Functional tea polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses functional tea polysaccharide and a preparation method and application thereof.The preparation method comprises the steps that coarse old tea leaves serve as raw materials and are subjected to superfine treatment, dewaxing and small molecule impurity pretreatment, then cellulase-papain composite wall breaking extraction is adopted, hydrogen peroxide is further adopted for treatment, and the tea polysaccharide high in yield and high in biological activity is obtained. The tea polysaccharide extracted by the method disclosed by the invention can slow down weight gain of an obesity C57BL / 6J mouse caused by high fat diet and improve glucose and lipid metabolism disorder, so that db / m mouse intestinal flora is metabolized to generate a new function of important biomarkers such as indole and the like, and high-quality reutilization of coarse and old tea leaves is realized.

Description

(1) Technical Field

[0001] The present invention relates to a functional tea polysaccharide, a preparation method thereof and an application thereof. (2) Background Art

[0002] As a popular beverage, tea is widely consumed globally and its consumption is increasing year by year. Every year, a large amount of fresh tea leaves in late spring, summer and autumn are discarded without being effectively utilized, which has become a major problem restricting the sustainable development of the tea industry. The fresh tea leaves in summer and autumn have a strong bitter taste, low resource utilization rate; there are few utilization channels and low product benefits. There is an urgent need to improve the utilization rate of discarded tea resources such as summer and autumn tea, and turn low-value discarded tea resources into high-value-added tea and deep-processed products. The discarded tea leaves in summer and autumn contain a large amount of insoluble components such as cellulose, hemicellulose and lignin. If soluble bioactive polysaccharides can be extracted from them and the technical bottleneck of difficult utilization of discarded tea resources can be broken through, the efficient and high-value utilization of discarded tea resources can be realized, laying a solid foundation for improving the overall efficiency of the tea industry.

[0003] Metabolic disorders are one of the most common and prevalent chronic diseases in the world, with the mortality rate continuously rising, including obesity, hyperlipidemia, diabetes, cardiovascular diseases, etc. Clinical and experimental evidence shows that high-fat diet (HFD) intake is the main factor in the development of insulin resistance and metabolic disorders in humans and animals. It is estimated that the global incidence of metabolic disorders is about 1 / 4, which is similar to the incidence of obesity and type 2 diabetes. The number of diabetes patients is estimated to be 463 million and is expected to reach 700 million by 2045. Due to the global health burden caused by metabolic diseases and the trend of younger incidence, treatment strategies have received extensive attention. However, current treatment methods cannot control metabolic disorders and pathological consequences, which requires more novel treatment strategies. Natural compounds extracted from plant resources have attracted extensive attention to discover new and effective candidates for preventing metabolic disorders with fewer side effects and more targets. Natural polysaccharides extracted from tea have continuously attracted the interest of researchers in recent years due to their good biocompatibility, safety and diverse biological functions. It has been found that they have functions such as regulating blood glucose levels, reducing blood lipid accumulation, and improving cardiovascular and cerebrovascular functions, and are expected to become ideal functional factors for improving metabolic disorders.

[0004] The content of tea polysaccharides in coarse and old tea leaves is much higher than that in tender tea leaves. In China and Japan, there is a common experience of treating diabetes by drinking coarse and old tea regularly. However, the cell walls of coarse and old tea leaves are tough and waxy, and it is very difficult to dissolve polysaccharides fully by conventional methods. The extraction of natural polysaccharides first requires cell wall disruption. Wall breaking is an important process for preparing polysaccharides from natural resources and has always been a bottleneck restricting the production of polysaccharides and the like, which is extremely challenging. The cell wall structure of tea leaves serves as its protective layer and barrier, and the degree of wall breaking will directly affect the polysaccharide yield and function. The biological activity of polysaccharides is closely related to their primary structure and spatial conformation. Once the structural characteristics of polysaccharides are damaged, their biological activity will decrease significantly or even be lost. Therefore, there is an urgent need for a polysaccharide extraction method that does not damage the integrity of the "active fragments" of polysaccharides during the tea wall-breaking process, has a high yield, does not introduce impurities, is pollution-free, has low equipment requirements and low energy consumption. Polysaccharide extraction methods usually include mechanical methods, physical methods, chemical methods, and biological enzyme methods, etc. Among them, most mechanical methods have problems such as high equipment requirements and high energy consumption. Physical methods are simple and easy to operate, but require a large enough temperature difference and are only suitable for laboratory operations. Chemical methods are likely to introduce impurities and have problems such as safety and environmental pollution. Exactly which method can more effectively maintain the biological activity of tea polysaccharides and how the extraction process affects the structure of tea polysaccharides have always been topics worthy of exploration.

[0005] In recent years, the fact that bioenzymes can accurately and efficiently break the cell wall at specific sites on the tea leaf cell wall under relatively compatible conditions has received wide attention. Since different types of enzymes have different wall-breaking sites and there are certain differences in the wall-breaking effects, the combination of two or more enzymes can achieve a better wall-breaking effect.

[0006] Therefore, it is necessary to find a method for efficiently extracting new bioactive extracts from coarse and old tea leaves. (III) Summary of the Invention

[0007] The object of the present invention is to provide a functional tea polysaccharide, its preparation method and application. The present invention uses coarse and old tea leaves as raw materials. After ultramicroization and pretreatment for removing wax and small molecule impurities, first, a composite cellulase-papain wall-breaking extraction is carried out. Taking the dissolution rate of tea polysaccharides as the response value, the optimal parameters of the biological composite enzyme treatment are optimized through single-factor experiments and Box-Behnken experimental design. Further treatment is carried out with hydrogen peroxide, and through single-factor experiments and Box-Behnken experimental design optimization, tea polysaccharides with high yield and strong biological activity are obtained. The tea polysaccharides extracted by the method of the present invention can slow down the weight gain of obese C57BL / 6J mice caused by a high-fat diet and improve the disorder of glucose and lipid metabolism, enabling new functions of important biomarkers such as indole to be produced by the intestinal flora metabolism of db / m mice, and realizing the high-quality reuse of coarse and old tea leaves.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides a functional tea polysaccharide, and the tea polysaccharide is prepared by the following method:

[0010] (1) Pretreatment: Take coarse and old tea leaf powder, stir and heat-extract it with DGRA solvent at 50 - 55 °C for 1.5 - 2.5 h, centrifuge (preferably centrifuge at 8000 r / min for 15 min), then reflux the filter residue with 70% ethanol by volume for 1 - 3 h, filter to remove substances such as lipids, small molecule impurities, alkaloids, and tea polyphenols surrounded outside the tea cells, and then dry the filter cake in the sun and crush it (preferably pass through a 200-mesh sieve) to obtain the pretreated powder of coarse and old tea; the DGRA solvent is an anhydrous ethanol solution of n-caprylic acid and n-decanoic acid;

[0011] (2) Composite enzymatic hydrolysis extraction: Add a composite enzyme of cellulase - papain to the pretreated powder of coarse and old tea, add deionized water, adjust the pH to 4.5 - 6.5 (preferably 5.0) with citric acid, and carry out enzymatic hydrolysis at 45 - 65 °C for 60 - 100 min. After the enzymatic hydrolysis is completed, put it into a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea pulp;

[0012] (3) Hydrogen peroxide synergistic extraction: Add a solution of hydrogen peroxide with a mass concentration of 30% and deionized water to the tea pulp in step (2), mix well, and carry out constant temperature water bath extraction at 60 - 100 °C and 200 rpm for 5 - 25 min; take out the mixed solution and centrifuge (centrifuge at 8000 r / min for 20 min) to obtain the supernatant; the supernatant is concentrated, ethanol-precipitated, and deproteinized to obtain tea polysaccharide.

[0013] Preferably, the coarse and old tea leaf powder in step (1) is prepared by the following method: Place the commercially available coarse and old tea leaves in an oven and dry them at a low temperature (preferably 60 °C) until the moisture content is below 9%, put them into a pulverizer to crush, and sieve to obtain coarse and old tea leaf powder with a mesh number > 150.

[0014] Preferably, in step (1), the molar ratio of n-caprylic acid to n-decanoic acid is 2 - 3:1, and the total volume ratio of n-caprylic acid and n-decanoic acid to anhydrous ethanol is 1:2.5 - 3.5.

[0015] Preferably, the composite enzyme of cellulase - papain in step (2) is composed of cellulase and papain mixed in a mass ratio of 2 - 4:1 (preferably 3:1), and the addition amount of the composite enzyme of cellulase - papain is 1.5 - 2.5% based on the mass of the pretreated powder of coarse and old tea, preferably 1.5%.

[0016] Preferably, in step (2), the volume of deionized water used is 25 - 45 mL / g based on the mass of the pretreated powder of coarse and old tea, preferably 35 mL / g; the enzymatic hydrolysis is preferably carried out at 55 °C for 80 min.

[0017] Preferably, the addition amount of the 30% hydrogen peroxide and deionized water solution in step (3) is such that the final mass concentration of hydrogen peroxide is 0.9% - 2.1%, preferably 1.5%.

[0018] Preferably, the extraction conditions in step (3) are 100 °C, 200 rpm, and extraction for 18 min.

[0019] Preferably, the method for concentrating and alcohol-precipitating the supernatant is as follows: The supernatant is rotary-evaporated to one-third of its original volume, cooled to 4 °C to obtain a concentrated solution; while stirring, 95% ethanol four times the volume of the concentrated solution is slowly added, and after alcohol-precipitating at 4 °C in a refrigerator for 12 h, centrifugation (preferably centrifuging at 3000 r / min in a centrifuge for 5 min) is performed, the precipitate is taken, deionized water is added, and protein is removed by the Sevag method to obtain tea polysaccharide.

[0020] Preferably, the protein removal step is as follows: One-fourth volume of Sevag reagent (chloroform: n-butanol = 4:1, v:v) is added to the precipitate and deionized water for protein removal. After sufficient shaking, it is allowed to stand for stratification, and the upper layer is taken and the above protein removal operation is repeated until the upper layer has no absorption peak at 280 nm; the supernatant after the last protein removal is rotary-evaporated to 25 - 35% of its original volume at 50 °C and dried at 60 °C to obtain tea polysaccharide.

[0021] The present invention also provides an application of the tea polysaccharide in the preparation of a weight loss product, and the weight loss product can slow down obesity induced by a high-fat diet.

[0022] The present invention also provides an application of the tea polysaccharide in the preparation of a preparation for improving glycolipid metabolism disorders, and the preparation can improve blood lipid abnormalities caused by a high-fat diet, slow down blood glucose elevation, and glycogen reduction.

[0023] The present invention also provides an application of the tea polysaccharide in the preparation of an intestinal flora regulator, and the regulator can improve the disorder state of the intestinal flora in diabetic mice and promote the proliferation of more beneficial bacteria.

