Method for preparing intermediate by reduced glutathione-indicated amino acid maillard reaction

Abstract

A method for preparing an intermediate by a reduced glutathione-indicated amino acid Maillard reaction is provided. The method includes a two-stage reaction at an increased temperature. A reduced glutathione is added after different times of a low-temperature reaction, and a subsequent Maillard reaction is effectively inhibited on a basis wherein a substance is interacted with an intermediate degradation product to reduce a formation of colored substances. Comparing with a browning of Maillard products after a high-temperature stage, a reaction time with a best color inhibition effect is found to be the optimal preparation condition of the intermediate, and the intermediate is prepared in an aqueous medium at a low temperature under this optimal preparation condition. The method uses the water soluble reduced glutathione as a tracer to improve a tracing accuracy comparing to cysteine.

Claims

1. A method for preparing an intermediate by a reduced glutathione-indicated amino acid Maillard reaction, comprising the following steps: 1) adding an amino acid and an aldose or ketose into water for a dissolution, and adjusting pH of a mixture to 6-8, wherein raw materials comprise by weight, 10 parts of amino acids, 10-40 parts of the aldose or the ketose and 200-1000 parts of the water; 2) placing the mixture obtained in step 1) in water bath at a constant-temperature in 80-100° C. for a first-stage Maillard reaction, and taking out an equal volume of 5-8 samples in sequence from the mixture during this first-stage Maillard reaction at the 10th-180th minutes at an interval of 10-20 minutes and immediately placing the 5-8 samples in an ice bath for cooling to terminate a reaction to obtain first-stage Maillard reaction solutions; 3) adding an equal amount of reduced glutathione into each sample of the 5-8 samples obtained in step 2) separately, re-adjusting the pH of the first-stage Maillard reaction solutions to 6-8 after uniform mixing, then transferring the first-stage Maillard reaction solutions into a temperature-resistant and pressure-resistant bottle for a second-stage high-temperature Maillard reaction at a same temperature in 110° C-130° C. for 60-180 minutes and placing the second-stage Maillard reaction solutions in ice bath for cooling to terminate the reaction to obtain the second-stage Maillard reaction solutions; 4) diluting the second-stage Maillard reaction solutions obtained in step 3) respectively, measuring absorbance value of each diluted second-stage Maillard reaction solution at a wavelength of 420 nm, drawing a curve diagram according to obtained absorbance values versus a corresponding reaction time of the diluted second-stage Maillard reaction in step 2), and determining the optimal reaction time under corresponding reaction conditions according to reaction time corresponding to lowest absorbance value; 5) repeating the operation of step 1): adding the amino acid and the aldose or the ketose into the water for the dissolution, and adjusting the pH of the mixed solution to 6-8, wherein amounts of the raw materials are based on the parts by weight used in step 1); 6) placing the mixed solution obtained in step 5) in water bath at the temperature used in step 2), wherein a thermal treatment time is the optimal reaction time in step and, immediately placing a product solution in ice bath for cooling to terminate the reaction to obtain a Maillard reaction intermediate solution; 7) concentrating the Maillard reaction intermediate solution of first-stage Maillard reaction solution obtained in step 6) under a reduced pressure and a low temperature to remove 80%-90% water and then purifying the Maillard reaction intermediate solution by a cation exchange resin to obtain a pure Amadori rearrangement product (ARP) or Heyns rearrangement product (HRP), wherein the amino acid in step 1) is one or more of alanine, glycine, cysteine and proline; the aldose or ketose in step 1) is one or more of ribose, xylose and fructose; an addition amount of the reduced glutathione in step 3) is 1%-2.5% w/v of a volume of the each sample of the 5-8 samples taken in step 2), wherein the 5-8 samples have the equal volume; and in step 7), a temperature is controlled to be 20-30° C. during a concentration under the reduced pressure, and a vacuum degree is 0.025-0.05 MPa.

