Method for preparing Maillard reaction intermediate based on gamma-aminobutyric acid and glucose

Abstract

The invention discloses a method for preparing a Maillard reaction intermediate based on gamma-aminobutyric acid and glucose, and belongs to the technical field of food production. According to the method, the Maillard reaction intermediate of gamma-aminobutyric acid and glucose is subjected to high-purity synthesis by adopting clean solvent water or edible alcohol, so that the method is green, environment-friendly and safe. The high-concentration ethanol can be combined with a low-temperature precipitation method to remove a part of amino acid impurities in a reaction system, so that the purity of the product is improved. The synthesis conditions are as follows: the reaction temperature is 75-90 DEG C, the pH value is 6.0-7.5, the vacuum degree is 500-950 mbar, and the reaction time is 20-60 minutes. According to the method disclosed by the invention, the Maillard reaction intermediate of gamma-aminobutyric acid and glucose can be synthesized at a relatively low temperature (less than or equal to 90 DEG C) within a shorter time (less than or equal to 1 hour), and the purity of the product reaches 97% or above. The production cost and the energy consumption are reduced to a great extent, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to a method for preparing Maillard reaction intermediates from γ-aminobutyric acid and glucose, belonging to the technical field of food production. Background Art

[0002] The saccharide compound - D-1-[(3-carboxypropyl)amino]-1-deoxyglucose has the specific structure shown as follows:

[0003]

[0004] It has been reported in the prior art WO99053930A1 that this compound has the function of inhibiting the growth of human prostate cancer cells and breast cancer cells. Currently, this compound is mainly obtained by extraction and separation from plants. For example, the pomegranate peel extract reported by Salem Elkahoui et al. (Investigation of the Valorization of Methanolic Extract of Punica granatum L. Peel in Terms of Phytochemical, Trace Element, Antioxidant Activities and ADMET Profile of Active Compounds. Pol. J. Environ. Stud. Vol. 33, 2024, 2587), the aqueous extract of Dioscorea opposita Thunb. reported by Xie et al. (Protection effect of Dioscoreae Rhizoma against ethanol-induced gastric injury in vitro and in vivo: A phytochemical and pharmacological study. Journal of Ethnopharmacology. Vol 333, 2024, 118427), etc. There has been no report on chemical synthesis methods.

[0005] Similar reactions, such as the Maillard reaction in food, involve the nucleophilic addition of the carbonyl group of reducing sugars in food to the amino group of amino acids, forming unstable Schiff bases, which are then rearranged to generate Maillard reaction intermediates - N-(1-deoxy-D-fructose-1-yl)-amino acids. These Maillard reaction intermediates have relatively stable physicochemical properties and the ability to release flavor substances. A variety of Maillard reaction intermediates have been proven to be safe and have functions beneficial to physical health, such as antioxidant, hypoglycemic, free radical scavenging, and blood pressure lowering effects. The Maillard reaction intermediate of histidine and glucose can synergistically inhibit tumor formation with lycopene. The Maillard reaction intermediate of arginine and glucose can significantly inhibit the level of angiotensin converting enzyme. The Maillard reaction intermediate of proline and glucose can regulate the immune level in the body. The Maillard reaction intermediate of lysine and glucose has an important impact on human intestinal health. The Maillard reaction intermediate of asparagine and glucose can inhibit the growth of Salmonella in the intestine. The Maillard reaction intermediate of aspartic acid and glucose can effectively reduce the degree of pulmonary edema. In addition, it has been reported that Maillard reaction intermediates have the ability to chelate metal ions such as Cu 2+ 、Fe 2+ and Zn 2+ and have a significant inhibitory effect on oxidation free radicals caused by metal ions.

[0006] Currently, the synthesis methods of Maillard reaction intermediates include organic synthesis and aqueous synthesis. Most organic solvent syntheses use toxic reagents such as methanol and N,N-dimethylformamide as reaction solvents, which cause great environmental pollution and the synthesized products cannot be used in the food industry. However, the existing aqueous phase methods (using water as the solvent system) are not applicable to the reaction system for synthesizing target carbohydrate compounds from γ-aminobutyric acid and glucose, and the yield of the target product is relatively low. Summary of the Invention

[0007]

Technical Problem

[0008] Provide a synthesis method for efficiently preparing Maillard reaction intermediates of high-purity γ-aminobutyric acid and glucose.

