Lycium barbarum polysaccharide fermentation product with effect of relieving rheumatoid arthritis and preparation method of lycium barbarum polysaccharide fermentation product

Abstract

The invention provides a lycium barbarum polysaccharide leavening with the effect of relieving rheumatoid arthritis and a preparation method of the lycium barbarum polysaccharide leavening, and belongs to the technical field of microbial fermentation. According to the application, the lycium barbarum polysaccharide is used as a unique carbon source, and the fermentation product obtained by fermenting the lycium barbarum polysaccharide through the Rombusia litubursia strain has a remarkable effect of eliminating arthrocele, can inhibit expression of inflammatory factors, improve the level of anti-inflammatory factors and relieve rheumatoid arthritis, and has an important application value.

Description

Technical Field

[0002] This invention belongs to the field of microbial fermentation, and specifically relates to a fermented product of wolfberry polysaccharide with the effect of relieving rheumatoid arthritis and its preparation method. Background Technology

[0003] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation, pannus proliferation, cartilage and subchondral bone degeneration, joint space narrowing, and joint deformities, severely reducing the quality of life for middle-aged and elderly individuals. Currently available treatments are expensive and have certain side effects. Exploring the active ingredients in food, especially medicinal and edible resources, and intervening through diet has become a new strategy for alleviating RA. Non-starch polysaccharides in food cannot be directly absorbed by the human body; instead, they enter the large intestine, are fermented by gut microbiota to produce various metabolites, and are then absorbed through the intestinal epithelium to exert their active effects.

[0004] Goji berries are the fruit of the Lycium barbarum plant, belonging to the Solanaceae family and the Lycium genus. They are a dual-use resource in my country, used for both medicinal and culinary purposes. Goji berry polysaccharides are the main functional components, primarily existing in acidic and neutral forms, and possess immunomodulatory and anti-inflammatory activities. Currently, goji berry polysaccharides have been shown to alleviate rheumatoid arthritis, but this requires the participation of specific gut microbiota to be converted into metabolites before they can exert their effects. However, the gut microbiota structure of rheumatoid arthritis patients changes, such as the loss of some gut bacteria, thus weakening the allergic effect of goji berry polysaccharides. In vitro fermentation of goji berry polysaccharides, administering them to the human body in the form of fermented products, not only avoids the negative effects of gut microbiota loss but also allows for more rapid absorption of metabolites, thereby enhancing efficacy.

[0005] Romboutsia lituseburensis It is an important member of the gut microbiota in humans and animals. Currently, although there are occasional studies on the fermentation of wolfberry polysaccharides using gut bacteria, the utilization of... Romboutsia lituseburensis Reports on fermented wolfberry polysaccharides have not yet been mentioned, and there are currently no fermented wolfberry polysaccharide products on the market that have the effect of relieving rheumatoid arthritis. Summary of the Invention

[0006] To address the aforementioned problems, this application aims to provide a method for preparing a fermented product of Lycium barbarum polysaccharides, the method comprising: Step 1: Prepare a culture medium containing wolfberry polysaccharides; Step 2: Activate Romboutsia lituseburensis The strain was inoculated into the culture medium containing Lycium barbarum polysaccharide prepared in step one, and anaerobic fermented at 35-40℃ and pH 6-6.5 to obtain the Lycium barbarum polysaccharide ferment.

[0007] In one embodiment, the fermentation conditions in step two are: temperature 36~38℃, pH 6.1~6.3.

[0008] In one embodiment, in step two, the duration of anaerobic fermentation is 10-35 h, preferably 16-32 h, more preferably 20-30 h, for example 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h.

[0009] In one embodiment, step two involves obtaining a fermentation broth after fermentation, and then freeze-drying the fermentation broth to obtain a freeze-dried powder based on wolfberry polysaccharides.

[0010] In one embodiment, the content of the wolfberry polysaccharide in the culture medium is 10~30 g / L, preferably 15~25 g / L, for example 15 g / L, 16 g / L, 20 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L.

