Application of Chinese chestnut non-starch polysaccharide

Abstract

The invention provides application of non-starch Chinese chestnut polysaccharide, the non-starch Chinese chestnut polysaccharide comprises polysaccharide CNP-1, polysaccharide CNP-2 and polysaccharide CNP-1, the weight-average molecular weight of the polysaccharide CNP-1 is 4.82 * 10 < 3 > kDa, the polysaccharide CNP-1 comprises glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride, the molar ratio of the glucose to the galactose to the arabinose to the rhamnose to the fructose to the fucose to the xylose to the glucosamine hydrochloride is 0.626: 0.121: 0.084: 0.071: 0.061: 0.023: 0.011: 0.003; the polysaccharide CNP-2b has the weight-average molecular weight of 1.25 * 10 < 4 > kDa and comprises glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride according to the molar ratio of (0.505): (0.161): (0.148): (0.077): (0.052): (0.025): (0.027): 0.003; the non-starch polysaccharide can regulate the intestinal flora abundance caused by colitis, improve the intestinal flora structure, maintain the intestinal steady state, relieve the injury degree of colon and ensure the body health.

Description

Technical Field

[0001] The present application relates to the technical field of health food, and in particular to the application of chestnut non-starch polysaccharide. Background Art

[0002] Chestnut non-starch polysaccharides, as a kind of plant polysaccharide with both medicinal and edible properties, promote human health. Chestnut non-starch crude polysaccharides have multiple pharmacological activities, such as lowering blood sugar, anti-oxidation, anti-tumor, anti-fatigue, and enhancing the body's immunity. However, the regulatory effect of chestnut non-starch polysaccharides on intestinal flora has not been reported, and there are few studies on the prevention and auxiliary treatment of intestinal inflammatory diseases, or the protective effect on the intestinal barrier. Summary of the invention

[0003] In view of this, the purpose of the present application is to propose an application of chestnut non-starch polysaccharides to regulate intestinal flora and relieve intestinal inflammation.

[0004] Based on the above objectives, the present application provides a non-starch chestnut polysaccharide, comprising:

[0005] Polysaccharide CNP-1a, with a weight average molecular weight of 4.82x10 3 kDa, including glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose, and glucosamine hydrochloride, with a molar ratio of 0.626:0.121:0.084:0.071:0.061:0.023:0.011:0.003;

[0006] Polysaccharide CNP-2b, with a weight average molecular weight of 1.25x10 4 kDa, including glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride with a molar ratio of 0.505:0.161:0.148:0.077:0.052:0.025:0.027:0.003.

[0007] Optionally, the neutral sugar in the chestnut non-starch polysaccharide is based on glucose as a reference standard, and the content is 57.19±0.21%, and the uronic acid in the chestnut non-starch polysaccharide is based on galacturonic acid as a reference standard, and the content is 0.45±0.83%.

[0008] Based on the same inventive concept, the present disclosure also provides a method for preparing chestnut non-starch polysaccharide, comprising:

[0009] Defatting: peel and slice fresh chestnuts, freeze-dry, and crush. Rinse the chestnut powder with a solvent to remove fat to obtain defatted chestnut dry powder;

[0010] Extraction: Take chestnut defatted dry powder and dissolve it in water, centrifuge to obtain supernatant, and concentrate to obtain the first chestnut non-starch polysaccharide extract;

[0011] Protein removal: adding protease to the first chestnut non-starch polysaccharide extract to decompose the protein, thereby obtaining a second chestnut non-starch polysaccharide extract;

[0012] Starch removal: adding amylase to the second chestnut non-starch polysaccharide extract to hydrolyze starch, centrifuging to obtain a supernatant, adding anhydrous ethanol to precipitate, and obtaining a chestnut non-starch polysaccharide precipitate;

[0013] Decolorization: the chestnut non-starch polysaccharide precipitate is redissolved to obtain the third chestnut non-starch polysaccharide solution, and a macroporous resin is added to the third chestnut non-starch polysaccharide solution for decolorization to obtain the fourth chestnut non-starch polysaccharide solution;

[0014] Removing small molecule impurities: dialyzing or ultrafiltration the fourth chestnut non-starch polysaccharide solution to remove impurities to obtain the fifth chestnut non-starch polysaccharide solution;

[0015] Concentration: concentrating the fifth chestnut non-starch polysaccharide solution and freeze-drying to obtain chestnut non-starch polysaccharide powder;

[0016] Separation and purification: The chestnut non-starch polysaccharide powder is dissolved in water, separated and purified to obtain chestnut non-polysaccharides CNP-1a and CNP-2b.

[0017] Optionally, the mass ratio of the defatted dry powder to the water is 1:20, the extraction temperature is 50° C., the number of extractions is 2 to 3 times, and the time for each extraction is 1.5 to 2 hours.

[0018] Optionally, in the protein removal process, the protease is papain, the amount of protease added is 20 U / g, and the pH of the first chestnut non-starch polysaccharide extract is adjusted to 6 and the temperature to 60°C.

