WO2021196572A1 - 富含岩藻糖的胞外多糖及其制备方法和应用 - Google Patents

富含岩藻糖的胞外多糖及其制备方法和应用 Download PDF

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WO2021196572A1
WO2021196572A1 PCT/CN2020/123082 CN2020123082W WO2021196572A1 WO 2021196572 A1 WO2021196572 A1 WO 2021196572A1 CN 2020123082 W CN2020123082 W CN 2020123082W WO 2021196572 A1 WO2021196572 A1 WO 2021196572A1
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fucose
fuco
eps
rich
extracellular polysaccharide
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PCT/CN2020/123082
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English (en)
French (fr)
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邹祥
李姗姗
王振宇
谢爱卿
夏海钦
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西南大学
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Priority claimed from CN202010234516.8A external-priority patent/CN111234047B/zh
Priority claimed from CN202010680173.8A external-priority patent/CN111728976B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

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  • the present invention relates to extracellular polysaccharides, in particular to fucose-rich extracellular polysaccharides, and also relates to a preparation method and application of extracellular polysaccharides.
  • EPS extracellular polysaccharides
  • EPS secreted by bacteria, yeasts, fungi or molds vary greatly in composition.
  • EPS contains one or more monosaccharide residues, which are linked by linear or branched chains.
  • xanthan gum has been used as a thickener, stabilizer, and emulsifier in the food industry, personal care products, and pharmaceutical industries.
  • Gellan gum is widely used as a food ingredient. Its special gelling properties make gellan gum a multifunctional additive for controlled drug release formulations and tissue engineering applications. In addition to being used as product ingredients, many EPS also exhibit significant immune stimulation, immune regulation, anti-tumor, anti-viral, anti-inflammatory and antioxidant activities. Despite the wide variety of EPS currently available, research interest in exploring new and versatile biopolymers is still growing.
  • fucose-containing extracellular polysaccharides have received special attention due to the unique functions of fucose.
  • Fucose is a rare L-configuration 6-deoxyhexose, usually found in microbial extracellular polysaccharides, brown algae and mammals. Fucose modification has been determined to be related to many biological functions, including immune regulation and cancer.
  • Sulfate-containing fucoidans which are rich in brown algae, have anticoagulant, antithrombotic, immunomodulatory, anticancer and antiproliferative activities.
  • the production of fucoidan in plants or algae is low, and its composition varies with climate and seasons.
  • the fucose-rich exopolysaccharides produced by them are considered to be a better alternative.
  • the fucose-containing exopolysaccharide with the highest yield in other reports was produced by Enterobacter A47, with a yield of 13.23g/L.
  • one of the objects of the present invention is to provide a fucose-rich extracellular polysaccharide;
  • the second object of the present invention is to provide a method for preparing the fucose-rich extracellular polysaccharide;
  • the third purpose is to provide the application of fucose-rich extracellular polysaccharides in the preparation of degradable antibacterial materials;
  • the fourth purpose of the present invention is to provide nano silver antibacterial films containing the fucose-rich extracellular polysaccharides;
  • the fifth object of the present invention is to provide a method for preparing the nano silver antibacterial film;
  • the sixth object of the present invention is to provide the application of the nano silver antibacterial film in the preparation of antibacterial materials;
  • the seventh object of the present invention is to provide a method based on the rich Fuco-oligosaccharides hydrolyzed by fucose-containing extracellular polysaccharides;
  • the eighth object of the present invention is to provide the application of the fuco-oligosacc
  • the present invention provides the following technical solutions:
  • the extracellular polysaccharide is produced by fermentation of Kosakonia sp. CCTCC M2018092.
  • the structural formula of the extracellular polysaccharide is as follows:
  • the method for preparing the fucose-rich extracellular polysaccharide is produced by fermentation of Kosakonia sp.CCTCC M2018092 strain.
  • the fermentation broth of Kosakonia sp.CCTCC M2018092 strain is adjusted to pH 1-5 with sulfuric acid, hydrolyzed at 50-80°C for 0-10 hours, filtered to remove the bacteria and calcium sulfate, and the cut-off flow is 10kDa
  • the ultrafiltration membrane filters to remove small molecules, and after the protein is removed, it is dialyzed in deionized water with a molecular weight cut-off of 8000-14000Mw, and lyophilized to obtain fucose-rich exopolysaccharides;
  • Nano silver antibacterial film containing the fucose-rich extracellular polysaccharide 4.
