A dark tea polysaccharide, its preparation method and application

Abstract

The present invention provides a dark tea polysaccharide, a preparation method thereof and an application thereof, belonging to the technical field of extraction of active ingredients of tea and efficacy research. The repeatable primary structural unit of the dark tea polysaccharide contains eight sugar residues, namely →4)-α-D-GalpA-6-O-Me-(1→, α-L-Araf-(1→, →3,4)-β-D-Galp-(1→, 3-O-Me-β-D-Galp-(1→, →4,6)-α-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→, →2,4)-α-L-Rhap-(1→, and the connection mode of the sugar residues is as shown in Formula 1. The dark tea polysaccharide has excellent prebiotic activity and the effects of reducing blood sugar and fat. The preparation method has mild process conditions, simple operation, high purity of the obtained polysaccharide and stable structure, and is suitable for industrial-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and efficacy research of active ingredients of tea, and particularly relates to a dark tea polysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Tea generally refers to a beverage made from the buds and leaves of the tea tree (an evergreen shrub of the genus Camellia, Camellia sinensis (L.) O. Kuntze) through processing. Because of its unique active functional components, unique aroma and taste, it is loved by more and more consumers and is currently one of the most popular low-calorie and alcohol-free beverages in the world. A large number of studies have shown that tea polysaccharide is the main functional component for tea to exert various biological activities such as antioxidant, anti-inflammatory, immunomodulatory and liver protection. In particular, the research on tea polysaccharide in regulating metabolic syndromes such as intestinal flora, lowering blood sugar and controlling obesity has attracted much attention, indicating that tea polysaccharide has broad application prospects as a prebiotic in the development of intestinal flora regulators and new drugs for lowering blood sugar and reducing fat. Dark tea is a special type of tea in China. Due to the particularity of microbial fermentation during the processing process, the structure and biological activity of its polysaccharide are different from those of other teas. At present, there has been no research on the dark tea polysaccharide fermented by pure bacteria of Eurotium cristatum, and the main active components and efficacy of the polysaccharide need to be clarified urgently.

[0003] The chemical structure of polysaccharide is the basis for its biological activity. At present, almost all the dark tea polysaccharides involved are crude polysaccharides extracted from traditional dark tea. So far, the structural characteristics and pharmacological effects of the dark tea polysaccharide fermented by Eurotium cristatum are still unclear, and there is a lack of a suitable extraction and purification process to prepare a dark tea polysaccharide with a clear chemical structure, uniform composition, prebiotic activity and blood sugar lowering and fat reducing functions. These all limit the research and development of dark tea polysaccharide in regulating intestinal flora imbalance and related health products and new drugs for lowering blood sugar and reducing fat. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a dark tea polysaccharide, a preparation method thereof, and an application thereof. The dark tea polysaccharide has a specific glycosidic bond structure and excellent intestinal probiotic activity and the effects of losing weight, lowering blood sugar and reducing fat.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a dark tea polysaccharide. The repeatable primary structural unit of the dark tea polysaccharide contains eight sugar residues: →4)-α-D-GalpA-6-O-Me-(1→, α-L-Araf-(1→, →3,4)-β-D-Galp-(1→, 3-O-Me-β-D-Galp-(1→, →4,6)-α-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→, →2,4)-α-L-Rhap-(1→. The connection mode of the sugar residues is shown in Formula 1:

[0007]

[0008] Preferably, the relative molecular mass of the dark tea polysaccharide is 1.28×10 4 Da. The dark tea polysaccharide is composed of rhamnose, arabinose, glucose, galactose and galacturonic acid. The molar ratio of rhamnose, arabinose, glucose, galactose and galacturonic acid is 12.35:23.23:21.25:31.90.

[0009] The present invention also provides a preparation method of the dark tea polysaccharide, comprising the following steps:

[0010] (1) Mix dark tea with absolute ethanol, filter to collect the precipitate, and dry to obtain dark tea powder;

[0011] (2) Mix the dark tea powder with water for extraction, filter to collect the filtrate to obtain a water extract of dark tea;

[0012] (3) Concentrate and centrifuge the water extract of dark tea, collect the supernatant concentrate, then mix it with absolute ethanol for alcohol precipitation, filter to collect the precipitate to obtain a dark tea alcohol precipitate;

[0013] (4) After mixing the dark tea alcohol precipitate with water, add protease for enzymatic hydrolysis. After enzymatic hydrolysis, add Sevag reagent to collect the supernatant polysaccharide solution, then adsorb it with macroporous resin overnight, collect the eluate, dialyze and dry to obtain crude dark tea polysaccharide;

[0014] (5) Subject the crude dark tea polysaccharide to anion exchange column chromatography for separation, collect the polysaccharide-containing eluate to obtain a dark tea polysaccharide fraction;

[0015] (6) Purify the dark tea polysaccharide fraction by gel column chromatography to obtain the dark tea polysaccharide.

[0016] Preferably, in step (1), the dark tea is derived from dark tea fermented by Eurotium cristatum; the material-liquid ratio of the dark tea to absolute ethanol is 1 g:15 - 25 mL; the mixing method is stirring at 20 - 30 °C and 100 - 140 rpm for 10 - 14 h;

[0017] In step (2), the material-liquid ratio of the dark tea powder to water is 1 g: 15-25 mL; the extraction temperature is 60-90 °C, the number of extractions is 2-3 times, and the extraction time for each extraction is independently 3-5 h;

[0018] In step (3), it is concentrated to 1 / 3-1 / 4 of the original volume of the aqueous extract of dark tea; the volume ratio of the supernatant concentrate to absolute ethanol is 1: 3-5.

[0019] Preferably, in step (4), the material-liquid ratio of the dark tea alcohol precipitate to water is 10 g: 90-110 mL; the protease includes papain, the enzymatic hydrolysis temperature is 35-40 °C, and the enzymatic hydrolysis time is 6-8 h; the Sevag reagent is prepared from chloroform and n-butanol in a volume ratio of 4: 1, and the number of times of protein removal using the Sevag reagent is 4-6 times, and the volume of the Sevag reagent added each time is 20-25 mL; the macroporous resin includes macroporous resin AB-8; the dialysis bag for dialysis is a dialysis bag with a molecular weight cut-off of 3400-3600 Da, and the dialysis time is 20-28 h.

[0020] Preferably, in step (5), the anion exchange column is DEAE seplife FF, the sample loading concentration is 40-100 mg / mL, the elution method is to perform water elution, 0.1 mol / L NaCl, 0.2 mol / L NaCl, and 0.3 mol / L NaCl gradient elution in sequence, the elution flow rate is 3-5 mL / min, and the elution time for each component is 40-70 min; the polysaccharide-containing eluate is the eluate obtained by eluting with 0.2 mol / L NaCl.

[0021] Preferably, in step (6), the column used for gel column chromatography is Sephacryl S-400HR, the sample loading concentration is 20-40 mg / mL, the eluent for chromatography is water, the elution flow rate is 0.5-1.5 mL / min, and the elution time is 4-6 h.

[0022] The present invention also provides an application of the above-mentioned dark tea polysaccharide or the dark tea polysaccharide prepared by the above-mentioned preparation method, and the application includes at least one of the following;

[0023] (a) The application of the dark tea polysaccharide in the preparation of intestinal flora regulators;

[0024] (b) The application of the dark tea polysaccharide in the preparation of hypoglycemic products;

[0025] (c) The application of the dark tea polysaccharide in the preparation of fat-reducing products.

