Functional tea polysaccharide, and preparation method and application thereof

Abstract

The application discloses a functional tea polysaccharide as well as a preparation method and application thereof. The tea polysaccharide is prepared by using coarse old tea leaves as raw materials, and the raw materials are pretreated by supermicronization and dewaxing and small molecule impurities, and then are extracted by adopting cellulase-papain compound wall breaking, and further treated by adopting hydrogen peroxide, so that the tea polysaccharide with high yield and strong biological activity is obtained. The tea polysaccharide extracted by the method can slow down the weight increase of obese C57BL / 6J mice caused by high-fat diet, improve glycolipid metabolism disorder, and make db / m mice intestinal flora metabolize to produce new functions of important biomarkers such as indole, so that the high-quality reuse of coarse old tea leaves is realized.

Description

(I) TECHNICAL FIELD

[0001] The present application relates to a functional tea polysaccharide, a preparation method and application thereof. (II) BACKGROUND

[0002] Tea is a popular beverage, widely consumed globally and consumption is increasing year by year. A large amount of tea fresh leaves in late spring and summer and autumn are abandoned and not effectively utilized, which has become a major problem restricting the sustainable development of the tea industry. Summer and autumn tea fresh leaves have heavy bitter taste and low resource utilization rate; the utilization approaches are few and the product benefits are low. It is urgent to improve the utilization rate of abandoned tea resources such as summer and autumn tea, so as to change the low-value abandoned tea resources into tea leaves and deep processing products with high added value. A large amount of insoluble components such as cellulose, hemicellulose and lignin are contained in summer and autumn abandoned tea leaves, if soluble bioactive polysaccharides can be extracted from them, the technical bottleneck of difficult utilization of abandoned tea resources can be broken through, and efficient and high-value utilization of abandoned tea resources can be realized, which lays a solid foundation for improving the overall efficiency of the tea industry.

[0003] Due to the global health burden caused by metabolic diseases and the trend of younger incidence rate, treatment strategies have attracted extensive attention, but current treatment methods cannot control metabolic disorders and pathological consequences, which requires novel treatment strategies. Natural compounds extracted from plant resources have attracted extensive attention to discover new and effective candidates to prevent metabolic disorders with less side effects and multiple targets. Natural polysaccharides extracted from tea leaves have good biocompatibility and safety and diverse biological functions, which have attracted the interest of researchers in recent years, and it is found that they have the functions of regulating blood glucose level, 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 polysaccharide in coarse old tea is much higher than that in tender tea leaves. In China and Japan, there is an experience of treating diabetes by frequently drinking coarse old tea. However, it is difficult to fully dissolve polysaccharide by using conventional methods because the cell wall of coarse old tea is tough and the tea leaves are waxy. The extraction of natural polysaccharide first needs to break the cell wall. The breaking of the cell wall is an important process for preparing polysaccharide from natural resources. The breaking of the cell wall has been a bottleneck for the production of polysaccharide and is extremely challenging. The structure of the cell wall of tea leaves becomes a protective layer and a barrier. Whether the cell wall is broken will directly affect the yield and function of polysaccharide. The biological activity of polysaccharide is closely related to the primary structure and spatial conformation of polysaccharide. Once the structural characteristics of polysaccharide are destroyed, the biological activity of polysaccharide will be significantly reduced or even lost. Therefore, it is urgent to develop a method for extracting polysaccharide from tea leaves, which does not destroy the integrity of the "active fragment" of polysaccharide, has high yield, does not introduce impurities, is pollution-free, has low requirement for equipment and low energy consumption. The extraction methods of polysaccharide usually include mechanical method, physical method, chemical method and biological enzyme method. The mechanical method has problems such as high requirement for equipment and high energy consumption. The physical method is simple and easy to operate, but needs a large temperature difference and is only suitable for laboratory operation. The chemical method easily introduces impurities and has problems such as safety and environmental pollution. It is a subject worth exploring to explore which method can more effectively maintain the biological activity of tea polysaccharide and how the extraction process affects the structure of tea polysaccharide.

[0005] In recent years, biological enzymes have attracted widespread attention due to the characteristics that they can precisely and efficiently break the cell wall at specific sites on the cell wall of tea leaves under relatively matched conditions. Because different types of enzymes have different breaking sites, the breaking effect has certain differences. Two or more enzymes can achieve better breaking effect.

[0006] Therefore, it is necessary to find a method for efficiently extracting new biologically active extract from coarse old tea leaves. (Three) Contents of the Invention

[0007] The purpose of the present application is to provide a functional tea polysaccharide and a preparation method and application thereof. The present application uses coarse old tea leaves as raw materials, and after ultra-fine and dewaxing and pretreatment of small molecule impurities, firstly, cellulase-papain compound is used for breaking the cell wall extraction, and the optimal parameters of biological compound enzyme treatment are optimized by single factor experiment and Box-Behnken experiment design with tea polysaccharide dissolution rate as response value. Further, hydrogen peroxide is used for treatment, and the optimal parameters are obtained by single factor experiment and Box-Behnken experiment design optimization, so as to obtain tea polysaccharide with high yield and strong biological activity. The tea polysaccharide extracted by the method of the present application can slow down the increase of body weight of obese C57BL / 6J mice caused by high-fat diet and improve the disorder of sugar and lipid metabolism, and realize the new function of intestinal flora metabolism of db / m mice to produce important biomarkers such as indole, so as to realize the high-quality reuse of coarse old tea leaves.

[0008] The technical scheme adopted by the present application is:

[0009] The application provides a kind of functional tea polysaccharide, the tea polysaccharide is prepared as follows:

[0010] (1) pretreatment: take coarse old tea powder, adopt DGRA solvent and stir hot dipping treatment 1.5-2.5 h under the condition of 50-55 ℃, centrifugal (preferably 8000 r / min centrifugal 15 min), filter residue is treated again with 70% ethanol reflux 1-3 h, filter, remove tea cell outer surrounding lipid, small molecule impurities, alkaloids, tea polyphenols and other substances, then the filter cake is dried and crushed (preferably pass 200 mesh screen), obtain coarse old tea pretreatment powder; the DGRA solvent is n-octanoic acid and n-decanoic acid anhydrous ethanol solution;

[0011] (2) composite enzymolysis extraction: add cellulase-papain composite enzyme to coarse old tea pretreatment powder, add deionized water, adjust pH 4.5-6.5 (preferably 5.0) with citric acid, enzymolysis under the condition of temperature 45-65 ℃ for 60-100 min, after enzymolysis, put into 100 ℃ boiling water bath for 3 min to kill enzyme, get tea slurry;

[0012] (3) hydrogen peroxide synergistic extraction: add 30% hydrogen peroxide and deionized water solution to step (2) tea slurry, mix, constant temperature water bath extraction under the condition of 60-100 ℃, 200 rpm for 5-25 min; take out the mixed solution, centrifugal (8000 r / min centrifugal 20 min), obtain supernatant; the supernatant is concentrated, alcohol precipitation, deproteinization, obtain tea polysaccharide.

[0013] Preferably, step (1) coarse old tea powder is prepared as follows: put the commercially available coarse old tea leaves in the oven and dry at low temperature (preferably 60 ℃) until the moisture content is below 9%, put into a pulverizer and crush, sieve, get coarse old tea powder with mesh number >150.

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

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

[0016] Preferably, the volume of deionized water in step (2) is 25-45 mL / g based on the mass of coarse old tea pretreatment powder, preferably 35 mL / g; the enzymolysis is preferably carried out at a temperature of 55 ℃ for 80 min.

[0017] Preferably, the solution of 30% hydrogen peroxide and deionized water is added in an amount such that the final concentration of hydrogen peroxide is 0.9%-2.1%, preferably 1.5%.

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

[0019] Preferably, the method for concentrating and alcohol precipitating the supernatant is as follows: the supernatant is concentrated by rotary evaporation to one-third of the original volume, cooled to 4℃, and a concentrated solution is obtained; 95% ethanol is slowly added to the concentrated solution in an amount of 4 times the volume of the concentrated solution while stirring, and the solution is placed in a refrigerator at 4℃ for alcohol precipitation for 12 h; then the solution is centrifuged (preferably at 3000 r / min for 5 min in a centrifuge), and the precipitate is taken and deproteinized by the Sevag method using deionized water to obtain tea polysaccharides.

[0020] Preferably, the deproteinization step is as follows: Sevag reagent (chloroform:n-butanol=4:1, v:v) is added to the precipitate and deionized water in an amount of one-fourth of the volume of the precipitate, and the solution is shaken thoroughly, then allowed to stand to separate into layers, and the upper layer is repeatedly subjected to the above deproteinization operation until there is no absorption peak at 280 nm in the upper layer; and the supernatant after the last deproteinization operation is rotary evaporated at 50℃ to 25-35% of the original volume, and dried at 60℃ to obtain tea polysaccharides.

[0021] The present application also provides a use of the tea polysaccharides 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 application also provides a use of the tea polysaccharides in the preparation of a preparation for improving sugar and lipid metabolism disorders, and the preparation can improve blood lipid abnormalities caused by a high-fat diet, slow down the increase in blood glucose, and reduce glycogen.

