EGCG-glucose adduct and preparation method and application thereof

文档序号：101834 发布日期：2021-10-15 浏览：40次中文

阅读说明：本技术 一种egcg-葡萄糖加合物及其制备方法和应用 (EGCG-glucose adduct and preparation method and application thereof ) 是由宛晓春张梁姜宗德于 2021-08-18 设计创作，主要内容包括：本发明提供了一种EGCG-葡萄糖加合物及其制备方法和应用,属于掩味剂技术领域。本发明提供的EGCG-葡萄糖加合物能够对茶叶或茶饮料的苦味或涩味有明显的抑制或掩盖作用,能够作为苦涩味掩味剂用于食品、饮料领域。本发明提供了上述EGCG-葡萄糖加合物的制备方法,本发明通过模拟茶叶的烘焙过程,在加热的条件下,EGCG中A环6位上的C和8位上的C与葡萄糖结合,发生亲核加成反应,在进一步的加热过程中,EGCG的C6和C8位上连接的葡萄糖基团还会脱水环化,经分离纯化后得到具有式I、式II、式III或式IV所示结构的EGCG-葡萄糖加合物。同时,本发明提供的制备方法操作简单,容易实施,成本较低。(The invention provides an EGCG-glucose adduct and a preparation method and application thereof, belonging to the technical field of taste masking agents. The EGCG-glucose adduct provided by the invention can obviously inhibit or mask the bitter taste or the astringent taste of tea or tea drinks, and can be used as a bitter and astringent taste masking agent in the fields of foods and drinks. The invention provides a preparation method of the EGCG-glucose adduct, which is characterized in that the baking process of tea leaves is simulated, under the heating condition, C on 6-position and C on 8-position of an A ring in the EGCG are combined with glucose to generate nucleophilic addition reaction, in the further heating process, glucose groups connected on C6 and C8 of the EGCG are also subjected to dehydration and cyclization, and the EGCG-glucose adduct with the structure shown in formula I, formula II, formula III or formula IV is obtained after separation and purification. Meanwhile, the preparation method provided by the invention is simple to operate, easy to implement and low in cost.)

1. An EGCG-glucose adduct having a structure represented by formula I, formula II, formula III or formula IV:

2. a process for the preparation of the EGCG-glucose adduct according to claim 1 comprising the steps of:

(1) mixing epigallocatechin gallate, glucose and water, and carrying out nucleophilic addition reaction under the heating condition to obtain a nucleophilic addition product, wherein the nucleophilic addition product comprises EGCG-glucose adducts with structures shown in formula I, formula II, formula III and formula IV;

(2) and separating and purifying the nucleophilic addition product to respectively obtain the EGCG-glucose adduct with the structure shown in the formula I, the formula II, the formula III or the formula IV.

3. The preparation method according to claim 2, wherein the mass ratio of epigallocatechin gallate to glucose is (1-5): (1-5);

the mass of the water is 10-200% of the total mass of the epigallocatechin gallate and the glucose.

4. The method according to claim 2, wherein the heating temperature is 100 to 200 ℃.

5. The method according to claim 2 or 4, wherein the time of the nucleophilic addition reaction is 10 to 120 min.

6. The method of claim 2, wherein the separation and purification comprises the steps of:

mixing the nucleophilic addition product with a polar solvent, and concentrating after mixing to obtain a solution to be separated;

performing first column chromatography separation on the liquid to be separated to obtain a first column chromatography separation product, wherein a filler of the first column chromatography separation is Sephadex LH-20 gel, and the elution condition is methanol isocratic elution;

performing thin-layer chromatography separation on the first-time column chromatography separation product, combining fractions after color development, and sequentially obtaining a thin-layer chromatography separation component 1, a thin-layer chromatography separation component 2, a thin-layer chromatography separation component 3 and a thin-layer chromatography separation component 4, wherein a developing agent for thin-layer chromatography separation is a mixed solution of chloroform, acetone and formic acid, and the volume ratio of chloroform to acetone to formic acid in the mixed solution is (11-13): 6-8): 1-3;