[0024] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0025] 1. The present invention uses a pretreatment method of a pulverization method and a DGRA solvent method, and a cell wall-breaking - extraction method of a composite bio-enzyme - hydrogen peroxide to extract tea polysaccharide. The technological process is green and environmentally friendly, the operation is simple and easy, and the polysaccharide dissolution rate can be as high as more than 25%. It is 3.371 times higher than the tea polysaccharide extracted by traditional hot water extraction, 1.472 times higher than the crude powder obtained by traditional mechanical pulverization, 1.341 times higher than that without DGRA solvent pretreatment, 2.912 times higher than that without hydrogen peroxide co-extraction, 1.673 times higher than that without composite enzyme co-extraction, 1.196 times higher than that with single cellulase co-extraction with hydrogen peroxide, and 1.307 times higher than that with single papain co-extraction with hydrogen peroxide.

[0026] 2. A large amount of wax components, as well as insoluble dietary fibers with dense structures such as cellulose, hemicellulose, and lignin, are contained in coarse and old tea leaves, and there are defects in their physicochemical and functional properties. Conventional methods for extracting tea polysaccharides have problems such as high energy consumption, insufficient cell wall breaking, and local high temperature. First, through the pretreatment method combining the pulverization method and the DGRA-ethanol solvent method, the lipids surrounded by extracellular lipids of coarse and old tea leaves, small molecule impurities, alkaloids, tea polyphenols and other substances can be fully removed. Furthermore, cellulase and papain, as different types of biological enzymes, can break the cell wall more effectively due to the differences in the cell wall breaking sites. 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 ·OH. Based on this, on the basis of the cell wall breaking-extraction by composite biological enzymes (the optimal action pH is 5.0, adjusted by citric acid), hydrogen peroxide can generate strong ·OH under the action of citric acid, which can further break the cell wall efficiently and has unique advantages such as decolorization. Combining the design method of BBD and the cell wall breaking morphology observed by SEM scanning electron microscope, it fully ensures the controllable breaking effect of the cell wall, forms a loose and porous state, is conducive to the effective dissolution of tea polysaccharides, does not damage the integrity of the "active fragments" of polysaccharides during the cell wall breaking-extraction process, has a high extraction rate, and at the same time has many advantages such as no impurity introduction, no pollution, low equipment requirements and low energy consumption in the whole operation process.

[0027] The tea polysaccharides extracted by the method of composite biological enzyme synergistic hydrogen peroxide have 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.

[0028] 3. The tea polysaccharides prepared by the method of the present invention have the new functions of slowing down the weight gain of obese mice induced by high-fat diet and improving the disorders of glucose and lipid metabolism, and enabling important biomarkers such as indole to be produced by the intestinal flora metabolism of db / m mice (indole can inhibit inflammation and maintain intestinal homeostasis). (IV) Description of the Drawings

[0029] Figure 1 The effects of different factors (temperature, pH, liquid-solid ratio, E / S, extraction time) on the dissolution rate of tea polysaccharides.

[0030] Figure 2 The single-factor trajectory diagram of composite enzyme optimization.

[0031] Figure 3 The effects of different factors (liquid-solid ratio, extraction time, hydrogen peroxide concentration and temperature) on the dissolution rate of tea polysaccharides.

[0032] Figure 4 The single-factor trajectory diagram of hydrogen peroxide optimization.

[0033] Figure 5 SEM images of the surface characteristics of raw tea powder (Note: from left to right, the magnification factors are 2.00kx and 5.00kx respectively).

[0034] Figure 6 SEM images of the surface characteristics of tea powder after treatment with composite enzyme - hydrogen peroxide (Note: from left to right, the magnification factors are 2.00kx and 5.00kx respectively).

[0035] Figure 7 Effect of tea polysaccharide on the dynamic body weight of high - fat diet mice.

[0036] Figure 8 Effect of tea polysaccharide on the obesity degree of high - fat diet mice.

[0037] Figure 9 Effect of tea polysaccharide on the dynamic blood glucose of high - fat diet mice.

[0038] Figure 10 Effect of tea polysaccharide on the liver glycogen of high - fat diet mice.

[0039] Figure 11 Effect of tea polysaccharide on the blood lipids (triglyceride, cholesterol, high - density lipoprotein and low - density lipoprotein) of high - fat diet mice.

[0040] Figure 12 GC - MS detection graphs of the in vitro fermentation metabolites of feces from db / m mice (from top to bottom are the normal control group, inulin group, 0.5mg / mL tea polysaccharide sample group, 1.0mg / mL tea polysaccharide sample group, 2.0mg / mL tea polysaccharide sample group and 4.0mg / mL tea polysaccharide sample group).

[0041] Figure 13 GC - MS detection graphs of the in vitro fermentation metabolites of feces from db / db mice (from top to bottom are the negative control group, inulin group, 0.5mg / mL tea polysaccharide sample group, 1.0mg / mL tea polysaccharide sample group, 2.0mg / mL tea polysaccharide sample group and 4.0mg / mL tea polysaccharide sample group). (V) Specific implementation manners

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

[0043] The reference standard for the improved phenol - sulfuric acid method described in the embodiments of the present invention: Spectrophotometry for the determination of the content of crude polysaccharide in Liubao tea - T / GBC 23 - 2024.

[0044] Example 1: Optimization of tea polysaccharide extraction process parameters

[0045] 1 Materials and reagents

[0046] Coarse and old tea leaves (provided by Hubei Huanggang Caotangchun Tea Co., Ltd.).

[0047] Hydrogen peroxide, phenol, sulfuric acid, Tween 80, potassium sodium tartrate, sodium sulfite, DNS (3,5-dinitrosalicylic acid), vitamin K1, hemin, 0.1 mol / L phosphate buffer (pH 6.8), ethanol, chloroform, butanol, etc. are all of analytical grade. GAM medium (without glucose and soluble starch) is purchased from Qingdao Haibo Biotechnology Co., Ltd. Inulin is produced by Beijing Tongrentang. Cellulase (activity 50 U / mg) and papain (10 U / mg) are both purchased from Yuanye Biotechnology Co., Ltd. Triglyceride assay kit (A110-2-1), low-density lipoprotein cholesterol assay kit (A113-1-1), total cholesterol assay kit (A111-2-1), high-density lipoprotein cholesterol assay kit (A112-1-1), liver glycogen assay kit (A043-1-1) and glucagon-like peptide-1 kit (H294-1) are all purchased from Nanjing Jiancheng Bioengineering Institute. The water used in the experiment is deionized water.

[0048] Male C57BL / 6J mice, db / m mice and db / db mice are all purchased from Shanghai Slake Laboratory Animal Co., Ltd., license number: SCXK (Shanghai) 2022-0004.

[0049] 2 Experimental instruments

[0050] JP-250A-2 type high-speed multi-functional pulverizer, Shanghai Jiupin Industry and Trade Co., Ltd.; XMTD-8222 electrothermal blast drying oven, Shanghai Jinghong Experimental Instrument Co., Ltd.; JA2003 electronic analytical balance, Shanghai Shunyu Hengping Scientific 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.; UV1800PC ultraviolet-visible spectrophotometer, Shanghai Aoxi Scientific Instrument Co., Ltd.; SHZ-D(Ⅲ) type circulating water multi-purpose vacuum pump, Zhengzhou Keli Instrument 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; Accu-Chek Bluetooth blood glucose meter, Abbott Diabetes Care (Hangzhou) Co., Ltd.; HBS-1096C microplate reader, Nanjing Detie Biotechnology Co., Ltd.

[0051] 3 Experimental methods

[0052] 3.1 Preparation of coarse and old tea leaf powder

[0053] Place the coarse and old tea leaves in an oven and dry them at a low temperature (60 °C) until the moisture content is below 9.0%. Crush them and pass through a 150-mesh sieve to obtain coarse and old tea leaf powder with a mesh size > 150 meshes.

[0054] 3.2 Pretreatment of Coarse and Old Tea Leaves

[0055] Prepare the DGRA solvent: Mix n-caprylic acid and n-decanoic acid in a molar ratio of 5:2, and magnetically stir at a constant temperature of 75 - 85 °C for 2 h until the solution is clear. Then, mix well with anhydrous ethanol in a volume ratio of 1:3 based on the total volume of n-caprylic acid and n-decanoic acid to prepare the DGRA solvent.

[0056] Take 1000 g of the coarse and old tea leaf powder prepared according to 3.1, add 3 L of the DGRA solvent, stir and heat-extract at 55 °C for 2 h, then centrifuge at 8000 r / min for 15 min. The filter residue is refluxed with a 70% ethanol aqueous solution by volume for 1 h, filtered to remove substances such as lipids, small molecule impurities, alkaloids, and tea polyphenols surrounded outside the tea cells. Then, dry the filter cake in the sun, crush it, and pass through a 200-mesh sieve to obtain 665 g of pretreated powder of coarse and old tea.

[0057] 3.3 Process Optimization of Extracting Tea Polysaccharides by Complex Enzyme Method

[0058] (1) Temperature

[0059] Weigh 2 g of the pretreated powder of coarse and old tea leaves prepared by the method in 3.2, add 0.03 g of a complex enzyme of cellulase - papain (mass ratio 3:1) according to an addition amount with E / S (mass percentage of the complex enzyme in the pretreated powder of coarse and old tea leaves) being 1.5%, add 70 mL of deionized water, with a liquid-to-solid ratio of 35:1 (mL / g), adjust the pH value of the solution to 5.5 with citric acid, control the temperature at 45 °C, 55 °C, 65 °C, 75 °C, or 85 °C respectively, and control the extraction time to 60 min. After the extraction is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 3 min. Centrifuge at 8000 rpm for 20 min, take 1.0 mL of the supernatant, and use the phenol - sulfuric acid method to detect the absorbance value of the supernatant at 480 nm. Calculate the polysaccharide concentration in the supernatant according to the standard curve of glucose concentration and absorbance value. The standard equation is y = 9.2133x + 0.1298, R 2 = 0.9986, where y represents the absorbance value and x represents the polysaccharide concentration, and calculate the extraction yield according to the following formula (1). The results are shown in Figure 1 .

[0060] Calculate the extraction yield of tea polysaccharides according to the following formula:

[0061] Extraction yield of tea polysaccharides (%) = (C × V × f) / m × 100% Formula (1)

[0062] In formula (1), C is the concentration of tea polysaccharide calculated from the standard equation (mg / mL); V is the volume of the supernatant (mL); f is the dilution factor; m is the mass of the pre-treated powder sample of old tea leaves (mg).

[0063] (2) pH

[0064] Fix the temperature in step (1) at 65 °C, and change the pH to pH 4.5, 5.0, 5.5, 6.0, and 6.5 respectively, with other operations remaining the same. The results are shown in Figure 1 .

[0065] (3) Fix the temperature in step (1) at 65 °C, and set the liquid-to-material ratios to 15:1, 25:1, 35:1, 45:1, 55:1, 65:1 mL / g respectively, with other operations remaining the same. The results are shown in Figure 1 .