[0019] Optionally, in the starch removal process, the amylase is α-amylase, the amount of amylase added is 12 U / g, the pH of the second chestnut non-starch polysaccharide extract is adjusted to 6, the temperature is 93° C., and the hydrolysis time is 0.5 h.

[0020] Optionally, in the decolorization process, the macroporous resin is an AB-8 macroporous activated resin, and the preparation method thereof comprises:

[0021] washing the AB-8 macroporous resin to obtain the impurity-free AB-8 macroporous resin;

[0022] The activated and impurity-removed AB-8 macroporous resin was soaked in anhydrous ethanol and then washed to obtain the AB-8 macroporous activated resin.

[0023] Based on the same inventive concept, the present disclosure also provides an application of chestnut non-starch polysaccharides to regulate the abundance of intestinal flora caused by colitis, improve the structure of intestinal flora to maintain intestinal homeostasis, reduce the degree of damage to the colon, and ensure the health of the body.

[0024] Optionally, the intestinal flora includes beneficial flora and harmful flora, the beneficial flora includes Muribaculaceae, Lachnospiraceae, Firmicutes and Fibrobacterium, and the harmful flora includes Proteobacteria, Escherichia coli-Shigella and Actinobacteria.

[0025] Optionally, chestnut non-starch polysaccharides can reduce the content of inflammatory factors IFN-γ, IL-1β, TNF-α, IL-6 caused by colitis, and increase the content of anti-inflammatory factor IL-10.

[0026] From the above, it can be seen that the present application provides a chestnut non-starch polysaccharide, which includes glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride, and the chestnut non-starch polysaccharide can significantly promote the proliferation of beneficial intestinal bacteria, inhibit the growth of various harmful bacteria, slow down the degree of damage to the colon and improve inflammation. Therefore, the chestnut non-starch polysaccharide in the present invention has important application value for the development of functional foods and food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 It is the elution curve of chestnut CNP-1a and CNP-2b separated and purified by DEAE-52 anion exchange column and Sephadex G-100 column chromatography;

[0029] Figure 2 This is the weight average molecular weight analysis chart of chestnut non-starch polysaccharides CNP-1a and CNP-2b;

[0030] Figure 3 This is the monosaccharide composition analysis diagram of chestnut non-starch polysaccharides CNP-1a and CNP-2b;

[0031] Figure 4 This is the infrared spectra of chestnut non-starch polysaccharides CNP-1a and CNP-2b;

[0032] Figure 5This is the analysis diagram of the methylation results of chestnut non-starch polysaccharide CNP-1a in Example 3 (a is the analysis diagram of Glcp-(1→, b is the analysis diagram of →6)-Glcp-(1→, and c is the analysis diagram of →3,6)-Glcp-(1→);

[0033] Figure 6 It is the nuclear magnetic resonance spectrum of the chestnut non-starch polysaccharide CNP-1a in Example 3 (a is 1H NMR spectrum, b is 13CNMR spectrum, c is HH-COSY spectrum, d is HSQC spectrum, e is HMBC spectrum, and f is NOESY spectrum);

[0034] Figure 7 This is a graph showing the effect of chestnut non-starch polysaccharides on the expression of immune factor IFN-γ;

[0035] Figure 8 This is a graph showing the effect of chestnut non-starch polysaccharides on the expression of inflammatory factor IL-1β;

[0036] Fig. 9 This is a graph showing the effect of chestnut non-starch polysaccharides on the expression of inflammatory factor IL-6;

[0037] Fig.10 This is a graph showing the effect of chestnut non-starch polysaccharides on the expression of inflammatory factor TNF α;

[0038] Fig.11 This is a graph showing the effect of chestnut non-starch polysaccharides on the expression of anti-inflammatory factor IL-10;

[0039] Fig.12 This is a diagram of the diversity and composition analysis of chestnut non-starch polysaccharides on the phylum level in DSS-induced ulcerative colitis mice;

[0040] Fig.13 This is a diversity and composition analysis of chestnut non-starch polysaccharides on the genus level in DSS-induced ulcerative colitis mice. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical scheme and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] The chestnut used in the embodiment of the present invention is the large green cracked chestnut.

[0043] Based on the background, chestnut (Castanea mollissima Blume) is a nut food with high nutritional value, known as "bread on the tree". China is the origin of chestnut and is also the world's largest producer and exporter of chestnut. The main production areas are concentrated in Hebei, Hubei, Shandong, Yunnan and Liaoning. The main planting areas of chestnuts in Hebei include the shallow hilly areas of Yanshan Mountain and Taihang Mountain. It is one of the local specialties with great advantages in the export of agricultural and sideline products in Hebei Province. Its output, export volume and quality are all ranked first in the country. Chestnut has high nutritional value and is rich in starch, protein, vitamins, fat and mineral elements. The nutrients that can be absorbed and utilized by the human body are as high as 98%, and it has medicinal value and is a nutritional health product. According to traditional Chinese medicine, chestnut is sweet and warm in nature, and has the functions of nourishing the stomach and spleen, nourishing the kidney and strengthening the tendons, promoting blood circulation, stopping bleeding and reducing swelling, and is beneficial to the prevention and treatment of hypertension and coronary heart disease. In addition to containing nutrients such as starch, vitamins, and trace elements, chestnuts are also rich in biologically active non-starch polysaccharides.

