A multi-target cascade inhibition method for blocking MeIQx formation and maintaining food quality

Abstract

The application provides an inhibition method for multi-target cascade blocking MeIQx formation and maintaining food quality, and belongs to the technical field, and comprises the following steps: adding GSH-lactose@UVC-GA complex to food raw materials; the complex inhibits MeIQx formation and maintains food quality through the following multi-target cascade blocking mechanism: directly degrading the generated MeIQx molecules; consuming the precursor substances required for MeIQx formation, including threonine, creatinine and glucose; blocking the Amadori rearrangement, Strecker degradation, hydroxy aldehyde condensation and / or imine-enamine tautomerism steps in the MeIQx formation cascade reaction path; removing the carbonyl intermediates in the MeIQx formation process, including glyoxal and methyl glyoxal; removing free radicals, reducing the lipid oxidation level and the protein oxidation degree. The application synchronously inhibits imine-enamine tautomerism, hydroxy propionaldehyde dehydration, Amadori rearrangement and Strecker degradation through the multi-target cascade blocking mechanism, significantly improves the key reaction energy barrier, and realizes the MeIQx inhibition effect of more than 90%.

Description

Technical Field

[0001] This invention relates to the field of technology, and more particularly to an inhibition method for blocking the formation of MeIQx through a multi-target cascade and maintaining food quality. Background Technology

[0002] Heat processing is a core technology in the food industry for imparting flavor, texture, and storage stability to food ingredients. However, the accompanying Maillard reaction, Strecker degradation, and free radical cascade reactions inevitably generate heterocyclic amines (HCAs), which are highly carcinogenic pollutants. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), as one of the most widely distributed and mutagenic heterocyclic amines in heat-processed foods, has been classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). Its accumulation in meat and seafood products has become a core global safety barrier restricting the greening and health-oriented upgrading of the food industry.

[0003] Current strategies for controlling MeIQx in the food system have long been constrained by paradigmatic technological bottlenecks, with traditional research failing to overcome the limitations of single function, single target, and single effect. Physical adsorption methods can only achieve the transfer of pollutants between different phases, but cannot achieve chemical degradation at the molecular level, and their targeting and removal efficiency are insufficient to meet the needs of large-scale processing. Chemical oxidation or strong reducing agent treatment can easily introduce exogenous toxic substances and significantly damage the nutritional structure and sensory quality of food, which contradicts the concept of green processing. Single-component natural inhibitors such as polyphenols and amino acids generally suffer from insufficient stability, limited target sites, and negative impacts on matrix pH and color, making it difficult to achieve efficient degradation and quality maintenance in a coordinated manner. Furthermore, although natural eutectic solvents (NADES) have been proven to be green and biocompatible functional assembly media, and glutathione, lactose, and UV-modified gallic acid (UVC-GA) possess the molecular potential to target and quench free radicals, nucleophilically block reactions, and stabilize the structural framework, research on the directional assembly of these three components into multifunctional catalytic clusters via NADES for blocking the MeIQx molecular-level cascade reaction remains completely lacking globally. Therefore, a multi-target cascade inhibition method is needed to block MeIQx formation and maintain food quality. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-target cascade inhibition method for MeIQx formation and maintaining food quality, thus solving existing technical problems. This invention uses a choline chloride-lactic acid type natural eutectic solvent as a green medium to directionally synthesize glutathione-lactose / UV-irradiated gallic acid clusters, thereby overcoming the technical bottlenecks of lack of synergistic effect in physical mixing of natural functional molecules and safety hazards associated with chemical cross-linking.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A multi-target cascade inhibition method for blocking MeIQx formation and maintaining food quality includes the following steps: Add the GSH-lactose@UVC-GA complex to food ingredients; The complex inhibits MeIQx formation and maintains food quality through the following multi-target cascade blocking mechanism: (a) Direct degradation of the generated MeIQx molecules; (b) Consuming the precursors required to form MeIQx, said precursors include threonine, creatine anhydride and glucose; (c) Blocking the Amadori rearrangement, Strecker degradation, aldol condensation and / or imine-enamine tautomerization steps in the MeIQx formation cascade pathway; (d) Remove carbonyl intermediates in the formation of MeIQx, including glyoxal and methylglyoxal; (e) Scavenging free radicals reduces lipid oxidation levels and protein oxidation.

