A yellow green tea polysaccharide and its extraction method and application

Abstract

The present invention relates to the technical field of active ingredient extraction, and specifically discloses a yellow green tea polysaccharide and its extraction method and application, comprising the following steps: S1. defatting and decolorizing yellow green tea powder and drying for standby use; S2. mixing tea powder with an extracting solution in an ultrasonic device to obtain an extraction mixture, and then adding a complex enzyme to the extraction mixture for extraction to obtain a first mixture; S3. filtering the first mixture, taking the supernatant, and concentrating it by rotary evaporation to obtain a second mixture; S4. precipitating the second mixture with alcohol, centrifuging, and re-dissolving the precipitate to obtain a third mixture; S5. adding sevage reagent to the third mixture to obtain a deproteinized mixture, and then rotary evaporating to obtain a concentrated solution; S6, dialyzing and drying the concentrated solution to obtain yellow green tea polysaccharide. In the present invention, the enzymatic extraction step and the ultrasonic extraction step are carried out simultaneously. By adjusting the relevant processing parameters, the two extraction methods can be coordinated with each other, so that the extraction efficiency of the yellow green tea polysaccharide is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of active ingredient extraction, and in particular to a 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 acids and proteins in tea. They have multiple beneficial effects on human health, such as anti-obesity activity, hypoglycemic activity, and lipid-lowering activity.

[0003] The main methods for extracting tea polysaccharides currently include hot water extraction, enzymatic extraction, and ultrasonic extraction. Hot water extraction is low-cost and simple to operate, but it is time-consuming and labor-intensive. Enzyme-assisted extraction can effectively catalyze the degradation of cell walls, allowing plant cells to release intracellular polysaccharides. This method can achieve tea polysaccharide extraction at a relatively low cost. In the prior art, ultrasonic extraction is often combined with thermal extraction to shorten the extraction time and increase 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 beneficial to the human body. However, the limited yield per acre of Zhonghuang No. 1 tea and its high price have hindered its widespread promotion and application. For this expensive tea, improving the extraction rate of nutrients and thus increasing its utilization efficiency remains a challenge. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for extracting polysaccharides from yellow green tea. 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:

[0007] A method for extracting xanthocyanidin green tea polysaccharides comprises the following steps: S1. crushing xanthocyanidin green tea, defatting and decolorizing the same, and drying for later use; S2. mixing xanthocyanidin green tea powder with an extracting solution in an ultrasonic device to obtain an extraction mixture, and then adding a complex enzyme containing cellulase and papain to the extraction mixture for extraction to obtain a first mixture; S3. filtering the first mixture, taking the supernatant, and concentrating it by rotary evaporation to obtain a second mixture; S4. adding ethanol to the second mixture for precipitation, centrifuging it, and re-dissolving the precipitate in water to obtain a third mixture; S5. adding sevage reagent to the third mixture to remove protein to obtain a deproteinized mixture, and then rotary evaporating the deproteinized mixture to remove the sevage reagent and concentrating it to obtain a concentrated solution; and S6. dialyzing the concentrated solution and drying it to obtain xanthocyanidin green tea polysaccharides.

[0008] As a further improvement of the present invention, in S2, the yellowed green tea powder and the extract are mixed at a liquid-to-solid ratio of 1: (10-20) to obtain the extraction mixture.

[0009] As a further improvement of the present invention, in S2, 3-7% of the complex enzyme is added to the extraction mixture, and the mass ratio of cellulase to papain in the complex enzyme is (2.5-3.5):1.

[0010] As a further improvement of the present invention, in S2, the first mixed solution is obtained by extracting at an extraction temperature of 60-80° C. and a pH value of 4.0-5.5 for 2-2.5 hours.

[0011] As a further improvement of the present invention, in S2, the ultrasonic frequency of the ultrasonic device is 80 kHz.

[0012] As a further improvement of the present invention, in S4, the volume ratio of the deproteinized mixture to the ethanol is 1:(4-5).

[0013] 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.

[0014] As a further improvement of the present invention, in S1, the yellowed green tea is crushed and passed through an 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.

[0015] In a second aspect, the present invention provides a xanthocyanidin green tea polysaccharide prepared by any one of the above-mentioned xanthocyanidin green tea polysaccharide extraction methods.

[0016] 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.

