Pyruvate-containing fucotetraose with intestinal barrier enhancing function and preparation method of pyruvate-containing fucotetraose

Abstract

The invention relates to the technical field of oligosaccharide preparation, and discloses pyruvate-containing fucotetraose with an intestinal barrier enhancing function and a preparation method of the pyruvate-containing fucotetraose, the structural main chain of the pyruvate-containing fucotetraose is alpha-L-Fucp-(1-> 3)-alpha-L-Fucp-(1-> 3)-beta-D-Glcp, and the side chain of the pyruvate-containing fucotetraose is formed by introducing 1, 2-D-Galp of which the 4 and 6 sites are substituted by pyruvate into a reaction kettle through a reaction between 1, 2-D-Galp and 1, 2-D-Galp. The second alpha-L-Fucp of the main chain is substituted in a 2, 4 connection mode, and the molecular formula is C27H44O21. The pyruvate-containing fucotetraose provided by the invention can promote intestinal goblet cell-like cells Ls174T to generate mucoprotein and enhance the barrier function of the intestinal tract; the safe concentration of the pyruvate-containing fucotetraose to Ls174T cells is 1 mg / mL, and after the pyruvate-containing fucotetraose with the concentration of 1 mg / mL acts on the Ls174T cells, the output of mucoprotein in a mucous layer is increased by 1.5 times or above.

Description

Technical Field

[0001] The present invention relates to the technical field of oligosaccharide preparation, and in particular to a pyruvic acid-containing fucotetraose having the function of enhancing intestinal barrier and a preparation method thereof. Background Art

[0002] Human milk oligosaccharides are important components of breast milk, including fucosyl oligosaccharides (2'-fucosyllactose, 3-fucosyllactose and lactose-N-neotetraose, etc.), which have a significant promoting effect on the health of infants and young children, such as regulating intestinal flora, strengthening intestinal barrier, improving immunity and cognitive ability, etc., and have extremely high application value. At present, fucosyl oligosaccharides in breast milk are the most studied natural fucosyl oligosaccharides. However, the fucose in these oligosaccharides is located at the non-reducing end of the sugar chain. The limited structure limits the wide application of fucosyl oligosaccharides in breast milk. Therefore, it is necessary to explore fucosyl oligosaccharides with diverse structures in order to develop functional products with different activities.

[0003] Bacterial exopolysaccharides are high molecular weight polysaccharide substances synthesized by bacteria during the culture process and secreted into the culture medium. Exopolysaccharides can be divided into homopolysaccharides and heteropolysaccharides according to their monosaccharide composition, among which heteropolysaccharides are usually composed of repeating units composed of a variety of different monosaccharides (glucose, galactose, uronic acid, mannose, rhamnose and fucose, etc.). Exopolysaccharides are widely used in food, cosmetics, petroleum and pharmaceutical industries. For example, xanthan gum, bacterial cellulose and fructan are used as thickeners, stabilizers and emulsifiers in industry. There are more than 200 bacteria that can produce exopolysaccharides containing fucose, and the structure of the exopolysaccharides containing fucose produced by each bacterium is also different. Therefore, bacterial sources are a new way to obtain new fucoidan oligosaccharides. Common polysaccharide degradation methods include physical degradation, chemical degradation and enzymatic degradation. Unlike the other two degradation methods, enzymatic degradation is characterized by high efficiency, specificity, mild conditions, and high product purity. However, it is difficult to obtain enzymes. For new extracellular polysaccharides, it is necessary to screen degrading bacteria from the environment and further extract the enzymes.

[0004] Ls174T cells are a human epithelial cell line isolated from the colon tissue of a 58-year-old patient with colon cancer. They have a characteristic epithelial morphology and grow in an adherent form. The cells can be used to simulate intestinal goblet cell-related models and evaluate mucin secretion. Mucin is the main component of the intestinal mucus layer and is essential for maintaining the intestinal barrier function. Mucin can isolate the host epithelium from intestinal microorganisms and prevent the invasion of pathogens. The mucus layer is divided into two layers: the outer layer is the natural habitat of symbiotic bacteria, and the inner layer is tightly attached to the epithelial cells, which act as a "filter" to prevent the infiltration of microorganisms and provide a relatively sterile environment for the intestinal epithelium. Studies have shown that 2'-fucosyllactose does not promote the secretion of mucin by Ls174T cells in a normal state. It mainly promotes intestinal health by regulating the composition of intestinal flora. Therefore, the development of a fucoidan oligosaccharide that can directly promote the secretion of mucin by Ls174T cells to enhance the intestinal barrier provides an important theoretical basis for the widespread application of fucoidan oligosaccharides. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a pyruvate-containing fucotetraose having enhanced intestinal barrier function and a preparation method thereof.

