Polysaccharide with intestinal flora regulating function and its products and applications

Abstract

The present invention discloses a polysaccharide having the function of regulating intestinal flora, and its products and applications, and relates to the field of biotechnology. The preparation method of the polysaccharide comprises the following steps: dissolving Flammulina velutipes bran polysaccharide in water, adjusting the pH to 2.8-3.2, adding H2O2 solution and ascorbic acid / FeCl3 to obtain a mixed solution; after ultrasonic treatment of the mixed solution, adjusting the pH to neutral to obtain a reaction solution; dialyzing, concentrating and freeze-drying the reaction solution to obtain a polysaccharide having the function of regulating intestinal flora. The present invention utilizes ultrasound-assisted combined H2O2‑Vc method or ultrasound-assisted combined H2O2‑Fe 3+ The degraded polysaccharide from Flammulina velutipes bran was prepared by the method. It was verified that the degraded polysaccharide from Flammulina velutipes bran can improve the abundance of intestinal flora, regulate the composition of intestinal flora, promote the growth of beneficial flora, and thus promote intestinal health.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to polysaccharides having the function of regulating intestinal flora and products and applications thereof. Background Art

[0002] Gut flora refers to the various microorganisms that live in the intestines and play a key role in the intestines. One of the main functions of intestinal flora is to help digest and absorb food. They help break down cellulose and other difficult-to-digest substances in food and produce nutrients, such as short-chain fatty acids, which are very important for our bodies. In addition, intestinal flora can also help synthesize vitamins and other beneficial compounds. Gut flora also plays a key role in the immune system. They interact with immune cells, help regulate immune responses, and maintain the balance of the immune system. A healthy intestinal flora can reduce potential inflammatory responses and help prevent allergies and autoimmune diseases. In addition, intestinal flora is closely related to heart health, body weight regulation, and emotional stability. Some studies have also shown that intestinal flora imbalance may be associated with many diseases, such as enteritis, irritable bowel syndrome, and inflammatory bowel disease.

[0003] As people's awareness of health increases, the balance of intestinal microecology has become a hot topic in recent years. The intestine is considered the second brain of the human body, and it is closely related to our digestive system, immune system and nervous system. The balance of intestinal microecology not only affects digestion and absorption and nutrient metabolism, but also plays an important role in immune function, mental health and the occurrence and development of chronic diseases. Therefore, it is very necessary to develop a product that can promote intestinal health by regulating intestinal flora. Summary of the invention

[0004] The purpose of the present invention is to provide a polysaccharide having the function of regulating intestinal flora and its products and applications to solve the problems existing in the above-mentioned prior art. The polysaccharide can improve the abundance of intestinal flora, regulate the composition of intestinal flora, promote the growth of beneficial flora, and thus promote intestinal health.

[0005] Flammulina velutipes is a widely cultivated edible and medicinal mushroom. With the widespread application of industrial cultivation technology of Flammulina velutipes, the yield of Flammulina velutipes residue (FVR), which is the waste after the fruiting body of edible mushrooms is harvested, has greatly increased. Flammulina velutipes residue polysaccharide (FVRP) is a polysaccharide extracted from FVR and is one of the main components of FVR. Based on FVRP, the present invention has developed a polysaccharide product that can promote intestinal health by regulating intestinal flora.

[0006] Based on this, the present invention provides the following solutions:

[0007] The present invention provides a polysaccharide FVRPV having the function of regulating intestinal flora, and the preparation method of the polysaccharide FVRPV comprises the following steps:

[0008] Dissolve the polysaccharide from Flammulina velutipes in water, adjust the pH to 2.8-3.2, and add H 2 O 2 solution and ascorbic acid to obtain a mixed solution;

[0009] After the mixed solution is subjected to ultrasonic treatment, the pH value is adjusted to neutral to obtain a reaction solution;

[0010] The reaction solution is dialyzed, concentrated and freeze-dried to obtain the polysaccharide FVRPV.

[0011] Furthermore, the H 2 O 2 The mass concentration of the solution is 30%, and the H 2 O 2 The volume fraction of the solution in the mixed solution is 0.030-0.040%;

[0012] The mass concentration of the ascorbic acid in the mixed solution is 0.025-0.035%.

[0013] Furthermore, the temperature of the ultrasonic treatment is 75-85° C., the power is 600 W, and the time is 20-40 min.

[0014] The present invention also provides a polysaccharide FVRPF having the function of regulating intestinal flora. The preparation method of the polysaccharide FVRPF comprises the following steps:

[0015] Dissolve the polysaccharide from Flammulina velutipes in water, adjust the pH to 2.8-3.2, and add H 2 O 2 Solution and FeCl 3 , to obtain a mixed solution;

[0016] After the mixed solution is subjected to ultrasonic treatment, the pH value is adjusted to neutral to obtain a reaction solution;

[0017] The reaction solution is dialyzed, concentrated and freeze-dried to obtain the polysaccharide FVRPF.

[0018] Furthermore, the H 2 O 2 The mass concentration of the solution is 30%, and the H 2 O 2 The volume fraction of the solution in the mixed solution is 1.10-1.15%;

[0019] The FeCl 3 The mass concentration in the mixed solution is 0.01-0.03%.

[0020] Furthermore, the temperature of the ultrasonic treatment is 75-85° C., the power is 600 W, and the time is 20-40 min.

[0021] The present invention also provides the use of the above-mentioned polysaccharide FVRPV and / or polysaccharide FVRPF in the preparation of products for regulating intestinal flora.

[0022] Furthermore, the product is a health product or a medicine.