[0023] The present application also provides a use of tea polysaccharides in the preparation of an intestinal flora modulator, and the modulator can improve the intestinal flora imbalance state of diabetic mice and promote the proliferation of more beneficial bacteria.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] 1. The present application adopts the pretreatment method of pulverization and DGRA solvent method, and the wall-breaking-extraction method of composite biological enzyme-hydrogen peroxide to extract tea polysaccharide. The process is green and environmentally friendly, easy to operate, and the polysaccharide dissolution rate can be as high as 25% or more. Compared with traditional hot water extraction of tea polysaccharide, it is improved by 3.371 times, compared with traditional mechanical crushing of coarse powder, it is improved by 1.472 times, compared with DGRA solvent pretreatment, it is improved by 1.341 times, compared with hydrogen peroxide extraction, it is improved by 2.912 times, compared with composite enzyme extraction, it is improved by 1.673 times, compared with single cellulase extraction with hydrogen peroxide, it is improved by 1.196 times, and compared with single papain extraction with hydrogen peroxide, it is improved by 1.307 times.

[0026] 2. For the large amount of wax components in coarse old tea leaves, as well as cellulose, hemicellulose, lignin and other structure dense insoluble dietary fibers, there are defects in physical and chemical properties and functional characteristics. The conventional method of extracting tea polysaccharide has problems such as high energy consumption, insufficient wall breaking, local high temperature, etc. The present application first removes the lipid surrounding the extracellular lipid of coarse old tea leaves, small molecule impurities, alkaloids, tea polyphenols and other substances by the pretreatment method of pulverization combined with DGRA-ethanol solvent method. Further, cellulase and papain as different types of biological enzymes can more effectively break the wall-extracting at different tea leaf wall breaking sites. Hydrogen peroxide is a clean and efficient oxidant and a typical environmental protection agent, and is also one of the most commonly used reagents for generating ·OH. Based on this, on the basis of composite biological enzyme (optimal action pH 5.0, citric acid adjustment) wall-breaking-extraction, hydrogen peroxide can generate strong ·OH under the action of citric acid, which can further effectively break the wall and has the advantages of unique decolorization, etc. Combined with the design method of BBD and the cell wall breaking morphology of SEM scanning electron microscope, the controllable breaking effect of cell wall is fully guaranteed, forming a loose and porous state, which is beneficial to the effective dissolution of tea polysaccharide. The wall-breaking-extraction process does not damage the integrity of the "active fragment" of polysaccharide, has high extraction rate, and has many advantages such as no introduction of impurities, no pollution, low requirement for equipment and low energy consumption.

[0027] The tea polysaccharide extracted by the method of composite biological enzyme combined with hydrogen peroxide has no odor and good sensory properties, and does not need to be decolorized. The low concentration of hydrogen peroxide used in the operation is easy to decompose into water and oxygen after the operation is completed, and will not leave any residues in the whole unit operation process.

[0028] 3. The tea polysaccharide prepared by the method of the present application has the new function of slowing down the weight gain of high-fat diet induced obese mice and improving glucose and lipid metabolism disorder, and making db / m mice intestinal flora metabolize important biomarkers such as indole (indole can inhibit inflammation and maintain intestinal homeostasis). (Four) Description of Drawings

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

[0030] Figure 2 , Single-factor trajectory chart of complex enzyme optimization.

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

[0032] Figure 4 , Single-factor trajectory chart of hydrogen peroxide optimization.

[0033] Figure 5 , Scanning electron microscope images of the surface characteristics of raw tea powder (Note: From left to right, the magnification is 2.00 kx and 5.00 kx, respectively).

[0034] Figure 6 , Scanning electron microscope images of the surface characteristics of tea powder after complex enzyme-hydrogen peroxide treatment (Note: From left to right, the magnification is 2.00 kx and 5.00 kx, respectively).

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

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

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

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

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

[0040] Figure 12 , GC-MS detection chart of fecal in vitro fermentation metabolites of db / m mice (From top to bottom, they are the normal 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, and 4.0 mg / mL tea polysaccharide sample group).

[0041] Figure 13 , GC-MS detection chart of fecal in vitro fermentation metabolites of db / db mice (From top to bottom, they are the 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, and 4.0 mg / mL tea polysaccharide sample group). (V) DETAILED DESCRIPTION

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

[0043] The improved phenol-sulfuric acid method described in the embodiments of the application refers to the standard: T / GBC 23-2024 Determination of Crude Polysaccharide Content in Liupu Tea by Spectrophotometry.

[0044] Example 1, optimization of tea polysaccharide extraction process parameters

[0045] 1. Materials and reagents

[0046] Crude old tea leaves (provided by Hubei Caotang Spring Tea Co., Ltd.).

[0047] Hydrogen peroxide, phenol, sulfuric acid, Tween 80, potassium sodium tartrate, sodium sulfite, DNS (3, 5-dinitrosalicylic acid), vitamin K1, hematin chloride, 0.1 mol / L phosphate buffer (pH 6.8), ethanol, chloroform, butanol, etc. are all analytical pure. GAM medium (without glucose and soluble starch) was purchased from Qingdao Haibo Biological Co., Ltd. Inulin was produced by Beijing Tongren Tang. Cellulase (activity 50 U / mg) and papain (10 U / mg) were purchased from Yuan Ye Biological Technology 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) were all purchased from Nanjing Jiancheng Biological Engineering Institute. The water used in the experiment was deionized water.

[0048] C57BL / 6J male mice, db / m mice and db / db mice were purchased from Shanghai Slaek Experimental Animal Responsibility Co., Ltd. License No.: SCXK (Shanghai) 2022-0004.

[0049] 2. Experimental instruments

[0050] JP-250A-2 high-speed multifunctional pulverizer, Shanghai Jiupin Trading Co., Ltd.; XMTD-8222 electric heating air drying oven, Shanghai Jinghong Experimental Instrument Co., Ltd.; JA2003 electronic analytical balance, Shanghai Sunway Hengping Scientific Instrument Co., Ltd.; L535-1 low-speed centrifuge, Hunan Xiangyi Experimental Instrument Development Co., Ltd.; TG16-WS table type high-speed centrifuge, Hunan Xiangyi Centrifuge Instrument Co., Ltd.; UV1800PC ultraviolet visible spectrophotometer, Shanghai Ouli Scientific Instrument Co., Ltd.; SHZ-D (III) circulating water type multi-purpose vacuum pump, Zhengzhou Keli Instrument and Equipment Co., Ltd.; VS-840K-U clean bench, Suzhou Antai Air Technology Co., Ltd.; intelligent biochemical incubator, Ningbo Haishu Saifu Experimental Instrument Factory; Keyence RE-52 rotary evaporator, Shanghai Yalong Biochemical Instrument Factory; Aikang Bluetooth blood glucose meter, Aikang Biotechnology (Hangzhou) Co., Ltd.; HBS-1096C enzyme-labeled analyzer, Nanjing Detong Biological Technology Co., Ltd.

[0051] 3 Experimental method

[0052] 3.1 Preparation of coarse old tea powder

[0053] The coarse old tea was placed in an oven at low temperature (60℃) and dried to a moisture content of less than 9.0%, then crushed through a 150-mesh sieve to obtain coarse old tea powder with a mesh size of >150.

[0054] 3.2 Pretreatment of coarse old tea

[0055] Preparation of DGRA solvent: Mix n-octanoic acid and n-decanoic acid at a molar ratio of 5:2, and stir magnetically at 75-85℃ for 2 h until the solution is clear. Then, mix thoroughly according to the volume ratio of n-octanoic acid and n-decanoic acid to absolute ethanol at 1:3 to obtain the DGRA solvent.

[0056] Take 1000 g of coarse old tea powder prepared according to 3.1, add 3 L of DGRA solvent, and stir at 55℃ for 2 h. Then, centrifuge at 8000 r / min for 15 min. The residue is treated with 70% ethanol aqueous solution by reflux for 1 h, filtered to remove lipids, small molecule impurities, alkaloids, tea polyphenols and other substances outside the tea cell wall. Then, the filter cake is dried and crushed through a 200-mesh sieve to obtain 665 g of coarse old tea pretreatment powder.

[0057] 3.3 Optimization of the process for extracting tea polysaccharides by composite enzyme method

[0058] (1) Temperature

[0059] Take 2 g of the crude old tea leaves pretreated powder prepared in Method 3.2, add 0.03 g of cellulase-papain (mass ratio 3:1) composite enzyme at an addition amount of 1.5% (mass percentage of composite enzyme in crude old tea leaves pretreated powder), add 70 mL of deionized water, liquid-to-material ratio is 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 at 60 min. After the extraction is completed, put it into a boiling water bath at 100°C for 3 min to inactivate the enzyme. Centrifuge at 8000 rpm for 20 min, take 1.0 mL of supernatant, and detect the absorbance value of the supernatant at 480 nm by phenol-sulfuric acid method. According to the standard curve of glucose concentration and absorbance value, calculate the polysaccharide concentration in the supernatant, the standard equation is y = 9.2133x + 0.1298, R 2 = 0.9986, y represents the absorbance value, x represents the polysaccharide concentration, and convert it into the extraction yield according to the following formula (1), the results are shown in Figure 1 .

[0060] The extraction yield of tea polysaccharide is calculated according to the following formula:

[0061] Tea polysaccharide extraction yield (%) = (C x V x f) / m x 100% Formula (1)

[0062] In formula (1), C is the tea polysaccharide concentration (mg / mL) converted by the standard equation; V is the volume of the supernatant (mL); f is the dilution factor; m is the mass of the crude old tea leaves pretreated powder sample (mg).