[0026] The present invention also provides a pharmaceutical composition for regulating intestinal flora and reducing blood sugar and fat, comprising the above-mentioned dark tea polysaccharide or the dark tea polysaccharide prepared by the above-mentioned preparation method and pharmaceutically acceptable excipients.

[0027] Preferably, the oral dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, dripping pill, pill or powder.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention provides a dark tea polysaccharide, its preparation method and application. The dark tea polysaccharide has a novel structure, a clear chemical structure and a uniform composition. It can significantly increase the relative abundances of intestinal probiotics Akkermansia, Bifidobacterium, Ruminococcus_torques_group and Faecalibaculum, and down-regulate the relative abundances of harmful bacteria genera Ileibacterium, Coriobacteriaceae_UCG-002, unclassified_f__Atopobiaceae and Lachnospiraceae_NK4A136_group. It inhibits the weight gain and blood sugar and lipid levels induced by high-fat diet, and itself has no toxic and side effects on normal organisms. It has excellent prebiotic activity and the effects of reducing blood sugar and fat. It solves the problem that dark tea lacks active ingredients with a single composition and a clear structure in the research and development of new drugs for regulating intestinal flora and reducing blood sugar and fat. It lays a reliable material foundation for the research on the structure-activity relationship of the intestinal probiotic activity and blood sugar and fat reduction of dark tea polysaccharide, and plays a promoting role in the research and development of polysaccharides as preparations for regulating flora disorders and new drugs for reducing blood sugar and fat.

[0030] The present invention provides a preparation method of the dark tea polysaccharide. The process of combining anion exchange resin and gel column chromatography is used to prepare a dark tea polysaccharide with intestinal probiotic activity and the functions of reducing blood sugar and fat. The process conditions are mild, the operation is simple, and the environment is friendly. The obtained polysaccharide has high purity and stable structure, and maximally maintains the activity of the polysaccharide, which is suitable for industrial-scale production.

[0031] The yield of the dark tea polysaccharide extracted by the method of the present invention is above 1.76%, and the purity of the dark tea polysaccharide is above 96.1%. Description of the Drawings

[0032] Figure 1It is the elution curve of dark tea polysaccharide DTPS1; A is the elution curve of dark tea polysaccharide DTPS1 on a DEAE seplife FF ion exchange chromatography column, where the red line represents four elution gradients of ultrapure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, and 0.3 mol / L NaCl solution, and the blue line is the sugar-containing elution peak eluted from each component; B is the elution curve of dark tea polysaccharide DTPS1 on a Sephacryl S-400HR gel column;

[0033] Figure 2 In it, A is the ultraviolet (UV) full wavelength scan chart of dark tea polysaccharide DTPS1, and B is the GPC-RI-MALS chromatogram, where the red line: multi-angle laser light scattering signal (RI); the blue line: differential signal (SLS);

[0034] Figure 3 It is the scanning electron microscope (SEM) image of dark tea polysaccharide DTPS1;

[0035] Figure 4 It is the Fourier transform infrared spectroscopy (FT-IR) chart of dark tea polysaccharide DTPS1;

[0036] Figure 5 It is the ion chromatography (IC) chart of the monosaccharide composition of dark tea polysaccharide DTPS1; A is the chromatogram of the monosaccharide standard; B is the monosaccharide chromatogram of dark tea polysaccharide DTPS1;

[0037] Figure 6 It is the TIC chart of the methylated alditol acetate derivative of dark tea polysaccharide DTPS1;

[0038] Figure 7 It is the nuclear magnetic resonance spectrum of dark tea polysaccharide DTPS1; A is the hydrogen spectrum of dark tea polysaccharide DTPS1; B is the carbon spectrum of dark tea polysaccharide DTPS1; C is the 1H-1H COSY spectrum of dark tea polysaccharide DTPS1; D is the NOESY spectrum of dark tea polysaccharide DTPS1; E is the HSQC spectrum of dark tea polysaccharide DTPS1; F is the HMBC spectrum of dark tea polysaccharide DTPS1;

[0039] Figure 8 It is the effect of dark tea polysaccharide DTPS1 on the diversity and richness of the intestinal flora of obese mice; A is the result of the Shannon index; B is the result of the Ace index; C is the result of the Chaos index; D is the result of the PcOA analysis; E is the result of the Venn analysis;

[0040] Figure 9Effect of dark tea polysaccharide DTPS1 on the abundances of beneficial and harmful bacteria in the gut microbiota of obese mice; A shows the results of the influence of different treatment groups on the community composition at the phylum level; B shows the results of the influence of different treatment groups on the community composition at the genus level; C shows the results of the influence of different treatment groups on the relative abundance of Faecalibaculum; D shows the results of the influence of different treatment groups on the relative abundance of Ruminococcus_torques_group; E shows the results of the influence of different treatment groups on the relative abundance of Bifidobacterium; F shows the results of the influence of different treatment groups on the relative abundance of Akkermansia; G shows the results of the influence of different treatment groups on the relative abundance of Ileibacterium; H shows the results of the influence of different treatment groups on the relative abundance of Lachnospiraceae-NK4A136-group; I shows the results of the influence of different treatment groups on the relative abundance of unclassified-f-Atopobiaceae; J shows the results of the influence of different treatment groups on the relative abundance of Coriobacteriaceae-UCG-002;

[0041] Figure 10 Regulatory effects of dark tea polysaccharide DTPS1 on body weight, blood lipids and blood glucose in obese mice; A shows the results of the influence of different treatment groups on the body weight of obese mice; B shows the results of the influence of different treatment groups on the body fat of obese mice; C shows the results of the influence of different treatment groups on the fat weight of obese mice; D shows the results of the influence of different treatment groups on the total cholesterol of obese mice; E shows the results of the influence of different treatment groups on the triglyceride of obese mice; F shows the results of the influence of different treatment groups on the low-density lipoprotein of obese mice; G shows the results of the influence of different treatment groups on the high-density lipoprotein of obese mice; H shows the results of the influence of different treatment groups on the blood glucose level of obese mice. Detailed implementation mode

[0042] The present invention provides a dark tea polysaccharide, and the repeatable primary structural unit of the dark tea polysaccharide contains eight sugar residues, namely →4)-α-D-GalpA-6-O-Me-(1→, α-L-Araf-(1→, →3,4)-β-D-Galp-(1→, 3-O-Me-β-D-Galp-(1→, →4,6)-α-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→, →2,4)-α-L-Rhap-(1→. The connection mode of the sugar residues is shown in Formula 1:

[0043]

[0044] In the present invention, the relative molecular mass of the dark tea polysaccharide is preferably 1.28×10 4Da, the dark tea polysaccharide preferably consists of rhamnose, arabinoglucose, galactose and galacturonic acid, and the molar ratio of rhamnose, arabinoglucose, galactose and galacturonic acid is 12.35:23.23:21.25:31.90. The dark tea polysaccharide is a single-component polysaccharide obtained by separating and purifying the dark tea fermented by Eurotium cristatum, and is named DTPS1.