  • the method for preparing the nano-silver antibacterial film is to mix the fucose-rich extracellular polysaccharide with AgNO 3 solution, synthesize nano-silver particles under ultraviolet excitation, and then add the extracellular polysaccharide solution to form a film to prepare nano-silver Antibacterial film.
  • the concentration of the fucose-rich extracellular polysaccharide is 0.01 ⁇ 0.50 mg/mL, the concentration of AgNO 3 is 2 mM; and the ultraviolet excitation is irradiated under a 354 nm ultraviolet lamp for 12 minutes.
  • the fuco-oligosaccharide is prepared by hydrolyzing the fucose-containing extracellular polysaccharide produced by Kosakonia sp.CCTCC M2018092 fermentation with trifluoroacetic acid.
  • the hydrolysis of trifluoroacetic acid involves adding extracellular polysaccharides with a mass fraction of 5% to trifluoroacetic acid so that the final concentration of trifluoroacetic acid is 0.1M, hydrolyzing at 100°C for at least 1 hour, and then removing trifluoride with a 200Da nanofiltration membrane. Acetic acid to obtain fuco-oligosaccharides.
  • the fuco-oligosaccharide is used in the preparation of prebiotics that promote carbohydrate metabolism, sugar biosynthesis and metabolism, reduce membrane transport function, enhance digestive system function, or reduce disease infection.
  • the fuco-oligosaccharide is used in promoting the production of short-chain fatty acids in the intestinal tract.
  • the short-chain fatty acid is acetic acid or propionic acid.
  • the fuco-oligosaccharide is used for improving the level of intestinal microflora.
  • the improvement of the level of intestinal microflora is to increase the level of Bacteroides and reduce the levels of Firmicutes and Proteobacteria.
  • the improvement of the level of intestinal microflora is to increase the levels of Parabacteroides, Bacteroides colonis and Prevotella, and reduce the levels of Lactobacillus and Bacteroides.
  • the present invention discloses a new type of fucose-rich EPS, produced by Kosakonia sp.CCCCC M2018092, its Mw is 3.65 ⁇ 105 Da, and the Mw of the isolated AH-EPS is 3.47 ⁇ 104 Da
  • AH-EPS is mainly composed of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvate, the molar ratio is about 2.03:1.00:1.18:0.64:0.67; through chemical analysis and NMR The analysis clarified the main link structure between sugar residues, and determined that the three-dimensional structure of AH-EPS is a triple-helical chain conformation.
  • the fucose-rich EPS of the present invention is hydrolyzed to obtain fuco-oligosaccharides.
  • Fuco-oligosaccharides have probiotic functions.
  • fuco-oligosaccharides can promote carbohydrate metabolism, sugar biosynthesis and metabolism, and reduce membrane transport functions.
  • the function of enhancing the function of the digestive system or reducing disease infection can promote the production of short-chain fatty acids in the intestine and improve the level of intestinal microflora. Therefore, it can prepare intestinal prebiotics, which can be applied to human health and livestock and poultry breeding.
  • Figure 1 shows the GPC diagram of the original EPS and AH-EPS.
  • Figure 2 is the analysis of the complete acid hydrolysate of AH-EPS (A: HPLC of the complete acid hydrolysate of AH-EPS; B: GC-MS total ion current chromatogram of the complete acid hydrolysate of AH-EPS).
  • Figure 3 shows the 1 H NMR spectrum and 13 C NMR spectrum of AH-EPS in D 2 O (A: 1 H NMR spectrum; B: 13 C NMR spectrum).
  • Figure 4 shows the two-dimensional nuclear magnetic spectrum analysis of AH-EPS (A: 2D 1 H/ 13 C HSQC spectrum; B: 2D 1 H/ 13 C HMBC spectrum).
  • Figure 5 shows the two-dimensional nuclear magnetic spectrum analysis of AH-EPS (A: 2D 1 H/ 1 H COYY spectrum; B: 2D 1 H/ 1 H NOESY spectrum).
  • Figure 7 shows the changes in the maximum absorption peaks of Congo Red, Congo Red + AH-EPS and Congo Red + Dextran complex in various concentrations of sodium hydroxide solution.