[0023] The present invention also provides a product for regulating intestinal flora, wherein the active ingredient comprises the above-mentioned polysaccharide FVRPV and / or polysaccharide FVRPF.

[0024] Furthermore, the product is a health product or a medicine.

[0025] The present invention discloses the following technical effects:

[0026] The present invention utilizes ultrasound-assisted combined H 2 O 2 -Vc method and ultrasound-assisted combined H 2 O 2 -Fe 3+ Two kinds of Flammulina velutipes degradable polysaccharides were prepared by this method. It was verified that these two kinds of Flammulina velutipes degradable polysaccharides can improve the abundance of intestinal flora, regulate the composition of intestinal flora, promote the growth of beneficial flora, and thus promote intestinal health.

[0027] The invention provides a new raw material basis and technical support for the development of products that promote intestinal health and has broad market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 Fourier transform infrared spectrum analysis of FVRP, FVRPV and FVRPF;

[0030] Figure 2 is the particle size distribution of FVRP;

[0031] Figure 3 is the particle size distribution of FVRPV;

[0032] Figure 4 is the particle size distribution of FVRPF;

[0033] Figure 5 is the potential distribution of FVRP;

[0034] Figure 6 is the potential distribution of FVRPV;

[0035] Figure 7 is the potential distribution of FVRPF;

[0036] Figure 8 is the average molecular weight of FVRPV and FVRPF;

[0037] Fig. 9 is the monosaccharide composition of FVRPs;

[0038] Fig.10 is the ABTS free radical scavenging ability of FVRPs;

[0039] Fig.11 is the DPPH radical scavenging ability of FVRPs;

[0040] Fig.12 is the reduction capacity of FVRPs;

[0041] Fig.13 is the hydroxyl radical scavenging ability of FVRPs;

[0042] Fig.14 The effect of FVRPs on the OD value of plant lactic acid bacteria;

[0043] Fig.15 The effect of FVRPs on the OD value of Lactobacillus rhamnosus;

[0044] Fig.16 Fourier transform infrared spectrum of FVRP after in vitro fermentation;

[0045] Fig.17 Fourier transform infrared spectrum of FVRPV after in vitro fermentation;

[0046] Fig.18 Fourier transform infrared spectrum of FVRPF after in vitro fermentation;

[0047] Fig.19 is the change of ABTS clearance rate of FVRPs after in vitro fermentation;

[0048] Fig. 20 is the change of reducing capacity of FVRPs after in vitro fermentation;

[0049] Fig.21is the change of DPPH clearance rate of FVRPs after in vitro fermentation;

[0050] Fig. 22 pH changes during in vitro fermentation of FVRPs;

[0051] Fig.23 Sob rarefaction curve plot for Alpha diversity analysis;

[0052] Fig.24 Shannon index graph for Alpha diversity analysis;

[0053] Fig.25 Simpson index plot for Alpha diversity analysis;

[0054] Fig.26 ACE Index graph for Alpha Diversity Analysis;

[0055] Fig. 27 Chao index graph for Alpha diversity analysis;

[0056] Fig.28 PCoA plot for Beta diversity analysis;

[0057] Fig.29 Sample-level cluster analysis for Beta diversity analysis;

[0058] Fig.30 This is a stacked diagram of the species distribution of the intestinal flora;

[0059] Fig.31 This is a stacked diagram of the distribution of intestinal flora genus and species;

[0060] Fig.32 is the branch diagram of LEfSe;

[0061] Fig.33 This is a heat map of the predicted intestinal flora functions based on KEGG. DETAILED DESCRIPTION

[0062] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0063] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0064] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0065] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0066] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0067] The preparation method of Flammulina velutipes fungus husk polysaccharide used in the present invention is as follows:

[0068] Flammulina velutipes waste was mixed with ultrapure water at a solid-liquid ratio of 1:50, and ultrasonic-assisted extraction was performed with an ultrasonic power of 540 W, an extraction temperature of 75 ° C, and an extraction time of 20 min. After the extraction, the filtrate was filtered to obtain a filtrate. Anhydrous ethanol was added to the filtrate for precipitation overnight, and the protein was removed by the Sevag method, and Flammulina velutipes waste polysaccharide (FVRP) was obtained by dialysis, concentration, and freeze-drying.

[0069] Example 1

[0070] (1) Dissolve the polysaccharide from Flammulina velutipes in ultrapure water to prepare a 5 mg / mL (4.5-6.5 mg / mL can achieve the same effect) FVRP solution, adjust the pH to 3.0 (2.8-3.2 can achieve the same effect), add 30% H 2 O 2 solution and ascorbic acid (Vc) to obtain a mixed solution; wherein 30% of H 2 O 2 The volume fraction of the solution in the mixed solution is 0.035% (0.030-0.040% can achieve the same effect), and the mass concentration of Vc in the mixed solution is 0.03% (0.025-0.035% can achieve the same effect).

[0071] (2) the mixed solution obtained in step (1) is subjected to ultrasonic treatment at 80° C. (75-85° C. can achieve the same effect) with a power of 600 W for 30 min (20-40 min can achieve the same effect), and then the pH is adjusted to neutral to obtain a reaction solution;

[0072] (3) The reaction solution obtained in step (2) was dialyzed using a 500Da dialysis bag, first dialyzed with running water for 48 hours, and then dialyzed with ultrapure water for 48 hours to obtain a retentate, which was concentrated and freeze-dried to obtain a degraded polysaccharide of Flammulina velutipes bran, named FVRPV.