2. The method according to claim 1, wherein cooling time in steps 2) and 3) is 10-30 minutes, and the first-stage Maillard reaction solutions are cooled to 10° C. or below to terminate the first-stage Maillard reaction.

3. The method according to claim 1, wherein the second-stage Maillard reaction solutions at the increased temperature in step 4) are diluted 2-50 times with distilled water.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a curve diagram showing a relationship between the absorbance value of a solution of second-stage Maillard reaction at increased temperature after dilution and the Maillard reaction time of a first stage in Example 1 of the present invention;

(2) FIG. 2 is a mass spectrogram of ARP prepared in Example 1 of the present invention;

(3) FIG. 3A shows a nuclear magnetic resonance hydrogen spectrogram of ARP prepared in Example 1;

(4) FIG. 3B shows a nuclear magnetic resonance carbon spectrogram of ARP prepared in Example 1;

(5) FIG. 4 is a curve diagram showing a relationship between the absorbance value of a solution of second-stage Maillard reaction at increased temperature after dilution and the Maillard reaction time of a first stage in Example 2 of the present invention;

(6) FIG. 5A is a total ion current chromatogram showing liquid chromatography-mass spectrometry characterization results of ARP prepared in Example 2 of the present invention;

(7) FIG. 5B is a mass spectrogram showing liquid chromatography-mass spectrometry characterization results of ARP prepared in Example 2 of the present invention;

(8) FIG. 6 is a curve diagram showing a relationship between the absorbance value of a solution of second-stage Maillard reaction at increased temperature after dilution and the Maillard reaction time of a first stage in Example 3 of the present invention;

(9) FIG. 7A is a total ion current chromatogram showing liquid chromatography-mass spectrometry characterization results of ARP prepared in Example 3 of the present invention;

(10) FIG. 7B is a mass spectrogram showing liquid chromatography-mass spectrometry characterization results of ARP prepared in Example 3 of the present invention;

(11) FIG. 8 is a curve diagram showing a relationship between the absorbance value of a solution of second-stage Maillard reaction at increased temperature after dilution and the Maillard reaction time of a first stage in Example 4 of the present invention;

(12) FIG. 9 is a mass spectrogram of HRP prepared in Example 4 of the present invention; and

(13) FIG. 10 is a curve diagram showing a relationship between the concentrations of ARP generated in a cysteine and xylose system and different low-temperature reaction times in Comparative Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(14) The following describes the present invention in detail with reference to the drawings and examples.

Example 1

(15) (1) 17.8 kg of alanine and 60 kg of xylose are dissolved in 1000 kg of water, the pH of the mixed solution is adjusted to 8.0, a reaction is carried out at 80° C. under a water bath condition, and 180 L of a sample is taken at 40 minutes, 60 minutes, 80 minutes, 100 minutes and 120 minutes separately and placed in an ice bath for cooling to terminate the reaction.

(16) (2) 1.8 kg of reduced glutathione is added into the five reaction solutions obtained above separately, the pH of the reaction solutions are re-adjusted to 8.0, and the reaction solutions are transferred into a temperature-resistant and pressure-resistant bottle, heated to 120° C. for a two-stage high-temperature Maillard reaction for 60 minutes and placed in an ice bath for cooling to terminate the reaction to obtain solutions of Maillard reaction at increased temperature.

(17) (3) The solutions of Maillard reaction at increased temperature are diluted 5 times separately, the absorbance value at a wavelength of 420 nm is measured, a curve diagram is drawn according to the absorbance value and the corresponding low-temperature reaction time in step (1), and the results are as shown in FIG. 1. It can be seen from FIG. 1 that the reaction time corresponding to the low absorbance value of the solutions of Maillard reaction at increased temperature is 80 minutes, the best color inhibition effect is achieved, and thus it can be determined that the optimal reaction time in the first reaction stage at 80° C. is 80 minutes.

