Method for preparing a mixture of mussel oligosaccharides and use of the product thereof in inflammatory bowel diseases

Abstract

This invention discloses a method for preparing a mixture of mussel oligosaccharides and the application of the product in inflammatory bowel disease. This invention uses mussel polysaccharide MP-1 as a raw material, processed by α-amylase or Bacillus subtilis (… Bacillus subtilis BNCC185269) obtained mussel oligosaccharide mixtures MOS-A and MOS-B composed of different oligosaccharides. Animal experiments have shown that the two oligosaccharide mixtures prepared by this invention can effectively alleviate intestinal inflammation. Therefore, the mussel oligosaccharide mixtures prepared by this invention are natural and safe drugs or foods for improving inflammatory bowel disease. They can effectively improve inflammatory bowel disease, are safe, non-toxic, and have no side effects. The raw materials are abundant, the steps are simple, the cost is low, and the effects are significant.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation method of mussel oligosaccharide mixture and the application of the product in inflammatory bowel disease. Background Technology

[0002] Mussels (Mytilus spp.), as an important marine economic shellfish, have long adapted to high osmotic pressure, high salinity, and complex microecological environments, evolving a unique and functionally specific system of metabolites. Traditional studies have widely reported the excellent activities of mussel polysaccharides in anti-inflammatory, antioxidant, and immunomodulatory effects, which are closely related to their complex spatial conformation and abundant functional groups. However, high molecular weight polysaccharides generally suffer from limitations such as limited solubility, low intestinal absorption, and low bioavailability, which to some extent restricts their development and application.

[0003] In recent years, with advancements in glycoscience and bioenzyme engineering, research focus has gradually shifted from polysaccharides to their degradation products—marine oligosaccharides. Compared to polysaccharides, oligosaccharides not only possess better water solubility and biocompatibility, but their smaller molecular weight also makes them more easily able to penetrate the intestinal mucosal epithelium. They can precisely intervene in the intestinal microenvironment through multiple mechanisms, such as regulating intestinal flora balance, inhibiting pro-inflammatory signaling pathways (e.g., NF-κB), and enhancing the expression of tight junction proteins (e.g., Occludin, ZO-1). Of particular note is that specific oligosaccharide fragments derived from mussels—such as glucosamine oligosaccharides linked by β-(1→4) glycosidic bonds or chitosan oligosaccharide analogs containing amino groups—have been shown to target macrophage polarization, converting them to the anti-inflammatory M2 type, thereby significantly reducing colonic mucosal damage and oxidative stress levels in inflammatory bowel disease (IBD) animal models. Therefore, the preparation of mussel oligosaccharide mixtures with well-defined structures and specific glycosidic bond types through controlled degradation technology has become a new trend in the development of marine functional foods and drugs.

[0004] Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by impaired intestinal epithelial barrier function and dysregulation of the immune response. Existing treatments, such as aminosalicylic acid preparations, glucocorticoids, and biologics, while capable of controlling symptoms to some extent, suffer from problems such as decreased response rates, high relapse rates, and potential side effects. Therefore, there is an urgent clinical need to develop a natural product that can safely and effectively repair the intestinal barrier and regulate immune homeostasis through multiple targets. Mussel oligosaccharides, with their natural marine origin, well-defined structural basis, and good intestinal targeting, hold promise as a highly valuable functional component or drug lead compound in IBD intervention strategies. Developing efficient preparation processes for these oligosaccharides and elucidating their application mechanisms in IBD not only represents a high-value utilization of marine carbohydrate resources but also provides novel solutions and scientific evidence for nutritional and pharmacological interventions in inflammatory bowel disease. Summary of the Invention

[0005] Purpose of the invention: The present invention addresses the problems of limited solubility, low intestinal absorption rate, and low bioavailability of prepared mussel polysaccharide MP-1. The technical problem to be solved is to provide a mixture of two mussel oligosaccharides that can improve inflammatory bowel disease.

[0006] Another technical problem that this invention aims to solve is to provide a method for preparing two mussel oligosaccharide mixtures.

[0007] The final technical problem to be solved by this invention is to provide the application of a mixture of two mussel oligosaccharides in the preparation of a medicament for the prevention or treatment of inflammatory bowel disease.

[0008] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a method for preparing a mussel oligosaccharide mixture, comprising the following steps: reacting α-amylase or... Bacillus subtilis BNCC185269 was added to mussel polysaccharide MP-1 for reaction. After the reaction, acetonitrile was added, the supernatant was collected by centrifugation, the acetonitrile was removed, and the mixture was lyophilized to obtain a mussel oligosaccharide mixture. The mussel polysaccharide MP-1 has an α-1,4-glycosidic bond as the main chain and α-1,6-glycosidic bonds as the branches. On average, there is one α-1,6-glucose branch for every 10 glucose main chains. The relative molecular weight is 312~533 kDa, and the configuration is an α-pyran-type dextran. Its structural formula is shown below:

[0009] .

[0010] The mass ratio of α-amylase to mussel polysaccharide MP-1 is 1:2 to 1:1. Bacillus subtilis BNCC185269 was revived and cultured in LB medium with an inoculum size of 5-10%, a revival time of 12-24 h, a pH of 7-7.5, and a mussel polysaccharide MP-1 concentration of 0.5-1.5 mg / mL.