[0009]

Technical Solution

[0010] The present invention develops a method for efficiently preparing Maillard reaction intermediates of high-purity γ-aminobutyric acid and glucose using water or a water-ethanol mixture as a solvent. This method is green, environmentally friendly, and the product has high purity, and can be applied in fields such as food and medicine. In the present invention, γ-aminobutyric acid and glucose are used as raw materials, with the molar ratio of glucose to amino acid being 1:1 to 2:1, and the amino acid addition amount in the solution being not less than 0.2 mol / L. After being fully dissolved (a suspension in an edible alcohol solution with an ethanol content > 60% v / v), an edible organic acid is used to adjust the pH to 6.0 - 7.5. The reaction is carried out at a temperature of 75 - 90°C and a vacuum degree of 500 - 950 mbar for 20 - 60 minutes. Compared with ordinary aqueous-phase synthesis, ethanol can precipitate a part of the amino acids, resulting in fewer amino acids in the product and higher purity of the target product; compared with organic-phase synthesis methods, it is more green, environmentally friendly, clean, and efficient.

[0011] The first object of the present invention is to provide a method for preparing Maillard reaction intermediates, which uses γ-aminobutyric acid and glucose as raw materials and reacts in water or an aqueous ethanol solution.

[0012] In one embodiment of the present invention, the structure of the Maillard reaction intermediate is as follows:

[0013]

[0014] In one embodiment of the present invention, the molar ratio of γ-aminobutyric acid to glucose is (1 - 2):1. Specifically, 1:1 can be selected.

[0015] In one embodiment of the present invention, the addition amount of γ-aminobutyric acid relative to water or an aqueous ethanol solution is not less than 0.2 mol / L.

[0016] In one embodiment of the present invention, the concentration of the aqueous ethanol solution is 0 - 95% v / v, and further preferably 50% - 95% v / v.

[0017] In one embodiment of the present invention, the reaction temperature in the aqueous ethanol solution is 75 - 90°C. Further preferably 80 - 90°C.

[0018] In one embodiment of the present invention, the reaction temperature in water is 80 - 120°C.

[0019] In one embodiment of the present invention, the reaction time is 20 - 100 minutes. Further preferably 20 - 60°C.

[0020] In one embodiment of the present invention, the reaction is carried out under normal pressure or in an environment with a vacuum degree of 500 - 950 mbar.

[0021] In one embodiment of the present invention, the reaction further includes adjusting the pH of the system to 6.0 - 8.0. Specifically, it can be 7.5 ± 0.1.

[0022] In one embodiment of the present invention, after the reaction is completed, it is cooled, and then filtered through an organic filter membrane to remove the precipitated amino acid impurities, and the filtrate is collected; the filtrate is loaded onto a cation exchange resin chromatography column, and first deionized water is used to wash away the remaining glucose in the sample. When there is no reducing sugar in the eluate, ammonia water is used for elution, and the eluate is collected; the collected eluate is detected by high performance liquid chromatography coupled with an evaporative light scattering detector, the amino acid content is distinguished by the external standard method, and the components with an amino acid content exceeding 2% are discarded, and the components with a Maillard reaction intermediate purity > 94% are combined.

[0023] In one embodiment of the present invention, the organic filter membrane is specifically a 0.22 μm polytetrafluoroethylene filter membrane or a 0.45 μm polytetrafluoroethylene filter membrane.

[0024] In one embodiment of the present invention, during the process of first using deionized water to wash away the remaining glucose in the sample, the flow rate of the deionized water is 0.2 - 1.0 mL / min.

[0025] In one embodiment of the present invention, during the process of using ammonia water for elution, the concentration of the ammonia water is 0.1 - 0.8 mol / L, and the flow rate of the ammonia water is 0.1 - 0.5 mL / min.