[0066] (4) E / S

[0067] Fix the temperature in step (1) at 65 °C, and add the cellulase-papain (mass ratio 3:1) complex enzyme at the addition amounts of E / S being 0.9%, 1.2%, 1.5%, 1.8%, or 2.1% respectively, with other operations remaining the same. The results are shown in Figure 1 .

[0068] (5) Extraction time

[0069] Fix the temperature in step (1) at 65 °C, and control the extraction time at 20, 40, 60, 80, 100, or 120 min respectively, with other operations remaining the same. The results are shown in Figure 1 .

[0070] (6) Response surface analysis experiment

[0071] On the basis of the above single-factor experiments, select the three factors (liquid-to-material ratio, extraction time, extraction temperature) that have the most significant influence on the extraction rate of tea polysaccharide as independent variables, and take the extraction rate of tea polysaccharide as the response value. Use Design-Expert.V8.0.6 software, adopt Box-Behnken Design negative (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.

[0072] Table 1 Factor level table for experimental design of optimizing the process of extracting tea polysaccharide by complex enzyme method

[0073] Encoded water A: Liquid-to-material ratio B: Extraction time (min) C: Extraction temperature (°C) -1 25 60 45 0 35 80 55 1 45 100 65

[0074] 3.4 Process optimization of treating tea polysaccharide with hydrogen peroxide

[0075] Weigh 2 g of the pre-treated powder of old and coarse tea leaves prepared by Method 3.2, add a cellulase - papain (mass ratio 3:1) complex enzyme at an addition amount of E / S of 1.5%, add deionized water, with a liquid - to - material ratio of 35 mL / g, adjust the pH value of the solution to 5.0 with citric acid, control the temperature at 55 °C, control the extraction time at 80 min, and after extraction, place it in a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea slurry.

[0076] Add hydrogen peroxide with a mass concentration of 30% and deionized water to the tea slurry. Respectively take temperature, liquid - to - material ratio, hydrogen peroxide concentration, and treatment time as single factors, place them in a constant - temperature oscillator for treatment, and conduct a single - factor experiment with the extraction yield of tea polysaccharide as the response value. The specific method is as follows:

[0077] (1) Liquid - to - material ratio: Add a solution of hydrogen peroxide with a mass concentration of 30% and deionized water to the tea slurry at liquid - to - material ratios of 10:1, 15:1, 20:1, 25:1, 30:1 (mL / g) respectively based on the mass of the pre - treated powder of old and coarse tea leaves for preparing the tea slurry, so that the final mass concentration of added hydrogen peroxide is 1.2% in all cases, pH 5.0, perform water - bath extraction in a constant - temperature oscillator at 90 °C and 200 rpm for 10 min, and centrifuge at 8000 rpm for 20 min. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol - sulfuric acid method, and convert it into the extraction yield.

[0078] (2) Treatment time: Fix the liquid - to - material ratio in step (1) at 20:1, and the extraction times are 5 min, 10 min, 15 min, 20 min, and 25 min respectively. Other steps are the same as in step (1).

[0079] (3) Hydrogen peroxide concentration: Fix the liquid - to - material ratio in step (1) at 20:1, and make the final mass concentration of added hydrogen peroxide be 0.3%, 0.6%, 0.9%, 1.2%, 1.5% respectively. Other steps are the same as in step (1).

[0080] (4) Treatment temperature: Fix the liquid - to - material ratio in step (1) at 20:1, and the temperatures are 60 °C, 70 °C, 80 °C, 90 °C, 100 °C respectively. Other steps are the same as in step (1).

[0081] On the basis of the above single - factor experiments, select the three factors (liquid - to - material ratio, extraction time, hydrogen peroxide concentration) that have the most significant influence on the extraction yield of tea polysaccharide as independent variables, and use the extraction yield of tea polysaccharide as the response value. Use Design - Expert.V8.0.6 software, adopt Box - Behnken Design (three - factor and three - level), 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 2.

[0082] Table 2 Factor - level table for experimental design of optimizing the process of treating tea polysaccharide with hydrogen peroxide

[0083] Encoded water A: Liquid-to-material ratio (mL / g) B: Extraction time (min) C: Hydrogen peroxide concentration (%) -1 10 5 0.9 0 20 15 1.2 1 30 25 1.5

[0084] 3.5 Scanning Electron Microscope (SEM) Observation of Raw Tea Powder and Treated Residue

[0085] Weigh 2 g of the pre-treated powder of coarse and old tea leaves prepared by method 3.2, add 0.03 g of a composite enzyme of cellulase - papain (mass ratio 3:1) at an addition amount of E / S of 1.5%, add 70 mL of deionized water, with a liquid-to-solid ratio of 35 mL / g, adjust the pH value of the solution to 5 with citric acid, control the temperature at 55 °C, control the extraction time to 80 min. After the extraction, place it in a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea slurry.

[0086] Add 40 mL of a solution of hydrogen peroxide and deionized water with a mass concentration of 30% to the tea slurry according to the amount of 20 mL / g of the pre-treated powder of coarse and old tea leaves used to prepare the tea slurry, so that the final mass concentration of added hydrogen peroxide is 1.2%, pH 5.0. Extract in a water bath at a temperature of 100 °C in a constant temperature oscillator for 18 min, and centrifuge at 8000 rpm for 20 min. After the supernatant is concentrated to 1 / 3 of the original volume, cool it to 4 °C, add 4 times the volume of 95% ethanol, let it stand at 4 °C for 12 h, then centrifuge at 3000 rpm for 10 min. Take the precipitate, dissolve it in deionized water, and use the Sevag method to remove proteins, that is, place the deionized water solution of the precipitate in a separatory funnel, add 1 / 4 of the volume of the Sevag reagent (chloroform: n-butanol = 4:1, v:v) of the deionized water solution of the precipitate, shake it vigorously up and down for 2 min, then place it on an iron stand and let it stand until it is layered up and down. Take the upper layer and detect whether there is an absorption peak at 280 nm. Continue to elute with the Sevag reagent until there is no absorption peak at 280 nm in the upper layer. Concentrate the upper layer to 25 - 35% of the original volume and dry it at 60 °C to obtain 0.5 g of tea polysaccharide, that is, the tea powder treated with the composite enzyme - hydrogen peroxide, which is used for subsequent detection of glycolipid metabolism and intestinal flora metabolites.

[0087] Take 0.2 g of tea polysaccharide, coat it on an MC1000 ion sputtering instrument (icn sputter), and observe the microscopic morphology in an itachi SU8010 type field emission scanning electron microscope (SEM). At the same time, take 0.2 g of the coarse and old tea powder in 3.1 (i.e., raw tea powder) for microscopic morphology observation.

[0088] 3.6 Effects of Tea Polysaccharide on Glycolipid Metabolism in Obese Mice

[0089] 3.6.1 Feeding and Grouping of Mice

[0090] Healthy male C57BL / 6 specific pathogen free mice at 5 - 6 weeks of age with uniform body weight were selected. The breeding temperature was 20℃±2℃, and the relative humidity was 55 - 60%. After the mice were purchased, they were fed freely during the adaptation period. One week later, the mice were randomly divided into 4 groups, with 5 mice in each group, including a normal control group (standard diet, normal saline), a negative control group (high - fat diet, normal saline), a high - dose sample group (high - fat diet, 300 mg / kg tea polysaccharide), and a low - dose sample group (high - fat diet, 100 mg / kg tea polysaccharide). The mice in the normal control group were fed the standard diet throughout the experiment, and the mice in the other groups were fed the high - fat diet (Research Diets D12492 60 kcal% Fat). The mice were fasted for 12 h with free access to water, weighed, and their initial body weights were recorded. Blood was taken from the tip of the tail and the initial blood glucose value of the mice was measured using a blood glucose meter. During the tea polysaccharide intervention period, the body weights and fasting blood glucose of the mice were monitored regularly.

[0091] 3.6.2 Detection of biochemical indexes

[0092] (1) Obesity degree

[0093] Calculation formula for obesity degree (%): [(average body weight of mice in the high - fat group - average body weight of mice in the normal diet group) / average body weight of mice in the normal diet group]×100%.

[0094] (2) Determination of indexes related to glycolipid metabolism

[0095] After 15 weeks of the animal experiment, the mice were fasted for 12 h with free access to water. Blood was taken from the orbital plexus vein, and the blood was collected in a 1.5 mL centrifuge tube. It was left standing at room temperature for 30 min, then centrifuged at 3500 rpm for 20 min at 4℃, and the upper - layer serum was taken and stored at - 80℃ for later use. The contents of triglyceride (TG), total cholesterol (TC), high - density lipoprotein (HDL), and low - density lipoprotein (LDL) in the mouse serum were measured using a biochemical test kit. For the kit from Nanjing Jiancheng Bioengineering Institute, the specific operation for index detection was carried out according to the instructions.

[0096] The mice were dissected to obtain the liver, which was rinsed on the surface with pre - cooled normal saline, dried, weighed, put into a 1.5 mL centrifuge tube, labeled, quickly frozen in liquid nitrogen, and stored in a - 80℃ refrigerator. An appropriate amount of liver was weighed according to the instructions, 9 times the weight of pre - cooled normal saline was added, and it was ground using a handheld high - speed homogenizer. After centrifugation, the supernatant was taken to prepare a 10% liver tissue homogenate, and the level of hepatic glycogen in the liver was detected using the kit from Nanjing Jiancheng Bioengineering Institute.

[0097] 3.7 Effects of tea polysaccharide on the metabolites of intestinal flora in diabetic mice

[0098] 3.7.1 Preparation of reagents

[0099] GAM basal medium: Weigh 49.0 g of GAM medium, heat and dissolve it in 1000 mL of deionized water, autoclave at 121 °C for 15 min. When cooled to about 50 °C, add 1 mL of sterile 0.1% vitamin K1 solution and 1 mL of hemin (5 mg / mL) to every 1000 mL of the medium, mix well and set aside.

[0100] Preparation of 0.1% vitamin K1 solution: Weigh 0.01 g of vitamin K1, place it in a conical flask, add 1 mL of Tween 80, gently shake the conical flask, and then stir with a glass rod to fully mix vitamin K1 and Tween 80. After adding a small amount of deionized water, stir and shake well, and make up the volume to 100 mL to obtain 0.1% vitamin K1 solution (it can be stored for 1 year in the dark at 2 - 8 °C).

[0101] Preparation of 5 mg / mL hemin: Weigh 0.5 g of hemin, add at least a small amount of deionized water, add 1 mL of 1 mol / L sodium hydroxide solution and stir to dissolve evenly, make up the volume to 100 mL to prepare 5 mg / mL hemin solution (store in the dark at 2 - 8 °C for 1 year).

[0102] Inulin refers to the polysaccharide substance derived from chicory tubers and is commercially available.