carrying out second column chromatography separation on the thin-layer chromatography separation component 1, continuously carrying out thin-layer chromatography separation and color development, and then sequentially obtaining a second column chromatography separation component 1, a second column chromatography separation component 2 and a second column chromatography separation component 3, wherein a filler for the second column chromatography separation is silica gel, an eluent is a mixed solution of chloroform, acetone and formic acid, and the volume ratio of chloroform to acetone to formic acid in the mixed solution is (11-13) to (6-8) to (1-3);

performing high performance liquid chromatography separation on the second column chromatography separation component 2 to sequentially obtain EGCG-glucose adducts with structures shown in formula I, formula II, formula III and formula IV;

the chromatographic column for high performance liquid chromatography separation is Agilent ZORBAX SB-Aq C₁₈The chromatographic column comprises a mobile phase A and a mobile phase B, wherein the phase A is 0.2% formic acid water, and the phase B is methanol;

the elution conditions of the high performance liquid chromatography separation are as follows:

0-4 minutes, 5% methanol water-20% methanol water;

20 to 23 percent of methanol water for 4 to 16 minutes;

16-19 minutes, 23% methanol water;

19-20 minutes, 23% -42% methanol water;

20-21 minutes, 42% methanol water-100% methanol;

for 21-22 minutes, 100% methanol-5% methanol water;

22-28 minutes, 5% methanol water.

7. Use of the EGCG-glucose adduct of claim 1 or the EGCG-glucose adduct prepared by the process of any one of claims 2 to 6 as a bitter taste masking agent.

8. Use according to claim 7, wherein the taste-masking agent is a taste-masking agent for tea or tea beverages.

9. A taste-masking agent, comprising the EGCG-glucose adduct of claim 1 or the EGCG-glucose adduct prepared by the preparation method of any one of claims 2 to 6 and edible auxiliary materials.

10. The taste masking agent according to claim 9, wherein the mass ratio of the EGCG-glucose adduct to the edible auxiliary material is 1: 1-300.

Technical Field

The invention relates to the technical field of taste masking agents, in particular to an EGCG-glucose adduct and a preparation method and application thereof.

Background

As one of three kinds of non-alcoholic beverages in the world, the tea leaves have unique flavor which is loved by consumers. Baking is an important processing procedure in the tea processing process, so that the moisture of the tea can be reduced, the tea can be stored conveniently, and the flavor of the tea can be improved. After the tea is baked at a high temperature of 80-160 ℃, the bitterness and astringency are obviously reduced, the rice crust aroma or baking aroma is obviously improved, and the taste becomes thick and mellow.

It has been found that the change of taste of tea leaves during baking is not differentiated from the conversion of catechins having bitter and astringent taste. If the baking process of the tea leaves can be simulated, chemical components with obvious inhibition effect on the bitter taste or the astringent taste of the tea leaves or the tea drinks can be obtained, and the method can make important contribution to the field of foods or drinks.

Disclosure of Invention

In view of the above, the present invention aims to provide an EGCG-glucose adduct, and a preparation method and application thereof. The EGCG-glucose adduct obtained by the invention has good masking effect on bitter taste or astringent taste.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an EGCG-glucose adduct which has a structure shown in a formula I, a formula II, a formula III or a formula IV:

the invention provides a preparation method of the EGCG-glucose adduct, which comprises the following steps:

Preferably, the mass ratio of the epigallocatechin gallate to the glucose is (1-5): (1-5);

the mass of the water is 10-200% of the total mass of the epigallocatechin gallate and the glucose.

Preferably, the heating temperature is 100-200 ℃.

Preferably, the time of the nucleophilic addition reaction is 10-120 min.

Preferably, the separation and purification comprises the following steps:

mixing the nucleophilic addition product with a polar solvent, and concentrating after mixing to obtain a solution to be separated;

performing second column chromatography separation on the thin-layer chromatography separation component 1, continuously performing thin-layer chromatography separation and color development, and sequentially obtaining a second column chromatography separation component 1, a second column chromatography separation component 2 and a second column chromatography separation component 3, wherein a filler of the second column chromatography separation is silica gel, an eluent is a mixed solution of chloroform, acetone and formic acid, and the volume ratio of chloroform, acetone and formic acid in the mixed solution is (11-13): 6-8): 1-3;

the elution conditions of the high performance liquid chromatography separation are as follows:

0-4 minutes, 5% methanol water-20% methanol water;

20 to 23 percent of methanol water for 4 to 16 minutes;

16-19 minutes, 23% methanol water;

19-20 minutes, 23% -42% methanol water;

20-21 minutes, 42% methanol water-100% methanol;

for 21-22 minutes, 100% methanol-5% methanol water;

22-28 minutes, 5% methanol water.

