Fermented nostoc commune polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of extraction, and discloses a preparation method of fermented nostoc commune polysaccharide, which comprises the following steps: crushing nostoc commune, and preparing crude nostoc commune polysaccharide by adopting a water extraction and alcohol precipitation method; inoculating lactobacillus reuteri into the fermentation culture medium added with the nostoc commune crude polysaccharide, and fermenting for 48 hours; centrifugally collecting fermentation liquor, adding 95% ethanol, and standing; centrifugally collecting precipitates, and dissolving with hot water; removing protein by using a Sevage reagent; and freeze-drying to obtain the fermented nostoc commune polysaccharide (FNVPS). The polysaccharide is mainly composed of galactose, glucose and xylose, the weight-average molecular weight is 670.78 kDa, and FNVPS has remarkable anti-inflammatory activity in a mouse ulcerative colitis model and can remarkably delay senescence on wild caenorhabditis elegans. The FNVPS has the characteristics of easily available raw materials, high yield and the like; the process is simple, large-scale industrial production can be realized, and the method has great development potential in functional food and medical raw materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction, and in particular relates to a fermented Nostoc commune polysaccharide, a preparation method thereof and an application thereof. Background Art

[0002] Biological modification can change the molecular structure of plant-based polysaccharides and improve related biological activities and functional properties. Common strategies for polysaccharide modification include physical, chemical and biological methods, among which biological methods have the advantages of environmental protection, mild reaction conditions and high efficiency. Related research shows that probiotic fermentation can change the molecular structure of polysaccharides and also improve the biological activity of polysaccharides through structural modification. In addition, probiotic fermentation has the advantages of environmental friendliness, low extraction temperature and energy saving, so it may be a green and efficient polysaccharide modification method with great development potential in the long run. At present, common probiotics used for fermentation include Lactobacillus, Bifidobacterium, Streptococcus and yeast, etc. In the previous research of this research group, a strain of Lactobacillus reuteri LRSY523 (patent number: CN114107088A) was obtained, which has the effects of protecting the intestinal barrier function, improving obesity and insulin resistance in mice fed a high-fat diet.

[0003] Polysaccharides, as recognized bioactive macromolecules that are non-toxic and have no side effects on the human body, can be used as antioxidants, anti-inflammatory agents and immunomodulators, etc. Nostoc commune, scientific name Nostoc commune Vaucher, the analysis results of nutritional components show that Nostoc commune contains about 20% polysaccharides. Research shows that Nostoc commune polysaccharides have antibacterial, anti-inflammatory, immunomodulatory, antitumor and other activities, but the effects of probiotic modification on the structure and biological activity of Nostoc commune polysaccharides have not been reported. At present, the related research on fermented polysaccharides mainly uses raw materials for probiotic fermentation, and then extracts the polysaccharides from the fermented raw materials for research on structure and biological activity. However, the present invention directly ferments the extracted crude Nostoc commune polysaccharide as a single carbon source with Lactobacillus reuteri LRSY523 to exclude the influence of other active components in the raw materials on the fermentation process, and can more intuitively study the influence of probiotic fermentation on the structure and activity of polysaccharides themselves. The present invention provides a new idea for the application of Nostoc commune in the fields of anti-inflammatory and anti-aging, and provides an important reference basis for the deep development and utilization of Nostoc commune resources.

[0004] Through retrieval, the following several patent disclosure documents related to the present invention patent application were found:

[0005] 1. A Nostoc commune antitumor polysaccharide and its preparation method (CN104193845A). The preparation method is to dry and crush pure natural Nostoc commune, weigh an appropriate amount, add distilled water for reflux extraction, concentrate the extract by rotary evaporation to an appropriate amount, add a 10.6% potassium ferrocyanide solution and a 21.9% zinc acetate solution in a volume ratio of 1:1 to precipitate proteins. After centrifugation, the supernatant is aspirated. After dialysis of the concentrated solution with distilled water, anhydrous ethanol is added to precipitate white flocculates, and after centrifugation, it is freeze-dried under vacuum to obtain Nostoc commune polysaccharide. In vitro antitumor experiments show that this polysaccharide can significantly inhibit the proliferation of human breast cancer cells without cytotoxicity, has good antitumor activity, and provides the possibility for the development of new anti-cancer health products or drugs.