  • Figure 8 shows the main structure of AH-EPS.
  • Figure 9 shows the preparation and antibacterial test of EPS/nanosilver film
  • A UV-visible spectra of silver nanoparticle solutions synthesized by AH-EPS with different concentrations
  • BC TEM images of AH-EPS@AgNP at different magnifications
  • D size distribution of AH-EPS@Ag NP obtained from TEM analysis
  • E pictures of EPS film and EPS/nanosilver film
  • F growth inhibition zone for Staphylococcus aureus produced by EPS/nanosilver film , Which contains 0.89% (a), 0.44% (b), 0.22% (c) and 0% (d) AH-EPS@Ag NPs).
  • Figure 11 is a GC-MS total ion chromatogram of the complete acid hydrolysate of oligosaccharides.
  • Figure 12 shows the effect of fuco-oligosaccharides on the pH of the fermentation broth.
  • Figure 13 shows the effect of fuco-oligosaccharides on short-chain fatty acids.
  • Figure 15 shows the level of intestinal flora in test-tube fermentation (SGCON is the control group and SGFOP is the fuco-oligosaccharide-added group).
  • Figure 17 shows the results of mucosal spherule simulation (A: lumen; B: mucosa; S, H, and J resolution represent ascending colon, transverse colon, and descending colon, the number behind represents fermentation days, and w represents stop adding samples).
  • Figure 18 shows the results of the simulated intestinal tract (A: intestinal phylum level; B: intestinal genus level; C: mucosal phylum level; D: intestinal mucosal genus level; S is ascending colon, H is transverse colon, and J is descending colon; 0 , 3, w respectively represent: control group, add fuco-oligosaccharide on the 3rd day, stop adding fuco-oligosaccharide).
  • Figure 20 shows the safety evaluation results of fuco-oligosaccharides (A: the effect of fuco-oligosaccharides on weight; B: the effect of fuco-oligosaccharides on brain and heart; C: the effect of fuco-oligosaccharides on colon length; D: The effect of fuco-oligosaccharide on liver weight; E: the effect of fuco-oligosaccharide on liver function and kidney function).
  • A the effect of fuco-oligosaccharides on weight
  • B the effect of fuco-oligosaccharides on brain and heart
  • C the effect of fuco-oligosaccharides on colon length
  • D The effect of fuco-oligosaccharide on liver weight
  • E the effect of fuco-oligosaccharide on liver function and kidney function).
  • EPS is fermented and produced by Kosakonia sp.CCTCC M2018092 strain under fed-batch conditions.
  • the specific steps are as follows: Take Kosakonia sp.CCTCC M2018092 strain in a 250mL shake flask containing 30mL medium and cultivate it at 30°C and 200rpm for 20 hours Then, 30ml of bacterial culture solution was transferred to a 15L fermenter for pre-growth culture at 30°C and 300rpm for 13 hours (aeration volume 1.5m3/h). After that, 3L of the pre-grown bacterial liquid was transferred to a 50L fermentor (containing 30L of medium) for fed-batch fermentation.
  • a peristaltic pump was used to add 200 g/L glucose solution in batches at a rate of 0.9-3.8 rpm 13 hours after the start of fermentation.
  • the aeration volume of the fermenter is 1.5m3/h, and the dissolved oxygen concentration is controlled above 10% by automatically adjusting the speed (300-550rpm) through the speed linkage.
  • the pH of the 50L fermentor was controlled at 7.0 by adding sodium hydroxide, and the temperature was controlled at 30°C.
  • Table 1 The composition of each medium during the cultivation process is shown in Table 1.
  • Method 1 After the fermentation is completed, the first route is to adjust the pH of the fermentation broth to 2.0 with sulfuric acid, and then centrifuge at 12000 rpm for 20 minutes to remove the bacteria and calcium sulfate; after the supernatant is removed by the Sevage method, the deionized water is dialyzed (retained The molecular weight is 8000-14000Mw) and then freeze-dried to obtain the original fermented polysaccharide (EPS) with a yield of 13.5g/L.
  • EPS polysaccharide
  • the weight average molecular weight (Mw) of EPS and AH-EPS is measured by gel permeation chromatography (GPC).
