Yellow green tea polysaccharide as well as extraction method and application thereof

Abstract

The invention relates to the technical field of active ingredient extraction, and particularly discloses yellow green tea polysaccharide as well as an extraction method and application thereof, and the extraction method comprises the following steps: S1, degreasing and decoloring yellow green tea powder, and drying for later use; s2, mixing the tea powder with the extracting solution in ultrasonic equipment to obtain an extracting mixed solution, and adding a compound enzyme into the extracting mixed solution for extracting to obtain a first mixed solution; s3, filtering the first mixed solution, taking supernate, and performing rotary evaporation and concentration to obtain a second mixed solution; s4, carrying out alcohol precipitation on the second mixed solution, centrifuging, and redissolving the precipitate to obtain a third mixed solution; s5, adding a sevage reagent into the third mixed solution to obtain a deproteinized mixed solution, and then performing rotary evaporation to obtain a concentrated solution; and S6, dialyzing the concentrated solution, and drying to obtain the yellowed green tea polysaccharide. According to the method, the enzymolysis extraction step and the ultrasonic extraction step are synchronously carried out, and the two extraction methods can be matched with each other by adjusting related processing parameters, so that the extraction efficiency of the yellowed green tea polysaccharide is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of active ingredient extraction, and in particular to yellowed green tea polysaccharide and an extraction method and application thereof. Background Art

[0002] Tea polysaccharides (TPS) are a class of macromolecular active substances composed of various monosaccharides, uronic acid and protein in tea. They have many beneficial effects on human health, such as anti-obesity activity, hypoglycemic activity, lipid-lowering activity, etc.

[0003] The main methods for extracting tea polysaccharides include hot water extraction, enzymatic extraction and ultrasonic extraction. Among them, hot water extraction is low in cost and simple to operate, but time-consuming and labor-intensive. Enzyme-assisted extraction can effectively catalyze the degradation of cell walls and release intracellular polysaccharides from plant cells. This method can achieve the extraction of tea polysaccharides at a lower cost. In the prior art, ultrasonic extraction is usually combined with thermal extraction to shorten the extraction time of tea polysaccharides and improve the extraction rate of tea polysaccharides.

[0004] Zhonghuang No. 1 yellow tea is a high-quality variety suitable for high-end green tea. It contains theanine, tea polysaccharides, tea polyphenols, catechin polymers and other nutrients that are beneficial to the human body. However, the yield per mu of Zhonghuang No. 1 tea is limited and its price is often high, so it has not been widely promoted and applied. For expensive tea, how to improve the extraction rate of nutrients and thus improve the utilization efficiency of tea has become a problem that needs to be solved. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for extracting yellow green tea polysaccharides. The extraction method is suitable for the extraction of yellow green tea. By combining enzymatic extraction with ultrasonic extraction, the extraction rate of tea polysaccharides in yellow green tea is greatly improved, thereby improving the utilization rate of yellow green tea leaves as raw materials, which is conducive to the promotion and use of yellow green tea.

[0006] The present invention is achieved through the following technical solutions: A method for extracting yellow green tea polysaccharide comprises the following steps: S1. crushing yellow green tea, defatting and decolorizing it, and drying it for later use; S2. mixing yellow green tea powder with an extracting solution in an ultrasonic device to obtain an extracting mixed solution, and then adding a complex enzyme containing cellulase and papain to the extracting mixed solution for extraction to obtain a first mixed solution; S3. filtering the first mixed solution, taking the supernatant and concentrating it by rotary evaporation to obtain a second mixed solution; S4. adding ethanol to the second mixed solution for precipitation, centrifuging it, taking the precipitate and re-dissolving it in water to obtain a third mixed solution; S5. adding sevage reagent to the third mixed solution to remove protein to obtain a deproteinized mixed solution, and then rotary evaporating the deproteinized mixed solution to remove the sevage reagent and concentrate it to obtain a concentrated solution; S6, dialyzing the concentrated solution and drying it to obtain yellow green tea polysaccharide.

[0007] As a further improvement of the present invention, in S2, the yellowed green tea powder and the extract are mixed in a ratio of liquid to material of 1: (10-20) to obtain the extraction mixture.

[0008] As a further improvement of the present invention, in S2, 3-7% of the composite enzyme is added to the extraction mixture, and the mass ratio of cellulase to papain in the composite enzyme is (2.5-3.5):1.