[0103] 3.7.2 Collection of fecal samples and in vitro fermentation

[0104] Select healthy male db / m mice and db / db mice at 5 - 6 weeks of age with uniform body weight, collect fresh fecal samples of the mice. The fecal samples are divided into db / m group (normal control group, inulin group, 0.5 mg / mL tea polysaccharide sample group, 1 mg / mL tea polysaccharide sample group, 2 mg / mL tea polysaccharide sample group, 4 mg / mL tea polysaccharide sample group) and db / db diabetic group (negative control group, inulin group, 0.5 mg / mL tea polysaccharide sample group, 1.0 mg / mL tea polysaccharide sample group, 2.0 mg / mL tea polysaccharide sample group, 4.0 mg / mL tea polysaccharide sample group) according to the source.

[0105] Add the fresh fecal samples of each group to sterile 0.1 mol / L phosphate buffer (pH 6.8), and process them into a fecal suspension of 10 g / 100 mL for inoculation into the prepared GAM basal medium.

[0106] In the db / m group, 500 μL of fecal suspension was inoculated into 5 mL of GAM basal medium for the normal control group; 500 μL of fecal suspension was inoculated into 5 mL of GAM basal medium containing 0.5 mg / mL inulin for the inulin group; 500 μL of fecal suspension was inoculated into 5 mL of GAM basal medium containing 0.5, 1, 2, and 4 mg / mL of tea polysaccharide for the 0.5 mg / mL tea polysaccharide sample group, 1 mg / mL tea polysaccharide sample group, 2 mg / mL tea polysaccharide sample group, and 4 mg / mL tea polysaccharide sample group respectively. The culture media of each group were first added into anaerobic tubes, sterilized at 115 °C for 30 min, and then the fecal suspension was added.

[0107] In the db / db diabetic group, 500 μL of fecal suspension was inoculated into 5 mL of GAM basal medium for the negative control group; 500 μL of fecal suspension was inoculated into 5 mL of GAM basal medium containing 0.5 mg / mL inulin for the inulin group; 500 μL of fecal suspension was inoculated into 5 mL of GAM basal medium containing 0.5, 1, 2, and 4 mg / mL of tea polysaccharide for the 0.5 mg / mL tea polysaccharide sample group, 1.0 mg / mL tea polysaccharide sample group, 2.0 mg / mL tea polysaccharide sample group, and 4.0 mg / mL tea polysaccharide sample group respectively. The culture media of each group were first added into anaerobic tubes, sterilized at 115 °C for 30 min, and then the fecal suspension was added.

[0108] 3.7.3 Detection of total sugar content

[0109] Each group was cultured in an anaerobic environment at 37 °C, and samples were taken at 0 h, 12 h, 24 h, and 48 h of fermentation to detect the contents of total sugar (phenol-sulfuric acid method) and reducing sugar (DNS colorimetric method).

[0110] Determination method of reducing sugar content by DNS colorimetric method: Prepare the color reagent. Add 6.3 g of DNS and 262 mL of 2 mol / L sodium hydroxide aqueous solution to 500 mL of aqueous solution containing 182 g of sodium potassium tartrate, then add 5 g of heavy phenol and 5 g of sodium sulfite, stir and dissolve. After cooling, add water to make up to 1000 mL to prepare 3,5-dinitrosalicylic acid reagent, and store it in a brown bottle for later use.

[0111] Drawing of the glucose standard curve: Respectively take 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of glucose standard solution (1 mg / mL) into 25-mL test tubes. Accurately add 2 mL of DNS reagent to each test tube, heat in a boiling water bath for 2 min, cool with running water, and make up to the 15-mL scale with water. Measure the absorbance at a wavelength of 540 nm. For the determination of the sample, appropriately dilute the sample solution to make the sugar concentration 0.1 - 1.0 mg / ml. Take 1.0 mL of the diluted sample into a 15-mL graduated test tube, add 2.0 mL of DNS reagent, boil in boiling water for 2 min, cool, and make up to 15 mL with water. Measure the absorbance at a wavelength of 540 nm. Calculate the content of reducing sugar in the sample according to the standard curve.

[0112] 3.7.4 Changes in metabolites

[0113] When fermenting for 48 h, take samples and detect the fermentation products of different groups by GC-MS. The GC determination conditions are as follows: The carrier gas is high-purity helium, the chromatographic column is DB-5ms (30.0 m × 250 μm, 0.25 μm), maintain at 50 °C for 2 min, increase to 100 °C at a rate of 8 °C / min and hold for 1 min, increase to 150 °C at a rate of 10 °C / min and hold for 1 min, increase to 250 °C at a rate of 20 °C / min and hold for 5.75 min. The MS determination conditions are as follows: EI source, the electron energy is 70 eV, the ion source temperature is selected as 230 °C, the interface temperature is 280 °C, and the program time is 26 min. All experiments are carried out under aseptic conditions.

[0114] 3.8 Data processing

[0115] The experimental data are expressed as Means ± SD. Use the homogeneity of variance test and LSD multiple comparison analysis in one-way analysis of variance (ANOVA) of IBM SPSS Statistics 25 software for significant difference analysis. p < 0.05 indicates a significant difference.

[0116] 4 Experimental results

[0117] 4.1 Single-factor experiment and response surface optimization of complex enzyme cell wall breaking - extraction of tea polysaccharide

[0118] 4.1.1 Single-factor experiment on the dissolution of tea polysaccharide

[0119] The effects of temperature, pH, liquid-to-solid ratio, ratio of complex enzyme to substrate (E / S), and extraction time on the extraction yield of tea polysaccharide are shown in Figure 1 .

[0120] From Figure 1It can be seen that with the increase of temperature, the extraction rate of tea polysaccharide increases. When the temperature reaches 55 °C, the extraction rate of tea polysaccharide is the highest. With the continuous increase of temperature, the extraction rate of tea polysaccharide shows a trend of first decreasing and then slightly increasing. It is speculated that the optimal action temperature of the composite enzyme is about 55 °C, and the increase of temperature will deviate from the optimal action temperature of the enzyme; the upward trend that appears when the temperature increases to 85 °C may be that the increase of temperature accelerates the dissolution of polysaccharide, but the effect of temperature increase is not as large as that of the enzyme. Therefore, 55 °C is selected as the central point for the subsequent optimization experiment.

[0121] With the increase of pH, the extraction rate of tea polysaccharide shows a trend of first increasing and then decreasing; when the pH is 5.0, the extraction rate of tea polysaccharide is the highest, but there is no significant difference from other pH values (except pH 6.5). There is no vertex for this factor on the response value, so it is not optimized. Therefore, the pH is determined to be 5.0 in the subsequent experiment.

[0122] When the liquid-to-solid ratio increases from 15 mL / g to 35 mL / g, the extraction rate of tea polysaccharide increases significantly; when the liquid-to-solid ratio of the system exceeds 35 mL / g, the extraction rate of tea polysaccharide shows a significant downward trend. Therefore, 35 mL / g is selected as the central point for the subsequent optimization experiment.

[0123] The extraction rate of tea polysaccharide shows a continuously significant upward trend with the increase of the ratio of composite enzyme to substrate (E / S). When the E / S increases to 1.5%, continuing to increase the E / S, although the extraction rate of tea polysaccharide shows an increasing trend, there is no significant difference from 1.5%. There is no central point for this factor on the response value, so it is not optimized. Considering the use cost, the E / S is determined to be 1.5% in the subsequent experiment.

[0124] With the increase of extraction time from 20 min to 80 min, the extraction rate of tea polysaccharide shows a continuously significant upward trend. When the time continues to increase to more than 80 min, the extraction rate of tea polysaccharide shows a significant downward trend. When the extraction time is 80 min, the extraction rate of tea polysaccharide is significantly higher than that at other times. Therefore, 80 min is used as the central point in the subsequent optimization experiment.

[0125] 4.1.2 Response surface optimization of tea polysaccharide dissolution

[0126] 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 3.

[0127] Table 3 Experimental design and results of composite enzyme breaking-wall - extracting tea polysaccharide

[0128]

[0129] Perform multiple regression fitting on the experimental data in Table 3 to obtain a regression equation with the extraction yield of tea polysaccharide (Y) as the response value:

[0130] Y = 8.68 + 0.215A + 0.030B - 0.087C + 0.003AB - 0.063AC - 0.081BC - 0.637A 2 - 0.207B 2 - 0.458C 2

[0131] Perform an analysis of variance on the regression equation, and the results are shown in Table 4.

[0132] Table 4 Regression negative analysis of compound enzyme cell wall breaking - extraction of tea polysaccharide

[0133]

[0134] The analysis in Table 4 shows that the p - value is used as a tool to check the significance of each coefficient. The negative p - value is 0.0068, indicating that the negative is significant (p < 0.01). R 2 is 0.9080, indicating that within the range of experimental parameters, the predicted value fits well with the simulation experimental value; the adjusted R 2 is 0.7896, indicating that the equation can explain 78.96% of the change in the response value, and the fitting degree is good. The p - value of the lack - of - fit term is 0.453, which is not significant, indicating that the residuals are caused by random errors and are not significant compared to the pure error (p > 0.05); the C.V value is 2.79%, indicating a relatively high negative credibility. Therefore, the experimental results can be predicted using this regression equation. Among the three factors, the linear term A and the quadratic terms A 2 and C 2 have a significant impact on the response value curve effect; the interaction terms AB, AC, and BC have no significant impact on the response value surface effect, indicating that there is no obvious synergistic effect among the liquid - to - material ratio, extraction time, and temperature. The order of the influence of the three factors on the extraction yield of tea polysaccharide is: A > C > B.

[0135] From the regression negative analysis, the interaction terms (AB, AC, BC) are all not significant, so there is no need to perform response surface and contour plot analysis. Perform a single - factor effect analysis through the dimensionality reduction analysis method, that is, fix the three factors in the regression equation at the zero level, and obtain the single - factor negatives of the three factors as follows:

[0136] Y A = 8.38 + 0.21A - 0.67A 2

[0137] Y B = 8.19 + 0.03B - 0.27B 2

[0138] YC = 8.30 - 0.086C - 0.50C 2

[0139] The single - factor trajectories of each factor obtained from the above equation are shown in Figure 2 .

[0140] Figure 2 It shows the effects of liquid - to - material ratio, extraction time and temperature on the extraction rate of tea polysaccharide. With the increase of the number of levels, the extraction rate of tea polysaccharide first increases significantly, then increases gently, and finally decreases significantly. There are maximum values in the influence of the three factors on the extraction rate of tea polysaccharide between - 1 and 1 levels. The curve corresponding to the liquid - to - material ratio changes most obviously in this range, and this steep curve indicates that the extraction rate of tea polysaccharide is most sensitive to this factor.

[0141] The optimal conditions obtained by negative optimization are: liquid - to - material ratio 1:36.689, time 81.468 min, extraction temperature 54.054 °C. Under these conditions, the theoretical dissolution rate of tea polysaccharide is 8.700%. Considering the actual laboratory conditions, the optimal adjustment is: liquid - to - material ratio 1:35, time 80 min, extraction temperature 55 °C. To further verify the effectiveness and accuracy of this negative result and the actual situation, three parallel experiments are carried out according to the selected extraction conditions. The extraction rate of tea polysaccharide can reach 8.717% ± 0.128%, 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.