[0011] In one embodiment, the Romboutsia lituseburensis The inoculation amount of the strain is 0.1% to 10% (v / v), preferably 0.5% to 8% (v / v), more preferably 1% to 5% (v / v), for example 1%, 2%, 3%, 4%, 5% (v / v).

[0012] In one embodiment, the culture medium further contains an anaerobic fermentation substrate, which includes any of the following components: peptone, beef meal, yeast powder, Tween, sodium acetate heptahydrate, dipotassium hydrogen phosphate heptahydrate, triammonium citrate, magnesium sulfate heptahydrate, and manganese sulfate tetrahydrate.

[0013] In one embodiment, the anaerobic fermentation medium preferably comprises the following components: 10 mg / mL peptone, 5 mg / mL beef meal, 4 mg / mL yeast extract, 1 mg / mL Tween 80, 5 mg / mL sodium acetate heptahydrate, 2 mg / mL dipotassium hydrogen phosphate heptahydrate, 2 mg / mL triammonium citrate, 0.2 mg / mL magnesium sulfate heptahydrate, and 0.05 mg / mL manganese sulfate tetrahydrate.

[0014] In one embodiment, the wolfberry polysaccharide is a polysaccharide mixture, wherein the monosaccharide composition of the polysaccharide includes mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose.

[0015] In one embodiment, the molecular weight of the wolfberry polysaccharide is not less than 3 kDa.

[0016] In one embodiment, the wolfberry polysaccharide is prepared by the following method: After adding protease to the wolfberry extract for enzymatic hydrolysis, the extract is precipitated with alcohol to obtain a precipitate. Monosaccharides and oligosaccharides are removed from the precipitate to obtain the wolfberry polysaccharide.

[0017] In one embodiment, the wolfberry extract is prepared by the following steps: dried wolfberries are pulverized, decolorized and degreased with ethanol solution, filtered to remove the supernatant, and the filter residue is naturally air-dried into powder; the powder is extracted by water extraction to obtain an aqueous extract, which is then concentrated to obtain the wolfberry extract.

[0018] In one embodiment, the ethanol solution used for decolorization and degreasing is preferably an ethanol solution with a concentration of 80% by volume.

[0019] In one embodiment, the decolorization and degreasing time with ethanol solution is 10-15 h, preferably 12 h.

[0020] In one embodiment, the water extraction method is performed by extracting 1 to 3 times, each time at 45 to 85°C for 180 to 300 min; preferably, it is performed by extracting 2 times, each time at 60°C for 240 min.

[0021] In one embodiment, the protease used for enzymatic hydrolysis includes, but is not limited to, papain, neutral protease, and bromelain, with papain being preferred.

[0022] In one embodiment, the enzymatic hydrolysis step is as follows: adding 0.5% protease and treating at 40-70°C for 6-18 hours, preferably at 55°C for 12 hours.

[0023] In one embodiment, the alcohol precipitation step is as follows: add 4 times the volume of anhydrous ethanol and precipitate at 4°C for 12 h.

[0024] In one embodiment, the precipitate is obtained by centrifugation after alcohol precipitation. Preferably, the centrifugation method is centrifugation at 8000×g for 10 min.

[0025] In one implementation, the method for removing monosaccharides and oligosaccharides is dialysis.

[0026] In one embodiment, the dialysis conditions are: dialysis using a 3 kDa semipermeable membrane for at least 36 h; preferably, dialysis using a 3 kDa semipermeable membrane for 48 h.

[0027] In one embodiment, the anaerobic fermentation can be carried out in an anaerobic fermenter.

[0028] Optionally, the anaerobic fermenter is incubated in a constant-temperature magnetically stirred water bath at 37±1℃, and nitrogen is introduced throughout the fermentation process to keep the anaerobic fermentation device anaerobic.

[0029] In one embodiment, step one involves sterilizing the culture medium containing wolfberry polysaccharides after preparation.

[0030] Preferably, the sterilization conditions are high-pressure sterilization at 210 kPa and 121°C for 15 min.