[0045] The present invention also provides a method for preparing dark tea polysaccharide, comprising the following steps:

[0046] (1) Mix dark tea with absolute ethanol, filter to collect the precipitate, and dry to obtain dark tea powder;

[0047] (2) Mix the dark tea powder with water for extraction, filter to collect the filtrate to obtain the water extract of dark tea;

[0048] (3) Concentrate and centrifuge the water extract of dark tea, collect the supernatant concentrate, then mix it with absolute ethanol for alcohol precipitation, filter to collect the precipitate to obtain the alcohol precipitate of dark tea;

[0049] (4) After mixing the alcohol precipitate of dark tea with water, add protease for enzymatic hydrolysis, add Sevag reagent after enzymatic hydrolysis to collect the supernatant polysaccharide solution, then adsorb it with macroporous resin overnight, collect the eluate, dialyze and dry to obtain the crude dark tea polysaccharide;

[0050] (5) Perform anion exchange column chromatography separation on the crude dark tea polysaccharide, collect the polysaccharide-containing eluate to obtain the dark tea polysaccharide fraction;

[0051] (6) Purify the dark tea polysaccharide fraction by gel column chromatography to obtain the dark tea polysaccharide.

[0052] In the present invention, dark tea is mixed with absolute ethanol, filtered to collect the precipitate, and dried to obtain dark tea powder. The dark tea is derived from the dark tea fermented by Eurotium cristatum; the dark tea is the dried, pulverized and sieved dark tea. The material-liquid ratio of the dark tea to absolute ethanol is preferably 1 g:15 - 25 mL, more preferably 1 g:17 - 23 mL, and even more preferably 1 g:20 mL; the mixing method is preferably stirring at 20 - 30 °C and 100 - 140 rpm for 10 - 14 h, more preferably stirring at 22 - 28 °C and 110 - 130 rpm for 11 - 13 h, and even more preferably stirring at 25 °C and 120 rpm for 12 h; the filtration can be selected as suction filtration. The drying method is preferably drying in an oven at 50 - 60 °C, more preferably drying in an oven at 52 - 58 °C, and even more preferably drying in an oven at 55 °C.

[0053] In the present invention, after obtaining the dark tea powder, the dark tea powder is mixed with water for extraction, and the filtrate is collected by filtration to obtain the aqueous extract of dark tea. The material-liquid ratio of the dark tea powder to water is preferably 1 g: 15-25 mL, more preferably 1 g: 16-24 mL, and even more preferably 1 g: 20 mL; the mixing method is not particularly limited, and stirring, vortexing, etc. can be used, that is, it can be mixed evenly. The extraction temperature is preferably 60-90 °C, more preferably 65-80 °C, and even more preferably 70-75 °C; the number of extractions is preferably 2-3 times, more preferably 3 times, and the extraction time for each extraction is independently preferably 3-5 h, more preferably 4 h; the extraction preferably uses stirring extraction, and the stirring speed is preferably 110-130 rpm / min, more preferably 115-125 rpm / min, and even more preferably 120 rpm / min.

[0054] In the present invention, after obtaining the aqueous extract of dark tea, the aqueous extract of dark tea is concentrated and centrifuged, and the supernatant concentrate is collected, then mixed with absolute ethanol for alcohol precipitation, and the precipitate is collected by filtration to obtain the alcohol precipitate of dark tea. The concentration is carried out by vacuum concentration, and the concentration is preferably to 1 / 3-1 / 4 of the original volume of the aqueous extract of dark tea; the volume ratio of the supernatant concentrate to absolute ethanol is preferably 1: 3-5, the volume ratio of the supernatant concentrate to absolute ethanol is preferably 1: 3.5-4.5, and even more preferably 1: 4; the centrifugation is preferably carried out at 4 °C and 9000-11000 rpm / min for 15-25 min. The alcohol precipitation temperature is preferably 4 °C, and the alcohol precipitation time is preferably 12-24 h. The filtration is carried out by filtration with gauze. After filtration, drying is carried out, and the drying is preferably carried out in an oven at 50-60 °C, more preferably in an oven at 52-58 °C, and even more preferably in an oven at 55 °C.

[0055] In the present invention, after obtaining the ethanol precipitation of dark tea, the ethanol precipitation of dark tea is mixed with water, protease is added for enzymatic hydrolysis, after enzymatic hydrolysis, Sevag reagent is added to collect the supernatant polysaccharide solution, then it is adsorbed by macroporous resin overnight, the eluate is collected, dialyzed and dried to obtain crude dark tea polysaccharide. The material-liquid ratio of the ethanol precipitation of dark tea to water is preferably 10 g: 90-110 mL, more preferably 10 g: 95-105 mL, and even more preferably 10 g: 100 mL; the protease includes papain, and the mass ratio of the ethanol precipitation of dark tea to papain is 3-5:1, more preferably 4:1; the temperature of enzymatic hydrolysis is preferably 35-40 °C, more preferably 36-38 °C, and even more preferably 37 °C, the time of enzymatic hydrolysis is preferably 6-8 h, more preferably 6.5-7.5 h, and even more preferably 7 h; the Sevag reagent is prepared from chloroform and n-butanol according to a volume ratio of 4:1, the number of times of protein removal using Sevag reagent is preferably 4-6 times, more preferably 5 times, and the volume of Sevag reagent added each time is preferably 20-25 mL; the macroporous resin preferably includes macroporous resin AB-8; the dialysis bag for dialysis is preferably a dialysis bag with a molecular weight cut-off of 3400-3600 Da, more preferably a dialysis bag with a molecular weight cut-off of 3450-3550 Da, and even more preferably a dialysis bag with a molecular weight cut-off of 3500 Da; the dialysis time is preferably 20-28 h, more preferably 22-26 h, and even more preferably 24 h. The drying preferably uses freeze-drying.

[0056] In the present invention, after obtaining the crude dark tea polysaccharide, the crude dark tea polysaccharide is separated by anion exchange column chromatography, and the polysaccharide-containing eluate is collected to obtain the dark tea polysaccharide fraction. The anion exchange column is preferably DEAE seplife FF, and the sample loading concentration is preferably 40-100 mg / mL, more preferably 45-60 mg / mL. The elution method is to perform water elution, 0.1 mol / L NaCl, 0.2 mol / L NaCl and 0.3 mol / L NaCl gradient elution in sequence, and the elution flow rate is preferably 3-5 mL / min, more preferably 3.5-4.5 mL / min, and even more preferably 4 mL / min. The elution time for each fraction is 40-70 min; the polysaccharide-containing eluate is the eluate obtained by 0.2 mol / L NaCl elution.

[0057] In the present invention, after obtaining the dark tea polysaccharide fraction, the dark tea polysaccharide fraction is purified by gel column chromatography to obtain dark tea polysaccharide. The column used for gel column chromatography is preferably Sephacryl S-400HR, the sample loading concentration is 20-40 mg / mL, the eluent for chromatography is preferably water, such as ultrapure water; the elution flow rate is preferably 0.5-1.5 mL / min, more preferably 1 mL / min; the elution time is preferably 4-6 h.

[0058] The extraction rate of the dark tea polysaccharide prepared by the present invention is 1.71%, the purity of the dark tea polysaccharide is above 96.1%, and the chemical structure of the dark tea polysaccharide is clear and the composition is uniform. The dark tea polysaccharide can increase the beneficial bacteria and reduce the relative abundance of harmful bacteria, and has intestinal probiotic activity and hypoglycemic and lipid-lowering functions.