[0044] Chestnut non-starch polysaccharides have the characteristics of biological macromolecules and have a complex structure. The structure of polysaccharides is affected by monosaccharide composition, sugar ring structure (five-membered ring or six-membered ring), terminal carbon glycosidic bond configuration (α or β), glycosidic bond attachment site, polysaccharide molecular chain length and its branching degree. Furthermore, the differences in higher-level structures such as the behavior and spatial conformation of chestnut non-starch polysaccharides in solution also lead to different pharmacological activities. The differences in polysaccharide structures are affected by sample sources and separation and purification methods. Therefore, different sources and extraction methods have a direct correlation with the physiological activity of the prepared polysaccharides.

[0045] Colitis is an inflammatory bowel disease (IBD), which is divided into ulcerative colitis (UC) and Crohn's disease (CD). It is an autoimmune disease associated with immune disorders, genetic susceptibility, and microbial population disorders. Globally, the prevalence of UC is higher than that of CD. UC is a chronic, continuous, diffuse inflammatory disease. It is caused by changes in the structure of the intestinal flora and changes in the permeability of the intestinal barrier. It occurs in the colon mucosa and is associated with the rectum. In recent years, the incidence rate in China has continued to increase, mainly affecting the colon mucosa, and lesions mostly occur in the distal colon. UC lasts for a long time and is prone to recurrence. It is a global disease that seriously affects the physical and mental health and quality of life of patients, and also imposes a heavy burden on society.

[0046] As a kind of plant polysaccharide with both medicinal and edible properties, chestnut non-starch polysaccharides promote human health. Modern medicine shows that chestnut non-starch crude polysaccharides have multiple pharmacological activities, such as lowering blood sugar, anti-oxidation, anti-tumor, anti-fatigue, and enhancing the body's immunity. However, the regulatory effect of chestnut non-starch polysaccharides on intestinal flora has not been reported, and there are few studies on the prevention and auxiliary treatment of intestinal inflammatory diseases such as UC, or the protective effect on the intestinal barrier.

[0047] The following is combined with Figure 1-13 The embodiments of the present application will be described in detail.

[0048] Example 1

[0049] A method for preparing chestnut non-starch polysaccharide comprises the following steps:

[0050] S101: defatting: peeling and slicing fresh chestnuts, freeze-drying, crushing, washing chestnut powder with a solvent, and removing fat to obtain defatted chestnut dry powder;

[0051] Specifically, fresh chestnuts collected from Chengde, Hebei Province were shelled and sliced, and put into a freeze dryer. The freeze-dried chestnuts were crushed with a grinder, passed through an 80-mesh sieve for later use, and 100 g of chestnut dry powder was taken and repeatedly rinsed and defatted with petroleum ether to obtain chestnut defatted dry powder.

[0052] S102: Extraction: Take defatted chestnut dry powder and add water to dissolve it, centrifuge to obtain a supernatant, and concentrate it to obtain a first chestnut non-starch polysaccharide extract;

[0053] Specifically, the defatted chestnut powder was dissolved in distilled water, the mass ratio of defatted powder to distilled water was 1:20, the extraction temperature was set at 50°C, the extraction time was 1.5 to 2 hours, and after centrifugation at 4000 r / min for 10 minutes, the supernatant was concentrated, the residue was repeatedly extracted 2 to 3 times under the same conditions, and the supernatants were combined to obtain the first chestnut non-starch polysaccharide extract.

[0054] S103: removing protein: adding protease to the first chestnut non-starch polysaccharide extract to decompose protein, thereby obtaining a second chestnut non-starch polysaccharide extract;

[0055] Specifically, the protease is papain, the amount of the protease added is 20 U / g, the pH of the first chestnut non-starch polysaccharide extract is adjusted to 6, the temperature is 60° C., and the decomposition time is 2 hours.

[0056] S104: removing starch: adding amylase to the second chestnut non-starch polysaccharide extract to hydrolyze starch, centrifuging to obtain a supernatant, adding anhydrous ethanol to precipitate, and obtaining a chestnut non-starch polysaccharide precipitate;

[0057] Specifically, the amylase is α-amylase, the amount of the amylase added is 12 U / g, the pH of the second chestnut non-starch polysaccharide extract is adjusted to 6, the temperature is 93° C., and the hydrolysis time is 0.5 h.