[0017] Ultrasonic extraction and enzymatic extraction are currently widely used in the extraction of natural plant polysaccharides. While there are cases where both methods are used sequentially, there are relatively few cases where they are used simultaneously. The extraction method described in the present invention can quickly and efficiently destroy the cell walls of etiolated green tea, thereby releasing the active ingredients within as much as possible.

[0018] The present invention utilizes a composite enzyme to decompose the cellulose and hemicellulose in the cell walls, allowing the tea polysaccharides encapsulated in the cell walls to be precipitated as much as possible; and utilizes the cavitation effect, thermal effect, and mechanical effect of ultrasound to assist enzymatic hydrolysis. Specifically, the present invention performs ultrasonic treatment on the yellowed green tea powder while cellulase and papain are performing enzymatic hydrolysis. When ultrasonic waves act on the tea cells, the tea cells are destroyed and torn, thereby forming a large number of small cavities thereon. These small cavities are torn open and then instantly closed, but during this period, a transient pressure of up to several thousand atmospheres is generated, which can instantly rupture the cell walls and organisms in the tea powder, forcing the tea polysaccharides to be released and flow out. At the same time, the cellulase and papain that decompose the cellulose and protein in the enzymatically hydrolyzed tea cells can also enter the cavities, further accelerating the decomposition of the cell tissue, thereby facilitating the release of tea polysaccharides, thereby greatly shortening the extraction time of the tea polysaccharides and improving the extraction efficiency of the tea polysaccharides.

[0019] The yellowed green tea polysaccharide prepared by the above extraction method can exist in a low molecular weight form, 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 discovered 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 Shigella spp. ( Escherichia_Shigella ) and Dorea ( Dorea ), so the inventors confirmed that yellow 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

[0020] The following drawings are provided for use in conjunction with preferred embodiments of the present invention to help understand the objects and advantages of the present invention, wherein:

[0021] Figure 1 This is a statistical chart showing the extraction rate of yellow green tea polysaccharide in comparative example 1 combined with the extraction rate of yellow green tea polysaccharide in example 1;

[0022] Figure 2This is a statistical chart showing the extraction rate of yellow green tea polysaccharide in comparative example 2 combined with the extraction rate of yellow green tea polysaccharide in example 1;

[0023] Figure 3 This is a statistical chart showing the extraction rate of yellow green tea polysaccharide in comparative example 3 combined with the extraction rate of yellow green tea polysaccharide in example 1;

[0024] Figure 4 This is a statistical chart showing the extraction rate of yellow green tea polysaccharide in comparative example 4 combined with the extraction rate of yellow green tea polysaccharide in example 1;

[0025] Figure 5 This is a statistical chart showing the extraction rate of yellow green tea polysaccharide in comparative example 5 combined with the extraction rate of yellow green tea polysaccharide in example 1;

[0026] Figure 6 It is a combination of molecular weight graph and IR spectrum graph of GTP;

[0027] Figure 7 The combined image of Congo red, scanning electron microscope and XRD patterns of GTP;

[0028] Figure 8 is the pH and OD during in vitro fermentation 600 Value result statistics chart;

[0029] Figure 9 This is a statistical chart showing the effects of yellow green tea polysaccharides on intestinal flora ASV;

[0030] Figure 10 This is the sequencing curve of the intestinal flora;

[0031] Figure 11 NMDS analysis of fecal fermentation liquid microbiome in different groups;

[0032] Figure 12 This is a statistical chart showing the effects of GTP on the intestinal flora at the phylum level;

[0033] Figure 13 The statistical diagram shows the effect of GTP on the genus level of intestinal flora;

[0034] Figure 14 Statistical chart of LEfSe analysis results among intestinal bacterial groups. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and implementation examples.

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, rather than to represent all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0037] Example 1:

[0038] This embodiment provides a method for extracting yellow green tea polysaccharides, comprising the following steps:

[0039] S1. The yellow green tea was crushed and passed through an 80-mesh sieve to obtain yellow green tea powder. The yellow green tea powder was soaked in 95% ethanol for 2 h for decolorization and defatting, and then removed and air-dried for later use;

[0040] S2. In an ultrasonic apparatus, at an ultrasonic frequency of 80 kHz, an extraction temperature of 80°C, and an extraction pH of 5.5, yellow green tea powder and an extract were prepared at a liquid-to-solid ratio of 1:20 g / mL to obtain an extraction mixture. Then, 7% of a complex enzyme was added to the extraction mixture and mixed for 2.5 hours to obtain a first mixture, wherein the complex enzyme comprised cellulase and papain in a mass ratio of 3:1.

