Tea leaf fixation treatment method based on light wave and microwave coupling

Abstract

The invention discloses a tea leaf fixation treatment method based on light wave and microwave coupling, and belongs to the technical field of tea leaf production. Through the synergistic effect of an immobilized enzyme technology, microwaves, light waves, frequency bands and steam, cell walls of tea cells are fully broken, so that beneficial components in the tea are more easily extracted and absorbed, nutritional components in the tea are cooperatively reserved in multiple steps, the nutritional value of the tea is increased, and the tea has a good health care effect. The treated tea leaves are mellow and smooth in mouth feel and have unique fragrance and flavor; the prepared immobilized enzyme has high adsorption capacity, TiO2 microspheres are coated with aminated SiO2, the surface active sites and adsorption area of the carrier are increased, and the space structure and chemical properties of the carrier are optimized by combining modification of acetylated distarch phosphate, so that the immobilized enzyme has higher affinity and specific adsorption capacity on a substrate, and the immobilized enzyme has a good application prospect. The enzymolysis efficiency is improved, the use amount of the enzyme is reduced, the production cost is reduced, and the immobilized enzyme can be recycled and reused.

Description

Technical Field

[0001] The invention belongs to the technical field of tea production, and in particular relates to a tea withering treatment method based on light wave and microwave coupling. Background Art

[0002] Tea leaf withering is a crucial step in the tea making process. Its core is to quickly passivate the activity of polyphenol oxidase in fresh leaves through high temperature, inhibit enzymatic oxidation reaction, and evaporate part of the water to soften the leaves, laying the foundation for subsequent rolling, drying and other processes. The traditional tea leaf withering process mainly includes typical methods such as frying, steaming, baking and sun drying.

[0003] Stir-frying is the most common way to kill green tea. Usually, the fresh leaves are placed in an iron pan or drum at 200-300℃ and stir-fried for 3-5 minutes, and the temperature of the leaves is quickly raised to above 75℃ through heat conduction. This method relies on the operator's experience in controlling the heat and the frequency of stir-frying, and is easy to form the unique "wok fragrance" of tea. The steaming method uses 100-120℃ steam treatment for 30-60 seconds to quickly kill enzymes through moist heat, and is often used in the process of retaining the bright green color of tea. The baking method uses hot air as the medium to gradually dehydrate the tea leaves at 80-120℃ for 10-30 minutes; while the sun-drying method uses sunlight to wither naturally, which takes up to several hours and is often seen in post-fermented teas such as Pu'er tea.

[0004] Although the above traditional methods have been used in tea processing for a long time, they still have certain limitations. For example, during the process of frying and steaming green tea, heat energy is transferred from the outside to the inside, which can easily lead to uneven heating of the leaves, charring of the outer layer and residual enzyme activity in the inner layer; baking and sun drying green tea may aggravate the oxidative degradation of heat-sensitive components such as tea polyphenols and vitamins due to the long processing time. In addition, the traditional process has limited physical wall-breaking effect on the cell wall of tea leaves, making it difficult to fully release active substances such as intracellular polysaccharides and flavonoids, which to a certain extent restricts the improvement of the nutritional value of tea. How to achieve efficient and uniform withering treatment while retaining the active ingredients of tea leaves to the maximum extent is still a technical difficulty that needs to be optimized in this field. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the present invention combines light wave and microwave coupling technology with immobilized enzyme technology to achieve efficient and uniform heating in the process of tea withering, thereby retaining the active ingredients in the tea to the maximum extent, fully releasing the beneficial substances in the tea cells, and significantly improving the nutritional value and taste quality of the tea.