[0009] As a further improvement of the present invention, in S2, the first mixed solution is obtained by extracting for 2 to 2.5 hours at an extraction temperature of 60 to 80° C. and a pH value of 4.0 to 5.5.

[0010] As a further improvement of the present invention, in S2, the ultrasonic frequency of the ultrasonic device is 80 kHz.

[0011] As a further improvement of the present invention, in S4, the volume ratio of the deproteinized mixed solution to the ethanol is 1:(4-5).

[0012] As a further improvement of the present invention, in S5, the volume ratio of chloroform to n-butanol in the sevage reagent is (4-5):1.

[0013] As a further improvement of the present invention, in S1, the yellowed green tea is crushed and passed through a 80-100 mesh sieve to obtain yellowed green tea powder, and the yellowed green tea powder is soaked in 95% ethanol for degreasing and decolorization, and then naturally air-dried.

[0014] In a second aspect, the present invention provides a yellow green tea polysaccharide, which is prepared by any one of the above-mentioned methods for extracting yellow green tea polysaccharides.

[0015] In a third aspect, the present invention provides an application of the above-mentioned yellowed green tea polysaccharide in the field of medicine for regulating intestinal flora and assisting weight loss.

[0016] In the prior art, ultrasonic extraction and enzymatic extraction have been widely used in the extraction process of natural plant polysaccharides. There are cases where the two methods are used in combination, but there are fewer cases where they are used simultaneously. The extraction method of the present invention can quickly and efficiently destroy the cell wall of yellowed green tea, so that the active ingredients therein can be released as much as possible.

[0017] The present invention uses a composite enzyme to decompose the cellulose and hemicellulose in the cell wall, so that the tea polysaccharides wrapped in the cell wall can be precipitated as much as possible; on the other hand, the cavitation effect, thermal effect and mechanical effect of ultrasound are used to assist enzymolysis. Specifically, the present invention performs ultrasonic treatment on the yellow green tea powder while cellulase and papain are performing enzymolysis. When the ultrasonic wave acts on the tea cells, the tea cells are destroyed and torn, and a large number of small holes are formed thereon. Such small holes will be closed instantly after being torn open, but an instantaneous pressure of up to several thousand atmospheres is generated during the process, which can cause the cell walls and organisms in the tea powder to rupture instantly, forcing the tea polysaccharides to be released and flow out; at the same time, the cellulase and papain that decompose the cellulose and protein of the enzymolysis tea cells can also enter the holes, further accelerating the decomposition of the cell tissue, thereby contributing to the release of tea polysaccharides, thereby greatly shortening the extraction time of tea polysaccharides and improving the extraction efficiency of tea polysaccharides.

[0018] The yellowed green tea polysaccharide prepared by the above extraction method can exist in the form of low molecular weight, and the low molecular weight polysaccharide can better maintain its original activity and is more easily absorbed by cells. At the same time, the inventors have found through experiments that the yellowed green tea polysaccharide extracted increases the relative abundance of beneficial bacteria in the intestinal flora, such as Bacteroides ( Bacteroides )、Lactococcus( Lactococcus ) and Faecalibacterium ( Faecalibacterium ) etc.; it also reduces the opportunistic pathogens such as Enterobacteriaceae and Shigella ( Escherichia_Shigella ) and Dorea ( Dorea ), so the inventors confirmed that yellowed green tea polysaccharides can be beneficial to the homeostasis of intestinal microorganisms, and can inhibit obesity and assist weight loss by regulating the human intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings are provided for combining with the preferred implementation examples of the present invention to help understand the purpose and advantages of the present invention, wherein: Figure 1 It is a statistical chart of the extraction rate test results of yellow green tea polysaccharide in comparative example 1 combined with the extraction rate test results of yellow green tea polysaccharide in example 1; Figure 2 This is a statistical chart of the extraction rate test results of yellow green tea polysaccharides in Comparative Example 2 combined with the extraction rate test results of yellow green tea polysaccharides in Example 1; Figure 3 This is a statistical chart of the extraction rate test results of yellow green tea polysaccharides in Comparative Example 3 combined with the extraction rate test results of yellow green tea polysaccharides in Example 1; Figure 4 This is a statistical chart of the extraction rate test results of yellow green tea polysaccharides in Comparative Example 4 combined with the extraction rate test results of yellow green tea polysaccharides in Example 1; Figure 5 This is a statistical chart of the extraction rate test results of yellow green tea polysaccharides in Comparative Example 5 combined with the extraction rate test results of yellow green tea polysaccharides in Example 1; Figure 6 It is a combination of molecular weight graph and IR spectrum graph of GTP; Figure 7 It is a combination of Congo red image, scanning electron microscope image and XRD spectrum of GTP; Figure 8 pH and OD during in vitro fermentation 600 Value result statistics chart; Fig. 9 This is a statistical chart showing the effect of yellow green tea polysaccharides on intestinal flora ASV; Fig.10 This is a sequencing curve diagram of the intestinal flora; Fig.11 NMDS analysis of fecal fermentation liquid microbiota in different groups; Fig.12 This is a statistical chart showing the effect of GTP on the level of intestinal flora; Fig.13 This is a statistical chart showing the effect of GTP on the genus level of intestinal flora; Fig.14 This is a statistical chart of the LEfSe analysis results among intestinal flora groups. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below based on the accompanying drawings and implementation examples.