[0059] Based on this, the present invention also provides an application of the above dark tea polysaccharide or the dark tea polysaccharide prepared by the above preparation method, and the application includes at least one of the following;

[0060] (a) The application of the dark tea polysaccharide in the preparation of an intestinal flora regulator;

[0061] (b) The application of the dark tea polysaccharide in the preparation of a hypoglycemic product;

[0062] (c) The application of the dark tea polysaccharide in the preparation of a fat-reducing product.

[0063] In the present invention, the regulator or product includes food, health products or drugs.

[0064] The present invention also provides a pharmaceutical composition for regulating intestinal flora and hypoglycemic and lipid-lowering, which includes the above dark tea polysaccharide or the dark tea polysaccharide prepared by the above preparation method and pharmaceutically acceptable excipients.

[0065] In the present invention, the oral dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, dripping pill, pill or powder. The excipients include one or several of excipient, lubricant, preservative, diluent, sweetener, binder, colorant, solubilizer and surfactant. The mass percentage content of the dark tea polysaccharide in the regulator or product is preferably 20-99%, more preferably 30-70%.

[0066] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] In the following embodiments, the dark tea fermented by Eurotium cristatum is provided by Huangshan Shuigong Tea Industry Co., Ltd. (SP17020). The DEAE seplife FF weak anion exchange column (26mm×400mm) is purchased from Suzhou Lanxiao Biotechnology Co., Ltd. The Sephacryl S-400HR gel separation column (26mm×1000mm) is purchased from GE Company.

[0068] Example 1 Preparation process of dark tea polysaccharide

[0069] (1) Take an appropriate amount of dark tea fermented by Eurotium cristatum, dry it at 60 °C, crush it with a pulverizer, sieve it through a 60-mesh sieve, add absolute ethanol according to the solid-liquid ratio of 1 g: 20 mL, stir at 25 °C and 120 rpm for 12 h, filter by suction, collect the precipitate, and dry it in an oven at 55 °C to obtain the decolorized and defatted dry dark tea powder;

[0070] (2) Mix the dark tea powder obtained in step (1) evenly with ultrapure water according to the solid-liquid ratio of 1 g: 20 mL, stir and extract at 70 °C and 120 rpm for 4 h, filter by suction, and collect filtrate a and residue a; Mix residue a evenly with ultrapure water according to the solid-liquid ratio of 1 g: 20 mL, stir and extract at 70 °C and 120 rpm for 4 h, filter by suction, and collect filtrate b and residue b; Mix residue b evenly with ultrapure water according to the solid-liquid ratio of 1 g: 20 mL, stir and extract at 70 °C and 120 rpm for 4 h, filter by suction, and collect filtrate c. Combine filtrate a to filtrate c to obtain the water extract of dark tea;

[0071] (3) Place the water extract of dark tea obtained in step (2) in a rotary evaporator (35 rpm, 55 °C, 0.09 - 0.10 MPa) for vacuum concentration to 1 / 4 of the original volume of the water extract of dark tea, centrifuge at 4 °C and 10000 rpm for 20 min, discard the precipitate, collect the supernatant concentrate, add 4 volumes of absolute ethanol (to make the final concentration of ethanol 80%), precipitate at 4 °C for 12 h, filter with gauze, collect the precipitate, and dry it in an oven at 55 °C to obtain the alcohol precipitate of dark tea;

[0072] (4) Take 10 g of the alcohol precipitate of dark tea obtained in step (3), add 100 mL of ultrapure water until completely dissolved, add 2.5 g of papain, enzymatically hydrolyze at 37 °C for 6 h, add 1 / 4 volume (25 mL) of Sevag reagent (chloroform / n-butanol volume ratio = 4 / 1) to remove protein 5 times, collect and combine the supernatant polysaccharide solution each time, then adsorb and elute it with macroporous resin AB-8 overnight for 13 h, collect the eluate, dialyze it with a dialysis bag with a molecular weight cut-off of 3500 Da for 24 h, and freeze-dry it at -50 °C and 10 Pa for 48 h to obtain the crude polysaccharide of dark tea after impurity removal.

[0073] (5) Dissolve the crude polysaccharide of dark tea obtained in step (4) with ultrapure water to prepare a 50 mg / mL tea polysaccharide solution, load it onto a DEAEseplife FF weak anion exchange column, and perform gradient elution with ultrapure water and 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, and 0.3 mol / L NaCl solution components at a flow rate of 4 mL / min. The elution time for each component is 50 min. Detect the polysaccharide content in the eluate by the phenol-sulfuric acid colorimetric method, collect the sugar-containing eluates of each component, and draw an elution curve (such asFigure 1 A), Collect the eluate of the 0.2 mol / L NaCl elution fraction, which is the dark tea polysaccharide fraction.

[0074] (6) Collect the dark tea polysaccharide fraction obtained in step (5) and perform gel column chromatography on a Sephacryl S-400HR column. The sample loading concentration is 20 mg / mL, and elute with ultrapure water at a flow rate of 1 mL / min for 250 min. Detect the polysaccharide content in the eluate by the phenol-sulfuric acid colorimetric method, collect all the polysaccharide-containing eluates within 250 min, and draw an elution curve (such as Figure 1 B). After concentration under reduced pressure, then dialysis for desalting, and vacuum freeze-drying to obtain dark tea polysaccharide DTPS1 (total extraction rate is 1.71%), and the purity of dark tea polysaccharide is 96.1%. Among them, the total extraction rate of dark tea polysaccharide (%) = M1÷M2×100, where M1 is the mass of dark tea polysaccharide (mg); M2 is the mass of dry tea (mg).

[0075] Preparation process of dark tea polysaccharide in Example 2

[0076] (1) Take an appropriate amount of dark tea fermented by Eurotium cristatum, dry it at 60 °C, crush it with a pulverizer, sieve it through a 60-mesh sieve, add anhydrous ethanol according to the solid-liquid ratio of 1 g:25 mL, stir at 20 °C and 140 rpm / min for 14 h, filter by suction, collect the precipitate, and place it in an oven at 60 °C to dry to obtain the decolorized and defatted dry dark tea powder;

[0077] (2) Mix the dark tea powder obtained in step (1) with ultrapure water according to the solid-liquid ratio of 1 g:15 mL, stir and extract at 60 °C and 130 rpm / min for 5 h, filter by suction, and collect filtrate a and residue a; Mix residue a with ultrapure water according to the solid-liquid ratio of 1 g:15 mL, stir and extract at 60 °C and 130 rpm / min for 5 h, filter by suction, and collect filtrate b and residue b; Mix residue b with ultrapure water according to the solid-liquid ratio of 1 g:15 mL, stir and extract at 60 °C and 130 rpm / min for 5 h, filter by suction, and collect filtrate c. Combine filtrate a to filtrate c to obtain the dark tea water extract;

[0078] (3) Place the dark tea water extract obtained in step (2) in a rotary evaporator (35 rpm / min, 55 °C, 0.09 - 0.10 MPa) and concentrate it under reduced pressure to 1 / 4 of the original volume of the dark tea water extract. Centrifuge at 4 °C and 9000 rpm / min for 25 min, discard the precipitate, collect the supernatant concentrate, add 4.5 times the volume of anhydrous ethanol, precipitate at 4 °C for 24 h, filter with gauze, collect the precipitate, and place it in an oven at 50 °C to dry to obtain the dark tea ethanol precipitate;

[0079] (4) Add 10 g of the ethanol-precipitated product of dark tea obtained in step (3) to 110 mL of ultrapure water until completely dissolved. Add 2.5 g of papain, and enzymatically hydrolyze at 35 °C for 8 h. Then add 1 / 4 volume (25 mL) of Sevag reagent (chloroform / n-butanol volume ratio = 4 / 1) to remove proteins 4 times. Collect and combine the supernatant polysaccharide solutions each time. Then, adsorb overnight for 12 h with macroporous resin AB-8, collect the eluate, dialyze with a dialysis bag with a molecular weight cut-off of 3450 Da for 28 h, and freeze-dry at -50 °C and 10 Pa for 48 h to obtain the impurity-removed crude dark tea polysaccharide.