[0006] In order to achieve the above purpose, the following technical scheme is adopted: The present invention provides a tea leaf withering treatment method based on light wave and microwave coupling, comprising the following steps:

[0007] (1) Pretreatment: Select fresh tea leaves that are free of pests and diseases, wash them, remove impurities and dust on the surface, and then screen them to remove leaves and impurities that do not meet the standards;

[0008] (2) Enzyme treatment: the pretreated tea leaves are mixed with a phosphate buffer solution having a pH of 7.6 to prepare an enzymatic solution, and then introduced into an immobilized enzyme reaction device for enzymatic hydrolysis at 30-50° C. and a flow rate of 5-20 BV / h for 1-2 h, wherein the immobilized column of the immobilized enzyme reaction device is filled with a mixed enzyme preparation, wherein the mixed enzyme preparation comprises ficin, cellulase, hemicellulase and pectinase, and the mixed enzyme preparation is obtained by mixing the above four enzymes with an immobilized enzyme carrier solution and immobilizing them;

[0009] (3) Microwave sterilization: The enzyme-treated tea leaves are placed in an environment with a temperature of 10-30°C and a humidity of 30-90% to cool for 0.5-3 hours, and then microwaved for 30-90 seconds using a microwave device with a frequency of 2450 MHz and a power of 10 kW, so that the internal temperature of the tea leaves quickly rises to 80-90°C;

[0010] (4) Light wave treatment: Use red light with a wavelength of 600-700nm and an intensity of 1000-1500lux to irradiate the tea leaves for 1-2min;

[0011] (5) Ultrasonic treatment: Use 20-50kHz ultrasonic wave band to treat tea leaves for 3-5 minutes;

[0012] (6) Steam treatment: Use steam at 100-120°C to kill the tea leaves for 5-8 minutes;

[0013] (7) Drying: Dry the steam-treated tea leaves at 60°C for 1 h;

[0014] The immobilized enzyme carrier solution is prepared by the following steps:

[0015] S1, add to ethanol, then add 0.1-0.5mol / L soluble zinc salt solution and complexing agent, ultrasonically treat at 30-60kHz for 15-30min, then filter, wash with deionized water 3 times to obtain TiO2 microspheres, add the TiO2 microspheres to 0.05-0.1mol / L sodium methylcellulose solution, then ultrasonically treat at 30-60kHz for 15-30min, add TEOS, anhydrous ethanol and 0.5-2mol / L ammonia water, first react at 200rpm for 2h, then switch to 400rpm for 5h, then filter out the solid, wash with ethanol 3 times to obtain amination SiO2 coated TiO2 microspheres;

[0016] S2. Add the amino SiO2-coated TiO2 microspheres into deionized water, perform ultrasonic treatment at 30-60kHz for 20-50min, then add acetylated distarch phosphate, stir at 95°C and 450-600rpm for 5h, cool to room temperature, filter out the precipitate, wash with deionized water 3 times, freeze-dry at 4°C, then add the obtained solid into a phosphate buffer solution with a pH of 7.6, add 0.05-0.2mol / L glutaraldehyde solution, and react at 25-30°C for 2-5h to obtain the immobilized enzyme carrier solution.

[0017] Furthermore, in the step (2), the mass ratio of the pretreated tea leaves to the phosphate buffer solution in the enzymatic hydrolysis solution is 1:3-7.

[0018] Furthermore, the mass ratio of ficin, cellulase, hemicellulase and pectinase is 2-6:1-5:1-5:1-5.

[0019] Furthermore, the total enzyme activity in the immobilized column is higher than 1000 U / g, and the packing density of the immobilized column is 0.5-1.0 g / mL.

[0020] Furthermore, in step S1, the mass ratio of ethanol, soluble zinc salt solution and complexing agent is 10-30:1-3:0.1-0.5.

[0021] Furthermore, in step S1, the mass ratio of TiO2 microspheres, sodium methylcellulose solution, TEOS, anhydrous ethanol and ammonia water is 1-2:10-20:1.5-3:5-20:0.1-0.3.

[0022] Furthermore, in step S2, the mass ratio of the aminated SiO2-coated TiO2 microspheres, deionized water, acetylated distarch phosphate, phosphate buffer solution and glutaraldehyde solution is 1-3:20-50:0.5-1.5:4-8:0.5-1.