The invention provides application of the EGCG-glucose adduct as a bitter taste masking agent.

Preferably, the taste-masking agent is a taste-masking agent of tea or tea beverages.

The invention provides a taste masking agent, which comprises the EGCG-glucose adduct and edible auxiliary materials.

Preferably, the mass ratio of the EGCG-glucose adduct to the edible auxiliary materials is 1: 1-300.

The invention provides an EGCG-glucose adduct which has a structure shown in a formula I, a formula II, a formula III or a formula IV. The EGCG-glucose adduct provided by the invention can obviously inhibit or mask the bitter taste or the astringent taste of tea or tea drinks, and can be used as a bitter and astringent taste masking agent in the fields of foods and drinks.

The invention provides a preparation method of the EGCG-glucose adduct, which is characterized in that the baking process of tea leaves is simulated, under the heating condition, C on 6-position and C on 8-position of an A ring in the EGCG are combined with glucose to generate nucleophilic addition reaction, in the further heating process, glucose groups connected on C6 and C8 of the EGCG are also subjected to dehydration and cyclization, and the EGCG-glucose adduct with the structure shown in formula I, formula II, formula III or formula IV is obtained after separation and purification. Meanwhile, the preparation method provided by the invention is simple to operate, easy to implement and low in cost.

Drawings

FIG. 1 is a flow chart of the separation and purification of example 1;

FIG. 2 shows the evaluation results of bitterness and astringency intensities of EGCG and glucose before and after baking;

FIG. 3 is a fragment ion diagram of EGCG-glucose adduct 1;

FIG. 4 is a fragment ion diagram of EGCG-glucose adduct 2;

FIG. 5 is a fragment ion diagram of EGCG-glucose adduct 3;

FIG. 6 is a fragment ion diagram of EGCG-glucose adduct 4;

FIG. 7 shows the results of UPLC-MS measurements of the nucleophilic addition product obtained in example 1 and a sample of yellow Camellia;

fig. 8 is a result of evaluation of the bitterness and astringency inhibition test of EGCG-glucose adduct.

Detailed Description

The invention provides an EGCG-glucose adduct which has a structure shown in a formula I, a formula II, a formula III or a formula IV:

the invention provides a preparation method of the EGCG-glucose adduct, which comprises the following steps:

The method comprises the steps of mixing epigallocatechin gallate, glucose and water, and carrying out nucleophilic addition reaction under the heating condition to obtain a nucleophilic addition product, wherein the nucleophilic addition product comprises EGCG-glucose adducts with structures shown in formula I, formula II, formula III and formula IV. In the invention, the purity of the epigallocatechin gallate and the glucose is preferably analytically pure, and the purity is preferably more than or equal to 97%.

In the present invention, the mass ratio of epigallocatechin gallate to glucose is preferably (1-5): (1-5), more preferably 1: 1. In the present invention, the mass of the water is 10% to 200%, more preferably 30% to 100%, of the total mass of epigallocatechin gallate and glucose.

In the present invention, the mixing means preferably includes vortex mixing and ultrasonic mixing which are sequentially performed. In the invention, the speed of vortex mixing is preferably 500r/min, and the time is preferably 2-3 min, and more preferably 2.5 min. In the invention, the power of the ultrasonic mixing is preferably 200W, the frequency is preferably 40kHz, and the time is preferably 2-3 min, and more preferably 2.5 min. The invention is beneficial to promoting the dissolution of the raw materials through the vortex mixing and the ultrasonic mixing.

In the invention, the heating mode is preferably oil bath heating, and the heating temperature is preferably 100-200 ℃, and more preferably 120-150 ℃.

In the invention, the time of the nucleophilic addition reaction is preferably 10-120 min, and more preferably 30-90 min. In the invention, under the heating condition, C on 6-position and C on 8-position of A ring in EGCG are combined with glucose to generate nucleophilic addition reaction, and in the further heating process, glucose groups connected with C6 and C8 of EGCG are also subjected to dehydration cyclization, and the EGCG-glucose adduct with the structure shown in formula I, formula II, formula III or formula IV is obtained after separation and purification.

