Modified tea residue dietary fiber capable of improving biological activity and application of modified tea residue dietary fiber

Abstract

The invention discloses a modified tea residue dietary fiber capable of improving biological activity and application of the modified tea residue dietary fiber. The modified tea residue dietary fiber is prepared by the following steps: pretreating tea residues by combining microwaves and bio-enzyme, extracting by using a H2O2 solution containing Vc-FeSO4, and precipitating and drying to obtain insoluble tea residue dietary fiber; and performing concentration, alcohol precipitation and deproteinization on the supernate to obtain the soluble tea residue dietary fiber. The dissolution rate, free radical scavenging ability, alpha-amylase inhibition ability and caenorhabditis elegans anti-stress ability of the tea residue SDF can be significantly improved by a free radical mediated modification technology treatment mode (plt; 0.05) of the method. The process is mild in condition, green and environment-friendly, and meets the production requirements of health-care food.

Description

(1) Technical Field

[0001] The present invention relates to a modified tea residue dietary fiber with improved biological activity and its application. (2) Background Art

[0002] As a natural and healthy beverage, tea has a long history of over a thousand years and is deeply loved by people. In 2020, the global tea consumption reached 6.3 million tons, and it is expected to increase to 7.4 million tons in the next five years. China is the largest tea-producing country in the world. In 2020, China's tea output was 2.986 million tons, an increase of 0.1926 million tons compared with the previous year, with an increase rate of 9.62%. The output of tea residue generated from tea production and consumption is very large. For every 1 ton of black tea produced, 30 - 50 kg of tea residue can be generated, and for every 1 ton of green tea produced, 30 kg of tea residue can be generated. According to incomplete statistics, only tea beverage production enterprises in China generate 180,000 - 200,000 tons of tea residue every year. Only a very small part of such a huge amount of tea residue is reasonably utilized, and most of it is treated as agricultural waste, which not only pollutes the environment but also causes a huge waste of biological resources. Paying more attention to the utilization of tea residue and improving the resource utilization rate can not only improve economic benefits but also cherish and protect environmental resources.

[0003] The tea residues after tea soaking contain abundant dietary fibers (DF) such as cellulose, hemicellulose, and lignin. The DF in tea residues mainly consists of insoluble dietary fiber (IDF). Research shows that the roles played by IDF and soluble dietary fiber (SDF) in the human body are different, and the physiological functions of DF are closely related to the ratio of the two. Compared with IDF, SDF from plants has important physiological functions and excellent physicochemical properties. For example, SDF can reduce lipid digestion and show better effects in controlling blood sugar. In addition, SDF has a greater ability to form gels and act as emulsifiers than IDF, which makes it easy to be incorporated into foods. Data indicate that the content of SDF in dietary fiber should reach more than 10% to be regarded as high-quality dietary fiber with strong physiological activity and health care functions. Although tea residues are rich in dietary fiber, they are mainly IDF, with a low SDF content and defective physicochemical and functional characteristics, unable to meet their applications in the fields of food, medicine, etc. Through reasonable and effective modification, not only can the connection bonds of DF components be broken and transformed into small molecules, enabling some IDF to be transformed into SDF, but also the dense network structure of DF can be changed into a loose network structure, with higher physicochemical properties such as water-binding capacity, swelling ratio, and adsorption capacity, and better physiological functions can be exerted. Therefore, finding the best modification method to increase the effective dissolution of SDF in tea residues and achieve a reasonable ratio of SDF > 10% in the DF composition will provide new ideas for the comprehensive utilization of tea residues, turn waste into treasure, increase their economic and nutritional value, and realize the high-value utilization of biological resources.

[0004] The modification methods of DF usually include mechanical method, physical method, chemical method, and biological enzyme method, etc. Among them, the mechanical method mostly has problems such as high equipment requirements, high energy consumption, insufficient cell wall breaking, and local high temperature. The physical method is simple and easy to operate, but requires a large enough temperature difference and is only suitable for laboratory operations. The chemical method is prone to introducing impurities and has problems such as safety and environmental pollution. The biological enzyme method has a high production cost and it is difficult for a single biological enzyme to effectively break the cell wall, and multiple enzymes need to be used in combination. Different modification methods are different in changing the corresponding physicochemical properties of polysaccharides. For example, ultrasonic modification can change the properties of polysaccharides such as destroying the molecular structure, decreasing the degree of polymerization, and reducing the molecular weight; the modified product of phosphoric acid esterification has an enhanced scavenging effect on hydroxyl radicals and also has a very weak scavenging ability on superoxide radicals of the original polysaccharide; etherification modification can improve the water solubility of hemicellulose and decrease its thermal stability. Various modification methods enable natural polysaccharides to have a wider application in the fields of food, medicine, materials, etc. Moist heat modification, citric acid esterification modification, oxidation modification, complexation modification, etc. have improved the utilization value of polysaccharides in the food field.

[0005] As a processing by-product of the most commonly consumed beverage daily, tea residue, it is of great significance and economic value to properly extract and optimize the modified tea residue polysaccharides. In view of the characteristics of high IDF content, poor palatability and low bioavailability in tea residue DF, this invention first pre-treats the tea residue, and then, taking the dissolution rate of SDF in tea residue as the response value, through a free radical-mediated modification method, on the basis of single factor experiments, supplemented by Box-Behnken experimental design optimization, obtains the optimal technological parameters for the effective dissolution of tea residue SDF, so as to obtain tea residue SDF with high yield and strong biological activities (such as antioxidant activities like ABTS and DPPH free radical scavenging abilities, α-amylase inhibitory ability and the ability to extend the lifespan of Caenorhabditis elegans under oxidative stress), as well as tea residue IDF with excellent physical and chemical properties, thus realizing the high-quality reuse of tea residue waste. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a modified tea residue dietary fiber and its application. Using tea residue as the raw material, after first pre-treating it by microwave combined with biological enzymes, then taking the dissolution rate of SDF in tea residue as the response value, through a free radical-mediated modification method, with Fe 2+ -Vc-H2O2 as the extractant, through single factor experiments and Box-Behnken experimental design optimization, obtains tea residue SDF with high yield and strong biological activities (such as antioxidant activities like ABTS and DPPH free radical scavenging abilities, α-amylase inhibitory ability and the ability to extend the lifespan of Caenorhabditis elegans under oxidative stress), as well as tea residue IDF with excellent physical and chemical properties.

[0007] The technical solution adopted by this invention is as follows:

[0008] This invention provides a modified tea residue dietary fiber with enhanced biological activities. The modified tea residue dietary fiber includes insoluble tea residue dietary fiber and soluble tea residue dietary fiber, and the modified tea residue dietary fiber is prepared by the following method:

[0009] (1) Weigh tea residue powder, add deionized water, microwave-treat it for 5 - 30 min under the condition of a power of 50 - 250 W, then add a mixture of hemicellulase and cellulase, adjust the pH to 4.8, enzymatically hydrolyze it for 0.5 - 2.5 h under the condition of a temperature of 30 - 70 °C. After the enzymatic hydrolysis ends, place it in a boiling water bath at 100 °C to inactivate the enzyme for 5 min. After adjusting the pH to 7.0, obtain tea slurry;

[0010] (2) Add Vc, FeSO4 and deionized water to a 30% H2O2 solution by mass concentration, fully dissolve and shake well to obtain an H2O2 solution containing Vc-FeSO4;

[0011] (3) Add all the H2O2 solution containing Vc-FeSO4 in step (2) to the tea slurry in step (1), mix well to obtain a tea residue-Vc-FeSO4-H2O2 mixture;

[0012] (4) Place the tea residue-Vc-FeSO4-H2O2 mixture obtained in step (3) into a constant temperature oscillator and oscillate it. Adjust the oscillator parameters to 50-100 °C and 100-200 rpm, and perform reciprocating gyratory oscillation for 20-120 min; Take out the mixture, centrifuge it (preferably centrifuge at 8000 r / min for 10 min) to obtain the supernatant and the residue; Dry the residue at 50 °C to obtain insoluble tea residue dietary fiber (IDF).

[0013] (5) Rotavapor-concentrate the supernatant obtained in step (4) to one-fifth of its original volume to obtain a concentrated solution; Slowly add 95% ethanol, which is 4 times the volume of the concentrated solution, while stirring, place it in a refrigerator at 4 °C for alcohol precipitation for 12 h, then carefully pour out the supernatant and retain the precipitate in the lower layer;

[0014] (6) Centrifuge the precipitate in the lower layer obtained in step (5) (preferably centrifuge at 1000 r / min for 5 min in a centrifuge), take the precipitate, add water, and rotavapor-concentrate it at 65 °C until the ethanol is completely evaporated to obtain a concentrated solution;

[0015] (7) Deproteinize the concentrated solution obtained in step (6) by the Sevag method to obtain soluble tea residue dietary fiber (SDF).

[0016] Preferably, the tea residue powder in step (1) is prepared as follows: Place the tea residue after extracting the tea soup in an oven and dry it at a low temperature (preferably 60 °C) until the moisture content is below 8%, put it into a pulverizer and pulverize it, and pass it through a 200-300 mesh sieve after pulverization to obtain the tea residue powder.

[0017] Preferably, the volume of deionized water used in step (1) is 20-70 mL / g based on the mass of the tea residue powder, preferably 50-70 mL / g, more preferably 60 mL / g; The mixture of hemicellulase and cellulase is composed of hemicellulase and cellulase mixed at a mass ratio of 4:1, and the mixture of hemicellulase and cellulase is 1-2% based on the mass of the tea residue powder, preferably 1.6%; The enzymatic hydrolysis temperature is preferably 50 °C for enzymatic hydrolysis for 2 h. The microwave treatment conditions are preferably 250 W for 20 min.

[0018] Preferably, the mass ratio of the 30% H2O2 solution in step (2) to the tea residue powder in step (1) is 5 - 8:1, preferably 6.4:1; the mass ratio of Vc to the tea residue powder in step (1) is 0.001 - 0.1:1, preferably 0.048:1; the mass ratio of FeSO4 to the tea residue powder in step (1) is 0.1 - 1:1, preferably 0.192:1; the deionized water is 1 - 10 mL / g based on the mass of the tea residue powder in step (1), preferably 2.36 mL / g; the mass ratio of Vc to FeSO4 is 1:4; the ratio of H2O2 in the 30% H2O2 solution to the total mass of Vc and FeSO4 is 8:1.

[0019] Preferably, the oscillation conditions in step (4) are 90 °C and 200 rpm, with reciprocating gyratory oscillation for 70 min.

[0020] Preferably, the protein removal step in step (7) is as follows: Add one - quarter volume of Sevag reagent (chloroform: n - butanol = 4:1, v:v) to the concentrated solution, keep it at a constant temperature of 37 °C and oscillate for 25 min, then pour the mixture into a centrifuge tube, centrifuge at 4000 r / min for 5 min, take the supernatant and repeat the above protein removal operation until there is no absorption peak at 280 nm in the upper layer; Rotate and evaporate the organic solvent from the supernatant after the last protein removal at 50 °C to obtain a purified concentrated solution of tea residue SDF; Freeze - dry the concentrated solution of tea residue SDF, with an initial temperature of - 30 °C, start heating when the vacuum degree is 80 Pa, keep it at - 20 °C for 1 h, then keep it for 1 h when the temperature rises by 10 °C each time, keep heating until 20 °C, and keep it for more than 24 h until the sample is dry (water content is below 8%) to obtain soluble tea residue dietary fiber.

[0021] The present invention also provides an application of the modified tea residue dietary fiber in the preparation of an antioxidant, and the modified tea residue dietary fiber is soluble tea residue dietary fiber.

[0022] The present invention also provides an application of the modified tea residue dietary fiber in the preparation of an α - amylase inhibitor, and the modified tea residue dietary fiber is soluble tea residue dietary fiber.

[0023] The present invention also provides an application of the modified tea residue dietary fiber in the preparation of a preparation for prolonging the lifespan of Caenorhabditis elegans under oxidative stress, and the modified tea residue dietary fiber is soluble tea residue dietary fiber.

[0024] The present invention also provides an application of the modified tea residue dietary fiber in flour - based products such as biscuits and noodles, and the modified tea residue dietary fiber is insoluble tea residue dietary fiber.