[0063] (2) pH

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

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

[0066] (4) E / S

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

[0068] (5) Extraction time

[0069] The temperature in step (1) was fixed at 65°C, and the extraction time was controlled at 20, 40, 60, 80, 100 or 120 min, respectively, and the other operations were the same, and the results are shown in Table 2. Figure 1 .

[0070] (6) Response surface analysis test

[0071] On the basis of the above single factor experiment, the three factors (solid-liquid ratio, extraction time, and extraction temperature) that most significantly affected the yield of tea polysaccharide were selected as independent variables, and the yield of tea polysaccharide was used as the response value. Using Design-Expert.V8.0.6 software, a Box-Behnken Design negative (three factors and three levels) was used, and the response surface analysis test was designed according to the principle of combined experiment design, and the factor levels of the response surface experiment are shown in Table 1.

[0072] Table 1 Factor level table for optimization of tea polysaccharide extraction process by composite enzyme method

[0073]

[0074] 3.4 Process optimization of hydrogen peroxide treated tea polysaccharide

[0075] 2 g of the crude old tea pretreated powder prepared by the method of 3.2 was weighed, and cellulase-papain (mass ratio 3:1) composite enzyme was added at an addition amount of 1.5% E / S, deionized water was added, the liquid-solid ratio was 35 mL / g, the pH value of the solution was adjusted to 5.0 with citric acid, the temperature was controlled at 55°C, and the extraction time was controlled at 80 min. After the extraction was completed, the enzyme was inactivated in a 100°C boiling water bath for 3 min to obtain tea slurry.

[0076] The tea slurry was added with hydrogen peroxide with a mass concentration of 30% and deionized water, and temperature, solid-liquid ratio, hydrogen peroxide concentration, and treatment time were used as single factors. The tea polysaccharide yield was used as the response value for single factor experiment in a constant temperature oscillator. The specific method is as follows:

[0077] (1) Liquid-solid ratio: 10:1, 15:1, 20:1, 25:1, and 30:1 (mL / g) liquid-solid ratio was added to the tea slurry prepared from the crude old tea pretreated powder, and the solution of hydrogen peroxide with a mass concentration of 30% and deionized water was added, so that the final mass concentration of hydrogen peroxide was 1.2%, and the pH value was 5.0. The extraction was carried out in a constant temperature oscillator at a temperature of 90°C and a speed of 200 rpm for 10 min, and centrifuged at 8000 rpm for 20 min. 1.0 mL of supernatant was taken, and the polysaccharide content in the supernatant was detected by phenol-sulfuric acid method, and the yield was calculated.

[0078] (2) Treatment time: The liquid-solid ratio of step (1) was fixed at 20:1, and the extraction time was 5 min, 10 min, 15 min, 20 min, and 25 min, respectively. Other steps were the same as step (1).

[0079] (3) Hydrogen peroxide concentration: The liquid-solid ratio of step (1) was fixed at 20:1, and the final concentration of hydrogen peroxide was 0.3%, 0.6%, 0.9%, 1.2%, and 1.5%, respectively. Other steps were the same as step (1).

[0080] (4) Treatment temperature: The liquid-solid ratio of step (1) was fixed at 20:1, and the temperature was 60°C, 70°C, 80°C, 90°C, and 100°C, respectively. Other steps were the same as step (1).

[0081] On the basis of the above single-factor experiment, the three factors (liquid-solid ratio, extraction time, and hydrogen peroxide concentration) that most significantly affected the yield of tea polysaccharide were selected as independent variables, and the yield of tea polysaccharide was used as the response value. Using Design-Expert.V8.0.6 software, a Box-Behnken Design negative (three factors and three levels) was used to design the response surface analysis experiment according to the principle of combination experiment design. The factor levels of the response surface experiment are shown in Table 2.

[0082] Table 2 Factor levels of the experimental design for optimizing the hydrogen peroxide treatment of tea polysaccharide

[0083]

[0084] 3.5 Scanning electron microscopy (SEM) observation of raw tea powder and treated residue

[0085] 2 g of the crude old tea leaf pretreated powder prepared by the method of 3.2 was weighed, 0.03 g of cellulase-papain (mass ratio 3:1) complex enzyme was added at an addition amount of 1.5%, 70 mL of deionized water was added, the liquid-solid ratio was 35 mL / g, the pH value of the solution was adjusted to 5 with citric acid, the temperature was controlled at 55°C, and the extraction time was controlled at 80 min. After extraction, the enzyme was inactivated in a 100°C boiling water bath for 3 min to obtain tea juice.

[0086] The tea polysaccharide 0.2 g was taken and coated on a MC1000 ion sputter, and the microscopic morphology was observed in an itachi SU8010 field emission scanning electron microscope (SEM). Meanwhile, 0.2 g of 3.1 coarse old tea powder (i.e. raw tea powder) was taken for microscopic morphology observation.

[0087] The tea polysaccharide 0.2 g was taken and coated on a MC1000 ion sputter, and the microscopic morphology was observed in an itachi SU8010 field emission scanning electron microscope (SEM). Meanwhile, 0.2 g of 3.1 coarse old tea powder (i.e. raw tea powder) was taken for microscopic morphology observation.

[0088] 3.6 Influence of tea polysaccharide on glycolipid metabolism of obese mice

[0089] 3.6.1 Raising and grouping of mice

[0090] Healthy male C57BL / 6 clean level mice aged 5-6 weeks with uniform body weight were selected. The feeding temperature was 20 ℃ ± 2 ℃, and the relative humidity was 55-60%. After the mice were purchased, they were adaptively fed with free diet and water, and after 1 week, the mice were randomly divided into 4 groups, 5 mice in each group, including a normal control group (standard feed, physiological saline), a negative control group (high-fat feed, physiological saline), a high-dose sample group (high-fat feed, tea polysaccharide 300 mg / kg), and a low-dose sample group (high-fat feed, tea polysaccharide 100 mg / kg). The normal control group of mice was fed with standard feed during the entire experiment, and the rest of the mice were fed with high-fat feed (Research Diets D12492 60 kcal% Fat). The mice were fasted without water for 12 h, weighed and recorded the initial body weight of the mice, and the tail tip was taken for blood and a blood glucose meter was used to determine the initial blood glucose value of the mice. During the tea polysaccharide intervention period, the body weight and fasting blood glucose of the mice were monitored regularly.

[0091] 3.6.2 Biochemical index detection

[0092] (1) Obesity degree

[0093] Obesity degree (%) calculation formula: [(average body weight of high-fat group mice - average body weight of normal diet group mice) / average body weight of normal diet group mice] x 100%.

[0094] (2) Determination of sugar and lipid metabolism related indexes

[0095] After the animal experiment lasted for 15 weeks, the animals were fasted for 12 hours without water, and blood was collected from the orbital plexus vein. The blood was collected in a 1.5 mL centrifuge tube, and the tube was left to stand at room temperature for 30 minutes. The tube was centrifuged at 3500 rpm for 20 minutes at 4°C. The supernatant was collected and stored at -80°C 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 biochemical test kits. The specific operation for index detection was performed according to the instructions of the kit from Nanjing Jiancheng Biological Engineering Institute.

[0096] The liver of the mouse was dissected, and the surface was rinsed with pre-cooled physiological saline. The water was absorbed, and the liver was weighed and placed in a 1.5 mL centrifuge tube. The tube was labeled and quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. The appropriate amount of liver was weighed according to the instructions, and 9 times the weight of pre-cooled physiological saline was added. The liver was ground using a handheld high-speed homogenizer. The supernatant was collected after centrifugation to obtain a 10% liver tissue homogenate. The level of liver glycogen in the liver was detected using a kit from Nanjing Jiancheng Biological Engineering Institute.

[0097] 3.7 Effect of tea polysaccharide on intestinal flora metabolites of diabetic mice

[0098] 3.7.1 Preparation of reagents

[0099] GAM base medium: weigh 49.0 g of GAM medium, dissolve in 1000 mL of deionized water, autoclave at 121°C for 15 minutes, and when the medium cools to about 50°C, add 1 mL of sterile 0.1% vitamin K1 solution and 1 mL of hematin chloride (5 mg / mL) per 1000 mL of medium, mix well, and reserve.

[0100] 0.1% Vitamin K1 solution preparation: weigh 0.01 g of vitamin K1 and 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 mix the vitamin K1 and Tween 80 thoroughly. After adding a small amount of deionized water, stir and shake until the volume reaches 100 mL. A 0.1% vitamin K1 solution is obtained (store at 2-8°C in the dark for up to 1 year).

[0101] 5 mg / mL hematin chloride preparation: 0.5 g of hematin chloride was weighed and added to a certain amount of deionized water, 1 mL of 1 mol / L sodium hydroxide solution was added, and the mixture was stirred and dissolved uniformly, and then water was added to 100 mL to prepare a 5 mg / mL hematin chloride solution (2-8°C, avoid light, and store for 1 year).

[0102] Inulin refers to polysaccharide substances derived from chicory tubers, which are commercially available.

[0103] 3.7.2 Fecal sample collection and in vitro fermentation

[0104] Healthy male db / m mice and db / db mice aged 5-6 weeks with uniform body weight were selected, and fresh fecal samples of the mice were collected. The fecal samples were 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, and 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, and 4.0 mg / mL tea polysaccharide sample group) according to the source.