[0011] The α-amylase is hydrolyzed at a temperature of 55-60°C for 6-8 hours, and the α-amylase is terminated by heating in boiling water for 10-20 minutes.

[0012] After enzymatic hydrolysis, acetonitrile is added and centrifuged at high speed to remove polysaccharides and enzymes. The high-speed centrifugation conditions are: rotation speed 8000 ~ 10000 rpm, temperature 3 ~ 5℃, time 15 ~ 30 min, followed by rotary evaporation for 10 ~ 15 min to remove acetonitrile.

[0013] Among them, the Bacillus subtilis OD of BNCC185269 bacterial culture 600 =0.5~0.6.

[0014] Among them, the Bacillus subtilis The reaction temperature of BNCC185269 and mussel polysaccharide MP-1 was 35~37℃, the reaction time was 6~10h, and the rotation speed was 150~200rpm.

[0015] Among them, the Bacillus subtilis After fermentation of BNCC185269 and mussel polysaccharide MP-1, the bacterial cells were removed by high-speed centrifugation and filtration. The high-speed centrifugation conditions were: rotation speed 3000~5000 rpm, temperature 3~5℃, time 15~30 min, and filtration membrane 0.22~0.44 μM.

[0016] The present invention also includes the mussel oligosaccharide mixture prepared by the aforementioned preparation method.

[0017] The mussel oligosaccharide mixture is either MOS-A or MOS-B, which are two different combinations of oligosaccharides. MOS-A includes glucose, maltose, maltotriose, maltotetraose, and maltopentose, while MOS-B includes glucose, isomaltose, maltose, maltotriose, maltotetraose, and maltopentose.

[0018] The present invention also includes the application of the mussel oligosaccharide mixture in the preparation of medicaments for the prevention or treatment of inflammatory bowel disease.

[0019] The preparation method of mussel oligosaccharide mixture MOS-A includes the following steps: adding α-amylase aqueous solution to mussel polysaccharide MP-1 aqueous solution for enzymatic hydrolysis, terminating the reaction, adding acetonitrile, centrifuging to collect the supernatant, removing acetonitrile by rotary evaporation, and lyophilizing to obtain mussel oligosaccharide mixture MOS-A.

[0020] The preparation method of mussel oligosaccharide mixture MOS-B includes the following steps: Bacillus subtilis After BNCC185269 was revived in LB medium, it was added to sterile water containing mussel polysaccharide MP-1 and cultured at room temperature. After centrifugation to remove the cells, the supernatant was collected by filtration membrane, acetonitrile was added, centrifuged to remove the remaining polysaccharide, and after rotary evaporation to remove acetonitrile, it was freeze-dried to obtain the mussel oligosaccharide mixture MOS-B.

[0021] The LB culture medium is composed of the following components per 1L of water: 10.0g tryptone, 5.0g yeast extract, and 10.0g sodium chloride.

[0022] This invention also includes the application of the mussel oligosaccharide mixtures MOS-A and MOS-B in the preparation of drugs for the prevention or treatment of inflammatory bowel disease. Preferably, the inflammatory bowel disease includes colitis. This invention utilizes rectal administration; a dosage of 10-20 mg / kg of both oligosaccharide mixtures achieves therapeutic efficacy, with 20 mg / kg demonstrating good efficacy comparable to that of a positive control drug.

[0023] The colitis mentioned includes, but is not limited to, ulcerative colitis.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention uses the novel mussel polysaccharide MP-1 as raw material, which is hydrolyzed by α-amylase or Bacillus subtilis Fermentation with BNCC185269, and animal experiments have demonstrated that the mussel oligosaccharide mixture prepared by this invention can effectively alleviate intestinal inflammation. Therefore, this mussel oligosaccharide mixture is a natural and safe drug or food for improving inflammatory bowel disease. It can effectively improve inflammatory bowel disease, is safe, non-toxic, and has no side effects. It is made from abundant raw materials, has a simple process, is inexpensive, and has significant effects. Attached Figure Description

[0025] Figure 1 The above is the HPGPC chromatogram of mussel polysaccharide MP-1 of the present invention;

[0026] Figure 2 The infrared spectrum analysis of mussel polysaccharide MP-1 of the present invention is shown below;

[0027] Figure 3 The HPLC chromatogram of the mussel oligosaccharide mixture MOS-A;

[0028] Figure 4 The HPLC chromatogram of the mussel oligosaccharide mixture MOS-B;

[0029] Figure 5 for Bacillus subtilis Single-factor experimental curves of BNCC185269 fermentation of MP-1;

[0030] Figure 6 for Bacillus subtilis Figure 1. Results of a three-factor, four-level response surface methodology experiment for fermenting MP-1 with BNCC185269;

[0031] Figure 7 The figure shows the effect of mussel oligosaccharides MOS-A and MOS-B intervention of the present invention on the disease activity index of DSS-induced ulcerative colitis mice;

[0032] Figure 8 The figure shows the effect of the mussel oligosaccharide mixtures MOS-A and MOS-B of the present invention on the body weight of mice with DSS-induced ulcerative colitis.