[0073] Example 2

[0074] (1) Dissolve the polysaccharide from Flammulina velutipes in ultrapure water to prepare a 5 mg / mL (4.5-6.5 mg / mL can achieve the same effect) FVRP solution, adjust the pH to 3.0 (2.8-3.2 can achieve the same effect), add 30% H 2 O 2 Solution and FeCl 3 , and a mixed solution was obtained; of which 30% H 2 O 2 The volume fraction of the solution in the mixed solution is 1.13% (1.10-1.15% can achieve the same effect), FeCl 3 The mass concentration in the mixed solution is 0.02% (0.01-0.03% can achieve the same effect).

[0075] (2) the mixed solution obtained in step (1) is subjected to ultrasonic treatment at 80° C. (75-85° C. can achieve the same effect) with a power of 600 W for 30 min (20-40 min can achieve the same effect), and then the pH is adjusted to neutral to obtain a reaction solution;

[0076] (3) The reaction solution obtained in step (2) was dialyzed using a 500Da dialysis bag, first dialyzed with running water for 48 hours, and then dialyzed with ultrapure water for 48 hours to obtain a retentate, which was concentrated and freeze-dried to obtain a degraded polysaccharide of Flammulina velutipes bran, named FVRPF.

[0077] Example 3

[0078] 1. Determination of physical and chemical properties

[0079] The total sugar content of FVRPs was determined by the phenol-sulfuric acid method using glucose as the standard; the reducing sugar content of FVRPs was determined by the DNS method; the protein content of FVRPs was determined by the Coomassie Brilliant Blue method, and the uronic acid content of FVRPs was determined by the m-hydroxybiphenyl method using bovine serum albumin as the standard and galacturonic acid as the standard; the total phenol content in FVRPs was determined by the Folin-phenol method using gallic acid as the standard.

[0080] The specific physical and chemical properties are shown in Table 1. The results show that the polysaccharide content of FVRPs is greater than 80%, contains a small amount of protein, contains a small amount of uronic acid, and is an acidic polysaccharide. It contains a small amount of total phenols and reducing sugars. According to the content, it is found that degradation does not change the physical and chemical properties of polysaccharides too much.

[0081] Table 1 Physicochemical properties of FVRPs

[0082]

[0083] 2. Infrared spectroscopy analysis

[0084] The FVRPs powder and spectral grade potassium bromide powder were mixed evenly and pressed into tablets. FT-IR analysis was performed using a Nexus 470 FT-IR spectrometer (Equx55, Brucher, Germany). After the spectrum was measured, Prism software was used for plotting and analysis.

[0085] Analysis results (see Figure 1 )show:

[0086] The infrared spectrum of FVRPs shows 2927cm -1 and 2364cm -1 The presence of an absorption peak at 1420 cm indicates the presence of aliphatic CH bonds and CH in the sample. -1 There is a broad absorption band near 1100-1010cm, which may be caused by the deformation vibration of carbon-hydrogen bonds. -1 In the range of 808 cm-1, two obvious absorption peaks were observed, which may be caused by the stretching vibration of CO bond or CN bond. -1 These characteristic peaks indicate that the glycosyl residues of FVRPs have α-type glycosidic bonds. 2 O 2 -Vc and ultrasonic assisted H 2 O 2 -Fe 3+ After treatment, the main functional groups of polysaccharides did not change significantly, which indicates that the two degradation methods did not change the infrared structural characteristics of polysaccharides.

[0087] 3. Particle size distribution and Zeta potential scanning

[0088] Accurately weigh 1.0 mg of FVRPs sample, add 1 mL of ultrapure water, and ultrasonically treat for 5 min to completely disperse the sample. Then, use a nanoparticle size potentiometer to measure the particle size distribution of FVRPs for three consecutive measurements.

[0089] Particle size is related to the bioavailability of polysaccharides. Particle size affects the metabolism of polysaccharides in the body. Reducing particle size is beneficial to polysaccharide metabolism. The results of the investigation on particle size and potential changes of FVRPs are shown in Table 2 and Figure 2-7 From Table 2 and Figure 2 According to the recorded data, the particle size of FVRP is mainly concentrated between 160-700nm, with an average particle size of 442.9nm; the particle size of FVRPV is ( Figure 3 ) The size ranges from 130 to 390 nm, with an average particle size of 305.2 nm; the particle size of FVRPF ( Figure 4 ) sizes are concentrated between 140-1173nm, with an average particle size of 226.5nm. These average particle size values ​​all fall within the range of 1-1000nm, which meets the common particle size standards of polysaccharides. Zeta potential can reflect the state of electrostatic charge on the surface of the sample solution. For polysaccharides, its Zeta potential is closely related to the stability of the solution system. Figures 5 to 7 The Zeta potential distribution of FVRPs was shown. The calculated Zeta potentials of FVRP, FVRPV, and FVRPF were -9.2mV, -8.04mV, and -7.8mV, respectively. The surface of the FVRPs solution was mainly negatively charged. Generally, the higher the absolute value of the Zeta potential, the more stable the system. This shows that the absolute value of the potential of FVRP is higher than that of FVRPV and FVRPF, indicating that degradation promotes the molecular ionization of polysaccharides, which enhances the electrostatic repulsion between particles and makes them easier to be absorbed and utilized.

[0090] Table 2 Particle size and potential changes of FVRPs

[0091]

[0092] Note: Lowercase letters indicate intra-group differences (P<0.05).

[0093] 4. Determination of relative molecular mass

[0094] Gel permeation chromatography was used in this experiment. A TSK-Gel G3000PW xL column was used for chromatographic analysis, the mobile phase was 0.1 mol / L sodium nitrate (containing 0.01% sodium azide), the column temperature was 35°C, the flow rate was 0.5 mL / min, and the detector was a differential detector. In order to draw a standard curve, each series of dextran standards (180, 2700, 5250, 9750, 13050, 36800 and 64650 Da) were analyzed, and the relative molecular mass of the sample was calculated by GPC software.