[0031] In one embodiment, the Romboutsia lituseburensis The strain was activated using the following method: Will Romboutsia lituseburensis The preserved bacterial strain was inoculated into an anaerobic fermentation medium supplemented with glucose, cultured and passaged, and then inoculated into a fresh anaerobic fermentation medium supplemented with Lycium barbarum polysaccharides, cultured and passaged again to obtain the activated strain. Romboutsia lituseburensis .

[0032] In a preferred embodiment, the Romboutsia lituseburensis The activation of the strain is carried out through the following steps: A. Romboutsia lituseburensis The preserved strain was inoculated into an anaerobic fermentation medium supplemented with glucose and cultured anaerobically at 37±1℃ for 18±2 h, and then passaged 1 to 3 times. B. Take the result from step A. Romboutsia lituseburensis They were inoculated into anaerobic fermentation medium supplemented with Lycium barbarum polysaccharide and cultured anaerobically at 37±1℃ for 24±2 h, and passaged 1 to 3 times.

[0033] In one embodiment, the activation medium in step A is obtained by adding 20 g / L glucose as a carbon source to the anaerobic fermentation medium.

[0034] In one embodiment, the activation culture medium in step B is obtained by adding Lycium barbarum polysaccharide as a carbon source to the anaerobic fermentation culture medium, wherein the amount of Lycium barbarum polysaccharide added is 10-30 g / L, preferably 20 g / L.

[0035] In one implementation, in step A and / or step B Romboutsia lituseburensis The inoculum size of the strain is 1-3% of the culture medium volume, preferably 2%.

[0036] On the other hand, this application also provides a fermented product of wolfberry polysaccharides prepared using the method described above.

[0037] In one embodiment, the Lycium barbarum polysaccharide ferment contains, in relative abundance, at least one or more of the following substances: tripeptide (glutamic acid-proline-glutamine) 7.2–7.5 wt%, N-γ-glutamylcysteine ​​5.8–6.0 wt%, 5'-(3',4'-dihydroxyphenyl)-γ-valerate sulfate 5.3–5.7 wt%, 4-methylpyridine 4.7–5.3 wt%, 2-[6-(4-chlorophenoxy)hexyl]epoxide-2-carboxylic acid 4.8–5.4 wt%, 2-fluoro-2′,3′-dideoxyadenosine 4.6–4.9 wt%, 4-hydroxyphenylacetonitrile 4.4–4.8 wt%, and biliverdin 2.6–3.2 wt%.

[0038] Among them, fermented wolfberry polysaccharides, which contain the above-mentioned substances, can eliminate joint swelling to a certain extent, reduce the expression of inflammatory factors, and thus alleviate rheumatoid arthritis.

[0039] On the other hand, this application also provides the use of the wolfberry polysaccharide ferment obtained by the method, or the use of the wolfberry polysaccharide ferment in the preparation of drugs, health foods and / or feeds that have the effect of relieving rheumatoid arthritis.

[0040] On the other hand, this application also provides a medicament for relieving rheumatoid arthritis, wherein the active ingredient of the medicament contains the fermented wolfberry polysaccharide obtained by the method.

[0041] In one embodiment, the content of the active ingredient Lycium barbarum polysaccharide fermentation product in the drug is 0.01% to 99.99%, where % is a mass percentage (w / w), volume percentage (v / v), mass-volume ratio (w / v), or volume-mass ratio (v / w).

[0042] In one embodiment, the drug may also contain a compound of wolfberry polysaccharide fermentation product with other components having the same or different biological activities.

[0043] In one embodiment, the dosage form of the drug includes powder, tablet, granule, capsule, solution, suspension, emulsion or lyophilized form.

[0044] In one embodiment, the health food includes solid beverages, liquid beverages, or semi-solid beverages.

[0045] In one embodiment, the feed is dry feed or wet feed.

