethyl acetate phase were used for analysis after vacuum concentration. The ethyl acetate phase was analyzed with thin-layer chromatography based on developing solvent (the petroleum ether: ethyl acetate = 1:4), the result demonstrated that products were not the raw material. Then, the ethyl acetate phase was separated through the thin-layer chromatography based on the developing solvent (the petroleum ether: ethyl acetate = 1:4) for two times, the products were joined in the silica gel column after separation (the eluent was ethyl acetate), the collected solution was used for related determination after vacuum concentration. NMR, FT-IR, GC-MS, and GC analysis were used for structure characterization.

2.4. Analysis of separated compounds

2.4.1. Sample analysis by Gas Chromatography (GC)

Samples were prepared according to a section of preparation of MRPs, followed by filtration through a 0.45 µm micropore filter before injecting into GC, which was performed using an Agilent Technologies 7820A (Agilent Technologies, U.S.A.) GC system, where a thermostatted column, i.e. Eclipse Plus Agilent 19091N-133 column (30 m × 250 mm, 0.25 µm), was used in combination with a Hydrogen fire Detector (FID). The analytical method was set up with an injector/detector temperature of 260°C, and a gradient oven temperature started from 35°C (kept for 5 min), followed by increasing with a rate of 5°C/min to 240°C (kept for 20 min). A 1 µL sample was injected each time with a 20:1 split ratio.

2.4.2. Sample analysis by Gas Chromatography-Mass Spectroscopy (GC-MS)

The chemical composition of the brown solution ethyl acetate extract was analyzed using an Agilent Technologies 7890A Series GC system coupled with a 5975C MS detector. The GC was employed with an Agilent INNOWAX column with 0.25 μm film thickness and 60 m × 0.25 mm (i.d.). The analytical method was set up with an injector temperature of 260°C, and a gradient oven temperature started from 50°C (kept for 2 min), followed by increasing with a rate of 5°C/min to 240°C (kept for 20 min). A 1 µL sample was injected each time with 1 mL/min flow rate of N₂ as carrier gas and a 10:1 split ratio. The Mass Spectroscopy condition was as follows: the transfer line between GC and mass detector was held at 280°C, followed by the detection with 35–550 a.m.u. scanning range and 70 eV ionization energy in the electron impact mode. Data were collected and processed using Xcalibur software, identifying volatiles by identifying the mass fragments from the sample and searching in the NIST11.L spectral library, and quantitative analysis of the peak area was conducted using integral calculus.

2.4.3. Sample analysis by Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR method was used to carry out infrared analysis, according to the method described by van der Venc et al. (2002). Approximately 20 mg of the extracted compounds blended with 180 mg of dried KBr (10%, w/w) were used for the spectroscopic measurements. The DRIFT mode of a VERTEX 70 FT-IR spectrometer (Bruker, Germany) was used to record the FT-IR spectra between 3800 and 400 cm⁻¹. The data for pure KBr were used to correct the background noise.

2.4.4. Sample analysis by Nuclear Magnetic Resonance (NMR)

The ¹H NMR, ¹³C NMR, and two-dimensional (2D NMR) correlation spectra were determined by a 400 MHz Agilent-NMR-vnmrs 400 spectrophotometer (Agilent Technologies, U.S.A.). The letters s, d, t, q, and m denote singlets, doublets, triplets, quartets, and multiplets, respectively, and the corresponding coupling constants are given in Hz; the internal standard used was TMS.

2.5. Antioxidant activity measurement

2.5.1. DPPH radical scavenging activity measurement

The DPPH radical scavenging activity of the MRPs was determined following the method described by Yen & Hsieh with slight modification (Yen & Hsieh, 1995). Briefly, a fresh DPPH in methanol solution (DPPH-MeOH) with 0.12 mM concentration was prepared before use and stored in the dark. An aliquot of 4.0 ml of DPPH-MeOH solution was mixed with 1.0 mL of 50-fold diluted MRP samples, and the mixtures were vortexed sharply and incubated for 30 min at room temperature and in a dark environment. The mixtures were measured using a spectrophotometer (Shimadzu UV 160A, Shimadzu Co., Kyoto, Japan) at 517 nm absorbance. The same preparation process was used for the control by substituting the MRP samples with distilled water. All samples were measured in triplicate. The percentage of DPPH radical-scavenging activity was determined by calculating the absorbance of the mixtures (Singh & Rajini, 2004).