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0022] Embodiment 1: This embodiment provides a method for extracting yellow green tea polysaccharides, comprising the following steps: S1. Grind the yellow green tea and pass it through an 80-mesh sieve to obtain yellow green tea powder, soak the yellow green tea powder in 95% ethanol for 2 h for decolorization and defatting, take it out and air-dry it for later use; S2. In an ultrasonic device, under the conditions of an ultrasonic frequency of 80 kHz, an extraction temperature of 80 °C, and an extraction pH of 5.5, the yellowed green tea powder and the extract were prepared in a ratio of 1:20 g / mL to obtain an extraction mixture, and then 7% of the composite enzyme was added to the extraction mixture and mixed for 2.5 h to obtain a first mixture, wherein the composite enzyme contained cellulase and papain, and the mass ratio of cellulase to papain was 3:1; S3. After filtering the first mixed solution, the supernatant was concentrated by rotary evaporation to obtain a second mixed solution; S4. Pour the second mixed solution into ethanol for precipitation, wherein the volume ratio of the deproteinized mixed solution to ethanol is 1:4, and after the precipitation is completed, the mixed solution is centrifuged, and the precipitate is re-dissolved in water to obtain a third mixed solution; S5. Add sevage reagent to the third mixed solution to remove protein to obtain a deproteinized mixed solution, and then perform rotary evaporation on the deproteinized mixed solution to remove the sevage reagent and concentrate to obtain a concentrated solution, wherein the sevage reagent contains chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is 4:1; S6. The concentrated solution is dialyzed with deionized water and then dried to obtain yellowed green tea polysaccharide (hereinafter referred to as GTP).

[0023] Preferably, in S5, vacuum freeze drying equipment is used to dry the yellowed green tea polysaccharide to obtain the yellowed green tea polysaccharide. This method can reduce the damage to the active substances in the yellowed green tea polysaccharide during the drying step.

[0024] Comparative Example 1: The difference between this comparative example and Example 1 is that in this example, the extraction step in S2 is performed at extraction temperatures of 40°C, 50°C, 60°C and 70°C, respectively.

[0025] Comparative Example 2: The difference between this comparative example and Example 1 is that in this example, the liquid-to-solid ratio of yellowed green tea powder to the extract is 1:10, 1:15, 1:25, 1:30 g / mL respectively to obtain an extraction mixed solution for the extraction step in S2.

[0026] Comparative Example 3: The difference between this comparative example and Example 1 is that, in this example, the extraction step in S2 is performed at extraction pH values ​​of 4, 4.5, 5 and 6, respectively.

[0027] Comparative Example 4: The difference between this comparative example and Example 1 is that, in this example, the extraction step in S2 is carried out under the conditions of adding the composite enzyme at concentrations of 1%, 3%, 5% and 9%, respectively.

[0028] Comparative Example 5: The difference between this comparative example and Example 1 is that, in this example, the extraction duration in S2 is 1 h, 1.5 h, 2 h and 3 h.

[0029] Comparative Example 6: Conventional ultrasonic extraction method was used to extract the xanthocyanidin green tea polysaccharide from the xanthocyanidin green tea powder of the same mass as that in Example 1.

[0030] Comparative Example 7: The xanthocyanidin green tea polysaccharide in the xanthocyanidin green tea powder of the same mass as that in Example 1 was extracted by conventional enzymatic extraction method.