[0046] This application has at least the following beneficial effects: 1. This application uses Lycium barbarum polysaccharide extracted from Lycium barbarum, a traditional medicinal and edible resource, as raw material. Compared with current drugs for treating rheumatoid arthritis, it has the advantages of being cheaper, safer, and having fewer side effects. 2. This application utilizes specific intestinal bacteria. Romboutsia lituseburensis In vitro fermentation can, on the one hand, replenish beneficial bacteria in the gut of patients with rheumatoid arthritis, avoiding the problem of reduced drug efficacy due to the lack of gut bacteria; on the other hand, the small molecules obtained by microbial fermentation are more easily absorbed by the human body and exert their effects. 3. This application uses Lycium barbarum polysaccharide as the sole fermentation carbon source substrate, processed by specific intestinal strains. Romboutsia lituseburensis The fermentation product obtained under specific conditions has a significantly increased abundance of metabolites related to rheumatoid arthritis. Furthermore, experimental results show that the fermentation product can reduce the expression of inflammatory factors by inhibiting pro-inflammatory pathways, thereby alleviating symptoms such as joint enlargement, redness, and swelling, and has a significant effect on relieving or improving rheumatoid arthritis. Attached Figure Description

[0047] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 The graph shows the joint diameter measurement results of RA rats in each example group during the efficacy experiment; Figure 2 The graph shows the arthritis scores of RA rats in each example group during the efficacy experiment. Figure 3 This is a graph showing the serum inflammatory factor levels of RA rats in each example group during the efficacy experiment. Detailed Implementation

[0049] The present invention is described below through specific embodiments. These embodiments should be understood as illustrative, not limiting, of the scope of the invention, which is primarily defined by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the spirit and scope of the invention, also fall within the scope of protection of the present invention.

[0050] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the reagents and raw materials used in this invention are commercially available. All chemical reagents involved in this invention are of analytical grade.

[0051] The anaerobic fermentation medium used in the following examples has the following components and dosages: 10 mg / mL peptone, 5 mg / mL beef meal, 4 mg / mL yeast powder, 1 mg / mL Tween 80, 5 mg / mL sodium acetate heptahydrate, 2 mg / mL dipotassium hydrogen phosphate heptahydrate, 2 mg / mL triammonium citrate, 0.2 mg / mL magnesium sulfate heptahydrate, 0.05 mg / mL manganese sulfate tetrahydrate.

[0052] The Lycium barbarum polysaccharides involved in the following examples were prepared using the following method: After crushing the wolfberry fruit, it was decolorized and defatted with 80% (v / v) ethanol for 12 h, and the supernatant was removed by filtration. The residue was extracted twice with distilled water (60℃, 240 min each time), and the supernatants from the two extractions were combined and concentrated by rotary evaporation. 0.5% (w / w) papain (activity ≥800 U / mg) was added and reacted at 65℃ for 12 h. Then, 4 volumes of anhydrous ethanol were added and the mixture was precipitated overnight at 4℃. The precipitate was collected by centrifugation. The precipitate was dissolved in ultrapure water and dialyzed through a 3 kDa semipermeable membrane for 48 h to remove monosaccharides and oligosaccharides. Finally, water-soluble wolfberry polysaccharides were obtained by freeze-drying.

[0053] The strains involved in the following examples Romboutsia lituseburensis for Romboutsia lituseburensis Laplanche and Saissac , 1948 (CCUG 18920), purchased through the Guangdong Provincial Microbiology Analysis and Testing Center of the Institute of Microbiology, Guangdong Academy of Sciences, with the original source being the Culture Collection Center of the University of Gothenburg, Sweden; Lactobacillus johnsonii The strain was obtained through the China Industrial Microbial Culture Collection Center. Lactobacillus johnsonii (ATCC 43121).