[0025] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0026] 1. The present invention adopts the pretreatment of microwave and biological enzymes, a green chemical modification method, which is simple and easy to operate, environmentally friendly. Combining with the molecular weight distribution range of polysaccharides, tea residue SDF with high purity can be prepared; the dissolution rate of SDF can be as high as over 19.75%, and it has high radical scavenging abilities such as ABTS and DPPH; it has strong α-amylase inhibitory ability; and it has good protective effects on C.elegans under oxidative stress conditions.

[0027] 2. A large amount of insoluble dietary fiber with dense structures such as cellulose, hemicellulose, and lignin exists in the tea residue after tea soaking, and there are defects in its physical and chemical and functional properties, as well as problems such as high energy consumption, insufficient cell wall breaking, and local high temperature in conventional modification methods. Microwave is a new heat source that can be used to produce hot compressed water. Through the intense friction generated by the dipole rotation of water molecules, rapid sample heating can be achieved; microwave not only has advantages in shortening the reaction time, but also has advantages in providing the hydrolysis of biomass rich in carbohydrates. Different types of biological enzymes such as cellulase and hemicellulase can more effectively modify DF due to the differences in the cell wall breaking sites of tea residue. Hydrogen peroxide is a clean and efficient oxidant and a typical environmental protection agent, and it is also one of the most commonly used reagents for generating oxygen free radicals. Based on this, on the basis of the pretreatment of microwave-assisted and biological enzyme methods for tea residue, using the fact that hydrogen peroxide can generate super strong free radicals under the action of reducing agents and organic acids, it can efficiently break the cell wall and has the unique advantages of decolorization and reducing the molecular weight of DF. Combining with the design method of BBD, while fully ensuring the controllable crushing effect of the cell wall and forming a loose and porous state, it is beneficial to the full dissolution of soluble SDF in tea residue DF, and to prepare functional SDF with a narrow molecular weight distribution, without destroying the integrity of the "active fragments" of SDF during the cell wall breaking process, and with high yield. At the same time, it has many advantages such as no impurity introduction, no pollution, low equipment requirements, and low energy consumption in the whole operation process.

[0028] 3. The product prepared by the free radical-mediated modification technology for treating tea residue has no peculiar smell and good sensory properties, and no post-treatment such as decolorization is required. The low-concentration hydrogen peroxide used in the operation is easily decomposed into water and oxygen after the operation, and no residues will be left during the whole unit operation process. Fe 2+ -Vc added during the operation helps the effective dissolution of SDF in tea residue and the enhancement of biological activity. The treatment method of the free radical-mediated modification technology can significantly improve the dissolution rate of tea residue SDF, the free radical scavenging abilities (ABTS + scavenging rate, DPPH free radical scavenging rate), α-amylase inhibitory ability, and the anti-stress ability of Caenorhabditis elegans (p<0.05). The dissolution rate of tea residue SDF, ABTS +, the DPPH free radical scavenging rate and α - amylase inhibition rate were increased by 2.69 times, 2.44 times, 2.09 times, and 1.55 times respectively compared with the tea residue SDF extracted by traditional hot water extraction; the average survival time of nematodes under hydrogen peroxide induction and methyl viologen induction was increased by 20.06% and 39.85% respectively compared with the tea residue SDF extracted by traditional hot water extraction. This modification process also had a good improvement effect on the physicochemical properties of IDF in tea residues. Moreover, the water - binding capacity of tea residue IDF passing through a 150 - mesh sieve after modification was 1.91 times that of the tea residue before modification, and the sodium cholate adsorption capacity was 32.04 times that before modification, both showing significant effects (p < 0.05). This process has mild conditions, is green and environmentally friendly, and meets the production requirements of health foods. (IV) Description of Drawings

[0029] Figure 1 , Effects of different microwave pretreatment conditions (microwave time, solid - liquid ratio, and microwave power) on the dissolution rate of tea residue SDF.

[0030] Figure 2 , Effects of different biological enzyme treatment conditions (enzymolysis time, temperature, and enzyme addition amount) on the dissolution rate of tea residue SDF.

[0031] Figure 3 , Effects of different extraction conditions (liquid - solid ratio, extraction time, temperature, and H2O2 concentration) on the extraction yield of tea residue SDF.

[0032] Figure 4 , Response surface and contour maps of the effects of different extraction factors on the dissolution rate of tea residue SDF.

[0033] Figure 5 , Effects of Vc addition amount on the dissolution rate of tea residue SDF.

[0034] Figure 6 , Effects of ferrous sulfate addition amount on the dissolution rate of tea residue SDF.

[0035] Figure 7 , Effects of Vc - FeSO4 addition amount on the dissolution rate of tea residue SDF.

[0036] Figure 8 , Effects of raw material particle size on the dissolution rate of tea residue SDF.

[0037] Figure 9 , Water - binding capacity, swelling ratio, oil - holding capacity, and sodium cholate adsorption capacity of tea residue IDF before and after modification.

[0038] Figure 10 , Scavenging abilities of tea residue SDF with different concentrations on DPPH, hydroxyl, ABTS, and PTIO free radicals.

[0039] Figure 11 , Inhibitory ability of tea residue SDF on α - amylase.

[0040] Figure 12 3. Effects of tea residue SDF on the survival rate of C. elegans under hydrogen peroxide-induced oxidative stress.

[0041] Figure 13 4. Effects of tea residue SDF on the survival rate of C. elegans under methyl viologen-induced oxidative stress. (V) Specific implementation manners

[0042] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0043] The modified phenol-sulfuric acid method described in the embodiments of the present invention refers to the standard: SN / T 4260-2015 Determination of crude polysaccharides in exported plant-derived foods.

[0044] Example 1 Optimization of the conditions for modifying tea residue dietary fiber

[0045] 1 Materials and reagents

[0046] Tea residue after extracting tea beverage (provided by Meitingbao Plant Technology China Co., Ltd.).

[0047] Hydrogen peroxide, ascorbic acid, phenol, sulfuric acid, soybean oil, 0.1 mol / L sodium phosphate buffer solution (pH 7.0), 1 mmol / L and 6 mmol / L sodium cholate aqueous solutions, 0.1 mol / L glucose aqueous solution, acarbose, potato starch, α-amylase (5 mU / mL), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxo-1-oxyl (PTIO), ethanol, potassium persulfate. Hemicellulase (activity 6 U / mg), cellulase (activity 50 U / mg), all purchased from Yuanye Biotechnology Co., Ltd. The water used in the experiment is deionized water.

[0048] N2 wild-type Caenorhabditis elegans and Escherichia coli OP50 were kindly provided by the College of Animal Sciences, Zhejiang University.

[0049] 2 Experimental instruments

[0050] JP-250A-2 High-Speed Multifunctional Pulverizer, Shanghai Jiupin Industry and Trade Co., Ltd.; FA224 Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.; HH-S Digital Display Constant Temperature Water Bath, Jintan Guowang Experimental Instrument Factory; SHA-B Digital Display Constant Temperature Oscillator, Changzhou Tianrui Instrument Co., Ltd.; L535-1 Low-Speed Centrifuge, Hunan Xiangyi Experimental Instrument Development Co., Ltd.; TG16-WS Tabletop High-Speed Centrifuge, Hunan Xiangyi Centrifuge Instrument Co., Ltd.; UV-Visible Spectrophotometer i3, Haineng Instrument; SHZ-D(Ⅲ) Type Circulating Water Multifunctional Vacuum Pump, Zhengzhou Keli Instrument and Equipment Co., Ltd.; VS-840K-U Clean Bench, Suzhou Antai Air Technology Co., Ltd.; Intelligent Biochemical Incubator, Ningbo Haishu Saifu Experimental Instrument Factory; Keyence RE-52 Rotary Evaporator, Shanghai Yarong Biochemical Instrument Factory; DHG-9240A Electrothermal Constant Temperature Forced Air Drying Oven, Shanghai Precision Experimental Equipment Co., Ltd.

[0051] 3 Experimental Methods

[0052] 3.1 Preparation of Tea Residue Powder

[0053] Place the tea residue in an oven and dry it at a low temperature (60 °C) until the moisture content is below 9.0%. Then crush it and pass it through sieves in sequence to obtain tea residue powder with mesh sizes of <60 mesh, 60 - 100 mesh, 100 - 150 mesh, 150 - 200 mesh, 200 - 300 mesh, and >300 mesh.

[0054] 3.2 Pretreatment of Tea Residue

[0055] (1) Weigh 10 g of tea residue powder (passed through a 150 - 200 mesh sieve). Using deionized water as the extraction solvent, take the liquid-to-solid ratio (20, 30, 40, 50, 60, 70 mL / g), treatment time (5, 10, 15, 20, 25, 30 min), and microwave power (50, 100, 150, 200, 250 W) as single factors, place the tea residue in a microwave extractor for treatment. After the treatment, centrifuge at 8000 rpm for 20 min, record the volume of the supernatant, and the precipitate is the tea residue after water extraction. Take 1.0 mL of the supernatant and use the modified phenol-sulfuric acid method to detect the SDF content. According to the SDF standard equation y = 9.4262x - 0.0363 (R 2 = 0.9998, where x represents the absorbance value and y represents the SDF concentration in mg / mL), obtain the SDF concentration and calculate the dissolution rate according to formula (1). Among them, when taking the liquid-to-solid ratio as a single factor, the treatment time is 15 min and the microwave power is 200 W; when taking the treatment time as a single factor, the liquid-to-solid ratio is 50 mL / g and the microwave power is 200 W; when taking the microwave power as a single factor, the liquid-to-solid ratio is 50 mL / g and the treatment time is 15 min.

[0056] (2) Take the tea residues after water extraction obtained by the treatment method with the highest SDF dissolution rate in step (1), and further add a mixture of hemicellulase and cellulase (mass ratio 4:1) in amounts of 0.4%, 0.8%, 1.2%, 1.6%, or 2.0% respectively according to E / S (the mass percentage of the enzyme in 10 g of tea residue powder), adjust the pH to 4.8, control the temperatures at 30 °C, 40 °C, 50 °C, 60 °C, or 70 °C respectively, and control the extraction times at 0.5 h, 1 h, 1.5 h, 2 h, or 2.5 h respectively. After the extraction is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 5 min. After adjusting the pH to 7.0, centrifuge at 8000 rpm for 20 min. Take 1.0 mL of the supernatant, and use the modified phenol-sulfuric acid method to detect the SDF concentration in the supernatant and convert it to the dissolution rate. Among them, when investigating the addition amount of the hemicellulase and cellulase mixture, the temperature is 50 °C and the extraction time is 2 h; when investigating the temperature, the addition amount of the hemicellulase and cellulase mixture is 1.2% and the extraction time is 2 h; when investigating the extraction time, the addition amount of the hemicellulase and cellulase mixture is 1.2% and the temperature is 50 °C.

[0057] Calculate the dissolution rate of SDF in tea residues according to the following formula:

[0058] SDF dissolution rate (%) = (C × V × f) / m × 100% Formula (1)

[0059] In Formula (1), C is the SDF concentration (mg / mL) calculated by the standard equation; V is the volume of the supernatant (mL); f is the dilution factor; m is the sample mass (mg).

[0060] 3.3 Process optimization for extracting SDF from tea residues by free radical-mediated modification technology

[0061] Weigh 10 g of tea residue powder (screened through 150 - 200 mesh), use deionized water as the extraction solvent, with a liquid-to-solid ratio of 50 mL / g. Place it in a microwave extractor, and after treating for 20 min at a microwave power of 250 W, add 0.16 g of a mixture of hemicellulase and cellulase with a mass ratio of 4:1 (the mass percentage of the enzyme in the tea residue powder is 1.6%), adjust the pH to 4.8, control the temperature at 50 °C, and the extraction time at 2 h. After the extraction is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 5 min. After adjusting the pH to 7.0, concentrate it to a water content of 90% to obtain 100 g of pretreated tea residue slurry.

[0062] Take the pretreated tea residue slurry, add hydrogen peroxide with a mass concentration of 30% and deionized water. Respectively take temperature, liquid-to-solid ratio, hydrogen peroxide concentration, and treatment time as single factors, place it in a constant temperature oscillator for treatment, and conduct a single-factor experiment with the SDF dissolution rate of tea residues as the response value. The specific method is as follows:

[0063] (1) Liquid-to-solid ratio: Weigh 10.0 g of the pretreated tea residue pulp prepared by the above method, and add hydrogen peroxide with a mass concentration of 30% and deionized water at liquid-to-solid ratios of 20:1, 30:1, 40:1, 50:1, and 60:1 (mL / g) respectively, so that the final mass concentration of the added hydrogen peroxide is 1.8%, pH 7.0. Extract in a water bath at 80 °C in a constant temperature oscillator for 80 min, and centrifuge at 8000 rpm for 20 min. Take 1.0 mL of the supernatant, and use the modified phenol-sulfuric acid method to detect the SDF content in the supernatant and convert it to the dissolution rate.