  • GPC gel permeation chromatography
  • 0.1M NaNO 3 and 500 ppm NaN 3 were used as eluents to separate samples with appropriate concentrations, and the results are shown in Figure 1.
  • the results showed that EPS produced from Kosakonia sp.
  • the monomer composition of AH-EPS was determined after complete acid hydrolysis.
  • the purified AH-EPS (5mg/ml) was dissolved in 5ml purified water, added with 0.1ml of trifluoroacetic acid and hydrolyzed at 120°C for 2h. After removing the trifluoroacetic acid, the sample was dissolved in 10mM sulfuric acid and analyzed by HPLC.
  • the monomer composition is determined by comparing the retention time with the retention time of each monomer standard. The result is shown in Figure 2 A. The results show that AH-EPS is composed of fucose, glucose, glucuronic acid and galactose.
  • thiol-acetate derivatization and GC-MS analysis further confirmed the above experimental results. That is, accurately weigh 14.6mg fucoidan and dissolve it in 0.5ml xylose solution (8g/L), then add 3ml TFA (2mol/L), press the cap and heat it in an oil bath at 120°C for 2 hours at 55°C. Blow dry under nitrogen. Add 2ml of ethanethiol and 1ml of trifluoroacetic acid, and magnetically stir in a water bath at 25°C for 25min.
  • GC-MS conditions are: Shimadzu (GCMS-QP2010, Japan), Rtx-5 capillary column (0.25mm ⁇ 30m), vaporization chamber temperature is 280°C, high purity helium is used as carrier gas, flow rate is 1ml/min; The sample volume is 0.3ul.
  • the result is shown in Figure 2 B.
  • the results show that AH-EPS is composed of fucose, glucose, glucuronic acid and galactose.
  • AH-EPS The content of glucuronic acid is 14.62%.
  • the absolute configuration of monosaccharides in AH-EPS is analyzed by GC-MS. After the determination of the modified (-)-2-butyl glycoside, AH-EPS was also found to be composed of L-fucose, D-glucose, D-galactose and D-glucuronic acid.
  • the retention time and ion fragments in the molecule were determined by GC-MS to determine the acetylation of uronic acid.
  • the results are shown in Table 1.
  • the results show that the fucose, glucose, galactose and glucuronic acid in AH-EPS
  • the molar ratio is 2.03:1.00:1.18:0.64.
  • AH-EPS Quantitative analysis of pyruvic acid in AH-EPS was performed on the Shimadzu (LC-16) HPLC system using an ultraviolet detector (SPD-16) at a wavelength of 215 nm.
  • SPD-16 ultraviolet detector
  • AH-EPS methylation-reduced AH-EPS.
  • AH-EPS 5mg/ml AH-EPS solution in 1mM oxalic acid, 0.1M sodium chloride, pH 3.0
  • the solution was then neutralized with NaOH, dialyzed against deionized water and freeze-dried.
  • uronic acid should be reduced before methylation, and AH-EPS without pyruvate is reduced by EDC to prepare uronic acid-reduced AH-EPS.
  • AH-EPS (5mg) is added to In 2mL 75% THF-0.1mol/L MES solution, adjust the pH to 4.75 with 10% Et3N, add EDC (20mg) and stir at ambient temperature for 1 hour. The reaction was then terminated with 2M acetic acid solution. The reaction solution was dialyzed for 24 using a dialysis bag with a cut-off volume of 3.5 kDa, and then freeze-dried. The lyophilized sample was re-dissolved in 1.0ml water, added 0.5ml 10% acetic acid-methanol solution, dried with nitrogen to remove the boric acid generated during the reduction process, continued to add 1.0ml 10% acetic acid-methanol solution, dried with nitrogen, repeat 3-4 Second-rate. Finally, add 0.5ml of methanol, dry with nitrogen, and repeat 3 times to ensure that the boric acid is removed. After the uronic acid reduction sample is obtained, it is dried at 60°C for 5 hours for methylation analysis.
  • AH-EPS is mainly composed of 1,4-linked fucose, 1,3,4-linked fucose, 1,3-linked glucose, 1,3-linked galactose and terminal galactose
  • the composition, the molar ratio is 1:1.02:1.63:0.33:0.68, and the AH-EPS chain is composed of the only branch point at the fucose residue C3.
  • pyruvate was deduced to be linked to terminal galactose.