[0095] Studies have shown that the average molecular weight of FVRP is 335.25 kDa. Figure 8 The average molecular weights of FVRPV and FVRPF were calculated to be 297.427 kDa and 256.057 kDa, respectively. This result indicates that after ultrasound-assisted H 2 O 2 -Vc and ultrasonic assisted H 2 O 2 -Fe 3+ The molecular weight of polysaccharides is reduced by the treatment. The reduction of the molecular weight of polysaccharides may enhance the antioxidant, prebiotic, anti-tumor and other biological activities of polysaccharides.

[0096] 5. Monosaccharide composition analysis

[0097] High performance gel permeation chromatography (HPGPC) was used to determine the monosaccharide composition. First, 10 mg of FVRPs was prepared into a 5 mg / mL solution, which was then hydrolyzed with trifluoroacetic acid. Then, under alkaline conditions, the degradation solution was derivatized with PMP and separated using a DionexCarboPac PA10 liquid chromatography column; the injection volume was 5 μL. Mobile phase A was 0.1 M NaOH, and mobile phase B was a mixed solution of 0.1 M NaOH and 0.2 M NaAc. During the experiment, the flow rate was maintained at 0.5 mL / min and the column temperature was maintained at 30 °C. The elution gradient was set to: 0 to 45 min, the ratio of phase A to phase B was maintained at 60:40 (V / V); at 45 min, the ratio of phase A to phase B was adjusted to the desired value.

[0098] The results of monosaccharide composition of FVRPs are shown in Fig. 9and Table 3. The results showed that FVRP, FVRPV and FVRPF are all heteropolysaccharides, and their monosaccharide compositions are similar. This finding indicates that the degradation treatment did not change the monosaccharide types of the polysaccharides. As shown in Table 3, FVRPs are mainly composed of 9 monosaccharides, including glucose (Glc) (8.3%, 14.53%, 9.91%), mannose (Man) (15.8%, 21.57%, 26.71%) and galacturonic acid (16.2%, 1.46%, 4.36%). There was no significant change in the monosaccharide composition, but the molar ratio of monosaccharides was different. After degradation treatment, the content of glucose and mannose increased, and the content of galacturonic acid decreased. This shows that degradation preferentially acts on the glycosidic bonds near galacturonic acid, while the glycosidic bonds near glucose and mannose are more stable. Therefore, ultrasonic-assisted H 2 O 2 -Vc and ultrasonic assisted H 2 O 2 -Fe 3+ The degradation system causes less damage to the basic structure of polysaccharides when processing them, and the reaction process is relatively stable.

[0099] Table 3 Monosaccharide composition of FVRPs

[0100]

[0101]

[0102] 6. Determination of the antioxidant activity of FVRPs in vitro

[0103] The ABTS scavenging rate, DPPH scavenging rate, reducing capacity and hydroxyl radical scavenging rate of FVRPs were determined, and vitamin C was used as a positive control. The specific steps are as follows:

[0104] (1) ABTS free radical scavenging activity: evaluated by absorbance at 734 nm. Dissolve 0.3841 g ABTS and 0.0662 g potassium persulfate in water and dilute to 100 mL to prepare ABTS base solution, which is stored in the dark at room temperature overnight; dilute the base solution to an OD value of approximately 0.750 ± 0.02 at 734 nm to serve as the working solution. Methods: Add 200 μL ABTS working solution to 50 μL FVRPs samples of different concentrations, react in the dark for 10 min, and measure the OD value at 734 nm three times. The absorbance of the sample plus ABTS is A1, the absorbance of the sample plus water is A2, the absorbance of ABTS plus water is A3, and the absorbance of water is A4.

[0105] ABTS free radical scavenging rate (%) = [1-(A1-A2) / (A3-A4)] × 100%.

[0106] (2) DPPH free radical scavenging activity: Take 200 μL of FVRPs solution, add 100 μL of DPPH solution, place in dark place for 10 min, and measure the OD 517 The values ​​were repeated 3 times and the average value was taken. A1 is the sample absorbance value of the sample solution plus DPPH-ethanol solution; A2 is the sample control group absorbance value of the sample solution 99.9% ethanol solution; A3 is the blank group absorbance value of distilled water plus DPPH solution; A4 is the blank group absorbance value of distilled water plus 99.9% ethanol solution.

[0107] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A3-A4] × 100%.

[0108] (3) Determination of reducing ability: FVRPs were prepared into sample solutions of different concentrations using distilled water. 50 μL of sample was taken and added with 50 μL of pH 6.6 phosphate buffer and 50 μL of 1% potassium ferrocyanide chloride solution. The solution was reacted in a 50°C water bath for 20 min. Then, 50 μL of 10% trichloroacetic acid and 50 μL of 0.1% ferric chloride were added. After standing for 10 min, the absorbance at 700 nm was measured. Water was used instead of the sample as the blank group, and deionized water was used instead of the potassium ferrocyanide solution as the control group. The reaction was repeated three times, and the data were recorded and the average value was taken.

[0109] (4) Hydroxyl radical scavenging effect: FVRPs were prepared into sample solutions of different concentrations using distilled water, and 9 mmol / L FeSO 4 and 9mmol / L salicylic acid-ethanol solution, 50μL each, and finally 1.2mmol / L H 2 O 2 The reaction was started with 50 μL of ethanol and the absorbance was measured at 510 nm. The reaction was allowed to react at 37 °C for 30 min, and the sample concentration absorbance A1 was measured at a wavelength of 510 nm. In addition, deionized water was used instead of H 2 O 2 Repeat the above operation to measure the absorbance value A2 of FVRPs itself, and at the same time replace the polysaccharide solution with water to measure the absorbance A0.