[0054] Example 1 This embodiment provides a wolfberry polysaccharide ferment, which is prepared by the following method: (1) Preparation of fermentation medium: Prepare anaerobic fermentation medium and add wolfberry polysaccharide to make the content of wolfberry polysaccharide 20 g / L; after mixing, adjust the pH to 6.2, and sterilize at high temperature and high pressure (sterilize at 121℃ for 15 min) to obtain fermentation medium; (2) Strains Romboutsia lituseburensisActivation: Romboutsia lituseburensis The preserved bacterial strain was inoculated into an anaerobic fermentation medium supplemented with glucose (20 g / mL) at a concentration of 2% (v / v), and cultured anaerobically at 37°C for 20 h. The culture was then subcultured twice. The culture was then inoculated into the fermentation medium obtained in step (1) at a concentration of 2% (v / v), and cultured anaerobically at 37°C for 26 h. This process was repeated twice to obtain the activated bacterial strain. Romboutsia lituseburensis ; (3) Preparation of Lycium barbarum polysaccharide fermentation product: The activated polysaccharide obtained in step (2) is used to prepare the fermentation product. Romboutsia lituseburensis The bacterial culture was added to the fermentation medium obtained in step (1) at an inoculation amount of 2% (v / v), and anaerobic fermentation was carried out at 37°C for 16 h to obtain the fermentation broth. After freeze-drying, the fermentation broth was freeze-dried into a powder and stored at -20°C.

[0055] Example 2 This embodiment provides a wolfberry polysaccharide fermentation product. The preparation method of this fermentation product is different from that of Example 1 in that the fermentation culture time in step (3) is extended from 16h to 24h, while the other steps are the same as those in Example 1.

[0056] Example 3 This embodiment provides a wolfberry polysaccharide ferment. The preparation method of this ferment is different from that of Example 1 in that the fermentation time in step (3) is extended from 16h to 32h, while the other steps are the same as those in Example 1.

[0057] Comparative Example 1 The preparation method of this comparative example is roughly the same as that of Example 2, except that in step (3), the activated... Romboutsia lituseburensis After the bacterial culture is inoculated into the fermentation medium, it is immediately pasteurized, that is, fermentation is not carried out, and only a mixture of wolfberry polysaccharide, culture medium and inactivated bacteria is obtained. The remaining steps are the same as in Example 2.

[0058] Comparative Example 2 The preparation method of this comparative example is roughly the same as that of Example 2, except that: no wolfberry polysaccharide was added when preparing the fermentation medium in step (1), that is, only the culture medium fermentation product of the bacteria. The other methods and steps are the same as those of Example 2.

[0059] Comparative Example 3 The preparation method of this comparative example is roughly the same as that of Example 2, except that the strain is used in steps (2) and (3). Romboutsia lituseburensis Replace with another strain that can survive in the intestines of mammals, including humans. Lactobacillus johnsonii (ATCC 43121), the remaining steps are the same as in Example 2.

[0060] Efficacy testing experiment Experimental Example 1: Determination of the relative content of substances related to the relief of rheumatoid arthritis in fermentation products The following experiments were conducted using samples prepared in Examples 1-3 and Comparative Examples 1-3.

[0061] The levels of substances associated with the relief of rheumatoid arthritis in each example sample were detected using an AB SCIEX ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry (UPLC-TripleTOF) system.

[0062] Sample preparation: Take 50 mg of fermentation broth and add 400 µL of extraction buffer [methanol:water = 4:1 (v:v)] containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine). Grind the mixture in a cryo-tissue homogenizer for 6 min (-10℃, 50 Hz), followed by low-temperature ultrasonic extraction for 30 min (5℃, 40 kHz). After extraction, allow the mixture to stand at -20℃ for 30 min, then centrifuge for 15 min (13000×g, 4℃). Collect the supernatant for liquid chromatography-mass spectrometry analysis. Additionally, 20 µL of supernatant from each sample was transferred and mixed to serve as the quality control (QC) sample.

[0063] Chromatographic conditions: An ACQUITYUPLC HSS T3 column (100 mm × 2.1 mm, pore size 1.8 µm) was used; elution was performed according to the gradient elution program described in Table 1. Mobile phase A consisted of 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B consisted of 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The flow rate was set to 0.40 mL / min, the injection volume to 10 μL, and the column temperature to 40℃.

[0064] Table 1 Gradient elution program

[0065] Mass spectrometry conditions: The sample was ionized by electrospray ionization, and mass spectrometry signals were acquired using positive and negative ion scanning modes, respectively. The scanning range was 50–1000 m / z, the ionization voltage (positive electrode) was 5000 V, the ionization voltage (negative electrode) was -4000 V, the declusivity voltage was 80 V, the spray gas was 50 psi, the auxiliary heating gas was 50 psi, the curtain gas was 30 psi, the ion source heating temperature was 500 °C, the cyclic collision energy was 20–60 V, and the cycle time was 510 ms.