[0006] 2. A method for extracting Dendrobium officinale polysaccharide by microbial fermentation (CN118745450A). This method uses microbial fermentation to extract Dendrobium officinale polysaccharide, which specifically includes the following steps: S1, extracting Dendrobium officinale polysaccharide by water extraction method, centrifuging to obtain the supernatant, and sterilizing it; S2, culturing and counting lactic acid bacteria, then inoculating the lactic acid bacteria into the sterilized supernatant, and conducting single-factor experiments and response surface experiments to ferment to obtain a fermentation broth; S3, purifying the fermented Dendrobium officinale polysaccharide; S4, measuring the molecular weight of the fermented Dendrobium officinale polysaccharide; S5, measuring biological activity. The beneficial effects of this invention are: selecting four factors of temperature, rotation speed, time, and strain inoculation amount for single-factor experiments, using the DPPH scavenging rate as an evaluation index to obtain three relatively optimal levels, and then designing a response surface experiment with four factors and three levels. Through response surface experiment analysis, the optimal conditions for the fermentation of Dendrobium officinale polysaccharide are: temperature 39°C, time 48h, rotation speed 150r / min, and strain inoculation amount 3%.

[0007] 3. Auricularia auricula polysaccharide, its application and preparation method (WO2024045207A1). Auricularia auricula polysaccharide, its application and preparation method belong to the technical field of polysaccharides. After defatting Auricularia auricula, it is preliminarily enzymatically hydrolyzed under the action of snailase, further degraded and extracted by the synergistic action of H2O2 and ultrasonic waves, deeply enzymatically hydrolyzed under the action of a composite enzyme, and fermented by a mixture of Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium longum. The fermented Auricularia auricula polysaccharide undergoes a phosphorylation reaction under the action of a phosphorylation reagent, and after further deproteinization and decolorization, it is chelated with zinc salt to obtain a polysaccharide-zinc complex, that is, Auricularia auricula polysaccharide. The preparation method is simple, the extraction efficiency is high, the obtained Auricularia auricula polysaccharide has high purity, good solubility, is easy to absorb, and has good physiological activities, including good anti-inflammatory, anti-aging, hypoglycemic effects, etc., has the effects of improving immunity, promoting intellectual development, reducing blood lipid, regulating total cholesterol, and effectively increasing beneficial cholesterol, and has broad application prospects.

[0008] By comparison, there are essential differences between this invention patent application and the above-mentioned patent disclosure documents.

[0009] Compared with the existing inventions, the present invention directly uses Nostoc commune polysaccharide as a carbon source to ferment Lactobacillus reuteri, reducing the influence of other nutritional components in the raw materials on the fermentation process, and enabling a more intuitive study of the effects of probiotic fermentation on the structure and activity of the polysaccharide itself. At the same time, the interaction between Lactobacillus reuteri and Nostoc commune polysaccharide is explored. On the one hand, Nostoc commune polysaccharide can promote the growth and reproduction of Lactobacillus reuteri, and on the other hand, the fermentation of Lactobacillus reuteri makes the polysaccharide significantly exhibit anti-inflammatory activity at the mouse level and anti-aging activity at the nematode level. Summary of the Invention

[0010] The object of the present invention is to overcome the deficiencies in the prior art and provide a method for fermenting Nostoc commune polysaccharide, a preparation method thereof, and an application thereof.

[0011] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0012] A preparation method for fermenting Nostoc commune polysaccharide, comprising the following steps:

[0013] First step: After pulverizing Nostoc commune, sieve it through a 50-mesh sieve to obtain Nostoc commune powder, and extract Nostoc commune polysaccharide by the water extraction and alcohol precipitation method to obtain crude Nostoc commune polysaccharide;

[0014] Second step: Inoculate Lactobacillus reuteri into the fermentation medium added with crude Nostoc commune polysaccharide and ferment for 48 h;

[0015] Third step: Centrifuge to collect the fermentation broth, and add ethanol with a volume percentage of 95% to the fermentation broth and let it stand;

[0016] Fourth step: Centrifuge to collect the precipitate and dissolve it in hot water;

[0017] Fifth step: Use Sevage reagent to remove proteins, and rotary evaporate to remove Sevage reagent;

[0018] Sixth step: Desalt with a dialysis bag with a molecular weight of 8 kDa, and then perform vacuum freeze-drying. The freeze-dried product is powdered to obtain fermented Nostoc commune polysaccharide, abbreviated as FNVPS.