[0058] S105: decolorization: the chestnut non-starch polysaccharide precipitate is redissolved to obtain a third chestnut non-starch polysaccharide solution, and a macroporous resin is added to the third chestnut non-starch polysaccharide solution for decolorization to obtain a fourth chestnut non-starch polysaccharide solution;

[0059] Specifically, in the decolorization process, the macroporous resin is AB-8 macroporous activated resin, and the preparation method thereof comprises:

[0060] Put AB-8 macroporous resin in a beaker, add distilled water to soak it to remove floating impurities, repeat this step until no impurities float out, and obtain the impurity-free AB-8 macroporous resin;

[0061] The AB-8 macroporous resin after impurity removal was activated and soaked in anhydrous ethanol for 12 hours for activation. After the activation, the AB-8 macroporous resin was repeatedly rinsed with distilled water until the washing liquid was no longer turbid or had no obvious ethanol smell, thereby obtaining the AB-8 macroporous activated resin.

[0062] S106: removing small molecule impurities: dialyzing or ultrafiltering the fourth chestnut non-starch polysaccharide solution to remove impurities to obtain a fifth chestnut non-starch polysaccharide solution;

[0063] Specifically, the dialysis bag or ultrafiltration membrane used for dialysis or ultrafiltration preferably has a molecular weight cutoff of 8000 to 14000 Da, and the dialysis time is 48 to 72 h.

[0064] S108: Concentration: Concentrating the fifth chestnut non-starch polysaccharide solution, and freeze-drying to obtain chestnut non-starch polysaccharide powder, namely CNP;

[0065] Specifically, the temperature of the fifth chestnut non-starch polysaccharide solution was adjusted to 60° C., and after being concentrated under reduced pressure, the solution was freeze-dried using a freeze dryer to obtain CNP.

[0066] S109: separation and purification: dissolving CNP in water, separating and purifying to obtain chestnut non-polysaccharide CNP-1a and CNP-2b.

[0067] Specifically, the steps include:

[0068] S1091: Activation and filling of DEAE-Cellulose 52 (DEAE-cellulose DE-52): DEAE-Cellulose 52 filler was fully swollen with ethanol aqueous solution for 24 hours, and the filler was washed in the order of 0.5 mol / L NaOH-deionized water-0.5 mol / L HCl-deionized water-0.5 mol / L NaOH-deionized water, and then filled into a medium-pressure chromatography column;

[0069] S1092: Activation and filling of Sephadex G-100 dextran (cross-linked dextran gel G-100): The Sephadex G-100 dextran filler was added to the ultrapure aqueous solution to fully swell for 24 hours, and then filled into the medium-pressure chromatography column after the filler was soaked;

[0070] S1093: Separation and purification of CNP using a DEAE-Cellulose 52 anion exchange column: CNP was dissolved in 5 times the amount of deionized water, loaded onto a DEAE52 anion exchange column treated in step S1091, eluted with 0 and 0.05 mol / L NaCl, and the eluate was collected. After color development using the phenol-sulfuric acid method, the absorbance was measured at 490 nm using an ultraviolet spectrophotometer, and an elution curve was drawn (refer to Figure 1 a), collect samples of two different components;

[0071] S1094: Separation and purification of CNP using Sephadex G-100 dextran column: The two samples obtained in step S1093 were eluted with deionized water, the eluate was collected, and the absorbance value was measured at 490 nm using an ultraviolet spectrophotometer after color development using the phenol-sulfuric acid method to draw an elution curve (refer to Figure 1 b and Figure 1 c), collecting samples, lyophilizing after dialyzing, and obtaining the two non-starch chestnut polysaccharides CNP-1a and CNP-2b.

[0072] Example 2

[0073] Determination of neutral sugar content and uronic acid content of CPN:

[0074] The neutral sugar content of CPN was determined by the phenol-sulfuric acid method, with glucose as the standard; the uronic acid content was determined by the sulfuric acid-carbazole method, with glucuronic acid as the standard. The specific contents are shown in the following table:

[0075] Table 1 Test results of physicochemical properties of chestnut non-starch polysaccharide CNP

[0076]

[0077] Example 3

[0078] Weight average molecular weight and composition of CNP-1a and CNP-2b:

[0079] The molecular weight and distribution of CNP-1a and CNP-2b were determined by high performance liquid gel chromatography (refer to Figure 2 ), chromatographic conditions: the chromatographic column is Shodex SB804HQ (8.0mm ID×300mm) gel column, the detector is a differential refractive index detector (RID), the flow rate is 1.0mL / min, and the mobile phase is ultrapure water. The result is that the weight average molecular weight of CNP-1a is 4.82x10 3 kDa, the weight average molecular weight of CNP-2b is 1.25x10 4 kDa.

[0080] The monosaccharide composition of CNP-1a and CNP-2b was determined by pre-column derivatization HPLC (refer to Figure 3 ), the detector was an electrochemical detector, the chromatographic column was Dionex CarbopacTM PA20 (3*150mm), the mobile phase was A: H2O, B: 15mmol / L NaOH, C: 15mmol / L NaOH and 100mmol / L NaAc, the flow rate was 0.3mL / min, the injection volume was 25μL, the column temperature was 30℃, and the detection wavelength was 245nm. The results showed that CNP-1a was composed of glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride with a molar ratio of 0.626:0.121:0.084:0.071:0.061:0.023:0.011:0.003, and the molar ratio analysis was shown in Table 2; CNP-2b was composed of glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride with a molar ratio of 0.505:0.161:0.148:0.077:0.052:0.025:0.027:0.003, and the molar ratio analysis was shown in Table 3.