[0142] 4.2 Single - factor experiments and response surface optimization of tea polysaccharide dissolution by hydrogen peroxide treatment technology

[0143] 4.2.1 Single - factor experiments of tea polysaccharide dissolution

[0144] The effects of liquid - to - material ratio, extraction time, hydrogen peroxide concentration and temperature on the extraction rate of tea polysaccharide are shown in Figure 3 .

[0145] From Figure 3 the influence of liquid - to - material ratio on the dissolution rate of tea polysaccharide, it can be seen that when the liquid - to - material ratio increases from 10 mL / g to 20 mL / g, the dissolution rate of tea polysaccharide increases significantly. When the liquid - to - material ratio of the system exceeds 20 mL / g, the dissolution rate of tea polysaccharide shows a significant downward trend. Therefore, 20 mL / g is selected as the center point for subsequent optimization experiments.

[0146] From Figure 3From the influence of extraction time on the dissolution rate of tea polysaccharide, it can be seen that as the extraction time increases from 5 min to 15 min, the dissolution rate of tea polysaccharide shows a significant upward trend. When the time continues to increase to 25 min, the dissolution rate of tea polysaccharide shows a significant downward trend. When the extraction time is 15 min, the dissolution rate of tea polysaccharide is higher than that in other time periods. Therefore, 15 min is used as the central point in the subsequent optimization experiments.

[0147] As Figure 3 From the influence of hydrogen peroxide concentration on the dissolution rate of tea polysaccharide, it can be seen that as the hydrogen peroxide concentration increases from 0.3% to 1.2%, the dissolution rate of tea polysaccharide continuously increases. When the hydrogen peroxide concentration exceeds 1.2%, the dissolution rate shows a downward trend. It indicates that hydrogen peroxide has an optimal concentration. Therefore, 1.2% hydrogen peroxide concentration is selected as the central point for the subsequent optimization experiments.

[0148] From Figure 3 From the influence of temperature on the dissolution rate of tea polysaccharide, it can be seen that the dissolution rate of tea polysaccharide continuously increases as the extraction temperature increases from 60 °C to 100 °C, indicating that the increase in temperature can promote the breaking of tea cell walls and the dissolution of polysaccharides. There is no vertex for this factor on the response value, so it is not optimized. Therefore, the temperature is determined to be 100 °C in the subsequent experiments.

[0149] 4.2.2 Response surface optimization of tea polysaccharide dissolution

[0150] 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 5.

[0151] Table 5 Response surface design and experimental results of tea polysaccharide dissolution

[0152]

[0153] According to the Box-Behnken Design, a total of 17 experiments were carried out for the three independent parameters optimized. The dissolution rate of tea polysaccharide varied greatly within the extraction condition range. By applying multiple regression analysis, the following second-order polynomial equation was obtained according to the response variable and test variables:

[0154] Y = 24.42 + 0.55A + 2.90B + 0.95C - 0.065AB - 0.20AC + 0.23BC - 1.14A 2 -5.03B 2 -1.77C 2

[0155] Where Y is the predicted extraction yield of tea polysaccharide, and A, B, and C are the coded variables of solid-liquid ratio, extraction time, and hydrogen peroxide concentration, respectively. Multiple regression fitting was performed on the experimental data in Table 5 to obtain a regression equation. Analysis of variance was carried out on the regression equation, and the results are shown in Table 6.

[0156] Table 6 Analysis of Variance of the Negative Regression Equation

[0157]

[0158]

[0159] Correlation Coefficient R of the Regression Equation 2 = 0.9879, indicating a good fit between the predicted value and the experimental value within the range of experimental parameters; Adjusted Correlation Coefficient R 2 adj = 0.9723, indicating that the equation can explain 97.23% of the response value changes and has a good fitting degree. The p-value was used as a tool to check the significance of each coefficient. The negative p-value < 0.0001 indicates that the negative fitting degree is very significant (p < 0.01). The p-value of the lack-of-fit term is 0.7325, indicating that the residuals are caused by random errors and are not significant relative to the pure error (p > 0.05); The coefficient of variation is 2.94%, indicating a relatively high negative credibility. Therefore, the regression equation can be used to predict the experimental results.

[0160] From the results of the analysis of variance, among the three factors, the linear terms A, B, C and the quadratic terms A 2 、B 2 、C 2 have extremely significant effects on the response value curve; the interaction terms AB, AC, and BC have no significant effects on the response value surface, indicating that there is no obvious synergistic effect between the solid-liquid ratio and hydrogen peroxide concentration, the solid-liquid ratio and extraction time, and the extraction time and hydrogen peroxide concentration. According to the F value, the order of the effects of the three factors on the extraction yield of tea polysaccharide is: B > C > A.

[0161] From the negative regression analysis, the interaction terms (AB, AC, BC) are all not significant, so there is no need to perform response surface and contour plot analysis. Single-factor effect analysis was carried out through the dimensionality reduction analysis method, that is, the three factors in the regression equation were fixed at the zero level, and the single-factor negatives of the three factors were obtained as follows:

[0162] Y A = 21.40 + 0.55A - 1.52A 2

[0163] Y B = 23.13 + 2.90B - 5.20B 2

[0164] Y C=21.68+0.95C-2.11C 2

[0165] According to the above equation, the single factor trajectory of each factor is shown in Figure 4 .

[0166] Figure 4 The results show that the influence of liquid-to-solid ratio, extraction time and hydrogen peroxide concentration on the extraction yield of tea polysaccharides shows that with the increase of the number of levels, the influence of the extraction time factor on the extraction yield of tea polysaccharides shows a trend of first increasing significantly and then decreasing significantly. The influence of the liquid-to-solid ratio and hydrogen peroxide concentration on the extraction yield of tea polysaccharides shows a trend of gently increasing and then gently decreasing. The influence of the three factors on the extraction yield of tea polysaccharides between the -1 and 1 levels all have maximum values. The curve corresponding to the extraction time changes most significantly within this range, and the steep curve shows that the response of the extraction yield of tea polysaccharides is most sensitive to this factor.

[0167] The best conditions for negative optimization were: liquid-to-solid ratio 22.074 mL / g, extraction time 17.929 min, hydrogen peroxide concentration 1.283%, under which the theoretical dissolution rate of tea polysaccharides was 25.039%. Combined with the actual laboratory conditions, the optimal adjustment was: liquid-to-solid ratio 20 mL / g, extraction time 18 min, hydrogen peroxide concentration 1.2%. In order to further verify the effectiveness and accuracy of the negative and actual conditions, three parallel experiments were carried out according to the optimal extraction conditions, and the dissolution rate of tea polysaccharides could reach 25.382% ± 0.424%, which was consistent with the predicted value, indicating that the response surface analysis method was reliable and well fitted with the actual situation, thus verifying the effectiveness of the regression equation.

[0168] 4.3 Observation of tea surface structural characteristics using scanning electron microscopy

[0169] The microstructure of raw tea powder and tea powder treated with compound enzyme-peroxide and perhydrogen under 2000 and 5000 times magnification is shown in Figure 2. Figure 5 and Figure 6 .Depend on Figure 5 The surface of the raw tea powder is rough, the structure is dense, and it overlaps like tiles. The surface of each tile is smooth and complete without any breakage or cracks. Figure 6 After the compound enzyme-peroxide perhydrogen treatment, the sample structure was greatly damaged and the structure was loose. The original tile-like structure became scattered fragments, and there were hole-like structures and deep cracks on the surface. It is speculated that the synergistic effect of compound enzyme-peroxide perhydrogen has a great degree of damage to the cell wall of tea powder. The SEM image is consistent with the extraction yield of tea polysaccharides, which can explain that the synergistic treatment of compound enzyme-peroxide perhydrogen can cause the drastic rupture of the cell wall structure of tea powder and promote the efficient dissolution of tea polysaccharides.

[0170] 4.4 Effects of tea polysaccharides on glucose and lipid metabolism in obese mice

[0171] 4.4.1 Dynamic Effects of Tea Polysaccharide on Body Weight and Blood Glucose in Mice Fed with High-Fat Diet

[0172] When the body weight of mice in the negative control group is 20% higher than that of the normal control group, it can be regarded as successful establishment of the obesity model. For example Figure 7 , after one week of adaptive feeding, the body weights of mice weighing 18 g - 20 g were 18.5 g - 20.5 g, and there were no significant differences in the body weights of mice in each group. During the process of feeding with high-fat diet, the body weights of mice in each group continued to increase. By the 14th week, the body weights of mice in the negative control group had increased to more than 34 g, while the body weights of mice in the normal control group were only 25 g. Compared with the negative control group, the body weights of mice in the low-dose and high-dose tea polysaccharide groups increased at almost the same rate in the first 4 weeks. After the 4th week, the increase in the body weights of mice in the tea polysaccharide group gradually became slower; among them, the body weights of mice in the high-dose tea polysaccharide group increased more slowly than those in the low-dose tea polysaccharide group. By the 14th week, the body weights of the mice were 30.7 g, showing a significant difference compared with the negative control group (p < 0.05). For example Figure 8 , the obesity levels of mice in each group showed an increasing trend during the process of feeding with high-fat diet. The obesity level of mice in the negative control group exceeded 20% at the 6th week, indicating that an obesity model had been formed. The obesity level of mice in the low-dose tea polysaccharide group was higher than that of the negative control group in the first 4 weeks but lower than 20%, and no obesity model was formed; from the 6th week to the 14th week at the end of the experiment, its obesity level was always lower than that of the negative control group. The obesity level of mice in the high-dose tea polysaccharide group was always lower than that of the negative control group throughout the experiment. Especially from the 6th week, its obesity level increased slowly and was significantly lower than that of the negative control group, indicating that tea polysaccharide has the effect of slowing down the increase in body weight of mice caused by high-fat diet. For example Figure 9 , during the process of feeding with high-fat diet, the blood glucose of mice in the negative control group showed an increasing trend, while the blood glucose of mice in other groups was always in a fluctuating state. In the first two weeks, the blood glucose of the 4 groups of mice was close and there were no differences. From the 6th week to the 14th week, the blood glucose of mice in the high-dose and low-dose tea polysaccharide groups was always lower than that of the negative control group, indicating that tea polysaccharide has the effect of slowing down the increase in blood glucose of mice caused by high-fat diet.

[0173] 4.4.2 Effects of Tea Polysaccharide on Hepatic Glycogen in Mice Fed with High-Fat Diet

[0174] Hepatic glycogen is a form of energy reserve and plays an important role in maintaining blood glucose stability, providing energy, and promoting liver metabolism and detoxification. The measurement results of hepatic glycogen content in each group of mice are as follows Figure 10 , among which the hepatic glycogen content of mice in the negative control group was significantly lower than that of mice in the normal group (p < 0.05). After intervention with high-dose and low-dose tea polysaccharide, the decrease in hepatic glycogen was alleviated. Especially for high-dose tea polysaccharide, the difference in hepatic glycogen content increased compared with the negative control group (p < 0.05).