[0064] (2) Treatment time: Weigh 10.0 g of the pretreated tea residue pulp prepared by the above method, add hydrogen peroxide with a mass concentration of 30% and deionized water at a liquid-to-solid ratio of 40:1 so that the final mass concentration of the added hydrogen peroxide is 1.8%, pH 7.0. In a constant temperature oscillator at 80 °C, after water bath extraction for 20 min, 40 min, 60 min, 80 min, 100 min, and 120 min, other steps are the same as in step (1).

[0065] (3) Hydrogen peroxide concentration: Weigh 10.0 g of the pretreated tea residue pulp prepared by the above method, add hydrogen peroxide with a mass concentration of 30% and deionized water at a liquid-to-solid ratio of 40:1 respectively, so that the final mass concentrations of the added hydrogen peroxide are 1.8%, 2.1%, 2.4%, 2.7%, 3.0%, and 3.3% respectively, pH 7.0. After water bath extraction at 80 °C in a constant temperature oscillator for 80 min. Other steps are the same as in step (1).

[0066] (4) Treatment temperature: Weigh 10.0 g of the pretreated tea residue pulp prepared by the above method, add hydrogen peroxide with a mass concentration of 30% and deionized water at a liquid-to-solid ratio of 40:1 so that the final mass concentration of the added hydrogen peroxide is 1.8%, pH 7.0. In a constant temperature oscillator at 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, and 100 °C, extract in a water bath for 80 min. Other steps are the same as in step (1).

[0067] On the basis of the above single-factor experiments, select the three factors (liquid-to-solid ratio, extraction time, H2O2 concentration) that have the most significant influence on the SDF yield of tea residue as independent variables, and use the SDF dissolution rate of tea residue as the response value. Use Design-Expert.V8.0.6 software, adopt the Box-Behnken Design model (three factors and three levels), and design a response surface analysis experiment according to the principle of combined experimental design. The factor levels of the response surface experiment are shown in Table 1.

[0068] Table 1 Factor level table for the experimental design of optimizing the process of treating tea residue polysaccharide by free radical-mediated H2O2-Vc modification technology

[0069] Coding level A: Liquid-to-material ratio B: Extraction time (min) <![CDATA[C: H2O2 concentration (%)]]> -1 50 60 2.1 0 60 80 2.7 1 70 100 3.3

[0070] 3.4 Particle Size of Raw Materials and Vc and Fe 2+ Effect on the Dissolution Rate of SDF from Tea Residue

[0071] (1) Vc and Fe 2+ Effect on the Dissolution Rate of SDF from Tea Residue

[0072] After obtaining the optimal parameters (liquid-to-solid ratio 40:1, final mass concentration of hydrogen peroxide added 1.8%, 80 °C, 80 min) through single-factor and response surface design in the method of 3.3, weigh 10.0 g of the pretreated tea residue slurry prepared by the method of 3.3, add hydrogen peroxide mixture and deionized water according to the liquid-to-solid ratio of 40:1 (mL / g), so that the final mass concentration of hydrogen peroxide mixture added is 1.8% in both cases, pH 7.0, water bath extraction in a constant temperature oscillator at 80 °C for 80 min, and centrifuge at 8000 rpm for 20 min. Take 1.0 mL of the supernatant, and detect the content of SDF in the supernatant by the modified phenol-sulfuric acid method and convert it into the dissolution rate.

[0073] The hydrogen peroxide mixture is one of the following: ① Hydrogen peroxide-Vc mixture: Add Vc at 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32 of the mass of hydrogen peroxide respectively. ② Add ferrous sulfate at 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32 of the mass of hydrogen peroxide respectively. ③ Add Vc-ferrous sulfate (mass ratio of Vc to ferrous sulfate is 1:4) at 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32 of the mass of hydrogen peroxide respectively.

[0074] (2) Effect of Particle Size of Raw Materials on the Dissolution Rate of SDF from Tea Residue

[0075] For the tea residue powders with mesh numbers of <60 mesh, 60 - 100 mesh, 100 - 150 mesh, 150 - 200 mesh, 200 - 300 mesh, >300 mesh prepared in 3.1, respectively take 10.0 g of the pretreated tea residue slurry prepared by the method of 3.3, add 30% hydrogen peroxide and deionized water according to the liquid-to-solid ratio of 40:1 (mL / g), so that the final mass concentration of hydrogen peroxide added is 1.8% in both cases, pH 7.0, water bath extraction in a constant temperature oscillator at 80 °C for 80 min, and centrifuge at 8000 rpm for 20 min. Take 1.0 mL of the supernatant, and detect the content of SDF in the supernatant by the modified phenol-sulfuric acid method and convert it into the dissolution rate.

[0076] 3.5 Modification Method of Tea Residue Dietary Fiber

[0077] According to the optimal extraction parameters optimized by response surface in 3.4, the extraction method of SDF from tea residue is as follows:

[0078] (1) Place the tea residue after extracting the tea soup in an oven and dry it at a low temperature of 60 °C until the moisture content is below 8%. Put it into a pulverizer and pulverize it. After pulverization, sieve it through a 200-300 mesh sieve to obtain tea residue powder for standby.

[0079] (2) Weigh 10 g of the tea residue powder from step (1), add 500 mL of deionized water, place it in a microwave extractor, and after treating it for 20 min under the condition of a power of 250 W, add 0.16 g of a hemicellulase and cellulase mixture (mass ratio 4:1), adjust the pH to 4.8, and enzymatically hydrolyze it at a temperature of 50 °C for 2 h. After the enzymatic hydrolysis ends, put it into a boiling water bath at 100 °C to inactivate the enzyme for 5 min. After adjusting the pH to 7.0, concentrate it to a water content of 90% to obtain the pretreated tea residue slurry.

[0080] (3) Weigh 64 g of a 30% H2O2 solution (containing 19.2 g of H2O2), add 0.48 g of Vc and 1.92 g of FeSO4 (H2O2 / Vc-FeSO4 = 8 / 1), add 23.6 g of deionized water, fully dissolve and shake well to obtain an H2O2 solution containing Vc-FeSO4.

[0081] (4) Add all the H2O2 solution containing Vc-FeSO4 from step (3) to the tea residue slurry from step (2), mix well to obtain a tea residue-Vc-FeSO4-H2O2 mixed solution. At this time, the liquid-solid ratio of the tea residue to the solution in the system is 1:18, and the mass concentration of H2O2 in the system is 10.07%.

[0082] (5) Place the tea residue-Vc-FeSO4-H2O2 mixed solution from step (4) in a constant temperature oscillator and oscillate it. Adjust the oscillator parameters to 90 °C and 200 rpm, and reciprocally oscillate it for 70 min. Take out the mixed solution, centrifuge it at 8000 r / min for 10 min to obtain the supernatant and the filter residue. Take the supernatant and measure its volume. The filter residue is IDF (dried at 50 °C, and 7.8 g can be obtained), which is used for the following experiments to measure its functional properties.

[0083] (6) Rotavaporize and concentrate the supernatant from step (5) to one-fifth of its original volume to obtain a concentrated solution. Slowly add 95% ethanol four times the volume of the concentrated solution while stirring, and place it in a refrigerator at 4 °C for alcohol precipitation for 12 h. After 12 h, carefully pour out the supernatant and retain the lower layer precipitate.

[0084] (7) Centrifuge the lower layer precipitate from step (6) in a centrifuge at 1000 r / min for 5 min. Take the precipitate, add an appropriate amount of water, and rotavaporize and concentrate it at 65 °C until the ethanol is completely evaporated to obtain a concentrated solution.

[0085] (8) Deproteinize the concentrate obtained in step (7). The specific steps are as follows: Place the concentrate in a glass bottle, add Sevag reagent (chloroform: n-butanol = 4:1, v:v) equal to one-fourth of the volume of the concentrate, oscillate at a constant temperature of 37 °C for 25 min, then pour the mixture into a centrifuge tube and centrifuge at 4000 r / min for 5 min. Take the supernatant and repeat the above deproteinization operation until there is no absorption peak at 280 nm in the upper layer. After rotating and evaporating the organic solvent from the supernatant after the last deproteinization at 50 °C, a purified tea residue SDF concentrate can be obtained. Freeze-dry the tea residue SDF concentrate. The initial temperature is -30 °C. Start heating when the vacuum degree reaches 80 Pa. Keep the temperature at -20 °C for 1 h, then keep the temperature for 1 h every time it rises by 10 °C until it reaches 20 °C, and keep it for more than 24 h until the sample is dry (water content below 8%) to obtain 1.975 g of modified tea residue SDF.

[0086] 3.6 Determination of Physicochemical Properties of Tea Residue IDF

[0087] 3.6.1 Preparation of Tea Residue IDF

[0088] The filter residue obtained in step (5) of method 3.5 is dried at 50 °C and then ground, and passed through 60, 100, 150, 200, and 250 mesh sieves respectively to obtain tea residue IDF with different mesh numbers.

[0089] 3.6.2 Water-Binding Capacity of Tea Residue IDF

[0090] Weigh 1.000 g of the tea residue before modification (prepared by method 3.1) and tea residue IDF with different mesh numbers after modification respectively, place them in beakers, add 50 mL of deionized water, let stand at 37 °C for 2 h, filter through a 0.75 mm nylon mesh until no water drops, weigh the mass of the wet tea residue and record it as M1, and dry it to a constant weight M2 at 110 °C. The water-binding capacity is (M1 - M2) / M2, unit: g / g.

[0091] 3.6.3 Swelling Ratio of Tea Residue IDF

[0092] Weigh 1.000 g (M0) of the tea residue before modification (prepared by method 3.1) and tea residue IDF with different mesh numbers after modification respectively into 10 mL graduated cylinders, and read the dry powder volume V0. Then take 0.1 g (M1) of IDF into the graduated cylinder, add water to 10 mL, mix well and let it settle naturally at 37 °C for 24 h, and read the volume V1 after the IDF swells. The swelling ratio is (M0V1 / M1V0) - 1, unit: mL / mL.

[0093] 3.6.4 Oil-Holding Capacity of Tea Residue IDF

[0094] Weigh 1.000 g of tea residue before modification (prepared by method 3.1) and tea residue IDF with different mesh numbers after modification (M1) into 50 mL centrifuge tubes, add 25 g of soybean oil, let it stand at 37 °C for 1 h, then centrifuge at 3000 rpm for 20 min. Pour off the upper layer of oil, filter the remaining oil and sample through a 0.75 mm wire mesh, and use filter paper to absorb the excess oil remaining on the surface of the filter residue. Weigh the mass as M2. The oil-holding capacity is (M2 - M1) / M1, unit: g / g.

[0095] 3.6.5 Sodium cholate adsorption capacity of tea residue IDF

[0096] Prepare 25 mL of 1 mmol / L and 6 mmol / L sodium cholate solutions respectively with 0.1 mol / L sodium phosphate buffer (pH 7.0). Add 0.50 g of tea residue before modification (prepared by method 3.1) and tea residue IDF with different mesh numbers after modification respectively. After shaking well, adjust the pH to 7.0 ± 0.2, place it in a shaker at 200 rpm, shake at 37 °C for 2 h, centrifuge at 4000 rpm for 20 min, take the supernatant and determine the residual sodium cholate concentration by the furfural colorimetric method, and calculate the sodium cholate adsorption capacity according to the concentration difference before and after the reaction.

[0097] 3.7 Determination of the molecular weight of tea residue SDF

[0098] Adopt high performance gel permeation chromatography (HPGPC), select a TSKgel Super Multi PW-M chromatographic column, detect with a differential refractive index detector, the mobile phase is pure water, the column temperature is 40 °C, and the flow rate is 0.6 mL / min. Prepare six Dextran standards with different standard molecular weights with deionized water, the concentration is 10 mg / mL for each, and the injection volume is 20 μL. The molecular weights of the six Dextran standards are Mw = 670000, Mw = 158100, Mw = 91100, Mw = 31200, Mw = 20100, Mw = 4300, Mw = 1200, Mw = 505 in turn. Take the retention time as the abscissa and LogM as the ordinate to draw a standard curve. Prepare 10 mg / mL tea residue SDF prepared by step (8) of method 3.5 with deionized water, inject the sample according to the above conditions, and obtain the elution time (T R ), and calculate the molecular weight of tea residue SDF through the regression equation of the standard curve.