[0006] Furthermore, the amount of the GSH-lactose@UVC-GA complex added is 0.01 to 5 wt% of the food raw material. Furthermore, the food ingredients are surimi products, meat products, dairy products, or grain products; the surimi products include squid surimi, tilapia surimi, and shrimp surimi. Furthermore, the preparation method of the GSH-lactose@UVC-GA complex includes the following steps: Step S1: Irradiate the gallic acid aqueous solution under a UVC light source to obtain a UVC-GA solution; Step S2: Glutathione is reacted with lactose in a solution to obtain a GSH-lactose derivative; Step S3: The GSH-lactose derivative obtained in step S2 and the UVC-GA solution obtained in step S1 are subjected to a self-assembly reaction in a natural eutectic solvent to obtain the GSH-lactose@UVC-GA complex. Furthermore, the wavelength of the UVC light source is 254 nm, and the average radiant intensity of the UVC light source is 4540 μW / cm². 2 The distance between the UVC light source and the liquid surface is set to 15 cm, and the irradiation time of the UVC light source is 6 h; the concentration of the gallic acid aqueous solution is 14 mmol / L.

[0007] Furthermore, the molar ratio of glutathione to lactose is 1:3; the solvent is deionized water; and the reaction is carried out in an oil bath at 130°C for 30 min.

[0008] Furthermore, the natural eutectic solvent is a mixture of choline chloride and lactic acid, wherein the molar ratio of choline chloride to lactic acid is 1:1 to 1:4; and 20 wt.% of deionized water is added to the mixture.

[0009] Furthermore, the molar ratio of the GSH-lactose derivative to the UVC-GA solution is 1:1; the self-assembly reaction is carried out under conditions of pH 3.0-6.0.

[0010] Furthermore, the self-assembly reaction temperature is 25-55°C; the self-assembly reaction time is 1-7 h. The present invention, by adopting the above-described technical solution, has the following beneficial effects: This invention utilizes the hydrogen bond network of the natural eutectic solvent of choline chloride / lactic acid to encapsulate and protect the active ingredient. At the same time, it uses UVC light treatment to photochemically modify gallic acid, generating additional free radical active sites and enhancing the intermolecular interaction between the active ingredient and MeIQx and its precursors.

[0011] The complex of this invention, while inhibiting the formation of MeIQx, effectively inhibits lipid oxidation and protein oxidation through its excellent free radical scavenging ability, and can simultaneously maintain the color quality and pH stability of food, thus achieving a synergistic unity between inhibiting the formation of harmful substances and maintaining food quality.

[0012] The complex of this invention achieves a systematic blockade of the MeIQx formation pathway through five synergistic mechanisms: direct degradation of already generated MeIQx; consumption of precursor substances threonine, creatinine, and glucose, cutting off the supply of raw materials for MeIQx synthesis from the source; blocking the Amadori rearrangement, Strecker degradation, aldol condensation, and imine-enamine tautomerism in the cascade reaction pathway; scavenging key carbonyl intermediates glyoxal and methylglyoxal; and scavenging free radicals and reducing lipid and protein oxidation.