After the nucleophilic addition product is obtained, the nucleophilic addition product is separated and purified to obtain the EGCG-glucose adduct with the structure shown in formula I, formula II, formula III or formula IV. In the present invention, the separation and purification preferably comprises the steps of:

and mixing the nucleophilic addition product with a polar solvent, and concentrating to obtain a solution to be separated.

In the present invention, the polar solvent is preferably one or more of methanol, ethanol, ethyl acetate, acetone and water, and more preferably methanol. The present invention utilizes the polar solvent to dissolve the nucleophilic addition product.

In the present invention, the volume ratio of the nucleophilic addition product to the polar solvent is preferably 1: 5. In the present invention, the concentration is preferably performed by concentrating the volume of the polar solvent to 20 to 30% of the original volume.

After the to-be-separated liquid is obtained, the to-be-separated liquid is subjected to first column chromatography separation to obtain a first column chromatography separation product.

In the invention, during the first column chromatography separation, the total sample volume of the liquid to be separated is preferably 10mL, the sample loading is preferably carried out for 3 times, and the sample loading volume is preferably 3-4 mL each time.

In the invention, the filler separated by the first column chromatography is Sephadex LH-20 gel, and the elution condition is methanol isocratic elution.

After the first column chromatography separation product is obtained, the thin-layer chromatography separation is carried out on the first column chromatography separation product, and a thin-layer chromatography separation component 1, a thin-layer chromatography separation component 2, a thin-layer chromatography separation component 3 and a thin-layer chromatography separation component 4 are sequentially obtained after color development.

In the invention, the developing solvent for the thin-layer chromatography is preferably a mixed solution of chloroform, acetone and formic acid, and the volume ratio of the chloroform, the acetone and the formic acid in the mixed solution is preferably (11-13): 6-8): 1-3, and more preferably 12:7: 2. In the present invention, the color developer for color development is preferably a vanillin sulfuric acid ethanol solution, and the mass concentration of the vanillin sulfuric acid ethanol solution is preferably 5%.

The thin-layer chromatography separation component 1 is subjected to second column chromatography separation, and the second column chromatography separation component 1, the second column chromatography separation component 2 and the second column chromatography separation component 3 are sequentially obtained after color development.

In the invention, the filler for the second column chromatography separation is preferably silica gel, and the particle size of the silica gel is preferably 100-200 meshes. In the invention, the elution phase of the second column chromatography separation is preferably a mixed solution of chloroform, acetone and formic acid, and the volume ratio of the chloroform, the acetone and the formic acid in the mixed solution is preferably (11-13): 6-8): 1-3, and more preferably 12:7: 2. In the present invention, the color developer for color development is preferably a vanillin sulfuric acid ethanol solution, and the mass concentration of the vanillin sulfuric acid ethanol solution is preferably 5%.

The second column chromatography separation component 2 is subjected to high performance liquid chromatography separation to sequentially obtain EGCG-glucose adducts with structures shown in formula I, formula II, formula III and formula IV.

In the invention, the chromatographic column for high performance liquid chromatography separation is Agilent ZORBAX SB-Aq C₁₈A chromatographic column, the size of the chromatographic column is 250 multiplied by 4.6mm, and the column diameter is 5 μm. In the present invention, the flow rate of the high performance liquid chromatography separation is preferably 1 mL/min.

In the invention, the mobile phase of the high performance liquid chromatography comprises an A phase and a B phase, wherein the A phase is formic acid water with the volume concentration of 0.2%, and the B phase is methanol;

the elution conditions of the high performance liquid chromatography separation are as follows:

0-4 minutes, 5% methanol water-20% methanol water;

20 to 23 percent of methanol water for 4 to 16 minutes;

16-19 minutes, 23% methanol water;

19-20 minutes, 23% -42% methanol water;

20-21 minutes, 42% methanol water-100% methanol;

for 21-22 minutes, 100% methanol-5% methanol water;

22-28 minutes, 5% methanol water;

the concentration of the methanol water is volume concentration.