[0033] Figure 9 This is a representative diagram showing the colon length in mice with DSS-induced ulcerative colitis treated by the intervention of the mussel oligosaccharide mixtures MOS-A and MOS-B of the present invention.

[0034] Figure 10 The figure shows the effect of the mussel oligosaccharide mixtures MOS-A and MOS-B of the present invention on the colon length of mice with DSS-induced ulcerative colitis.

[0035] Figure 11 The figure shows the effect of the mussel oligosaccharide mixtures MOS-A and MOS-B of the present invention on the histopathological effects of colon tissue in mice with DSS-induced ulcerative colitis. Detailed Implementation

[0036] Example 1

[0037] 1. Preparation of mussel polysaccharide MP-I

[0038] Take 250g of thawed mussel meat (purchased from various aquaculture farms in Rizhao, Shandong), homogenize it, add 20 times the amount of water (about 5L), heat to a gentle boil for 2 hours, filter through 4 layers of gauze, and concentrate to 500mL by rotary evaporation. Add 1% papain (5g / 500mL) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: G2227454) to the concentrate, incubate in a 60℃ water bath for 3 hours, and gently boil for 15 minutes to inactivate the enzyme and obtain the enzymatic hydrolysate. Add 500mL of Sevage reagent (chloroform: n-butanol = 4:1) to the enzymatic hydrolysate and shake vigorously for 10 minutes. After standing and separating the layers, discard the lower layer. Add 1 / 4 volume (250mL) of Sevage reagent to the supernatant, centrifuge at 4000rpm for 10 minutes, and repeat until no protein layer remains. Concentrate the solution to 200-300mL, add 750ml of pure ethanol, let stand for 2 hours, centrifuge at 4000rpm for 10 minutes, and discard the supernatant. The precipitate was washed twice with 750 ml of 75% ethanol and once with acetone. After centrifugation, crude polysaccharide was obtained, dried, and weighed to obtain 5 g of mussel crude polysaccharide.

[0039] Dissolve 1.5g of crude mussel polysaccharide in 5mL of pure water to a concentration of 0.3g / mL. Centrifuge at 4000rpm for 5min and collect the supernatant mussel polysaccharide solution. Prepare elution buffers of varying concentrations: 0.1M NaCl, 0.2M NaCl, and 0.5M NaCl, pH=7. Prepare an equilibration solution containing 2M NaCl and 0.2M NaOH, pH=13.

[0040] First, wash the DEAE Sepharose FF anion exchange column (packing material company: Cytiva; packing material model: Cytiva Sweden AB SE-75184 Uppsala Sweden; column height 27cm, inner diameter 2.6cm, packing volume 90ml) with equilibration solution of 2M NaCl and 0.2M NaOH for 2-3 column volumes, then wash with pure water until the mobile phase pH=7.

[0041] Next, 5 mL of crude mussel polysaccharide solution was loaded onto the plate at a flow rate of 1 mL / min. Elution was performed sequentially with four gradients of elution buffers: 0 M NaCl, 0.1 M NaCl, 0.2 M NaCl, and 0.5 M NaCl. Each gradient elution took approximately 6 hours. The eluent was collected, with a total elution time of 24 hours. 200 μL of each eluent was added to 200 μL of phenol solution (5% by mass). Then, 1 mL of concentrated sulfuric acid was added to each sugar tube on an ice pack. After mixing, the tube was boiled for 20 minutes and then immediately cooled for 20 minutes. 200 μL of the solution from each sugar tube was transferred to a 96-well plate (n=3, three parallel samples) and detected using a microplate reader at 490 nm. A distinct sugar peak appeared with the 0.1 M NaCl elution solution. The average value of each tube's reading was used to plot an elution curve. The peak sugar tube from the 0.1 M NaCl elution was collected and freeze-dried to obtain mussel polysaccharide. The weight of polysaccharide eluted with 0.1M NaCl was 1g, with a yield of 70%.

[0042] To further improve purity, mussel polysaccharide was purified using a Sepharose CL-6B molecular sieve chromatography column. After washing the column with pure water, 100 mg of freeze-dried mussel polysaccharide was dissolved in 5 mL of pure water (concentration 20 mg / mL) and loaded with 2.5 mL of water each time, for a total of 5 mL. Elution was then performed with 0.05 M NaCl solution at a constant flow rate of 0.3 mL / min, one tube every 10 minutes, for a total of 40 tubes. The sugar distribution of these 40 samples was measured using the sulfuric acid-phenol method. Samples containing sugar were freeze-dried to obtain mussel polysaccharide MP-1, weighing 85 mg with a purity of 99%. It is a dextran with a molecular weight of 312–533 kDa, predominantly composed of α-1,4-glycosidic bonds with a small amount of α-1,6-glycosidic bonds as branches, and a repeating unit of 9. The content was relatively high, with a total yield of 56%. %.

[0043] 2. Determination of total sugar content of mussel polysaccharide MP-I

[0044] The total sugar content of mussel polysaccharides was determined using the sulfuric acid-phenol method. A 5% phenol solution and a 0.1 mg / mL standard glucose solution were prepared. 10 mg of mussel polysaccharide MP-I sample was accurately weighed, transferred to a 100 mL volumetric flask, and diluted to volume with water to a concentration of 0.1 mg / mL. This solution was then used as the sample determination solution.

