Color generation substance, preparation method and application of color generation substance in color mixing of tea beverage and food

Abstract

The invention discloses a color generation substance, a preparation method and application of the color generation substance in color mixing of tea beverages and food, and the preparation method comprises the following steps: S1, mixing according to a proportion to prepare a phosphate buffer solution with a certain pH value; s2, weighing catechin substances, dissolving the catechin substances in the phosphate buffer solution obtained in the step S1, and fully dissolving the catechin substances to prepare a catechin buffer solution system; s3, heating the buffer system in the step S2 at high temperature to promote chemical reaction in the system; and S4, rapidly cooling by using an ice-water bath after heating is finished, and adding a protective solution to stop the reaction, so as to obtain a colored system containing the coloring substance. On the basis of an in-vitro simulation system, low-molecular-weight color generation substances are found, an efficient preparation method is established, the influence rule of concentration change on color generation is studied, the antioxidant capacity of the color generation substances is proved, and a material and theoretical basis is provided for quality regulation and control of tea and development and utilization of natural pigment substances; the color generation substance can be applied to color mixing of tea beverages and food.

Description

Technical Field

[0001] The invention belongs to the field of analysis and preparation of natural products, and specifically relates to a method for preparing a catechin non-enzymatic reaction color product and its application in coloring tea beverages and foods. Background Art

[0002] Catechins are a class of polyphenols widely found in plants and are one of the important components that determine the color, aroma and taste of tea. Phenotype catechins are relatively abundant in tea (Camellia sinensis) and have many biological activities that are beneficial to health, such as antioxidant, anti-tumor, anti-inflammatory, antibacterial and antiviral. Heat treatment is a relatively important process in tea processing. It can not only accelerate the loss of fresh leaves’ green grass smell and reduce water content, but also promote the formation of flavor substances. With the increase of heating and baking temperature and the extension of heating time, the tea soup gradually changes from light yellow-green to orange-red, and the dry tea changes from brown to dark brown. This may be because heating promotes the Maillard reaction and caramelization reaction. It may also be because the structure of catechins is unstable under the action of heat, and oxidative degradation and isomerization of polyphenols occur to form pigment substances, which together affect the change of tea color. This type of natural coloring substance obtained by simple reaction is more suitable for development and utilization in food.

[0003] In recent years, the use of in vitro simulation systems to study the non-enzymatic reactions of catechins has proven that catechins mainly undergo isomerization, degradation and polymerization reactions during the heating process. Since the color characteristics of pigments in tea are highly related to their molecular structure and degree of conjugation, the research and preparation of coloring substances mainly focus on oxidative polymerization reactions to generate polymers with relatively large molecular weights. However, problems such as unstable structures and long reaction times have led to major limitations on the application of natural pigments in tea. The discovery of low molecular weight coloring substances based on in vitro simulation systems and the establishment of efficient preparation methods are of great significance for promoting the research on thermal reaction products in tea processing, improving the color quality of tea, and the development and utilization of food coloring. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a method for preparing a non-enzymatic reaction color product of catechins and its application in tea beverage and food coloring. Through the synthetic preparation process described in the present invention, a coloring compound is obtained, and the coloring substance has biological activity and has application prospects in color presentation, and can be used in the material basis research of tea and the beverage and food industry or the biopharmaceutical industry.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] In one aspect, the method for preparing a coloring substance comprises the following process steps:

[0007] Step 1: Select a suitable phosphate buffer system and mix and prepare a phosphate buffer with appropriate pH according to the proportion;

[0008] Step 2: Weigh catechins and dissolve them in the phosphate buffer obtained in step 1, and fully dissolve them to prepare a catechin buffer system with an appropriate concentration;

[0009] Step 3: heating the catechin buffer system in step 2 at high temperature to promote the chemical reaction in the system;

[0010] Step 4: After heating, cool rapidly in an ice water bath, add a protective liquid to stop the reaction, and obtain a colored system containing a coloring substance;

[0011] Step 5: Use reversed liquid chromatography to separate and purify the colored substances in the colored system, select octadecyl bonded stationary phase filler as the chromatographic separation material, weigh 320g of filler (particle size 10μm), add 700mL of isopropanol, stir and disperse to mix, and fill the slurry into a dynamic axial column (DAC column) with a diameter of 50mm. The column bed height is 260mm, and the mobile phase A is 0.05% trifluoroacetic acid aqueous solution (v / v), and the mobile phase B is methanol. The mobile phase gradient elution mode is 0-10min, 8-15% B; 10-40min, 15-25% B; 40-45min, 25-100% B; 45-60min, 100% B. The flow rate is set to 80mL / min, the column temperature is room temperature, the detection wavelength is 320nm, and the injection volume is 125mL. Collect fractions according to the retention time of the sample liquid phase preparation spectrum, and collect the 20-30min segment fraction to obtain a fraction rich in coloring substances;

[0012] Step 6: Pre-freeze the fraction in a -80°C refrigerator, transfer it to a vacuum freeze dryer, set the freezing temperature to -80°C, and perform reduced pressure freeze drying to remove the solvent to obtain a colored substance.