[0175] 4.4.3 Effects of Tea Polysaccharide on Blood Lipids in Mice Fed with High-Fat Diet

[0176] Obesity is accompanied by dyslipidemia. The abnormal increase in triglyceride (TG), total cholesterol (TC) levels and low-density lipoprotein cholesterol (LDL-C) in the blood, along with the abnormal decrease in high-density lipoprotein cholesterol (HDL-C), is called dyslipidemia. As Figure 11 , compared with the normal control group, the TG level in the negative control group of mice increased significantly. Compared with the negative control group, both high- and low-dose tea polysaccharide groups could reduce the TG level in mice, especially the low-dose tea polysaccharide significantly reduced the TG level. The triglyceride-glucose index (TyG) is an effective model for predicting the risks of cardiovascular and cerebrovascular diseases and diabetes, and its calculation is as follows: TyG = Ln[TG (mg / dl) × FPG (mg / dl) / 2], where FPG is the fasting blood glucose. Through calculation, it can be seen that the TyG value of the negative control group of mice was the highest, reaching 7.25, and the TyG values of the high- and low-dose tea polysaccharide groups of mice were both lower than 7, indicating that tea polysaccharide can effectively reduce the risks of cardiovascular and cerebrovascular diseases and diabetes by reducing the TG level.

[0177] From Figure 11 , compared with the normal control group, the TC level and LDL-C level in the negative control group of mice increased significantly. Compared with the negative control group, both high- and low-dose tea polysaccharide groups could reduce the TC level and LDL-C level in mice, and the reduction effect was concentration-dependent. Compared with the normal control group, the HDL-C level in the negative control group of mice decreased significantly. Compared with the negative control group, both high- and low-dose tea polysaccharide groups could increase the HDL-C level in mice, and the increase effect was also concentration-dependent. It shows that tea polysaccharide has the effect of improving dyslipidemia in mice caused by high-fat diet.

[0178] 4.5 Fermentation Products of Tea Polysaccharide after Fermentation by Intestinal Flora

[0179] 4.5.1 Changes in Total Sugar and Reducing Sugar of Tea Polysaccharide after Fermentation by Intestinal Flora

[0180] The dynamic changes of total sugar and reducing sugar of tea polysaccharide fermented by the intestinal flora of db / m mice and db / db mice at different time points are shown in Tables 7, 8, 9 and 10.

[0181] Table 7 shows the effects of the intestinal flora of normal mice on the total sugar consumption during the fermentation of different carbon sources. In the normal control group, the positive control (inulin) group, and the tea polysaccharide groups at different concentrations, the total sugar content was the highest at 0 h, and there was no difference among the groups. As the fermentation time extended, the total sugar content in each group continuously decreased, indicating that the carbon source in the culture medium was continuously metabolized and consumed by the intestinal bacteria. At 24 h of fermentation, the total sugar content in the low-concentration (0.5 mg / mL) tea polysaccharide group was the highest. Subsequently, in order of total sugar content, they were the normal control group, the inulin group, the 1 mg / mL tea polysaccharide group, the 4 mg / mL tea polysaccharide group, and the 2 mg / mL tea polysaccharide group, indicating that the low-concentration tea polysaccharide had little effect on the intestinal bacteria's fermentation of the carbon source. As the concentration of tea polysaccharide increased, the fermentation effect increased continuously and was better than that of the positive control. At 48 h of fermentation, the total sugar content in the normal control group, the inulin group, and the low-concentration (0.5 mg / mL) tea polysaccharide group decreased to the lowest level, indicating that the carbon source in the culture medium was basically consumed. However, when the tea polysaccharide concentration was higher than 1 mg / mL, the total sugar concentration showed an increasing trend instead, possibly because the tea polysaccharide promoted the proliferation of more beneficial bacteria in the mouse intestine, thereby promoting the conversion of undigested dietary fiber in the feces into total sugar.

[0182] Table 8 shows the effects of the intestinal flora of spontaneously diabetic db / db mice on the total sugar consumption during the fermentation of different carbon sources. Similarly, in the negative control group, the inulin group, and the tea polysaccharide groups at different concentrations, the total sugar content was the highest at 0 h, and there was no difference among the groups. As the fermentation time extended, the total sugar content in each group continuously decreased, indicating that the carbon source in the culture medium was continuously metabolized and consumed by the intestinal bacteria. At 12 h of fermentation, there was no difference in the total sugar content among the groups except for the 4 mg / mL tea polysaccharide group. At 24 h of fermentation, the total sugar content in the inulin group, the low-concentration (0.5 mg / mL) tea polysaccharide group, and the 2 mg / mL tea polysaccharide group was significantly higher than that in other groups. The total sugar content in the 1 mg / mL tea polysaccharide group and the 4 mg / mL tea polysaccharide group was significantly lower than that in the negative control group, indicating that the high-concentration tea polysaccharide had promoted the proliferation of probiotics in the intestine of diabetic mice, but its effect did not show a concentration-dependent relationship. At 48 h of fermentation, the total sugar content in the negative control group did not change significantly compared with that at 24 h, indicating that the intestinal flora of diabetic mice was dysregulated and could not normally ferment the carbon source in the culture medium. The total sugar content in the inulin group and the high-concentration (>1 mg / mL) tea polysaccharide group was significantly lower. In particular, the total sugar content in the 4 mg / mL tea polysaccharide group was not different from that in the inulin group, indicating that the high-concentration tea polysaccharide could improve the dysregulated state of the intestinal flora in diabetic mice and promote the proliferation of more beneficial bacteria.

[0183] Table 9 shows the effects of the intestinal flora of normal mice on the fermentation of different carbon sources to produce reducing sugars. Within 24 h of fermentation, no reducing sugars were detected in each group, indicating that the carbohydrate substances in each group had not been degraded to produce reducing sugars. At 48 h of fermentation, reducing sugars were detected in each group, and there was no difference between groups, indicating that the intestinal bacteria of normal mice had consumed the carbon sources. Table 10 shows the effects of the intestinal flora of spontaneous db / db diabetic mice on the fermentation of different carbon sources to produce reducing sugars. Similarly, within 24 h of fermentation, no reducing sugars were detected in each group, indicating that the carbohydrate substances in each group had not been degraded to produce reducing sugars. At 48 h of fermentation, reducing sugars were detected in each group. There was no significant difference in the reducing sugar content between the negative control group and the low-concentration (0.5 mg / mL and 1 mg / mL) tea polysaccharide groups; the reducing sugar content in the inulin group and the high-concentration (2 mg / mL and 4 mg / mL) tea polysaccharide groups was significantly higher than that in the negative control group, especially in the 4 mg / mL tea polysaccharide group. It is speculated that more reducing sugars were produced by the degradation of carbohydrate substances by intestinal bacteria.

[0184] Table 7 Dynamic changes in total sugars during the fermentation of tea polysaccharides by the intestinal flora of db / m mice at different time points

[0185] Normal control group Inulin group 0.5 mg / mL tea polysaccharide 1 mg / mL tea polysaccharide 2 mg / mL tea polysaccharide 4 mg / mL tea polysaccharide 0h 6.535 6.680 6.754 6.043 6.673 6.467 12h 2.026 3.147 4.020 3.062 4.477 2.641 24h 2.125 1.739 2.974 1.619 1.106 1.350 48h 0.979 1.160 1.683 1.618 1.582 1.609

[0186] Table 8 Dynamic changes in total sugars during the fermentation of tea polysaccharides by the intestinal flora of db / db mice at different time points

[0187] Negative control group Inulin group 0.5 mg / mL tea polysaccharide 1 mg / mL tea polysaccharide 2 mg / mL tea polysaccharide 4 mg / mL tea polysaccharide 0h 7.809 7.917 7.955 7.888 7.912 8.197 12h 5.346 5.361 5.583 5.679 5.456 6.616 24h 3.359 4.512 4.311 3.009 4.748 2.358 48h 3.240 1.655 3.146 2.462 2.644 1.775

[0188] Table 9 Dynamic changes in reducing sugars during the fermentation of tea polysaccharides by the intestinal flora of db / m mice at different time points

[0189] Normal control group Inulin group 0.5 mg / mL tea polysaccharide 1 mg / mL tea polysaccharide 2 mg / mL tea polysaccharide 4 mg / mL tea polysaccharide 0h ― ― ― ― ― ― 12h ― ― ― ― ― ― 24h ― ― ― ― ― ― 48h 1.368 1.400 1.414 1.420 1.383 1.486

[0190] Table 10 Dynamic changes in reducing sugars during the fermentation of tea polysaccharides by the intestinal flora of db / db mice at different time points

[0191] Negative control group Inulin group 0.5 mg / mL tea polysaccharide 1 mg / mL tea polysaccharide 2 mg / mL tea polysaccharide 4 mg / mL tea polysaccharide 0h ― ― ― ― ― ― 12h ― ― ― ― ― ― 24h ― ― ― ― ― ― 48h 1.326 1.502 1.412 1.403 1.508 1.639

[0192] 4.5.2 Changes in metabolites after the glycolysis of tea polysaccharides by intestinal flora

[0193] The metabolites of the intestinal flora fermentation of mice in different experimental groups were detected by GC-MS, as shown in Figure 12 and Figure 13 .

[0194] By comparing with the NIST 20 mass spectrometry database (NIST / EPA / NIH Mass Spectral Library), in db / m mice, compounds such as 2,3-butanediol, tridecane, tetradecane, pentadecane, hexadecane, 3-tert-butyl-6-octen-1-ol, and heptyl hexacosyl ether were produced in all 5 experimental groups. However, indole and 4-tert-butylcyclohexanone were also produced in the tea polysaccharide sample groups at 2.0 mg / mL and 4.0 mg / mL. In db / db mice, compounds such as tridecane, tetradecane, pentadecane, hexadecane, n-decanol, 3-tert-butyl-6-octen-1-ol, and heptyl hexacosyl ether were produced in all 5 experimental groups. However, 4-tert-butylcyclohexanone was also produced in the inulin group and the 4.0 mg / mL polysaccharide sample group, and 2,3-butanediol was also produced in the 1.0 mg / mL tea polysaccharide sample group. And dodecamethyldihydrohexasiloxane was produced in the negative control group, which is an environmental pollutant and was not found in other groups.