[0099] 3.8 Determination of the functional properties of tea residue SDF

[0100] For the tea residue SDF prepared by step (8) of method 3.5, measure the following functional property indexes.

[0101] 3.8.1 Determination of the inhibitory ability of tea residue SDF on α-amylase

[0102] Prepare tea residue SDF solutions with different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL) using deionized water. Take 100 μL of tea residue SDF solutions with different concentrations respectively, add 0.4 mL of 0.02 mol / L sodium phosphate buffer (pH 6.9) and 100 μL of α-amylase (enzyme activity ≥ 5000 U / g) to each, and mix. Incubate in a water bath at 37 °C for 10 min. Then add 200 μL of 1% potato starch aqueous solution with mass concentration to each, continue to incubate in a water bath at 37 °C for 10 min, and then add 1 mL of DNS and boil in a water bath for 5 min. After cooling to room temperature, add 9 mL of distilled water for dilution, measure the absorbance at 540 nm, and calculate the α-amylase inhibition rate according to formula (3). Under the same conditions, use the aqueous solution of acarbose with the same concentration to replace the tea residue SDF solutions with different concentrations as the positive control.

[0103] α-amylase inhibition rate (%) = (A1 - A0) / A1 × 100, formula (3)

[0104] In formula (3), A0 is the absorbance of the SDF sample, and A1 is the absorbance of the positive control.

[0105] 3.8.2 Detection of in vitro antioxidant indexes

[0106] Respectively prepare test solutions with different concentrations (0.0625, 0.125, 0.25, 0.5, and 1 mg / mL) using deionized water from the supernatant in step (5), the concentrated solution in step (7), and the purified tea residue SDF concentrated solution in step (8) of method 3.5, and measure the following antioxidant indexes.

[0107] (1) Determination of DPPH free radical scavenging rate

[0108] Add 0.394 mg of DPPH to 10 mL of absolute ethanol and mix well to prepare a 0.1 mmol / L DPPH ethanol solution, so that its absorbance at 520 nm is between 0.6 - 1.0, and store it in the dark. Take 2 mL of the DPPH ethanol solution and mix it with 2 mL of test solutions with different concentrations on a vortex oscillator, react in the dark at 25 °C for 30 min, measure the absorbance at 520 nm, and record it as A1; use deionized water to replace the DPPH ethanol solution as a control, and record it as A2; use deionized water to replace the test solution as a blank, and record it as A0; use Vc and tea polyphenol aqueous solutions with the same concentration as the test solution as positive controls. Calculate the DPPH free radical scavenging rate according to formula (4).

[0109] DPPH free radical scavenging rate = (1 - (A1 - A2) / A0) × 100%, formula (4).

[0110] (2) Determination of hydroxyl radical scavenging rate

[0111] Dissolve 0.2504 g of ferrous sulfate in water and make up the volume to 100 mL in a volumetric flask to prepare a 9 mmol / L ferrous sulfate solution. Dissolve 0.1243 g of salicylic acid in ethanol and make up the volume to 100 mL in a volumetric flask to prepare a 9 mmol / L salicylic acid-ethanol solution. Take 1 mL of 30% hydrogen peroxide solution, add water and make up the volume to 1000 mL in a volumetric flask to prepare an 8.8 mmol / L hydrogen peroxide solution.

[0112] Take 1 mL of the test solution with different concentrations and place it in a test tube. Then, successively add 1 mL of 9 mmol / L ferrous sulfate solution, 1 mL of 9 mmol / L salicylic acid-ethanol solution, and 1 mL of 8.8 mmol / L hydrogen peroxide solution. After shaking well and sealing, react in a 37 °C constant temperature water bath for 30 min. Measure the absorbance at 510 nm and record it as A1; use deionized water to replace the hydrogen peroxide solution as a control and record it as A2; use deionized water to replace the test solution as a blank and record it as A0; use Vc and tea polyphenol aqueous solutions with the same concentration as the test solution as positive controls. Calculate the hydroxyl radical scavenging rate according to formula (5).

[0113] Hydroxyl radical scavenging rate % = [1 - (A1 - A2) / A0] × 100% Formula (5).

[0114] (3) Determination of ABTS radical scavenging rate

[0115] Preparation of ABTS stock solution: Prepare a 2.6 mmol / L potassium persulfate and 7.4 mmol / L ABTS solution with deionized water, mix them in equal volumes, and let it stand at room temperature in the dark for 24 h to obtain the ABTS stock solution.

[0116] Preparation of ABTS test solution: Dilute the ABTS stock solution with deionized water until the absorbance at 734 nm is 0.700 ± 0.020, which is the ABTS test solution.

[0117] Add 2 mL of the ABTS test solution to different concentrations of the test solution, mix well, react at room temperature in the dark for 6 min, measure the absorbance at 734 nm, and record it as A; use aqueous solutions of Vc and tea polyphenols with the same concentration to replace the test solution as positive controls. Use deionized water to replace the test solution as a blank control and record it as A0. Calculate the ABTS radical scavenging rate according to formula (6).

[0118] ABTS radical scavenging rate = (1 - A / A0) × 100% Formula (6)

[0119] In formula (6), A = sample + ABTS test solution; A0 = deionized water + ABTS test solution.

[0120] (4) Determination of PTIO radical scavenging rate

[0121] Dissolve 3 mg of PTIO solid in 20 mL of deionized water, and ultrasonicate for 5 min to dissolve it to obtain a PTIO test solution. Take 160 μL of the PTIO test solution into a 96-well plate, add 40 μL of deionized water to make the total volume 200 μL, measure its absorbance at a wavelength of 560 nm, and adjust by dilution with water so that the absorbance of the PTIO test solution is 0.1 ± 0.01. Define the absorbance here as A0.

[0122] After adjusting the pH of the PTIO test solution to pH = 7, add the test solutions with different concentrations, mix well, and react at room temperature for 24 h. Record the absorbance value as A. Aqueous solutions of Vc and tea polyphenols with the same concentration are used as positive controls. Calculate the PTIO free radical scavenging rate according to formula (7).

[0123] Scavenging rate = (1 - A / A0) × 100% Formula (7).

[0124] 3.9 Detection of in vivo biological activity indicators

[0125] Preparation of Caenorhabditis elegans growth medium (NGM medium): 1000 mL of NGM medium contains 2.5 g of peptone, 3 g of NaCl, 17 g of agar, 25 mL of PBS buffer (pH 6.0, 1 M), and 975 mL of deionized water. After autoclaving, add 1 mL of cholesterol aqueous solution (5 mg / mL), 1 mL of MgSO4 aqueous solution (1 M), and 1 mL of CaCl2 aqueous solution (1 M).

[0126] Preparation of LB medium: 1000 mL of LB medium contains 25 g of LB broth and 1000 mL of deionized water.

[0127] Preparation of lysis solution: 3.5 mL of sterile water, 0.3 mL of NaOH (5 mol / l), 1.2 mL of NaClO (6%), prepare it immediately before use.

[0128] Preparation of M9 buffer: 1000 mL of M9 buffer contains 15.12 g of Na2HPO4·12H2O, 3 g of KH2PO4, 5 g of NaCl, 0.25 g of MgSO4·7H2O, and 1000 mL of deionized water.

[0129] E. coli OP50 culture solution: Inoculate E. coli OP50 into the sterilized LB medium, and place it in a shaker at 37 °C for 24 h to obtain the E. coli OP50 culture solution.

[0130] Culture of Caenorhabditis elegans: First, spread the E. coli OP50 culture solution onto the NGM medium, inoculate Caenorhabditis elegans (C. elegans) N2, and incubate at a constant temperature of 20 °C for 72 h. Then, a large number of adult worms and larvae can be seen on the medium.

[0131] Synchronization of Caenorhabditis elegans: Select an NGM plate with a large number of worms and good growth. Pipette 1 mL of M9 buffer onto the plate and gently shake to wash the worms on the plate. Then, transfer the worm suspension to a 1.5 mL EP tube. After the worms settle naturally, use a pipette to aspirate the upper liquid, leaving the lower worms. Repeat the above worm-washing steps 3 - 4 times until most of the worms on the NGM plate are transferred to the EP tube. Discard the supernatant, resuspend the worms in 0.25 mL of ddH2O, add 0.15 mL of lysis solution, and let it stand at room temperature for 5 min to lyse. Centrifuge at 4000 rpm for 1 min, discard the supernatant, and then wash repeatedly with 1 mL of M9 buffer until there is no smell of sodium hypochlorite. Transfer the lysed C. elegans eggs to an NGM plate without E. coli OP50 spread on it. After drying the plate in a laminar flow hood, seal it with a sealing film and transfer it to an incubator at a constant temperature of 20 °C for 12 - 18 h. Then, wash the L1-stage larvae with M9 buffer again and inoculate them onto the NGM medium spread with E. coli OP50, and culture at 20 °C for 48 h to synchronize the L4-stage worms.

[0132] Effect of tea residue SDF on the hydrogen peroxide stress response of Caenorhabditis elegans. Set up negative control groups, positive control groups, and sample groups on NGM medium plates spread with E. coli OP50. Add 100 μL of M9 buffer to the negative control group, add 100 μL of glutathione aqueous solution (1 mg / mL) to the positive control group, and add 100 μL of tea residue SDF solutions with different concentrations (0.125, 0.25, 0.5, 1 mg / mL) to the sample groups. Pick and inoculate synchronized L4-stage worms onto the plates and culture for 48 h. Then, pick 30 worms from each group and transfer them to a 96-well plate containing 3 mM hydrogen peroxide, and culture at 20 °C. Observe the survival of Caenorhabditis elegans every 1 h. The experiment continues until the last Caenorhabditis elegans dies. Determine death when the Caenorhabditis elegans does not move when touched with a platinum wire.

[0133] Effect of tea residue SDF on the methyl viologen stress response of Caenorhabditis elegans. NGM medium plates coated with E. coli OP50 were set up with a negative control group, a positive control group, and a sample group. The negative control group was added with 100 μL of M9 buffer, the positive control group was added with 100 μL of Vc aqueous solution (1 mg / mL), and the sample group was added with 100 μL of tea residue DF solutions at different concentrations (0.125, 0.25, 0.5, 1 mg / mL). Synchronized L4-stage nematodes were picked and inoculated onto the plates for 48 h of culture. Then, 30 worms were picked from each group and transferred to a 96-well plate added with 75 mM methyl viologen for culture at 20 °C. The survival of Caenorhabditis elegans was observed every 1 h and recorded, and the experiment continued until the last Caenorhabditis elegans died. When the Caenorhabditis elegans still did not move after being touched with a platinum wire, it was determined to be dead.

[0134] 4 Experimental results

[0135] 4.1 Effect of microwave on the dissolution of SDF during tea residue pretreatment

[0136] The effects of liquid-to-solid ratio, treatment time, and microwave power on the dissolution rate of tea residue SDF are shown in Figure 1 .

[0137] From Figure 1 the effect of microwave treatment time on the dissolution rate of tea residue SDF, it can be seen that as the microwave treatment time increases from 5 min to 20 min, the dissolution rate of tea residue SDF shows an increasing trend. When the treatment time exceeds 20 min, the increase in the dissolution rate of tea residue SDF becomes gentle. There is no significant difference among the levels after 20 min, indicating that the extension of microwave treatment time has little effect on the dissolution rate of tea residue SDF.

[0138] From Figure 1 the effect of liquid-to-solid ratio on the dissolution rate of tea residue SDF, it can be seen that as the liquid-to-solid ratio increases from 20 mL / g to 60 mL / g, the dissolution rate of tea residue SDF first shows a rapid increase and then a slow increase trend. When the liquid-to-solid ratio continues to increase, the dissolution rate of tea residue SDF shows a decreasing trend.

[0139] As Figure 1 shown by the effect of microwave power on the dissolution rate of tea residue SDF, the dissolution rate of tea residue SDF continuously increases as the microwave power increases from 50 W to 250 W. When the microwave power reaches 250 W, the dissolution rate of tea residue SDF reaches 4.69% ± 0.189%. This shows that the higher the microwave power, the stronger its internal heating effect, the better the cell wall breaking effect, and the easier the dissolution rate of tea residue SDF is to dissolve.