[0013] Furthermore, the phosphate buffer system in step 1 refers to a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer system, or a disodium hydrogen phosphate-citric acid buffer system, and the appropriate pH refers to a pH value of 6 to 8.

[0014] Furthermore, the catechins in step 2 refer to epicatechin gallate and epigallocatechin gallate.

[0015] Furthermore, the appropriate concentration in step 2 is 300-800 mg / mL, preferably 500 mg / mL.

[0016] Furthermore, the high temperature heating in step 3 refers to heating at 80-120°C.

[0017] Furthermore, in step 4, the ice water bath is at 0-4°C.

[0018] Furthermore, in step 4, the protective solution is a mixed solution of 70% methanol water, 1% hydrochloric acid water, 0.4% ascorbic acid and 28.6% water, and the ratio of the protective solution to the reaction solution is 1:1 (v / v).

[0019] Furthermore, the pre-freezing time in step 6 is 4 to 24 hours.

[0020] On the other hand, the present invention also provides a coloring substance prepared by the above method, wherein the chemical structural formula of the coloring substance is as follows:

[0021]

[0022] The above compound is named as: 4,7-dihydroxycoumarin, yellow powder solid ( Figure 1 ).

[0023] Possible potential reaction mechanisms:

[0024]

[0025] EGCG first easily degallic acidizes to form EGC in an alkaline environment, and then undergoes oxidation on the B ring to form an o-quinone structure (intermediate A), which is further oxidized, hydrated and ring-opened to form intermediate B. Intermediate B is hydrolyzed to obtain intermediate C, and intermediate C undergoes esterification to form intermediate D with a coumarin core. The hydroxyl group on the structure of intermediate D shifts to form the coloring substance 4,7-dihydroxycoumarin.

[0026] Furthermore, the coloring substance monomer solution exhibits the following coloring properties and L*a*b* values ​​as the concentration changes (eg Figure 2 , Figure 3 shown): Figure 3 The color performance of the coloring substance in terms of brightness (L*), red-green (a*) and yellow-blue (b*) is described. As the concentration of the coloring substance 4,7-dihydroxycoumarin gradually increases, the color of the solution gradually changes from colorless to yellow, orange-red to reddish brown. The L value corresponding to the brightness of the reaction solution gradually decreases, and the a value and b value gradually increase, respectively reflecting that the red and yellow degrees of the solution gradually deepen.

[0027] Furthermore, the coloring substance has certain antioxidant activity. Figure 4 and Figure 5 This indicates the antioxidant activity of color-producing substances. Figure 4 It is the ABTS free radical scavenging ability of the coloring substance at different concentrations; Figure 5 is the DPPH radical scavenging ability under different concentrations of coloring substances; with the gradual increase of the concentration of 4,7-dihydroxycoumarin in the solution, the ABTS free radical scavenging ability and the DPPH free radical scavenging ability both gradually increased, proving that the pigment has certain antioxidant activity and its antioxidant ability is positively correlated with the monomer concentration of the coloring substance.

[0028] In a third aspect, the present invention further proposes the use of the coloring substance in coloring tea beverages and foods.

[0029] In the present invention, according to the reaction characteristics of epicatechin gallate and epigallocatechin gallate, an in vitro monomer reaction system is constructed, and a compound that has not been reported and has a contribution to color is discovered. The color change of the reaction system is further analyzed, and the pigment substance is identified by using a colorimeter and an ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometer. The influence of concentration changes on the color of the substance is summarized, and it is proved that the substance has antioxidant ability, which provides a certain material and theoretical basis for the quality control of tea and the development and utilization of natural pigment substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Color substance map;

[0031] Figure 2 Visual diagram of the color of the coloring substance at different concentrations;

[0032] Figure 3 L*a*b* values ​​of coloring substances at different concentrations;

[0033] Figure 4 ABTS free radical scavenging ability of color-producing substances at different concentrations;

[0034] Figure 5 DPPH free radical scavenging ability of coloring substances at different concentrations;

[0035] Figure 6 Color change diagram of in vitro reaction system with reaction time;

[0036] Figure 7 Ultra-high performance liquid chromatography-high resolution mass spectrometry primary negative ion mode spectrum of the color-producing substance;