[0006] In order to achieve the above-mentioned object, the technical scheme of the present invention is: a pyruvate-containing fucotetraose having the function of enhancing intestinal barrier, wherein the main chain of the pyruvate-containing fucotetraose is α-L-Fucp-(1→3)-α-L-Fucp-(1→3)-β-D-Glcp, and the side chain is β-D-Galp substituted by pyruvate at positions 4 and 6, which replaces the second α-L-Fucp in the main chain by 1,4 connection, and the molecular formula is C 27 H 44 O 21 .

[0007] Another technical solution of the present invention is: a method for preparing the pyruvate-containing fucotetraose having the function of enhancing the intestinal barrier, comprising the following steps:

[0008] (1) fermenting Clavibacter michiganensis CN14 to prepare exopolysaccharide;

[0009] (2) fermenting Pectobacterium carotovorum D14 to prepare a crude enzyme solution;

[0010] (3) The crude enzyme solution obtained in step (2) is used to degrade the extracellular polysaccharide obtained in step (1), and the oligosaccharide components with a molecular weight less than 1000 Da are collected by high temperature enzyme inactivation and graded alcohol precipitation, and the oligosaccharide components are freeze-dried to obtain pyruvate-containing fucose.

[0011] Furthermore; the Michigan Bacillus CN14 described in step (1) has a deposit number of CGMCC No.31378 and was deposited at the General Microbiology Center of the China Culture Collection Administration on July 19, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] Furthermore, the carrot soft rot pectin bacteria D14 described in step (2) has a preservation number of CGMCC No.31377, and was deposited in the General Microbiology Center of the China Culture Collection Administration on July 19, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] Furthermore, the fermentation method for preparing extracellular polysaccharides in step (1) is as follows: inoculating Michigan Bacillus CN14 into a liquid fermentation medium for oscillating fermentation, the inoculation amount of the bacteria is 0.5% to 2% (v / v), the fermentation temperature is 25 to 30°C, the oscillation speed is 150 to 200 rpm, the fermentation time is 48 to 120 hours, and after the fermentation is completed, centrifugation is performed to collect the supernatant, and the supernatant is concentrated and dialyzed to remove small molecular impurities, and freeze-dried to obtain extracellular polysaccharides; the liquid fermentation medium formula is: 30 g / L glucose, 4 g / L yeast extract, 5 g / L calcium carbonate, 3 g / L potassium dihydrogen phosphate, 3 g / L potassium hydrogen phosphate, 1 g / L sodium chloride, and the pH is 7.0±0.2.

[0014] Furthermore, the dialysis bag has a molecular weight cut-off of 10,000Da.

[0015] Furthermore, the molar percentage of fucose in the extracellular polysaccharide is 46.27% to 52.13%, and the molar percentage of pyruvic acid is 10.2 to 20.12% (w / w).

[0016] Furthermore, the method for preparing crude enzyme solution by fermentation in step (2) is as follows: inoculating carrot soft rot pectin bacillus D14 into a fermentation medium for fermentation, the inoculation amount of the bacteria is 0.5% to 2% (v / v), the fermentation temperature is 30 to 40°C, the fermentation time is 48 to 120 hours, and after the fermentation is completed, the supernatant is collected by centrifugation to obtain a crude enzyme solution; the fermentation medium comprises the following components in weight percentage: 0.5% of the extracellular polysaccharide prepared in step (1), 0.5% of yeast extract, 0.5% of peptone, 0.1% of sodium chloride, 0.3% of dipotassium hydrogen phosphate, and 0.3% of potassium dihydrogen phosphate.

[0017] Furthermore, step (3) is specifically as follows: the extracellular polysaccharide prepared in step (1) is configured with ultrapure water to form an extracellular polysaccharide solution with a mass concentration of 0.5% to 3%, and then the crude enzyme solution obtained in step (2) is used to degrade the extracellular polysaccharide solution, wherein the volume ratio of the crude enzyme solution to the extracellular polysaccharide solution is 1:(3 to 5), the degradation temperature is 20 to 45°C, the pH is 4 to 8, the degradation time is 12 to 96 hours, and the oligosaccharide components with a molecular weight less than 1000Da are collected by high temperature enzyme inactivation and graded alcohol precipitation, and pyruvate-containing fucose is obtained after freeze-drying.

[0018] Another technical solution of the present invention is: an application of the pyruvate-containing fucotetraose in enhancing the intestinal barrier.