[0026] In one embodiment of the present invention, the preparation method specifically includes the following steps:

[0027] (1) Add equimolar amounts of γ-aminobutyric acid and glucose to 0 - 95% v / v edible alcohol, stir and heat in a water bath for a period of time, stop heating and terminate the reaction;

[0028] (2) Place the solution obtained in step (1) at 0 - 4 °C for a period of time, take out the supernatant and filter it through a 0.22 μm organic filter membrane to remove the precipitated amino acid impurities;

[0029] (3) Load the filtrate obtained in step (2) onto a cation exchange resin chromatography column, first use deionized water with a flow rate of 0.2 - 1.0 mL / min to wash away the remaining glucose in the sample. When there is no reducing sugar in the eluate, use ammonia water with a concentration of 0.1 - 0.8 mol / L for elution, and collect one tube every 10 mL;

[0030] (4) Detect the eluate collected in step (3) by high performance liquid chromatography coupled with an evaporative light scattering detector, distinguish the amino acid content by the external standard method, and discard the components with an amino acid content exceeding 2%, and combine the components with a Maillard reaction intermediate purity > 97%;

[0031] (5) Freeze-dry the combined components in step (4) in a vacuum freeze dryer to obtain a high-purity Maillard reaction intermediate product powder;

[0032] (6) Grind the powder obtained in step (5) and prepare it into an aqueous solution of 1 mg / L, and detect it by ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry in the ESI positive ion mode to confirm that its mass spectrum peak contains a quasi-molecular ion peak of m / z = 266 and a [M+H-H2O] peak of m / z = 248. + peak;

[0033] (7) Take the powder obtained in step (5), grind it, dissolve it in 0.5 ml of deuterium oxide and put it into a nuclear magnetic tube, and perform structural analysis by 600 MHz nuclear magnetic hydrogen spectrum and carbon spectrum to confirm that the α-carbon of the amino group of the amino acid has a nuclear magnetic shift.

[0034] In one embodiment of the present invention, the chromatographic column of the high performance liquid chromatograph tandem evaporative light scattering detector for detecting the sample purity is an amide column, and the hydrophilic chromatography mode is adopted.

[0035] In one embodiment of the present invention, the mobile phase of the liquid chromatography is pure acetonitrile and an aqueous solution of 10 mmol / L ammonium formate.

[0036] The second object of the present invention is a method for increasing the content of Maillard reaction intermediates in food, which is to heat and react food rich in γ-aminobutyric acid and glucose in water or edible alcohol for a period of time.

[0037] In one embodiment of the present invention, the concentration of ethanol in the edible alcohol is 0-95% v / v. Further, 50%-95% v / v is optional.

[0038] In one embodiment of the present invention, the reaction temperature in the ethanol aqueous solution is 75-90°C. Further, 80-90°C is optional.

[0039] In one embodiment of the present invention, the reaction temperature in water is 80-120°C.

[0040] In one embodiment of the present invention, the reaction time is 20-100 minutes. Further, 20-60°C is optional.

[0041] The beneficial effects of the present invention:

[0042] The present invention provides a chemical synthesis method for efficiently preparing Maillard reaction intermediates of high-purity γ-aminobutyric acid and glucose. The method of the present invention synthesizes Maillard reaction intermediates of γ-aminobutyric acid and glucose at a lower temperature (≤90 °C) and in a shorter time (≤1 h), thereby greatly reducing production costs and energy consumption, and enabling the product purity to reach over 94% and up to over 97% at most, and the yield to reach over 43% and up to over 70% at most. Description of the Drawings

[0043] Figure 1 It is a comparative diagram of the results of the yield (A), A 420 value (B) and the system pH (C) of Maillard reaction intermediates of γ-aminobutyric acid and glucose synthesized with edible alcohol at a concentration of 95% v / v ethanol as the solvent at different temperatures.

[0044] Figure 2 It is the chromatogram and mass spectrum of Maillard reaction intermediates of γ-aminobutyric acid and glucose on an ultra-high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer (UPLC-QTOF-MS). Among them, A is the total ion current chromatogram, B is the first-order mass spectrum in the ESI+ mode, and C is the second-order mass spectrum at a collision voltage of 6 V.

[0045] Figure 3 It is the nuclear magnetic resonance spectrum of Maillard reaction intermediates of γ-aminobutyric acid and glucose. Among them, A is 1 the 1H NMR spectrum, and B is 13 the 13C NMR spectrum.

[0046] Figure 4 It is a schematic diagram of the pathway for γ-aminobutyric acid and glucose to be converted into Maillard reaction intermediates.