  • AH-EPS (56mg) was dissolved in 50ml sodium periodate solution (0.015M) and stored in a refrigerator at 4°C. Take 0.2ml solution to 50ml with purified water at an interval of 12h, and dilute the absorbance of the solution at 233nm. After the absorbance was stable for 126 hours, 4 ml of ethylene glycol was added to terminate the reaction, and a small amount of the aqueous solution was analyzed for formic acid by HPLC. The remaining reactants were dialyzed with purified water (with a cut-off volume of 8000 MW) and then lyophilized. Add 3ml of sodium borohydride solution (26g/L) to the lyophilized product and reduce at room temperature for 22h. The reduced product was added to 2ml TFA (3M) and hydrolyzed in an oil bath at 120°C for 2 hours, dried with nitrogen and re-dissolved in the mobile phase for HPLC analysis.
  • TFA sodium borohydride
  • High performance liquid chromatography (HPLC) analysis conditions Xtimate (Welch) Sugar-H column (7.8mm ⁇ 300mm, 5 ⁇ m) column; column temperature: 40°C; flow rate: 0.5ml/min; mobile phase: 10Mm sulfuric acid; detector : Differential detector (RI-201H). Injection volume: 15 ⁇ L.
  • B) shows two CH 3 signals of fucose ( ⁇ 15.32 and 15.49 ppm), one CH 3 signal of the acetone substituent ( ⁇ 25.04 ppm) and the acetone substituent and glucose
  • the two C O group signals of aldehyde acid substituents ( ⁇ 175.99 and 176.36ppm), in The free hydroxyl C-6 signals of glucose and galactose were observed in the meantime.
  • Residue G represents the acetone substituent.
  • the methyl proton signal of pyruvic acid (1.45 ppm) is coupled with the 13 C signal at 102.04 ppm (O-C-O group of pyruvate).
  • the results show that pyruvate participates in the formation of six-membered cyclic ketals including O-4 and O-6 positions.
  • the C-5 of fucose was further coupled with the anomeric protons of residue C ( ⁇ 5.35ppm) and residue E ( ⁇ 4.99ppm), indicating that residues C and E are fucose residues ( Figure 5, A ).
  • the C-4 of fucose in residue C is coupled with the anomeric proton of residue E, which indicates that residue C is 1,4- ⁇ -L-Fucp, that is, there is ⁇ 4)- ⁇ -L-Fucp- (1 ⁇ 4)- ⁇ -L-Fucp-(1 ⁇ link.
  • residue E is coupled with the anomeric proton of residue F, that is, there is ⁇ 3)- ⁇ -D-Glcp- (1 ⁇ 4)- ⁇ -L-Fucp-(1 ⁇ 4)- ⁇ -L-Fucp-(1 ⁇ link.
  • NOESY spectrum shows H-3 at residue F and residue C There is a residue cross peak between the H-1, which determines the Fucp-(1 ⁇ 3)-Glcp link and ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ 4)- ⁇ -L-Fucp-(1 ⁇ 4)- ⁇ -L-Fucp-(1 ⁇ )- ⁇ -L-Fucp-(1 ⁇ is a repeating backbone unit.
  • FT-IR FT-IR in the scanning range of 4000cm -1 to 400cm -1, DSC in the ambient air atmosphere at 30 deg.] C to 400 deg.] C at a heating rate 10 ° K / min of.
  • the peaks at 2928 and 2858 cm -1 are characteristic peaks of sugar, which are specifically attributed to the stretching vibration of CH.
  • the peak at 1097 cm -1 reflects the tensile vibrations of C–O–H and C–O–C, while the peak at 885 cm -1 indicates the presence of ⁇ -glycosidic bonds.
  • the DSC curve of AH-EPS ( Figure 6, B) shows a broad endothermic peak at 94.5°C, which is the result of the dehydration process. The temperature was further increased, and an exotropic zone with a maximum of 356.5°C was observed. This may be due to changes in the three-dimensional structure and oxidation of AH-EPS.
  • Polysaccharide chains exhibit different three-dimensional structures, such as triple helical chains, single random coiled chains and random coiled chains.