[0019] The beneficial effects of the present invention are as follows: the pyruvate-containing fucotetraose of the present invention can promote the intestinal goblet cell-like cells Ls174T to produce mucin and enhance the intestinal barrier function; the safe concentration of the pyruvate-containing fucotetraose for Ls174T cells is 1 mg / mL, and after 1 mg / mL of pyruvate-containing fucotetraose acts on Ls174T cells, the amount of mucin produced in the mucus layer is increased by more than 1.5 times. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the elution curve of the degradation product purified by Bio-rad gel P4 in Example 4;

[0021] Figure 2 HPLC chart of monosaccharide composition of the purified product of Example 5;

[0022] Figure 3 This is the HPLC chart of the pyruvic acid content of the purified product in Example 5;

[0023] Figure 4 This is the ESI-MS image of the purified product of Example 5;

[0024] Figure 5 This is the ESI-CID-MS / MS image of the purified product of Example 5;

[0025] Figure 6 This is a one-dimensional NMR image of the purified product of Example 5; Figure 6 -A is the hydrogen nuclear magnetic resonance spectrum, Figure 6 -B is the carbon NMR spectrum;

[0026] Figure 7 This is the 2D NMR COSY spectrum of the purified product of Example 5;

[0027] Figure 8 This is the 2D NMR HSQC spectrum of the purified product of Example 5;

[0028] Fig. 9This is the two-dimensional nuclear magnetic HMBC spectrum of the purified product of Example 5;

[0029] Fig.10 The effect of different concentrations of pyruvate-containing fucose on the cytotoxicity of Ls174T cells in Example 6;

[0030] Fig.11 This is Example 7, which shows the effect of pyruvate-containing fucose on the content of Mucin2 produced by Ls174T cells. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0032] Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by conventional technicians in the field to which the present invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0033] Example 1: Screening of Bacillus michiganensis and optimization of exopolysaccharide preparation conditions

[0034] The rotten tomato leaves were collected and immersed in sterile phosphate buffer (135mmol / L NaCl, 2.7mmol / L KCl, 1.5mmol / L KH 2 PO 4 and 8mmol / LK 2 HPO 4 , pH 7.2), and after 20 minutes, evenly spread the soaking liquid on a solid culture medium (yeast extract 5g / L, peptone 10g / L, glucose 5g / L, gentamicin 20μg / mL, polymyxin B 30μg / mL, agar 15g / L, pH 7.0±0.2) plate, and culture in a 27℃ incubator for 48h. Then pick a single colony and inoculate it into a liquid culture medium (yeast extract 5g / L, peptone 10g / L, glucose 5g / L, gentamicin 20μg / mL, polymyxin B 30μg / mL, pH7.0±0.2), and enrich and culture it in a 27℃ incubator. When its OD 600The supernatant was removed by centrifugation and the bacterial precipitate was sent to Qingdao Ruiboxingke Biotechnology Co., Ltd. for 16S RNA sequencing. Finally, a Clavibacter michiganensis was obtained, named Clavibacter michiganensis CN14, and deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number of CGMCC No. 31378 and the deposit date of July 19, 2024.

[0035] Michigan rod bacillus CN14 with a preservation number of CGMCC No. 31378 is inoculated into a liquid fermentation medium, and the fermentation medium formula is as follows: 30 g / L glucose, 4 g / L yeast extract, 5 g / L calcium carbonate, 3 g / L potassium dihydrogen phosphate, 3 g / L potassium hydrogen phosphate, 1 g / L sodium chloride, and the pH is 7.0±0.2. The inoculation amount of the bacteria is 0.5% to 2% (v / v), the fermentation temperature is 25 to 30° C., the shaking speed is 150 to 200 rpm, and the fermentation time is 48 to 120 hours. After the fermentation, the culture medium was centrifuged at 8,000 rpm for 30 min in a centrifuge, the bacterial precipitate was discarded, the supernatant was collected, and it was concentrated to 1 / 4 of the original volume by rotary evaporation at 50°C. The liquid after rotary evaporation was then used to remove small impurities through a dialysis bag with a molecular weight cutoff of 10,000 Da, and then the extracellular polysaccharide was freeze-dried using a vacuum freeze dryer and placed in a -4°C refrigerator for use.