[0031] Results test: 1. Extraction rate test of yellow green tea polysaccharide: The extraction rate of yellow green tea polysaccharides was tested for the GTP extracted in Example 1 and Comparative Examples 1 to 5. The testing step firstly requires weighing the yellow green tea powder before extraction to obtain the "mass of the original tea sample", and then mainly using the phenol-sulfuric acid method to test the content of the extracted GTP to obtain the "mass of tea polysaccharides in the extract", and finally calculating the yellow green tea polysaccharide extraction rate by the following formula: 2. Characterization and detection of the structure of yellow green tea polysaccharides: The total sugar content in the GTP prepared in Example 1 was determined by the phenol-sulfuric acid method; the reducing sugar content in GTP was determined by the dinitrosalicylic acid method; the protein content in GTP was determined by the BCA method; the uronic acid content in GTP was determined by the m-hydroxybiphenyl method, and the molecular weight of GTP was determined by high performance liquid chromatography.

[0032] At the same time, an infrared spectrometer was used to detect the infrared spectrum of the polysaccharide, a scanning electron microscope (SEM) was used to analyze the microstructure of the polysaccharide, Congo red was used to determine the triple helix structure of the polysaccharide, and an X-ray diffraction experiment was used to determine whether there was a crystal structure in the polysaccharide.

[0033] 3. Detection of the effect of yellow green tea polysaccharides on intestinal flora: A. Culture medium preparation: Accurately weigh 0.45 g KH2PO4, 0.45 g K2HPO4, 0.05 g NaCl, 0.064 g CaCl2·2H2O, 0.09 g MgSO4·7H2O, 2.5 g yeast extract, 10 g tryptone, 2 mL heme (5 mg / mL), 1 g L-cysteine, 200 μL vitamin I (consisting of 2 mg cobalamin (VB 12 ), 2 mg biotin (VH), 10 mg folic acid, 6 mg p-aminobenzenesulfonic acid, and 30 mg pyridoxamine (VB6) were dissolved in deionized water and the volume was adjusted to 40 mL to prepare), dissolved in deionized water and the volume was adjusted to 1 L to prepare the culture medium.

[0034] B. Preparation of test group: The prepared culture medium was divided into 6 portions, and each portion was filled with 5 mL of 25 mL vials using a peristaltic pump under nitrogen atmosphere. After high pressure steam sterilization, a single fermentation culture medium was obtained. A total of 6 groups were designed for the experiment, and the grouping and naming are shown in Table 1. Among them, the enzymatic polysaccharide group was added with a corresponding amount of yellowed green tea polysaccharide; the inulin group was a positive control group with added carbon source.

[0035] Table 1 In vitro fermentation experiment design C. Preparation of human feces fermentation liquid: In this test, a total of 12 volunteers were recruited. All volunteers had no digestive system diseases, fasting blood sugar less than 100 mg / dL (excluding prediabetes or diabetes), no history of smoking, no current medication history, and no antibiotics in the past 3 months. Volunteers were divided according to body mass index (BMI), with BMI < 25 divided into normal weight group, a total of 6 cases, and BMI ≥ 25 divided into overweight / obese group (OB), a total of 6 cases. Fecal specimens were collected from volunteers. The fecal specimens were diluted with sterilized phosphate buffer solution (PBS) to obtain a 10% fecal suspension (w / v). After centrifugation of the fecal suspension (speed 300 rpm, temperature 6 ℃, duration 5 min), 0.5 mL of the supernatant was inoculated into the culture medium, shaken and then exhausted with an air bag. Placed in a 37 ℃ constant temperature anaerobic incubator for 24 h. 1 mL of fermentation liquid was taken at 0, 12, and 24 h after the start of fermentation to determine relevant indicators.

[0036] C. pH and OD of fermentation broth 600 Value determination: Take appropriate amount of fermentation solution from each group at different times and measure its pH value with a pH meter.

[0037] OD of fecal in vitro fermentation600 When testing the OD value, set the wavelength of the spectrophotometer to 600nm, and then measure the absorbance value of the sample to obtain the OD value. 600 This value is used to evaluate the growth, metabolic activity and fermentation process of microorganisms in the fermentation product. 600 By monitoring the values, we can understand the growth of microbial communities under different conditions and thus evaluate the stability and efficiency of fermentation.