  • Congo red can form special complexes with triple helix polysaccharides in alkaline solution. As the concentration of NaOH increases, the maximum absorption wavelength ( ⁇ max ) will be red-shifted. As shown in Figure 7, when the NaOH concentration is close to 0.2M, the ⁇ max of Congo Red increases to the maximum. In the presence or absence of dextran, the ⁇ max of Congo Red gradually decreases with the increase of NaOH concentration until it reaches a constant value. This result indicates that AH-EPS has a triple helical chain conformation.
  • AH-EPS solution Mix the AH-EPS solution and 2mM AgNO 3 solution of different concentrations (0.01mg/mL, 0.03mg/mL, 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.50mg/mL) into 5mL water and shake for 30 minutes .
  • the absorption spectra of silver nanoparticle solutions prepared with different concentrations of AH-EPS are shown in A in FIG. 9. The results showed that when the concentration of AH-EPS was 0.05 mg/mL, the UV-vis spectrum of the solution showed the strongest absorption peak of silver nanoparticles at 423 nm. Therefore, 0.05 mg/mL AH-EPS was used to prepare silver nanoparticles for further characterization and application. The reason for this phenomenon is that AH-EPS is rich in -OH groups, which can reduce Ag + to Ag 0 . The ratio of reducing agent and stabilizer in the reaction has a significant effect on the morphology and size distribution of silver nanoparticles.
  • AH-EPS@Ag NPs The TEM image of silver nanoparticles prepared with 0.05 mg/mL AH-EPS confirms that the silver nanoparticles covered by AH-EPS (AH-EPS@Ag NPs) are spherical with an average diameter of 20 nm ( Figure 9, B ⁇ D).
  • EPS films and EPS/nano silver films containing different amounts of AH-EPS@AgNP were prepared ( Figure 9, E).
  • the antibacterial activity of EPS/nanosilver film was analyzed by disc diffusion method, and obvious inhibition zone appeared around EPS/nanosilver film, indicating that AH-EPS@Ag NPs effectively inhibited the growth of Staphylococcus aureus (Figure 9, F).
  • AH-EPS is mainly composed of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvate, and the molar ratio is about 2.03:1.00:1.18:0.64:0.67.
  • the main link structure between sugar residues was also clarified by chemical analysis and NMR analysis, as shown in Figure 8, and the three-dimensional structure of AH-EPS is a triple-helical chain conformation.
  • the purified AH-EPS can be further used as a reducing agent and stabilizer to prepare uniform silver nanoparticles (15-30nm) without any other solvents and reagents.
  • an EPS film containing AH-EPS@Ag NPs was established, and it showed strong antibacterial activity against Streptococcus aureus.
  • This antibacterial film only contains silver nanoparticles and polysaccharides, making it a strong application potential in the development of new biodegradable antibacterial materials.
  • the hydrolyzed polysaccharide prepared in Example 1 was dissolved in deionized water to form a polysaccharide aqueous solution with a mass fraction of 5%, trifluoroacetic acid was added to make the final concentration 0.1M, and hydrolyzed in an oil bath at 100°C for 0.5h, 1h, 1.5h, 2h, 3h, the obtained polysaccharide hydrolysate was removed using a 200Da nanofiltration membrane to remove trifluoroacetic acid, concentrated by rotary evaporation at 40°C to a small volume, and lyophilized to obtain an oligosaccharide sample.
  • the molecular weight was determined by high performance liquid chromatography. The column was G3000PWXL.
  • the column temperature was 40°C
  • the mobile phase was 0.1M NaNO 3
  • the injection volume was 15 ⁇ l.
  • the results are shown in Table 4. The results showed that the extraction rate of fuco-oligosaccharides in the removal of trifluoroacetic acid using 200Da nanofiltration membrane was 93%, and the extraction rate of fuco-oligosaccharides could be higher while removing trifluoroacetic acid.
  • the molecular weight of the polysaccharide is 47916Da, and the PDI (polymer dispersion) is relatively high, indicating that the molecular weight of the sample is extremely heterogeneous.
  • the molecular weights of fuco-oligosaccharides are obtained after trifluoroacetic acid hydrolysis for 0.5h, 1h, 1.5h, 2h, and 3h. They are 14813Da, 1702Da, 2650Da, 7528Da, and 1875Da.
  • the molecular weight does not decrease after 1h of hydrolysis, indicating that the glycosidic bonds of polysaccharide molecules that can be broken by trifluoroacetic acid at this concentration have reached the upper limit.