[0080] (5) Dissolve the crude dark tea polysaccharide obtained in step (4) with ultrapure water solvent to prepare a 100 mg / mL tea polysaccharide solution. Load it onto a DEAE seplife FF weak anion exchange column, and perform gradient elution with ultrapure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, and 0.3 mol / L NaCl solution components at a flow rate of 4.5 mL / min in sequence. The elution time for each component is 40 min. Detect the polysaccharide content in the eluate by the phenol-sulfuric acid colorimetric method, and collect the eluate of the 0.2 mol / L NaCl elution component to obtain the dark tea polysaccharide component.

[0081] (6) Collect the dark tea polysaccharide component obtained in step (5) and perform gel column chromatography on a Sephacryl S-400HR column. The sample loading concentration is 40 mg / mL, and elute with ultrapure water at a flow rate of 1.5 mL / min for 240 min. Detect the polysaccharide content in the eluate by the phenol-sulfuric acid colorimetric method, collect all the polysaccharide-containing eluates, and plot an elution curve. After concentration under reduced pressure, then dialyze to remove salts, and vacuum freeze-dry to obtain dark tea polysaccharide.

[0082] Preparation process of dark tea polysaccharide in Example 3

[0083] (1) Take an appropriate amount of dark tea fermented by Eurotium cristatum, dry it at 60 °C, crush it with a pulverizer, sieve it through a 60-mesh sieve, add anhydrous ethanol according to a solid-liquid ratio of 1 g:15 mL, stir at 30 °C and 100 rpm for 10 h, filter by suction, collect the precipitate, and dry it in an oven at 50 °C to obtain the decolorized and defatted dry dark tea powder;

[0084] (2) Mix the dark tea powder obtained in step (1) with ultrapure water according to a solid-liquid ratio of 1 g:25 mL, stir and extract at 90 °C and 110 rpm for 3 h, filter by suction, and collect filtrate a and residue a; mix residue a with ultrapure water according to a solid-liquid ratio of 1 g:25 mL, stir and extract at 90 °C and 110 rpm for 3 h, filter by suction, and collect filtrate b and residue b. Combine filtrates a to b to obtain the water extract of dark tea;

[0085] (3) The aqueous extract of dark tea obtained in step (2) was placed in a rotary evaporator (35 rpm / min, 55 °C, 0.09 - 0.10 MPa) and concentrated under reduced pressure to 1 / 4 of the original volume of the aqueous extract of dark tea. It was centrifuged at 4 °C and 11,000 rpm / min for 15 min. The precipitate was discarded, and the supernatant concentrate was collected. 3.5 times the volume of absolute ethanol was added, and after precipitation at 4 °C for 12 h, it was filtered through gauze. The precipitate was collected and dried in an oven at 60 °C to obtain the alcohol-precipitated product of dark tea.

[0086] (4) 10 g of the alcohol-precipitated product of dark tea obtained in step (3) was added with 90 mL of ultrapure water until completely dissolved, and 2 g of papain was added. After enzymatic hydrolysis at 40 °C for 7 h, 1 / 4 volume (25 mL) of Sevag reagent (chloroform / n-butanol volume ratio = 4 / 1) was added to remove protein 6 times. The supernatant polysaccharide solutions each time were collected and combined, and then adsorbed by macroporous resin AB-8 overnight for 15 h. The eluate was collected and dialyzed with a dialysis bag with a molecular weight cut-off of 3550 Da for 20 h, and freeze-dried at -50 °C and 10 Pa for 48 h to obtain the crude polysaccharide of dark tea after impurity removal.

[0087] (5) The crude polysaccharide of dark tea obtained in step (4) was configured into a 40 mg / mL tea polysaccharide solution with ultrapure water as the solvent, and loaded onto a DEAE seplife FF weak anion exchange column. Gradient elution was carried out with ultrapure water, 0.1 mol / L NaCl solution, 0.2 mol / L NaCl solution, and 0.3 mol / L NaCl solution components at a flow rate of 3.5 mL / min. The elution time for each component was 70 min. The polysaccharide content in the eluate was detected by the phenol-sulfuric acid colorimetric method, and the eluate of the 0.2 mol / L NaCl elution component was collected to obtain the polysaccharide component of dark tea.

[0088] (6) The polysaccharide component of dark tea obtained in step (5) was subjected to Sephacryl S-400HR gel column chromatography. The sample loading concentration was 30 mg / mL, and elution was carried out with ultrapure water at a flow rate of 0.5 mL / min for 360 min. The polysaccharide content in the eluate was detected by the phenol-sulfuric acid colorimetric method, and all the polysaccharide-containing eluates were collected to draw an elution curve. After concentration under reduced pressure, dialysis was carried out to remove salts, and vacuum freeze-drying was carried out to obtain dark tea polysaccharide.

[0089] Example 4: Physicochemical properties of the dark tea polysaccharide DTPS1 prepared in Example 1

[0090] (1) Determination of carbohydrate content in the dark tea polysaccharide DTPS1

[0091] The phenol-sulfuric acid method was used to determine the carbohydrate content of DTPS1 obtained in Example 1. It was detected by a spectrophotometer at 490 nm. The carbohydrate content in DTPS1 was 96.1 ± 0.52%.

[0092] (2) UV Spectral Analysis of Dark Tea Polysaccharide DTPS1

[0093] The dark tea polysaccharide DTPS1 was scanned by a full-wavelength scanning microplate reader in the wavelength range of 190 - 400 nm. As Figure 2 shown in A, there are no absorption peaks of pigments, proteins, and nucleic acids in the scanning spectrum of dark tea polysaccharide DTPS1, indicating that DTPS1 contains almost no pigments, proteins, and nucleic acids.

[0094] (3) Determination of the Homogeneity and Weight-Average Molecular Weight of Dark Tea Polysaccharide DTPS1

[0095] The absolute molecular weight and molecular weight distribution of dark tea polysaccharide DTPS1 were determined by high-performance gel permeation chromatography - refractive index - multi-angle laser light scattering method (GPC-RI-MALS). GPC-RI-MALS detection conditions: Liquid phase system: UltiMate3000 (Thermo, USA); Refractive index detector: OPTILAB T-REX (Wyatt technology, CA, USA); Eighteen-angle laser light scattering detector: DAWN HELEOS-II (Wyatt technology, CA, USA); Gel exclusion chromatography columns Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) in series; Mobile phase: 0.1 M NaNO3 solution; Flow rate: 0.6 mL / min; Column temperature: 45 °C; Injection volume: 100 μL.