[0081] Table 2 Molar ratio of monosaccharide composition of CNP-1a

[0082]

[0083] Table 3 Molar ratio of monosaccharide composition of CNP-2b

[0084]

[0085] CNP-1a and CNP-2b were determined by infrared spectroscopy. Samples were pressed into KBr pellets and analyzed by Fourier transform infrared spectrometer at 4000-400 cm 1 Infrared spectrum scanning is performed within the range of Figure 4 ).

[0086] Figure 4 is the infrared spectrum of the chestnut non-starch polysaccharide CNP-1a and CNP-2b, such as Figure 4 As shown, 3420.8cm -1 (CNP-1a), 3411cm -1 The broad strong absorption peak at (CNP-2b) is caused by -OH stretching vibration; 2924.6cm -1 (CNP-1a), 2929.5cm -1 The absorption peak at (CNP-2b) is related to the asymmetric stretching vibration of CH; 1641.5cm -1 (CNP-1a), 1644cm -1(CNP-2b) is the stretching vibration peak caused by polysaccharide bound water or C=O carbonyl, which is consistent with the monosaccharide composition results. CNP-1a and CNP-2b are at 1419.1 cm －1 and 1421.6cm －1 The weak absorption peak at 950-1200 cm -1 The three characteristic peaks between them are caused by the stretching vibration of COH and COC glycosidic bonds, which indicates that CNP-1a and CNP-2b have pyranose ring configurations; 920.6cm -1 (CNP-1a), 913.2cm -1 The characteristic absorption peak at (CNP-2b) indicated that it had an α-type glycosidic bond.

[0087] Example 4

[0088] Structural Identification of Chestnut Non-Starch Polysaccharide CNP-1a

[0089] (1) Analysis of methylation results

[0090] Weigh the CNP-1a sample (2-3 mg) and place it in a glass reaction bottle, add 1 mL of anhydrous DMSO, quickly add the anhydrous alkaline solution of methylation reagent A, seal, dissolve under ultrasound, and then add the methylation reagent B iodomethane solution. React in a magnetic stirring water bath at 30 ° C for 60 minutes. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction, and dialyze in a 1000Da dialysis bag for 24 hours and then freeze-dry. Accurately weigh 2 mg of the dialyzed sample and 200 mg of potassium bromide, press into tablets, and the blank control is made of potassium bromide powder tablets. Place them in Fourier transform infrared spectrometer FT-IR650 (Tianjin Gangdong Technology Development Co., Ltd.) for scanning and recording, and make sure that the methylation is complete before doing subsequent experiments.

[0091] Take the methylated polysaccharide, add 1ml of 2mol / L trifluoroacetic acid (TFA) to hydrolyze for 90min, and evaporate to dryness on a rotary evaporator. Add 2ml of double distilled water to the residue, reduce it with 60mg of sodium borohydride for 8h, add glacial acetic acid to neutralize, rotary evaporate, dry in an oven at 101℃, then add 1ml of acetic anhydride to acetylate, react at 100℃ for 1h, and cool. Then add 10ml of pure water to terminate the reaction.

[0092] The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, and the upper aqueous solution was removed. This was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid phase vial. The acetylated product sample was analyzed using a Thermo scientific, 1300-7000 gas chromatography-mass spectrometer (analysis chart refer to Figure 5 );

[0093] GC-MS conditions: HP-INNOWAX column 30m*0.32mm*0.25um; programmed temperature conditions: starting temperature 140°C, heating to 230°C at 1°C / min; injection port temperature 250°C, detector temperature 250°C, carrier gas helium, flow rate 1mL / min.

[0094] The results of methylation analysis of CNP-1a are shown in Table 4. The results show that the glycosidic bond type of CNP-1a is relatively simple, consisting of three types of glycosidic bonds: Glup-(1→, →6-Glcp-(1→, →3,6)-Glcp-(1→, with molar ratios of 0.101, 0.740 and 0.159.

[0095] Table 4 Methylation analysis results of CNP-1a

[0096]

[0097] (2) Nuclear magnetic resonance spectroscopy analysis

[0098] Weigh 50 mg of CNP-1a sample, dissolve it in 0.5 ml of heavy water and freeze-dry it. Then, the freeze-dried powder is dissolved in 0.5 ml of heavy water again, and freeze-dried again, and the above process is repeated to fully exchange active hydrogen. Then the sample is dissolved in 0.5 ml of heavy water, and placed in a 600 MHz nuclear magnetic resonance instrument at room temperature 25°C to measure 1H NMR spectrum, 13C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0099] The 1H NMR spectrum, 13C NMR spectrum, HH-COSY spectrum, HMBC spectrum, NOESY spectrum and HSQC spectrum of CNP-1a are shown in Figure 6 shown.