[0041] S3. The first mixed solution was filtered and the supernatant was concentrated by rotary evaporation to obtain a second mixed solution;

[0042] S4. The second mixture was poured into ethanol for precipitation, wherein the volume ratio of the deproteinized mixture and ethanol was 1:4. After the precipitation was completed, the mixture was centrifuged, the precipitate was re-dissolved in water to obtain a third mixture;

[0043] S5. Sevage reagent is added to the third mixed solution to remove protein to obtain a deproteinized mixed solution, and the deproteinized mixed solution is then subjected to rotary evaporation to remove the sevage reagent and concentrated to obtain a concentrate, wherein the sevage reagent comprises chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is 4:1;

[0044] S6. The concentrated solution is dialyzed with deionized water and then dried to obtain yellowed green tea polysaccharide (hereinafter referred to as GTP).

[0045] 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.

[0046] Comparative Example 1:

[0047] 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.

[0048] Comparative Example 2:

[0049] The difference between this comparative example and Example 1 is that in this example, the liquid-to-solid ratio of the yellowed green tea powder to the extract is 1:10, 1:15, 1:25, and 1:30 g / mL, respectively, to prepare an extraction mixture for performing the extraction step in S2.

[0050] Comparative Example 3:

[0051] 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.

[0052] Comparative Example 4:

[0053] The difference between this comparative example and Example 1 is that, in this example, the extraction step in S2 is performed under the conditions of adding the complex enzyme at concentrations of 1%, 3%, 5% and 9%, respectively.

[0054] Comparative Example 5:

[0055] 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.

[0056] Comparative Example 6:

[0057] 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.

[0058] Comparative Example 7:

[0059] Conventional enzymatic 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.

[0060] Results test:

[0061] 1. Extraction rate test of yellow green tea polysaccharide:

[0062] The extraction rate of yellow green tea polysaccharides was tested for the GTP extracted in Example 1 and Comparative Examples 1 to 5. This testing step first 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". Finally, the yellow green tea polysaccharide extraction rate was calculated using the following formula:

[0063]

[0064] 2. Structural characterization of yellow green tea polysaccharides:

[0065] The total sugar content of the GTP prepared in Example 1 was determined by the phenol-sulfuric acid method; the reducing sugar content of the GTP was determined by the dinitrosalicylic acid method; the protein content of the GTP was determined by the BCA method; the uronic acid content of the GTP was determined by the m-hydroxybiphenyl method, and the molecular weight of the GTP was determined by high performance liquid chromatography.

[0066] 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.

[0067] 3. Detection of the effects of yellow green tea polysaccharides on intestinal flora:

[0068] A. Culture medium preparation:

[0069] 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 (composed 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 the culture medium.

[0070] B. Preparation of the experimental group:

[0071] The prepared culture medium was divided into six portions and filled into 25 mL vials using a peristaltic pump under nitrogen atmosphere, with each portion filled with 5 mL. After autoclaving, individual fermentation cultures were prepared. Six groups were designed for this experiment; the groups and nomenclature are shown in Table 1. The enzymatic polysaccharide group included the addition of a corresponding amount of yellowed green tea polysaccharide; the inulin group served as a positive control group supplemented with a carbon source.

[0072] Table 1 In vitro fermentation experiment design

[0073]

[0074] C. Preparation of human feces fermentation liquid:

[0075] Twelve volunteers were recruited for this study. All volunteers had no digestive system diseases, fasting blood glucose levels below 100 mg / dL (excluding prediabetes or diabetes), no history of smoking, no current medication use, and no antibiotic use within the past three months. Volunteers were divided according to body mass index (BMI): 6 subjects with a BMI <25 were classified as normal weight, and 6 subjects with a BMI ≥25 were classified as overweight / obese (OB). Fecal specimens were collected from the volunteers. The stool specimens were diluted with sterile phosphate-buffered saline (PBS) to obtain a 10% (w / v) stool suspension. After centrifugation (300 rpm, 6°C, 5 min), 0.5 mL of the supernatant was inoculated into culture medium, shaken, and then deflated using an air bag. The culture was incubated in a 37°C anaerobic incubator for 24 h. 1 mL of fermentation broth was collected at 0, 12, and 24 h after the start of fermentation for determination of relevant parameters.