[0096] The results are as Figure 2 shown in B. Dark tea polysaccharide DTPS1 is a homogeneous polysaccharide, and the weight-average molecular weight is: 1.28×10 4 Da.

[0097] (4) Apparent Morphology Analysis of Dark Tea Polysaccharide DTPS1

[0098] The apparent morphology of dark tea polysaccharide DTPS1 was analyzed by high-resolution field emission scanning electron microscopy. An appropriate amount of dark tea polysaccharide DTPS1 was placed on a conductive carbon tape, and after spraying gold on the surface of the polysaccharide sample with a sputtering instrument, the apparent morphology was observed using a scanning electron microscope.

[0099] The results are as Figure 3 shown. The surface of dark tea polysaccharide DTPS1 is uniform and smooth, presenting an irregular flaky shape.

[0100] (5) Characteristic Group Analysis of Dark Tea Polysaccharide DTPS1

[0101] The characteristic groups of dark tea polysaccharide DTPS1 were scanned and analyzed using a Nicolet iZ-10 Fourier transform infrared spectrometer. Weigh 10 mg of dark tea polysaccharide DTPS1 and 200 mg of potassium bromide, mix them evenly, grind, dry, fix, and press them into a 1-mm-thick sheet, then scan and analyze them on the machine. The scanning range is 4000 cm -1 ~400 cm -1 .

[0102] The results are as Figure 4 shown. Dark tea polysaccharide DTPS1 has typical polysaccharide characteristic peaks. The strong absorption peak generated at 3419 cm -1 is the stretching vibration of O-H; the absorption peak at 2924 cm -1 belongs to the stretching vibration of C-H; the absorption peak at 1617.33 cm -1 belongs to the stretching vibration of C=O; the absorption peak at 1403 cm -1 belongs to the bending vibration of C-H; the absorption peak at 1143 cm -1 belongs to the two stretching vibrations of C-O-C and C-O-H in the pyranose ring of the sugar residue; the absorption peaks formed at 958 cm -1 and 638 cm -1 indicate that the polysaccharide contains β-configured glycosidic bonds and α-configured glycosidic bonds.

[0103] Example 5: Chemical structure identification of dark tea polysaccharide DTPS1 prepared in Example 1

[0104] (1) Monosaccharide composition of dark tea polysaccharide DTPS1

[0105] The monosaccharide composition of dark tea polysaccharide DTPS1 was determined using a Thermo ICS5000 ion exchange chromatography system and a pulsed amperometric detector. Accurately weigh 5 mg of sample 1 and place it in an ampoule bottle. Add 2 mol / L trifluoroacetic acid (TFA) and hydrolyze it at 121 °C for 2 h. After drying by nitrogen blowing, transfer it to a chromatographic vial for testing. Separately weigh the following monosaccharide standards, including fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (GalA), glucuronic acid (GlcA), mannuronic acid (ManA), and guluronic acid (GulA), and prepare monosaccharide mixed standard solutions with different concentration gradients (5, 10, 20, 25, 30 μg / mL) respectively. According to the absolute quantification method, calculate the molar mass ratio of different monosaccharide compositions in the sample.

[0106] Detection conditions: Chromatographic column: Dionex Carbopac TMPA20 (3 × 150 mm, 10 μm); Mobile phase: A: H2O; B: 0.1 M NaOH; C: 0.1 M NaOH, 0.2 M NaAc; Flow rate: 0.5 mL / min; Injection volume: 5 μL; Column temperature: 30 °C; Elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0107] The results are as Figure 5 shown. The molar mass ratio of rhamnose (Rha), arabinose (Ara), galactose (Gal), and galacturonic acid (GalA) in the monosaccharide composition of DTPS1 is 12.35:23.23:21.25:31.90.

[0108] (7) Analysis of the glycosidic bond type of dark tea polysaccharide DTPS1

[0109] Gas chromatography - mass spectrometry (GC - MS) was used to analyze the glycosidic bond type of dark tea polysaccharide DTPS1. Weighed 20 mg of dark tea polysaccharide DTPS1 and placed it in a glass reaction flask, added 1 mL of ultrapure water to dissolve it, then added 1 - cyclohexyl - 2 - morpholinoethyl carbodiimide methyl p - toluenesulfonate (30 mg / mL, 1 mL), and reacted with magnetic stirring at 37 °C for 2 h. Added 1 mL of 2 M imidazole, divided the sample into two equal parts, and added NaBH4 (30 mg / mL, 1 mL) and NaBD4 (30 mg / mL) respectively, and reacted for 3 h. Added glacial acetic acid to terminate the reaction. The reaction solution was dialyzed against running water for 48 h, concentrated, and freeze - dried to obtain the carbonyl - reduced sample. Took the polysaccharide samples before and after carbonyl reduction and placed them in ampoules, added imidazole and NaBD4, imidazole and NaBH4 respectively, and reacted for 3 h. Added 100 μL of 2 M TFA, and reacted at 121 °C for 90 min. Evaporated to dryness at 30 °C, added 50 μL of 2 M ammonia water, 50 μL of 1 M NaBD4, mixed well, and reacted at room temperature for 2.5 h. Added 20 μL of acetic acid to terminate the reaction, dried under nitrogen, washed twice with 250 μL of methanol, and dried under nitrogen. Added 250 μL of acetic anhydride, vortex - mixed well, and reacted at 100 °C for 2.5 h. Added 1 mL of water and let it stand for 10 min. Added 500 μL of dichloromethane (CH2Cl2), vortex - mixed well, centrifuged, discarded the aqueous phase, repeated the water wash 3 times, took the lower - layer CH2Cl2 phase, and detected it by GC - MS.

[0110] GC-MS detection conditions: The gas chromatography used an Agilent 7890A detection system. The chromatographic column was BPX70 (30m × 0.25mm × 0.25μm). The injection volume was 1 μL, the split ratio was 10:1, the carrier gas was high-purity helium. The initial temperature of the column oven was maintained at 140 °C for 2.0 min, then programmed to rise to 230 °C at a rate of 3 °C / min and held for 3 min. The mass spectrometry used an Agilent 5977B quadrupole mass spectrometry detection system, equipped with an electron impact ionization source (EI) and a MassHunter workstation. The mass scanning range (m / z) was 50 - 350.

[0111] The results are as Figure 6 shown. In the total ion current chromatogram of the methylation product of the dark tea polysaccharide DTPS1 of the present invention, eight methylation derivative ion peaks appeared. According to the retention time of each peak, by online searching each mass spectrum, the glycosidic bond types and molar ratios of DTPS1 were analyzed and obtained, as shown in Table 1. The results were consistent with the monosaccharide composition.