[0047] Figure 5 It is a comparative diagram of the results of the yield (A), A 420 value (B) and the system pH (C) of Maillard reaction intermediates of γ-aminobutyric acid and glucose synthesized with water as the solvent at different temperatures. Detailed Embodiments

[0048] Identification method for Maillard reaction intermediates of γ-aminobutyric acid and glucose:

[0049] ARP is identified by ultra-high performance liquid chromatography-mass spectrometry (Maldi Synapt Q-Tof, waters, USA) and a BEH C18 chromatographic column (1.7 μm, 2.1 mm × 50 mm, waters, USA). UPLC-MS / MS analysis is performed using the positive ion electrospray ionization mode.

[0050] Liquid phase conditions: Mobile phase: Phase A is acetonitrile, Phase B is 0.1% formic acid in water; Column temperature: 35°C, Flow rate: 0.3 mL / min; Injection volume: 1 μL, Gradient elution conditions: Initial condition, Phase B is 100%; From 1 minute to 5 minutes, Phase B changes from 100% to 90%; From 5 minutes to 6 minutes, Phase B changes from 90% to 50%; From 6 minutes to 7 minutes, Phase B changes from 50% to 20%; From 7 minutes to 9 minutes, Phase B remains at 50%; From 9 minutes to 9.5 minutes, Phase B changes from 20% to 100%; From 9.5 minutes to 13 minutes, Phase B remains at 100%. Desolvation gas temperature, 400°C; Gas flow rate, 700 L / h; Cone voltage, 20 V; Cone gas flow rate, 50 L / h; Collision energy, 6 V; Detector voltage, 1800 V; Mass range, 20 - 1000 m / z.

[0051] The synthetic route of the Maillard reaction intermediate involved in the present invention is as follows:

[0052]

[0053] Example 1: A method for synthesizing the Maillard reaction intermediate of γ-aminobutyric acid and glucose using edible alcohol with an ethanol concentration of 95% v / v as the solvent

[0054] Glucose and γ-aminobutyric acid were added to 50 mL of edible alcohol with an ethanol concentration of 95% v / v. Their molar concentrations were the same, both being 0.02 mol / L. And at 25 °C, the reaction solution was controlled at pH 7.5 ± 0.1 with NaOH solution (6 M); the reaction solution was added to a 100 mL pressure-resistant glass bottle with a PTFE cork, a rotor was added, and it was immersed in an oil bath. Then magnetic stirring was started, and the temperature was maintained at 80 °C and 90 °C. After reacting for 20 minutes, 40 minutes, 60 minutes, 80 minutes, and 100 minutes respectively, the pressure-resistant bottle was taken out and placed in an ice-water mixture to terminate the reaction. After cooling, a 0.45 μm polytetrafluoroethylene filter membrane was used to filter out the precipitated amino acids in the solution, and the filtrate was preserved. 10 mL of the filtrate was put into a 1.3 cm * 30 cm chromatography column containing DOWEX 50WX8 hydrogen-form ion exchange resin, and then deionized water was used to wash it at a flow rate of 0.06 L / h (equivalent to 1 mL / min). Until the eluent could not change the color of the TTC reagent (2% - 3% 2,3,5-triphenyltetrazolium chloride methanol solution and 6 mol / L NaOH solution), ammonia water with a concentration of 0.1 mol / L was used to elute the target substance at a speed of 0.03 L / h (equivalent to 0.5 mL / min), and an automatic collector was used to collect every 10 ml in a centrifuge tube. The fractions that could make the TTC change color were preserved. Immediately, a LC-20A liquid phase detector equipped with a Shimadzu ELSD-LTII was used, and the chromatographic column was an Xbridge Amide column (5 μm, 4.6 mm × 250 mm, Waters, Milford, MA, USA) to detect different fractions. The components with an amino acid content > 2% were discarded, and the remaining parts were combined and freeze-dried to obtain the Maillard reaction intermediate product powder.

[0055] Among them, the liquid phase conditions were as follows: the injection volume was 10 μL. Mobile phase A was an ultrapure aqueous solution of ammonium formate at 10 mmol / L, mobile phase B was chromatographically pure acetonitrile, and the flow rate was 0.8 ml / min. The gradient elution was as follows: 80% B - 70% B from 0 - 18 minutes, 70% B from 18 - 23 minutes, 70% B - 80% B from 23 - 25 minutes, 80% B from 25 - 28 minutes. The parameters of the evaporative light scattering detector were as follows: the nitrogen pressure was 315 kPa, the evaporation temperature was 50 °C, and the gain value was 6.