[0100] The hydrogen spectrum signals are mainly concentrated between 3.0 and 5.5 ppm, δ3.2-4.0 ppm is the sugar ring proton signal, and the signal peaks of the main terminal proton peaks δ5.23, 4.89, and 5.24 are concentrated in the 4.3-5.5 ppm region.

[0101] Carbon spectrum analysis in 13C NMR (201MHz, D2O): Nuclear magnetic carbon spectrum signals are mainly concentrated between 60-120ppm. By observing the carbon spectrum, it can be seen that the main anomeric carbon signal peaks δ99.12, 100.76, 100.33 anomeric carbon regions are mainly between δ93 and 105. And the main signal peaks of δ72.76, 74.65, 70.998, 71.48, 66.83, 74.45, 77.46, 74.19, 70.66, 66.85, 70.84, 74.03, 71.39, 72.87, 61.67 are distributed in the 60-85ppm region. According to the monosaccharide composition results, the polysaccharide is composed of glucose, indicating that the polysaccharide is mainly dextran.

[0102] Through the HSQC spectrum, we can observe that the anomeric carbon signal is δ99.12, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.89. Through HH-COSY, the signals of H1-2 are 4.89 / 3.48, the signals of H2-3 are 3.48 / 3.63, the signals of H3-4 are 3.63 / 3.42, and the signals of H3-4 are 3.42 / 3.82. We can infer that H1-5 are δ4.89, 3.48, 3.63, 3.42, and 3.82, respectively. The NOESY spectrum observes that δ4.89 has correlation peaks with 3.90 and 3.66, and the corresponding H6 of C6 at 66.83ppm is 3.90 and 3.66ppm. The corresponding C1-6 are 99.12, 72.76, 74.65, 70.99, 71.48, and 66.83; therefore, the signal should be attributed to the glycosidic bond→6)-α-Glcp-(1→.

[0103] Through the HSQC spectrum, we can observe that the anomeric carbon signal is δ100.76, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.89. Through HH-COSY, the signals of H1-2 are 5.23 / 3.49, the signals of H2-3 are 3.49 / 3.74, the signals of H3-4 are 3.74 / 3.41, the signals of H3-4 are 3.63 / 3.41, and the signals of H4-5 are 3.41 / 3.81. We can infer that H1-5 are δ5.23, 3.49, 3.74, 3.41, and 3.81, respectively. The NOESY spectrum observes that δ4.89 has correlation peaks with 3.90 and 3.66, and C6 is 66.85ppm, corresponding to H6 at 3.91 and 3.65ppm. The corresponding C1-6 are δ100.76, 74.45, 81.82, 74.19, 70.66, 66.85; therefore, the signal should be attributed to the glycosidic bond →3,6)-α-Glcp-(1→. According to similar rules and combined with HMBC and NOESY, all glycosidic bond signals are assigned, and the results are shown in Table 5:

[0104] Table 5 Hydrogen and carbon signal assignments

[0105]

[0106] In the HMBC spectrum, we attributed the glycosidic bond signals of the polysaccharide according to the one-dimensional and two-dimensional NMR spectra. The anomeric hydrogen of the glycosidic bond →6)-α-D-Glcp-(1→ has a related signal peak with its own C6, and the anomeric carbon has a related signal peak with its own H6, indicating the existence of a linking mode of →6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→.

[0107] In addition, the anomeric hydrogen of →6)-α-D-Glcp-(1→) and C6 of the glycosidic bond →3,6)-α-D-Glcp-(1→) have correlation peaks, indicating the presence of a glycosidic bond →6)-α-D-Glcp-(1→3,6)-α-D-Glcp-(1→.

[0108] The anomeric hydrogen of α-D-Glcp-(1→) has a correlation peak with the C3 of the glycosidic bond →3,6)-α-D-Glcp-(1→. At the same time, the anomeric C1 of α-D-Glcp-(1→) has a correlation peak with the H3 of the glycosidic bond →3,6)-α-D-Glcp-(1→, indicating the existence of the glycosidic bond α-D-Glcp-(1→3,6)-α-D-Glcp-(1→.

[0109] In the NOESY spectrum, there are correlation peaks at H6 of →6)-α-D-Glcp-(1→ and →3,6)-α-D-Glcp-(1→, indicating the existence of a glycosidic bond →6)-α-D-Glcp-(1→3,6)-α-D-Glcp-(1→. Combined with methylation analysis and hydrogen spectrum integration, it was found that the ratio of (the sum of T-Glcp+1-3-6Glcp) to 1-4Glcp was 1:30.