[0013] This invention utilizes a multi-target simultaneous inhibition mechanism of the cascade reaction of MeIQx formation through the GSH-lactose@UVC-GA complex. By raising the reaction activation barrier of key steps, it inhibits the transformation processes of core intermediates such as imine-enamine tautomerism, hydroxypropionaldehyde dehydration, Amadori rearrangement, and Strecker degradation at the source, achieving a paradigm shift from passive scavenging to active pathway intervention. Simultaneously, it demonstrates that this material possesses a triple function: highly efficient catalytic degradation of MeIQx, inhibition of lipid and protein oxidation, and maintenance of stable sensory quality in food, successfully solving the common industry problem of the difficulty in achieving both effectiveness and quality protection in traditional control strategies. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on MeIQx yield; Figure 2 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on threonine production; Figure 3 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on creatinine production; Figure 4 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on glucose production. Figure 5 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA composite of the present invention on total aldehyde yield; Figure 6 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA composite of the present invention on TBARS yield; Figure 7 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on the carbonyl valence formation. Figure 8 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on the intermediate content; Figure 9 This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on the content of precursor substances; Figure 10 This is a characterization diagram of the degradation process of MeIQx catalyzed by the GSH-lactose@UVC-GA complex of the present invention; Figure 11 This is an analytical diagram showing the blocking effect of the GSH-lactose@UVC-GA complex of the present invention on the cascade reaction during the degradation of MeIQx; Figure 12This is a schematic diagram illustrating the effect of the GSH-lactose@UVC-GA complex of the present invention on the degradation and quality characteristics of MeIQx in three types of fish paste. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0016] Example 1. Preparation of GSH-lactose@UVC-GA complex The preparation method of GSH-lactose @ UVC-GA complex includes the following steps: Step S1: Irradiate the gallic acid aqueous solution under a UVC light source to obtain a UVC-GA solution; The UVC light source has a wavelength of 254 nm and an average radiant intensity of 4540 μW / cm². 2 The distance between the UVC light source and the liquid surface is set to 15cm, and the irradiation time of the UVC light source is 6 hours. The concentration of the gallic acid aqueous solution was 14 mmol / L.

[0017] Step S2: Glutathione is reacted with lactose in a solution to obtain a GSH-lactose derivative; The molar ratio of glutathione to lactose is 1:3; The solvent is deionized water, and the reaction is carried out in an oil bath at 130°C for 30 min.

[0018] Step S3: The GSH-lactose derivative obtained in step S2 and the UVC-GA solution obtained in step S1 are subjected to a self-assembly reaction in a natural eutectic solvent to obtain the GSH-lactose@UVC-GA cluster. The natural eutectic solvent is a mixture of choline chloride and lactic acid, wherein the molar ratio of choline chloride to lactic acid is 1:1 to 1:4. Add 20 wt.% of deionized water to the mixture; The molar ratio of the GSH-lactose derivative to the UVC-GA solution is 1:1; the self-assembly reaction is carried out under conditions of pH 3.0-6.0. The self-assembly reaction temperature is 25-55°C; the self-assembly reaction time is 1-7 h.

[0019] 2. Evaluation of the multi-target cascade blocking effect of the complex on MeIQx A systematic evaluation was conducted on the multi-target cascade blocking effect of the GSH-lactose@UVC-GA complex prepared above on MeIQx.

[0020] 2.1 Construction of the MeIQx Simulation System Add 5.0 mL of phosphate buffer (pH 8.0) to a high-temperature and high-pressure reaction flask, followed by 0.4 mmol of threonine, 0.4 mmol of creatinine, 0.2 mmol of glucose, and 200.0 μL of soybean oil. Heat the reaction system in oil baths at 150℃, 180℃, and 210℃ for 8 min, respectively. After the reaction is terminated, immediately quench the reaction with an ice-water bath to prepare the MeIQx simulation system, which is then stored for later use.

[0021] 2.2 Four groups of experiments were set up respectively. Control group: MeIQx simulation system + no added complex + no light exposure GA group: MeIQx simulation system + 1 mmol gallic acid UVC-GA group: MeIQx simulation system + 1 mmol UVC-treated gallic acid Experimental group: MeIQx simulation system + 1 mmol GSH-lactose@UVC-GA complex.

[0022] 2.3 Sample preparation and MeIQx quantification 1.0 mL of the simulated MeIQx reaction solution from each treatment group was transferred, and the samples were extracted with an equal volume of ethyl acetate (5 min each time, repeated 3 times). After purification using an Oasis MCX solid-phase extraction column, the MeIQx content was quantitatively analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) in multiple reaction monitoring mode. The degradation rate was calculated using the following formula: MeIQx degradation rate = (MeIQx concentration in control group) (Residual MeIQx concentration in the treatment group) / (MeIQx concentration in the control group) × 100% 3. The effect of the complex on the consumption of MeIQx precursor materials The precursors threonine, creatinine, and glucose were quantified by HPLC. Carbonyl intermediates (glyoxal and methylglyoxal) were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and headspace gas chromatography-mass spectrometry (GC-MS).