[0110] Hydroxyl radical scavenging rate (%) = [1-(A1-A2) / A0] × 100%.

[0111] ABTS free radical and DPPH free radical are widely used to evaluate the overall antioxidant capacity of compounds. FVRPs showed significant ABTS free radical and DPPH free radical scavenging activities, which increased in a dose-dependent manner (see Fig.10 and Fig.11). The ABTS removal rates of FVRP, FVRPV and FVRPF at 4.0 mg / mL were 89.27%, 96.06% and 99.3%, respectively, and the DPPH removal rates were 60.18%, 66.37% and 72.56%, respectively. Reducing power is one of the important indicators that directly reflects antioxidant capacity. The absorbance value of the reaction system at 700 nm indicates the reducing capacity of the antioxidant. The larger the absorbance value, the stronger the reducing capacity of the sample. Fig.12 As shown in the figure, with the increase of polysaccharide concentration, the reducing power OD values ​​of FVRP, FVRPV and FVRPF at 4.0 mg / mL reached 0.5, 0.8 and 2 respectively. Hydroxyl free radicals are considered to be the most toxic reactive oxygen species and can cause peroxidation damage to biological organisms. Fig.13 As shown in the data, with the increase of polysaccharide concentration, the hydroxyl radical scavenging effect of FVRPs was enhanced. At 4.0 mg / mL, the hydroxyl radical scavenging effects of FVRP, FVRPV and FVRPF reached 30%, 35% and 63%, respectively. The above results indicate that degradation can improve the antioxidant activity of polysaccharides.

[0112] Example 3 Effect of FVRPs on the growth of probiotics and determination of related indicators

[0113] (1) Determination of probiotic growth activity

[0114] FVRP, FVRPV, and FVRPF (2 mg / mL) were added to 4 mL MRS broth containing 0.5% glucose. 2 mg / mL fructooligosaccharide (FOS) was used as a positive control. Two activated probiotic strains, Lactobacillus plantarum and Lactobacillus rhamnosus, were selected for the experiment. Their initial OD 600 The values ​​were all adjusted to 0.2. Next, 2.0% inoculum of the two probiotics was taken and added to 4 mL of carbon-free MRS medium containing FVRP, FVRPV, and FVRPF (2 mg / mL). Subsequently, these culture media were placed in an anaerobic incubator and cultured at 37°C. In order to determine the specific effects of FVRP, FVRPV, and FVRPF on the growth of probiotics, the optical density OD of the two lactobacilli was measured at four time points during the culture process: 0h, 12h, 24h, 36h, and 48h. 600 .

[0115] Fig.14The promotion effect of the three polysaccharides on the growth of Lactobacillus plantarum was demonstrated in detail. Within 0 to 48 hours, the three polysaccharides all showed significant growth-promoting effects. In the 0 to 12 hour stage, the growth-promoting effect of FVRPV was significantly better than that of FVRP, FVRPF, and FOS (a common prebiotic), which were all higher than CK. Entering the 12 to 36 hour stage, the growth-promoting effects of FVRPV and FVRP were significantly better than those of FVRPF and FOS, which were both higher than CK. By 48 hours, FVRPV was significantly better than FVRP and FVRPF.

[0116] Fig.15 The results showed that the three polysaccharides promoted the growth of Lactobacillus rhamnosus. FVRP, FVRPV and FVRPF can effectively promote the growth of Lactobacillus rhamnosus. It can be found that the growth-promoting effect of FOS was significantly higher than that of other groups at 0 to 12 hours. Subsequently, the growth-promoting effects of FVRPF and FOS gradually became consistent, and were significantly higher than FVRP and FVRPV at the 24 to 36 hour stage. By the end of the 48-hour experiment, FVRPF and FOS were significantly better than FVRP and FVRPV. Both were significantly better than CK.

[0117] (2) Acid production and short-chain fatty acids (SCFAs) content determination

[0118] 1 mL of the polysaccharide solution of each group at 48 h was mixed with 0.2 mL of 25% metaphosphoric acid solution, and then centrifuged at 10,000 r / min for 15 min to obtain the supernatant. After that, the samples were analyzed by gas chromatography using a Nukol capillary column (30 m × 0.32 mm × 0.25 μm) as the chromatographic column, and the analysis time for each sample lasted 32 min.

[0119] Table 4 shows the concentration of SCFAs produced by plant lactic acid bacteria in different groups, mainly producing acetic acid and propionic acid. As important SCFAs, acetic acid and propionic acid have a positive effect on the growth of probiotics and the health of the host. In this example, it was found that the acetic acid content and total SCFAs of the FVRPF group and the FVRPV group after degradation were significantly higher than those of the FVRP. Table 5 shows the concentration of SCFAs produced by rhamnosus lactic acid bacteria in different groups, mainly producing acetic acid, propionic acid, isobutyric acid, and isovaleric acid. Isobutyric acid and isovaleric acid, two short SCFAs, may have adverse effects on the host and easily cause dysfunction of the host's fermentation and digestion processes. In terms of isobutyric acid concentration, the isobutyric acid concentration of the FVRP group was 0.66±0.034μmol / g, which was significantly lower than that of the CK group and the FOS group. No isobutyric acid was detected in the FVRPF and FVRPV groups. In terms of isovaleric acid concentration, the FVRPV group was also significantly lower than the isovaleric acid concentration of the FVRP group, lower than the CK group and the FOS group but higher than the FVRPF group.