[0019] Further, in the second step, the fermentation medium is prepared as follows: For every 20 g of crude Nostoc commune polysaccharide, 10 g of peptone, 2 g of trisodium citrate, 0.2 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 0.05 g of manganese sulfate, and 1000 mL of distilled water are mixed and then autoclaved to obtain it. The sterilization temperature is 121 °C and the time is 15 min;

[0020] In the second step, the inoculation is carried out according to an inoculation amount of 5%, the fermentation temperature is 37 °C, the shaker speed is 200 rpm / min, and the time is 48 h.

[0021] Further, in the second step, the Lactobacillus reuteri is Lactobacillus reuteri LRSY523.

[0022] Further, in the third step, the centrifugation speed and time are: 8000 rpm / min, 15 min; the addition amount of the 95% ethanol is four times the volume of the fermentation broth; the standing time is 12 h.

[0023] Further, in the fourth step, the centrifugation speed and time are: 8000 rpm / min, 15 min; the rotary evaporation temperature is 65 °C;

[0024] Alternatively, in the fifth step, the Sevage reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, and the volume ratio of the supernatant to the Sevage reagent is 5:1; the rotary evaporation temperature is 50 °C.

[0025] Further, the molecular weight of the fermented Nostoc commune polysaccharide is 670.78 kDa, and the monosaccharide composition is mannose 2.33%, glucuronic acid 1.56%, glucose 27.93%, galactose 43.32%, xylose 17.71%, and arabinose 6.75%.

[0026] The fermented Nostoc commune polysaccharide prepared by the above preparation method.

[0027] Further, at the animal model level, it was found that the fermented Nostoc commune polysaccharide FNVPS can effectively alleviate acute colitis caused by DSS and significantly reduce the secretion of inflammatory factors; at the Caenorhabditis elegans level, it was found that the fermented Nostoc commune polysaccharide FNVPS can effectively delay the aging-related indicators of nematodes, indicating that the fermented Nostoc commune polysaccharide has the potential to be developed into functional foods, cosmetics, and pharmaceutical raw materials for anti-inflammatory and anti-aging purposes.

[0028] Use of the fermented Nostoc commune polysaccharide as described above in the preparation of anti-inflammatory functional foods and / or cosmetics and / or foods and / or drugs.

[0029] Use of the fermented Nostoc commune polysaccharide as described above in the preparation of anti-aging functional foods and / or cosmetics and / or foods and / or drugs.

[0030] The advantages and positive effects achieved by the present invention are:

[0031] 1. In the present invention, the preparation method of fermented Nostoc commune polysaccharide is to crush and sieve Nostoc commune, extract crude Nostoc commune polysaccharide and use it as the sole carbon source to prepare a culture medium, inoculate Lactobacillus reuteri for fermentation. After fermentation, the fermentation broth is collected by centrifugation, and the polysaccharide in the fermentation broth is separated by ethanol precipitation. After removing ethanol with a rotary evaporator, protein is removed by the Sevage method, the Sevage reagent is removed by rotary evaporation, and after dialysis and lyophilization, fermented Nostoc commune polysaccharide (FNVPS) is obtained. The polysaccharide is mainly composed of galactose, glucose, xylose and arabinose, and the weight-average molecular weight distribution range is 670.78 kDa. The FNVPS of the present invention has the characteristics of easy availability of raw materials and high yield; the process is simple, can be mass-produced industrially, and has great development potential in functional foods and pharmaceutical raw materials.