[0036] Monosaccharide composition determination method: accurately weigh 8 mg of extracellular polysaccharide sample and place it in an ampoule tube, add 2 mL of 2 mol / L trifluoroacetic acid (TFA), seal it with an alcohol burner, and hydrolyze it at 110°C for 6 hours. Then, add 3 times the volume of methanol after cooling to room temperature, and remove TFA with a rotary evaporator to obtain the acid hydrolysis product. Prepare 1 mol / L fucose monosaccharide standard solution and the acid hydrolysis product together for PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization. The derivatization method is: take 100 μL of fucose monosaccharide standard solution and the acid hydrolysis product and mix them with 210 μL of 0.3 mol / L NaOH solution, add 200 μL of 0.5 mol / L PMP methanol solution, mix them evenly, place them in a 70°C oven for reaction for 60 minutes, cool them at room temperature after the reaction, and add 210 μL of 0.3 mol / L HCl to neutralize the excess alkali. Add 3 mL of dichloromethane to the mixture for extraction, shake and let stand, discard the lower layer after separation, repeat this extraction process 3 times. Filter the upper layer with a 0.22 μm microporous membrane and perform HPLC analysis.

[0037] Liquid phase instrument: Agilent 1260 high performance liquid chromatograph;

[0038] Liquid phase column model: Agilent ZORBAX 300XDB-C18;

[0039] Liquid phase conditions: 0.05 mol / L KH 2 PO 4 Phosphate buffer (pH 6.7) / CH 3 CN (83:17, V:V) was the mobile phase; the column temperature was 35°C; the flow rate was 1 mL / min; the injection volume was 20 μL; and the detection wavelength was 245 nm.

[0040] Pyruvic acid content determination method: prepare the acid hydrolysis product according to the extracellular polysaccharide degradation method in the above monosaccharide composition determination method, adjust the pH of the acid hydrolysis product to 3.0 with 1 mol / L sodium hydroxide, centrifuge at 8,000 rpm for 10 min, and filter the supernatant with a 0.22 μm microporous filter membrane for HPLC analysis.

[0041] Liquid phase instrument: Agilent 1260 high performance liquid chromatograph;

[0042] Liquid column model: Shodex KC-811;

[0043] Liquid phase conditions: 0.1% phosphoric acid aqueous solution as mobile phase; column temperature of 50°C; flow rate of 0.7 mL / min; injection volume of 20 μL; detection wavelength of 210 nm.

[0044] The results are shown in Table 1. The culture conditions (inoculum size, fermentation temperature, oscillation speed, fermentation time) have an impact on the yield of extracellular polysaccharides, the molar proportion of fucose in the monosaccharide composition, and the mass content of pyruvic acid. According to these three indicators of extracellular polysaccharides, the inoculation size was 0.5%, the fermentation temperature was 28°C, the oscillation speed was 180rpm, and the fermentation time was 120h. Under these culture conditions, the extracellular polysaccharide yield obtained was 7.44g / L, the molar proportion of fucose was 48.54mol%, and the pyruvic acid content was 20.12% (w / w).

[0045] Table 1 shows the optimization of exopolysaccharide production conditions and the yield, fucose molar ratio and pyruvic acid content under the corresponding conditions

[0046]

[0047] Example 2: Screening of degradation bacteria and preparation of crude enzyme solution

[0048] Use ultrapure water to prepare an extracellular polysaccharide solution with a mass concentration of 2%, and add additional yeast extract with a mass concentration of 0.25% and peptone with a mass concentration of 0.25%. Store it in an open environment at 30°C, and measure the reducing sugar production and the viscosity of the sugar solution every 24 hours. Method for measuring the amount of reducing sugar production: Use fucose of different concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) as a standard solution, take 0.5 mL of standard solutions of different concentrations and samples every 24 hours, add 0.5 mL DNS, and use distilled water plus DNS as a blank. After fully mixing, heat in a boiling water bath for 5 minutes, then place it in cold water to cool to room temperature, use distilled water to make up to 10 mL, and finally measure the OD on a UV spectrophotometer. 520 , the amount of reducing sugar produced was calculated according to the standard curve. The viscosity of the sugar solution was measured with a rotational viscometer. When the viscosity was reduced to a level close to that of the aqueous solution and the amount of reducing sugar produced exceeded 2 mg / mL, bacteria were screened from the sugar solution. A strain of bacteria capable of degrading extracellular polysaccharides was screened, and 16S RNA sequencing revealed that it was Pectobacterium carotovorum, named Pectobacterium carotovorum D14, and deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a storage address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a storage number of CGMCC No. 31377 and a storage date of July 19, 2024.