(1) $\mathrm{Radical}\mathrm{scavenging}\mathrm{activity}\left(\mathrm{\%}\right)=\left[\left(A_C-A_S\right)/A_C\right]\times 100,$(1)

where A_s is the absorbance of the sample at 517 nm and A_c is the absorbance of the control at 517 nm.

2.5.2. ABTS radical scavenging activity measurement

The ABTS cation radical scavenging activity of the MRPs was determined using the method described by Re & Hwang in a modified version (Hwang et al., 2011; Re et al., 1999). The oxidation of ABTS by potassium persulfate led to the formation of blue/green color. When adding antioxidants to the system, ABTS·+ radicals will be quenched and decolorized, which can be spectrophotometrically monitored at 734 nm. The results can be indicated as (mM Trolox)/(mL of MRPs). Briefly, 2.45 mM potassium persulfate was mixed with 7 mM ABTS stock solution and then incubated at room temperature and in a dark environment for 12–16 h to generate ABTS·+ radicals, which are stable for 2 days. Then, 5 mM phosphate buffered saline (pH 7.4) was used to dilute the ABTS·+ radicals to obtain a solution with an absorbance of 0.70 ± 0.02 at 734 nm and further equilibrated at 30°C. The photometric assay was conducted by mixing a 3 mL diluted ABTS·+ solution with 50 µL of MRP samples for 45 sec and then measuring the absorbance at 734 nm after 6-min incubation. A control measurement was prepared using distilled water as a sample. The percentage of ABTS radical-scavenging activity was determined by calculating the absorbance of the mixtures. All samples were measured in triplicate.

(2) $\mathrm{ABTS}\mathrm{radical}\mathrm{scavenging}\mathrm{activity}\left(\mathrm{\%}\right)\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\left[\left(A_0-A_S\right)/A_0\right]\mathit{\hspace{0.17em}}\times \mathit{\hspace{0.17em}}100,$(2)

Where A₀ is the absorbance without the sample and As is the absorbance with the sample.

2.5.3. Reducing capacity measurement

The reducing capacity of MRP samples was determined using the method described by Oyaizu (Oyaizu, 1988) with slight modification. First, mix 1.0 mL of MRP sample (50-fold dilution) with 1.0 mL of 1% potassium ferricyanide (K₃Fe (CN)₆) and 1.0 mL of 0.2 M sodium phosphate buffer (pH 6.6). The mixture was then heated at 50°C for 20 min in a temperature-controlled water bath and cooled down to room temperature before adding 1.0 ml of 10% trichloroacetic acid. Afterward, the mixtures were separated by centrifugation at 3000 rpm and room temperature for 10 min, 1.0 mL of supernatant was extracted and mixed with 200 µL of 0.1% FeCl₃ and 1.0 mL of distilled water. The control sample was prepared by using distilled water instead of 1% potassium ferricyanide. The reducing capacity of the reaction mixture was assessed using a spectrophotometer at 700 nm. Absorbance was taken as a measure of the reducing power, with the lowest value being used as the initial reference value, and an increase in absorbance is indicative of an increase in reducing capacity. All samples were analyzed in triplicate.

(3) $\mathrm{Reducing}\mathrm{capacity}\left(\mathrm{\%}\right)\mathit{\hspace{0.17em}}=\mathit{\hspace{0.17em}}\left[\left(A_C-A_S\right)/A_C\right]\mathit{\hspace{0.17em}}\times \mathit{\hspace{0.17em}}100,$(3)

where A_s is the absorbance of the sample at 700 nm and A_c is the absorbance of the control at 700 nm.

2.6. Statistical analysis

All experimental data were expressed as mean ± standard deviation (SD). Means and standard deviations were calculated for each treatment using Origin 2018 and GraphPad Prism 9. The statistical analysis was performed using a statistical software package (Mathematics 4.0, Microsoft Corp., Redmond, Washington, U.S.A.). A value of p < .05 was considered to be significant.