[0140] Among the above three factors for optimization, only the liquid-to-solid ratio showed a turning point in the effect on the dissolution rate of SDF from tea residues. The other two factors did not show a turning point among the selected levels. Therefore, further response surface optimization was not required. For subsequent experiments, a liquid-to-solid ratio of 50 mL / g was selected, and the tea residues were treated under the conditions of a microwave power of 250 W for 20 min as the microwave pretreatment conditions.

[0141] 4.2 Effect of Biological Enzymes on the Dissolution of SDF during Tea Residue Pretreatment

[0142] The effects of enzymolysis time, temperature, and enzyme dosage (E / S) on the dissolution rate of SDF from tea residues are shown in Figure 2 .

[0143] From Figure 2 the effect of enzymolysis time on the dissolution rate of SDF from tea residues, it can be seen that as the enzymolysis time increased from 0.5 h to 2 h, the dissolution rate of SDF from tea residues showed a continuously significant increasing trend. When the enzymolysis time continued to increase, the dissolution rate of SDF from tea residues also increased, but the increase amplitude was very small, and there was no significant difference compared with that at 2 h, indicating that the continued enzymolysis time had little effect on the dissolution rate of SDF from tea residues.

[0144] From Figure 2 the effect of enzymolysis temperature on the dissolution rate of SDF from tea residues, it can be seen that as the temperature increased from 30 °C to 50 °C, the dissolution rate of SDF from tea residues showed a rapid upward trend. When the temperature continued to increase to 70 °C, the dissolution rate of SDF from tea residues first increased gently and then showed a downward trend.

[0145] From Figure 2 the effect of enzyme dosage on the dissolution rate of SDF from tea residues, it can be seen that the dissolution rate of SDF from tea residues continuously and significantly increased as the enzyme dosage increased from 0.4% to 1.6%. When the enzyme dosage continued to increase, the increase in the dissolution rate of SDF from tea residues showed a gentle trend.

[0146] Among the above three factors for optimization, only the enzymolysis showed a turning point in the effect on the dissolution rate of SDF from tea residues. The other two factors did not show a turning point among the selected levels. Therefore, further response surface optimization was not required. For subsequent experiments, an enzymolysis temperature of 50 °C, an enzyme dosage of 1.6%, and an enzymolysis time of 2 h were selected as the enzymolysis pretreatment conditions.

[0147] 4.3 Single-Factor Experiments and Response Surface Optimization of the Dissolution of SDF from Tea Residues by Free Radical-Mediated H2O2-Vc Modification Treatment Technology

[0148] 4.3.1 Single-Factor Experiments on the Dissolution of SDF from Tea Residues

[0149] The effects of liquid-to-solid ratio, extraction time, temperature, and H2O2 concentration on the extraction yield of SDF from tea residues are shown in Figure 3 .

[0150] From Figure 3From the influence of the liquid-solid ratio, it can be seen that when the liquid-solid ratio increases from 20 mL / g to 60 mL / g, the dissolution rate of SDF from tea residues increases significantly. When the liquid-solid ratio of the system exceeds 60 mL / g, the dissolution rate of SDF from tea residues shows a significant downward trend. Therefore, 60 mL / g is selected as the central point for subsequent optimization experiments.

[0151] From Figure 3 From the influence of the extraction time, it can be seen that as the extraction time increases from 20 min to 60 min, the dissolution rate of SDF from tea residues shows a trend of significant increase followed by significant decrease. When the time continues to increase to 120 min, the dissolution rate of SDF from tea residues shows a trend of significant increase followed by significant decrease again. When the extraction time is 80 min, the dissolution rate of SDF from tea residues is significantly higher than the yields at other times. Therefore, 80 min is used as the central point in subsequent optimization experiments.

[0152] From Figure 3 From the influence of the temperature, it can be seen that the dissolution rate of SDF from tea residues increases continuously as the extraction temperature increases from 50 °C to 90 °C, indicating that the increase in temperature can promote the breakage of the tea residue cell wall and the dissolution of SDF. When the temperature exceeds 90 °C, the dissolution rate of SDF from tea residues shows a downward trend. Therefore, the extraction temperature is determined to be 90 °C in subsequent experiments.

[0153] As Figure 3 From the influence of the H2O2 concentration, it can be seen that as the H2O2 concentration increases from 1.8% to 2.7%, the dissolution rate of SDF from tea residues increases continuously. When the H2O2 concentration exceeds 2.7%, the dissolution rate shows a continuous downward trend. This indicates that H2O2 has an optimal concentration. Therefore, 2.7% of E / S is selected as the central point for subsequent optimization experiments.

[0154] 4.3.2 Response surface optimization of SDF dissolution from tea residues

[0155] Based on the analysis of the single-factor experiment results, according to the central composite experimental design principle, using Design-Expert 8.0.6 software, the experimental design and results are shown in Table 2.

[0156] Table 2 Response surface design and experimental results for SDF extraction from tea residues and experimental results

[0157]

[0158] According to the Box-Behnken Design model, a total of 17 experiments were conducted on the three independent parameters for optimization. The dissolution rate of SDF from tea residues varied greatly within the extraction condition range. By applying multiple regression analysis, the following second-order polynomial equation was obtained based on the response variable and test variables:

[0159] Y = 13.19 + 0.13A - 0.17B + 1.14C - 0.073AB + 0.79AC + 0.31BC - 2.43A 2 - 1.24B 2 + 0.31C 2

[0160] Where Y is the predicted extraction yield of tea residue SDF, and A, B, and C are the coded variables of solid-liquid ratio, extraction time, and H2O2 concentration, respectively. Multiple regression fitting was performed on the experimental data in Table 2 to obtain the regression equation. Analysis of variance was carried out on the regression equation, and the results are shown in Table 3.

[0161] Table 3 Analysis of variance of the model regression equation

[0162]

[0163]

[0164] Correlation coefficient R of the regression equation 2 = 0.9353, indicating that the predicted value fits well with the experimental value within the range of experimental parameters; adjusted correlation coefficient R 2 adj = 0.8521, indicating that the equation can explain 85.21% of the response value change and has a good fitting degree. The p-value was used as a tool to check the significance of each coefficient. The p-value of the model was 0.0021, indicating that the fitting degree of the model was very significant (p < 0.01). The p-value of the lack-of-fit term was 0.1854, indicating that the residuals were caused by random errors and were not significant relative to the pure error (p > 0.05); the coefficient of variation was 5.82%, indicating that the model had a high credibility. Therefore, the experimental results can be speculated using this regression equation.

[0165] From the results of the analysis of variance, among the three factors, the linear term C and the quadratic terms A 2 and B 2 had a very significant effect on the response value curve effect; the interaction terms AB, AC, and BC had no significant effect on the response surface effect, indicating that there was no obvious synergistic effect between the solid-liquid ratio and H2O2 concentration, the solid-liquid ratio and extraction time, and the extraction time and H2O2 concentration. The order of the influence of the three factors on the extraction yield of tea residue SDF was: C > B > A.

[0166] According to the regression equation, contour plots and response surfaces were made using Design-Expert.V8.0.6 software as Figure 4 .

[0167] A steep response surface and dense contour lines indicate that the factor has a significant effect on the response value. As Figure 4, the AC interaction effect response surface is relatively steep, and the dense contour lines indicate that the interaction between the solid-liquid ratio and the H2O2 concentration is relatively large; on the contrary, a gentle response surface slope and sparse contour lines indicate that the influence of the factors on the response value is not significant. Figure 4 In Figure 4 , the response surface slopes of AB and BC are relatively gentle and the contour lines are relatively sparse, indicating that the interactions between the solid-liquid ratio and the extraction time, and between the extraction time and the H2O2 concentration are relatively insignificant. This result is consistent with the results of the variance analysis.

[0168] The optimal conditions optimized by the model are as follows: the liquid-solid ratio is 61.69 mL / g, the extraction time is 73.29 min, and the H2O2 concentration is 3.29%. Under these conditions, the dissolution rate of SDF from tea residues reaches 14.2526%. Considering the actual laboratory conditions, the optimal adjustment is: the liquid-solid ratio is 60 mL / g, the extraction time is 70 min, and the H2O2 concentration is 3.2%. To further verify the effectiveness and accuracy of the model and the actual situation, three parallel experiments were carried out according to the optimized extraction conditions. The dissolution rate of SDF from tea residues can reach 13.81%, which is consistent with the predicted value, indicating that the response surface analysis method is reliable and fits well with the actual situation, thus verifying the effectiveness of the regression equation.

[0169] 4.4 Vc and Fe 2+ And the influence of the raw material particle size on the dissolution rate of SDF from tea residues

[0170] When the Vc addition amounts are 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 of the mass of hydrogen peroxide respectively, the influence on the dissolution rate of SDF from tea residues is shown in Figure 5 . From Figure 5 , it can be seen that when the Vc addition amount is 1 / 2 of the mass of hydrogen peroxide, the dissolution rate of SDF from tea residues is the highest. As the Vc addition amount decreases, the dissolution rate of SDF from tea residues continuously decreases, but when the addition amount is in the range of 1 / 4 - 1 / 16, the decrease amplitude is not large. When the addition amount decreases to 1 / 32, the dissolution rate of SDF from tea residues significantly decreases. It shows that a certain amount of Vc can significantly promote the dissolution of SDF from tea residues.

[0171] When the ferrous sulfate addition amounts are 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 of the mass of hydrogen peroxide respectively, the influence on the dissolution rate of SDF from tea residues is shown in Figure 6 . From Figure 6 , it can be seen that when the ferrous sulfate addition amount is 1 / 2 of the mass of hydrogen peroxide, the dissolution rate of SDF from tea residues is the highest. As the ferrous sulfate addition amount decreases, the dissolution rate of SDF from tea residues shows a trend of first decreasing, then increasing, and then decreasing again. When the addition amount decreases to 1 / 32, the dissolution rate of SDF from tea residues significantly decreases. Compared with Figure 6 , the increase in the dissolution rate of SDF from tea residues by adding ferrous sulfate is not as good as that of Vc.

[0172] When the addition amounts of Vc-FeSO4 (mass ratio of Vc to FeSO4 is 1:4) are 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 of the mass of hydrogen peroxide respectively, the effects on the dissolution rate of SDF from tea residues are shown in Figure 7 . From Figure 7 , it can be seen that adding Vc-FeSO4 to the system can significantly increase the dissolution rate of SDF from tea residues. When the addition amount of Vc-FeSO4 is 1 / 2 of the mass of hydrogen peroxide, the dissolution rate of SDF from tea residues is the highest. As the addition amount of Vc-FeSO4 decreases to 1 / 8, the dissolution rate of SDF from tea residues shows a slight downward trend, which is equivalent to the effect when the addition amount is 1 / 2. When the addition amount continues to decrease to 1 / 32, the dissolution rate of SDF from tea residues decreases significantly. By comparing with Figure 6 , Figure 7 , as well as considering factors such as the usage cost of Vc and FeSO4 and the taste of Vc, Vc-FeSO4 (mass ratio of Vc to FeSO4 is 1:4) is added to the system, and its addition amount is 1 / 8 of the mass of hydrogen peroxide.

[0173] The effects of raw material particle size on the dissolution rate of SDF from tea residues are shown in Figure 8 . From Figure 8 , it can be seen that the raw material particle size has a great influence on the dissolution rate of SDF from tea residues. When the raw material mesh number < 60 meshes, the dissolution rate of SDF from tea residues is relatively low. As the mesh number increases to 60 - 100 meshes, the dissolution rate of SDF from tea residues increases significantly. When the mesh number continues to increase to 100 - 150 meshes and 150 - 200 meshes, the dissolution rate of SDF from tea residues shows a slight downward trend, but is higher than that when the raw material mesh number < 60 meshes. When the raw material mesh number continues to increase to 200 - 300 meshes, the dissolution rate of SDF from tea residues increases significantly and reaches the highest. When the raw material mesh number further increases > 300 meshes, the dissolution rate of SDF from tea residues shows an obvious downward trend. In summary, the raw material mesh number of 200 - 300 meshes is selected.

[0174] 4.5 Functional characteristics of IDF from modified tea residues

[0175] The filter residues obtained in step (5) of 3.5 are dried at 50 °C and then pulverized, and sieved through 60, 100, 150, 200, and 250 mesh sieves respectively to obtain tea residue IDF with different mesh numbers. The water-holding capacity, swelling ratio, oil-holding capacity, and sodium cholate adsorption capacity of the tea residues before modification (prepared by the method in 3.1) and the tea residue IDF with different mesh numbers after modification are as Figure 9 shown.