[0112] Table 1 Glycosidic bond types of dark tea polysaccharide DTPS1

[0113]

[0114] Example 6: Analysis of the chemical structure characteristics of the dark tea polysaccharide DTPS1 prepared in Example 1

[0115] The chemical structure of the dark tea polysaccharide DTPS1 was further characterized by a nuclear magnetic resonance spectrometer. 50 mg of the dark tea polysaccharide DTPS1 was completely dissolved in heavy water (D2O), freeze-dried, and transferred to a nuclear magnetic tube after repeated exchanges three times. Using a Bruker AVANCE NEO 500 MHz nuclear magnetic resonance spectrometer, one-dimensional 1 1H spectrum, 13 13C spectrum, and two-dimensional COSY, HSQC, HMBC, and NOESY spectra were scanned.

[0116] The results are as Figure 7 shown. The dark tea polysaccharide DTPS1 is composed of a repeating structural unit of eight sugar residues: →4)-α-D-GalpA-6-O-Me-(1→, α-L-Araf-(1→, →3,4)-β-D-Galp-(1→, 3-O-Me-β-D-Galp-(1→, →4,6)-α-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→, →2,4)-α-L-Rhap-(1→, which is basically consistent with the methylation structure. The chemical shift analysis of the sugar residues is shown in Table 2. The connection mode of the sugar residues is shown in Formula 1:

[0117]

[0118] Table 2 Sugar residues 1 H and 13 Chemical shift of C

[0119]

[0120]

[0121] Example 7: Intestinal beneficial activity of dark tea polysaccharide DTPS1 prepared in Example 1

[0122] A high-fat diet was used to induce an obesity model in mice. The mice were given oral administration of dark tea polysaccharide DTPS1 during the high-fat diet period to analyze the effect of dark tea polysaccharide DTPS1 on the intestinal flora of mice fed a high-fat diet.

[0123] (1) Animal experiment design and methods

[0124] SPF C57BL / 6J male mice (6 weeks old, 20±2g) were purchased from Henan Sikebes Biotechnology Co., Ltd. (SYXK (Yu) 2020-0005). After one week of adaptive feeding under standard conditions in the SPF animal room, all mice were randomly divided into 6 groups (n=10), and the groups were set up as follows: ND group (normal diet plus oral gavage of normal saline); ND+DTPS1 group (normal diet plus oral gavage of 200mg / kg BW DTPS1); HFD group (high-fat diet plus oral gavage of normal saline); HFD+DTPS1 group (high-fat diet plus oral gavage of 200mg / kg BW DTPS1). Each group was gavaged once a day, with a gavage volume of 0.1mL / 10g, for 12 consecutive weeks. During the feeding period, the daily physiological indicators of each group of mice were regularly detected. After 12 weeks, the mice were killed by CO2 asphyxiation, and the cecal contents and related organ indicators of each group of mice were collected for subsequent analysis and detection.

[0125] (2) Determination of intestinal flora

[0126] After the experiment, the cecal contents of each group of mice were collected, placed in sterile EP tubes, and then quickly frozen in liquid nitrogen and stored at -80°C. The samples were sent to Meiji Biotechnology for total DNA extraction. After verifying the DNA concentration and purity, targeted 16s rDNA PCR amplification was performed, and the PCR amplification products were recovered. After purification and quantification, the library was constructed according to the Illumina MiSeq PE300 sequencing platform protocol. After splicing and filtering the raw sequencing data, valid data was obtained, and related analyses such as ASV clustering and species classification were performed on the Meiji Biotechnology I-Sanger online analysis platform.

[0127] (3) Experimental results

[0128] Analyze the effect of DTPS1 on the composition of the intestinal flora of mice by 16S rRNA sequencing. The results of intestinal flora diversity and clustering analysis are as follows Figure 8 shown. The results of the Ace, Chao, and Shannon indices combined with the Venn diagram showed that a high-fat diet led to a decrease in the abundance and diversity of intestinal microorganisms in mice. After DTPS1 intervention, the richness and diversity of the flora in the ND group and the HFD group of mice were significantly increased.

[0129] Furthermore, the classification and composition of the intestinal microorganisms of mice in each group were analyzed using a community histogram. At the phylum level, as shown in Figure 9 A, the ratio of Firmicutes to Bacteroidetes (F / B) in the intestinal tract of mice was analyzed. It was found that the F / B value of the HFD group of mice was significantly higher than that of the ND group, and the F / B value of the HFD group of mice was significantly decreased after DTPS1 intervention. It is considered that an increase in the F / B value may be beneficial to energy absorption and fat storage, leading to weight gain and obesity. The present invention found that DTPS1 can reverse the increase in the F / B value of the intestinal flora of obese mice caused by a high-fat diet, indicating that DTPS1 can improve obesity by regulating the F / B value.

[0130] The changes in the intestinal flora of mice in each group at the genus level are as follows Figure 9As shown in B-J, compared with the HFD group, oral gavage of DTPS1 significantly increased the relative abundances of the beneficial bacteria Akkermansia, Bifidobacterium, Ruminococcus_torques_group, and Faecalibaculum, and downregulated the relative abundances of the harmful bacteria Ileibacterium, Coriobacteriaceae_UCG-002, unclassified_f__Atopobiaceae, and Lachnospiraceae_NK4A136_group. Both Ruminococcus_torques_group and Faecalibaculum have been reported to be short-chain fatty acid (SCFA) producers. Studies have shown that the main fermentation product of Ruminococcaceae is acetic acid, which can improve the body's gastrointestinal dysfunction by enhancing the intestinal immune defense ability. Faecalibaculum is a Gram-positive obligate anaerobe with high fermentation ability, which can increase the production of short-chain fatty acids, especially butyric acid. The increase in Akkermansia abundance has been proven to effectively maintain the integrity of the intestinal barrier and reduce intestinal inflammation. In addition, Bifidobacterium, as an intestinal probiotic, can help the host improve the intestinal flora structure and intestinal microenvironment. In summary, dietary supplementation with DTPS1 can balance intestinal homeostasis and improve the intestinal flora dysbiosis in obese mice induced by the HFD group by increasing the richness and diversity of intestinal microorganisms in mice, upregulating the abundance of probiotics, and downregulating the abundance of harmful bacteria.

[0131] Example 8: Hypoglycemic and Lipid-Lowering Activity of the Dark Tea Polysaccharide DTPS1 Prepared in Example 1

[0132] Using a high-fat diet-induced mouse obesity model, mice were given oral gavage of the dark tea polysaccharide DTPS1 during the high-fat diet period, and the effects of DTPS1 on blood glucose and blood lipids in high-fat diet mice were analyzed.

[0133] (1) Monitoring of Mouse Body Weight

[0134] During the experiment, the mental state, activity, hair glossiness and other routine basic indicators of the mice were observed daily, and the body weight, diet, and water intake changes of each group of mice were regularly monitored and recorded every week, and the body weight change curve was plotted.

[0135] (2) Body Fat Monitoring

[0136] A small animal in vivo composition analyzer was used to detect the ratio of fat rate to lean meat rate in the body composition of each group of mice.

[0137] (3) Determination of Fat Organ Index

[0138] After sacrificing the mice, the epididymal fat, inguinal fat, perirenal fat, etc. of the mice were taken out separately, weighed, and the fat organ index was calculated. The organ index = (organ weight / animal weight) × 100%.

[0139] (4) Determination of blood glucose

[0140] In the week before the end of the experiment, the mice in each group were fasted overnight for 12 h, blood was taken from the tip of the tail, and the fasting blood glucose concentration was measured using a blood glucose meter.