[0066] Data Processing: After the initial LC-MS data was processed, the raw data were imported into the metabolomics processing software Progenesis QI for baseline filtering, peak identification, integration, retention time correction, and peak alignment. This resulted in a data matrix of retention time, mass-to-charge ratio, and peak intensity. Missing values ​​were removed using the 80% rule, and then filled with the minimum value from the original matrix. The response intensity of the sample mass spectrometry peaks was normalized using summation normalization to obtain a normalized data matrix. Variables with a relative standard deviation (RSD) > 30% for QC samples were removed, and logarithmic transformation (log10) was performed to obtain the final data matrix used for subsequent analysis. The mass spectrometry information was then matched with the public metabolic databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) to obtain metabolite information.

[0067] The contents of substances related to the relief of rheumatoid arthritis in the samples prepared in Examples 1-3 and Comparative Examples 1-3 are shown in Table 2.

[0068] Table 2. Relative content (wt%) of substances related to alleviating rheumatoid arthritis in samples from each experimental group.

[0069] As shown in Table 2, compared with the control group, the content of metabolites that alleviate rheumatoid arthritis in the fermentation products of Examples 1-3 was significantly increased after fermentation for different times. Furthermore, the content of each metabolite accumulated more in the fermentation products obtained around 24 hours of fermentation, and no significant increase in metabolites was observed after further extending the fermentation time.

[0070] Experimental Example 2: Efficacy determination in relieving rheumatoid arthritis The following experiments were conducted using samples prepared in Examples 1-3 and Comparative Examples 1-3.

[0071] All procedures in this experiment were approved by the Animal Experiment Ethics Committee of Jinan University. Sixty-four specific pathogen-free (SPF) female Wistar rats (7-8 weeks old) were housed in an SPF-grade room with temperature control (23±2℃) and humidity control (55%±5%), and were exposed to light for 12 hours. The rats were randomly divided into eight groups of eight each: control group (Con), model group (Mod), Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. After the acclimatization period, the rats were fed standard AIN-93M feed and distilled water containing a compound antibiotic (1 g / L ampicillin, 1 g / L neomycin, and 0.5 g / L metronidazole), with free access to food and water. From day 11 until the end of the experiment, rats in Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group were administered 2 mL of the corresponding fermentation product by gavage, while rats in the other groups were administered an equal volume of distilled water by gavage. On days 17 and 24 of the experiment, except for the control group (Con) rats, the other rats were injected intradermally with an equal volume of 0.2 μL of a mixture of emulsified bovine type II collagen and complete Freund's adjuvant, while the control group rats were injected with an equal volume of physiological saline. Furthermore: From day 17 until the end of the experiment, the diameter of the rat's paw was measured twice a week using vernier calipers. The results are as follows: Figure 1 As shown.

[0072] On day 31 of the experiment, arthritis scores were assessed on the hind paws of each rat, after which the rats were fasted overnight. The scoring criteria are shown in Table 3. Table 3. Rat Arthritis Index Scoring Criteria

[0073] The scoring results are as follows Figure 2 As shown.

[0074] On day 32, anesthesia was administered via intraperitoneal injection of sodium pentobarbital (40 mg / kg body weight), and blood was drawn from the abdominal aorta to collect serum for the detection of inflammatory factors. The results are as follows: Figure 3 As shown.

[0075] Combination Figures 1 - 3The results showed that, compared with the control group, the rats in the Example group showed greater improvement in joint diameter, arthritis index score, and inflammatory factor content, especially Example 2. Furthermore, compared with Example 1, Example 2 showed a significant reduction in inflammatory factors TNF-α, IL-17A, and IL-1β; however, there was no significant difference in the effect of Example 2 and Example 3 in alleviating rheumatoid arthritis. Therefore, considering both efficacy and cost, the fermented product obtained in Example 2 (fermentation time of 24 h) has a good effect in alleviating rheumatoid arthritis, and extending the fermentation time did not significantly enhance the efficacy. Moreover, comparing the results of Example 2 and Comparative Example 3 showed that the polysaccharides of Lycium barbarum... Romboutsia lituseburensis Fermentation ratio Lactobacillus johnsonii Fermented substances have a more significant effect in relieving rheumatoid arthritis.