[0023] Furthermore, the soluble zinc salt is one of zinc nitrate, zinc chloride, zinc sulfate, zinc phosphate, zinc lactate, zinc acetate or zinc citrate.

[0024] Furthermore, the complexing agent is one of disodium ethylenediaminetetraacetate, formamide, acetamide, propionamide, isobutylamide, ethylenediamine, 1,2-propylenediamine, 1,4-butylenediamine or 1,10-decanediamine.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention uses the synergistic effect of immobilized enzyme technology, microwaves, light waves, frequency bands and steam to fully rupture the cell walls of tea cells, making it easier to extract and absorb the beneficial ingredients in the tea leaves;

[0027] (2) In the traditional method of withering tea leaves at high temperatures, the tea polyphenols, amino acids and other nutrients in the tea leaves will be oxidized and decomposed in large quantities due to the long-term heat action. The microwave withering method used in the present invention can quickly raise the internal temperature of the tea leaves to an appropriate range in a short time, thereby reducing the high temperature action time. The light wave treatment uses red light of a specific wavelength and intensity to gently stimulate the tea leaves cells without causing excessive heat damage, thereby promoting physiological and biochemical reactions in the cells. The ultrasonic treatment and steam treatment are also carried out under relatively mild conditions. The multi-step synergy retains the nutrients in the tea leaves to the maximum extent, which not only improves the nutritional value of the tea leaves, but also makes the treated tea leaves taste more mellow and smooth, with a unique aroma and flavor, thus satisfying the consumer's demand for high-quality tea leaves.

[0028] (3) The immobilized enzyme prepared by the present invention has a high adsorption capacity. During the preparation process, the surface active sites and adsorption area of ​​the carrier are increased by aminated SiO2 coating of the TiO2 microspheres; combined with the modification of acetylated distarch phosphate, the spatial structure and chemical properties of the carrier are further optimized, so that the immobilized enzyme has a stronger affinity and specific adsorption ability for the substrate. The immobilized enzyme with high adsorption capacity can more efficiently bind to the tea substrate during the enzymatic hydrolysis process, thereby improving the enzymatic hydrolysis efficiency, reducing the amount of enzyme used, and reducing the production cost. At the same time, it is also beneficial to the recovery and reuse of the immobilized enzyme, thereby improving the economy and sustainability of the entire withering treatment method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of the tea leaf withering method based on light wave and microwave coupling of the present invention;

[0030] Figure 2 The tea polyphenols, amino acid content and phenol-to-ammonia ratio test results of various embodiments and comparative examples of the present invention are shown in FIG.

[0031] Figure 3 The thermal stability test results of the immobilized enzymes in Examples 1-3 of the present invention and the free enzyme in Comparative Example 1;

[0032] Figure 4 This is the test result of the reuse efficiency of the immobilized enzyme in Example 1 of the present invention.

[0033] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0035] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0036] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0037] Example 1

[0038] Tea withering treatment method based on light wave and microwave coupling

[0039] (1) Pretreatment: Select fresh tea leaves that are free of pests and diseases, wash them, remove impurities and dust on the surface, and then screen them to remove leaves and impurities that do not meet the standards;

[0040] (2) Enzyme treatment: The pretreated tea leaves were mixed with a phosphate buffer solution having a pH of 7.6 at a mass ratio of 1:7 into an enzymatic solution, and the mixture was introduced into an immobilized enzyme reaction device for enzymatic hydrolysis at 50° C. and a flow rate of 20 BV / h for 2 h. The immobilized column of the immobilized enzyme reaction device was filled with a mixed enzyme preparation, which included ficin, cellulase, hemicellulase and pectinase. The mixed enzyme preparation was obtained by mixing and immobilizing the above four enzymes with an immobilized enzyme carrier solution. The mass ratio of ficin, cellulase, hemicellulase and pectinase was 6:5:5:5. The immobilized enzyme carrier solution was prepared by the following steps:

[0041] S1: add it to ethanol, then add 0.5mol / L zinc nitrate solution and complexing agent disodium ethylenediaminetetraacetate, the mass ratio of ethanol, zinc nitrate and disodium ethylenediaminetetraacetate is 30:3:0.5, ultrasonically treat at 60kHz for 30min, then filter, wash with deionized water 3 times to obtain TiO2 microspheres, add TiO2 microspheres to 0.1mol / L sodium methylcellulose solution, then ultrasonically treat at 60kHz for 30min, add TEOS, anhydrous ethanol and 2mol / L ammonia water, the mass ratio of TiO2 microspheres, sodium methylcellulose solution, TEOS, anhydrous ethanol and ammonia water is 2:20:3:20:0.3, react at 200rpm for 2h, then switch to 400rpm for 5h, then filter out the solid, wash with ethanol 3 times to obtain amino SiO2-coated TiO2 microspheres;

[0042] S2: The aminated SiO2-coated TiO2 microspheres were added to deionized water, ultrasonically treated at 60kHz for 50min, and then acetylated distarch phosphate was added. After stirring at 95°C and 600rpm for 5h, the mixture was cooled to room temperature, the precipitate was filtered out, washed with deionized water for 3 times, and freeze-dried at 4°C. The obtained solid was then added to a phosphate buffer solution with a pH of 7.6, and then a 0.2mol / L glutaraldehyde solution was added. The immobilized enzyme carrier solution was obtained by reacting at 30°C for 5h. The mass ratio of aminated SiO2-coated TiO2 microspheres, deionized water, acetylated distarch phosphate, phosphate buffer solution and glutaraldehyde solution was 3:50:1.5:8:1. The total enzyme activity in the immobilized column was higher than 1000U / g, and the filling density of the immobilized column was 1.0g / mL.

[0043] (3) Microwave sterilization: The enzyme-treated tea leaves are placed in an environment with a temperature of 30°C and a humidity of 90% for 3 hours, and then microwaved for 90 seconds using a microwave device with a frequency of 2450 MHz and a power of 10 kW, so that the internal temperature of the tea leaves quickly rises to 90°C;

[0044] (4) Light wave treatment: Use red light with a wavelength of 700nm and an intensity of 1500lux to irradiate the tea leaves for 2min;

[0045] (5) Ultrasonic treatment: Use 50kHz ultrasonic wave band to treat tea leaves for 5 minutes;

[0046] (6) Steam treatment: Use 120°C steam to kill the tea leaves for 8 minutes;

[0047] (7) Drying: Dry the steam-treated tea leaves at 60°C for 1 h.

[0048] Example 2

[0049] Tea withering treatment method based on light wave and microwave coupling

[0050] (1) Pretreatment: Select fresh tea leaves that are free of pests and diseases, wash them, remove impurities and dust on the surface, and then screen them to remove leaves and impurities that do not meet the standards;

[0051] (2) Enzyme treatment: The pretreated tea leaves were mixed with a phosphate buffer solution having a pH value of 7.6 at a mass ratio of 1:3 into an enzymatic solution, and the mixture was introduced into an immobilized enzyme reaction device for enzymatic hydrolysis at 30° C. and a flow rate of 5 BV / h for 1 h. The immobilized column of the immobilized enzyme reaction device was filled with a mixed enzyme preparation, which included ficin, cellulase, hemicellulase and pectinase. The mixed enzyme preparation was obtained by mixing and immobilizing the above four enzymes with an immobilized enzyme carrier solution. The mass ratio of ficin, cellulase, hemicellulase and pectinase was 2:1:1:1. The immobilized enzyme carrier solution was prepared by the following steps:

[0052] S1: add it to ethanol, then add 0.1mol / L zinc chloride solution and complexing agent ethylenediamine, the mass ratio of ethanol, zinc chloride solution and ethylenediamine is 10:1:0.1, ultrasonic treatment is carried out at 30kHz for 15min, then filtered, washed with deionized water for 3 times to obtain TiO2 microspheres, add TiO2 microspheres to 0.05mol / L sodium methylcellulose solution, then ultrasonic treatment is carried out at 30kHz for 15min, TEOS, anhydrous ethanol and 0.5mol / L ammonia water are added, the mass ratio of TiO2 microspheres, sodium methylcellulose solution, TEOS, anhydrous ethanol and ammonia water is 1:10:1.5:5:0.1, react at 200rpm for 2h, then switch to 400rpm for 5h, then filter out the solid, wash with ethanol 3 times to obtain amino SiO2-coated TiO2 microspheres;

[0053] S2: The aminated SiO2-coated TiO2 microspheres were added to deionized water, ultrasonically treated at 30kHz for 20min, and then acetylated distarch phosphate was added. After stirring at 95℃ and 450rpm for 5h, the mixture was cooled to room temperature, the precipitate was filtered out, washed with deionized water for 3 times, and freeze-dried at 4℃. The obtained solid was then added to a phosphate buffer solution with a pH of 7.6, and then a 0.05mol / L glutaraldehyde solution was added. The solution was reacted at 25℃ for 2h to obtain an immobilized enzyme carrier solution. The mass ratio of aminated SiO2-coated TiO2 microspheres, deionized water, acetylated distarch phosphate, phosphate buffer solution and glutaraldehyde solution was 1:20:0.5:4:0.5. The total enzyme activity in the immobilized column was higher than 1000U / g, and the filling density of the immobilized column was 0.5g / mL.

[0054] (3) Microwave sterilization: The enzyme-treated tea leaves are placed in an environment with a temperature of 10°C and a humidity of 30% to cool for 0.5 h, and then microwaved for 30 s using a microwave device with a frequency of 2450 MHz and a power of 10 kW, so that the internal temperature of the tea leaves quickly rises to 80°C;

[0055] (4) Light wave treatment: Use red light with a wavelength of 600nm and an intensity of 1000lux to irradiate the tea leaves for 1min;

[0056] (5) Ultrasonic treatment: Use 20kHz ultrasonic wave band to treat tea leaves for 3 minutes;

[0057] (6) Steam treatment: Use steam at 100°C to kill the tea leaves for 5 minutes;

[0058] (7) Drying: Dry the steam-treated tea leaves at 60°C for 1 h.

[0059] Example 3

[0060] Tea withering treatment method based on light wave and microwave coupling

[0061] (1) Pretreatment: Select fresh tea leaves that are free of pests and diseases, wash them, remove impurities and dust on the surface, and then screen them to remove leaves and impurities that do not meet the standards;

[0062] (2) Enzyme treatment: The pretreated tea leaves were mixed with a phosphate buffer solution having a pH value of 7.6 at a mass ratio of 1:5 into an enzymatic solution, and the mixture was introduced into an immobilized enzyme reaction device for enzymatic hydrolysis at a temperature of 40° C. and a flow rate of 12 BV / h for 1.5 h. The immobilized column of the immobilized enzyme reaction device was filled with a mixed enzyme preparation, which included ficin, cellulase, hemicellulase and pectinase. The mixed enzyme preparation was obtained by mixing and immobilizing the above four enzymes with an immobilized enzyme carrier solution. The mass ratio of ficin, cellulase, hemicellulase and pectinase was 4:3:3:3. The immobilized enzyme carrier solution was prepared by the following steps:

[0063] S1: add it to ethanol, then add 0.3 mol / L zinc acetate solution and complexing agent 1,4-butanediamine, the mass ratio of ethanol, zinc acetate solution and 1,4-butanediamine is 20:2:0.3, ultrasonic treatment is carried out at 45 kHz for 30 minutes, then filtered, washed with deionized water for 3 times to obtain TiO2 microspheres, add TiO2 microspheres to 0.07 mol / L sodium methyl cellulose solution, then ultrasonic treatment is carried out at 45 kHz for 30 minutes, TEOS, anhydrous ethanol and 1.2 mol / L ammonia water are added, the mass ratio of TiO2 microspheres, sodium methyl cellulose solution, TEOS, anhydrous ethanol and ammonia water is 1.5:15:2.2:12:0.2, react at 200 rpm for 2 hours, then switch to 400 rpm for 5 hours, then filter out the solid, wash with ethanol 3 times to obtain amino SiO2-coated TiO2 microspheres;