[0141] (5) Detection of blood lipid indexes

[0142] After collecting the blood of the mice in each experimental group using heparin sodium tubes, the blood was centrifuged at 3500 r / min for 15 min at 4 °C to separate the plasma. According to the instructions of the biochemical kit, the blood lipid indexes such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-c), and high-density lipoprotein (HDL-c) in the plasma were detected.

[0143] (6) Experimental results

[0144] After 12 weeks of feeding, by comparing the changes in the body weight growth curves among groups, it was found that the HFD group significantly increased the body weight of the mice compared with the ND group, while oral supplementation of DTPS1 significantly inhibited the body weight gain of the HFD mice ( Figure 10 A); further, a small animal body fat composition analyzer was used to monitor the body composition of the mice in each group. The results of body composition detection showed that the body fat rate of the HFD mice was significantly higher than that of the ND mice ( Figure 10 B), and after DTPS1 intervention, the body fat rate of the HFD mice was significantly decreased. The analysis results of the weights of adipose tissue in different parts of the mice in each group showed that after DTPS1 intervention, the weights and volumes of white adipose tissues such as epididymal fat (eWAT), inguinal fat (iWAT), and perirenal fat (pWAT) of the high-fat diet-induced obese mice could be significantly decreased ( Figure 10 C); by measuring the plasma biochemical indexes of the mice in each group, the results showed that compared with the ND group mice, the levels of TC, TG, and LDL-c in the plasma of the HFD group mice were significantly increased, and the level of HDL-c was significantly decreased, indicating that the blood lipid function in the mice induced by high-fat diet was abnormal. After 12 weeks of DTPS1 intervention, the contents of TC, TG, and LDL-C in the plasma of the HFD mice were significantly decreased, and the level of HDL-c was increased ( Figure 10 D - G); the changes in the fasting blood glucose of the mice in each group were detected using a blood glucose meter in combination with the corresponding blood glucose test strips. Compared with the ND mice in the normal diet group, long-term high-fat diet significantly increased the fasting blood glucose level of the HFD group mice. After 12 weeks of FOFT intervention, it was found that DTPS1 could significantly decrease the increase in blood glucose level induced by high-fat diet ( Figure 10 H).

[0145] The above results reveal that DTPS1 can significantly reduce the increase in body weight, blood glucose, and blood lipid levels induced by a high-fat diet, and has excellent effects on weight loss, blood glucose reduction, and blood lipid reduction.

[0146] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A dark tea polysaccharide, characterized in that, The repeatable primary structural unit of the dark tea polysaccharide contains eight sugar residues: →4)-α-D-GalpA-6-O-Me-(1→, α-L-Araf-(1→, →3,4)-β-D-Galp-(1→, 3-O-Me-β-D-Galp-(1→, →4,6)-α-D-Galp-(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp-(1→, →2,4)-α-L-Rhap-(1→. The linkage of the sugar residues is shown in Formula 1: The relative molecular mass of the dark tea polysaccharide is 1.28×10 4 Da. The dark tea polysaccharide is composed of rhamnose, arabinose, glucose, galactose and galacturonic acid, and the molar ratio of rhamnose, arabinose, glucose, galactose and galacturonic acid is 12.35:23.23:21.25:31.

90.

2. The preparation method of the dark tea polysaccharide according to claim 1, characterized in that, It includes the following steps: (1) Mix dark tea with absolute ethanol, filter to collect the precipitate, dry it to obtain dark tea powder; (2) Mix the dark tea powder with water for extraction, filter to collect the filtrate to obtain the water extract of dark tea; (3) Concentrate and centrifuge the water extract of dark tea, collect the supernatant concentrate, then mix it with absolute ethanol for alcohol precipitation, filter to collect the precipitate to obtain the alcohol precipitate of dark tea; (4) After mixing the alcohol precipitate of dark tea with water, add protease for enzymatic hydrolysis. After enzymatic hydrolysis, add Sevag reagent to collect the supernatant polysaccharide solution, then adsorb it with macroporous resin overnight, collect the eluate, dialyze and dry it to obtain the crude dark tea polysaccharide; (5) Subject the crude dark tea polysaccharide to anion exchange column chromatography separation, collect the polysaccharide-containing eluate to obtain the dark tea polysaccharide fraction; (6) Purify the dark tea polysaccharide fraction by gel column chromatography to obtain the dark tea polysaccharide.

3. The preparation method according to claim 2, characterized in that, In step (1), the dark tea is derived from dark tea fermented by Eurotium cristatum; the material-liquid ratio of the dark tea to absolute ethanol is 1 g: 15-25 mL; the mixing method is stirring treatment at 20-30 °C and 100-140 rpm for 10-14 h; In step (2), the material-liquid ratio of the dark tea powder to water is 1 g: 15-25 mL; the extraction temperature is 60-90 °C, the number of extractions is 2-3 times, and the extraction time for each time is independently 3-5 h; In step (3), it is concentrated to 1 / 3-1 / 4 of the original volume of the water extract of dark tea; the volume ratio of the supernatant concentrate to absolute ethanol is 1: 3-5.

4. The preparation method according to claim 2, characterized in that, In step (4), the material-liquid ratio of the alcohol precipitate of dark tea to water is 10 g: 90-110 mL; the protease includes papain, the enzymatic hydrolysis temperature is 35-40 °C, and the enzymatic hydrolysis time is 6-8 h; the Sevag reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4: 1, and the number of times of protein removal with Sevag reagent is 4-6 times, and the volume of Sevag reagent added each time is 20-25 mL; the macroporous resin includes macroporous resin AB-8; the dialysis bag for dialysis is a dialysis bag with a molecular weight cut-off of 3400-3600 Da, and the dialysis time is 20-28 h.

5. The preparation method according to claim 2, characterized in that, In step (5), the anion exchange column is DEAEseplife FF, the sample loading concentration is 40-100 mg / mL, the elution is performed by sequentially performing water elution, 0.1 mol / L NaCl, 0.2 mol / L NaCl, and 0.3 mol / L NaCl gradient elution, the elution flow rate is 3-5 mL / min, and the elution time for each component is 40-70 min; the polysaccharide-containing eluate is the eluate obtained by 0.2 mol / L NaCl elution.

6. The preparation method according to claim 2, characterized in that, In step (6), the column used for gel column chromatography is Sephacryl S-400HR, the sample loading concentration is 20-40 mg / mL, the eluent for chromatography is water, the elution flow rate is 0.5-1.5 mL / min, and the elution time is 4-6 h.

7. Use of the dark tea polysaccharide according to claim 1 or the dark tea polysaccharide prepared by the preparation method according to any one of claims 2 to 6, characterized in that, The application includes at least one of the following; (a) The application of the dark tea polysaccharide in the preparation of an intestinal flora regulator; (b) The application of the dark tea polysaccharide in the preparation of a hypoglycemic product; (c) The application of the dark tea polysaccharide in the preparation of a fat-reducing product.

8. A pharmaceutical composition for regulating intestinal flora and reducing blood sugar and fat, characterized in that, It includes the dark tea polysaccharide described in claim 1 or the dark tea polysaccharide prepared by the preparation method described in any one of claims 2-6 and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, wherein The oral dosage form of the pharmaceutical composition includes powder, tablet, solution, granule, dripping pill, pill or powder.

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