[0076] As can be seen from the above embodiments, the wolfberry polysaccharide provided in this application Romboutsia lituseburensis Fermented substances can significantly increase the content of beneficial metabolites, have a significant effect on eliminating joint swelling, and can alleviate rheumatoid arthritis by inhibiting the expression of inflammatory factors, increasing the level of anti-inflammatory factors.

[0077] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing a fermented product of Lycium barbarum polysaccharides, characterized in that, The method includes: Step 1: Prepare a culture medium containing wolfberry polysaccharides; Step 2: Activate Romboutsia lituseburensis The strain was inoculated into the culture medium containing Lycium barbarum polysaccharide prepared in step one, and anaerobic fermented at 35-40℃ and pH 6-6.5 to obtain the Lycium barbarum polysaccharide ferment.

2. The method for preparing Lycium barbarum polysaccharide fermentation product according to claim 1, characterized in that, The duration of anaerobic fermentation in step two is 15-35 hours.

3. The method for preparing Lycium barbarum polysaccharide fermentation product according to claim 1, characterized in that, The content of the wolfberry polysaccharide in the culture medium is 10~30 g / L; And / or, the Romboutsia lituseburensis The inoculation amount of the strain is 0.1%~10% (v / v).

4. The method for preparing Lycium barbarum polysaccharide fermentation product according to claim 1, characterized in that, The culture medium also contains an anaerobic fermentation substrate, which includes any of the following components: peptone, beef meal, yeast powder, Tween, sodium acetate heptahydrate, dipotassium hydrogen phosphate heptahydrate, triammonium citrate, magnesium sulfate heptahydrate, and manganese sulfate tetrahydrate.

5. The method for preparing Lycium barbarum polysaccharide fermentation product according to claim 1, characterized in that, The wolfberry polysaccharide is prepared by the following method: adding protease to wolfberry extract for enzymatic hydrolysis, followed by alcohol precipitation to obtain a precipitate, and removing monosaccharides and oligosaccharides from the precipitate to obtain the wolfberry polysaccharide.

6. The method for preparing Lycium barbarum polysaccharide fermentation product according to claim 1, characterized in that, The molecular weight of the wolfberry polysaccharide is not less than 3 kDa.

7. The wolfberry polysaccharide fermentation product prepared by any one of claims 1 to 6.

8. The fermented product of Lycium barbarum polysaccharides according to claim 7, characterized in that, Based on relative abundance, the fermented Lycium barbarum polysaccharide contains at least one or more of the following substances: tripeptide (glutamic acid-proline-glutamine) 7.2–7.5 wt%, N-γ-glutamylcysteine ​​5.8–6.0 wt%, 5'-(3',4'-dihydroxyphenyl)-γ-valerol sulfate 5.3–5.7 wt%, 4-methylpyridine 4.7–5.3 wt%, 2-[6-(4-chlorophenoxy)hexyl]epoxide-2-carboxylic acid 4.8–5.4 wt%, 2-fluoro-2′,3′-dideoxyadenosine 4.6–4.9 wt%, 4-hydroxyphenylacetonitrile 4.4–4.8 wt%, and biliverdin 2.6–3.2 wt%.

9. The use of the fermented Lycium barbarum polysaccharide obtained by any of the methods described in claims 1-6, or the fermented Lycium barbarum polysaccharide as described in claim 7 or 8, in the preparation of drugs, health foods, and / or feeds that have the effect of relieving rheumatoid arthritis.

10. A drug for relieving rheumatoid arthritis, characterized in that, The active ingredient of the drug contains a fermented product of Lycium barbarum polysaccharides prepared by any one of the methods described in claims 1-6, or a fermented product of Lycium barbarum polysaccharides as described in claim 7 or 8.

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