[0037] Figure 8 Ultra-high performance liquid chromatography-high resolution mass spectrometry primary negative ion mode spectra of the color-producing substance and 4,7-dihydroxycoumarin standard;

[0038] Fig. 9 Ultra-high performance liquid chromatography-high resolution mass spectrometry secondary negative ion mode spectra of the color-producing substance and 4,7-dihydroxycoumarin standard;

[0039] Fig.10 Changes in the content of coloring substances in oxidized solutions of EGCG monomers treated with different methods;

[0040] Fig.11 The difference in the content of coloring substances in green tea before and after roasting;

[0041] Fig.12 The difference in the content of coloring substances in beauty tea before and after roasting. DETAILED DESCRIPTION

[0042] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0043] Example 1

[0044] The method for preparing a coloring substance comprises the following process steps:

[0045] Step 1: Prepare 0.2M disodium hydrogen phosphate solution and 0.1M citric acid solution respectively, and mix them in proportion to prepare a phosphate buffer solution with a pH of 8;

[0046] Step 2: Weigh 250 mg of epigallocatechin gallate and dissolve it in 500 mL of phosphate buffer to prepare a 500 mg / L epigallocatechin gallate buffer system;

[0047] Step 3: Take 10 mL of epigallocatechin gallate buffer and heat it in a water bath at 80 °C for 0, 15, 30, 60, and 90 min;

[0048] Step 4: After heating, use a 0°C ice-water mixture to quickly cool to room temperature, take 5 mL of the reaction solution and add 5 mL of the protective solution to stop the reaction;

[0049] The color of the in vitro reaction system changes with different reaction times (0, 15, 30, 60, 90 min), such as Figure 6 As shown: as the reaction time increases, the color of the reaction system changes from colorless to yellow to red and gradually deepens, proving that coloring substances are generated in the system and that the coloring substances can accumulate in the reaction system as the reaction time increases.

[0050] Example 2

[0051] The method for preparing a coloring substance comprises the following process steps:

[0052] Step 1: Prepare 0.2M disodium hydrogen phosphate and sodium dihydrogen phosphate solutions respectively, and mix them in proportion to prepare phosphate buffers with pH values ​​of 7 and 8 respectively;

[0053] Step 2: Weigh 250 mg of epigallocatechin gallate and dissolve it in 500 mL of phosphate buffer to prepare a 500 mg / L epigallocatechin gallate buffer system;

[0054] Step 3: Take 10 mL of epigallocatechin gallate buffer and heat it in a water bath at 80°C for 0, 15, 30, 60, and 90 min respectively;

[0055] Step 4: After heating, cool rapidly in an ice-water bath with a 0°C ice-water mixture, take 5 mL of the reaction solution and add 5 mL of the protective solution to stop the reaction.

[0056] The above colored system was detected in negative ion mode by ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry, and a low molecular weight colored substance ( Figure 7 ), analyze the properties of the color-producing substance and compare with the standard ( Figure 8 , Fig. 9 ). The content of coloring substances in the oxidized solution of EGCG monomers treated with different treatments in the in vitro reaction system is shown in Fig.10 As shown. It is proved that the coloring substance in the reaction system includes a substance with a mass-to-charge ratio of 177.0184 (MH), namely 4,7-dihydroxycoumarin. The content of the coloring substance has the same trend as the color change of the reaction system, which further proves that the substance contributes to the color change of the reaction system during the reaction.

[0057] Example 3

[0058] Step 1: Spread out and dry the spring and autumn tea leaves at room temperature (20-25°C) for 12 hours, steam (100°C, 90 seconds) or stir-fry (280°C, 90 seconds), roll for 30 minutes, and then dry for 45 minutes to obtain the final sample with a moisture content of 6%;

[0059] Step 2: The steamed youth tea and stir-fried youth tea samples obtained in step 1 were not treated (control) and roasted at 120°C for 4 hours (roasting); the steamed autumn tea samples were not treated (control), roasted at 120°C for 4 hours (light roasting), and roasted for 8 hours (heavy roasting);

[0060] Step 3: Take 3 g of the tea sample obtained in step 2, brew it with 100°C hot water for 4 min (tea to water ratio 1:50), and observe the color of the tea soup;

[0061] Step 4: Detect the tea soup obtained in step 3 using ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry.

[0062] The differences in the content of coloring substances in tea samples treated with different methods and the changes in the color of tea soup are as follows: Fig.11 As shown in the figure, for spring tea, after different withering methods and then roasting, the content of 4,7-dihydroxycoumarin increased significantly and the color of the tea soup became darker; for tea leaves of different seasons, during the tea making process of spring tea and autumn tea, after the roasting process, the content of 4,7-dihydroxycoumarin increased significantly and was positively correlated with the degree of roasting. This shows that the roasting process is conducive to the formation of 4,7-dihydroxycoumarin in tea leaves and thus affects the color of tea soup.