[0045] Pipette 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, and 0.6 ml of 0.1 mg / ml standard glucose solution into colorimetric tubes, respectively, and add distilled water to each tube to a final volume of 2.0 ml. Add 1.0 ml of 5% phenol to the solution, then quickly add 5.0 ml of concentrated sulfuric acid. Vortex to mix thoroughly, let stand for 10 minutes, and then place the colorimetric tubes in a shaking, constant-temperature metal bath at 80°C for 15 minutes. Measure the absorbance at 490 nm using a UV-Vis spectrophotometer. Use 1.0 ml of water as a blank, following the same colorimetric procedure. Plot the glucose concentration on the x-axis and the absorbance value on the y-axis to create a standard curve.

[0046] Pipette 1.0 ml of the sample solution into a colorimetric tube and follow the steps above to measure the absorbance value.

[0047] Based on the steps described above, a standard curve is constructed, yielding the equation: y = 0.065x - 0.006, R0. 2 =0.999, the total sugar content of mussel polysaccharides is 99.08%.

[0048] Table 1 Total sugar content of mussel polysaccharide MP-I

[0049]

[0050] 3. Molecular weight determination of mussel polysaccharide MP-I

[0051] The molecular weight and distribution of the prepared mussel polysaccharide MP-I were determined by HPGPC, and detection was performed using a tandem high-performance gel permeation chromatography (HPLC) column. The chromatographic conditions were as follows: Instrument: Waters high-performance liquid chromatograph; Detector: Waters differential detector; Column: three 8×300 mm polymer-based water-soluble SEC (GFC) columns in series; Mobile phase: 0.05 M NaCl solution; Flow rate: 0.65 ml / min; Column temperature: 40 ºC; Injection volume: 30 μl. The molecular weight results of mussel polysaccharide MP-I are shown in Table 2.

[0052] Table 2. Molecular weight results of mussel polysaccharide MP-I

[0053]

[0054] like Figure 1The purified mussel polysaccharide was a homogeneous polysaccharide, and its molecular weight was 312~533 kDa, as shown in Table 2.

[0055] 4. Determination of monosaccharide components of mussel polysaccharide MP-I

[0056] The monosaccharide fraction of mussel polysaccharide MP-I, prepared by high performance liquid chromatography, was determined by adding 5 mg of the sample to 1 mL of 2 M trifluoroacetic acid (TFA) solution and heating at 121 °C for 2 hours. Nitrogen gas was then introduced, and the mixture was dried. Methanol was added for washing, and the mixture was dried again. This methanol washing process was repeated 2-3 times to obtain a 5 mg / mL polysaccharide hydrolysate.

[0057] Take 0.2 mL of 5 mg / mL polysaccharide hydrolysate into a stoppered conical centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution for PMP derivatization, vortex mix, and react in a 70 ℃ water bath for 1 h. After the reaction is complete, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add 1 mL of chloroform and vortex extract 3 times to remove excess PMP, discard the chloroform layer, take 0.3 mL and add water to make up to 1 mL.

[0058] The chromatographic conditions were as follows: Instrument: Thermo U3000 liquid chromatography system; Detector: DAD detector; Column: ZORBAX EclipseXDB-C18; Mobile phase: acetonitrile: phosphate buffer (potassium dihydrogen phosphate 12 g / L, pH adjusted to 6.8 with 2 M NaOH) isocratic elution, with a volume ratio of acetonitrile to phosphate buffer of 17:83; Flow rate: 0.8 ml / min; Column temperature: 30 ºC; Detection wavelength: 250 nm; Injection volume: 10 μL.

[0059] The monosaccharide composition of mussel polysaccharide MP-I is shown in Table 3. The monosaccharide composition of this mussel polysaccharide MP-I is 100% glucose.

[0060] Table 3. Results of monosaccharide composition of mussel polysaccharide MP-I

[0061]

[0062] 5. Methylation determination of mussel polysaccharide MP-I

[0063] The methylation determination of the prepared mussel polysaccharide MP-I was performed on an Agilent 7890A-5977B gas chromatography-mass spectrometry (GC-MS) system. The sample was pretreated according to the following steps: 1 mg of mussel polysaccharide MP-I was dissolved in 1 mL of DMSO; 30 mg of NaOH was added and incubated for 30 min; 250 μL of iodomethane solution was added, nitrogen was introduced, and the reaction was carried out in the dark for 1 h; another 250 μL of iodomethane solution was added and the reaction was carried out for 1 h; 1 mL of water and 2 mL of dichloromethane were added, vortexed, centrifuged, and the aqueous phase was discarded; the mixture was washed three times with water; the lower dichloromethane phase was aspirated and dried under nitrogen; 1 mL of 2M TFA was added, and the reaction was carried out at 121 °C for 120 min; the mixture was dried under nitrogen at 30 °C; 1 mL of freshly prepared 1 M NaBD4 (Anhui Zesheng Technology Co., Ltd.; product number: E0900270250; 1 M NaBD4 was prepared by dissolving in ammonia water) was added. The sample was incubated with magnetic stirring at room temperature for 2.5 hours; the reaction was terminated by adding 300 μl of acetic acid, and dried under nitrogen; the sample was dried twice under nitrogen at 40°C with 2 ml of 5% (vol / vol) acetic acid methanol (5% (vol / vol) acetic acid dissolved in methanol is used to remove excess reducing agent NaBD4 and its products), then washed with 2 ml of methanol and dried twice under nitrogen at 40°C; 1.5 mL of acetic anhydride was added, vortexed, and reacted at 100°C for 2.5 h; 2 ml of water was added and allowed to stand for 10 min; 1 mL of dichloromethane was added, vortexed, centrifuged, and the aqueous phase was discarded. The washing was repeated three times with water; the lower dichloromethane phase was collected and analyzed.