[0105] The fresh fecal samples of each group were added to sterile 0.1 mol / L phosphate buffer (pH 6.8) to prepare a 10 g / 100 mL fecal suspension, which was used to inoculate the prepared GAM basic medium.

[0106] In the db / m group, 500 μL of fecal suspension was inoculated into 5 mL of GAM basic medium in the normal control group; 500 μL of fecal suspension was inoculated into 5 mL of GAM basic medium containing 0.5 mg / mL inulin in the inulin group; 500 μL of fecal suspension was inoculated into 5 mL of GAM basic medium containing 0.5, 1, 2, and 4 mg / mL tea polysaccharide in 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 medium of each group was first added to an anaerobic tube and sterilized at 115°C for 30 min, and then the fecal suspension was added.

[0107] In the db / db diabetes 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, or 4 mg / mL 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 medium was first added to an anaerobic tube and sterilized at 115°C for 30 min, and then the fecal suspension was added.

[0108] 3.7.3 Total sugar content detection

[0109] Each group was placed in a 37°C anaerobic environment for culture, and the total sugar (phenol-sulfuric acid method) and reducing sugar (DNS colorimetric method) contents were detected at 0 h, 12 h, 24 h, and 48 h of fermentation, respectively.

[0110] DNS colorimetric method for determining reducing sugar content: Prepare the chromogenic agent by adding 6.3 g of DNS and 262 mL of 2 mol / L sodium hydroxide aqueous solution to 500 mL of water containing 182 g of potassium sodium tartrate, then adding 5 g of heavy phenol and 5 g of sodium sulfite, stirring to dissolve, cooling, and diluting with water to 1000 mL. The 3,5-dinitrosalicylic acid reagent is prepared and stored in a brown bottle for later use.

[0111] Preparation of glucose standard curve: Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution (1 mg / mL) into 25 mL test tubes, respectively, accurately add 2 mL of DNS reagent, heat in a boiling water bath for 2 min, cool with running water, and make up to 15 mL with water. Measure the absorbance at 540 nm wavelength. For sample determination, dilute the sample solution appropriately 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 a boiling water bath for 2 min, cool, and make up to 15 mL with water. Measure the absorbance at 540 nm wavelength. Calculate the reducing sugar content in the sample according to the standard curve.

[0112] 3.7.4 Metabolite changes

[0113] At 48 h of fermentation, samples were taken and the fermentation products of different groups were detected by GC-MS. The GC detection conditions were as follows: the carrier gas was high-purity helium, the chromatographic column was DB-5ms (30.0 m x 250 μm, 0.25 μm), 50°C was maintained for 2 min, then increased to 100°C at a rate of 8°C / min, maintained for 1 min, then increased to 150°C at a rate of 10°C / min, maintained for 1 min, then increased to 250°C at a rate of 20°C / min, and maintained for 5.75 min. The MS detection conditions were as follows: EI source, electron energy of 70 eV, ion source temperature of 230°C, interface temperature of 280°C, and program time of 26 min. All experiments were performed under sterile conditions.

[0114] 3.8 Data processing

[0115] The experimental data were expressed as Means ± SD, and the significant difference analysis was performed by one-way ANOVA and LSD multiple comparison analysis using IBM SPSS Statistics 25 software. The difference was significant at p < 0.05.

[0116] 4 Experimental results

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

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

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

[0120] As can be seen from Figure 1 , with the increase of temperature, the extraction rate of tea polysaccharide increased, and when the temperature reached 55°C, the extraction rate of tea polysaccharide was the highest. With the continuous increase of temperature, the extraction rate of tea polysaccharide showed a trend of decline and slight increase, which was speculated to be that the optimal action temperature of the compound enzyme was about 55°C, and the increase of temperature would deviate from the optimal action temperature of the enzyme; the upward trend at a temperature of 85°C was probably due to the fact that the increase of temperature accelerated the dissolution of polysaccharide, but the effect of temperature increase was not as great as that of the enzyme. Therefore, 55°C was selected as the center point for the subsequent optimization experiment.

[0121] With the increase of pH, the extraction rate of tea polysaccharide showed a trend of first increase and then decrease; when the pH was 5.0, the extraction rate of tea polysaccharide was the highest, but there was no significant difference between it and other pH values (except pH 6.5). This factor did not have a vertex for the response value, so it was not optimized, and therefore the pH was determined as 5.0 in the subsequent experiment.

[0122] When the liquid-material ratio increased from 15 mL / g to 35 mL / g, the yield of tea polysaccharide was significantly increased; when the liquid-material ratio of the system was more than 35 mL / g, the yield of tea polysaccharide showed a significant downward trend. Therefore, 35 mL / g was selected as the center point of the subsequent optimization experiment.

[0123] The yield of tea polysaccharide showed a significant upward trend with the increase of the ratio of complex enzyme to substrate (E / S). When E / S increased to 1.5%, the yield of tea polysaccharide showed an increasing trend, but there was no significant difference with 1.5%. This factor had no center point on the response value, and was not optimized. From the perspective of cost, E / S was determined as 1.5% in the subsequent experiment.

[0124] With the increase of extraction time from 20 min to 80 min, the yield of tea polysaccharide showed a significant upward trend. When the time continued to increase to more than 80 min, the yield of tea polysaccharide showed a significant downward trend. When the extraction time was 80 min, the yield of tea polysaccharide was significantly higher than that of other times. Therefore, 80 min was used as the center point in the subsequent optimization experiment.

[0125] 4.1.2 Response surface optimization of tea polysaccharide dissolution

[0126] Based on the analysis of single factor experiment results, according to the principle of central composite experiment design, Design-Expert 8.0.6 software was used for experiment design and results, as shown in Table 3.

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

[0128]

[0129] The experimental data in Table 3 were subjected to multiple regression fitting, and the regression equation with tea polysaccharide yield (Y) as the response value was obtained:

[0130] Y=8.68+0.215A+0.030B-0.087C+0.003AB-0.063AC-0.081BC-0.637A²-0.207B²-0.458C²

[0131] The regression equation was subjected to variance analysis, and the results are shown in Table 4.

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

[0133]

[0134] The analysis in Table 4 shows that the p-value was used as a tool to check the significance of each coefficient, and the negative p-value was 0.0068, indicating that the negative result was significant. p <0.01). R² is 0.9080, indicating that the predicted value fits the simulated experimental value well within the experimental parameter range; the adjusted R² is 0.7896, indicating that the equation can explain 78.96% of the response value variation, and the fit is good. The p-value of the lack-of-fit term is 0.453, which is not significant, indicating that the residual is caused by random error, and it is not significant relative to the pure error. p >0.05); the CV value was 2.79%, indicating a high confidence level for a negative result. Therefore, this regression equation can be used to predict the experimental results. Among the three factors, the linear term A and the quadratic term A... 2 C 2 The interaction terms AB, AC, and BC have a significant effect on the response value curve effect; however, they do not have a significant effect on the response value surface effect, indicating that there is no significant synergistic effect between the liquid-to-solid ratio, extraction time, and temperature. The order of influence of the three factors on the extraction yield of tea polysaccharides is: A > C > B.

[0135] The negative regression analysis showed that the interaction terms (AB, AC, BC) were all insignificant, therefore response surface methodology and contour plotting were not required. One-way effect analysis was performed using dimensionality reduction, fixing the three factors in the regression equation at the zero level. The one-way negative regression results for the three factors are as follows:

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

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

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

[0139] Based on the above equations, the single-factor trajectories of each factor are shown below. Figure 2 .

[0140] Figure 2 The results show that the extraction yield of tea polysaccharides is affected by the liquid-to-solid ratio, extraction time, and temperature. With increasing level number, the extraction yield initially increases significantly, then the increase slows down, and finally decreases significantly. All three factors have maximum effects on the extraction yield of tea polysaccharides within the range of -1 to 1. The curve corresponding to the liquid-to-solid ratio shows the most significant change within this range, and this steep curve indicates that the extraction yield of tea polysaccharides is most sensitive to this factor.

[0141] The optimal conditions for negative-response extraction were: a material-to-liquid ratio of 1:36.689, an extraction time of 81.468 min, and an extraction temperature of 54.054℃. Under these conditions, the theoretical dissolution rate of tea polysaccharides was 8.700%. Based on actual laboratory conditions, the optimal conditions were further adjusted to: a material-to-liquid ratio of 1:35, an extraction time of 80 min, and an extraction temperature of 55℃. To further verify the effectiveness and accuracy of the negative-response extraction method, three parallel experiments were conducted under the optimized extraction conditions. The polysaccharide yield from the extracted tea polysaccharides reached 8.717% ± 0.128%, consistent with the predicted value. This indicates that the response surface methodology is reliable and fits the actual situation well, thus verifying the effectiveness of the regression equation.

[0142] 4.2 Single-factor experiments and response surface methodology optimization for the dissolution of tea polysaccharides using hydrogen peroxide treatment technology

[0143] 4.2.1 Single-factor experiment on the dissolution of tea polysaccharides

[0144] The effects of liquid-to-solid ratio, extraction time, hydrogen peroxide concentration, and temperature on the extraction yield of tea polysaccharides are shown in the figure. Figure 3 .

[0145] Depend on Figure 3 The effect of the liquid-to-solid ratio on the dissolution rate of tea polysaccharides was investigated. It was found that as the liquid-to-solid ratio increased from 10 mL / g to 20 mL / g, the dissolution rate of tea polysaccharides increased significantly. However, when the liquid-to-solid ratio exceeded 20 mL / g, the dissolution rate of tea polysaccharides showed a significant decreasing trend. Therefore, 20 mL / g was selected as the center point for subsequent optimization experiments.