[0049] Carrot soft rot pectin bacteria D14 was inoculated with 1% (v / v) of the inoculum into the fermentation medium (extracellular polysaccharide 0.5%, yeast extract 0.5%, peptone 0.5%, sodium chloride 0.1%, dipotassium hydrogen phosphate 0.3%, potassium dihydrogen phosphate 0.3%), and fermented at 37°C for 48h. After the fermentation, the supernatant was collected at 8,000rpm for 20min at 4°C to obtain the crude enzyme solution. The sample was prepared with ultrapure water with a mass concentration of 2% of the exopolysaccharide solution, and the sample and the crude enzyme solution were prepared at a solid-liquid ratio of 5:1. The pH and culture temperature of the solution were adjusted according to Table 2. The reaction time was 2h, and the effect of the crude enzyme solution on the enzyme activity at different pH and different temperatures was determined. The enzyme activity was determined by using different concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) of fucose as standard solution, taking 0.5 mL of standard solution of different concentrations and samples treated under different conditions, adding 0.5 mL of DNS, and using distilled water plus DNS as blank. After fully mixing, the mixture was heated in a boiling water bath for 5 min, and then placed in cold water to cool to room temperature, and then diluted to 10 mL with distilled water. Finally, the OD was measured on a UV spectrophotometer. 520 The enzyme activity unit is defined as the amount of enzyme that produces 1 μg of fucose per minute. The results are shown in Table 2. When the pH is 7.0 and the culture temperature is 30°C, the highest enzyme activity is 680U.

[0050] Table 2 shows the enzyme activity of crude enzyme solution of Pectinobacterium carotovora D14 at different pH and temperature

[0051]

[0052]

[0053] Example 3: Optimization of preparation conditions of degradation products

[0054] According to the crude enzyme solution preparation method of Example 2, the crude enzyme solution was prepared, and the extracellular polysaccharide solutions with mass concentrations of 0.5%, 1%, 2%, and 3% were respectively prepared with ultrapure water. The crude enzyme solution was used to degrade the extracellular polysaccharide solutions of different mass concentrations. The degradation conditions were: the volume ratio of the crude enzyme solution to the extracellular polysaccharide solution was 1:3, 1:4, and 1:5, respectively, the degradation temperature was 30°C, the pH was 7, and the reaction time was 12h, 24h, 48h, 72h, and 96h, respectively. The collected degradation solution was boiled in a 100°C boiling water bath for 5min to terminate the enzymolysis reaction. The molecular weight of the degradation products under different degradation conditions was determined by liquid phase, and the yield of degradation products below 1000Da was calculated. The results are shown in Table 3. The degradation conditions with the highest yield of degradation products with a molecular weight less than 1000Da are an extracellular polysaccharide concentration of 1%, a volume ratio of the crude enzyme solution to the extracellular polysaccharide solution of 1:4, and a reaction time of 48h. The liquid phase conditions are as follows:

[0055] Liquid phase instrument: Agilent 1260 high performance liquid chromatograph; differential detector

[0056] HPLC column model: TSK-gel G4000PWXL

[0057] Liquid phase conditions: 0.2 mol / L NaNO 3 Solution, 0.01 mol / L NaH 2 PO 4 The solution was the mobile phase; the column temperature was 30°C; the flow rate was 0.5 mL / min; the injection volume was 10 μL; and the differential temperature was 35°C.

[0058] Table 3 shows the enzyme activity of crude enzyme solution of Pectinobacterium carotovora D14 at different pH and temperature

[0059]

[0060]

[0061] Example 4: Purification of degradation products

[0062] According to the optimized degradation product preparation conditions of Example 2 and Example 3, the extracellular polysaccharide was degraded to obtain a degradation solution. The degradation solution was centrifuged at 8,000 rpm for 20 minutes, the supernatant was concentrated to 1 / 5 of the original volume, 3 volumes of 95% ethanol were added, and the mixture was allowed to stand at 4°C for 12 hours, centrifuged at 8,000 rpm for 10 minutes, the supernatant was concentrated to 1 / 10 of the original volume, 6 volumes of 95% ethanol were added, the mixture was allowed to stand at 4°C for 12 hours, centrifuged at 8,000 rpm for 10 minutes, the supernatant was concentrated to remove excess ethanol, and the degradation product was obtained after vacuum freeze drying and stored at 4°C for future use.

[0063] The degradation products of the above graded alcohol precipitation were separated and purified using Bio-rad gel P4 filler (ID: 90cm×1.0cm), 50mg / mL degradation product solution (sugar solution) was prepared with ultrapure water, the sample volume was 0.5mL 50mg / mL sugar solution, the mobile phase was 0.2mol / L ammonium bicarbonate, the flow rate was 0.1mL / min, one tube was collected every 5min, the total sugar content of each tube was determined by phenol-sulfuric acid curve and the elution curve was obtained (elution time was the horizontal axis, OD490 was the vertical axis), as shown in Figure 1 As shown, there is only one sugar component in the degradation product.