[0110] In summary, it can be inferred that the main glycosidic bond structure of the polysaccharide is: the main chain connection mode is the glycosidic bond of →6)-α-D-Glcp-(1→, and the terminal α-D-Glcp-(1→ is connected to the main chain through the O-3 bond. The structural formula of CNP-1a is as follows:

[0111]

[0112] Example 5

[0113] Analysis of the effect of chestnut non-starch polysaccharide CNP on the mouse model of ulcerative colitis induced by sodium dextran sulfate:

[0114] 1. Experimental grouping and treatment

[0115] (1) A total of 72 SPF-grade C57BL / 6 male rats, aged 6-8 weeks and weighing 18-22 g, were purchased and randomly divided into 6 groups after adaptive feeding in an SPF environment for one week: blank group, model group, positive control group, CNP-L group, CNP-M group and CNP-H group.

[0116] (2) Experimental group treatment

[0117] Blank group: no medication, no modeling, and normal feeding;

[0118] Model group: Dextran sulfate sodium DSS modeling, normal feeding;

[0119] Positive control group: the same model as the model group, 5-aminosalicylic acid administration and feeding + normal feeding;

[0120] CNP-L group: The mice were modeled in the same manner as the model group, and were given 200 mg / kg / day of chestnut non-starch polysaccharide CNP (prepared in Example 1) and fed normally.

[0121] CNP-M group: The mice were modeled in the same manner as the model group, and were given 400 mg / kg / day of chestnut non-starch polysaccharide CNP (prepared in Example 1) and fed normally.

[0122] CNP-L group: The mice were modeled in the same manner as the model group, and were given 600 mg / kg / day of chestnut non-starch polysaccharide CNP (prepared in Example 1) and fed normally.

[0123] Dextran sulfate sodium DSS was used to establish an ulcerative colitis (UC) animal model. The UC modeling method was as follows: after one week of drug intervention, dextran sulfate sodium DSS was used for modeling, and drug administration was continued during modeling for another week to complete the modeling.

[0124] Experimental methods

[0125] (1) During the experiment, the food intake, water intake, body weight, stool consistency and bleeding status of the mice were recorded every day, and the disease activity index (DAI) score was calculated. The formula was (body weight score + stool status score + bloody stool score) / 3, as shown in Table 6.

[0126] Table 6 Mouse disease activity index score table

[0127]

[0128] (2) After the experiment, blood was collected from the eyeballs, and the venous blood of the mice in the above groups was placed for 15 minutes. The serum was collected by refrigerated centrifugation (4000r / min, 10min) at 4°C using a high-speed refrigerated centrifuge and stored in a refrigerator at 80°C. Subsequently, the levels of serum IFN-γ, IL-1β, IL 6, TNFα, and IL-10 were detected using an enzyme-linked immunosorbent assay kit (Shanghai ELISA Biotechnology).

[0129] (3) The cecal contents of each group of mice were collected, treated with liquid nitrogen and stored in a refrigerator at 80°C. The DNA of the cecal contents was extracted, and the structural characteristics of the related intestinal flora were compared by 16s RNA sequencing combined with bioinformatics analysis.

[0130] 3. Experimental results

[0131] (1) The mice in the experimental group that were modeled with sodium dextran sulfate lost weight, had increased DAI scores, were thin, and had dry and dull fur, indicating that the ulcerative colitis mouse model was successfully established.

[0132] (2) Figure 7 , Figure 8 , Fig. 9 , Fig.10 and Fig.11 The figures show the effects of chestnut non-starch polysaccharide CNP on the expression of immune inflammatory factors IFN-γ, IL-1β, IL 6, TNFα and IL-10. It can be seen from the figure that chestnut non-starch polysaccharide CNP can down-regulate the levels of inflammatory factors IFN-γ, IL-1β, IL 6 and TNFα in UC mice, and up-regulate the level of anti-inflammatory factor IL-10, indicating that the chestnut non-starch polysaccharide CNP of the present invention can improve the inflammatory condition of UC mice induced by DSS by regulating the inflammatory signaling pathway.

[0133] (3) Fig.12 and Fig.13 These are the diversity and composition analysis diagrams of chestnut non-starch polysaccharide CNP on the phylum and genus levels of intestinal flora in UC mice. It can be seen from the figure that compared with the model group, after CNP intervention, the relative abundance of Bacteroidetes and norank_f_Muribaculaceae (Muribaculaceae) in the mouse intestine increased, while the relative abundance of Proteobacteria and Escherichia coli Shigella decreased, indicating that chestnut non-starch polysaccharide CNP can effectively improve the structure of intestinal flora and regulate the decrease in intestinal flora abundance caused by colitis.

[0134] In summary, the experimental results show that the present invention adopts the above-mentioned chestnut non-starch polysaccharide and its preparation method and uses it as an application to improve the intestinal flora structure and regulate the decrease in intestinal flora abundance caused by colitis. The prepared chestnut non-starch polysaccharide has good water solubility and no toxic side effects. The chestnut non-starch polysaccharide improves sodium dextran sulfate-induced ulcerative colitis through anti-inflammatory immunity and intestinal flora regulation, has the function of being utilized by intestinal flora, increasing the abundance of beneficial flora, and thus regulating intestinal flora, and is used in functional foods related to improving or treating diseases related to intestinal flora disorders.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. An application of chestnut non-starch polysaccharide, characterized in that: Regulate the abundance of intestinal flora caused by colitis, improve the structure of intestinal flora to maintain intestinal homeostasis, slow down the degree of damage to the colon, and ensure the health of the body.