[0064] S2: Add the aminated SiO2-coated TiO2 microspheres to deionized water, ultrasonically treat at 45kHz for 35min, then add acetylated distarch phosphate, stir at 95℃ and 520rpm for 5h, cool to room temperature, filter out the precipitate, wash with deionized water 3 times, freeze-dry at 4℃, then add the obtained solid to a phosphate buffer solution with a pH of 7.6, then add 0.1mol / L glutaraldehyde solution, react at 28℃ for 3.5h to obtain an immobilized enzyme carrier solution, the mass ratio of aminated SiO2-coated TiO2 microspheres, deionized water, acetylated distarch phosphate, phosphate buffer solution and glutaraldehyde solution is 2:30:1:5:0.8, the total enzyme activity in the immobilized column is higher than 1000U / g, and the filling density of the immobilized column is 0.75g / mL;

[0065] (3) Microwave sterilization: The enzyme-treated tea leaves were placed in an environment with a temperature of 20°C and a humidity of 60% for 1.75 hours, and then microwaved for 60 seconds using a microwave device with a frequency of 2450 MHz and a power of 10 kW, so that the internal temperature of the tea leaves quickly rose to 85°C;

[0066] (4) Light wave treatment: Use red light with a wavelength of 650nm and an intensity of 1250lux to irradiate the tea leaves for 1.5min;

[0067] (5) Ultrasonic treatment: Use 35kHz ultrasonic wave band to treat tea leaves for 4 minutes;

[0068] (6) Steam treatment: Use steam at 110°C to kill the tea leaves for 6.5 min;

[0069] (7) Drying: Dry the steam-treated tea leaves at 60°C for 1 h.

[0070] Comparative Example 1

[0071] In this comparative example, the pretreated tea leaves were mixed with a mixed enzyme at a ratio of 80:1, wherein the mass ratio of ficin, cellulase, hemicellulase and pectinase in the mixed enzyme was 4:3:3:3, and the rest were the same as in Example 3.

[0072] Comparative Example 2

[0073] In this comparative example, the tea leaves were not sterilized by microwave after cooling, and the rest were the same as those in Example 3.

[0074] Comparative Example 3

[0075] In this comparative example, no light wave treatment was performed, and the rest was the same as in Example 3.

[0076] Comparative Example 4

[0077] In this comparative example, no ultrasonic treatment was performed, and the rest was the same as in Example 3.

[0078] Comparative Example 5

[0079] In this comparative example, no steam treatment was performed, and the rest was the same as in Example 3.

[0080] Results Analysis

[0081] Test Example 1

[0082] Tea sensory evaluation

[0083] The tea leaves prepared in each embodiment and comparative example were subjected to sensory evaluation according to GB / T 23776-2018 “Tea Sensory Evaluation Method” and scored on a percentage basis. The results are shown in Table 1.

[0084] Table 1 Sensory evaluation results of tea leaves of Examples 1-3 and Comparative Examples 1-5

[0085]

[0086]

[0087] It can be seen from the sensory evaluation results in Table 1 that the tea leaves of Examples 1-3 have obtained high scores in terms of appearance, color, aroma, taste and soup color, especially Example 3 is particularly outstanding in terms of appearance, color and aroma. In contrast, the scores of Comparative Examples 1-5 are generally low. During the high-temperature treatment process, the traditional method of withering will cause a large amount of oxidation and decomposition of tea polyphenols, amino acids and other nutrients in the tea leaves due to long-term thermal action, affecting the aroma, taste and soup color. The microwave withering used in the present invention can quickly raise the internal temperature of the tea leaves to a suitable range in a short time, reducing the high-temperature action time; light wave treatment uses red light of a specific wavelength and intensity to gently stimulate tea cells without causing excessive thermal damage, and promote physiological and biochemical reactions in the cells; ultrasonic treatment and steam treatment are also carried out under relatively mild conditions, and multiple steps are coordinated to maximize the retention of nutrients in the tea leaves, so that the treated tea leaves have a strong aroma, a mellow taste and a good soup color.