[0120] The above results all indicate that degradation treatment of FVRP may enhance its prebiotic activity.

[0121] Table 4 Changes in SCFAs production by plant lactic acid bacteria promoted by three polysaccharides

[0122]

[0123] Note: Lowercase letters indicate intra-group differences (P<0.05).

[0124] Table 5 Changes of SCFA production by three polysaccharides in lactic acid bacteria

[0125]

[0126] Note: Lowercase letters indicate intra-group differences (P<0.05).

[0127] Example 4 FVRPs in vitro fermentation test

[0128] 1. In vitro fermentation sample processing

[0129] First, scrape and mix the volunteers' fresh feces in equal amounts, quickly add them to sterile saline, and prepare a feces mixture with a solid-liquid ratio of 10% (w / v). Next, homogenize the mixture for 1 minute, and then centrifuge it at 2300rpm and 4°C for 5 minutes to collect the supernatant. This preparation process must be carried out in a refrigerator or low-temperature environment, and the total duration should not exceed 15 minutes to ensure the freshness and activity of the fecal slurry. The collected supernatant is immediately placed in an anaerobic tank and stored at -80°C for future use.

[0130] The culture medium was prepared, and its components were as follows: 0.04 g / L potassium dihydrogen phosphate, 2.0 g / L peptone, 0.02 g / L hemin chloride, 2.0 g / L yeast extract, 0.01 g / L calcium chloride, 0.1 g / L sodium chloride, 2.0 mL / L Tween 80, 0.04 g / L potassium dihydrogen phosphate, 0.5 g / L bile salt, 0.01 g / L magnesium sulfate heptahydrate, 2 g / L sodium bicarbonate, 0.5 g / L cysteine ​​hydrochloride, 10 μL vitamin K1 (equivalent to 0.0097 g), and 1.0 mL / L 1% resin azure solution. Then, 10.0 mg / mL FVRP, FVRPV, FVRPF and oligofructose (FOS) were added respectively, of which the FOS group was the positive control group and the group without addition was the CK group. Finally, sterilization was performed at 121°C for 20 min.

[0131] The experiment was divided into five groups, and each group took 50mL of culture medium and used a 100mL shake flask. In each group of shake flasks, 5mL of 10% fecal slurry was added. Subsequently, all shake flasks were fermented in an anaerobic environment at 37°C. At the key time points of the fermentation process (0h, 6h, 12h, 24h, 48h), fermentation samples were collected respectively, and 2mL of supernatant was taken to determine the physicochemical properties, acid production and intestinal flora, and 10mL of supernatant was freeze-dried to determine the infrared spectrum.

[0132] 2. Changes in the physicochemical properties of FVRPs after in vitro fermentation

[0133] For a total of 25 samples from the FVRP, FVRPV, FVRPF, CK and FOS groups at different time periods during in vitro fermentation, the polysaccharide content, total sugar content, total reducing sugar content and total uronic acid content were determined respectively, using the same method as Example 3.

[0134] Table 6 shows the changes in the content of total sugar, reducing sugar and uronic acid in the experimental culture medium, and evaluates the utilization of polysaccharides by intestinal microorganisms. The concentrations of total sugar, reducing sugar and uronic acid in the FVRPs group decreased, and it is speculated that the structural differences of the three polysaccharides may lead to their different performances in the process of intestinal microbial utilization. These differences further affect the rate of fermentation reaction and the generation of products. In summary, FVRPs can be utilized by intestinal flora, and its utilization process is affected by the physical and chemical properties of polysaccharides.

[0135] Table 6 Changes in physicochemical properties of three polysaccharides after in vitro fermentation

[0136]

[0137]

[0138] Note: Capital letters indicate differences between groups, and lowercase letters indicate differences within groups (P<0.05).

[0139] 3. Analysis of infrared spectra of FVRPs after in vitro fermentation

[0140] For a total of 15 samples from the FVRP, FVRPV, and FVRPF groups in in vitro fermentation, infrared spectra were measured respectively, and the measurement method was consistent with the method in Example 3.

[0141] Infrared spectroscopy analysis showed that FVRP( Fig.16 )、FVRPV( Fig.17 )、FVRPF( Fig.18 ) Infrared changes within 0 to 48 hours of in vitro fermentation. At about 3400cm -1 There is a broad and strong absorption peak at 2927cm, which is attributed to the stretching vibration of hydroxyl (OH). -1 and 2364cm -1The absorption peaks at 1420 cm-1 and 1420 cm-2 correspond to the stretching vibration and deformation vibration of aliphatic CH. -1 The broad absorption band can be attributed to the deformation vibration of carbon-hydrogen bonds. -1 Two absorption peaks were observed in the range of 808cm-1, which may be caused by the stretching vibration of CO bond or CN bond. -1 The results showed that the infrared spectra of FVRP, FVRPV and FVRPF did not show changes in characteristic peaks at 0, 6, 12, 24 and 48 h as the in vitro fermentation time changed, indicating that their structures did not change significantly during the fermentation process. These results indicate that, except for some possible breakage of glycosidic bonds, FVRP, FVRPV and FVRPF showed stability during the fermentation process.

[0142] 4. Determination of antioxidant capacity of FVRPs after in vitro fermentation

[0143] The determination method continued to use the antioxidant determination method of Example 3, and the ABTS clearance rate, DPPH clearance rate and reducing capacity of the samples after in vitro fermentation of FVRP, FVRPV and FVRPF were determined.