[0064] The chromatographic parameters were as follows: Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas was high-purity helium (purity not less than 99.999%), flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, split injection was used, the split ratio was 10:1, and the solvent delay was 2.2 min. The temperature program was: 50℃ held for 1.0 min, increased to 130℃ at 50℃ / min, increased to 230℃ at 3℃ / min, and held for 2 min.

[0065] The mass spectrometry parameters are as follows: Agilent Technologies (China) Co., Ltd. quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source has an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode is full scan (SCAN), and the mass scan range (m / z) is 30-600.

[0066] The glycosidic bond linkage mode of mussel polysaccharide MP-I was analyzed based on the methylation results, and the results are shown in Table 4:

[0067] Table 4. Analysis of glycosidic bond linkage modes of mussel polysaccharide MP-I

[0068]

[0069] The main chain of the mussel polysaccharide MP-I is composed of α-1,4-glycosidic bonds, with α-1,6-glycosidic bonds serving as branches in a ratio of 9:2, and there are an average of 9 glucose molecules between two branches.

[0070] 6. Infrared spectral analysis of mussel polysaccharide MP-I

[0071] The prepared mussel polysaccharide MP-I was analyzed by Fourier transform infrared spectroscopy. The steps were as follows: 2 mg of dried mussel polysaccharide MP-I sample was weighed into a mortar, 200 mg of KBr powder was added and ground evenly, then compressed into tablets. The sample was scanned using a Fourier transform infrared microscope with a wavelength range of 4000~400 cm⁻¹. -1 Record the infrared spectrum.

[0072] like Figure 2 The infrared spectral analysis results of mussel polysaccharide MP-I are as follows: The polysaccharide sample MP-I exhibits a characteristic absorption peak at 3397.21 cm⁻¹. -1 A strong and broad absorption peak exists at 2929.46 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity peak is the absorption peak of the CH stretching vibration of the methine (-CH2); 1647.17 cm⁻¹ -1 The absorption peak at 1450-1200 cm⁻¹ is the hydration vibration peak of polysaccharides. -1 The absorption peak is at 1416.77 cm⁻¹. -1 1367.48 cm -1 1239.01 cm -1The peak at 1154.961 cm⁻¹ represents the angular vibration absorption of CH₄, which, along with the stretching vibration of CH₄, constitutes the characteristic absorption of the sugar ring; 1154.961 cm⁻¹ represents the stretching vibration of pyranose COC; 1150-1010 cm⁻¹ -1 The two strong absorption peaks between, at 1081.21 cm⁻¹ -1 1023.02 cm -1 The presence of pyranoside was confirmed again at point 1, and the absorptions at these three sites were due to the bending vibrations of the CO bond in the COH or COC structure; 929.98 cm⁻¹ -1 The peak at 848.48 cm⁻¹ shows the asymmetric ring stretching vibration of the pyran ring. -1 The peak represents the CH-angle vibration of the diastereomer of the α-terminal group of pyranose; 762.32 cm⁻¹ -1 The peaks of symmetrical ring stretching vibrations of the pyran ring are shown.

[0073] The above results further demonstrate that mussel polysaccharide MP-I has a typical pyranose ring structure and exhibits the characteristics of α-glycosidic bonds, 848 cm⁻¹ -1 The characteristic peaks at this location are consistent with the α-1,4-glycosidic and α-1,6-glycosidic bond linkages observed in methylation analysis. (1150-1010 cm⁻¹) -1 The COC vibration peak supports the presence of the glucose pyran ring.

[0074] Example 2: Preparation of mussel oligosaccharide mixture MOS-A by enzymatic hydrolysis

[0075] Accurately weigh 30 mg of the mussel polysaccharide MP-1 sample prepared in Example 1 and place it in a 10 mL centrifuge tube. Simultaneously, accurately weigh 25 mg of α-amylase into another 10 mL centrifuge tube. Then add 30 mL of pure water to this tube and vortex intermittently to fully dissolve the α-amylase. After complete dissolution, transfer 5 mL of the prepared enzyme solution to the centrifuge tube containing the MP-1 sample. Mix repeatedly by pipetting to completely dissolve the mussel polysaccharide MP-1, forming a homogeneous enzymatic reaction system. Next, set and stabilize the water bath temperature at 60°C. Fix the centrifuge tube containing the above enzymatic reaction system to a float, ensuring the liquid level in the tube is completely submerged in the water bath, and begin the enzymatic reaction. The enzymatic reaction of MP-1 and α-amylase continues for 6 hours, during which the centrifuge tube is manually shaken once every hour. After the reaction is complete, place the centrifuge tube in a boiling water bath for 15 minutes to terminate the enzyme reaction. The inactivated sample is stored at 4°C for later use.