  • the molecular weight increased abnormally after 2h of hydrolysis, indicating acid hydrolysis Polysaccharides are unstable.
  • the quantitative correction factors of fucose, glucose, galactose, and glucuronic acid in fuco-oligosaccharides were determined by the internal standard method. Accurately weigh 14.6mg fuco-oligosaccharides and dissolve them in 0.5mL xylose solution (8g/L), then add 3mL TFA (2mol/L), press the cap and heat in an oil bath at 120°C for 2h, and blow with nitrogen at 55°C Dry. Add 2mL ethanethiol and 1mL TFA and stir magnetically in a water bath at 25°C for 25 min.
  • Example 3 Test tube simulated intestinal fermentation of fuco-oligosaccharides
  • the medium components are as follows: peptone 2g/L; yeast extract 2g/L; NaCl 0.1g/L; K 2 HPO 4 0.04 g/L; KH 2 PO 4 0.04 g/L; MgSO 4 ⁇ 7H 2 O 0.01g/L; CaCl 2 ⁇ 6H 2 O 0.01g/L; NaHCO 3 2g/L; Tween 80 2mL/L, add heme at 0.02g/L after sterilization; 10mL/L vitamin K 1 ; 0.5g/L bile salt; 0.5g/L cysteine hydrochloride, adding fuco-oligosaccharide according to the mass fraction of 0.1%.
  • Short-chain fatty acid extraction Take 1ml of supernatant, centrifuge at 6000rpm/min for 10min, aspirate the supernatant, add 100 ⁇ L of concentrated hydrochloric acid and 5mL of ether and mix well, extract at room temperature for 20 minutes, then centrifuge at 5000r/min for 10 minutes at 4°C, and collect Supernatant. Transfer the supernatant to another tube, add 500 ⁇ L of 1M NaOH and mix well, extract at room temperature for 20 minutes and centrifuge at 5000r/min for 10 minutes at 4°C to collect the lower aqueous phase. Transfer the lower aqueous phase to another tube, add 100 ⁇ L of concentrated hydrochloric acid and mix well, and the resulting sample is filtered with a 0.22 ⁇ m filter membrane and then analyzed by high performance liquid chromatography.
  • the amplification system is (20 ⁇ L): 4 ⁇ L 5 ⁇ FastPfu Buffer, 2 ⁇ L of 2.5mM dNTPs, 0.8 ⁇ L of forward and reverse primers (5 ⁇ M), 0.4 ⁇ L of FastPfu polymerase, 10ng DNA template; the amplification program is: 95°C pre Denature for 2 minutes; denature at 95°C for 30 seconds, and anneal at 55°C for 30 seconds; extend at 72°C for 30 seconds; extend at 72°C for 5 minutes; cycle 25 times.
  • the amplicons were recovered and extracted by gel after 2% agarose gel electrophoresis, purified using AxyPrep DNA gel extraction kit according to the manufacturer's instructions, and quantified using QuantiFluor TM -ST (Promega, US).
  • the purified amplicons were pooled in a sequencing pool of equal molecular weight, and paired sequencing (2 ⁇ 300) was performed on the Illumina MiSeq of Chengdu Luoning Biotechnology Co., Ltd.
  • Example 4 CDMN simulated intestinal fermentation
  • Configure intestinal fermentation medium 2L (g/L): corn starch 8.0g/L; peptone 3.0g/L; yeast extract 4.5g/L; tryptone 3.0g/L; mucin 0.5g/L; L -Cysteine hydrochloride 0.8g/L; No. 3 bile salt 0.4g/L; Heme 0.05g/L; Sodium chloride 4.5g/L; Tween 80 1.0mL/L; Potassium chloride 2.5g /L; potassium dihydrogen phosphate 0.4g/L; magnesium chloride hexahydrate 4.5g/L; calcium chloride hexahydrate 0.2g/L; trace elements 2mL/L.
  • Trace element stock solution (g/L): Magnesium sulfate heptahydrate 3.0; Ferrous sulfate heptahydrate 0.1; Calcium chloride dihydrate 0.1; Manganese chloride tetrahydrate 0.32; Cobalt sulfate heptahydrate 0.18; Copper sulfate pentahydrate 0.01; Water zinc sulfate 0.18; hexahydrate nickel chloride 0.092.