[0076] C. pH and OD of fermentation broth 600 Value determination:

[0077] Take appropriate amount of fermentation solution from each group at different times and measure its pH value with a pH meter.

[0078] OD of fecal in vitro fermentation 600 When detecting 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.

[0079] D. Determination of Short-Chain Fatty Acids (SCFAs):

[0080] (1) The samples obtained at different times in each group were centrifuged (speed of 12000 rpm, duration of 4 min), 500 μL of the supernatant was taken, 100 μL of crotonic acid-metaphosphoric acid solution was added, the mixture was thoroughly mixed, and the mixture was placed in a -80 ℃ refrigerator for acidification for 24 h. After acidification, the mixture was thawed at 4 ℃ and centrifuged again (speed of 10000 rpm). The supernatant was filtered through a 0.22 μm water-based microporous membrane.

[0081] (2) Gas chromatography detection of the fermentation broth was performed using a DB-FFAP gas chromatography column (0.32 mm × 30 m × 0.5 μm) with crotonic acid as the internal standard.

[0082] 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 volume 1 μL.

[0083] E. Total DNA extraction and 16S rDNA high-throughput sequencing

[0084] 1.5 mL of the fermentation broth after 24 h of fermentation was centrifuged and genomic DNA was extracted from the precipitate. After extracting the total DNA of the sample, primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT -3') were designed. The DNA was amplified by PCR and purified, quantified and normalized to form a sequencing library. The library that passed the quality inspection was sequenced using Illumina Novasek 6000. The sequencing data was 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 microbial communities 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.

[0085] Test results:

[0086] 1. Test results of the extraction rate of yellow green tea polysaccharides:

[0087] 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 .

[0088] like Figure 1As shown in the figure, when the extraction temperature is low, the inventors speculate that enzymatic extraction is dominant. Therefore, when the extraction temperature exceeds 50°C, enzyme activity may decrease, leading to a decrease in polysaccharide extraction yield. In the 60-80°C range, the extraction yield of yellow green tea polysaccharides increases with increasing temperature. The inventors speculate that ultrasonic extraction is dominant at this time, and the increase in temperature leads to a large amount of GTP leaching, which in turn increases the extraction yield of yellow green tea polysaccharides. However, after testing, the extraction yield of yellow green tea polysaccharides decreases with increasing temperature above 80°C. This may be because above 80°C, enzyme activity decreases significantly and no longer cooperates with ultrasonic extraction. Therefore, at this temperature, only ultrasonic extraction is effective, resulting in a decrease in the extraction yield of yellow green tea polysaccharides.

[0089] like Figure 2 As shown in the results, the extraction yield of polysaccharides from yellow tea powder steadily increased within a liquid-to-liquid ratio of 1:(10-20) g / mL for yellow tea powder and extract, reaching its highest yield at a liquid-to-liquid ratio of 1:20 g / mL. The inventors speculate that this is due to the increased amount of extraction solvent, which increases the contact area between the yellow tea powder and the solvent, leading to the dissolution of water-soluble polysaccharides. However, when the liquid-to-liquid ratio reaches 1:25 g / mL, the extraction yield of polysaccharides from yellow tea decreases as the liquid-to-liquid ratio increases further. The inventors speculate that the addition of large amounts of solvent affects the degree of polysaccharide gelation and its rheological properties, reducing the mass transfer of the raw material.

[0090] like Figure 3 As shown in the results, the extraction rate of xanthocyanidin polysaccharides increased with the increase of extraction pH from 4.0 to 5.5, and reached its highest extraction rate at 5.5. However, after the pH exceeded 5.5, the extraction rate of xanthocyanidin polysaccharides decreased with increasing pH. The inventors speculate that this may be because the enzyme activity is reduced at higher pH values, which makes the extraction method lose the auxiliary function of enzymatic hydrolysis extraction, resulting in a lower extraction rate.