(18) An intermediate is prepared at the selected temperature and optimum time, further concentrated at a low temperature and then separated and purified by hydrogen type cation exchange resin to obtain a pure intermediate (ARP) of an alanine-xylose system, which is then freeze dried to obtain a solid sample. The obtained solid is dissolved in water and analyzed by using a mass spectrometry technology to obtain a mass spectrogram as shown in FIG. 2. The structural characterization of the solid is carried out by nuclear magnetic resonance to obtain a nuclear magnetic resonance spectrogram as shown in FIG. 3A and FIG. 3B.

Example 2

(19) (1) 8 kg of cysteine and 19.8 kg of xylose are dissolved in 800 kg of water, the pH of the mixed solution is adjusted to 7.5, a reaction is carried out at 100° C. under a water bath condition, and 130 L of a sample is taken at 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes separately and placed in an ice bath for cooling to terminate the reaction.

(20) (2) 1.3 kg of reduced glutathione is added into the six reaction solutions obtained above separately, the pH of the reaction solutions are re-adjusted to 7.5, and the reaction solutions are transferred into a temperature-resistant and pressure-resistant bottle, heated to 130° C. for a two-stage high-temperature Maillard reaction for 90 minutes and placed in an ice bath for cooling to terminate the reaction to obtain solutions of Maillard reaction at increased temperature.

(21) (3) The solutions of Maillard reaction at increased temperature are diluted twice separately, the absorbance value at a wavelength of 420 nm is measured, a curve diagram is drawn according to the absorbance value and the corresponding low-temperature reaction time in step (1), and the results are as shown in FIG. 4. It can be seen from FIG. 4 that the reaction time corresponding to the low absorbance value of the solutions of Maillard reaction at increased temperature is 40 minutes, the best color inhibition effect is achieved, and thus it can be determined that the optimal reaction time in the first reaction stage at 100° C. is 40 minutes.

(22) An intermediate is prepared at the selected temperature and optimum time, further concentrated at a low temperature and then separated and purified by hydrogen type cation exchange resin to obtain a pure intermediate (ARP) of a cysteine-xylose system, which is then freeze dried to obtain a solid sample. The obtained solid is dissolved in water and analyzed by using a high performance liquid chromatography-mass spectrometry analysis technology to obtain a total ion current chromatogram and a mass spectrogram which are as shown in FIG. 5A and FIG. 5B.

Example 3

(23) (1) 15 kg of glycine and 60 kg of ribose are dissolved in 1000 kg of water, the pH of the mixed solution is adjusted to 6.0, a reaction is carried out at 90° C. under a water bath condition, and 150 L of a sample is taken at 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes and 120 minutes separately and placed in an ice bath for cooling to terminate the reaction.

(24) (2) 3 kg of reduced glutathione is added into the six reaction solutions obtained above separately, the pH of the reaction solutions are re-adjusted to 6.0, and the reaction solutions are transferred into a temperature-resistant and pressure-resistant bottle, heated to 110° C. for a two-stage high-temperature Maillard reaction for 120 minutes and placed in an ice bath for cooling to terminate the reaction to obtain solutions of Maillard reaction at increased temperature.

(25) (3) The solutions of Maillard reaction at increased temperature are diluted 50 times separately, the absorbance value at a wavelength of 420 nm is measured, a curve diagram is drawn according to the absorbance value and the corresponding low-temperature reaction time in step (1), and the results are as shown in FIG. 6. It can be seen from FIG. 6 that the reaction time corresponding to the low absorbance value of the solutions of Maillard reaction at increased temperature is 60 minutes, the best color inhibition effect is achieved, and thus it can be determined that the optimal reaction time in the first reaction stage at 90° C. is 60 minutes.

(26) An intermediate is prepared at the selected temperature and optimum time, further concentrated at a low temperature and then separated and purified by hydrogen type cation exchange resin to obtain a pure intermediate (ARP) of a glycine-ribose system, which is then freeze dried to obtain a solid sample. The obtained solid is dissolved in water and analyzed by using a high performance liquid chromatography-mass spectrometry analysis technology to obtain a total ion current chromatogram and a mass spectrogram which are as shown in FIG. 7A and FIG. 7B.