[0195] Currently, there is still a lack of relevant research data on the specific effects and functions of compounds such as 4-tert-butylcyclohexanone and heptyl hexacosyl ether on the human body. However, indole produced in the tea polysaccharide sample group has functions such as maintaining blood glucose stability, antibacterial and anti-inflammatory effects, and assisting in reducing cholesterol. It is the main bacterial metabolite of tryptophan produced by various Bacteroides and Enterobacteriaceae bacteria, and can down-regulate the expression of pro-inflammatory cytokines and enhance the intestinal barrier. In recent years, with the gradual in-depth study of tryptophan microbial metabolites, researchers have also found that indole and its derivatives are essential signaling molecules in the microbiota-gut-brain axis and can affect brain function and behavior. Tryptophan metabolites have been identified as potent biomarkers for type 2 diabetes complications. Cohort studies have shown that the reduction of aryl hydrocarbon receptor (AHR) ligand production by the gut microbiota is a key factor in the pathogenesis of metabolic syndromes such as obesity and diabetes. These ligands include tryptophan metabolites such as indole (Ind), indole-3-acrylic acid (IA), indole-3-propionic acid (IPA), indole-3-acetic acid (IAA), and indole-3-aldehyde (IAld). The loss of the protective role of AHR in promoting repair, inhibiting inflammation, and maintaining intestinal homeostasis leads to this pathogenesis. In addition, 2,3-butanediol produced in the tea polysaccharide sample group is an important energy source and platform compound, and many microorganisms such as Enterobacter, Bacillus, and Serratia can be used to produce 2,3-butanediol. 2,3-butanediol has effects such as moisturizing, hydrating, antibacterial, and anti-inflammatory on the human body. There is also research showing that gastrointestinal microbiota contribute to the phenotypic characteristics of reduced serum cholesterol in Fmo5- / - mice, and 2,3-butanediol is identified as a potential drug for reducing plasma cholesterol. [1] In summary, after the tea polysaccharide is fermented by the intestinal flora of db / m and db / db mice, important metabolites such as indole and 2,3-butanediol with functions for the human body can be produced.

[0196] References:

[0197] [1]Sunil Veeravalli,Dorsa Varshavi,Flora H Scott,Dorna Varshavi,FrankS Pullen,Kirill Veselkov,Ian R Phillips,Jeremy R Everett,Elizabeth AShephard.Treatment of wild-type mice with2,3-butanediol,a urinary biomarkerof Fmo5- / -mice,decreases plasma cholesterol and epididymal fat deposition[J].Front Physiol.,2022,13:859681.doi:10.3389 / fphys.2022.859681.

[0198] Comparative Example 1: Direct hot water extraction

[0199] The coarse and old tea leaves were placed in an oven and dried at a low temperature (60 °C) until the moisture content was below 9.0%, then crushed and sieved to obtain coarse and old tea powder with a mesh size > 150 mesh. 2 g of the coarse and old tea powder was added to 110 mL of deionized water according to a solid-liquid ratio of 1 g:55 mL, placed in a constant temperature oscillator and oscillated. The oscillator parameters were adjusted to 100 °C and 200 rpm, and reciprocating rotary oscillation extraction was carried out for 100 min. Centrifugation was carried out at 8000 r / min for 20 min, 1.0 mL of the supernatant was taken, and the polysaccharide content in the supernatant was detected by the phenol-sulfuric acid method and converted into the extraction yield. The results are shown in Table 11.

[0200] Table 11 Effects of different extraction processes on the extraction yield of tea polysaccharides

[0201]

[0202] As can be seen from Table 11, different extraction processes have a great influence on the extraction yield of tea polysaccharides. The extraction process of composite enzyme synergistic hydrogen peroxide can significantly improve the extraction yield of tea polysaccharides, which is 3.371 times higher than that of tea polysaccharides extracted by traditional hot water.

[0203] Comparative Example 2: Effect of the mesh size of coarse and old tea powder on the yield of tea polysaccharides

[0204] In Method 3.1 of Example 1, the tea powder was changed to be sieved through a 20-mesh coarse sieve, and other operations were the same as the optimal conditions of Method 3.1, 3.2, 3.3, and 3.4 in Example 1. The detection and calculation method of the extraction yield of tea polysaccharides was the same as that in Example 1. The results are shown in Table 12.

[0205] Table 12 Influence of Different Particle Sizes of Tea Powder on the Extraction Yield of Tea Polysaccharides

[0206]

[0207] As can be seen from Table 12, the particle size of the ground tea has a great influence on the extraction yield of tea polysaccharides. The pretreatment method of passing through a 200-mesh sieve after grinding can increase the extraction yield of tea polysaccharides by 1.472 times compared with the coarse powder obtained by traditional mechanical grinding.

[0208] Comparative Example 3 Influence of Pretreatment on the Yield of Tea Polysaccharides

[0209] Using the method 3.1, and the optimal conditions of 3.3 and 3.4 in Example 1, omitting 3.2, the detection and calculation method of the extraction yield of tea polysaccharides is the same as that in Example 1, and the results are shown in Table 13.

[0210] Table 13 Influence of Different Pretreatments on the Extraction Yield of Tea Polysaccharides

[0211]

[0212] As can be seen from Table 13, the pretreatment method of the raw tea powder has a great influence on the extraction yield of tea polysaccharides. The pretreatment method with DGRA solvent can increase the extraction yield of tea polysaccharides by 1.341 times compared with the method without pretreatment. At the same time, the pumpkin yellow pigment prepared by DGRA solvent pretreatment can be used as a natural colorant for food additives and as a functional pigment for health foods or wellness foods.

[0213] Comparative Example 4 Influence of Hydrogen Peroxide Synergistic Extraction on the Yield of Tea Polysaccharides

[0214] Using the optimal conditions of 3.1, 3.2 and 3.3 in the method of Example 1, omitting 3.4, the detection and calculation method of the extraction yield of tea polysaccharides is the same as that in Example 1, and the results are shown in Table 14.

[0215] Table 14 Influence of Different Extraction Processes on the Extraction Yield of Tea Polysaccharides

[0216]

[0217] As can be seen from Table 14, the extraction process of tea has a great influence on the extraction yield of tea polysaccharides. The extraction method of composite enzyme synergistic hydrogen peroxide can increase the extraction yield of tea polysaccharides by 2.912 times compared with the extraction without hydrogen peroxide synergistic extraction.

[0218] Comparative Example 5 Influence of Composite Enzyme Extraction on the Yield of Tea Polysaccharides

[0219] Using the optimal conditions of 3.1, 3.2 and 3.4 in the method of Example 1, omitting 3.3, the detection and calculation method of the extraction yield of tea polysaccharides is the same as that in Example 1, and the results are shown in Table 15.

[0220] Table 15 Influence of Different Extraction Processes on the Extraction Yield of Tea Polysaccharides

[0221]

[0222] As can be seen from Table 15, the extraction process of tea has a great influence on the extraction yield of tea polysaccharides. The extraction method of combined enzymes synergistic with hydrogen peroxide can increase the extraction yield of tea polysaccharides by 1.673 times compared with the extraction without combined enzyme synergism.

[0223] Comparative Example 6 Influence of Enzymes on the Polysaccharide Yield

[0224] Adopt 3.1 and 3.2 in the method of Example 1, change the cellulase - papain under the optimal conditions in 3.3 to an equal amount of cellulase, 3.4 optimal conditions, and the detection and calculation methods of the extraction yield of tea polysaccharides are the same as those in Example 1. The results are shown in Table 16.

[0225] Table 16 Influence of Different Extraction Processes on the Extraction Yield of Tea Polysaccharides

[0226]

[0227] As can be seen from Table 16, the extraction process of tea has a great influence on the extraction yield of tea polysaccharides. The extraction method of combined enzymes synergistic with hydrogen peroxide can increase the extraction yield of tea polysaccharides by 1.196 times compared with the extraction with single cellulase synergistic with hydrogen peroxide.

[0228] Comparative Example 7 Influence of Enzymes on the Polysaccharide Yield

[0229] Adopt 3.1 and 3.2 in the method of Example 1, change the cellulase - papain under the optimal conditions in 3.3 to an equal amount of papain, 3.4 optimal conditions, and the detection and calculation methods of the extraction yield of tea polysaccharides are the same as those in Example 1. The results are shown in Table 17.

[0230] Table 17 Influence of Different Extraction Processes on the Extraction Yield of Tea Polysaccharides

[0231]

[0232] As can be seen from Table 17, the extraction process of tea has a great influence on the extraction yield of tea polysaccharides. The extraction method of combined enzymes synergistic with hydrogen peroxide can increase the extraction yield of tea polysaccharides by 1.307 times compared with the extraction with single papain synergistic with hydrogen peroxide.

[0233] Example 2

[0234] (1) Place the coarse and old tea leaves in an oven and dry them at a low temperature (60 °C) until the moisture content is below 9.0%, then crush them through a 150 - mesh sieve to obtain coarse and old tea leaf powder with a mesh number > 150.

[0235] (2) Preparation of DGRA solvent: Mix n-caprylic acid and n-decanoic acid in a molar ratio of 3:1, and magnetically stir at a constant temperature of 75 - 85 °C for 2 h until the solution becomes clear. Then, mix the total volume of n-caprylic acid and n-decanoic acid with absolute ethanol in a volume ratio of 2:5 and mix well to obtain the DGRA solvent.

[0236] Take 2 g of the coarse and old tea powder prepared in step (1), add 6 mL of the DGRA solvent, stir and heat-extract at 55 °C for 2 h, then centrifuge at 8000 r / min for 15 min. The filter residue is then refluxed with 70% ethanol by volume for 2 h, filtered, the filter cake is dried in the sun and then crushed, and passed through a 200-mesh sieve to obtain 1.25 g of the pretreated coarse and old tea powder.

[0237] (3) Add 1 g of the pretreated coarse and old tea powder to 0.015 g of a composite enzyme of cellulase and papain (mass ratio of 5:2, and the addition amount of the composite enzyme accounts for 1.5% of the mass of the pretreated coarse and old tea powder), add 25 mL of deionized water, with a solid-liquid ratio of 1:25 g / mL, adjust the pH to 4.5 with citric acid, and enzymatically hydrolyze at 55 °C for 60 min. After the enzymatic hydrolysis is completed, it is placed in a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea slurry.

[0238] (4) For all the tea slurry in step (3), add 1.65 mL of hydrogen peroxide with a mass concentration of 30% and 28.35 mL of deionized water according to a liquid-solid ratio of 30:1 (mL / g) of the pretreated coarse and old tea powder, so that the final mass concentration of the added hydrogen peroxide is 0.9%, pH 4.5. Place it in a constant temperature oscillator and oscillate, adjust the oscillator parameters to 100 °C and 200 rpm, and reciprocally oscillate for 25 min; take out the mixed solution and centrifuge (centrifuge at 8000 r / min for 20 min) to obtain the supernatant. Take 1.0 mL of the supernatant and detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and calculate the extraction yield according to Example 1, which is 23.213%.

[0239] Example 3

[0240] (1) Place the coarse and old tea leaves in an oven and dry at a low temperature (60 °C) until the moisture content is below 9.0%, then crush and pass through a 150-mesh sieve to obtain coarse and old tea powder with a mesh number > 150.