[0038] D. Determination of short-chain fatty acids (SCFAs): (1) Centrifuge the samples obtained at different times in each group (speed 12000 rpm, duration 4 min), take 500 μL of the supernatant, add 100 μL of crotonic acid-metaphosphoric acid solution, mix thoroughly and place in a -80 ℃ refrigerator for acidification for 24 h. After acidification, thaw at 4 ℃, centrifuge again (speed 10000 rpm), and take the supernatant to pass through a 0.22 μm water system microporous filter membrane; (2) Gas phase detection of fermentation broth: DB-FFAP gas chromatography column (0.32 mm × 30 m × 0.5 μm) was used for detection with crotonic acid as the internal standard.

[0039] The operating conditions of the gas chromatograph were as follows: injection port temperature 250 °C, column oven starting temperature 75 °C, temperature increased at 20 °C / min to 180 °C and maintained for 1 min, then increased at 40 °C / min to 220 °C and maintained for 1 min, FID detector temperature 250 °C, and sample loading volume 1 μL.

[0040] E. Total DNA extraction and 16S rDNA high-throughput sequencing 1.5 mL of fermentation broth after 24 h of fermentation was centrifuged and genomic DNA was extracted by precipitation. After extracting the total DNA of the sample, the primers were designed as 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT -3'), which were amplified by PCR and purified, quantified and normalized to form a sequencing library. The qualified library was sequenced using Illumina Novasek 6000. The sequencing data were analyzed using the online platform BMKCloud (https: / / www.biocloud.net). QIIME software was used for β diversity analysis, and non-metric multi-dimensional scaling (NMDS) analysis based on the Bray curtis algorithm was used. Community structure diagrams were generated based on the phylum and genus levels of species. The linear discriminant analysis effect size (LEfSe) analysis was used to compare the intestinal microbiota with significant differences between groups, and the Kruskal-Wallis test ( p <0.05) and LDA score>3.5 were used for screening, and the LDA value distribution histogram and evolutionary branch diagram were drawn.

[0041] Test results: 1. Test results of extraction rate of yellow green tea polysaccharides: The statistical graphs composed of the test results of Comparative Examples 1 to 5 and the test results of Example 1 are shown as follows: Figures 1 to 5 as shown in .

[0042] like Figure 1 As shown in the figure, when the extraction temperature is low, the inventors speculate that enzymatic extraction is the main method at this time. Therefore, when the extraction temperature exceeds 50 ℃, the enzyme activity may be reduced, which in turn leads to a decrease in the polysaccharide extraction rate. In the range of 60~80 ℃, the extraction rate of yellow green tea polysaccharides increases with the increase of temperature. The inventors speculate that ultrasonic extraction is the main method at this time. With the increase of temperature, GTP is leached in large quantities, which in turn increases the extraction rate of yellow green tea polysaccharides. After testing, after the temperature exceeds 80 ℃, the extraction rate of yellow green tea polysaccharides decreases with the increase of temperature. This may be because after exceeding 80 ℃, the activity of the enzyme is greatly reduced and can no longer cooperate with ultrasonic extraction. Therefore, only ultrasonic extraction is effective at this temperature, and the extraction rate of yellow green tea polysaccharides is also reduced.

[0043] like Figure 2As shown in , in the range of 1: (10~20) g / mL of the liquid-to-solid ratio of yellow green tea powder to the extract, the extraction rate of yellow green tea polysaccharides steadily increased and reached the highest at a liquid-to-solid ratio of 1:20 g / mL. The inventors speculate that this is due to the increase in the extraction solvent, which increases the contact area between the yellow green tea and the solvent and dissolves the water-soluble polysaccharides. However, when the liquid-to-solid ratio reaches 1:25 g / mL, the extraction rate of yellow green tea polysaccharides decreases as the liquid-to-solid ratio further increases. The inventors speculate that the addition of a large amount of solvent will affect the degree of polysaccharide gelation and its rheological properties, reducing the mass transfer of the raw materials.

[0044] like Figure 3 As shown in , when the extraction pH value increases from 4.0 to 5.5, the extraction rate of yellow green tea polysaccharides increases with the increase of the extraction pH value, and when the extraction pH value reaches 5.5, the extraction rate of yellow green tea polysaccharides reaches the highest. However, after the pH exceeds 5.5, the extraction rate of yellow green tea polysaccharides decreases with the increase of pH. The inventors speculate that this may be due to the reduction of enzyme activity at a higher pH value, which makes the extraction method lose the auxiliary assistance of enzymatic extraction, thereby reducing the extraction rate.