3. Results and discussion

3.1. Separation and purification of the active component from MRPs

MRPs mixture was extracted with methylene chloride and ethyl acetate in turn (Y. L. Zhang & Zhang, 2014), and the ethyl acetate phase was analyzed with thin-layer chromatography based on the developing solvent (the petroleum ether: ethyl acetate = 1:4). The extracted MRPs mixture was then injected into the silica gel column for separation by using ethyl acetate as the eluent. After purification, the standard substance was dissolved in ultrapure water, making up different mass concentrations (0.0001, 0.0005, 0.002, 0.008, 0.01 mg/kg). The standard curve was then established by employing Gas Chromatography (GC) analysis. The mass concentration ranged from 0.0001 to 0.01 mg/kg, and the equation of linear regression was y = (1.1 × 1010) ×+8.6 × 105 (R² = 0.9984) which showed that there were good linear relationships between chromatographic peak area and concentration of the standard substance (Figure 1).

Figure 1. Standard curve of the separated and purified component.

3.2. Content changes in the separated and purified component

The separated and purified component (SPC) was considered as the standard substance. The standard substance (represented by a mass concentration of 0.01 mg/kg) of GC is shown in Figure S1. The content of the standard substance in the ethyl acetate crude products and the original products are shown in Figures S2 and S3, respectively. We found out the percentage of the standard substance in the original products and ethyl acetate crude products from GC analysis, which was also relevant to the value of antioxidant activity. From Figure S2, we know that the content of the target component in the ethyl acetate crude products was 60.5%. From Figure S3, we could know that the target component in the original products was 0.55%.

3.3. GC-MS analysis

GC-MS analysis identified the major aromatic compounds present in the MRPs. This method is quick and straightforward for identifying unknown compounds. Separated and purified component in the ethyl acetate phase was extracted from the browned xylose-lysine solutions which were further analyzed by GC-MS. The total ion current of the separated and purified component from GC-MS analysis is shown in Figure S4A. High-resolution Mass Spectrometry data of the separated and purified component is shown in Figure S4B. The spectra showed the peak position and peak intensity of the characteristic compounds, and then we could know the relative molecular mass of the compound is 114. With the relevant information from the GC-MS spectrogram and the High-resolution Mass Spectrometry data, we could confirm that the relative molecular mass of the compound is 114, and its chemical formula is C₅H₆O₃.

3.4. FT-IR analysis

Polymeric molecules, such as proteins, are tricky to analyze by spectroscopic method, caused by their complicated molecular structures, which affect the detection of molecular vibrations arising from many atoms. FT-IR spectroscopy is a valuable tool for the analysis of protein-carbohydrate systems, as it allows for the distinct identification of carbohydrate and protein components, due to their different spectral fingerprints in the mid-infrared range and their chemical fingerprints do not overlap significantly (Farhat et al., 1998; F.-L. Gu et al., 2010; Mondaca-Navarro et al., 2020). The strong amide I and II bands located between 1650 and 1540 cm⁻¹ in proteins are their specific features. In the mid-infrared spectrum, the most intense bands are often referred to as the “saccharide” bands, which are caused by the vibration modes of the stretching of C-O and C-C bonds, as well as the bending mode of C-H bonds, which are usually located in the region of 1180–953 cm⁻¹ in carbohydrates (Iconomidou et al., 2000; Lin et al., 1999). The presence of these absorptions is usually weak in the spectra of most proteins (Caillard et al., 2009). The infrared spectral changes of the separated and purified component are shown in Figure 2. Regarding the infrared spectrum of the separated and purified component, the assignments of several peaks have already been clarified. In this study, the absorption peaks at 2955 and 1460 cm⁻¹ were attributed to the observed primary methyl and methylene groups, respectively. The adsorption bands at around 1697 and 1641 cm⁻¹ were attributed to the C=C stretching was also observed. Moreover, the changes in the 1300–1000 cm-1 regions could be attributed to the C-O-C and C=O of the ester group asymmetric stretching vibration (1300–1150 cm-1 the stronger) and symmetric stretching vibration (1300–1150 cm⁻¹ the weaker). Finally, the infrared spectra displayed the infrared spectral changes of the separated and purified component, and there was a C-OH (primary alcohol) band at 1048 cm⁻¹ (Doroshenko et al., 2013). The infrared spectral changes of the separated and purified component were consistent with the results of GC-MS, NMR and High-resolution Mass Spectrometry.

Figure 2. FT-IR spectroscopy of the separated and purified component.