[0088] Test Example 2

[0089] Determination of tea polyphenols, amino acid content and phenol-amino acid ratio in tea

[0090] According to GB / T 8313-2002, the tea polyphenols content and amino acid content were tested and the phenol-amino acid ratio was calculated. The results are shown in Figure 2 .

[0091] from Figure 2 It can be seen from the results that the tea polyphenols and amino acid contents of Examples 1-3 are significantly higher than those of Comparative Examples 1-5, and the phenol-ammonia ratio is reduced. This shows that the present invention not only improves the tea polyphenols and amino acid contents in tea leaves, but also reduces the phenol-ammonia ratio through the synergistic effect of immobilized enzyme, microwave sterilization, light wave treatment, ultrasonic treatment and steam treatment, so that the taste of tea leaves is mellower and the bitterness is reduced. This further proves that the multi-step coordinated treatment method of the present invention can not only quickly passivate the polyphenol oxidase activity in tea leaves and inhibit enzymatic oxidation reactions, but also retain the nutrients in tea leaves to the maximum extent under mild treatment conditions. The lower phenol-ammonia ratio makes tea leaves rich in trace elements and mellow in taste, and reduces bitterness, which meets the demand of consumers for high-quality tea leaves.

[0092] Test Example 3

[0093] Immobilized enzyme performance test

[0094] According to the thermal stability of the immobilized enzymes in Examples 1-3 and the free enzyme in Comparative Example 1, and the reuse efficiency of the immobilized enzyme in Example 1, the results are shown in Table 1. Figure 3 and Figure 4 .

[0095] from Figure 3 and Figure 4It can be seen from the results that the immobilized enzymes of Examples 1-3 have high thermal stability and enzyme activity, and the immobilized enzyme of Example 1 still has high enzyme activity after being reused 10 times. This is because in the process of preparing the immobilized enzyme, the surface active sites and adsorption area of ​​the carrier are increased by aminated SiO2 coating of the TiO2 microspheres; combined with the modification of acetylated distarch phosphate, the spatial structure and chemical properties of the carrier are further optimized, so that the immobilized enzyme has a stronger affinity and specific adsorption capacity for the substrate. The immobilized enzyme with high adsorption capacity can more efficiently bind to the tea substrate during the enzymolysis process, thereby improving the enzymolysis efficiency. In addition, Example 1 still has a high enzyme activity after being reused 10 times, which is conducive to the recovery and reuse of the immobilized enzyme, improves the economy and sustainability of the entire withering treatment method, and reduces production costs.