[0076] Next, acetonitrile was added to the enzymatic hydrolysate at a volume ratio of 1:1, and the mixture was thoroughly mixed. The mixture was then centrifuged at 4°C and 10,000 rpm / min for 10 minutes to remove MP-1 and α-amylase precipitates, and the supernatant was collected. The supernatant was then rotary evaporated to remove acetonitrile, pre-frozen at -80°C for 20 hours, and then transferred to a freeze dryer for 48 hours to obtain the white MOS-A final product. The final weight of white MOS-A was 22 mg, and the yield of MOS-A was 73.3%. To determine the composition of MOS-A, a standard was used for comparison with MOS-A.

[0077] Next, a preparative liquid chromatography system was used. The analytical amino column was replaced with a semi-preparative amino column, and the same mobile phase as the analytical column was used. Standards for glucose, maltose, maltotriose, maltotetraose, maltopentose, and maltohexaose were injected. The elution times of the semi-preparative column were observed and compared with subsequent samples. Samples were injected using a 100 μL syringe, the liquid chromatography chromatogram was saved, and the samples were stored at 4°C. For detailed results, please refer to [link to relevant documentation]. Figure 3 The mussel oligosaccharide mixture MOS-A eluted at 13.576, 16.433, 20.291, 31.431, and 38.719 min, corresponding to the elution times of glucose, maltose, maltotriose, maltotetraose, and maltopentose, respectively. This indicates that the mussel oligosaccharide mixture MOS-A contains at least glucose, maltose, maltotriose, maltotetraose, and maltopentose.

[0078] The HPLC analysis conditions for the mussel oligosaccharide mixture MOS-A were as follows: Instrument: Jasco PU-2080 high performance liquid chromatograph; Detector: RI-2031 Plus differential refractive index detector; Column: Ultimate - XB-NH2 column (4.6*250mm, 5μM); Mobile phase: 60% acetonitrile solution (60% volume acetonitrile, 40% volume water); Column temperature: 30℃; Flow rate: 1mL / min; Isocratic elution was performed, injection volume: 20μl, and total time was 50min.

[0079] Example 3: Preparation of mussel oligosaccharide mixture MOS-B by fermentation method

[0080] Take 2ml of glycerin for storage Bacillus subtilisBNCC185269 was added to 20 ml of LB liquid medium and cultured in a shaker at 37°C and 180 rpm for 12 hours to obtain the Bacillus subtilis seed culture required for fermentation. After culturing, the seed culture was centrifuged at 37°C and 4000 rpm for 10 minutes, the LB supernatant was discarded, and the precipitated bacterial cells were retained. The cells were then washed once with sterile physiological saline under aseptic conditions and transferred to sterile MP-1 specially prepared medium. At this point, the OD of the bacterial culture was... 600 =0.5~0.6. The preparation conditions of the sterile MP-1 special culture medium are as follows: accurately weigh 100 mg of mussel polysaccharide MP-1 sample prepared in Example 1, dissolve it in pure water, and make up to 100 ml. Sterilize it in a high-temperature sterilizer at 121°C for 15 min, and then sterilize it under ultraviolet light and cool it to room temperature.

[0081] The MP-1 specially prepared medium inoculated with the bacteria was then fermented at 37°C and 180 rpm. During fermentation, samples were taken periodically to monitor the formation of oligosaccharides in the fermentation broth. After 8 hours of fermentation, the fermentation broth was centrifuged at 4°C and 4000 rpm for 20 minutes, the supernatant was collected, the bacterial precipitate was removed, and the supernatant was filtered sequentially through 0.45 μm and 0.22 μm microporous membranes to obtain a clear, sterile supernatant. The collected clear supernatant was rotary evaporated, and acetonitrile was added at a supernatant-acetonitrile volume ratio of 1:1. The mixture was then centrifuged at 4°C and 10000 rpm for 30 minutes to remove the polysaccharide precipitate, and the supernatant was collected. Acetonitrile was removed by rotary evaporation of the supernatant and pre-frozen in an ultra-low temperature freezer at -80°C for 20 hours. Then, it was transferred to a freeze dryer and freeze-dried for 48 hours to obtain the white mussel oligosaccharide mixture MOS-B final product. The weight of the obtained mussel oligosaccharide mixture MOS-B was 49.55 mg, and the yield of MOS-B was 49.55%.