[0023] 4. Inhibitory effect of the complex on lipid oxidation and protein oxidation 4.1 Lipid oxidation level (TBARS method) Weigh 200 mg of sample, add 10 mL of 7.5% trichloroacetic acid, homogenize under ice bath conditions, and centrifuge at 10000 r / min for 15 min. Take 4 mL of the supernatant and mix with an equal volume of 0.02 mol / L thiobarbituric acid, heat in a boiling water bath for 40 min, cool, and measure the absorbance at 532 nm. Use 1,1,3,3-tetraethoxypropane as a standard. The results are expressed as mg MDA / kg.

[0024] 4.2 Protein carbonyl content (DNPH derivatization method) Weigh 100 mg of sample and homogenize with 10 mL of 0.1 mol / L phosphate buffer (pH 7.4) to extract protein. Centrifuge and collect the supernatant. Take 0.5 mL of the supernatant and react it with 1 mL of 10 mmol / L DNPH (dissolved in 2 mol / L HCl) and 2 mol / L HCl in the dark for 60 min. Add 1 mL of 20% trichloroacetic acid to precipitate the protein. Wash the precipitate three times with ethanol-ethyl acetate (1:1, v / v), redissolve it in 1.5 mL of 6 mol / L guanidine hydrochloride, and measure the absorbance at 370 nm, according to ε = 22000 L·mol⁻¹. -1 ·cm -1 Calculations were performed, and the results are expressed as nmol / mg protein.

[0025] 4.3 Total sulfhydryl content of protein (DTNB method) Take 1.0 mL of protein extract, add 2.9 mL of Tris-urea-EDTA buffer (pH 8.0) and 40 μL of 10 mmol / L DTNB, react at room temperature in the dark for 30 min, and measure the absorbance at 412 nm, according to ε = 14150 L·mol⁻¹. -1 ·cm -1 Calculations were performed, and the results are expressed as nmol / mg protein.

[0026] 5. Recyclability test of the compound After the reaction in the experimental group was terminated, the GSH-lactose@UVC-GA complex was recovered by centrifugation and washed three times with ultrapure water. After freeze-drying, 10 mg of the lyophilized sample was weighed and dissolved in 10.0 mL of 0.1 mol / L phosphate buffer (pH 7.4) to prepare a stock solution with a concentration of 1.0 mg / mL (equivalent to 1 mmol / L). This stock solution was reused for degradation experiments for a total of 5 cycles. The MeIQx degradation rate was determined using the same method as described above.

[0027] 6. The free radical scavenging activity of the complex and intermolecular interactions Electron paramagnetic resonance (EPR) spectroscopy was used to detect free radicals, with 5,5-dimethyl-1-pyrrololine N-oxide (DMPO, final concentration 50 mmol / L) as the free radical scavenger. 5.0 mg of each experimental group sample was weighed and dissolved in 10.0 mL of ultrapure water to prepare a 0.5 mg / mL solution. 200 μL of the reaction system was analyzed using a Bruker EMXnano spectrometer with the following parameters: central magnetic field 3425 G, scan width 100 G, microwave power 10 mW, modulation frequency 100 kHz, and modulation amplitude 1 G. Free radical scavenging activity was determined by DPPH and ABTS methods, using 0.2 mg / mL diluted samples and vitamin C (as a positive control). Absorbance was measured at 517 nm (dark reaction 30 min) and 734 nm (dark reaction 6 min), respectively. Radical scavenging rate (%) = ×100. Intermolecular interactions were characterized by the shifts of characteristic functional groups using FTIR analysis. 1.0 mg of each sample was weighed and mixed with KBr at a mass ratio of 1:100, compressed into tablets, and analyzed using a Nicolet iS50 spectrometer at 4000-400 cm⁻¹. -1 A total of 32 scans were performed within the wavenumber range.