3.5. NMR analysis

The structure of the separated and purified component (3-hydroxy-4-methyl-2(5 H)-furanone) was further determined by the combination of¹H NMR (Figure S5), ¹³C NMR (Figure S6) and two-dimensional NMR (Figure 3) analysis. The NMR spectra of the active component were described as follows: ¹H NMR (400 MHz, d, ppm): 8.33 (s, 1 H, H-OH), 4.52 (s, 2 H, H-5), 2.13 (s, 3 H, CH₃); ¹³C NMR (100 MHz, d, ppm) 195.45 (C-2), 172.97 (C-1), 134.52 (C-3), 72.54 (C-4), 13.12 (CH₃).

Figure 3. Two-dimensional correlation NMR spectra of the separated and purified component.

3.6. Measurement of the radical scavenging activity

3.6.1. Changes in the DPPH radical scavenging activity

The hydrogen-donating ability of antioxidants has been widely studied through the use of free DPPH radical scavenging activities (Lertittikul et al., 2007), which have been utilized to measure the purity of antioxidant compounds in food materials, fruit extracts and plants (Wong et al., 2006). The antioxidant activity of MRPs in sugar-amino acid model systems has been the focus of many studies. In these studies, Yen and Hsieh (Yen & Hsieh, 1995) have reported that MRPs from xylose-lysine system displayed DPPH radical scavenging activity. When antioxidants donate hydrogen to scavenge the DPPH radical, the formation of a stable DPPH-H molecule results in a color change from purple to yellow (Shon et al., 2003). The changes in the DPPH radical scavenging activity of the separated and purified component, the ethyl acetate crude products, the original products and natural antioxidants are depicted in Figure 4. We found that V_C has the highest DPPH radical scavenging activity, and the separated and purified component has the relatively high DPPH radical scavenging activity (86.1% vs 90.5% of V_C) from Figure 4.

Figure 4. The changes in the DPPH radical scavenging activity. Control: blank without active antioxidant component; OP: original products; CP: crude products extracted by ethyl acetate; SPC: separated and purified component; V_B6: vitamin B6; V_C: vitamin C.

3.6.2. Changes in the ABTS radical scavenging activity

The ABTS method is a widely used technique for verifying the antioxidant activity of MRPs, which is performed by calculating the percentage of the absorbance inhibition at 734 nm caused by the reduction of the radical cation. The total antioxidative capacity of beverages, aqueous mixtures and pure substances can be determined by the generation of the ABTS radical cation and further monitored by spectrophotometric method. The ABTS radical scavenging activity in the separated and purified component was higher than in those of the other components as shown in Figure 5. This test result was similar to the DPPH free radical scavenging percentage clearance, indicating that the separated and purified component has strong antioxidant activity.

Figure 5. The changes in the ABTS radical scavenging activity. Control: blank without active antioxidant component; OP: original products; CP: crude products extracted by ethyl acetate; SPC: separated and purified component; V_B6: vitamin B6; V_C: vitamin C.

3.7. Changes in the reducing capacity

The reduction of ferric iron (Fe³⁺) to ferrous iron (Fe²⁺) is commonly used to determine the reducing capacity of the antioxidant active components isolated from MRPs, which could detect the presence of ferrous iron (Fe²⁺) by the Prussian blue absorbance under 700 nm. Therefore, the reducing capacity of MRPs can be reflected by the increase in absorbance, which can be used to measure the efficacy of their reducing power. Our reports confirmed that the reducing capacity of MRPs increases as the heating time increases, which is demonstrated by an increase in absorbance at 700 nm. The separated and purified component exhibited the greatest reducing capacity, even higher than V_B6 and V_C (Figure 6).

Figure 6. The changes in reducing capacity. Control: blank without active antioxidant component; OP: original products; CP: crude products extracted by ethyl acetate; SPC: separated and purified component; V_B6: vitamin B6; V_C: vitamin C.