[0146] from Figure 3 The effect of extraction time on the dissolution rate of tea polysaccharides was investigated. A significant increase in dissolution rate occurred as the extraction time increased from 5 min to 15 min. However, a significant decrease in dissolution rate occurred when the extraction time continued to increase to 25 min. The dissolution rate of tea polysaccharides was highest at an extraction time of 15 min compared to other time periods. Therefore, 15 min was used as the center point in subsequent optimization experiments.

[0147] like Figure 3 The effect of hydrogen peroxide concentration on the dissolution rate of tea polysaccharides was investigated. The dissolution rate increased continuously as the hydrogen peroxide concentration increased from 0.3% to 1.2%, but decreased when the concentration exceeded 1.2%. This indicates that hydrogen peroxide has an optimal concentration. Therefore, a hydrogen peroxide concentration of 1.2% was selected as the center point for subsequent optimization experiments.

[0148] from Figure 3The effect of temperature on the dissolution rate of tea polysaccharide was that the dissolution rate of tea polysaccharide increased continuously with the increase of extraction temperature from 60 to 100°C, indicating that the increase of temperature could promote the breakage of tea cell wall and the dissolution of polysaccharide. There was no vertex for the response value, so the temperature was determined as 100°C in the subsequent experiment.

[0149] 4.2.2 Response surface optimization of tea polysaccharide dissolution

[0150] Based on the analysis of single factor experiment results, according to the principle of central composite design experiment, Design-Expert 8.0.6 software was used for experiment design and results, as 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, 17 experiments were carried out for the optimization of three independent parameters, and there was a great change in the dissolution rate of tea polysaccharide within the extraction condition range. Through the application of multiple regression analysis, the following quadratic polynomial equation was obtained according to the response variable and test variable:

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

[0156] Table 6 Variance analysis of negative regression equation

[0157]

[0158] The correlation coefficient R of the regression equation 2 = 0.9879, indicating that the predicted value and the test value fit well within the test parameter range; the adjusted correlation coefficient R 2 adj = 0.9723, indicating that the equation can explain 97.23% of the response value change, and the fitting degree is good. The p value is used as a tool to check the significance of each coefficient, and the negative p value < 0.0001, indicating that the negative fitting degree is very significant. p<0.01). The p-value for the lack-of-fit term is 0.7325, indicating that the residuals are caused by random error, which is not significant relative to the pure error. p The variance was >0.05, and the coefficient of variation was 2.94%, indicating a high degree of confidence in the negative result. Therefore, this regression equation can be used to predict the experimental results.

[0159] The results of the analysis of variance show that the linear terms A, B, and C of the three factors are similar to the quadratic term A. 2 B 2 C 2 The interaction terms AB, AC, and BC have a highly significant effect on the response value curve effect; however, they do not significantly affect the response value surface effect, indicating that there is no significant synergistic effect between the material-liquid ratio and hydrogen peroxide concentration, the material-liquid ratio and extraction time, or the extraction time and hydrogen peroxide concentration. Based on the F-value, the order of influence of the three factors on the extraction yield of tea polysaccharides is: B > C > A.

[0160] The negative regression analysis showed that the interaction terms (AB, AC, BC) were all insignificant, therefore response surface methodology and contour plotting were not required. One-way effect analysis was performed using dimensionality reduction, fixing the three factors in the regression equation at the zero level. The one-way negative regression results for the three factors are as follows:

[0161] Y A = 21.40 + 0.55A - 1.52A 2

[0162] Y B = 23.13 + 2.90B - 5.20B 2

[0163] Y C = 21.68 + 0.95C - 2.11C 2

[0164] Based on the above equations, the single-factor trajectories of each factor are shown below. Figure 4 .

[0165] Figure 4 The results show that the effects of liquid-to-solid ratio, extraction time, and hydrogen peroxide concentration on the extraction yield of tea polysaccharides exhibit a trend of first significantly increasing and then significantly decreasing with increasing levels. The effects of liquid-to-solid ratio and hydrogen peroxide concentration on the extraction yield of tea polysaccharides both show a trend of gradual increase followed by gradual decrease. All three factors have maximum effects on the extraction yield of tea polysaccharides within the range of -1 to 1 levels. The curve corresponding to extraction time shows the most significant change within this range, and this steep curve indicates that the extraction yield of tea polysaccharides is most sensitive to this factor.

[0166] The optimal conditions for negative optimization were: liquid material ratio 22.074 mL / g, extraction time 17.929 min, hydrogen peroxide concentration 1.283%, and the theoretical dissolution rate of tea polysaccharide under these conditions was 25.039%. Combined with the actual laboratory conditions, the optimal adjustment was: liquid material 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 situation, three parallel experiments were carried out according to the optimal extraction conditions, and the dissolution rate of tea polysaccharide could reach 25.382% ± 0.424%, which was consistent with the predicted value. It was proved that the response surface analysis method was reliable, and the actual situation was well fitted, thereby verifying the effectiveness of the regression equation.

[0167] 4.3 Scanning electron microscopy observation of tea surface structure characteristics

[0168] The microscopic structure of raw tea powder and composite enzyme-peroxide hydrogen treated tea powder under 2000 and 5000 times is shown in Figure 5 and Figure 6 . From Figure 5 , the surface of raw tea powder is rough and the structure is dense, like overlapping tiles, and each tile surface is smooth and complete without cracks and cracks. From Figure 6 , the structure of the sample after composite enzyme-peroxide hydrogen treatment is greatly damaged, and the structure is loose, the original tile structure becomes scattered fragments, the surface has hole structure and deep cracks, which is speculated to be the synergistic effect of composite enzyme-peroxide hydrogen on the cell wall of tea powder. The SEM image is consistent with the dissolution rate of tea polysaccharide, which can explain that the synergistic treatment of composite enzyme-peroxide hydrogen can cause severe rupture of the cell wall structure of tea powder and promote the efficient dissolution of tea polysaccharide.

[0169] 4.4 Effect of tea polysaccharide on glucose and lipid metabolism in obese mice

[0170] 4.4.1 Dynamic effect of tea polysaccharide on body weight and blood glucose of mice fed with high-fat diet

[0171] The body weight of the negative control group mice was 20% higher than that of the normal control group, which was considered as successful modeling of obesity. For example Figure 7, the body weight of the mice was 18.5 g-20.5 g, and there was no significant difference in the body weight of the mice in each group. During the high-fat diet feeding process, the body weight of the mice in each group continued to increase, and by the 14th week, the body weight of the mice in the negative control group had increased to more than 34 g, while the body weight of the mice in the normal control group was only 25 g. Compared with the negative control group, the body weight of the mice in the low-dose and high-dose tea polysaccharide groups increased at nearly the same speed in the first 4 weeks, and the body weight of the mice in the tea polysaccharide groups gradually slowed down after the 4th week. Among them, the body weight of the mice in the high-dose tea polysaccharide group was more slowly than that in the low-dose tea polysaccharide group, and by the 14th week, the body weight of the mice was 30.7 g, which was significantly different from that of the negative control group (p < 0.05). As Figure 8 , the obesity degree of the mice in each group showed a trend of continuous increase during the high-fat diet feeding process, and the obesity degree of the mice in the negative control group exceeded 20% at the 6th week, indicating that an obesity model had been formed. The obesity degree of the mice in the low-dose tea polysaccharide group was higher than that in the negative control group in the first 4 weeks, but it was lower than 20%, and an obesity model had not been formed. From the 6th week to the end of the experiment at the 14th week, the obesity degree of the mice was always lower than that of the negative control group. The obesity degree of the mice in the high-dose tea polysaccharide group was always lower than that of the negative control group during the entire experiment, especially after the 6th week, the obesity degree increased slowly and was significantly lower than that of the negative control group, indicating that tea polysaccharide had the effect of slowing down the increase of body weight of mice caused by high-fat diet. Figure 9 , during the high-fat diet feeding process, the blood glucose of the mice in the negative control group showed a trend of continuous increase, and the blood glucose of the mice in the other groups was always in a fluctuating state. In the first two weeks, the blood glucose of the four groups of mice was close, with no difference. From the 6th week to the 14th week, the blood glucose of the mice in the high-dose and low-dose tea polysaccharide groups was always lower than that in the negative control group, indicating that tea polysaccharide had the effect of slowing down the increase of blood glucose of mice caused by high-fat diet.

[0172] 4.4.2 Effect of tea polysaccharide on liver glycogen of mice fed with high-fat diet

[0173] Liver glycogen is a form of energy reserve, which plays an important role in maintaining blood glucose stability, providing energy, and promoting liver metabolism and detoxification. The determination results of liver glycogen content of mice in each group are as follows Figure 10 , the liver glycogen content of the mice in the negative control group was significantly lower than that in the normal group (p < 0.05). After the intervention of high-dose and low-dose tea polysaccharide, the decrease of liver glycogen was alleviated, and the difference in liver glycogen content between the high-dose tea polysaccharide group and the negative control group increased (p < 0.05).