[0241] (2) Preparation of DGRA solvent: Mix n-caprylic acid and n-decanoic acid in a molar ratio of 2:1, and magnetically stir at a constant temperature of 75 - 85 °C for 2 h until the solution becomes clear. Then, mix the total volume of n-caprylic acid and n-decanoic acid with absolute ethanol in a volume ratio of 2:7 and mix well to obtain the DGRA solvent.

[0242] Take 2 g of the coarse and old tea leaf powder prepared in step (1), add 6 mL of DGRA solvent, stir and heat-extract at 55 °C for 2.5 h, centrifuge at 8000 r / min for 15 min, reflux the filter residue with 70% ethanol by volume for 1.5 h, filter, dry the filter cake and then crush it, and pass through a 200-mesh sieve to obtain 1.25 g of the pre-treated powder of coarse and old tea.

[0243] (3) Add 1 g of the pre-treated powder of coarse and old tea to 0.015 g of the composite enzyme of cellulase - papain (mass ratio is 7:2, and the addition amount of the composite enzyme accounts for 1.5% of the mass of the pre-treated powder of coarse and old tea), add 45 mL of deionized water, the solid-liquid ratio is 1:45 g / mL, adjust the pH to 4.5 with citric acid, enzymolyze at 45 °C for 100 min, after the enzymolysis is completed, put it into a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea slurry.

[0244] (4) For all the tea slurry in step (3), add 2.2 mL of hydrogen peroxide with a mass concentration of 30% and 7.8 mL of deionized water according to the liquid-solid ratio of 10:1 (mL / g) of the pre-treated powder of coarse and old tea, so that the final mass concentration of the added hydrogen peroxide is 1.2%, pH 4.5, put it into a constant temperature oscillator and oscillate, adjust the oscillator parameters to 100 °C, 200 rpm, and reciprocally oscillate for 15 min; take out the mixed solution, centrifuge (centrifuge at 8000 r / min for 20 min) to obtain the supernatant. Take 1.0 mL of the supernatant, detect the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and convert it into the extraction yield according to Example 1, which is 23.976%.

[0245] Example 4

[0246] (1) Place the coarse and old tea leaves in an oven and dry them at a low temperature (60 °C) until the moisture is below 9.0%, crush and pass through a 150-mesh sieve to obtain coarse and old tea leaf powder with a mesh number > 150.

[0247] (2) Prepare DGRA solvent: Mix n-caprylic acid and n-decanoic acid according to a molar ratio of 5:2, stir magnetically at a constant temperature of 75 - 85 °C for 2 h until the solution is clear, and then mix well with anhydrous ethanol according to a volume ratio of 1:3 based on the total volume of n-caprylic acid and n-decanoic acid to prepare DGRA solvent.

[0248] Take 2 g of the coarse and old tea leaf powder prepared in step (1), add 6 mL of DGRA solvent, stir and heat-extract at 55 °C for 1.5 h, centrifuge at 8000 r / min for 15 min, reflux the filter residue with 70% ethanol by volume for 2.5 h, filter, dry the filter cake and then crush it, and pass through a 200-mesh sieve to obtain 1.25 g of the pre-treated powder of coarse and old tea.

[0249] (3) Add 1 g of pre-treated coarse and old tea powder to 0.021 g of a composite enzyme of cellulase - papain (mass ratio 5:2, and the addition amount of the composite enzyme accounts for 2.1% of the mass of the pre-treated coarse and old tea powder), add 35 mL of deionized water, with a solid - liquid ratio of 1:35 g / mL, adjust the pH to 4.5 with citric acid, enzymatically hydrolyze at 65 °C for 80 min. After the enzymatic hydrolysis is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea slurry.

[0250] (4) For all the tea slurry in step (3), add 2.75 mL of hydrogen peroxide with a mass concentration of 30% and 17.25 mL of deionized water according to a liquid - solid ratio of 20:1 (mL / g) based on the pre-treated coarse and old tea powder, so that the final mass concentration of added hydrogen peroxide is 1.5%, pH 4.5. Place it in a constant - temperature oscillator and oscillate. Adjust the oscillator parameters to 100 °C and 200 rpm, and reciprocally oscillate for 5 min; take out the mixed solution, centrifuge (centrifuge at 8000 r / min for 20 min) to obtain the supernatant. Take 1.0 mL of the supernatant, and use the phenol - sulfuric acid method to detect the polysaccharide content in the supernatant. Calculate the extraction yield according to Example 1, which is 23.521%.

[0251] Example 5

[0252] (1) Place the coarse and old tea leaves in an oven and dry them at a low temperature (60 °C) until the moisture content is below 9.0%, then crush them and pass through a 150 - mesh sieve to obtain coarse and old tea powder with a mesh number > 150.

[0253] (2) Prepare the DGRA solvent: Mix n - octanoic acid and n - decanoic acid according to a molar ratio of 3:1, magnetically stir at a constant temperature of 75 - 85 °C for 2 h until the solution is clear, and then fully mix the total volume of n - octanoic acid and n - decanoic acid with absolute ethanol according to a volume ratio of 2:5 to prepare the DGRA solvent.

[0254] Take 2 g of the coarse and old tea powder prepared in step (1), add 6 mL of the DGRA solvent, stir and heat - soak at 55 °C for 1.5 h, then centrifuge at 8000 r / min for 15 min. The filter residue is then reflux - treated with 70% ethanol by volume for 2.5 h, filtered, the filter cake is dried in the sun and then crushed, and passed through a 200 - mesh sieve to obtain 1.25 g of pre - treated coarse and old tea powder.

[0255] (3) Add 1 g of the pre - treated coarse and old tea powder to 0.018 g of a composite enzyme of cellulase - papain (mass ratio 3:1, and the addition amount of the composite enzyme accounts for 1.8% of the mass of the pre - treated coarse and old tea powder), add 25 mL of deionized water, with a solid - liquid ratio of 1:25 g / mL, adjust the pH to 4.5 with citric acid, enzymatically hydrolyze at 55 °C for 60 min. After the enzymatic hydrolysis is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme for 3 min to obtain tea slurry.

[0256] (4) All the tea slurry in step (3) was added with 2.2 mL of hydrogen peroxide with a mass concentration of 30% and 27.8 mL of deionized water at a liquid-to-solid ratio of 30:1 (mL / g) of the coarse and old tea pretreatment powder, so that the final mass concentration of the added hydrogen peroxide was 1.2%, pH 4.5. It was placed in a constant temperature oscillator and oscillated. The oscillator parameters were adjusted to 100 °C and 200 rpm, and reciprocating oscillation was carried out for 15 min; the mixed solution was taken out and centrifuged (centrifuged at 8000 r / min for 20 min) to obtain the supernatant. Take 1.0 mL of the supernatant and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant. The extraction yield was calculated according to Example 1 and was 24.163%.

[0257] The tea polysaccharides prepared in Examples 1-5 were detected for mouse glycolipid metabolism and intestinal flora metabolites by the method of Example 1. The results showed that important metabolites such as indole and 2,3-butanediol with functions for the human body could be produced.

Claims

1. A functional tea polysaccharide, characterized in that, The tea polysaccharide is prepared by the following method: (1) Pretreatment: Take coarse and old tea leaf powder, and perform stirring heat extraction treatment with DGRA solvent at 50 - 55°C for 1.5 - 2.5 h, then centrifuge. The filter residue is further subjected to reflux treatment with 70% ethanol by volume for 1 - 3 h, filtered, and the filter cake is dried in the sun and then pulverized to obtain the pretreated powder of coarse and old tea. The DGRA solvent is an anhydrous ethanol solution of n - octanoic acid and n - decanoic acid; (2) Composite enzymatic hydrolysis extraction: Add a composite enzyme of cellulase - papain to the pretreated powder of coarse and old tea, add deionized water, adjust the pH to 4.5 - 6.5 with citric acid, and perform enzymatic hydrolysis at 45 - 65°C for 60 - 100 min. After the enzymatic hydrolysis is completed, it is placed in a boiling water bath at 100°C to inactivate the enzyme for 3 min to obtain tea pulp; (3) Hydrogen peroxide - assisted extraction: Add a solution of hydrogen peroxide with a mass concentration of 30% and deionized water to the tea pulp in step (2), mix well, and perform constant - temperature water - bath extraction at 60 - 100°C and 200 rpm for 5 - 25 min; Take out the mixed solution and centrifuge to obtain the supernatant. The supernatant is concentrated, precipitated with alcohol, and de - proteinized to obtain the tea polysaccharide.

2. The tea polysaccharide according to claim 1, wherein The coarse and old tea leaf powder in step (1) is prepared by the following method: Place the commercially available coarse and old tea leaves in an oven and dry them at a low temperature until the moisture content is below 9%, put them into a pulverizer to pulverize, and sieve to obtain coarse and old tea leaf powder with a mesh number > 150 meshes.

3. The tea polysaccharide according to claim 1, characterized in that, In step (1), the molar ratio of n - octanoic acid to n - decanoic acid is 2 - 3:1, and the total volume ratio of n - octanoic acid and n - decanoic acid to anhydrous ethanol is 1:2.5 - 3.

5.

4. The tea polysaccharide according to claim 1, wherein In step (2), the composite enzyme of cellulase - papain is composed of cellulase and papain mixed in a mass ratio of 2 - 4:1, and the addition amount of the composite enzyme of cellulase - papain is 1.5 - 2.5% based on the mass of the pretreated powder of coarse and old tea.

5. The tea polysaccharide according to claim 1, wherein In step (2), the volume of deionized water used is 25 - 45 mL / g based on the mass of the pretreated powder of coarse and old tea; The enzymatic hydrolysis is carried out at 55°C for 80 min.

6. The tea polysaccharide according to claim 1, wherein In step (3), the addition amount of the solution of 30% hydrogen peroxide and deionized water makes the final mass concentration of hydrogen peroxide 0.9% - 2.1%.

7. The tea polysaccharide according to claim 1, characterized in that, The methods for concentrating and precipitating the supernatant with alcohol are as follows: Rotate - evaporate the supernatant to one - third of its original volume, cool it to 4°C to obtain the concentrated solution; Slowly add 95% ethanol with a volume 4 times that of the concentrated solution while stirring, place it in a refrigerator at 4°C for alcohol precipitation for 12 h, then centrifuge. Take the precipitate, add deionized water, add one - quarter volume of Sevag reagent to the precipitate and deionized water for de - proteinization, shake well, let it stand and layer, take the upper layer and repeat the above de - proteinization operation until there is no absorption peak at 280 nm in the upper layer; Rotate - evaporate the supernatant after the last de - proteinization at 50°C to 25 - 35% of its original volume, and dry it at 60°C to obtain the tea polysaccharide.

8. Use of the tea polysaccharide according to claim 1 in the preparation of weight - loss products.

9. Use of the tea polysaccharide according to claim 1 in the preparation of preparations for improving glycolipid metabolism disorders.

10. Use of the tea polysaccharide according to claim 1 in the preparation of intestinal flora regulators.

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