[0082] To determine the composition of MOS-B, PMP methanol derivatization was performed. The specific steps were as follows: 0.2 mL of an aqueous solution of 5 mg / mL mussel oligosaccharide mixture MOS-B was added to a stoppered conical centrifuge tube. 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution were added for PMP derivatization. After vortexing, the mixture was reacted in a 70 °C water bath for 1 h. After the reaction was complete, 0.2 mL of 0.5 mol / L hydrochloric acid was added to neutralize the added sodium hydroxide. 1 mL of chloroform was added for vortex extraction three times to remove excess PMP. After discarding the chloroform layer, 0.3 mL of the extract was diluted to 1 mL with water to obtain the sample solution. The obtained sample solution was injected into the chromatographic system using a 100 μL syringe. The corresponding elution fraction was collected according to the target time determined by the retention time of the standard. The preparative chromatogram was saved, and the collected sample fraction was temporarily stored at 4 °C. Specific results are shown in [link to results]. Figure 4 Compared with the control, the mussel oligosaccharide mixture MOS-B showed six peaks: peak 1 corresponds to glucose, peak 3 to maltotriose, peak 4 to maltose, peak 5 to maltotetraose, and peak 6 to maltopentose. Since the polysaccharide MP-1 used in fermentation has a β-1,6 glycosidic bond structure and undergoes allosteric changes during fermentation, peak 2 is presumably an isomaltose structure.

[0083] Based on the above preparation, the preparation process of the mussel oligosaccharide mixture MOS-B was optimized with optimal yield as the indicator. Single-factor experiments were conducted on shaking speed (180, 190, 200, 210, 220 rpm / min), initial pH (6.5, 7.0, 7.5, 8.0, 8.5), fermentation temperature (31℃, 33℃, 35℃, 37℃, 39℃), raw material concentration (0.5, 1.0, 1.5, 2.0, 2.5 mg / ml), and fermentation time (4h, 6h, 8h, 10h, 12h). The optimal single-factor results for each condition were found to be a shaking speed of 200 rpm / min, an initial pH of 7.5, a temperature of 37℃, a raw material concentration of 1 mg / ml, and a fermentation time of 8h. Regarding the selection of the fermentation medium, using a sterile aqueous solution containing 1 mg / ml MP-1 was superior to using a solution containing 1 mg / ml MP-1. The carbon-free medium (MM) conditions for MP-1 are superior. The formulation of the carbon-free medium (MM) (500 mL) is as follows: 1.4 g Na₂HPO₄, 0.25 g (NH₄)₂SO₄, 0.0005 mg CuCl₂·2H₂O, 0.015 mg H₃BO₃, 0.1 mg FeSO₄·7H₂O, 0.5 mg KH₂PO₄, 0.0015 mg MnCl₂·4H₂O, 0.001 mg NiCl₂·6H₂O, 0.25 mg Na₂EDTA, 0.01 mg CoCl₂·6H₂O, 0.005 mg ZnSO₄·7H₂O, and 0.0015 mg Na₂MoO₄·2H₂O. Detailed results can be found in [link to results]. Figure 5 Ultimately, fermentation temperature, feedstock concentration, and fermentation time were determined to have the most significant impact on the yield of MOS-B. Response surface methodology (RSM) experiments were conducted on these three factors using a three-factor, four-level orthogonal design. The factors and levels are shown in Table 5. The optimal preparation process parameters were determined to be: fermentation temperature 37.8℃, fermentation time 9.2 h, and feedstock concentration 0.8 mg / ml. Detailed results are available in [Table 5]. Figure 6 Based on the optimal parameters determined by response surface methodology, the weight of the mussel oligosaccharide mixture MOS-B was 50.08 mg, and the yield of MOS-B was 50.08%.

[0084] Table 5 Factors and Levels in Orthogonal Experiments

[0085]

[0086] The HPLC chromatographic conditions for the mussel oligosaccharide mixture MOS-B were as follows: Instrument: Shimadzu SPD-M20A high-performance liquid chromatograph; Detector: UV detector; Column: YMC-Pack-ODS-A column; Mobile phase: A was acetonitrile, B was 0.05M KH2PO4 (pH=6.8); Column temperature: 35℃; Flow rate: 1mL / min; Injection volume: 20μL. The HPLC program for fermenting MOS-B is shown in Table 6.

[0087] Table 6. HPLC program for fermentation of MOS-B

[0088]

[0089] Example 4: Effect of mussel oligosaccharide mixture on DSS-induced ulcerative colitis in mice

[0090] (1) Experimental animals: 40 male C57BL / 6J mice, aged 6-8 weeks and weighing 20±2 g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0091] (2) Animal grouping: After one week of acclimatization, animals were randomly grouped into 5 groups (8 animals / group): Control group, Model group, 5-ASA (aminosalicylic acid, Merck, catalog number A3537) treatment group, mussel oligosaccharide mixture MOS-A treatment group (20 mg / kg) and mussel oligosaccharide mixture MOS-B treatment group (20 mg / kg).

[0092] (3) Animal modeling and drug administration: The control group mice received standard drinking water, while the other groups of mice received drinking water with 3% DSS added to induce acute ulcerative colitis for 7 days. One day after modeling, the mussel oligosaccharide mixture MOS-A and mussel oligosaccharide mixture MOS-B treatment groups were slowly injected 100 μL of mussel oligosaccharide mixture MOS-A and mussel oligosaccharide mixture MOS-B (20 mg / kg) into the rectum daily, respectively. The 5-ASA positive control group was slowly injected 100 μL of 5-ASA (20 mg / kg) into the rectum daily.