[0032] 2. At the animal model level, it was found in the present invention that fermented Nostoc commune polysaccharide FNVPS can effectively relieve acute colitis caused by DSS and significantly reduce the secretion of inflammatory factors. At the level of Caenorhabditis elegans, it was found that fermented Nostoc commune polysaccharide FNVPS can effectively delay the aging-related indicators of nematodes. The fermented Nostoc commune polysaccharide (FNVPS) of the present invention has the characteristics of easy availability of raw materials, high yield and strong drug stability, and can be administered orally to increase the administration route; the process is simple and suitable for large-scale industrial production. It shows that this FNVPS has the potential to be developed into functional foods, cosmetics and pharmaceutical raw materials for anti-inflammatory and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the growth curve diagram of different culture media (Figure A), different strains (Figure B) and molecular weight diagram (Figure C) in the present invention;

[0034] Figure 2 It is the photo diagram of the FNVPS freeze-dried powder sample in the present invention;

[0035] Figure 3 It is the liquid phase diagram of the monosaccharide composition of FNVPS in the present invention;

[0036] Figure 4 It is the diagram of the effect of FNVPS on the body weight (Figure B) and colon length (Figure C) of mice with acute colitis in the present invention; among them, Figure A is the schematic diagram of the animal experiment design;

[0037] Figure 5 It is the diagram of the effect of FNVPS on inflammatory factors in the colon tissue of mice with acute colitis in the present invention;

[0038] Figure 6 It is the diagram of the effect of FNVPS on the lifespan of Caenorhabditis elegans in the present invention;

[0039] Figure 7 It is the diagram of the effect of FNVPS on lipofuscin in Caenorhabditis elegans in the present invention. Detailed implementation mode

[0040] The present invention will be further described below in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0041] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically noted in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the application date of the present invention for implementation.

[0042] A preparation method of fermented nostoc commune polysaccharide comprises the following steps:

[0043] First step: After pulverizing nostoc commune, passing through a 50-mesh sieve to obtain nostoc commune powder, and extracting nostoc commune polysaccharide by water extraction and alcohol precipitation to obtain crude nostoc commune polysaccharide;

[0044] Second step: Inoculating Lactobacillus reuteri into the fermentation medium added with crude nostoc commune polysaccharide and fermenting for 48 h;

[0045] Third step: Centrifuging to collect the fermentation broth, adding ethanol with a volume percentage of 95% to the fermentation broth and standing;

[0046] Fourth step: Centrifuging to collect the precipitate and dissolving it in hot water;

[0047] Fifth step: Removing proteins with Sevage reagent and rotary evaporating to remove Sevage reagent;

[0048] Sixth step: Desalting with a dialysis bag with a molecular weight of 8 kDa, and then performing vacuum freeze-drying. The freeze-dried powder is the fermented nostoc commune polysaccharide, abbreviated as FNVPS.

[0049] Preferably, in the second step, the fermentation medium is prepared according to 20 g of crude nostoc commune polysaccharide, 10 g of peptone, 2 g of trisodium citrate, 0.2 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 0.05 g of manganese sulfate, and 1000 mL of distilled water. After mixing, it is sterilized under high pressure to obtain it. The sterilization temperature is 121 °C and the time is 15 min;

[0050] In the second step, the inoculation is carried out according to an inoculation amount of 5%, the fermentation temperature is 37 °C, the shaker speed is 200 rpm / min, and the time is 48 h.

[0051] Preferably, in the second step, the Lactobacillus reuteri is Lactobacillus reuteri LRSY523.

[0052] Preferably, the centrifugation speed and time in the third step are: 8000 rpm / min, 15 min; the addition amount of the 95% ethanol is four times the volume of the fermentation broth; the standing time is 12 h.

[0053] Preferably, the centrifugation speed and time in the fourth step are: 8000 rpm / min, 15 min; the rotary evaporation temperature is 65 °C;

[0054] Alternatively, in the fifth step, the Sevage reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, and the volume ratio of the supernatant to the Sevage reagent is 5:1; the rotary evaporation temperature is 50 °C.

[0055] Preferably, the molecular weight of the fermented Nostoc commune polysaccharide is 670.78 kDa, and the monosaccharide composition is mannose 2.33%, glucuronic acid 1.56%, glucose 27.93%, galactose 43.32%, xylose 17.71%, arabinose 6.75%.