As a specific example of the invention, during the high performance liquid chromatography separation process, 13.046min elution sample is EGCG-glucose adduct represented by the structure shown in formula I; 15.147min, eluting the sample to obtain EGCG-glucose adduct represented by the structure shown in formula II; 18.709min, eluting the sample to obtain EGCG-glucose adduct represented by the structure shown in formula III; 20.237min, the sample eluted is EGCG-glucose adduct represented by the structure shown in formula IV.

In the invention, the detection method of the EGCG-glucose adduct is preferably an UPLC-MS method, the flow rate during detection is preferably 1mL/min, and the mobile phase: phase A is 0.2% formic acid water, phase B is methanol, and elution conditions are as follows:

0-5 minutes, 5% methanol water-20% methanol water;

5-16 minutes, 20% methanol water-25% methanol water;

16-25 minutes, 25% methanol water;

25-38 minutes, 25% methanol water-45% methanol water;

38-40 minutes, 45% methanol water-100% methanol;

40-42 minutes, 100% methanol-5% methanol water;

42-55 minutes, 5% methanol water;

the concentration of the methanol water is volume concentration.

The invention provides application of the EGCG-glucose adduct as a bitter taste masking agent. In the present invention, the taste-masking agent is preferably a taste-masking agent of tea leaves or tea drinks.

The invention provides a taste masking agent, which comprises the EGCG-glucose adduct and edible auxiliary materials.

In the invention, the edible auxiliary materials are preferably one or more of starch, dextrin, lactose, mannitol and cellulose. In the invention, the mass ratio of the EGCG-glucose adduct to the edible auxiliary material is preferably 1: 1-300, more preferably 1: 10-200, and further preferably 1: 50-100.

The EGCG-glucose adduct provided by the present invention, the preparation method and the application thereof are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of EGCG-glucose adduct

(1) Weighing 5g of EGCG and 5g of glucose (analytical grade standard, purity 95%) in test tubes, adding 10ml of pure water (pH 8), vortexing for 3 minutes, performing ultrasound for 3 minutes, and transferring to an oil bath pan at 150 ℃ to heat for 60 minutes;

(2) dissolving a nucleophilic addition product of EGCG and glucose by using a pure methanol solution, wherein the methanol solution is dissolved for a small amount of times, completely dissolving the product, combining the methanol solutions, and concentrating under reduced pressure to 10ml to obtain the nucleophilic addition product;

(3) dissolving a nucleophilic addition product of EGCG and glucose by using a pure methanol solution, and then passing the nucleophilic addition product dissolved by the methanol solution through a Sephadex LH-20 gel column, and loading the product for 3 times, wherein the volume of each loading is 3-4 ml; the Sephadex LH-20 gel column is subjected to isocratic elution by using a pure methanol solution, then different component samples obtained by methanol elution are quickly separated by a thin-layer chromatography plate, chloroform/acetone/formic acid (12:7:2, v/v/v) is used as a developing agent, color development is carried out by using a sulfuric acid ethanol solution, and spots on the silica gel plate after color development are combined to obtain 4 components (Fr.1-Fr 4). And then, carrying out dry loading on the component Fr.1, passing through a silica gel column (100-200 meshes), wherein the eluent of the silica gel column is chloroform/acetone/formic acid (12:7:2, v/v/v), rapidly separating the sample eluted by the eluent by using a thin layer chromatography plate, developing the color by using a vanillin sulfuric acid ethanol solution, and combining spots on the developed silica gel plate to obtain the components Fr.1-Fr 3. Separating the fraction Fr.2 obtained by silica gel column separation with high performance liquid equipment, wherein the high performance liquid equipment adopts AgilentZORBAX SB-Aq C separation chromatographic column₁₈A (250X 4.6mm, 5 μm) column, flow rate 1ml/min, mobile phase methanol (phase B) and 0.2% formic acid (phase A),the elution conditions were: gradient elution for 0-4 min, 5% methanol water-20% methanol water; 4-16 minutes, 20% methanol water-23% methanol water; 16-19 minutes, 23% methanol water; 19-20 minutes, 23% methanol water-42% methanol water; 20-21 minutes, 42% methanol water-100% methanol; 21-22 minutes, 100% methanol-5% methanol water; 22-28 min, 5% methanol water.