2. The use of a chestnut non-starch polysaccharide according to claim 1, characterized in that: The intestinal flora includes beneficial flora and harmful flora, wherein the beneficial flora includes bacteria of the family Muribaculaceae, Lachnospiraceae, Firmicutes and Fibrobacterium, and the harmful flora includes Proteobacteria, Escherichia coli-Shigella and Actinobacteria.

3. The use of a chestnut non-starch polysaccharide according to claim 2, characterized in that: It reduces the levels of inflammatory factors IFN-γ, IL-1β, TNF-α, and IL-6 caused by colitis, and increases the level of anti-inflammatory factor IL-10.

4. The use of a chestnut non-starch polysaccharide according to claim 1, characterized in that: Chestnut non-starch polysaccharides include: Polysaccharide CNP-1a, with a weight average molecular weight of 4.82x10 3 kDa, including glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose, and glucosamine hydrochloride, with a molar ratio of 0.626:0.121:0.084:0.071:0.061:0.023:0.011:0.003; Polysaccharide CNP-2b, with a weight average molecular weight of 1.25x10 4 kDa, including glucose, galactose, arabinose, rhamnose, fructose, fucose, xylose and glucosamine hydrochloride with a molar ratio of 0.505:0.161:0.148:0.077:0.052:0.025:0.027:0.

003.

5. The use of a chestnut non-starch polysaccharide according to claim 4, characterized in that: The neutral sugar in chestnut non-starch polysaccharide was 57.19±0.21% with glucose as the reference standard, and the uronic acid in chestnut non-starch polysaccharide was 0.45±0.83% with galacturonic acid as the reference standard.

6. The use of a chestnut non-starch polysaccharide according to claim 4, characterized in that: The preparation method of chestnut non-starch polysaccharide comprises: Defatting: peel and slice fresh chestnuts, freeze-dry, and crush. Rinse the chestnut powder with a solvent to remove fat to obtain defatted chestnut dry powder; Extraction: Take chestnut defatted dry powder and dissolve it in water, centrifuge to obtain supernatant, and concentrate to obtain the first chestnut non-starch polysaccharide extract; Protein removal: adding protease to the first chestnut non-starch polysaccharide extract to decompose the protein, thereby obtaining a second chestnut non-starch polysaccharide extract; Starch removal: adding amylase to the second chestnut non-starch polysaccharide extract to hydrolyze starch, centrifuging to obtain a supernatant, adding anhydrous ethanol to precipitate, and obtaining a chestnut non-starch polysaccharide precipitate; Decolorization: the chestnut non-starch polysaccharide precipitate is redissolved to obtain the third chestnut non-starch polysaccharide solution, and a macroporous resin is added to the third chestnut non-starch polysaccharide solution for decolorization to obtain the fourth chestnut non-starch polysaccharide solution; Removing small molecule impurities: dialyzing or ultrafiltration the fourth chestnut non-starch polysaccharide solution to remove impurities to obtain the fifth chestnut non-starch polysaccharide solution; Concentration: concentrating the fifth chestnut non-starch polysaccharide solution and freeze-drying to obtain chestnut non-starch polysaccharide powder; Separation and purification: The chestnut non-starch polysaccharide powder is dissolved in water, separated and purified to obtain chestnut non-polysaccharides CNP-1a and CNP-2b.

7. The use of a chestnut non-starch polysaccharide according to claim 6, characterized in that: In the extraction step, the mass ratio of the defatted dry powder to the water is 1:20, the extraction temperature is 50° C., the extraction times are 2 to 3 times, and the time for each extraction is 1.5 to 2 hours.

8. The use of chestnut non-starch polysaccharide according to claim 6, characterized in that: In the protein removal process, the protease is papain, the amount of protease added is 20 U / g, and the pH of the first chestnut non-starch polysaccharide extract is adjusted to 6 and the temperature to 60°C.

9. The use of a chestnut non-starch polysaccharide according to claim 6, characterized in that: In the starch removal process, the amylase is α-amylase, the amount of amylase added is 12 U / g, the pH of the second chestnut non-starch polysaccharide extract is adjusted to 6, the temperature is 93° C., and the hydrolysis time is 0.5 h.

10. The use of chestnut non-starch polysaccharide according to claim 6, characterized in that: In the decolorization process, the macroporous resin is AB-8 macroporous activated resin, and the preparation method thereof comprises: washing the AB-8 macroporous resin to obtain the impurity-free AB-8 macroporous resin; The activated and impurity-removed AB-8 macroporous resin was soaked in anhydrous ethanol and then washed to obtain the AB-8 macroporous activated resin.

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