[0063] Example 4

[0064] Step 1: withering, greening, piling, killing and drying the fresh leaves of Meiren Tea to obtain the final sample;

[0065] Step 2: roast the beauty tea samples obtained in step 1 at 105°C for 0, 2, 3, and 4 hours respectively;

[0066] Step 3: Take 3 g of the tea sample obtained in step 2, brew it with 100°C hot water for 5 min (tea:water ratio 1:50), and observe the color of the tea soup;

[0067] Step 4: Detect the tea soup obtained in step 3 using ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry.

[0068] The differences in the content of coloring substances in tea samples treated with different methods and the changes in the color of tea soup are as follows: Fig.12 As shown in the figure, as the roasting time increases, the content of 4,7-dihydroxycoumarin in the beauty tea sample gradually increases, causing the color of the tea soup to gradually change to dark red.

[0069] It should be noted that the 4,7-dihydroxycoumarin standard refers to the purchased 4,7-dihydroxycoumarin with a concentration greater than 98%.

[0070] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a coloring substance, characterized in that: The process steps include: S1: Select a phosphate buffer system and mix and prepare a phosphate buffer with a certain pH according to the proportion; S2: Weighing catechins and dissolving them in the phosphate buffer obtained in step S1 to fully dissolve them to prepare a catechin buffer system with a certain concentration; S3: heating the catechin buffer system in step S2 at high temperature to promote the chemical reaction in the system; S4: After heating, the mixture is rapidly cooled in an ice water bath, and a protective liquid is added to stop the reaction, thereby obtaining a colored system containing a coloring substance; S5: separating and purifying the colored substance in the colored system by reversed phase liquid chromatography to obtain a fraction rich in the colored substance; S6: Pre-freeze the fraction in a -80°C refrigerator, transfer to a vacuum freeze dryer, set the freezing temperature to -80°C, and freeze-dry under reduced pressure to remove the solvent to obtain a colored substance.

2. The method for preparing a coloring substance according to claim 1, characterized in that: The phosphate buffer system in step S1 is one of a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system, a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer system, and a disodium hydrogen phosphate-citric acid buffer system, and the certain pH value is a pH value of 6 to 8.

3. The method for preparing a coloring substance according to claim 1, characterized in that: In step S2, the catechin substances are epicatechin gallate and epigallocatechin gallate.

4. The method for preparing a coloring substance according to claim 1, characterized in that: The concentration of the catechin buffer system in step S2 is 300-800 mg / mL, the high temperature heating in step S3 is 80-120° C., and the ice water bath in step S4 is 0-4° C.

5. The method for preparing a coloring substance according to claim 1, characterized in that: The separation and purification in step S5 is specifically as follows: selecting an octadecyl bonded stationary phase filler as a chromatographic separation material, weighing a certain amount of filler, adding isopropanol, stirring and dispersing, and filling the slurry into a dynamic axial column (DAC column) with a diameter of 50 mm, a column bed height of 260 mm, mobile phase A is 0.05% trifluoroacetic acid aqueous solution (v / v), mobile phase B is methanol, and the mobile phase gradient elution mode is 0-10min, 8-15% B; 10-40min, 15-25% B; 40-45min, 25-100% B; 45-60min, 100% B; the flow rate is set to 80mL / min, the column temperature is room temperature, the detection wavelength is 320nm, and the injection volume is 125mL; fractions are collected according to the retention time of the sample liquid phase preparation spectrum, and the 20-30min segment fraction is collected to obtain a fraction rich in coloring substances.

6. The method for preparing a coloring substance according to claim 1, characterized in that: The protective solution in step S4 is a mixed solution of 70% methanol water, 1% hydrochloric acid water, 0.4% ascorbic acid and 28.6% water, and the volume ratio of the protective solution to the reaction solution is 1:

1.

7. The method for preparing a coloring substance according to claim 1, characterized in that: The pre-freezing time in step S6 is 4 to 24 hours.

8. A coloring substance obtained by the preparation method according to any one of claims 1 to 7, characterized in that: The coloring substance is a yellow powdery solid with antioxidant activity, and its chemical structure is as follows:

9. A color-producing substance according to claim 8, characterized in that: The coloring substance is dissolved in an organic solvent to obtain a monomer solution having the following coloring characteristics as the concentration changes: as the concentration of the coloring substance 4,7-dihydroxycoumarin gradually increases, the color of the solution gradually changes from colorless to yellow, orange-red to reddish brown, and methanol is preferably the organic solvent.

10. Use of the coloring substance according to claim 8 or 9 in coloring tea beverages and foods.

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