EGCG-glucose adduct 1 (6-C-sulfonated product of (-) -epigallocatechin-3-O-gate) (retention time 13.046 minutes, mass 6.1 mg), EGCG-glucose adduct 2 (8-C-hindered byproduct of (-) -epigallocatechin-3-O-gate) (retention time 15.147 minutes, mass 10.0 mg), EGCG-glucose adduct 3(8-C-Glucosyl- (-) -epigallocatechin-3-O-gate) (retention time 18.709 minutes, mass 6.3 mg), EGCG-glucose adduct 4 (6/8-C-glucopyranosyl- (-) -epigallocatechin-3-O-gate) (retention time 20.237 minutes, mass 17.5 mg) were obtained in this order.

The flow chart of the separation and purification is shown in figure 1.

Property verification of EGCG-glucose adduct

The characteristics of the EGCG-glucose adduct 1 are as follows:

1) a brown-yellow amorphous powder soluble in methanol and deuterated acetone;

2)、UVλ_max(nm)：240，280；

3) LC-MS (negative ion mode): 763.1728([ M-H) } M/z]^-，C₃₄H₃₆O₂₀ ^-Theoretical calculation of 763.1799);

4) the NMR spectrum data are shown in Table 1.

The characteristics of the EGCG-glucose adduct 2 are as follows:

1) a brown-yellow amorphous powder soluble in methanol and deuterated acetone;

2)、UVλ_max(nm)：240，280；

3) LC-MS (negative ion mode): 763.1741([ M-H) } M/z]^-，C₃₄H₃₆O₂₀ ^-Theoretical calculation of 763.1799);

4) the NMR spectrum data are shown in Table 1.

The characteristics of the EGCG-glucose adduct 3 are as follows:

1) a brown-yellow amorphous powder soluble in methanol and deuterated acetone;

2)、UVλmax(nm)：240，280；

4) LC-MS (negative ion mode): 619.1304([ M-H) } M/z]^-，C₂₈H₂₈O₁₆ ^-Theoretical calculation of 619.1377);

the characteristics of the EGCG-glucose adduct 4 are as follows:

1) a brown-yellow amorphous powder soluble in methanol and deuterated acetone;

2)、UVλ_max(nm)：240，280；

3) LC-MS (negative ion mode): 781.1862([ M-H) } M/z]^-，C₃₄H₃₈O₂₁ ^-Theoretical calculation of 781.1905);

4) the NMR spectrum data are shown in Table 1.

TABLE 1 NMR spectroscopy data for EGCG-glucose adducts

Note:¹h NMR and¹³c NMR was measured at 600MHz with delta units in ppm, solvents were deuterated acetone and deuterated deuterium heavy water, and the blank of the table indicates no data here.

As can be seen from table 1, the structures of EGCG-glucose adduct 1, EGCG-glucose adduct 2, EGCG-glucose adduct 3, and EGCG-glucose adduct 4 correspond to the structures shown in formula I, formula II, formula III, and formula IV.

Performance testing

Experiment of inhibiting bitter and astringent taste intensity of EGCG-glucose adduct

Establishment of taste panel: according to the Chinese traditional tea sensory evaluation standard (GB/T23776-2018), 10 trained panelists evaluated the bitterness and astringency intensity of the samples before and after EGCG-glucose simulated baking. All panelists had 9 training sessions within 3 weeks before the evaluation. For more accurate evaluation of bitterness and astringency intensity and for quantification, EGCG aqueous solutions with different concentrations (0, 1, 2, 3, 4 and 5mM) were used for bitterness and astringency evaluation training, and the corresponding bitterness and astringency scores were 0, 1, 2, 3, 4 and 5 respectively.

Baking reaction of EGCG and glucose:

weighing 50 mg of EGCG in a test tube, adding 200 microliters of pure water, swirling for 2-3 minutes to fully dissolve and mix the EGCG, transferring the sample into a 150 ℃ oil bath pan to heat for 60 minutes, then immediately taking out the sample, dissolving the sample by using the pure water after cooling, transferring the sample into a 50ml volumetric flask, and then performing constant volume (named as R-EGCG); unbaked EGCG was used as a control, dissolved in purified water and taken up in a 50ml volumetric flask (named NR-EGCG).