[0045] like Figure 4 As shown in , when the concentration of the compound enzyme added to the extraction mixture increases from 1% to 7%, the extraction rate of yellow green tea polysaccharides increases with the increase of the compound enzyme concentration, and when the compound enzyme concentration is 7%, the extraction rate of yellow green tea polysaccharides reaches the highest. However, when the compound enzyme concentration exceeds 7%, as the enzyme concentration increases, the extraction rate of yellow green tea polysaccharides decreases instead. The inventor speculates that this may be due to the fact that the substrate concentration and the enzyme reaction reach a threshold value due to the enzymatic reaction, or that the product of the enzymatic reaction causes competitive inhibition with certain substances similar to the substrate, affecting the enzymatic reaction.

[0046] like Figure 5 As shown in the figure, the extraction rate of yellow green tea polysaccharides increases with the extension of extraction time. When the enzymatic hydrolysis time is 2.5 h, the extraction rate of yellow green tea polysaccharides reaches the highest. However, when the extraction time is further increased to 3 h, the extraction rate of yellow green tea polysaccharides decreases. The inventors speculate that it may be due to the long extraction time, which leads to the decomposition of some yellow green tea polysaccharides, thereby reducing the total amount of yellow green tea polysaccharides obtained.

[0047] The extraction rate of yellow green tea polysaccharide in Example 1 was calculated to be 27.79%; the extraction rate of yellow green tea polysaccharide in Comparative Example 6 was calculated to be 17.98%; and the extraction rate of yellow green tea polysaccharide in Comparative Example 7 was calculated to be 21.54%. It can be seen that in the method used in the present invention, by correctly controlling the parameters, the two extraction methods of enzymatic extraction and ultrasonic extraction are coordinated with each other, and the extraction effect of yellow green tea is greatly improved, thereby improving the utilization rate of the raw material yellow green tea.

[0048] 2. Results of structural characterization of yellow green tea polysaccharides: like Figure 6 As shown in A, the molecular weight of GTP is relatively small. The inventors speculate that it may be due to the effects of enzymatic hydrolysis and ultrasound that the polysaccharide exists in the form of small molecules, and the polysaccharide in this form can retain biological activity and improve its cellular absorption rate. Figure 6 B is the infrared spectrum of GTP. Analysis shows that GTP contains characteristic peaks of OH, CH, -COOH and COC functional groups, indicating that GTP contains certain uronic acid and β-pyranose configurations.

[0049] like Figure 7 As shown in Figure A, there is a triple helix structure in GTP, which the inventors speculate may be the premise for some substances in GTP to have physiologically active functions. Crystal structure is usually used to evaluate the crystallization characteristics of polysaccharides and predict their physical properties, such as flexibility, swelling ability and solubility. Figure 7 B is the XRD diffraction pattern of GTP. The diffraction peak at 2θ=21.32° is sharp and strong, indicating that GTP contains microcrystals with larger particle size. GTP has a narrow and sharp characteristic peak at 26.54°, proving that GTP has a crystalline structure. There is a slightly smaller diffraction absorption peak in the range of 40°~50°, which is related to the aldehyde acid in the acidic polysaccharide. Figure 7 C~F in the figure are scanning electron microscope images of GTP, and GTP exists in irregular flakes, in which there are obvious slender chain-like knots and obvious relatively tight spherical connections. The microstructure of the yellow green tea polysaccharide extracted by the ultrasound-enzymatic hydrolysis synergistic technology used in the present invention is significantly different from the tea polysaccharides extracted in previous studies. It presents a complex winding structure, indicating that the internal voids are large and the active area is easily exposed, which makes the yellow green tea polysaccharide have a higher physiological activity. The inventor speculates that it may be related to the degradation of macromolecular polysaccharides and the rupture of polymer molecular chains by the action of ultrasound and enzymes.