[0091] like Figure 4 As shown in Figure 1, as the concentration of the complex enzyme added to the extraction mixture increased from 1% to 7%, the extraction rate of yellow green tea polysaccharides increased with the increase in complex enzyme concentration, reaching its highest extraction rate at 7%. However, when the complex enzyme concentration exceeded 7%, the extraction rate of yellow green tea polysaccharides actually decreased as the enzyme concentration increased. The inventors speculate that this may be due to the enzymatic reaction causing the substrate concentration and enzyme reaction to reach a threshold, or the product of the enzymatic reaction and certain substances similar to the substrate causing competitive inhibition, affecting the enzymatic reaction.

[0092] like Figure 5As shown in Figure 1, the extraction rate of xanthocyanidin polysaccharides increased with the extension of extraction time, reaching its highest extraction rate at 2.5 hours. However, when the extraction time was further increased to 3 hours, the extraction rate of xanthocyanidin polysaccharides actually decreased. The inventors speculate that this may be due to the excessively long extraction time, which caused some xanthocyanidin polysaccharides to be decomposed, thereby reducing the total amount of xanthocyanidin polysaccharides obtained.

[0093] The calculated extraction rate of the yellow green tea polysaccharide in Example 1 was 27.79%; the calculated extraction rate of the yellow green tea polysaccharide in Comparative Example 6 was 17.98%; and the calculated extraction rate of the yellow green tea polysaccharide in Comparative Example 7 was 21.54%. Thus, it can be seen that the method used in the present invention, through the correct control of parameters, promotes the mutual cooperation of the two extraction methods of enzymatic extraction and ultrasonic extraction, achieves a significant improvement in the extraction effect of the yellow green tea, and thus increases the utilization rate of the raw material yellow green tea.

[0094] 2. Structural characterization test results of yellow green tea polysaccharide:

[0095] like Figure 6 As shown in Figure A, the molecular weight of GTP is relatively small. The inventors speculate that this may be due to the effects of enzymatic hydrolysis and ultrasound, which causes the polysaccharide to exist in the form of small molecules. In this form, the polysaccharide can retain its 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.

[0096] like Figure 7 As shown in Figure A, GTP contains a triple helical structure, which the inventors speculate may be the prerequisite for the physiological activity of some substances in GTP. Crystal structure is often used to evaluate the crystallization characteristics of polysaccharides and predict their physical properties, such as flexibility, swelling capacity, 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 in the acidic polysaccharide. Figure 7Figures C-F are scanning electron microscope images of GTP, showing that GTP exists as irregular flakes with distinct elongated chain-like junctions and relatively tight spherical connections. The microstructure of the etiolated green tea polysaccharide extracted using the ultrasound-enzymatic hydrolysis synergistic technique used in this invention differs significantly from that of tea polysaccharides extracted in previous studies. Its complex, entangled structure suggests that its internal voids are relatively large, exposing active areas and contributing to its high physiological activity. The inventors speculate that this may be due to the action of ultrasound and enzymes, which degrade the macromolecular polysaccharide and break down the polymer chains.

[0097] 3. Test results of the effect of yellow green tea polysaccharides on intestinal flora:

[0098] like Figure 8 As 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 600 After 12 h, the OD values ​​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 will 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.

[0099] Table 2 Statistics of SCFAs content during fermentation

[0100]

[0101] Table 2 shows the SCFA content at different fermentation times, which was used to evaluate the in vitro fermentation performance of GTP in feces from different populations and the utilization of polysaccharides by intestinal bacteria. As shown in Table 2, among all SCFAs produced by intestinal microorganisms through GTP fermentation, acetate was the most abundant, followed by propionate and butyrate. In the normal-weight and obese groups, the addition of GTP or INL significantly increased the levels of total acid, acetate, and propionate after 12 and 24 h of fermentation compared with the control group (p < 0.05). Butyrate also increased slightly with increasing fermentation time, while the levels of isobutyrate, isovalerate, and valerate remained largely unchanged compared with 0 h. In summary, the addition of inulin and the yellowed green tea polysaccharide GTP enables intestinal microbiota to utilize polysaccharides for growth.

[0102] Depend on Figure 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. Figure 9 As shown in B, the total number of ASVs in the six groups is 136. Figure 9 The number of ASVs in the normal-weight and obese groups in Figures C and D shows that the number of ASVs increased in all groups after the addition of inulin and yellow green tea GTP, especially in the obese group, where the number of ASVs increased significantly after the addition of GTP. Furthermore, the INL and OIN groups had the most abundant characteristic ASVs, followed by the NP and OP groups, indicating that the addition of polysaccharides significantly affected the intestinal microbiome.