[0064] Example 5: Structural analysis of degradation products

[0065] (1) Monosaccharide composition analysis

[0066] The monosaccharide composition of the product purified by the Bio-rad gel P4 filler was detected according to the method of high performance liquid chromatography analysis of monosaccharide composition in Example 1. The results are as follows: Figure 2 As shown, the purified product contains only glucose, galactose and fucose, and the molar ratio of glucose, galactose and fucose is close to 1:1:2.

[0067] (2) Analysis of pyruvic acid content

[0068] The pyruvic acid content of the product purified by the Bio-rad gel P4 filler was detected according to the method of HPLC analysis of pyruvic acid content in Example 1. The results are as follows: Figure 3 As shown, the standard curve (Y = 9908X-209.89, R 2 =0.9988), the sample peak time was 10.117min, and the pyruvic acid content was calculated by substituting the peak area into the standard curve formula to be 12.37%.

[0069] (3) Mass spectrometry

[0070] The molecular weight of the purified product from the Bio-rad gel P4 filler was accurately determined using an Agilent 6460 triple quadrupole mass spectrometer by electrospray ionization (ESI-MS) in negative ion mode combined with collision-induced dissociation tandem mass spectrometry (ESI-CID-MS / MS). A mixed solvent of acetonitrile and water (volume ratio 1:1) was used as the mobile phase, the flow rate was set at 0.4 mL / min, the nebulizer pressure was set at 40 psi, and the capillary voltage was adjusted to 3500 V. Argon was used as the collision gas, and the collision energy was between 10-55 eV. ESI-MS analysis results Figure 4 As shown in Figure 2, the molecular weight of the purified product is 703.64, which is speculated to be a tetrasaccharide containing pyruvate (theoretical molecular weight is 704.66) with one H removed. + The ESI-CID-MS / MS analysis results are as follows: Figure 5 As shown in the figure, most of m / z 703.64 becomes m / z 249.07, which is speculated to be glucose or galactose with a molecular weight of 180.16 plus a pyruvic acid (180.16+88.06-18.01=250.21) and then a H + , which indicates that pyruvate is linked to glucose or galactose, not fucose.

[0071] (4) NMR analysis of fucoidan oligosaccharides

[0072] Take 20 mg of the single-component sample purified from the above Bio-rad gel P4 filler, first dissolve it in 0.5 mL of heavy water and then freeze-dry it. The freeze-dried sample is dissolved in 0.5 mL of heavy water again and calibrated with reference to the chemical shift of acetone. The obtained NMR spectra are collected on an Agilent DD2-500 instrument, with the operating frequency set to 500 MHz and performed at room temperature (25°C). Figure 6 to Figure 9 As shown, it is determined that pyruvic acid is connected to galactose, and galactose is located on the side chain. The main chain structure is fucose → fucose → glucose. The specific structure is as follows:

[0073]

[0074] Example 6: Determination of cytotoxicity of pyruvate-containing fucose to Ls174T cells

[0075] Cell culture and passaging

[0076] The complete medium for Ls174T cells includes the following components by volume percentage: 89% RMPI 1640 medium, 10% special grade fetal bovine serum and 1% double antibody (penicillin and streptomycin). Ls174T cells were inoculated into a T25 culture flask, 5 mL of complete medium was added, and the culture medium was incubated at 37°C with a volume fraction of 5% CO 2Culture in an incubator for 48 hours, change the culture medium every 24 hours, and subculture when the cell proliferation density is about 80%. Digest the cells with 1 mL of 0.25% trypsin containing EDTA for 2 minutes, digest and separate the cells from the wall of the T25 culture flask, add 2 mL of complete culture medium, centrifuge at 1000 rpm for 3 minutes, re-dissolve the cell pellet with complete culture medium, and subculture at a subculture ratio of 1:2.

[0077] Preparation of pyruvate-containing fucoidate solution

[0078] The product purified by the Bio-rad gel P4 filler was prepared into 0.5, 1.0, 2.0, 5.0, 10.0 mg / mL pyruvate-fucotetraose solutions using RMPI 1640 medium, and filtered using a 0.22 μm filter for later use.