[0176] The water-holding capacity refers to the ability of a certain amount of sample to bind water without external forces (except gravity and atmospheric pressure). Dietary fiber with high water-holding capacity can prevent food shrinkage and change the viscosity of food. From Figure 9It can be seen that the water-binding capacities of tea residue before modification and IDF of tea residue with different mesh numbers after modification are 2.14, 3.35, 4.09, 3.22, 3.77 and 2.92 g / g, respectively. Compared with the tea residue before modification, when the mesh number of IDF of tea residue after modification is below 250 mesh, the water-binding capacity can increase significantly (p<0.05); when the mesh number of IDF of tea residue after modification is above 250 mesh, although the water-binding capacity can be increased, there is no difference compared with the tea residue before modification (p>0.05).

[0177] The swelling ratio reflects the ability of dietary fiber to swell after absorbing water. From Figure 9 , compared with the tea residue before modification, when the mesh number of IDF of tea residue after modification is 150 mesh, the swelling ratio can increase significantly (p<0.05); there is no difference in the swelling ratio of IDF of tea residue after modification at other mesh numbers compared with the tea residue before modification (p>0.05). There are many hydrophilic groups in the dietary fiber structure, and it has good hydration ability (such as water-binding capacity and swelling ratio). Its hydration properties are related to its own structure, porosity, particle size and other factors. When the particle size of dietary fiber decreases, the volume expansion and extension during water dissolution produce a greater volume effect, making its hydration ability increase significantly; however, when the particle size of dietary fiber further decreases, the hydration ability shows a downward trend because its network structure is damaged and it cannot support a larger space due to mutual stacking.

[0178] The oil-holding capacity is mainly related to factors such as the particle size, surface properties, and hydration properties of dietary fiber. From Figure 9 , compared with the tea residue before modification, the oil-holding capacity of IDF of tea residue after modification shows a trend of first increasing and then decreasing with the increase of mesh number, but there is no difference compared with the tea residue before modification (p>0.05). The reason may be that when the particle size is large, there are few effective binding groups with oil, and when the particle size decreases, the specific surface area gradually increases, making the exposure rate of hydrophilic groups in the IDF molecule increase, and at the same time, agglomeration occurs, and its ability to adsorb oil will also decrease. However, generally, the oil-holding capacity of tea residue IDF is not strong.

[0179] Bile salts refer to a class of amphoteric macromolecules with a steroid nucleus structure from the body's bile and are an oil emulsifier. As the main organic solute in bile, it plays an important role in the digestion and absorption of fat, the absorption of fat-soluble vitamins, and cholesterol metabolism. From Figure 9 It can be seen that the adsorption capacity of tea residue before modification for sodium cholate is only 1.8 mg / g, while the adsorption capacity of IDF of tea residue with different mesh numbers after modification for sodium cholate increases significantly compared with that before modification (p<0.05), and the highest adsorption capacity can reach 57.67 mg / g, which is 32.04 times that before modification. The downward trend that appears when the mesh number of tea residue IDF is 250 may be caused by agglomeration due to too small particle size.

[0180] 4.6 Biological activities of SDF in tea residue after modification

[0181] 4.6.1 Free radical scavenging ability of tea residue SDF in vitro

[0182] The tea residue SDF was prepared according to the method in 3.5. The scavenging abilities of DPPH, hydroxyl, ABTS and PTIO free radicals are shown in Figure 10 . The free radical scavenging abilities of the obtained products at each step in the purification and preparation process of tea residue SDF were detected. In the figure, the supernatant refers to the supernatant in step (5) of the method in 3.5, the alcohol precipitation refers to the concentrated solution in step (7) of the method in 3.5, and the deproteinization refers to the purified concentrated solution of tea residue SDF in step (8) of the method in 3.5. To avoid the influence of residual tea polyphenols in the raw tea residue on the experimental results, Vc and tea polyphenols were selected as positive controls at the same time.

[0183] It can be seen from Figure 10 that within the experimental concentration range (0.125 - 1.0 mg / mL), the scavenging rates of tea residue SDF prepared by different purification processes for DPPH, hydroxyl, ABTS, and PTIO free radicals all showed a concentration-dependent relationship, that is, with the increase of the concentration of tea residue SDF, its scavenging effects on the 4 free radicals showed an increasing trend. Among them, the tea residue SDF in the supernatant had the best scavenging abilities for DPPH, hydroxyl, ABTS and PTIO (acidic) free radicals. When the concentration reached 1.0 mg / mL, its scavenging abilities for DPPH, ABTS and PTIO (acidic) free radicals were comparable to those of the positive controls Vc and tea polyphenols, and its scavenging ability for hydroxyl free radicals was better than that of tea polyphenols. The concentrated solution and the purified concentrated solution of tea residue SDF had good scavenging abilities for active nitrogen free radicals (DPPH and ABTS free radicals). When the concentration reached 1.0 mg / mL, their free radical scavenging abilities were comparable to those of the positive controls Vc and tea polyphenols; the concentrated solution = and the purified concentrated solution of tea residue SDF had slightly weaker scavenging effects on active oxygen free radicals (hydroxyl and PTIO free radicals) than the supernatant and the positive control Vc, but their scavenging effects on hydroxyl free radicals were comparable to those of tea polyphenols. It shows that the free radical-mediated modification treatment method will not damage the integrity of the "active fragments" of polysaccharides, and the modified tea residue SDF has extremely strong antioxidant ability, suggesting that it plays an important role in scavenging toxic free radicals and other reactive oxygen species formed in cell metabolism.

[0184] 4.6.2 Inhibitory ability of tea residue SDF on α-amylase

[0185] α-Amylase is one of the key enzymes for starch conversion in the body and can catalyze the hydrolysis of α-1,4-glycosidic bonds. It has been found that inhibitors of α-amylase have inhibitory effects on the activities of salivary and pancreatic α-amylase in the digestive tract, can prevent the conversion and utilization of starch and other carbohydrates in the diet, and have hypoglycemic effects. For example, the α-amylase inhibitor hypoglycemic drug acarbose has been widely used clinically for the treatment of type 2 diabetes. The tea residue SDF was prepared according to the method in 3.5. The inhibitory ability of the modified tea residue SDF on α-amylase is shown in Figure 11。Acarbose was selected as the positive control. From Figure 11 it can be seen that both the inhibitory ability of tea residue SDF and acarbose on α-amylase showed a concentration-dependent relationship. In the low concentration range (0.2 - 0.4 mg / mL), the inhibitory ability of acarbose on α-amylase was better than that of tea residue SDF; as the concentration increased, in the high concentration range (0.6 - 1.0 mg / mL), the inhibitory ability of tea residue SDF on α-amylase was better than that of acarbose. This indicates that the modified tea residue SDF has the effect of inhibiting the activity of α-amylase and curbing the degradation of starch carbohydrates into glucose.

[0186] 4.6.3 Effect of tea residue SDF on the survival rate of C.elegans in oxidative stress state

[0187] The tea residue SDF was prepared according to the method in 3.5, and its effect on the survival rate of C.elegans in the oxidative stress state induced by hydrogen peroxide is shown in Figure 12 and Table 4. Glutathione (GSH) was selected as the positive control.

[0188] Hydrogen peroxide can diffuse in cells and tissues. In the living system, as a precursor of free radicals, hydrogen peroxide can react with metal ions to produce ·OH through the Fenton reaction, and induce an intracellular oxidative stress response in nematodes. From Figure 12 and Table 4, the maximum survival time (Tmax) of nematodes in the negative (M9) group under hydrogen peroxide stress was only 300 min, and the positive substance (GSH) could extend the Tmax of nematodes to 420 min. After hydrogen peroxide treatment for 120 min, all concentrations of tea residue SDF (0.125 - 1.0 mg / mL) could shift the growth curve of nematodes under hydrogen peroxide stress to the right, and the Tmax was extended to more than 360 min. The average survival time of nematodes in the M9 group was 172.2 ± 18.6 min, and that in the GSH group was 271.8 ± 40.2 min. The average survival times of nematodes in different concentration sample groups were all significantly higher than those in the M9 group, which were 190.2 ± 22.8 min, 229.8 ± 25.2 min, 244.2 ± 40.2 min, and 241.8 ± 37.8 min respectively. The effect was the best when the sample concentration was 0.5 mg / mL, which could extend the average lifespan of nematodes by 1.42 times. Compared with the negative control group, all concentrations of tea residue SDF selected in the experiment could extend the average survival time of nematodes in the stress state. Among them, medium and high concentration tea residue SDF (0.25 - 1.0 mg / mL) could significantly extend the average lifespan of nematodes (p < 0.05), while low concentration (0.125 mg / mL) tea residue SDF had a weak effect on the survival time of nematodes (p > 0.05). This indicates that higher concentration of tea residue SDF can enhance the antioxidant stress ability of nematodes treated with hydrogen peroxide.

[0189] Table 4 Effects of tea residue SDF on the average survival rate of C. elegans under hydrogen peroxide-induced oxidative stress

[0190]

[0191] Note: "*" indicates significant difference compared with the negative control group (p < 0.05)

[0192] Tea residue SDF was prepared according to method 3.5, and its effects on the survival rate of C. elegans under methyl viologen-induced oxidative stress are shown in Figure 13 Table 5. Glutathione (GSH) was selected as the positive control.

[0193] Methyl viologen can induce mitochondrial peroxidation to produce oxidative stress. Under the stress of methyl viologen, the average survival time of nematodes in the negative control group was only 141.0 ± 15.6 min. The maximum survival time (T max ) of nematodes in the methyl viologen stress negative (M9) group was only 360 min, and the positive substance (GSH) extended the T max of nematodes to 480 min. After treatment with methyl viologen for 120 min, all concentrations of tea residue SDF (0.125 - 1.0 mg / mL) could shift the growth curve of nematodes under hydrogen peroxide stress to the right, and the Tmax was extended to more than 420 min. The average survival time of nematodes in the M9 group was 172.2 ± 18.6 min, and the average survival time of nematodes in the GSH group was 271.8 ± 40.2 min. The average survival times of nematodes in different concentration sample groups were all significantly higher than that in the M9 group, which were 255.6 ± 25.2 min, 247.8 ± 10.2 min, 289.2 ± 28.2 min, and 301.8 ± 9.6 min respectively. The effect was the best when the sample concentration was 1.0 mg / mL, which could extend the average lifespan of nematodes by 1.75 times. Compared with the negative control group, all concentrations of tea residue SDF (0.125 - 1.0 mg / mL) selected in the experiment could significantly extend the average survival time of nematodes under stress (p < 0.05), indicating that tea residue SDF can enhance the antioxidant stress ability of nematodes treated with methyl viologen.

[0194] Table 5 Effects of tea residue SDF on the average survival rate of C. elegans under methyl viologen-induced oxidative stress

[0195]

[0196] Note: "*" indicates significant difference compared with the negative control group (p < 0.05)

[0197] Comparative Example 1

[0198] The tea residues after extracting the tea soup were placed in an oven and dried at a low temperature (60 °C) until the moisture content was below 9.0%, then crushed and passed through a 200 - 300 mesh sieve to obtain tea residue powder; 10 g of tea residue powder (200 - 300 mesh sieve) was added with 600 mL of deionized water according to a solid - liquid ratio of 1 g:60 mL, placed in a constant - temperature oscillator and oscillated. The oscillator parameters were adjusted to 90 °C and 200 rpm, and reciprocating rotary oscillation extraction was carried out for 70 min. Centrifugation was carried out at 8000 r / min for 10 min, the supernatant was taken, rotary evaporation was used to concentrate it to one - fifth of the original volume to obtain a concentrated solution; while stirring, 95% ethanol four times the volume of the concentrated solution was slowly added, and it was placed in a 4 °C refrigerator for alcohol precipitation for 12 h. After 12 h, protein was removed by the method 3.5 in Example 1 until there was no absorption peak at 280 nm. After concentrating to a volume of 30 mL, freeze - drying (initial temperature of - 30 °C, vacuum degree of 80 Pa) was carried out to obtain 0.613 g of tea residue SDF by water extraction method. The yield, free radical scavenging ability, α - amylase inhibition rate and in - vivo biological activity analysis and testing of tea residue SDF were the same as those in Example 1, and the results are shown in Table 6.