[0050] 3. Test results of the effect of yellow green tea polysaccharides on intestinal flora: like Figure 8As shown in Figures A and B, there was almost no difference in pH between the NC and OC groups. The pH values ​​of the NC and OC groups gradually decreased during the fermentation process. After the addition of GTP and INL, the pH value decreased significantly (p < 0.05) and decreased significantly to 4.76 (INL group), 5.58 (NP group), 5.34 (OIN group), and 5.82 (OP group) at 12 h. Figure 8 As shown in C and D, at 0 h, the OD of all groups 600 There was no significant difference (p>0.05). After 12 h of fermentation, the OD of all groups 600 After 12 h, the OD of INL and OIN groups increased significantly (p<0.05). 600 The OD of NC and OC groups was higher than that of NP and OP groups, and continued to increase steadily. 600 The growth rate was slow during the whole fermentation process and was significantly lower than that of the NP and OP groups (p<0.05). The addition of GTP reduced the pH value of the fermentation solution, indicating that metabolites such as organic acids produced during polysaccharide fermentation would lead to changes in the living environment of intestinal flora. 600 The increase in the value indicated that the addition of GTP increased the density of microorganisms in the fermentation broth and promoted the growth of intestinal flora.

[0051] Table 2 Statistics of SCFAs content during fermentation Table 2 shows the content of SCFAs at different fermentation times, which is used to evaluate the in vitro fermentation performance of GTP in feces of different populations and the utilization level of polysaccharides by intestinal bacteria. As shown in Table 2, among all SCFAs produced by intestinal microorganisms in the fermentation of GTP, acetate has the highest content, followed by propionate and butyrate. After adding GTP or INL to the normal weight group and the obese group, the content of total acid, acetate and propionate increased significantly after 12 h and 24 h of fermentation compared with the control group (p<0.05), and the content of butyrate also increased slightly with the increase of fermentation time, while the content of isobutyrate, isovalerate and valerate did not change much compared with 0 h. In summary, after adding inulin and yellowed green tea polysaccharide GTP, intestinal flora can utilize polysaccharides for growth.

[0052] Depend on Fig. 9 A shows that a total of 4099 ASVs were obtained from the six groups of samples, of which 863 and 980 were in the NC and OC groups, respectively. Fig. 9 As shown in B, the total number of ASVs in the six groups is 136. Fig. 9The number of ASVs in the normal weight group and the obese group in C and D shows that after adding inulin and yellow green tea GTP, the number of ASVs in each group increased, especially after adding GTP in the obese group, the number of ASVs increased significantly. In addition, the number of characteristic ASVs in the INL group and the OIN group was the most abundant, followed by the NP group and the OP group, indicating that the addition of polysaccharides caused a significant impact on intestinal microorganisms.

[0053] like Fig.10 As shown in A~B, both the dilution curve and the Shannon index curve tend to be flat, reflecting the species diversity and richness in the sample and covering most of the microbial species information. The sample sequence is sufficient and meets the requirements for further analysis. Fig.10 As shown in C, there are obvious differences in richness and uniformity among the groups, indicating that the sequencing depth is sufficient. Fig.10 As shown in D, the species accumulation curve tends to flatten out during the sequencing process, indicating that the sequencing library is large enough to cover most of the bacterial diversity of all samples.

[0054] Principal component analysis of intestinal microorganisms, such as Fig.11 As shown in Figure 2, NMDS analysis showed that the intestinal flora structure of GTP and INL was different in the fecal fermentation liquid of normal weight and obese people. This indicates that the addition of GTP changes the microbial composition in the human intestine and there are certain differences with INL.

[0055] The top 10 most abundant phyla were evaluated at the phylum level (e.g. Fig.12 The microbial phyla with higher relative abundance are Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteriota. Fig.12 B shows that the addition of GTP and INL had little effect on Firmicutes, and there was no significant difference in relative abundance between the groups ( p >0.05); from Fig.12 As shown in C, after adding GTP or INL, the relative abundance of Bacteroidota in the corresponding fermentation broth increased significantly ( p <0.05). The ratio of Firmicutes to Bacteroidota (F / B) can be used as an indicator of intestinal microbial imbalance and obesity. In the NW and OB groups, the F / B value was significantly reduced after the addition of GTP and INL (e.g. Fig.12 E), indicating that the fermentation substrates GTP and INL can inhibit obesity to a certain extent. Fig.13 As shown, GTP increased the relative abundance of beneficial bacterial genera, such as Bacteroides ( Bacteroides )、Lactococcus( Lactococcus) and Faecalibacterium ( Faecalibacterium ) etc.; it also reduces harmful bacteria such as Enterobacter-Shigella ( Escherichia_Shigella ) and Dorea ( Dorea )'s relative abundance.