Example 4

(27) (1) 20 kg of proline and 20 kg of fructose are dissolved in 400 kg of water, the pH of the mixed solution is adjusted to 7.0, a reaction is carried out at 100° C. under a water bath condition, and 80 L of a sample is taken at 100 minutes, 120 minutes, 140 minutes, 160 minutes and 180 minutes separately and placed in an ice bath for cooling to terminate the reaction.

(28) (2) 20.0 kg of reduced glutathione is added into the five reaction solutions obtained above separately, the pH of the reaction solutions are re-adjusted to 7.0, and the reaction solutions are transferred into a temperature-resistant and pressure-resistant bottle, heated to 120° C. for a two-stage high-temperature Maillard reaction for 180 minutes and placed in an ice bath for cooling to terminate the reaction to obtain solutions of Maillard reaction at increased temperature.

(29) (3) The solutions of Maillard reaction at increased temperature are diluted twice separately, the absorbance value at a wavelength of 420 nm is measured, a curve diagram is drawn according to the absorbance value and the corresponding low-temperature reaction time in step (1), and the results are as shown in FIG. 8. It can be seen from FIG. 8 that the reaction time corresponding to the low absorbance value of the solutions of Maillard reaction at increased temperature is 140 minutes, the best color inhibition effect is achieved, and thus it can be determined that the optimal reaction time in the first reaction stage at 100° C. is 140 minutes.

(30) An intermediate is prepared at the selected temperature and optimum time, further concentrated at a low temperature and then separated and purified by hydrogen type cation exchange resin to obtain a pure intermediate (HRP) of a proline-fructose system, which is then freeze dried to obtain a solid sample. The obtained solid is dissolved in water and analyzed by using a mass spectrometry technology to obtain a mass spectrogram as shown in FIG. 9.

Comparative Example 1

(31) 8 kg of cysteine and 19.8 kg of xylose are dissolved in 800 kg of water, the pH of the mixed solution is adjusted to 7.5, a reaction is carried out at 100° C. under a water bath condition, a small amount of a sample is taken at different times and placed in an ice bath for cooling to terminate the reaction, the change rule of intermediate content at different reaction times is determined by high performance liquid chromatography, and the results are as shown in FIG. 10.

(32) It can be seen from FIG. 10 that at the beginning of a low-temperature reaction stage, the cumulative amount of an intermediate in the system is gradually increased, and the content of an intermediate tends to be stable after 40 minutes, which corresponds to the lowest color point in FIG. 4. That is, in the cysteine-xylose system, the time for mass production of an intermediate is 40 minutes.

(33) The experimental water in the above examples and comparative example is distilled water, aldose or ketose and amino acids are of food grade, chemical reagents used in high performance liquid chromatography-mass spectrometry analysis experiments are chromatographically pure, and the remaining chemical reagents are analytically pure. The detection conditions of high performance liquid chromatography are as follows: a chromatographic column CSHC18, a mobile phase containing acetonitrile and 0.1% formic acid water, a flow rate of 0.3 mL/min, gradient elution and a column temperature of 45° C. The conditions of mass spectrometry analysis are as follows: an ESI+ mode, a detector voltage of 1.8 kV, a capillary voltage of 3.5 kV, a cone voltage of 20 V and an extraction voltage of 7 V. The electron source temperature and the desolvation gas temperature are 100° C. and 400° C. respectively, the gas flow rate is 700 L/h, and the cone gas flow rate is 50 L/h. A sample is scanned in a mass ratio range of m/z20-1000, the scanning time is 1 second, and the scanning time delay is 0.1 second. A separated pure intermediate is dissolved in D.sub.2O and analyzed by a nuclear magnetic resonance instrument, and the test temperature is 298 K.