[0174] 4.4.3 Effect of tea polysaccharide on blood lipids of mice fed with high-fat diet

[0175] Obesity is accompanied by dyslipidemia, abnormal increase of triglyceride (TG), total cholesterol (TC) and low-density lipoprotein (LDL-C) in blood, and abnormal decrease of high-density lipoprotein (HDL-C) is called dyslipidemia. For example Figure 11 Compared with the normal control group, the TG level of the negative control group increased. Compared with the negative control group, the high and low dose of tea polysaccharide could reduce the TG level of mice, especially the low dose of tea polysaccharide could significantly reduce the TG level. The triglyceride-glucose index (TyG) is an effective model for predicting the risk of cardiovascular and cerebrovascular diseases and diabetes, which is calculated as follows: TyG = Ln [TG (mg / dl) x FPG (mg / dl) / 2], wherein FPG is fasting blood glucose. By calculation, the TyG value of the negative control group mice was the highest, reaching 7.25, and the TyG values of the high and low dose of tea polysaccharide groups were all lower than 7, indicating that tea polysaccharide could effectively reduce the risk of cardiovascular and cerebrovascular diseases and diabetes by reducing the TG level.

[0176] By Figure 11 Compared with the normal control group, the TC level and LDL-C level of the negative control group mice increased significantly. Compared with the negative control group, the high and low dose of tea polysaccharide groups could reduce the TC level and LDL-C level of mice, and the reduction effect had a concentration-dependent relationship. Compared with the normal control group, the HDL-C level of the negative control group mice decreased significantly. Compared with the negative control group, the high and low dose of tea polysaccharide groups could increase the HDL-C level of mice, and the increase effect also had a concentration-dependent relationship. It is shown that tea polysaccharide has the effect of improving the dyslipidemia of mice caused by high-fat feed.

[0177] 4.5 Fermentation products of tea polysaccharide after fermentation by intestinal flora

[0178] 4.5.1 Changes of total sugar and reducing sugar after fermentation of tea polysaccharide by intestinal flora

[0179] The dynamic changes of total sugar and reducing sugar at different time points of fermentation of tea polysaccharide by intestinal flora of db / m mice and db / db mice are shown in Tables 7, 8, 9 and 10.

[0180] Table 7 is the effect of normal mouse intestinal flora on the fermentation consumption of total sugar of different carbon sources. The total sugar content of the normal control group, the positive control (inulin) group and the different concentrations of tea polysaccharide groups is the highest at 0 h, and there is no difference between groups; with the extension of fermentation time, the total sugar content of each group decreases continuously, indicating that the carbon source in the culture medium is continuously metabolized and consumed by intestinal bacteria. At 24 h of fermentation, the total sugar content of the low concentration (0.5 mg / mL) tea polysaccharide group is the highest, followed by 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 in turn, indicating that low concentration tea polysaccharide has less effect on intestinal bacteria fermentation carbon source, and the effect increases with the increase of tea polysaccharide concentration and is better than the positive control. At 48 h of fermentation, the total sugar content of the normal control group, the inulin group and the low concentration (0.5 mg / mL) tea polysaccharide group is the lowest, indicating that the carbon source in the culture medium is basically consumed; but when the concentration of tea polysaccharide is higher than 1 mg / mL, the total sugar concentration shows an increasing trend, which may be that tea polysaccharide promotes the proliferation of more beneficial bacteria in the mouse intestine, and thus promotes the conversion of undigested dietary fiber in feces into total sugar.

[0181] Table 8 is the effect of intestinal flora of spontaneous db / db diabetic mice on the fermentation consumption of total sugar of different carbon sources. Similarly, the total sugar content of the negative control group, the inulin group and the different concentrations of tea polysaccharide groups is the highest at 0 h, and there is no difference between groups; with the extension of fermentation time, the total sugar content of each group decreases continuously, indicating that the carbon source in the culture medium is continuously metabolized and consumed by intestinal bacteria. At 12 h of fermentation, there is no difference in total sugar content between the groups except the 4 mg / mL tea polysaccharide group; at 24 h of fermentation, the total sugar content of the inulin group, the low concentration (0.5 mg / mL) tea polysaccharide group and the 2 mg / mL tea polysaccharide group is significantly higher than that of the other groups, and the total sugar content of the 1 mg / mL tea polysaccharide group and the 4 mg / mL tea polysaccharide group is significantly lower than that of the negative control group, indicating that high concentration tea polysaccharide has promoted the proliferation of probiotics in the intestine of diabetic mice, but the effect is not concentration-dependent. At 48 h of fermentation, the total sugar content of the negative control group has no significant change compared with that at 24 h, indicating that the intestinal flora of diabetic mice is disordered and cannot normally ferment the carbon source in the culture medium. The total sugar content of the inulin group and the high concentration (> 1 mg / mL) tea polysaccharide group is significantly low, especially the total sugar content of the 4 mg / mL tea polysaccharide group has no difference with that of the inulin group, indicating that high concentration tea polysaccharide can improve the disordered state of intestinal flora of diabetic mice and promote the proliferation of more beneficial bacteria.

[0182] Table 9 is the effect of normal mouse intestinal flora on the production of reducing sugar by fermentation of different carbon sources. Within 24 h of fermentation, no reducing sugar was detected in each group, indicating that the sugar substances in each group had not yet been degraded to produce reducing sugar. At 48 h of fermentation, reducing sugar was detected in each group, and there was no difference between groups, indicating that the intestinal flora of normal mice had consumed the carbon source. Table 10 is the effect of intestinal flora of spontaneous db / db diabetic mice on the production of reducing sugar by fermentation of different carbon sources. Similarly, within 24 h of fermentation, no reducing sugar was detected in each group, indicating that the sugar substances in each group had not yet been degraded to produce reducing sugar. At 48 h of fermentation, reducing sugar was detected in each group, and there was no significant difference in reducing sugar content between the negative control group, the low concentration (0.5 mg / mL and 1 mg / mL) tea polysaccharide group; the inulin group and the high concentration (2 mg / mL and 4 mg / mL) tea polysaccharide group had significantly higher reducing sugar content than the negative control group, especially the 4 mg / mL tea polysaccharide group, which was speculated to be the fermentation of sugar substances by intestinal bacteria to produce more reducing sugar.

[0183] Table 7 Dynamic changes in total sugar in intestinal flora of db / m mice fermented with tea polysaccharide at different time points

[0184]

[0185] Table 8 Dynamic changes in total sugar in intestinal flora of db / db mice fermented with tea polysaccharide at different time points

[0186]

[0187] Table 9 Dynamic changes in reducing sugar in intestinal flora of db / m mice fermented with tea polysaccharide at different time points

[0188]

[0189] Table 10 Dynamic changes in reducing sugar in intestinal flora of db / db mice fermented with tea polysaccharide at different time points

[0190]

[0191] 4.5.2 Changes in metabolic products after fermentation of tea polysaccharide by intestinal flora

[0192] The metabolic products of intestinal flora of mice in different experimental groups after fermentation were detected by GC-MS, and the results are shown in Table 11. Figure 12 Compared with Figure 13 .

[0193] By comparing with NIST 20 mass spectrometry database (NIST / EPA / NIH Mass Spectral Library), 2,3-butanediol, tridecane, tetradecane, pentadecane, hexadecane, 3-tert-butyl-6-octene-1-ol, heptyl hexacosanyl ether and other compounds were produced in the 5 experimental groups of db / m mice, but indole and 4-tert-butyl cyclohexanone were also produced in the 2.0 mg / mL and 4.0 mg / mL polysaccharide sample groups. In db / db mice, tridecane, tetradecane, pentadecane, hexadecane, n-decanol, 3-tert-butyl-6-octene-1-ol, heptyl hexacosanyl ether and other compounds were produced in the 5 experimental groups, but 4-tert-butyl cyclohexanone 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 polysaccharide sample group. The negative control group produced dodecamethylhexa-siloxane, an environmental pollutant, which was not found in other groups.

[0194] At present, there is still a lack of relevant research data on the specific effects and functions of 4-tert-butyl cyclohexanone, heptyl hexacosanyl ether and other compounds on the human body. However, indole produced by the polysaccharide sample group has the functions of maintaining blood glucose stability, anti-bacterial and anti-inflammatory, and auxiliary reduction of cholesterol, etc., and is the main bacterial metabolite of tryptophan produced by various Bacteroides and Enterobacteriaceae, which can down-regulate the expression of pro-inflammatory cytokines and enhance the intestinal barrier. In recent years, with the gradual deepening of the research on microbial metabolites of tryptophan, researchers have also found that indole and its derivatives are essential signaling molecules in the microbial-gut-brain axis, which can affect brain function and behavior. Tryptophan metabolites have been identified as potent biomarkers for complications of type 2 diabetes. Cohort studies have shown that the reduction of gut microbiota in the production of aryl hydrocarbon receptor (AHR) ligands 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-acrylate (IA), indole-3-propionate (IPA), indole-3-acetate (IAA), and indole-3-aldehyde (IAld). The loss of the protective effect of AHR in promoting repair, inhibiting inflammation and maintaining intestinal homeostasis leads to this pathogenesis. In addition, 2,3-butanediol produced by the polysaccharide sample group is an important energy substance and platform compound, and many microorganisms such as Enterobacter, Bacillus, Serratia, etc. can be used to produce 2,3-butanediol. 2,3-butanediol has the functions of moisturizing, hydrating, antibacterial, anti-inflammatory, etc. on the human body. Studies have shown that the gastrointestinal microbiota contributes to the phenotypic characteristics of reduced serum cholesterol in Fmo5- / - mice, and 2,3-butanediol is a potential drug for reducing plasma cholesterol [1] . In summary, after the intestinal flora of db / m and db / db mice are fermented by tea polysaccharide, important metabolites such as indole and 2,3-butanediol with functions for the human body can be produced.