[0028] 7. Kinetic and thermodynamic analysis of the complex The catalytic degradation kinetics of MeIQx in different treatment groups were determined using the initial rate method. First, 10.0 mg of lyophilized sample was dissolved in 10.0 mL of phosphate buffer (pH 7.4) to prepare a 1 mmol / L stock solution. The initial concentration [E]0 of the active component was determined using the BCA method. In a 1.0 mL reaction system, a final concentration of 0.1 mmol / L of sample and a MeIQx solution with a concentration gradient of 0.05–0.8 mmol / L were added, and the mixture was incubated at 37 °C under 365 nm UV light. Samples were taken at time points of 0, 2, 5, 10, 15, 20, and 30 min. After quenching the reaction with ice water, the residual amount of MeIQx was quantified using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The maximum reaction rate (V0) was calculated by fitting the data using the Lineweaver-Burk double reciprocal method. max ), Mie constant (K m ) and catalytic constant (k) catKinetic parameters such as kinetic parameters were determined. All experiments were independently repeated three times. Differential scanning calorimetry (DSC) was used to evaluate the thermal stability and thermodynamic parameters of the complex. Each sample was mixed with MeIQx at a 1:1 molar ratio and dissolved, then pre-frozen and freeze-dried until constant weight. 5–10 mg of sample was placed in an aluminum crucible, using an empty crucible as a reference, and measurements were taken within a temperature range of 30–250 °C at a heating rate of 10 °C / min and a nitrogen flow rate of 50 mL / min. The transition temperature (Tm) and enthalpy change (ΔH) were analyzed using the accompanying software, and the entropy change (ΔS) and Gibbs free energy change at 298 K were calculated using thermodynamic formulas. Each sample was measured three times, and the average value was taken.

[0029] 8. Blocking effect of the complex on the cascade reaction pathway Dissolve 10.0 mL of each sample solution in 10.0 mL of 0.1 mol / L phosphate buffer (pH 8.0) to prepare a 1.0 mg / mL test solution. Take 100 μL aliquots at time points 0, 1, 2, 4, 6, and 8 minutes. Amadori rearrangement and Strecker degradation reactions were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Aldol condensation products were determined by high-performance liquid chromatography (HPLC), and the imine-enamine tautomerism process was monitored by UV-Vis spectroscopy. Optimization calculations were performed using Gaussian 16 at the B3LYP / 6-311G (d, p) level, and molecular electrostatic potential and reduced density gradient analysis were conducted using Multiwfn software.

[0030] 9. Application and quality evaluation of the complex in the surimi model 9.1 Sample Preparation Fish paste samples (including squid paste, tilapia paste, and shrimp paste) were thawed at 4°C, and visible connective tissue was then removed. 10.0 g of fish paste, along with 1% (w / w) of various antioxidants by weight of the fish paste, was accurately weighed and thoroughly mixed with the fish paste homogenate. The experiment included 15 treatment groups: CG group (three types of fish paste, no antioxidants added), BHT group (three types of fish paste, with added butylated hydroxytoluene), VC group (three types of fish paste, with added vitamin C), GA group (three types of fish paste, with added gallic acid), and EG group (three types of fish paste, with added GSH-lactose@UVC-GA).

[0031] Fish paste was molded into a disc shape in a glass petri dish (4 cm in diameter, 0.4 cm in height) and baked in an oven preheated to 230 °C for 5 min on each side. The fish paste was then mixed with 20 mL of 1 mol / L sodium hydroxide and stirred thoroughly for 3 min to form a thick paste. 20 mL of n-hexane was added and stirred vigorously. The mixture was centrifuged at 4000 g for 8 min to defatted the paste, and the upper n-hexane layer was discarded. The sample was extracted with an equal volume of ethyl acetate (5 min each time, repeated 3 times) and purified using an Oasis MCX solid-phase extraction column. 9.2 Determination of Oxidation Degree and Quality Indicators After heat treatment at 180℃ for 8 min, the residual MeIQx content, lipid oxidation degree, and protein oxidation degree of each treatment group were measured using the same methods as described above. Color was measured using a colorimeter (Konica Minolta, Japan) under a D65 standard light source, determining the L*, a*, and b* values. pH was measured using a pH meter after homogenizing the sample with deionized water at a 1:9 mass ratio.

[0032] 10. Results The results show that heating temperature has a significant impact on all indicators.