[0199] Table 6 Yield and in - vitro and in - vivo biological activity indexes of tea residue SDF prepared by different treatments

[0200]

[0201] As can be seen from Table 6, the treatment method of tea residues has a great influence on the extraction yield and in - vitro biological activity of SDF. The free - radical - mediated modification treatment can significantly improve the extraction yield, free - radical scavenging ability, α - amylase inhibition rate and antioxidant stress ability of tea residue SDF. Among them, the extraction yield, ABTS + , DPPH free - radical scavenging rate and α - amylase inhibition rate of tea residue SDF prepared by free - radical - mediated modification are 2.69 times, 2.44 times, 2.09 times and 1.55 times higher than those of tea residue SDF extracted by traditional hot water extraction, respectively; the average survival time of nematodes under hydrogen peroxide induction and methyl viologen induction is 20.06% and 39.85% higher than that of tea residue SDF extracted by traditional hot water extraction, respectively.

[0202] Comparative Example 2

[0203] In Example 1, the tea residue powder was changed to be passed through a 60 - mesh coarse sieve, and other operations were the same as those in method 3.5 of Example 1. The extraction yield, free - radical scavenging ability, α - amylase inhibition rate and in - vivo biological activity analysis and testing of tea residue SDF were the same as those in Example 1, and the results are shown in Table 7.

[0204] Table 7 Yield and in - vitro and in - vivo biological activity indexes of tea residue SDF prepared by different treatments

[0205]

[0206] As can be seen from Table 7, the particle size of the crushed tea residue has a certain impact on the extraction yield and in vitro biological activity of SDF. The pretreatment method of passing through a 200-300 mesh sieve after crushing can increase the extraction yield, ABTS + , DPPH free radical scavenging rate and α-amylase inhibition rate of SDF extracted from tea residue by traditional mechanical crushing by 1.85 times, 1.22 times, 1.11 times and 1.14 times respectively; the average survival time of nematodes induced by hydrogen peroxide and methyl viologen is 4.86% and 10.92% higher than that of SDF extracted from tea residue by traditional hot water extraction respectively.

[0207] Comparative Example 3

[0208] Change the tea residue pretreatment in Example 1 to: take 10 g of tea residue powder (200-300 mesh sieve), without biological enzyme pretreatment, and directly use Vc-FeSO4-H2O2 modification treatment after microwave pretreatment. The other steps are the same as Method 3.5 in Example 1. The extraction yield, free radical scavenging ability, α-amylase inhibition rate and in vivo biological activity analysis and testing of tea residue SDF are the same as those in Example 1. The results are shown in Table 8.

[0209] Table 8 Yield and in vitro and in vivo biological activity indexes of tea residue SDF prepared by different treatments

[0210]

[0211] As can be seen from Table 8, the pretreatment method of tea residue has a certain impact on the extraction yield and in vitro biological activity of SDF. The pretreatment method combining microwave and biological enzyme can increase the extraction yield, ABTS + , DPPH free radical scavenging rate and α-amylase inhibition rate of SDF extracted from tea residue by microwave pretreatment by 1.35 times, 1.26 times, 1.09 times and 1.23 times respectively; the average survival time of nematodes induced by hydrogen peroxide and methyl viologen is 2.24% and 4.72% higher than that of SDF extracted from tea residue by traditional hot water extraction respectively.

[0212] Comparative Example 4

[0213] Change the tea residue pretreatment in Example 1 to: take 10 g of tea residue powder (200-300 mesh sieve), without microwave pretreatment, and directly use Vc-FeSO4-H2O2 modification treatment after biological enzyme pretreatment. The other steps are the same as Method 3.5 in Example 1. The extraction yield, free radical scavenging ability, α-amylase inhibition rate and in vivo biological activity analysis and testing of tea residue SDF are the same as those in Example 1. The results are shown in Table 9.

[0214] Table 9 Yield and in vitro and in vivo biological activity indexes of tea residue SDF prepared by different treatments

[0215]

[0216]

[0217] As can be seen from Table 9, the pretreatment method of tea residue has a certain influence on the extraction yield and in vitro biological activity of SDF. The pretreatment method combining microwave and biological enzyme can increase the extraction yield, ABTS + , DPPH free radical scavenging rate and α-amylase inhibition rate of SDF extracted from tea residue pretreated by biological enzyme method by 1.49 times, 1.18 times, 1.07 times and 1.14 times respectively; the average survival time of nematodes induced by hydrogen peroxide and methyl viologen is increased by 1.76% and 3.89% respectively compared with SDF of tea residue extracted by traditional hot water extraction.

[0218] Comparative Example 5

[0219] According to the pretreatment and extraction methods in Method 3.5 of Example 1, but first perform biological enzyme pretreatment and then microwave pretreatment, and other operations are the same. The extraction yield, free radical scavenging ability, α-amylase inhibition rate and in vivo biological activity analysis and testing of tea residue SDF are the same as those in Example 1, and the results are shown in Table 10.

[0220] Table 10 Yield and in vitro and in vivo biological activity indexes of SDF prepared from tea residue by different treatments

[0221]

[0222] As can be seen from Table 10, the pretreatment method of tea residue has a certain influence on the extraction yield and in vitro biological activity of SDF. The pretreatment method of microwave first and then biological enzyme can increase the extraction yield, ABTS + , DPPH free radical scavenging rate and α-amylase inhibition rate of SDF extracted from tea residue pretreated by biological enzyme method first and then microwave by 1.33 times, 1.10 times, 1.08 times and 1.15 times respectively; the average survival time of nematodes induced by hydrogen peroxide and methyl viologen is increased by 2.72% and 3.46% respectively compared with SDF of tea residue extracted by traditional hot water extraction.

[0223] Comparative Example 6

[0224] According to the pretreatment and modification methods in Method 3.5 of Example 1, after pretreatment by microwave combined with biological enzyme method, only hydrogen peroxide is added, and Vc and FeSO4 are not added, and other operations are the same. The extraction yield, free radical scavenging ability, α-amylase inhibition rate and in vivo biological activity analysis and testing of tea residue SDF are the same as those in Example 1, and the results are shown in Table 11.

[0225] Table 11 Yield and in vitro and in vivo biological activity indexes of SDF prepared from tea residue by different treatments

[0226]

[0227] As can be seen from Table 11, the extraction process of tea residue has a certain impact on the extraction rate of SDF and its antioxidant activity in vitro. The extraction method with the addition of Vc and FeSO4 during extraction can improve the extraction rate of SDF from tea residue and its biological activities in vivo and in vitro to a certain extent. Compared with the extraction without Vc and FeSO4, the extraction rate, ABTS + , DPPH free radical scavenging rate and α-amylase inhibition rate increased by 1.26 times, 1.68 times, 1.75 times and 1.20 times respectively; the average survival time of nematodes induced by hydrogen peroxide and methyl viologen increased by 14.27% and 35.40% respectively.

[0228] Example 2

[0229] (1) Place the tea residue after extracting the tea soup in an oven and dry it at a low temperature until the moisture content is below 8%. Put it into a pulverizer and pulverize it. After pulverization, sieve it through a 200-300 mesh sieve to obtain tea residue powder.

[0230] (2) Accurately weigh 10 g of tea residue powder (200-300 mesh sieve), add 600 mL of deionized water, place it in a microwave extractor, and after treating it for 30 min under the condition of a power of 250 W, add 0.20 g of a hemicellulase and cellulase mixture (mass ratio 4:1), adjust the pH to 4.8, and enzymatically hydrolyze it at a temperature of 60 °C for 2.0 h. After the enzymatic hydrolysis is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 5 min. After adjusting the pH to 7.0, obtain tea slurry.

[0231] (3) Weigh 50 g of a 30% H2O2 solution (containing 15 g of H2O2), add 0.375 g of Vc and 1.5 g of FeSO4, and then add 52.125 g of deionized water. After fully dissolving and shaking well, obtain an H2O2 solution containing Vc-FeSO4.

[0232] (4) Add all the H2O2 solution containing Vc-FeSO4 in step (3) to the tea slurry in step (2), mix well to obtain a tea residue-Vc-FeSO4-H2O2 mixture. At this time, the solid-liquid ratio of the tea residue to the solution in the system is 1:70, and the mass concentration of H2O2 in the system is 2.1%.

[0233] (5) Place the mixed tea residue-Vc-FeSO4-H2O2 mixture after mixing in step (4) in a constant temperature oscillator and oscillate it. Adjust the oscillator parameters to 90 °C and 200 rpm, and reciprocate and oscillate for 100 min. Take out the mixture, centrifuge it at 8000 r / min for 10 min, collect the supernatant and the filter residue, dry the filter residue at 50 °C, and obtain 7.7 g of tea residue IDF. The water-binding capacity of tea residue IDF is 3.92 g / g, the swelling ratio is 13.09 g / g, the oil-holding capacity is 1.15 g / g, and the sodium cholate adsorption capacity is 55.27 mg / g.

[0234] (6) Take the supernatant from step (5), rotary evaporate and concentrate it to one-fifth of the original volume to obtain a concentrated solution. While stirring, add 4 times the volume of 95% ethanol to the concentrated solution. After standing at 4 °C for 12 h, carefully pour out the supernatant and retain the precipitate at the bottom layer.

[0235] (7) Centrifuge the precipitate at the bottom layer in step (6) at 1000 rpm for 5 min. Take the precipitate, add an appropriate amount of water, and rotary evaporate and concentrate it at 65 °C until the ethanol is completely evaporated to obtain a concentrated solution.

[0236] (8) For the concentrated solution in step (7), perform protein deproteinization using the Sevag method. Specifically: Place the concentrated solution in a separating funnel, add one-fourth of the volume of Sevag reagent (chloroform: n-butanol = 4:1, v:v) of the concentrated solution, keep it at a constant temperature of 37 °C and oscillate for 25 min. Then pour the mixed solution into a centrifuge tube and centrifuge at 4000 r / min for 5 min. Take the supernatant and repeat the above protein deproteinization operation until there is no absorption peak at 280 nm in the upper layer. After rotary evaporating the organic solvent from the supernatant at 50 °C, obtain a purified concentrated solution of tea residue SDF. Use the method 3.5 of Example 1 for freeze-drying (the initial temperature is -30 °C and the vacuum degree is 80 Pa) to obtain 1.951 g of tea residue SDF.

[0237] Detected by the method of Example 1, the average yield of SDF is 19.51%, the ABTS + scavenging rate is 77.61%, the DPPH free radical scavenging rate is 89.27%, the α-amylase inhibition rate is 63.15%, the average survival time of nematodes induced by hydrogen peroxide is 238.3 min, and the average survival time of nematodes induced by methyl viologen is 299.1 min.

[0238] Example 3

[0239] (1) Place the tea residue after extracting the tea soup in an oven and dry it at a low temperature of 60 °C until the moisture content is below 8%. Put it into a pulverizer and pulverize it. After pulverization, sieve it through a 200-300 mesh sieve to obtain tea residue powder.

[0240] (2) Accurately weigh 10 g of tea residue powder (200-300 mesh sieve), add 700 mL of deionized water, place it in a microwave extraction instrument, and treat it for 30 min under the condition of a power of 250 W. Then add 0.20 g of a hemicellulase and cellulase mixture (mass ratio 4:1), adjust the pH to 4.8, and enzymatically hydrolyze it at a temperature of 70 °C for 2.0 h. After the enzymatic hydrolysis is completed, put it into a boiling water bath at 100 °C to inactivate the enzyme for 5 min, and adjust the pH to 7.0 to obtain tea slurry.

[0241] (3) Weigh 80 g of H2O2 solution with a mass concentration of 30% (containing 24 g of H2O2), add 0.6 g of Vc, 2.4 g of FeSO4 and 17 g of deionized water. After fully dissolving and shaking well, obtain an H2O2 solution containing Vc-FeSO4.

[0242] (4) Add all the solution containing Vc-FeSO4 in step (3) to the tea slurry in step (2) to obtain a tea residue-Vc-FeSO4-H2O2 mixture. At this time, the solid-liquid ratio of the tea residue to the solution in the system is 1:80, and the mass concentration of H2O2 in the system is 3.0%.

[0243] (5) Put the tea residue-Vc-FeSO4-H2O2 mixture after mixing in step (4) into a constant temperature oscillator and oscillate. Adjust the oscillator parameters to 80 °C and 200 rpm, and reciprocally oscillate for 60 min. Take out the mixture, centrifuge at 8000 r / min for 10 min, collect the supernatant and the filter residue, dry the filter residue at 50 °C to obtain 7.8 g of tea residue IDF. The water-binding capacity of tea residue IDF is 4.01 g / g, the swelling ratio is 13.14 g / g, the oil-holding capacity is 1.09 g / g, and the sodium cholate adsorption capacity is 56.14 mg / g.