[0056] The distribution histogram and cladogram of LDA values ​​are shown in Fig.14 As shown in A~B. The number of bacterial species with LDA value (Log10)>3.5 in each group was plotted as a bar graph. There were 3, 9, 1, 5, 16 and 2 species with significant differences in the NC group, INL group, NP group, OC group, OIN group and OP group, respectively. The species branch evolution diagram showed that there were large differences in the distribution of bacterial communities in the six groups. At the phylum level, the Proteobacteria in the INL group was the most significantly different from the other groups. At the class level, the Proteobacteria in the INL group Gammaproteobacteria and Clostridia in the OIN group, respectively, can be used as biomarkers with significant differences at this level. Blautiah and Lachnoclostridium , OC group Dorea , FAMILy_ ХШ _UCG_001 , NC group Prevotella 、INL group Lactococcus and Incertae_Sedis , which can be used as a biomarker with significant differences at this level. unclassified_ Subdoligranulum and Dorea_longicate , NP group Bifidobacterium_longum It can be used as a biomarker indicating that they have significant differences at this level.

[0057] In summary, it can be seen that yellow green tea polysaccharide can be beneficial to the homeostasis of intestinal microorganisms, can inhibit obesity by regulating the intestinal flora of normal and obese people, and is beneficial to human health. Therefore, the inventors determined that yellow green tea polysaccharide can be used in the field of drugs related to intestinal flora regulation and weight loss assistance.

[0058] Finally, it should be noted that the above implementation cases are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned implementation cases, those skilled in the art should understand that they can still modify the technical solutions recorded in the aforementioned implementation cases, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the implementation cases of the present invention.

Claims

1. A method for extracting yellow green tea polysaccharides, characterized in that: Contains: S1. The yellow green tea is crushed, defatted, decolorized, and dried for later use; S2. In an ultrasonic device, the yellow green tea powder is mixed with the extract to obtain an extract mixture, and then a complex enzyme containing cellulase and papain is added to the extract mixture to extract to obtain a first mixture; S3. After filtering the first mixed solution, the supernatant was concentrated by rotary evaporation to obtain a second mixed solution; S4. The second mixed solution was added to ethanol for precipitation, and then centrifuged and the precipitate was re-dissolved in water to obtain a third mixed solution; S5. Sevage reagent is added to the third mixed solution to remove protein to obtain a deproteinized mixed solution, and then the deproteinized mixed solution is subjected to rotary evaporation to remove the sevage reagent and concentrated to obtain a concentrate; S6, dialyzing and drying the concentrated solution to obtain yellowed green tea polysaccharide.

2. The method for extracting yellow green tea polysaccharides according to claim 1, characterized in that: In S2, the yellowed green tea powder and the extract are mixed in a ratio of liquid to material of 1: (10-20) to obtain the extraction mixture.

3. The method for extracting yellow green tea polysaccharides according to claim 2, characterized in that: In S2, 3-7% of the composite enzyme is added to the extraction mixture, and the mass ratio of cellulase to papain in the composite enzyme is (2.5-3.5):

1.

4. The method for extracting yellow green tea polysaccharides according to claim 3, characterized in that: In S2, the first mixed solution is obtained by extracting at an extraction temperature of 60-80° C. and an extraction pH of 4.0-5.5 for 2-2.5 hours.

5. The method for extracting yellow green tea polysaccharides according to claim 1, characterized in that: In S2, the ultrasonic frequency of the ultrasonic device is 80 kHz.

6. The method for extracting yellow green tea polysaccharides according to claim 1, characterized in that: In S4, the volume ratio of the deproteinized mixed solution to the ethanol is 1:(4-5).

7. The method for extracting yellow green tea polysaccharides according to claim 1, characterized in that: In S5, the volume ratio of chloroform to n-butanol in the sevage reagent is (4-5):

1.

8. The method for extracting yellow green tea polysaccharides according to claim 1, characterized in that: In S1, the yellow green tea is crushed and passed through a 80-100 mesh sieve to obtain yellow green tea powder, and the yellow green tea powder is soaked in 95% ethanol for degreasing and decolorization, and then naturally air-dried.

9. A yellow green tea polysaccharide, characterized in that: The polysaccharide is prepared by the method for extracting yellowed green tea polysaccharides according to any one of claims 1 to 8.

10. Use of the yellowed green tea polysaccharide as claimed in claim 9 in the field of medicine for regulating intestinal flora and assisting weight loss.

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