[0056] The fermented Nostoc commune polysaccharide prepared by the above preparation method.

[0057] Preferably, at the animal model level, it was found that the fermented Nostoc commune polysaccharide FNVPS can effectively alleviate acute colitis caused by DSS and significantly reduce the secretion of inflammatory factors; at the Caenorhabditis elegans level, it was found that the fermented Nostoc commune polysaccharide FNVPS can effectively delay the aging-related indicators of nematodes, indicating that the fermented Nostoc commune polysaccharide has the potential to be developed into functional foods, cosmetics and pharmaceutical raw materials for anti-inflammatory and anti-aging.

[0058] Use of the fermented Nostoc commune polysaccharide as described above in the preparation of anti-inflammatory functional foods and / or cosmetics and / or foods and / or drugs.

[0059] Use of the fermented Nostoc commune polysaccharide as described above in the preparation of anti-aging functional foods and / or cosmetics and / or foods and / or drugs.

[0060] Specifically, the relevant preparation and detection are as follows:

[0061] Example 1: Preparation method and molecular weight determination of Nostoc commune fermented polysaccharide (FNVPS)

[0062] (1) After pulverizing Nostoc commune, sieve it through a 50-mesh sieve to obtain Nostoc commune powder. Weigh 20 g of the Nostoc commune powder passing through a 50-mesh sieve, add 700 mL of distilled water, heat and stir at 90 °C for 2 h, and centrifuge at 8000 rpm for 15 min after cooling to room temperature to obtain the supernatant.

[0063] (2) Add 95% ethanol solution with a volume four times that of the supernatant to the supernatant, precipitate at 4 °C for more than 12 h, and centrifuge at 8000 rpm for 15 min to obtain the precipitate.

[0064] (3) Add the above precipitate to 100 mL of distilled water and heat it at 60 °C to dissolve it into a polysaccharide solution, so that the ethanol completely volatilizes. Lyophilize to obtain crude Nostoc commune polysaccharide.

[0065] (4) Prepare a fermentation medium according to 20 g of crude Nostoc commune polysaccharide, 10 g of peptone, 2 g of ammonium citrate, 0.2 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 0.05 g of manganese sulfate, and 1000 mL of distilled water; at the same time, also prepare a medium with glucose as the carbon source, and other components are the same, the difference is that the crude Nostoc commune polysaccharide in the fermentation medium is replaced by glucose. Sterilize at 121 °C under high pressure for 15 min.

[0066] (5) Inoculate Lactobacillus reuteri LRSY523 and Lactobacillus plantarum respectively with an inoculum size of 5%, and ferment at 37 °C in a shaker at 200 rpm for 48 h, and draw a growth curve. As Figure 1 shown, compared with the glucose medium, the Nostoc commune polysaccharide medium has an obvious promoting effect on the growth of Lactobacillus reuteri.

[0067] (6) Heat the fermentation broth at 90 °C and stir for 2 h, cool it to room temperature, and then centrifuge at 8000 rpm for 15 min to retain the supernatant.

[0068] (7) Add an ethanol solution with a volume percentage of 95% that is 4 times the volume of the supernatant to the supernatant, precipitate at 4 °C for more than 12 h, centrifuge at 8000 rpm for 15 min, and retain the precipitate.

[0069] (8) Add the above precipitate to 50 mL of distilled water and heat it at 60 °C to dissolve it into a polysaccharide solution, so that the ethanol completely volatilizes, and obtain a crude fermented Nostoc commune polysaccharide solution.

[0070] (9) The volume ratio of the above crude fermented Nostoc commune polysaccharide solution to Sevage reagent (Sevage reagent is prepared by mixing chloroform and n-butanol according to a volume ratio of 4:1) is 5:1. Vigorously shake it in a separatory funnel for 10 min, let it stand for 10 h, then drain the lower organic phase and the middle denatured free protein, and repeatedly add Sevage reagent and shake until there is no obvious protein layer.