[0144] like Fig.19 As shown, the maximum ABTS clearance rate of the FVRP group could reach 82.21%, the maximum ABTS clearance rate of the FVRPV group could reach 95.8%, the maximum ABTS clearance rate of the FVRPF group could reach 99.7%, while the maximum ABTS clearance rate of the CK group could reach 43.02%, and the maximum clearance rate of the FOS group could reach 65.48%. Fig. 20 It represents the reducing power of fermentation broth in different groups. The maximum reducing power of FVRP group can reach 2.23, the maximum reducing power of FVRPV group can reach 2.6, the reducing power of FVRPF group can reach 3.4, the reducing power of CK group can reach 2.3, and the maximum reducing power of FOS can reach 2.4. Fig.21 It is the result of the measurement of DPPH scavenging ability. The maximum DPPH clearance rate of FVRP group can reach 84.8%, the maximum DPPH clearance rate of FVRPV group can reach 90.5%, the maximum DPPH clearance rate of FVRPF group can reach 83.9%, while the maximum DPPH clearance rate of CK group can reach 75.05%, and the maximum DPPH clearance rate of FOS group can reach 90.5%.

[0145] In this embodiment, it was found that the FVRPF group was greater than the FVRPV group and greater than the FVRP group. The FVRPF group showed better ABTS clearance rate and reducing power, while the DPPH clearance rate showed that the clearance ability of FVRPV was stronger, greater than that of FVRPF and FVRP.

[0146] 5. Determination of pH after in vitro fermentation of FVRPs

[0147] The acid production of FVRPs after in vitro fermentation was tested. Fig. 22 As shown in the figure, at 0h, the pH values ​​of all experimental groups were roughly between 8.0 and 9.0. As the fermentation progressed, the pH values ​​of each experimental group were observed to gradually decrease. The pH values ​​of all groups decreased from 0 to 12h, and the pH values ​​of FVRPV and FVRPF decreased more slowly than other groups from 12 to 24h, and all groups decreased from 24 to 48h. After 48h of fermentation, the pH values ​​of FVRPs, FOS, and CK all dropped to the lowest level. It can be found that among the experimental groups of FVRPs, the pH value of FVRPF decreased the most, followed by FVRPV, and finally FVRP.

[0148] 6. Changes of SCFAs in FVRPs in vitro fermentation products

[0149] The determination method of SCFAs was consistent with that in Example 3. Table 7 shows that after 48 h of in vitro fermentation, the concentrations of SCFAs produced by each group showed significant differences, with 7.69±0.16 μmol / g in the FVRP group, 4.43±0.04 μmol / g in the FVRPV group, and 3.49±0.03 μmol / g in the FVRPF group, all significantly higher than 1.41±0.56 μmol / g in the CK group.

[0150] Table 7 Changes of SCFAs of three polysaccharides after 48 h in vitro fermentation

[0151]

[0152] Note: Lowercase letters indicate intra-group differences (P<0.05).

[0153] 7. Effects of FVRPs in vitro fermentation on intestinal flora

[0154] The intestinal flora of 25 samples (5 replicates per group) from FVRP, FVRPV, FVRPF, CK, and FOS groups after 48 h of in vitro fermentation was determined. The ITS2 region of fungi and the V3-V4 region of 16S rDNA of bacteria were amplified and sequenced. The species distribution histogram was established based on the results of feature analysis. LEfSe analysis determined the statistically significant differences in biomarkers between the groups and analyzed the differential microbial communities. The relationship between species and biochemical indicators was determined by Spearman analysis. The differential functional genomics of the samples was analyzed using PICUSt software.

[0155] (1) Alpha diversity analysis

[0156] Fig.23 It can be seen that the five groups of rarefaction curves are all flat, indicating that the sequencing depth is large enough for subsequent research. Shannon index and Simpson index represent species richness and species evenness, and ACE index and Chao1 index represent species richness and diversity at the OTU level. Shannon ( Fig.24 )、Simpson( Fig.25 ) index initially increased significantly and then stabilized, indicating that the amount of sequencing data was large enough. ACE index ( Fig.26 ) and Chao Index ( Fig. 27 ) showed that compared with the FOS group, the ACE index and Chao index of the FVRPF, FVRPV and CK groups increased significantly. Compared with the CK group, the index of the FVRP and FVRPV groups decreased, while the index of the FVRPF group increased. Compared with the FVRP, the index of the FVRPV and FVRPF groups increased significantly, and the increase in the FVRPF group was more significant. The above results show that FVRPs can improve the abundance of intestinal flora.

[0157] (2) Beta diversity analysis

[0158] Beta diversity analysis can be used to compare the similarities of different samples in species diversity. Results ( Fig.28 ) showed that FVRP, FVRPV, and FVRPF groups could change the composition of intestinal flora. Sample hierarchical cluster analysis ( Fig.29 ), the FVRP, FVRPV, and FVRPF groups are close in distance and have small differences; the polysaccharide group is far from CK and has large differences; the polysaccharide group is far from FOS and has large differences. This indicates that the samples in each sample group have similar abundance at the OTU level, there are differences in diversity among the sample types, the number of sequencing is large enough, and the three polysaccharides have a significant improvement effect on the intestinal flora.

[0159] (3) Species composition analysis

[0160] From the species distribution stacking diagram ( Fig.30 ) It can be seen that at the level of intestinal flora, the five groups mainly contain five dominant bacterial phyla. It has been reported that an increase in the ratio of Bacteroidetes to Firmicutes (B / F) is associated with a reduced risk of insulin resistance and weight gain. Combined with the results of Firmicutes, the B / F ratio increased in the fermentation group with the addition of FVRPV. Therefore, FVRPV may have an anti-obesity effect. FVRP, FVRPV and FVRPF can regulate the composition of intestinal flora, promote the growth of beneficial bacteria, and suggest that the FVRPV component has potential anti-obesity effects.