[0093] (4) DAI score: During the experiment, the three clinical symptoms of UC were assessed daily according to DAI, including the percentage of weight loss, stool viscosity, and rectal bleeding. The DAI score is the sum of the daily weight loss score, stool viscosity score, and rectal bleeding score. DAI score indicators: the percentage of weight loss (0 points: no change in weight, 1 point: 1%-5% decrease, 2 points: 5%-10% decrease, 3 points: 10%-20% decrease, 4 points: >20%), stool consistency (0 points: normal, 2 points: slightly moist but not attached to the perianal area, 4 points: diarrhea), and rectal bleeding (0 points: normal, 2 points: slight bleeding, 4 points: hematochezia) are the sum of the three scores.

[0094] (5) Animal sampling: Eight hours after the last administration, mice were euthanized by cervical dislocation. The peritoneal cavity of the mice was exposed, and the colon and rectum were separated. The length of the colon and rectum was measured and photographed. Subsequently, pre-cooled physiological saline was repeatedly injected into the intestinal lumen to remove the contents. Approximately 1 cm of the distal colon was excised and fixed in 4% paraformaldehyde solution for histopathological examination.

[0095] (6) Experimental results:

[0096] ① Disease Activity Index (DAI) score for mouse colitis: See details below. Figure 7 and Figure 8 Compared to the control group, mice given 3% DSS in their drinking water exhibited acute clinical symptoms, including weight loss, altered fecal characteristics, and severe bloody stools, which were statistically reflected in increased DAI scores. Compared to the model group, the 5-ASA group, the mussel oligosaccharide mixture MOS-A group, and the mussel oligosaccharide mixture MOS-B group all significantly improved the clinical symptoms of ulcerative colitis in mice and reduced DAI scores.

[0097] ② Measurement of mouse colon length: See details below. Figure 9 and Figure 10 Compared with the control group, mice treated with DSS showed significant congestion, extensive mucosal ulceration, and shortened length in the colorectal tissue. Compared with the model group, rectal injection of the mussel oligosaccharide mixture MOS-B significantly reversed intestinal congestion and shortened colorectal length in mice, suggesting that the mussel oligosaccharide mixture MOS-B can improve the severity of UC and has a protective effect comparable to that of positive control drugs.

[0098] ③ Histopathological evaluation of mouse colon tissue: See details below. Figure 11Compared with the control group, DSS intervention led to damage to the colonic epithelial mucosa, necrosis of crypt structures, and extensive infiltration of inflammatory cells. However, compared with the model group, rectal injection of the mussel oligosaccharide mixture MOS-B significantly alleviated the damage to colonic tissue structure and reduced inflammatory cell infiltration, suggesting that the mussel oligosaccharide mixture MOS-B has a protective effect against DSS-induced colonic tissue pathological damage, and that MOS-B possesses a protective effect comparable to that of the positive control drug.

Claims

1. A method for preparing a mixture of mussel oligosaccharides, characterized in that, Includes the following steps: Will Bacillus subtilis The activated bacterial solution of BNCC185269 was added to mussel polysaccharide MP-1 for reaction. The concentration of mussel polysaccharide MP-1 was 0.5~1.5 mg / mL, the reaction temperature was 35~37℃, and the reaction time was 6~10 h. After the reaction was completed, the bacterial precipitate was removed by high-speed centrifugation, and the solution was filtered through a filter membrane to obtain a clear sterile supernatant. The collected clear supernatant was rotary evaporated and an equal volume of acetonitrile was added. The polysaccharide precipitate was removed by centrifugation, and the supernatant was collected. The acetonitrile was removed by rotary evaporation of the supernatant, and then the solution was transferred to a freeze dryer for freeze drying to obtain the mussel oligosaccharide mixture. The Bacillus subtilis The activated bacterial solution of BNCC185269 is Bacillus subtilis BNCC185269 was obtained by resuscitation and culture in LB medium. Bacillus subtilis OD of activated bacterial solution of BNCC185269 600 =0.5~0.6; During the resuscitation culture process, the... Bacillus subtilis The inoculum size of BNCC185269 was 5-10%, the recovery time was 12-24 hours, and the pH was 7-7.

5. The mussel polysaccharide MP-1 has an α-1,4-glycosidic bond as the main chain and α-1,6-glycosidic bonds as branches, with an average of one α-1,6-glucose branch per 10 glucose main chains. Its relative molecular weight is 312-533 kDa, and its structural formula is shown below: 。 2. The method for preparing the mussel oligosaccharide mixture according to claim 1, characterized in that, The high-speed centrifugation conditions are: rotation speed 3000~5000 rpm, temperature 3~5℃, time 15~30 min; the pore size of the filter membrane is 0.22~0.45 μM.

3. The mussel oligosaccharide mixture prepared by the preparation method according to any one of claims 1 to 2.

4. The mussel oligosaccharide mixture according to claim 3, characterized in that, The mussel oligosaccharide mixture is mussel oligosaccharide mixture MOS-B, which includes glucose, isomaltose, maltose, maltotriose, maltotetraose and maltopentose.

5. The use of the mussel oligosaccharide mixture according to claim 3 or 4 in the preparation of a medicament for the prevention or treatment of inflammatory bowel disease, wherein the inflammatory bowel disease is ulcerative colitis.

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