[0056] An ultraviolet spectrophotometer and a pH meter were used to detect the A 420 and pH of the synthesis system, and the detection results were as Figure 1 shown. Figure A shows that in edible alcohol, although only increased by 10 °C, the peak production time was advanced by 40 minutes, and the production at 40 minutes at 90 °C was 198% of the highest production at 80 °C. The characteristic absorption A of melanoidins within 20 - 60 minutes 420Relatively low, and the production of Maillard reaction intermediates increases. This indicates that 20 - 60 minutes at 80 - 90 °C is the appropriate reaction time.

[0057] Specific yield and purity results are shown in Table 1.

[0058] Table 1

[0059] Synthesis method Reaction temperature °C Reaction time min Yield HPLC purity 1 80 20 43.16％ 97.73％ 2 80 40 55.72％ 97.94％ 3 80 60 68.15％ 96.15％ 4 90 20 51.27％ 94.19％ 5 90 40 70.14％ 97.66％ 6 90 60 48.46％ 98.03％

[0060] Result characterization:

[0061] 1. Identification method for pure Maillard reaction intermediates of γ-aminobutyric acid and glucose by ultra-high performance liquid chromatography - mass spectrometry:

[0062] Take 1 mg of the freeze-dried powder of the product obtained by Method 5 and make up the volume to 100 mL. Take 1 mL of the solution, pass it through a 0.22 μm aqueous filter membrane, and then load it into a 1.5 mL liquid phase injection vial for waiting for detection.

[0063] Instrument: Waters high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometer, chromatographic column: BEH C18 (1.7 μm, 2.1 mm × 150 mm)

[0064] Liquid phase conditions: Column temperature: 35 °C, flow rate: 0.3 mL / min; injection volume: 1 μL; mobile phase A is acetonitrile, and mobile phase B is 0.1% formic acid by volume fraction. Using the method of gradient elution, the gradient change of mobile phase A is as follows: from 0 minute to 2 minutes, the volume fraction of A phase is 0%; from 2 minutes to 5 minutes, the volume fraction of A phase changes from 0% to 10%; from 5 minutes to 7 minutes, the volume fraction of A phase changes from 10% to 40%; from 7 minutes to 8 minutes, the volume fraction of A phase changes from 40% to 80%; from 8 minutes to 8.10 minutes, the volume fraction of A phase changes from 80% to 0%; after 8.10 minutes, the volume fraction of A phase remains 0%, and the total running time is 10 minutes

[0065] Mass spectrometry conditions: Electrospray ionization source (ESI), positive ion ionization mode 400 °C; capillary voltage: 2.5 kV; desolvation gas flow rate: 600 L / h; cone voltage: 20 V; cone gas flow rate: 50 L / h; collision energy: 6.0 eV; m / z: 100 - 1000; detection voltage: 1600 V; high-purity nitrogen gas (99.999%). Use MassLynx software to process the data.

[0066] The mass spectrometry detection results are as Figure 2 shown. The quasi-molecular ion peak [M + H] of the Maillard reaction intermediate of γ-aminobutyric acid and glucose can be clearly seen in the first-level mass spectrum + (m / z = 266) and the dehydration peak [M + H - H2O] +(m / z = 248), and characteristic peaks of hexose-derived Maillard reaction intermediates [M+H-3H2O-H2CO] can be found in the secondary mass spectrometry + (m / z = 182).

[0067] 2. Identification method for pure products of Maillard reaction intermediates of γ-aminobutyric acid and glucose by nuclear magnetic resonance spectrometer:

[0068] Take the freeze-dried powder (5 - 35 mg) of the product obtained by Method 5 and dissolve it in 500 μL of D2O. After complete dissolution, transfer it to a nuclear magnetic resonance tube. Using a fully digital nuclear magnetic resonance spectrometer and a 5 mm Pabbo probe, 1H nuclear magnetic resonance spectra and 13C nuclear magnetic resonance spectra at 298 K were obtained on a Bruker DRX 400 MHz spectrometer (Bruker BioSpin, Germany), and the results were analyzed using MestReNova software. The spectra are as Figure 3 shown. In the proton spectrum, hydrogens with chemical shifts of about 1.9 and 2.3 for γ-aminobutyric acid can be seen, as well as the hydrogen at about 3.1 where the α-carbon of the amino group has changed. In the carbon spectrum, 10 peaks corresponding to 10 carbons of the Maillard reaction intermediate of γ-aminobutyric acid and glucose can be seen.