4. Discussion

The Maillard reaction is a reaction between a carbonyl compound and an amino compound. Most foods contain amino and carbonyl groups, so this reaction is easy to occur, which not only affects the color of food but also has an important impact on the flavor of food (Doroshenko et al., 2013; Wong et al., 2006) and has been widely used in the food industry. Since Maillard reaction products (MRPs) are mixtures containing a large number of complex products, it is impractical to measure the antioxidant activity of each compound in the mixture to identify the compound with the main antioxidant activity. Therefore, it is necessary to find a practical method to identify the key substances that give rise to antioxidant activity in complex mixtures. In recent years, although a large amount of literature has reported the antioxidant activities of amino acid-reducing sugar simulation systems (Du et al., 2004; Mondaca-Navarro et al., 2017), protein-reducing sugar simulation systems (Chen et al., 2012; Y. F. Wang et al., 2010; H. Y. Wang et al., 2011), peptide-reducing sugar simulation systems (Botsoglou et al., 2002; R. Y. Gu et al., 2008; Pei et al., 2011; Wang & Xu, 2008), and Maillard reaction products in food systems (Manzocco et al., 2001; Morales & Jiménez-Pérez, 2004b); however, only a few studies have investigated how to elucidate the preliminary mechanism of antioxidant activity of Maillard reaction products. Studies have found that furanone compounds were detected in the Maillard reaction products of sugar (xylose or glucose)-glycine model systems and xylose-lysine model systems using NMR and mass spectrometry, and proved to have antioxidant activity, which is consistent with our research result (Ames et al., 1999; Bailey et al., 2000). In our study, xylose-lysine was used as the substrate to simulate the Maillard reaction. Under the basic conditions of its optimal antioxidant activity, the component with antioxidant activity was separated and purified by different solvent systems, polarity, molecular weight, etc., and the structural characterization of the separated and purified component was studied by nuclear magnetic, infrared, mass spectrometry and gas chromatography analysis, and proved they have strong antioxidant activity. It provides a theoretical basis for the application of Maillard reaction products as natural antioxidants in industry. It establishes a bridge for the development of new natural antioxidants compared with traditional antioxidants.

Some studies on Maillard reaction products have shown that the products obtained from the model Maillard reaction have a variety of biological activities, such as antimutagenic effects and hypotensive effects (Borrelli & Fogliano, 2005; Finot et al., 1900; Y. M. Kim et al., 2004; Rufia´n-Henares & Morales, 2007; Somoza, 2005; Wagner et al., 2007; Wijewickreme et al., 1997a). It is worth noting that the antioxidant activity of Maillard reaction products has the potential to replace traditional chemical synthesis of antioxidants, which is because Maillard reaction products are a type of compounds formed in the process of food processing and storage, which can be considered natural, and the commonly used traditional antioxidants are mostly some chemical synthesis of phenolic substances. Such as BHT, BHA, TBHQ, tocopherol, and so on. In recent years, some studies have shown that chemically synthesized antioxidants such as BHT and BHA may have carcinogenic effects, so they have been banned by many countries, and TBHQ has also been restricted. Therefore, the research and development of natural antioxidants has become a hot spot at home and abroad. In our study, the antioxidant capacity (free radical scavenging capacity, reducing capacity, and metal ion chelation capacity) of the initial Maillard reaction crude product, ethyl acetate crude product, separated and purified components, and some natural antioxidants were determined, and it was verified that the antioxidant capacity of the separated and purified components and the standard products was similar. This provides a theoretical basis for the development and research of new natural antioxidants. Due to the limitations of experimental conditions, time, and many other aspects, this experiment only conducted a preliminary study on the antioxidant activity of the reaction product, its cytotoxicity or safety for possible consumption was not evaluated, and many issues require further research.

5. Conclusions

Research into the separation and determination of antioxidant substances in MRPs is necessary due to the varied antioxidant activities with different MRPs in different MR conditions. This study demonstrated that the separated and purified component had a potent antioxidant activity. In conclusion, the potential of MRP as a novel antioxidant has been evaluated through a review of its functional properties. This research of Maillard reaction products as an antioxidant in industry provides the theoretical basis for the application, and at the same time, the bridges were established for developing new natural antioxidants compared with traditional antioxidants.

Author contributions

Conceptualization, Lu-Yan Zhang and Yin-Liang Zhang; methodology, Lu-Yan Zhang and Guo-Qing Shi; investigation, Lu-Yan Zhang; data curation, Lu-Yan Zhang and Yan Zhang; writing – original draft preparation, Lu-Yan Zhang and Yan Zhang; writing – review and editing, Lu-Yan Zhang, Yin-Liang Zhang, Yan Zhang and Pengfei Zhou; funding acquisition, Lu-Yan Zhang. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

We thank the analysis and testing center of Zhengzhou University of Light Industry for helping with the GC, GC-MS, FT-IR, and NMR spectra collection.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19476337.2024.2348739

Additional information

Funding

This work was funded by the PhD student Fund at the University of Camerino and the Basic and Frontier Program Research Project of Henan Province [grant 112300410145].