Weighing 50 mg of EGCG and glucose in a test tube respectively, adding 200 microliters of pure water into the test tube, swirling for 2-3 minutes to fully dissolve and mix the EGCG and the glucose, transferring the sample into a 150 ℃ oil bath pan to heat for 60 minutes, then immediately taking out the sample, dissolving the sample by using the pure water after cooling, transferring the sample into a 50ml volumetric flask, and then carrying out constant volume (named as R- (EGCG + Glu)); unbaked EGCG and glucose were used as controls, and dissolved in purified water and made to a volume of 50ml in a volumetric flask (named (NR- (EGCG + Glu)).

And in sample group 3, 50 mg of glucose is weighed in a test tube, 200 microliters of pure water is added, the vortex is carried out for 2-3 minutes, the glucose is fully dissolved and uniformly mixed, the sample is transferred to a 150 ℃ oil bath pot to be heated for 60 minutes, then the sample is immediately taken out, the sample is dissolved by using the pure water after being cooled, the sample is transferred to a 50ml volumetric flask, 50 mg of EGCG is added, and then the dissolution constant volume is carried out (named as (R-Glu) + EGCG). The samples before and after baking EGCG and glucose were evaluated for bitterness and astringency intensity, and the results are shown in fig. 2. In FIG. 2, (a) is a graph showing the change in bitterness intensity before and after baking of EGCG-glucose, and (b) is a graph showing the change in astringency intensity before and after baking of EGCG-glucose.

The taste evaluation result shows that the bitter taste and the astringent taste of EGCG and glucose before and after baking are obviously different, and the bitter taste and the astringent taste are obviously reduced after baking. The bitter taste and the astringent taste intensity before and after the EGCG baking are not obvious, which indicates that the degradation or isomerization products of the EGCG after the baking have little influence on the bitter taste and the astringent taste. In order to demonstrate that the bitterness and astringency intensity of EGCG and glucose is significantly reduced after baking, the bitterness and astringency intensity of EGCG are not inhibited or masked by the baked product of glucose. The baked glucose was dissolved in water, and the bitterness and astringency intensities were re-evaluated by adding an equal amount of EGCG, and the change in bitterness and astringency intensities was found to be insignificant. Whereas the bitterness and astringency of the R- (EGCG + Glu) sample were significantly lower than those of the (R-Glu) + EGCG sample. Therefore, the significant reduction in bitterness and astringency of EGCG and glucose after baking is due to the formation of EGCG-glucose adducts after the baking treatment. After the tea leaves are baked at a high temperature, the bitterness and astringency are remarkably reduced, probably because the catechin compounds (EGCG) having higher intensity of bitterness and astringency form catechin-sugar adducts with sugar (glucose), and the bitterness and astringency of the tea leaves are suppressed or shielded. The tea beverage may be heated at high temperature during processing, and catechin-sugar adduct may be formed in the tea beverage to inhibit or mask bitterness and astringency of the tea beverage. Therefore, the EGCG-glucose adduct can be developed into an inhibitor or a masking agent for bitter taste or astringent taste of tea or tea drinks, and has wide application prospect in the field of foods or drinks.

Detection of EGCG-glucose adducts

Weighing 100 mg of the yellow and big tea leaves (powder) respectively, placing the yellow and big tea leaves (powder) into a 5ml centrifuge tube, adding 3 ml of 70% methanol solution, uniformly swirling, carrying out ultrasonic extraction at normal temperature for 10 minutes, centrifuging for 10 minutes at 5000 r/min, and taking supernatant to a 10ml volumetric flask. Extracting the tea residue twice with 70% methanol, repeating the above steps, mixing the extractive solutions, diluting with 70% methanol to a volume of 10ml, shaking, passing through 0.22 μm organic microporous membrane as mother liquor, and testing three samples.

The detection method of the UPLC-MS is adopted, the flow rate is 1ml/min (the tee joint is needed when the detection analysis is carried out because the flow rate is larger), and the mobile phase: phase A is 0.2% formic acid water, phase B is methanol, and elution conditions are as follows: 0-5 minutes, 5% methanol water-20% methanol water; 5-16 minutes, 20% -25% methanol water; 16-25 minutes, 25% methanol water; 25-38 minutes, 25% methanol water-45% methanol water; 38-40 minutes, 45% methanol water-100% methanol; 40-42 minutes, 100% methanol-5% methanol water; 42-55 minutes, 5% methanol water.