[0071] (10) Dialyze with a dialysis bag with a molecular weight of 8 kDa to remove small molecules, and lyophilize to obtain Nostoc commune fermented polysaccharide (FNVPS) powder.

[0072] (11) Use gel exclusion method to determine the relative molecular mass of the Nostoc commune fermented polysaccharide (FNVPS) obtained in Example 1.

[0073] The sample was dissolved by heating with 0.1 M Na2NO3 to prepare a 5 mg / mL solution. Chromatographic conditions: Chromatographic column: TSK-Gel G3000PWxL chromatographic column (7.5 mm × 300 mm); Mobile phase: 0.1 mol / L Na2NO3; Column temperature: 40 °C; Flow rate: 0.5 mL / min; Detector: Differential refractive index detector. Preparation of the standard curve: Chromatographic analysis was performed on each dextran series standard (10, 70, 100, 300, 500 kDa) in turn, and the relative molecular mass of the sample was calculated.

[0074] It can be seen from Figure 1 comparing the polysaccharides fermented by the two Lactobacillus strains that the polysaccharide components fermented by Lactobacillus reuteri are significantly more homogeneous.

[0075] The physical picture of FNVPS is as shown in Figure 2 the figure, which is a white flocculent solid.

[0076] Example 2: Determination of the chemical composition and monosaccharide composition of FNVPS

[0077] (1) The total sugar content of the polysaccharide was determined by the phenol-sulfuric acid method, the protein content was determined by the BCA method, and the uronic acid content was determined by the m-hydroxybiphenyl method.

[0078] (2) The monosaccharide composition of FNVPS obtained in Example 1 was determined by the PMP pre-column derivation method.

[0079] Weigh 5 mg of the polysaccharide sample, dissolve it with 4 M trifluoroacetic acid (TFA), hydrolyze it at 110 °C for 4 h, cool it to room temperature, and add 1 mL of methanol (to remove TFA) and vacuum dry it repeatedly (preferably more than 3 times). The sample was dissolved with 1 mL of ultrapure water, an appropriate amount was taken, and an equal volume of 0.3 M NaOH solution and PMP-methanol solution were added, and derivatized in a water bath at 70 °C for 2 h. After cooling to room temperature, an equal volume of 0.3 M HCl solution was added, and then extracted with 1 mL of chloroform 3 times. After retaining the aqueous layer to obtain the monosaccharide sample, high performance liquid chromatography analysis was used. The standard product does not need to be degraded and can be directly derivatized. The derivatization method is the same as that of the sample.

[0080] Chromatographic conditions: Chromatographic column: Thermo C18 chromatographic column (250 mm × 4.6 mm); Mobile phase: A is potassium dihydrogen phosphate buffer solution (0.1 mol / L), B is acetonitrile, Flow rate: 1 mL / min; Column temperature 25 °C; Detector: Ultraviolet detector (254 nm). The liquid phase separation gradient is as follows: 0 min - 18 min, the linear gradient of solution A ranges from 82% to 50%; 19 min - 35 min, the linear gradient of solution A ranges from 60% to 18%; 36 min - 60 min, the linear gradient of solution A remains 18%. After comparing the retention time with the monosaccharide standard reference substance, the percentage of monosaccharide composition was calculated by peak area integration.

[0081] The results showed that as Figure 3 shown, the molecular weight of FNVPS was 670.78 kDa, and the monosaccharide composition was mannose 2.33%, glucuronic acid 1.56%, glucose 27.93%, galactose 43.32%, xylose 17.71%, and arabinose 6.75%.

[0082] Table 1 Chemical composition and monosaccharide composition of fermented Nostoc commune polysaccharide

[0083]

[0084] Example 3: Effects of FNVPS on body weight and colon length of mice with acute colitis

[0085] Experimental grouping: blank control group (Con), model group (Mod), polysaccharide drug treatment group (FNVPS).