[0195] References:

[0196] [1] Sunil Veeravalli, Dorsa Varshavi, Flora H Scott, Dorna Varshavi, Frank S Pullen, Kirill Veselkov, Ian R Phillips, Jeremy R Everett, Elizabeth A Shephard. Treatment of wild-type mice with 2,3-butanediol, a urinary biomarker of Fmo5 - / - mice, decreases plasma cholesterol and epididymal fat deposition[J]. Front Physiol., 2022, 13: 859681. doi: 10.3389 / fphys.2022.859681.

[0197] Comparative Example 1, direct hot water extraction

[0198] The crude old tea leaves were placed in an oven for low temperature (60°C) drying until the moisture content was below 9.0%, crushed and sieved to obtain crude old tea powder with a mesh size >150. 2 g of crude old tea powder was added to 110 mL of deionized water at a solid-liquid ratio of 1 g:55 mL, and placed in a constant temperature oscillator. The oscillator parameters were adjusted to 100°C and 200 rpm, and the extraction was carried out by reciprocating rotary oscillation for 100 min. The mixture was centrifuged at 8000 r / min for 20 min, and 1.0 mL of supernatant was taken for determination of the polysaccharide content in the supernatant by the phenol-sulfuric acid method, and the extraction yield was calculated. The results are shown in Table 11.

[0199] Table 11 Effect of different extraction processes on the extraction yield of tea polysaccharides

[0200]

[0201] 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 combined with hydrogen peroxide can significantly improve the extraction yield of tea polysaccharides, which is 3.371 times higher than that of traditional hot water extraction.

[0202] Comparative Example 2, effect of crude old tea leaf powder mesh size on tea polysaccharide yield

[0203] The tea powder in Example 1 Method 3.1 was replaced with a 20-mesh coarse sieve, and the other operations were the same as the optimal conditions of Example 1 Methods 3.1, 3.2, and 3.3 and 3.4. The tea polysaccharide extraction yield detection and calculation method was the same as Example 1, and the results are shown in Table 12.

[0204] Table 12 Effect of different tea powder particle size on tea polysaccharide extraction yield

[0205]

[0206] As shown in Table 12, the crushing particle size of tea has a great influence on the extraction yield of tea polysaccharide. The pretreatment method of crushing through a 200-mesh sieve can increase the extraction yield of tea polysaccharide by 1.472 times compared with the traditional mechanical crushing of coarse powder.

[0207] Comparative Example 3, Effect of Pretreatment on Tea Polysaccharide Yield

[0208] Example 1 Method 3.1, and 3.3 and 3.4 optimal conditions, omit 3.2, tea polysaccharide extraction yield detection and calculation method is the same as Example 1, the results are shown in Table 13.

[0209] Table 13 Effect of different pretreatment on tea polysaccharide extraction yield

[0210]

[0211] As shown in Table 13, the pretreatment method of raw tea powder has a great influence on the extraction yield of tea polysaccharide. The DGRA solvent pretreatment method can increase the extraction yield of tea polysaccharide by 1.341 times compared with the non-pretreatment method. 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 food or rehabilitation food.

[0212] Comparative Example 4, Effect of Hydrogen Peroxide Synergistic Extraction on Tea Polysaccharide Yield

[0213] Example 1 Method 3.1, 3.2 and 3.3 optimal conditions, omit 3.4, tea polysaccharide extraction yield detection and calculation method is the same as Example 1, the results are shown in Table 14.

[0214] Table 14 Effect of different extraction processes on tea polysaccharide extraction yield

[0215]

[0216] As shown in Table 14, the extraction process of tea has a great influence on the extraction yield of tea polysaccharide. The extraction method of composite enzyme synergistic hydrogen peroxide can increase the extraction yield of tea polysaccharide by 2.912 times compared with the non-hydrogen peroxide synergistic extraction.

[0217] Comparative Example 5, Effect of Composite Enzyme Extraction on Tea Polysaccharide Yield

[0218] Using the optimal conditions of 3.1, 3.2 and 3.4 in the method of Example 1, omitting 3.3, the extraction rate of tea polysaccharide was detected and calculated in the same way as in Example 1, and the results are shown in Table 15.

[0219] Table 15 Influence of different extraction processes on the extraction rate of tea polysaccharide

[0220]

[0221] As can be seen from Table 15, the extraction process of tea leaves has a great influence on the extraction rate of tea polysaccharide. The extraction method of composite enzyme combined with hydrogen peroxide can increase the extraction rate of tea polysaccharide by 1.673 times compared with the extraction without composite enzyme.

[0222] Comparative Example 6, Influence of enzyme on polysaccharide yield

[0223] Using 3.1, 3.2 in the method of Example 1, replacing cellulase-papain under the optimal conditions of 3.3 with an equal amount of cellulase, and 3.4 optimal conditions, the extraction rate of tea polysaccharide was detected and calculated in the same way as in Example 1, and the results are shown in Table 16.

[0224] Table 16 Influence of different extraction processes on the extraction rate of tea polysaccharide

[0225]

[0226] As can be seen from Table 16, the extraction process of tea leaves has a great influence on the extraction rate of tea polysaccharide. The extraction method of composite enzyme combined with hydrogen peroxide can increase the extraction rate of tea polysaccharide by 1.196 times compared with the extraction of single cellulase combined with hydrogen peroxide.

[0227] Comparative Example 7, Influence of enzyme on polysaccharide yield

[0228] Using 3.1, 3.2 in the method of Example 1, replacing cellulase-papain under the optimal conditions of 3.3 with an equal amount of papain, and 3.4 optimal conditions, the extraction rate of tea polysaccharide was detected and calculated in the same way as in Example 1, and the results are shown in Table 17.

[0229] Table 17 Influence of different extraction processes on the extraction rate of tea polysaccharide

[0230]

[0231] As can be seen from Table 17, the extraction process of tea leaves has a great influence on the extraction rate of tea polysaccharide. The extraction method of composite enzyme combined with hydrogen peroxide can increase the extraction rate of tea polysaccharide by 1.307 times compared with the extraction of single papain combined with hydrogen peroxide.

[0232] Example 2

[0233] (1) Put the coarse old tea leaves into an oven for low temperature (60°C) drying until the moisture is below 9.0%, crush and pass through a 150-mesh sieve to obtain coarse old tea leaf powder with a mesh size > 150.

[0234] (2) Prepare the DGRA solvent: mix n-octanoic acid and n-decanoic acid at a molar ratio of 3:1, and magnetically stir at 75-85°C for 2 h until the solution is clear. Then, fully mix the n-octanoic acid and n-decanoic acid with anhydrous ethanol at a volume ratio of 2:5 to obtain the DGRA solvent.

[0235] Take 2 g of the coarse old tea leaf powder prepared in step (1), add 6 mL of the DGRA solvent, and stir at 55°C for 2 h. Then, centrifuge at 8000 r / min for 15 min, and treat the residue with 70% ethanol by reflux for 2 h. Filter, dry the filter cake, crush, and pass through a 200-mesh sieve to obtain 1.25 g of coarse old tea pretreated powder.

[0236] (3) Add 1 g of the coarse old tea pretreated powder to 0.015 g of cellulase-papain complex enzyme (mass ratio 5:2, and the amount of complex enzyme added is 1.5% of the mass of the coarse old tea pretreated powder), add 25 mL of deionized water, and adjust the pH to 4.5 with citric acid. Enzymatically hydrolyze at 55°C for 60 min, then put it into a 100°C boiling water bath for 3 min to inactivate the enzyme, and obtain tea slurry.

[0237] (4) Add 1.65 mL of 30% hydrogen peroxide and 28.35 mL of deionized water to the tea slurry obtained in step (3) at a liquid-solid ratio of 30:1 (mL / g) of the coarse old tea pretreated powder, so that the final concentration of hydrogen peroxide is 0.9%, and the pH is 4.5. Put it into a constant temperature oscillator, adjust the oscillator parameters to 100°C and 200 rpm, and oscillate reciprocally for 25 min. Take out the mixture, centrifuge (8000 r / min for 20 min), and 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 yield according to Example 1, which is 23.213%.

[0238] Example 3

[0239] (1) Put the coarse old tea leaves into an oven for low temperature (60°C) drying until the moisture is below 9.0%, crush and pass through a 150-mesh sieve to obtain coarse old tea leaf powder with a mesh size > 150.

[0240] (2) Preparation of DGRA solvent: mix n-octanoic acid and n-decanoic acid in a molar ratio of 2:1, and stir magnetically at a constant temperature of 75-85°C for 2 h until the solution is clear. Then mix the total volume of n-octanoic acid and n-decanoic acid with absolute ethanol in a volume ratio of 2:7 to obtain the DGRA solvent.

[0241] Take 2 g of the crude old tea powder prepared in step (1), add 6 mL of the DGRA solvent, and stir at 55°C for 2.5 h. Centrifuge at 8000 r / min for 15 min, and then treat the residue with 70% ethanol by reflux for 1.5 h. Filter, dry the filter cake, and crush it to pass through a 200-mesh sieve to obtain 1.25 g of the crude old tea pretreated powder.