[0161] (4) Important analysis of differences between groups

[0162] Depend on Fig.31From the stacked graph of species distribution at the genus level shown in the figure, it can be seen that the top 10 dominant genera at the genus level of intestinal flora are Escherichia-Shigella, Bacteroides, Lactobacillus, Alcaligenes, Parabacteroides, Fusobacterium, Phascolarctobacterium, Pseudomonas, Acinetobacter, and Enterococus. Fig.32 ) It can be seen that there are certain differences in the microbial composition of the FVRP, FVRPV and FVRPF groups, which may be due to changes in molecular weight and glycosidic bonds after degradation. These changes are considered to be key factors in the chemical properties of polysaccharides and are expected to improve or change the resident intestinal bacteria. It is speculated that the degradation of FVRP can promote intestinal health by increasing the proportion of probiotics.

[0163] (5) Important analysis of differences between groups - KEGG

[0164] The results of KEGG functional gene prediction are as follows Fig.33 As shown in the results, FVRP, FVRPV, FVRPF and FOS significantly downregulated 19 metabolic pathways that were upregulated in CK, involving key biological processes such as carbohydrates, cofactors and vitamins, terpenoids and polyketides. At the same time, it was also found that the biosynthesis of other secondary metabolites and infectious disease pathways were downregulated in the CK group, while they were enriched under the treatment of the three polysaccharides and FOS. Comparing the performance of the three polysaccharides, CK and FOS in key metabolic pathways, FVRPF showed the most significant inhibitory effect in key pathways such as signal transduction, membrane transport, lipid metabolism, amino acid metabolism and carbohydrate metabolism, which was better than FOS, FVRPV and FVRP were better than CK. The CK group showed high activity in the process of cell growth and death, while the treatment of the three polysaccharides and FOS upregulated its activity to varying degrees, among which FVRPV and FOS had a more significant upregulation effect, followed by FVRPF and FVRP, suggesting that FVRP, FVRPV and FVRPF may affect the overall metabolic state by regulating cell growth and death.

[0165] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polysaccharide FVRPV having the function of regulating intestinal flora, characterized in that: The preparation method of the polysaccharide FVRPV comprises the following steps: Dissolve the Flammulina velutipes chaff polysaccharide in water, adjust the pH to 2.8-3.2, add H2O2 solution and ascorbic acid to obtain a mixed solution; After the mixed solution is subjected to ultrasonic treatment, the pH value is adjusted to neutral to obtain a reaction solution; The reaction solution is dialyzed, concentrated and freeze-dried to obtain the polysaccharide FVRPV; The preparation method of Flammulina velutipes chaff polysaccharide comprises the following steps: mixing Flammulina velutipes chaff and ultrapure water at a solid-liquid ratio of 1:50, performing ultrasonic-assisted extraction, wherein the ultrasonic power is 540 W, the extraction temperature is 75° C., and the extraction time is 20 min; filtering after the extraction to obtain a filtrate, adding anhydrous ethanol to the filtrate for alcohol precipitation overnight, deproteinizing by a Sevag method, and obtaining the Flammulina velutipes chaff polysaccharide by dialysis, concentration and freeze-drying; The mass concentration of the H2O2 solution is 30%, and the volume fraction of the H2O2 solution in the mixed solution is 0.030-0.040%; The mass concentration of ascorbic acid in the mixed solution is 0.025-0.035%; The temperature of the ultrasonic treatment is 75-85°C, the power is 600 W, and the time is 20-40 min.

2. A polysaccharide FVRPF having the function of regulating intestinal flora, characterized in that: The preparation method of the polysaccharide FVRPF comprises the following steps: Dissolve the polysaccharide from Flammulina velutipes fungus husk in water, adjust the pH to 2.8-3.2, add H2O2 solution and FeCl3 to obtain a mixed solution; After the mixed solution is subjected to ultrasonic treatment, the pH value is adjusted to neutral to obtain a reaction solution; The reaction solution is dialyzed, concentrated and freeze-dried to obtain the polysaccharide FVRPF; The preparation method of Flammulina velutipes chaff polysaccharide comprises the following steps: mixing Flammulina velutipes chaff and ultrapure water at a solid-liquid ratio of 1:50, performing ultrasonic-assisted extraction, wherein the ultrasonic power is 540 W, the extraction temperature is 75° C., and the extraction time is 20 min; filtering after the extraction to obtain a filtrate, adding anhydrous ethanol to the filtrate for alcohol precipitation overnight, deproteinizing by a Sevag method, and obtaining the Flammulina velutipes chaff polysaccharide by dialysis, concentration and freeze-drying; The mass concentration of the H2O2 solution is 30%, and the volume fraction of the H2O2 solution in the mixed solution is 1.10-1.15%; The mass concentration of FeCl3 in the mixed solution is 0.01-0.03%; The temperature of the ultrasonic treatment is 75-85°C, the power is 600 W, and the time is 20-40 min.

3. Use of the polysaccharide FVRPV as claimed in claim 1 and / or the polysaccharide FVRPF as claimed in claim 2 in the preparation of products for regulating intestinal flora.

4. The use according to claim 3, characterized in that: The product is a health product or a medicine.

5. A product for regulating intestinal flora, characterized in that: The active ingredient comprises the polysaccharide FVRPV according to claim 1 and / or the polysaccharide FVRPF according to claim 2.

6. The product according to claim 5, characterized in that The product is a health product or a medicine.

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