[0033] Heating at 150℃, 180℃, and 210℃ for 4 minutes respectively, the MeIQx content in the GSH-lactose@UVC-GA experimental group was significantly lower than that in the CG group (control group), GA group, and UVC-GA group, reaching the lowest value among all experimental groups. Figure 1 As shown, this complex exhibits a specific response to the regulation of MeIQx precursor content. Specifically, under the same heating conditions, the contents of key precursors such as threonine, creatinine, and glucose in the GSH-lactose@UVC-GA group were significantly lower than in the other experimental groups. Furthermore, there is a synergistic effect between temperature increase and precursor degradation; at 210℃, the precursor content significantly decreased, such as... Figure 2-4 As shown. Conversely, creatinine content, total aldehyde value, TBARS, and carbonyl value all increased significantly with increasing temperature, such as... Figure 5-7 As shown, this indicates that not only does the MeIQx precursor accumulate continuously during this process, but the lipid oxidation reaction also tends to be more intense.

[0034] The results showed that in the MeIQx simulated reaction system, the degradation rate of MeIQx in all treatment groups reached its highest under the condition of heating at 180°C for 8 min. Under this representative condition (180°C, 8 min), significant differences were observed among the four treatment groups. Compared to the CG group, the GA group and the UVC-GA group both showed a certain degree of inhibitory effect on MeIQx and its precursors; while in the GSH-lactose@UVC-GA group, the contents of MeIQx, glucose, creatinine, and threonine were reduced to the lowest levels. Figure 9 As shown. Similarly, the content of Merard reaction intermediates (such as aldehydes and pyrazines) in this group was also significantly lower than in other groups, such as... Figure 8 As shown.

[0035] Multi-omics characterization results showed that the GSH-lactose@UVC-GA group exhibited a significant advantage in degrading MeIQx compared to the CG, GA, and UVC-GA groups. HPLC analysis of the reaction products revealed the presence of MeIQx in all treatment groups. Figure 10 As shown in a. The GSH-lactose@UVC-GA group had the lowest MeIQx residue, at only 9.23 ng / mL, significantly lower than the CG group (125.74 ng / mL), the GA group (84.84 ng / mL), and the UVC-GA group (55.41 ng / mL). The MeIQx residue rate in this group was significantly lower than in the other groups. Figure 10 As shown in b.

[0036] The GSH-lactose@UVC-GA group exhibited characteristic absorption peaks of amide I and II bands and aromatic C=C bonds. The intermolecular interactions of hydrogen bonds, disulfide bonds, and amino groups in this group were significantly stronger than those in other control groups. Figure 10 As shown in c. Regarding free radical activity, EPR detection showed that the free radical scavenging ability of each group was negatively correlated with the signal intensity of active free radicals. The GSH-lactose@UVC-GA group had significantly higher scavenging rates for DPPH and ABTS free radicals than other groups, while its carbon-center free radical, ·OH, and O2 scavenging rates were significantly higher. - The signal strength was the lowest among all groups, such as Figure 10 As shown in d. Quantitative analysis of Maillard reaction activity showed that the amount of Maillard reaction products generated in the GSH-lactose@UVC-GA group was significantly lower than that in other control groups. Specifically, the relative abundance of GSH-MeIQx adducts, GA-MeIQx adducts, and MeIQx derivatives (such as 5-OH-MeIQx, 4-OH-MeIQx, etc.) were all the lowest. Figure 10As shown in e. Furthermore, through evaluation of the recycling performance of the complex, it was found that GSH-lactose@UVC-GA maintained a high activity retention rate, inhibition rate, and recovery rate after five cycles of degradation experiments, with no significant decrease in any performance index, demonstrating good stability, such as... Figure 10 As shown in e.

[0037] Monitoring the dynamic changes of imine-enamine tautomers using UV-Vis spectroscopy, such as Figure 11 As shown in Figure a, in the simulated reaction system at 180°C, the CG group showed a typical enhanced aldehyde condensation absorption peak, while the absorption peak of the GSH-lactose@UVC-GA group hardly shifted, indicating that it effectively suppressed the initiation of tautomerism. In contrast, the GA and UVC-GA groups only showed partial inhibition.