[0244] (6) Take the supernatant in step (5) and rotary evaporate and concentrate it to one-fifth of the original volume to obtain a concentrated solution. While stirring, add 4 times the volume of 95% ethanol to the concentrated solution. After standing at 4 °C for 12 h, carefully pour out the supernatant and retain the precipitate at the bottom layer.

[0245] (7) Place the precipitate at the bottom layer in step (6) in a centrifuge, centrifuge at 1000 rpm for 5 min, take the precipitate, add an appropriate amount of water, and rotary evaporate and concentrate it at 65 °C until the ethanol is completely evaporated to obtain a concentrated solution.

[0246] (8) Use the sevag method described in method 3.5 of Example 1 to remove protein from the concentrated solution in step (7) to obtain 1.907 g of tea residue SDF. The average yield of SDF is 19.07%, the ABTS + scavenging rate is 76.32%, the DPPH free radical scavenging rate is 88.71%, the α-amylase inhibition rate is 62.34%, the average survival time of nematodes induced by hydrogen peroxide is 237.4 min, and the average survival time of nematodes induced by methyl viologen is 298.6 min.

[0247] Example 4

[0248] (1) Place the tea residue after extracting the tea soup in an oven and dry it at a low temperature of 60 °C until the moisture content is below 8%. Put it into a pulverizer and pulverize it. After pulverization, sieve it through a 200-300 mesh sieve to obtain tea residue powder.

[0249] (2) Weigh accurately 10 g of tea residue powder (screened through 200 - 300 mesh), add 500 mL of deionized water, place it in a microwave extractor, and after treating for 20 min under the condition of a power of 250 W, add 0.16 g of a hemicellulase - cellulase mixture (mass ratio 4:1), adjust the pH to 4.8, and carry out enzymatic hydrolysis at a temperature of 50 °C for 2.5 h. After the enzymatic hydrolysis is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 5 min, and after adjusting the pH to 7.0, obtain tea slurry.

[0250] (3) Weigh 60 g of a 30% H₂O₂ solution (containing 18 g of H₂O₂), add 0.45 g of Vc and 1.8 g of FeSO₄, add 94.42 g of deionized water, fully dissolve and shake well to obtain an H₂O₂ solution containing Vc - FeSO₄.

[0251] (4) Add all the H₂O₂ solution containing Vc - FeSO₄ in step (3) to the tea slurry in step (2), mix well to obtain a tea residue - Vc - FeSO₄ - H₂O₂ mixture. At this time, the solid - liquid ratio of the tea residue to the solution in the system is 1:65, and the mass concentration of H₂O₂ in the system is 2.7%.

[0252] (5) Place the well - mixed tea residue - Vc - FeSO₄ - H₂O₂ mixture in a constant - temperature oscillator and oscillate. Adjust the oscillator parameters to 100 °C and 200 rpm, and carry out reciprocating and swirling oscillation for 100 min. Take out the mixture, centrifuge at 8000 r / min for 10 min, collect the supernatant and the filter residue, dry the filter residue at 50 °C to obtain 7.7 g of tea residue IDF. The water - binding capacity of tea residue IDF is 3.97 g / g, the swelling ratio is 13.02 g / g, the oil - holding capacity is 1.13 g / g, and the sodium cholate adsorption capacity is 56.21 mg / g.

[0253] (6) Take the supernatant in step (5) and rotary evaporate and concentrate it to one - fifth of the original volume to obtain a concentrated solution. While stirring, add 4 times the volume of 95% ethanol to the concentrated solution, let it stand at 4 °C for 12 h, then carefully pour out the supernatant and retain the lower - layer precipitate.

[0254] (7) Place the lower - layer precipitate in step (6) in a centrifuge, centrifuge at 1000 rpm for 5 min, take the precipitate, add an appropriate amount of water, and rotary evaporate and concentrate it at 65 °C until the ethanol is completely evaporated to obtain a concentrated solution.

[0255] (8) Use the sevag method described in method 3.5 of Example 1 to remove proteins from the concentrated solution in step (7) to obtain 1.957 g of tea residue SDF. The average yield of SDF is 19.57%, ABTS +The clearance rate was 77.92%, the DPPH free radical scavenging rate was 89.31%, the α-amylase inhibition rate was 63.71%, the average survival time of nematodes under hydrogen peroxide induction was 240.2 min, and the average survival time of nematodes under methyl viologen induction was 300.4 min.

[0256] Example 5

[0257] (1) Place the tea residue after extracting the tea soup in an oven and dry it at a low temperature of 60 °C until the moisture content is below 8%. Put it into a pulverizer and pulverize it. After pulverization, sieve it through a 200-300 mesh sieve to obtain tea residue powder.

[0258] (2) Accurately weigh 10 g of tea residue powder (200-300 mesh sieve), add 500 mL of deionized water, place it in a microwave extractor, and after treating it for 25 min under the condition of a power of 250 W, add 0.16 g of a mixture of hemicellulase and cellulase (mass ratio 4:1), adjust the pH to 4.8, and enzymatically hydrolyze it for 2.0 h at a temperature of 60 °C. After the enzymatic hydrolysis is completed, put it into a boiling water bath at 100 °C to inactivate the enzyme for 5 min. After adjusting the pH to 7.0, obtain tea slurry.

[0259] (3) Weigh 50 g of a 30% H2O2 solution (containing 15 g of H2O2), add 0.375 g of Vc, 1.5 g of FeSO4, and 2.0 g of deionized water. After fully dissolving and shaking well, obtain an H2O2 solution containing Vc-FeSO4.

[0260] (4) Add all the H2O2 solution containing Vc-FeSO4 in step (3) to the tea slurry in step (2), mix well to obtain a tea residue-Vc-FeSO4-H2O2 mixed solution. At this time, the material-liquid ratio of the tea residue to the solution in the system is 1:53, and the mass concentration of H2O2 in the system is 2.7%.

[0261] (5) Put the mixed tea residue-Vc-FeSO4-H2O2 solution after mixing into a constant temperature oscillator and oscillate it. Adjust the oscillator parameters to 90 °C and 200 rpm, and reciprocally oscillate for 80 min. Take out the mixed solution, centrifuge it at 8000 r / min for 10 min, collect the supernatant and the filter residue, dry the filter residue at 50 °C, and obtain 7.8 g of tea residue IDF. The water-binding capacity of tea residue IDF is 4.03 g / g, the swelling ratio is 13.15 g / g, the oil-holding capacity is 1.06 g / g, and the sodium cholate adsorption capacity is 55.27 mg / g.

[0262] (6) Take the supernatant in step (5) and rotary evaporate and concentrate it to one-fifth of the original volume to obtain a concentrated solution. While stirring, add 4 times the volume of 95% ethanol to the concentrated solution. After standing at 4 °C for 12 h, carefully pour out the supernatant and retain the lower-layer precipitate.

[0263] (7) Centrifuge the lower precipitate in step (6) at 1000 rpm for 5 min. Take the precipitate, add an appropriate amount of water, and rotary evaporate and concentrate it at 65 °C until ethanol is completely evaporated to obtain a concentrated solution.

[0264] (8) Use the sevag method described in Method 3.5 of Example 1 to remove proteins from the concentrated solution in step (7) to obtain 1.922 g of tea residue SDF. The average polysaccharide yield is 19.22%. ABTS + scavenging rate is 77.81%, DPPH radical scavenging rate is 89.04%, α-amylase inhibition rate is 63.62%, the average survival time of nematodes under hydrogen peroxide induction is 238.9 min, and the average survival time of nematodes under methyl viologen induction is 299.3 min.

Claims

1. A modified tea residue dietary fiber with improved biological activity, characterized in that, The modified tea residue dietary fiber includes insoluble tea residue dietary fiber and soluble tea residue dietary fiber, and the modified tea residue dietary fiber is prepared according to the following method: (1) Weigh tea dregs powder, add deionized water, microwave for 5-30 min at a power of 50-250 W, add a mixture of hemicellulase and cellulase, adjust the pH to 4.8, and perform enzymolysis at a temperature of 30-70° C. for 0.5-2.5 h. After the enzymolysis is completed, place the mixture in a 100° C. boiling water bath to inactivate the enzyme for 5 min, and adjust the pH to 7.0 to obtain tea slurry; (2) Add Vc, FeSO4 and deionized water to a 30% H2O2 solution, fully dissolve and shake well to obtain a H2O2 solution containing Vc-FeSO4; (3) adding all the H2O2 solution containing Vc-FeSO4 in step (2) to the tea slurry in step (1), mixing them evenly to obtain a tea residue-Vc-FeSO4-H2O2 mixed solution; (4) placing the tea residue-Vc-FeSO4-H2O2 mixture from step (3) into a constant temperature oscillator for oscillation, adjusting the oscillator parameters to 50-100°C, 100-200 rpm, and oscillating back and forth for 20-120 min; taking out the mixture, centrifuging it, obtaining a supernatant and a filter residue; drying the filter residue at 50°C to obtain insoluble tea residue dietary fiber; (5) Concentrating the supernatant from step (4) by rotary evaporation to one fifth of the original volume to obtain a concentrated solution; While stirring, slowly add 95% ethanol (4 times the volume of the concentrate) and place in a 4°C refrigerator for alcohol precipitation for 12 hours. Carefully pour out the supernatant and retain the lower sediment. (6) Centrifuging the lower precipitate from step (5), adding water to the precipitate and concentrating it by rotary evaporation at 65°C until the ethanol is completely evaporated to obtain a concentrated solution; (7) Deproteinizing the concentrated solution from step (6) using the Sevag method to obtain soluble tea residue dietary fiber.

2. The modified tea residue dietary fiber according to claim 1, wherein Step (1) tea residue powder is prepared as follows: the tea residue after extracting tea soup is placed in an oven and dried at low temperature until the moisture content is below 8%, and then placed in a grinder for crushing. After crushing, the tea residue is sieved through a 200-300 mesh sieve to obtain tea residue powder.

3. The modified tea residue dietary fiber according to claim 1, wherein The volume of deionized water in step (1) is 20-70 mL / g based on the mass of the tea dregs powder; the hemicellulase and cellulase mixture is prepared by mixing hemicellulase and cellulase in a mass ratio of 4:1, and the hemicellulase and cellulase mixture is 1-2% based on the mass of the tea dregs powder.

4. The modified tea residue dietary fiber according to claim 1, characterized in that, The mass ratio of the H2O2 solution with a mass concentration of 30% in step (2) to the tea residue powder in step (1) is 5-8:1; the mass ratio of the Vc to the tea residue powder in step (1) is 0.001-0.1:1; the mass ratio of the FeSO4 to the tea residue powder in step (1) is 0.1-1:1; and the deionized water is 1-10 mL / g based on the mass of the tea residue powder in step (1).

5. The modified tea residue dietary fiber according to claim 1, characterized in that, Step (7) The protein removal step is as follows: Add one-fourth volume of Sevag reagent to the concentrated solution. After constant temperature oscillation at 37 °C for 25 min, pour the mixture into a centrifuge tube and centrifuge at 4000 r / min for 5 min. Take the supernatant and repeat the above protein removal operation until there is no absorption peak at 280 nm in the upper layer. After rotary evaporation of the organic solvent from the supernatant after the last protein removal at 50 °C, a concentrated solution is obtained. Freeze-dry the concentrated solution. The initial temperature is -30 °C. Start heating when the vacuum degree is 80 Pa. Keep heating at -20 °C for 1 h, then keep for 1 h when the temperature rises by 10 °C each time until the temperature reaches 20 °C and keep for more than 24 h until the water content of the sample is below 8% to obtain soluble tea residue dietary fiber. The Sevag reagent is chloroform: n-butanol with a volume ratio of 4:

1.

6. Use of the modified tea residue dietary fiber according to claim 1 in the preparation of an antioxidant.

7. Use of the modified tea residue dietary fiber according to claim 1 in the preparation of an α-amylase inhibitor.

8. Use of the modified tea residue dietary fiber according to claim 1 in the preparation of a preparation for prolonging the lifespan of Caenorhabditis elegans under oxidative stress.

9. The application according to claim 8, wherein The modified tea residue dietary fiber is soluble tea residue dietary fiber.

10. Use of the modified tea residue dietary fiber according to claim 1 in flour products.

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