[0079] Toxicity test

[0080] According to the above-mentioned "Cell Culture and Passaging" method, the cells with a proliferation density of more than 80% in the T25 culture flask were digested with trypsin and added to complete medium. After centrifugation, they were resuspended in complete medium. 10 μL of the cell suspension was added to a hemocytometer for counting to obtain the cell concentration. The cell concentration was adjusted to 2.5×10 5 / mL, add 100μL of cell suspension to 96-well plates and culture for 24h. When it fills the wells, aspirate the culture medium and gently wash the floating cells with 200μL PBS. Add 100μL of pyruvate-fucotetraose solution with different concentrations (0.5, 1.0, 2.0, 5.0, 10.0mg / mL) and RMPI 1640 culture medium without sugar as the control group, culture for 24h, and wash twice with 200μL PBS. Prepare RMPI 1640 culture medium / CCK-8 solution as experimental reagents at a volume ratio of 9:1, add 100μL of experimental reagents to each well, incubate at 37℃ for 2h, and measure the absorbance at 450nm. The formula for calculating cell survival rate is:

[0081] Cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100

[0082] Where: OD 样品 OD is the absorbance of the substance to be tested in the reaction system; 对照 OD is the absorbance of the control group without sugar in the reaction system; 空白 It is the absorbance of the reaction system without inoculated cells.

[0083] like Fig.10As shown, 0.5 mg / mL and 1 mg / mL pyruvate-fucotetraose solutions had no toxic effect on cells. In the environment of 2 mg / mL pyruvate-fucotetraose solution, the cell survival rate was about 80%, which was higher than 2 mg / mL pyruvate-fucotetraose solution, where the cell survival rate was less than 50%. Therefore, 1 mg / mL pyruvate-fucotetraose solution was selected for subsequent experiments.

[0084] Example 7: Effect of pyruvate-containing fucose on mucin production by Ls174T cells

[0085] According to the method of "Cell Culture and Subculturing" in Example 6, the cells with a proliferation density of more than 80% in the T25 culture flask were digested with trypsin and added with complete medium. After centrifugation, they were resuspended with complete medium. The cell concentration was adjusted to 2.5×10 5 / mL, 1mL of cell suspension was added to 12-well plates and cultured for 24h. RMPI 1640 medium was used to prepare 1mg / mL pyruvate-fucotetraose solution, and the medium without sugar was used as the control group, and cultured in the incubator for another 24h. After the culture, the supernatant and cells were collected. The supernatant can be used to directly measure Mucin2. After the cells are collected, the cell wall needs to be broken with lysis solution. The specific steps are: the experiment is carried out on ice throughout the process, the cell surface is washed twice with pre-cooled PBS solution, and then 100μL of lysis solution is added to each well, lysed for 5min, and then the cells are scraped with a cell scraper and collected in a 1.5mL EP tube. Centrifuge at 10000rpm for 5min at 4℃ to collect the cell supernatant.

[0086] Kit: Shanghai ELISA Human Mucin 2 (Mucin2) ELISA kit.

[0087] The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay. Samples, standards, and HRP-labeled detection antibodies are added to the coated microwells pre-coated with human mucin 2 capture antibodies at once. After incubation and thorough washing, the substrate 3,3',5,5'-tetramethylbenzidine (TMB) is used for color development. TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The depth of the color is positively correlated with the concentration of Mucin 2.

[0088] Standard concentrations: 2, 4, 8, 16, 32, 64 μg / mL Mucin2.

[0089] Steps: Before use, the kit needs to be taken out of the refrigerator 20 minutes in advance to equilibrate to room temperature, and the microwell ELISA plate needs to be equilibrated at room temperature for 60 minutes in advance; 50 μL of sample and standards of different concentrations are added to the microwell plate, and 100 μL of detection antibody labeled with horseradish peroxidase is added to the blank well, and the reaction wells are sealed with a sealing film, and reacted at 37°C for 60 minutes; the liquid is discarded, patted dry on absorbent paper, 350 μL of washing solution is added to each well, and the well is allowed to stand for 1 minute, the washing solution is removed, and the well is patted dry on absorbent paper, and this is repeated 5 times; 50 μL of substrate A and B are added to each well, and the well is incubated at 37°C in the dark for 15 minutes; 50 μL of stop solution is added to each well, and the OD value of each well is measured at a wavelength of 450 nm within 15 minutes.

[0090] The results are as follows Fig.11 As shown, whether it is intracellular or extracellular, the addition of pyruvate-containing fucotetraose can significantly produce more Mucin2, among which the intracellular Mucin2 production is 1.5 times that of the control group, and the extracellular Mucin2 production is 1.9 times that of the control group. The increase in Mucin2 production is beneficial to enhancing the intestinal barrier function, improving the composition of intestinal microorganisms, promoting the adhesion of probiotics, regulating immune response, etc. Therefore, pyruvate-containing fucotetraose has the effect of enhancing the intestinal barrier.