[0038] Hydroxypropionaldehyde, as an important intermediate in the Maillard reaction, undergoes a dehydration process as follows: Figure 11 As shown in b, UVC-GA treatment significantly slowed down the dehydration rate, while the GSH-lactose@UVC-GA group almost completely inhibited the dehydration reaction, confirming that this cluster structure has a stronger inhibitory effect on heat-induced dehydration. The difference in inhibition was further assessed by measuring the amount of aldehydes generated in the Strecker degradation pathway, such as... Figure 12 As shown in c. Aldehyde accumulation was most significant in the CG group, further decreased in the GA group, slightly reduced in the UVC-GA group, and lowest in the GSH-lactose@UVC-GA treatment group. The Amadori rearrangement is a key step in heterocyclic amine formation. NMR spectra show, as... Figure 11 As shown in Figure e, the rearrangement product signal of the CG group was significant, while no obvious resonance peak was observed in the GSH-lactose@UVC-GA group, indicating that the cluster effectively blocked the rearrangement pathway. Although the UVC-GA group had a certain inhibitory effect, it was still inferior to the treatment with the composite cluster.

[0039] To elucidate the inhibition mechanism at the molecular level, the molecular electrostatic potentials of each reaction intermediate were calculated, such as... Figure 11 As shown in d, GSH-lactose@UVC-GA treatment causes a redistribution of the negative potential region on the surface of the key intermediate, reducing its electrophilicity and thus inhibiting the progress of subsequent reactions. The reaction pathway inhibition effect heatmap is shown in... Figure 11As shown in f, the inhibition rates of the GSH-lactose@UVC-GA group for each reaction step (46.0%–53.1%) were significantly higher than those of the GA group (13.9%–16.7%) and the UVC-GA group (29.3%–33.2%), with the best blocking effects on imine-enamine tautomerism (53.1%) and hydroxypropionaldehyde dehydration (50.9%). The cascade reaction progress comparison diagram further shows that, compared with the control group, the concentrations of MeIQx formation, Strecker degradation products, and Amadori rearrangement products in the GSH-lactose@UVC-GA group were significantly reduced within 8 min (reductions of 42.9%, 49.5%, and 46.0%, respectively), indicating that the GSH-lactose@UVC-GA cluster can effectively block multiple key nodes of the cascade reaction. Energy barrier change radar diagram, as shown... Figure 11 As shown in g, the activation energy changes of each step were quantified. Compared with the control group, the GA, UVC-GA, and GSH-lactose@UVC-GA clusters all increased the free energy barrier of each step of MeIQx formation in a gradient manner (p<0.001), with the GSH-lactose@UVC-GA group showing the largest increase (22.9%–29.6%), increasing the energy barriers for hydroxypropionaldehyde dehydration and Strecker degradation by 29.6% and 25.6%, respectively. The results indicate that this cluster can simultaneously increase the activation energy barrier of multiple key steps, especially showing the most significant inhibition of dehydration and degradation reactions.

[0040] In summary, GSH-lactose@UVC-GA effectively blocks the MeIQx generation pathway in the early stages of the cascade reaction through a multi-target intervention mechanism. Its comprehensive inhibitory efficacy is significantly better than that of gallic acid or UVC treatment alone, providing an efficient strategy for the control of heterocyclic amines.

[0041] The results showed that the GSH-lactose@UVC-GA complex exhibited significant catalytic degradation of MeIQx in all three types of fish paste. The residual levels of MeIQx in the experimental groups (EG-P, EG-T, EG-S) were significantly lower than those in the blank control group (CG) and the positive controls for BHT, VC, and GA, and were the lowest among all treatment groups, confirming that the complex possesses excellent MeIQx degradation capabilities. Figure 12 As shown in a.

[0042] In the process of inhibiting lipid oxidation, the thiobarbituric acid reactive substances (TBARS) value was used as the evaluation index. The GSH-lactose@UVC-GA complex exhibited the most superior antioxidant performance. The TBARS values ​​of all experimental fish surimi groups were significantly lower than those of other treatment groups, specifically: 0.272 mg MDA / kg for squid surimi EG-S group, 0.202 mg MDA / kg for tilapia surimi EG-T group, and 0.238 mg MDA / kg for shrimp surimi EG-P group. This result indicates that the complex can effectively inhibit the lipid oxidation process in fish surimi. Figure 12 As shown in b. Regarding protein oxidation, using protein carbonyl content as an evaluation index, the carbonyl formation in the experimental group was the lowest among all treatment groups, indicating a significant inhibition of protein carbonyl formation and effective mitigation of protein oxidative degradation, such as... Figure 12 As shown in c.