[0069] Above, it can be proved that the Maillard reaction intermediate with the shown structure was successfully synthesized by the above method.

[0070] Example 2: Synthesis of Maillard reaction intermediates of γ-aminobutyric acid and glucose in aqueous solution.

[0071] Add glucose and γ-aminobutyric acid to 10 mL of deionized water, with the same molar concentration of 0.02 mol / L for both. At 25 °C, use NaOH solution (6 M) to control the reaction solution at pH 7.5 ± 0.1. Add the solution with a rotor to a 100 mL pressure-resistant glass bottle with a PTFE cork and immerse it in an oil bath. Then turn on magnetic stirring, keep the temperature at 80 °C, 90 °C, 100 °C, 110 °C, and 120 °C, and the vacuum degree at 100 mbar. After reacting for 20 minutes, 40 minutes, 60 minutes, 80 minutes, and 100 minutes respectively, take out the pressure-resistant bottle and put it into an ice-water mixture to terminate the reaction. Use an ultraviolet spectrophotometer and a pH meter to detect the A 420 and pH of the synthesis system, and the detection results are as Figure 5 shown. Even the peak yield at the highest temperature of 120 °C in the aqueous solution is less than half of that of 95% v / v edible alcohol with ethanol concentration, indicating that ethanol is crucial for the efficient synthesis of high-purity Maillard reaction intermediate products of γ-aminobutyric acid and glucose.

[0072] In summary, the present invention uses clean solvents such as water or edible alcohol to synthesize the Maillard reaction intermediate of γ-aminobutyric acid and glucose with high purity, which is green, environmentally friendly and safe. High-concentration ethanol can also combine with the method of low-temperature precipitation to remove a part of amino acid impurities in the reaction system, thereby improving the purity of the product.

[0073] Furthermore, the method of the present invention uses edible alcohol as a solvent to synthesize the Maillard reaction intermediate of γ-aminobutyric acid and glucose at a lower temperature (≤90 °C) and in a shorter time (≤1 h), and the purity of the product can reach more than 97%. This greatly reduces the production cost and energy consumption, and has broad application prospects.

[0074] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for preparing Maillard reaction intermediates, which uses γ-aminobutyric acid and glucose as raw materials and reacts in water or an ethanol aqueous solution.

2. The method according to claim 1, characterized in that, The structure of the Maillard reaction intermediate is shown as follows:

3. The method according to claim 1, wherein The molar ratio of γ-aminobutyric acid to glucose is (1 - 2):

1.

4. The method according to claim 1, wherein The concentration of the ethanol aqueous solution is 0 - 95% v / v.

5. The method according to claim 1, wherein The concentration of the ethanol aqueous solution is 50% - 95% v / v.

6. The method according to claim 1, characterized in that, When reacting in the ethanol aqueous solution, the temperature is 75 - 90 °C and the reaction time is 20 - 60 minutes.

7. The method according to claim 1, wherein When reacting in the ethanol aqueous solution, the temperature is 90 °C and the reaction time is 40 minutes.

8. The method according to claim 1, characterized in that, The reaction also includes adjusting the pH of the system to 6.0 - 8.

0.

9. The method according to any one of claims 1-8, characterized in that, After the reaction is completed, it is cooled, and then filtered using an organic filter membrane to remove the precipitated amino acid impurities, and the filtrate is collected; the filtrate is loaded onto a cation exchange resin chromatography column, and deionized water is first used to elute the residual glucose in the sample. When there is no reducing sugar in the eluate, ammonia water is used for elution, and the eluate is collected; the collected eluate is detected by high performance liquid chromatography tandem evaporative light scattering detector, the amino acid content is distinguished by the external standard method, and the components with an amino acid content exceeding 2% are discarded, and the components with a Maillard reaction intermediate purity > 97% are combined.

10. A method for increasing the content of Maillard reaction intermediates in food, characterized in that, It is to heat and react a food rich in γ-aminobutyric acid and glucose in water or edible alcohol for a period of time.

Images