[0096] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0097] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for withering tea leaves based on light wave and microwave coupling, characterized in that: The following steps are involved: (1) Pretreatment: Select fresh tea leaves that are free of pests and diseases, wash them, remove impurities and dust on the surface, and then screen them to remove leaves and impurities that do not meet the standards; (2) Enzyme treatment: the pretreated tea leaves are mixed with a phosphate buffer solution having a pH of 7.6 to prepare an enzymatic solution, and then introduced into an immobilized enzyme reaction device for enzymatic hydrolysis at 30-50° C. and a flow rate of 5-20 BV / h for 1-2 h, wherein the immobilized column of the immobilized enzyme reaction device is filled with a mixed enzyme preparation, wherein the mixed enzyme preparation comprises ficin, cellulase, hemicellulase and pectinase, and the mixed enzyme preparation is obtained by mixing the above four enzymes with an immobilized enzyme carrier solution and immobilizing them; (3) Microwave sterilization: The enzyme-treated tea leaves are placed in an environment with a temperature of 10-30°C and a humidity of 30-90% to cool for 0.5-3 hours, and then microwaved for 30-90 seconds using a microwave device with a frequency of 2450 MHz and a power of 10 kW, so that the internal temperature of the tea leaves quickly rises to 80-90°C; (4) Light wave treatment: Use red light with a wavelength of 600-700nm and an intensity of 1000-1500lux to irradiate the tea leaves for 1-2 minutes; (5) Ultrasonic treatment: Use 20-50 kHz ultrasonic waves to treat tea leaves for 3-5 minutes; (6) Steam treatment: Use steam at 100-120°C to kill the tea leaves for 5-8 minutes; (7) Drying: Dry the steam-treated tea leaves at 60°C for 1 h; The immobilized enzyme carrier solution is prepared by the following steps: S1, add to ethanol, then add 0.1-0.5mol / L soluble zinc salt solution and complexing agent, ultrasonically treat at 30-60kHz for 15-30min, then filter, wash with deionized water 3 times to obtain TiO2 microspheres, add the TiO2 microspheres to 0.05-0.1mol / L sodium methylcellulose solution, then ultrasonically treat at 30-60kHz for 15-30min, add TEOS, anhydrous ethanol and 0.5-2mol / L ammonia water, first react at 200rpm for 2h, then switch to 400rpm for 5h, then filter out the solid, wash with ethanol 3 times to obtain amination SiO2 coated TiO2 microspheres; S2. Add the amino SiO2-coated TiO2 microspheres into deionized water, perform ultrasonic treatment at 30-60kHz for 20-50min, then add acetylated distarch phosphate, stir at 95°C and 450-600rpm for 5h, cool to room temperature, filter out the precipitate, wash with deionized water 3 times, freeze-dry at 4°C, then add the obtained solid into a phosphate buffer solution with a pH of 7.6, add 0.05-0.2mol / L glutaraldehyde solution, and react at 25-30°C for 2-5h to obtain the immobilized enzyme carrier solution.

2. The method for withering tea leaves based on light wave and microwave coupling according to claim 1, characterized in that: In the step (2), the mass ratio of the pretreated tea leaves to the phosphate buffer solution in the enzymatic hydrolysis solution is 1:3-7.

3. The method for withering tea leaves based on light wave and microwave coupling according to claim 2, characterized in that: The mass ratio of the ficinase, the cellulase, the hemicellulase and the pectinase is 2-6:1-5:1-5:1-5.

4. The method for withering tea leaves based on light wave and microwave coupling according to claim 3, characterized in that: The total enzyme activity in the immobilized column is higher than 1000 U / g, and the filling density of the immobilized column is 0.5-1.0 g / mL.

5. The method for withering tea leaves based on light wave and microwave coupling according to claim 4, characterized in that: In the step S1, the mass ratio of ethanol, soluble zinc salt solution and complexing agent is 10-30:1-3:0.1-0.

5.

6. The method for withering tea leaves based on light wave and microwave coupling according to claim 5, characterized in that: In the step S1, the mass ratio of TiO2 microspheres, sodium methylcellulose solution, TEOS, anhydrous ethanol and ammonia water is 1-2:10-20:1.5-3:5-20:0.1-0.

3.

7. The method for withering tea leaves based on light wave and microwave coupling according to claim 6, characterized in that: In the step S2, the mass ratio of the aminated SiO2-coated TiO2 microspheres, deionized water, acetylated distarch phosphate, phosphate buffer solution and glutaraldehyde solution is 1-3:20-50:0.5-1.5:4-8:0.5-1.

8. The method for withering tea leaves based on light wave and microwave coupling according to claim 7, characterized in that: The soluble zinc salt is one of zinc nitrate, zinc chloride, zinc sulfate, zinc phosphate, zinc lactate, zinc acetate or zinc citrate.

9. The method for withering tea leaves based on light wave and microwave coupling according to claim 8, characterized in that: The complexing agent is one of disodium ethylenediaminetetraacetate, formamide, acetamide, propionamide, isobutylamide, ethylenediamine, 1,2-propylenediamine, 1,4-butylenediamine or 1,10-decanediamine.

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