Claims

1. A pyruvate-containing fucoidan that enhances intestinal barrier function, characterized in that: The main chain of the pyruvate-containing fucotetraose is α-L-Fucp-(1→3)-α-L-Fucp-(1→3)-β-D-Glcp, and the side chain is β-D-Galp substituted by pyruvate at positions 4 and 6, which replaces the second α-L-Fucp in the main chain through a 1,4 connection. The molecular formula is C 27 H 44 O 21 .

2. A method for preparing pyruvate-containing fucotetraose having the function of enhancing intestinal barrier as claimed in claim 1, characterized in that: The following steps are involved: (1) fermenting Clavibacter michiganensis CN14 to prepare exopolysaccharide; (2) fermenting Pectobacterium carotovorum D14 to prepare a crude enzyme solution; (3) The crude enzyme solution obtained in step (2) is used to degrade the extracellular polysaccharide obtained in step (1), and the oligosaccharide components with a molecular weight less than 1000 Da are collected by high temperature enzyme inactivation and graded alcohol precipitation, and the oligosaccharide components are freeze-dried to obtain pyruvate-containing fucose.

3. The preparation method according to claim 2, characterized in that: The Michigan bacillus CN14 described in step (1) has a deposit number of CGMCC No. 31378 and was deposited in the General Microbiology Center of the China Culture Collection Administration on July 19, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

4. The preparation method according to claim 2, characterized in that: The carrot soft rot pectin bacteria D14 described in step (2) has a preservation number of CGMCC No. 31377 and was deposited in the General Microbiological Center of the China Culture Collection Administration on July 19, 2024. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

5. The preparation method according to claim 2, characterized in that: The fermentation method for preparing extracellular polysaccharides in step (1) is as follows: inoculating Michigan Bacillus CN14 into a liquid fermentation medium for oscillating fermentation, the inoculation amount of the bacteria is 0.5% to 2% (v / v), the fermentation temperature is 25 to 30°C, the oscillation speed is 150 to 200 rpm, the fermentation time is 48 to 120 hours, and after the fermentation is completed, centrifugation is performed to collect the supernatant, and the supernatant is concentrated and dialyzed to remove small molecular impurities, and freeze-dried to obtain extracellular polysaccharides; the liquid fermentation medium formula is: 30 g / L glucose, 4 g / L yeast extract, 5 g / L calcium carbonate, 3 g / L potassium dihydrogen phosphate, 3 g / L potassium hydrogen phosphate, 1 g / L sodium chloride, and the pH is 7.0±0.

2.

6. The preparation method according to claim 5, characterized in that: The dialysis used a dialysis bag with a molecular weight cut-off of 10,000 Da.

7. The preparation method according to claim 2, characterized in that: The molar proportion of fucose in the extracellular polysaccharide is 46.27% to 52.13%, and the molar proportion of pyruvic acid is 10.2% to 20.12% (w / w).

8. The preparation method according to claim 2, characterized in that: The method for preparing crude enzyme liquid by fermentation in step (2) is as follows: inoculating carrot soft rot pectin bacillus D14 into a fermentation medium for fermentation, wherein the inoculation amount of the bacteria is 0.5% to 2% (v / v), the fermentation temperature is 30 to 40° C., the fermentation time is 48 to 120 hours, and after the fermentation is completed, the supernatant is collected by centrifugation to obtain the crude enzyme liquid; the fermentation medium comprises the following components in weight percentage: 0.5% of the extracellular polysaccharide prepared in step (1), 0.5% of yeast extract, 0.5% of peptone, 0.1% of sodium chloride, 0.3% of dipotassium hydrogen phosphate, and 0.3% of potassium dihydrogen phosphate.

9. The preparation method according to claim 2, characterized in that: Step (3) specifically comprises: preparing the extracellular polysaccharide prepared in step (1) into an extracellular polysaccharide solution with a mass concentration of 0.5% to 3% with ultrapure water, and then degrading the extracellular polysaccharide solution with the crude enzyme solution obtained in step (2), wherein the volume ratio of the crude enzyme solution to the extracellular polysaccharide solution is 1:(3 to 5), the degradation temperature is 20 to 45° C., the pH is 4 to 8, the degradation time is 12 to 96 hours, and oligosaccharide components with a molecular weight less than 1000 Da are collected by high-temperature enzyme inactivation and graded alcohol precipitation, and pyruvate-containing fucose is obtained after freeze-drying.

10. Use of the pyruvate-containing fucotetraose according to claim 1 in enhancing the intestinal barrier.

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