[0043] In terms of fish paste color stability, the GSH-lactose@UVC-GA complex was superior to the blank control group and all positive control groups. The L*, a*, and b* values ​​of the experimental group fish paste were closer to the initial levels of fresh fish paste, with the smallest color change, indicating that this complex can effectively maintain the color stability of shrimp, tilapia, and squid fish paste. Figure 12 As shown in df. The pH changes of the three types of fish paste before and after the reaction (0 h, 4 h) in different treatment groups were measured. The initial pH of the different fish pastes differed, with tilapia fish paste having a lower pH than squid and shrimp fish paste. In the positive control group, the BHT group did not affect the pH of the aqueous phase and showed no significant difference from the blank control group (p>0.05); the VC group significantly decreased the pH of all fish paste systems (p<0.05); the GA group had the lowest pH among all groups (5.98-6.32), significantly lower than the other groups (p<0.05); the pH of the experimental groups significantly increased, and their pH values ​​were not significantly different from the CG and BHT groups (p>0.05). Figure 12 As shown in gi.

[0044] The results of this study indicate that GSH-lactose@UVC-GA not only efficiently catalyzes the degradation of MeIQx, but also has antioxidant functions, thereby comprehensively improving the quality of surimi products.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach, characterized in that: Includes the following steps: Add the GSH-lactose@UVC-GA complex to food ingredients; The complex inhibits MeIQx formation and maintains food quality through the following multi-target cascade blocking mechanism: (a) Direct degradation of the generated MeIQx molecules; (b) Consuming the precursors required to form MeIQx, said precursors include threonine, creatine anhydride and glucose; (c) Blocking the Amadori rearrangement, Strecker degradation, aldol condensation and / or imine-enamine tautomerization steps in the MeIQx formation cascade pathway; (d) Remove carbonyl intermediates in the formation of MeIQx, including glyoxal and methylglyoxal; (e) Scavenging free radicals reduces lipid oxidation levels and protein oxidation.

2. The method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach according to claim 1, characterized in that: The amount of the GSH-lactose@UVC-GA complex added is 0.01 to 5 wt% of the food raw material.

3. The method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach according to claim 1, characterized in that: The food ingredients are surimi products, meat products, dairy products, or grain products; the surimi products include squid surimi, tilapia surimi, and shrimp surimi.

4. The method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach according to claim 1, characterized in that: The preparation method of the GSH-lactose@UVC-GA complex includes the following steps: Step S1: Irradiate the gallic acid aqueous solution under a UVC light source to obtain a UVC-GA solution; Step S2: Glutathione is reacted with lactose in a solution to obtain a GSH-lactose derivative; Step S3: The GSH-lactose derivative obtained in step S2 and the UVC-GA solution obtained in step S1 are subjected to a self-assembly reaction in a natural eutectic solvent to obtain the GSH-lactose@UVC-GA complex.

5. The method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach according to claim 4, characterized in that: The UVC light source has a wavelength of 254 nm and an average radiant intensity of 4540 μW / cm². 2 The distance between the UVC light source and the liquid surface is set to 15 cm, and the irradiation time of the UVC light source is 6 h; the concentration of the gallic acid aqueous solution is 14 mmol / L.

6. The method for inhibiting the formation of MeIQx and maintaining food quality through a multi-target cascade approach according to claim 4, characterized in that: The molar ratio of glutathione to lactose is 1:3; the solvent is deionized water; and the reaction is carried out in an oil bath at 130°C for 30 min.

7. The method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach according to claim 4, characterized in that: The natural eutectic solvent is a mixture of choline chloride and lactic acid, wherein the molar ratio of choline chloride to lactic acid is 1:1 to 1:4; 20 wt.% of deionized water is added to the mixture.

8. The method for inhibiting the formation of MeIQx and maintaining food quality through a multi-target cascade approach according to claim 4, characterized in that: The molar ratio of the GSH-lactose derivative to the UVC-GA solution is 1:1; the self-assembly reaction is carried out under conditions of pH 3.0-6.

0.

9. The method for inhibiting MeIQx formation and maintaining food quality through a multi-target cascade approach according to claim 4, characterized in that: The self-assembly reaction temperature is 25-55°C; the self-assembly reaction time is 1-7 h.

Images