[0086] Experimental method: Mice were housed in a SPF-level breeding room at the China Institute of Radiation in Shanxi Province. The environmental temperature was 24 ± 2 °C, the humidity was 40% - 70%, and the light was cycled for 12 h / 12 h of darkness. The food and water provided for the mice were autoclaved, and the cage bedding was changed twice a week. After one week of adaptation and growth, the mice were randomly divided into three groups, with 10 mice in each group, numbered and weighed. Mice in the control group and the model group were gavaged with 200 μL of 0.01 M PBS; the FNVPS group was gavaged with 200 mg / kg of FNVPS based on the body weight of the mice; the control group mice drank normal water, and the model group and the FNVPS group drank normal water for one week, then drank water containing 2.5% DSS (dextran sulfate sodium salt) for 9 days, and then drank normal water for 2 days. The experiment lasted for 11 days, during which the body weight of the mice was recorded daily, and the colon length was measured after dissection.

[0087] The results showed that as Figure 4 shown, the colon length of the control group was 6.33 cm, and the body weight increased slightly compared with the beginning of the experiment. In contrast, the body weight of the model group decreased significantly to 70.85%, and the colon shortened to 4.47 cm, while the colon length of the polysaccharide drug treatment group was 5.40 cm and the body weight decreased to 75.98%, with a slower trend and a significant recovery compared with the model group. It was shown that FNVPS could relieve acute colitis in mice induced by DSS.

[0088] Example 4: Effects of FNVPS on inflammatory factors in colon tissues of mice with acute colitis

[0089] Experimental method: After collecting and weighing the colon tissues of mice in each group of Example 3, add precooled PBS of 0.01 M at a ratio of 1 g:10 mL, and freeze-grind to homogeneity on a tissue grinder. After centrifuging at 2000 rpm / min at low temperature for 20 min, collect the supernatant and aliquot and store it frozen at -20°C. Perform ELISA experiments according to the operation instructions of the kit, detect the OD value with an enzyme-labeled instrument and conduct subsequent analysis.

[0090] The results showed that: as Figure 5 shown, FNVPS could effectively regulate the secretion of inflammation-related factors, and TNF-α, IL-6, and IL-1β decreased by 14.5%, 43.5%, and 38.3% respectively compared with the model group, thus alleviating acute colitis in mice.

[0091] Example 5: Effect of FNVPS on the lifespan of Caenorhabditis elegans;

[0092] Experimental method: Collect a large number of Caenorhabditis elegans in the egg-laying period with M9 buffer in a 1.5 mL centrifuge tube, wash 3-4 times to remove impurities and food, discard the supernatant after the last wash, and add 1 mL of Caenorhabditis elegans lysate (the lysate is a mixture of sodium hydroxide, sodium hypochlorite and water in a mass ratio of 1:2:7) to lyse. Centrifuge at 5000 rpm for 1 min, discard the supernatant; wash with M9 5-6 times to remove the residual lysate. Collect the bottom nematode fragments and eggs, drop them on a clean nematode growth medium (NGM medium), and incubate overnight in a 20°C incubator. Synchronized Caenorhabditis elegans (i.e., nematodes after synchronization) can be obtained the next day. Divide the L4-stage larvae of Caenorhabditis elegans into two groups, with 100 in each group; they are the blank control group and the experimental group; place the experimental group on an NGM plate containing 1 mg / mL FNVPS for culture; place the control group on an NGM plate without drugs for culture at 20°C. Start counting the lifespan of nematodes in each group, which is recorded as day 0 of the experiment at this time. Next, transfer the plates every 2 days and record the number of dead Caenorhabditis elegans until all the tested nematodes die.

[0093] The results showed that: as Figure 6 shown, the lifespan of FNVPS was 4 days longer than that of the control group, indicating that it could significantly extend the lifespan of nematodes.

[0094] Example 6: Effect of FNVPS on lipofuscin in Caenorhabditis elegans

[0095] Experimental method: Take the synchronized nematodes in Example 5, and pick 30 nematodes respectively and place them in the blank control group and the FNVPS group. Set 3 parallel experiments in each group; transfer the petri dishes every 24 h during the egg-laying period, and transfer them every 48 h after the egg-laying period; after culturing for 10 days, randomly pick 10 nematodes and place them in a 10% levamisole solution by mass to anesthetize the nematodes; observe the fluorescence intensity of lipofuscin in the nematodes using a fluorescence microscope.