[0103] like Figure 10 As shown in A~B, the dilution curve and the Shannon index curve are both flat, reflecting the species diversity and richness in the sample and covering the vast majority of microbial species information. The sample sequence is sufficient and meets the requirements for further analysis. Figure 10 As shown in C, there are obvious differences in richness and uniformity among the groups, indicating that the sequencing depth is sufficient. Figure 10 As shown in D, the species accumulation curve tends to flatten during the sequencing process, indicating that the sequencing library is large enough to cover most of the bacterial diversity of all samples.

[0104] Principal component analysis of intestinal microorganisms, such as Figure 11 NMDS analysis revealed that the intestinal microbial communities in fecal fermentation broths from normal-weight and obese individuals differed between GTP and INL. This suggests that GTP supplementation alters the microbial composition in the human gut and creates differences with INL.

[0105] The top 10 most abundant phyla were evaluated at the phylum level (e.g. Figure 12The microbial phyla with higher relative abundance are Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteriota. Figure 12 B shows that the addition of GTP and INL has little effect on Firmicutes, and there is no significant difference in the relative abundance between the groups ( p >0.05); from Figure 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. Figure 12 E), indicating that the fermentation substrates GTP and INL can inhibit obesity to a certain extent. Figure 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.

[0106] The distribution histogram and evolutionary branch diagram of LDA values ​​are shown in Figure 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 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.

[0107] In summary, it can be seen that etiolated green tea polysaccharides can promote intestinal microbial homeostasis and can inhibit obesity by regulating the intestinal flora of both normal and obese people, thus benefiting human health. Therefore, the inventors have determined that etiolated green tea polysaccharides can be used in the field of drugs related to intestinal flora regulation and weight loss assistance.

[0108] 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 above implementation cases, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above 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. An application of yellow green tea polysaccharide in the preparation of drugs for regulating intestinal flora and assisting weight loss, characterized in that: Lutinoic green tea polysaccharide is prepared by the following extraction method: S1. The yellow green tea is crushed, defatted, decolorized, and dried for later use; S2 in an ultrasonic device, the yellow green tea powder and the extract were mixed to obtain an extract mixture, and then to the extract mixture was added a complex enzyme containing cellulase and papain to extract to obtain a first mixture; S3. The first mixed solution was filtered and the supernatant was concentrated by rotary evaporation to obtain a second mixed solution; S4. The second mixture was added to ethanol for precipitation, and then centrifuged and the precipitate was re-dissolved in water to obtain a third mixture; S5. Sevage reagent was added to the third mixture to remove protein to obtain a deproteinized mixture, and the deproteinized mixture was then subjected to rotary evaporation to remove sevage reagent and concentrated to obtain a concentrate; S6, dialyzing the concentrated solution and drying it to obtain yellowed green tea polysaccharide.

2. The use of a yellow green tea polysaccharide according to claim 1 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S2, the yellowed green tea powder and the extract are mixed in a liquid-to-solid ratio of 1:(10-20) to obtain the extraction mixture.

3. The use of a yellow green tea polysaccharide according to claim 2 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S2, 3-7% of the complex enzyme is added to the extraction mixture, wherein the mass ratio of cellulase to papain in the complex enzyme is (2.5-3.5):

1.

4. The use of a yellow green tea polysaccharide according to claim 3 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S2, the first mixed solution is obtained by extracting at an extraction temperature of 60 to 80° C. and an extraction pH of 4.0 to 5.5 for 2 to 2.5 hours.

5. The use of a yellow green tea polysaccharide according to claim 1 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S2, the ultrasonic frequency of the ultrasonic device is 80 kHz.

6. The use of a yellow green tea polysaccharide according to claim 1 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S4, the volume ratio of the deproteinized mixed solution to the ethanol is 1:(4-5).

7. The use of a yellow green tea polysaccharide according to claim 1 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S5, the volume ratio of chloroform to n-butanol in the sevage reagent is (4-5):

1.

8. The use of a yellow green tea polysaccharide according to claim 1 in the preparation of a drug for regulating intestinal flora and assisting weight loss, characterized in that: In S1, the yellow green tea is crushed and passed through an 80-100 mesh sieve to obtain yellow green tea powder, which is then soaked in 95% ethanol for degreasing and decolorization, and then naturally air-dried.

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