The nucleophilic addition product obtained in example 1 and the big yellow tea sample are subjected to UPLC-MS detection by taking EGCG-glucose adduct 1, EGCG-glucose adduct 2, EGCG-glucose adduct 3 and EGCG-glucose adduct 4 as the standard samples, wherein the fragment-ion diagram of EGCG-glucose adduct 1 is shown in fig. 3, the fragment-ion diagram of EGCG-glucose adduct 2 is shown in fig. 4, the fragment-ion diagram of EGCG-glucose adduct 3 is shown in fig. 5, the fragment-ion diagram of EGCG-glucose adduct 4 is shown in fig. 6, and the UPLC-MS detection of the nucleophilic addition product and the big yellow tea sample is shown in fig. 7. As can be seen from fig. 7, in the negative ion mode, the mass-to-charge ratio of the EGCG-glucose adduct 1 detected in the tea leaves is 763.1728, and the retention time is 12.453 minutes; the mass-to-charge ratio of the EGCG-glucose adduct 2 was 763.1741, retention time 14.326 minutes; the mass-to-charge ratio of the EGCG-glucose adduct 3 was 619.1304, retention time 17.636 minutes; the mass to charge ratio of EGCG-glucose adduct 4 was 781.1862 with a retention time of 18.300 minutes.

Tea soup bitterness and astringency inhibition test

Weighing 3g of green tea in an evaluation cup, adding 150mL of boiled water for brewing for 5 minutes, separating tea water, cooling tea soup, adding 4, 8 and 12mL of 10mg/mL EGCG-glucose adduct aqueous solution (the mass ratio of EGCG-glucose adduct 1, EGCG-glucose adduct 2, EGCG-glucose adduct 3 and EGCG-glucose adduct 4 in the aqueous solution is 1:1:1:1), taking the tea soup and 12mL of pure water as a control, stirring and uniformly mixing, and then respectively carrying out bitterness and astringency grading (n is 3) on each sample. The evaluation results are shown in fig. 8, in which (a) is a bitter taste evaluation result and (b) is an astringent taste evaluation result in fig. 8. As can be seen from fig. 8, as the amount of EGCG-glucose adduct added was increased, both bitterness and astringency of the tea soup were significantly reduced. Thus, the EGCG-glucose adduct has the effect of masking or inhibiting the bitterness and astringency intensity of the tea soup.

Caffeine, EGCG and quinic acid bitterness and astringency inhibition tests

Respectively preparing 100mL of 5mM caffein, EGCG and quinic acid solutions; and 5mM solutions of EGCG adduct 1, EGCG adduct 2, EGCG adduct 3, and EGCG adduct 4 were prepared each at 25 mL. Referring to the above method for scoring the intensity of bitterness and astringency of EGCG, 5mM caffeine, EGCG and quinic acid solution gave a bitterness or astringency score of 5. And then respectively mixing the caffein solution, the EGCG solution and the quinic acid solution with the EGCG-glucose adduct solution according to the volume ratio of 5:1, and then respectively carrying out bitter taste evaluation or astringent taste evaluation on each mixed sample by an evaluation team member. Since caffeine is mainly bitter, quinic acid is mainly astringent, and EGCG is mainly bitter and astringent, the evaluation results of the bitterness and astringency inhibition tests for caffeine, EGCG and quinic acid are shown in table 2. As can be seen from table 2, the EGCG-glucose adduct can significantly suppress the bitter taste of caffeine, the bitter and astringent taste of EGCG and the astringent taste of quinic acid; and the EGCG-glucose adduct 3 and the EGCG-glucose adduct 4 have better inhibition effect. Thus, the EGCG-glucose adduct can really mask or inhibit the bitterness and astringency of caffeine, EGCG and quinic acid.

TABLE 2 evaluation results of EGCG-glucose adducts for bitterness or astringency inhibition of caffeine, EGCG and quinic acid

Note: the blank in the table indicates no data. In the table, a, b and c in the above table respectively represent P < 0.05, P < 0.01 and P < 0.001.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

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EGCG-glucose adduct and preparation method and application thereof

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