[0096] The results show that: as Figure 7 shown, compared with the control group, the fluorescence intensity in the nematodes of the medicated group decreased by 9.39%, which was significantly weakened, indicating that FNVPS can effectively delay aging.

[0097] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes, and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A preparation method of fermented nostoc commune polysaccharide, characterized in that: It includes the following steps: The first step: After pulverizing the nostoc commune, sieve it through a 50-mesh sieve to obtain nostoc commune powder, and extract nostoc commune polysaccharide by the water extraction and alcohol precipitation method to obtain crude nostoc commune polysaccharide; The second step: Inoculate Lactobacillus reuteri into the fermentation medium added with crude nostoc commune polysaccharide and ferment for 48 h; The third step: Centrifuge to collect the fermentation broth, add ethanol with a volume percentage of 95% to the fermentation broth and let it stand; The fourth step: Centrifuge to collect the precipitate and dissolve it in hot water; The fifth step: Remove proteins with Sevage reagent and rotary evaporate to remove the Sevage reagent; The sixth step: Desalt with a dialysis bag with a molecular weight cut-off of 8 kD, and then carry out vacuum freeze-drying. The freeze-dried product is in powder form, which is the fermented nostoc commune polysaccharide, abbreviated as FNVPS.

2. The preparation method of fermented nostoc commune polysaccharide according to claim 1, characterized in that: In the second step, the fermentation medium is prepared as follows: For every 20 g of crude nostoc commune polysaccharide, 10 g of peptone, 2 g of trisodium citrate, 0.2 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 0.05 g of manganese sulfate, and 1000 mL of distilled water. After mixing, sterilize it under high pressure to obtain it. The sterilization temperature is 121 °C and the time is 15 min; In the second step, the inoculation is carried out according to an inoculation amount of 5%, the fermentation temperature is 37 °C, the shaker speed is 200 rpm / min, and the time is 48 h.

3. The preparation method of fermented nostoc commune polysaccharide according to claim 1, wherein: In the second step, the Lactobacillus reuteri is Lactobacillus reuteri LRSY523.

4. The preparation method of fermented nostoc commune polysaccharide according to claim 1, characterized in that: In the third step, the centrifugation speed and time are: 8000 rpm / min, 15 min; the addition amount of the 95% ethanol is four times the volume of the fermentation broth; the standing time is 12 h.

5. The preparation method of fermented nostoc commune polysaccharide according to claim 1, wherein: In the fourth step, the centrifugation speed and time are: 8000 rpm / min, 15 min; the rotary evaporation temperature is 65 °C; Alternatively, in the fifth step, the Sevage reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1, and the volume ratio of the supernatant to the Sevage reagent is 5:1; the rotary evaporation temperature is 50 °C.

6. The preparation method of the fermented nostoc commune polysaccharide according to any one of claims 1 to 5, characterized in that: The molecular weight of the fermented nostoc commune polysaccharide is 670.78 kDa, and the monosaccharide composition is mannose 2.33%, glucuronic acid 1.56%, glucose 27.93%, galactose 43.32%, xylose 17.71%, and arabinose 6.75%.

7. The fermented nostoc commune polysaccharide prepared by the preparation method according to any one of claims 1 to 6.

8. The fermented nostoc commune polysaccharide according to claim 7, wherein: It was found at the animal model level that the fermented nostoc commune polysaccharide FNVPS can effectively alleviate acute colitis caused by DSS and significantly reduce the secretion of inflammatory factors; it was found at the Caenorhabditis elegans level that the fermented nostoc commune polysaccharide FNVPS can effectively delay the aging-related indicators of nematodes, indicating that this fermented nostoc commune polysaccharide has the potential to be developed into functional foods, cosmetics and pharmaceutical raw materials for anti-inflammatory and anti-aging.

9. Use of the fermented nostoc commune polysaccharide according to claim 7 in the preparation of anti-inflammatory functional foods and / or cosmetics and / or foods and / or drugs.

10. Use of the fermented nostoc commune polysaccharide according to claim 7 in the preparation of anti-aging functional foods and / or cosmetics and / or foods and / or drugs.

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