[0242] (3) Add 1 g of the crude old tea pretreated powder to 0.015 g of a cellulase-papain composite enzyme (mass ratio 7:2, and the amount of the composite enzyme added accounts for 1.5% of the mass of the crude old tea pretreated powder), add 45 mL of deionized water, and adjust the pH to 4.5 with citric acid. Enzymatically hydrolyze at 45°C for 100 min. After the enzymatic hydrolysis is completed, put it in a 100°C boiling water bath to inactivate the enzyme for 3 min to obtain tea slurry.

[0243] (4) Add 2.2 mL of 30% hydrogen peroxide and 7.8 mL of deionized water to the tea slurry obtained in step (3) in a liquid-solid ratio of 10:1 (mL / g) of the crude old tea pretreated powder, so that the final concentration of hydrogen peroxide is 1.2% and the pH is 4.5. Put it in a constant-temperature oscillator and adjust the oscillator parameters to 100°C and 200 rpm for reciprocating oscillation for 15 min. Take out the mixture and centrifuge (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. According to the conversion in Example 1, the yield is 23.976%.

[0244] Example 4

[0245] (1) Dry the crude old tea leaves in an oven at a low temperature (60°C) until the moisture content is below 9.0%, crush them to pass through a 150-mesh sieve, and obtain crude old tea powder with a mesh size of >150.

[0246] (2) Preparation of DGRA solvent: mix n-octanoic acid and n-decanoic acid in a molar ratio of 5:2, and stir magnetically at a constant temperature of 75-85°C for 2 h until the solution is clear. Then mix the total volume of n-octanoic acid and n-decanoic acid with absolute ethanol in a volume ratio of 1:3 to obtain the DGRA solvent.

[0247] Take 2 g of the crude old tea powder prepared in step (1), add 6 mL of DGRA solvent, stir at 55°C for 1.5 h, then centrifuge at 8000 r / min for 15 min, filter the residue, and then treat it with 70% ethanol by reflux for 2.5 h. Filter the cake, dry it in the sun, crush it, and pass it through a 200 mesh sieve to obtain 1.25 g of crude old tea pretreated powder.

[0248] (3) Add 1 g of the crude old tea pretreated powder to 0.021 g of cellulase-papain complex enzyme (mass ratio 5:2, the amount of complex enzyme added is 2.1% of the mass of the crude old tea pretreated powder), add 35 mL of deionized water, the solid-liquid ratio is 1:35 g / mL, adjust the pH to 4.5 with citric acid, and enzymatically hydrolyze at 65°C for 80 min. After the enzymatic hydrolysis is completed, put it in a 100°C boiling water bath to inactivate the enzyme for 3 min to obtain tea slurry.

[0249] (4) Add 2.75 mL of 30% hydrogen peroxide and 17.25 mL of deionized water to the tea slurry obtained in step (3) according to the solid-liquid ratio of 20:1 (mL / g) of crude old tea pretreated powder, so that the final concentration of hydrogen peroxide is 1.5%, and the pH is 4.5. Put it in a constant temperature oscillator and adjust the oscillator parameters to 100°C and 200 rpm for reciprocating rotary oscillation for 5 min. Take out the mixed solution and centrifuge (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. According to the conversion of Example 1, the yield is 23.521%.

[0250] Example 5

[0251] (1) Dry the crude old tea leaves in an oven at a low temperature (60°C) until the moisture content is below 9.0%, crush them to pass through a 150 mesh sieve, and obtain crude old tea powder with a mesh size of >150.

[0252] (2) Prepare DGRA solvent: Mix n-octanoic acid and n-decanoic acid at a molar ratio of 3:1, and stir at 75-85°C for 2 h until the solution is clear. Then mix thoroughly with anhydrous ethanol according to the volume ratio of the total volume of n-octanoic acid and n-decanoic acid to anhydrous ethanol at 2:5 to obtain the DGRA solvent.

[0253] Take 2 g of the crude old tea powder prepared in step (1), add 6 mL of DGRA solvent, stir at 55°C for 1.5 h, then centrifuge at 8000 r / min for 15 min, filter the residue, and then treat it with 70% ethanol by reflux for 2.5 h. Filter the cake, dry it in the sun, crush it, and pass it through a 200 mesh sieve to obtain 1.25 g of crude old tea pretreated powder.

[0254] (3) 1 g of the crude old tea treatment powder was added to 0.018 g of a cellulase-papain composite enzyme (mass ratio 3:1, composite enzyme added amount accounting for 1.8% of the mass of the crude old tea treatment powder), 25 mL of deionized water was added, the solid-liquid ratio was 1:25 g / mL, the pH was adjusted to 4.5 with citric acid, and enzymolysis was performed at 55°C for 60 min. After the enzymolysis was completed, the enzyme was inactivated by placing the mixture in a 100°C boiling water bath for 3 min, and tea slurry was obtained.

[0255] (4) The tea slurry obtained in step (3) was added to 2.2 mL of hydrogen peroxide with a mass concentration of 30% and 27.8 mL of deionized water, so that the final mass concentration of hydrogen peroxide was 1.2% and the pH was 4.5. The mixture was placed in a constant-temperature oscillator, and the oscillator parameters were adjusted to 100°C and 200 rpm for reciprocating rotary oscillation for 15 min. The mixture was removed and centrifuged (8000 r / min for 20 min) to obtain a supernatant. 1.0 mL of the supernatant was taken, and the polysaccharide content in the supernatant was detected by the phenol-sulfuric acid method. The yield was calculated according to the method of Example 1 to be 24.163%.

[0256] The tea polysaccharides prepared in Examples 1-5 were subjected to mouse sugar lipid metabolism and intestinal flora metabolite detection by the method of Example 1, and the results showed that they could all produce important metabolites such as indole and 2,3-butanediol, which are beneficial to human body.

Claims

1. A method for preparing functional tea polysaccharides, characterized in that, The tea polysaccharide was prepared according to the following method: (1) Pretreatment: Take coarse old tea powder, use DGRA solvent to stir and soak at 50-55℃ for 1.5-2.5 h, centrifuge, filter residue is then refluxed with 70% ethanol for 1-3 h, filtered, the filter cake is dried and crushed to obtain coarse old tea pretreated powder; the DGRA solvent is an anhydrous ethanol solution of octanoic acid and decanoic acid; (2) Compound enzymatic hydrolysis extraction: Add a compound enzyme of cellulase-papain to the coarse old tea pretreatment powder, add deionized water, adjust the pH to 4.5-6.5 with citric acid, and enzymatically hydrolyze for 60-100 min at a temperature of 45-65℃. After the enzymatic hydrolysis is completed, put it in a 100℃ boiling water bath for 3 min to inactivate the enzyme and obtain tea pulp. (3) Hydrogen peroxide co-extraction: Add a solution of 30% hydrogen peroxide and deionized water to the tea pulp in step (2), mix well, and extract in a constant temperature water bath at 60-100℃ and 200 rpm for 5-25 min; take out the mixture, centrifuge, and obtain the supernatant; concentrate, precipitate with alcohol, and remove protein from the supernatant to obtain tea polysaccharides.

2. The method for preparing tea polysaccharides as described in claim 1, characterized in that, Step (1) The coarse old tea powder is prepared as follows: commercially available coarse old tea leaves are placed in an oven and dried at a low temperature until the moisture content is below 9%. The leaves are then crushed in a pulverizer and sieved to obtain coarse old tea powder with a mesh size > 150 mesh.

3. The method for preparing tea polysaccharides as described in claim 1, characterized in that, Step (1) The molar ratio of octanoic acid to decanoic acid is 2-3:1, and the volume ratio of the total volume of octanoic acid and decanoic acid to the volume of anhydrous ethanol is 1:2.5-3.

5.

4. The method for preparing tea polysaccharides as described in claim 1, characterized in that, The cellulase-papain complex enzyme in step (2) is made by mixing cellulase and papain at a mass ratio of 2-4:

1. The amount of cellulase-papain complex enzyme added is 1.5-2.5% based on the mass of the coarse old tea pre-treatment powder.

5. The method for preparing tea polysaccharides as described in claim 1, characterized in that, Step (2) The volume of deionized water used is 25-45 mL / g based on the mass of the coarse old tea pretreatment powder; the enzymatic hydrolysis is carried out at a temperature of 55℃ for 80 min.

6. The method for preparing tea polysaccharides as described in claim 1, characterized in that, Step (3) The amount of 30% hydrogen peroxide and deionized water solution added makes the final mass concentration of hydrogen peroxide 0.9%-2.1%.

7. The method for preparing tea polysaccharides as described in claim 1, characterized in that, The method for obtaining tea polysaccharides by concentrating, precipitating with alcohol, and deproteinizing the supernatant is as follows: The supernatant is concentrated to one-third of its original volume by rotary evaporation and cooled to 4°C to obtain a concentrated solution; while stirring, 95% ethanol with a volume of 4 times that of the concentrated solution is slowly added, and the mixture is placed in a refrigerator at 4°C for alcohol precipitation for 12 hours. After centrifugation, the precipitate is collected and deionized water is added. One-quarter volume of Sevag reagent is added to the precipitate and deionized water to remove proteins. After thorough shaking, the mixture is allowed to stand and separate into layers. The upper layer is collected and the above deproteinization operation is repeated until there is no absorption peak at 280 nm in the upper layer; the supernatant after the last deproteinization is rotary evaporated at 50°C to 25-35% of its original volume and dried at 60°C to obtain tea polysaccharides.

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