  • Intestinal bacterial liquid Collect 20g of feces from two normal young (24-year-old) males, dissolve them in 160ml PBS solution, filter with three layers of gauze to remove solid insolubles, and obtain fecal bacterial liquid. Fecal microorganisms are inoculated with 10% The amount is connected to 3 fermentation tanks, and 15 mucosal pellets are added to each tank.
  • the automatic pH control system supplements 0.5mol/L NaOH solution and 0.5mol/L HCl to adjust the fermentation pH, and relies on the heating and condensation system to keep the fermentation temperature constant at 37°C.
  • each fermenter In order to control the strict anaerobic environment of fermentation, each fermenter is ventilated with nitrogen in the morning, mid- and late every day to exhaust the air in the fermenter. After 24 hours of inoculation and culture, in order to maintain the normal growth of microorganisms, 300 mL of nutrients were supplied and discharged daily to maintain the fermentation volume unchanged. 3 new mucosal pellets were used to replace the mucosal pellets in the tank every day to simulate mucosal regeneration.
  • 16s rRNA sequencing The microbial genome is extracted from human feces using Tiangen Kit.
  • the V4-V5 region of the bacterial 16S rRNA gene was amplified by PCR, and the amplification primers were: 338F: 5'-barcode-ACTCCTACGGGAGGCAGCA-3'; 806R: 5'-GGACTACHVGGGTWTCTAAT-3', where barcode is a unique eight for each sample Base sequence.
  • the amplification system is (20 ⁇ L): 4 ⁇ L of 5 ⁇ FastPfu Buffer, 2 ⁇ L of 2.5mM dNTPs, 0.8 ⁇ L of forward and reverse primers (5 ⁇ M), 0.4 ⁇ L of FastPfu polymerase, 10ng of DNA template.
  • the amplification procedure is as follows: 95°C for 2min; 95°C for 30s; 55°C for 30s; 72°C for 30s; 72°C for 5min; 25 cycles.
  • Illumina MiSeq high-throughput sequencing the amplicons were recovered and extracted by 2% agarose gel electrophoresis, and purified using the AxyPrep DNA gel extraction kit according to the manufacturer's instructions, and used QuantiFluor TM -ST (Promega, US) for quantification. The purified amplicons were pooled in a sequencing pool of equal molecular weight, and paired sequencing (2 ⁇ 300) was performed on the Illumina MiSeq of Chengdu Luoning Biotechnology Co., Ltd.
  • the RDP classifier http://rdp.cme.msu.edu/ was used to analyze and annotate the phylogenetic relationship of each 16S rRNA gene sequence in the silva (SSU115) 16S rRNA database with a confidence threshold of 70%.
  • FIG 18 A is the level of the intestinal phylum. It can be seen that after three days of adding fuco-oligosaccharides, the levels of Firmicutes and Proteobacteria in the transverse colon decreased, and the levels of Bacteroidetes ) Increased, and the addition of fuco-oligosaccharides was stopped, the transverse colon and descending colon Bacteroidetes were maintained at a relatively stable level.
  • B is the intestinal genus level.
  • D indicates that Prevotella (p ⁇ 0.05) increased the most in the ascending colon after the addition of fuco-oligosaccharides to the mucosa, Bacteroidetes (p ⁇ 0.05) increased in the transverse colon, and Lactobacillus (p ⁇ 0.05) The level decreased in each intestinal segment; after the addition of fuco-oligosaccharides was stopped, the level of Lactobacillus (Lactobacillus) in the ascending colon and descending colon mucosa rose.
  • Stool collection fix the mouse, lift its tail, gently press the mouse's lower abdomen with your fingers, collect fresh feces in the correspondingly numbered plastic tube with a lid, seal it immediately, place the small tube in an ice box, and store all samples
  • the tube is stored in a low-temperature refrigerator at -80°C for later use.
  • GTT Gamma glutamyltransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase

Abstract

本发明公开了富含岩藻糖的胞外多糖及其制备方法和应用，由L-岩藻糖、D-葡萄糖、D-半乳糖、D-葡萄糖醛酸和丙酮酸单元组成，胞外多糖具有还原性，可作为还原剂和稳定剂，通过绿色的方法制备了尺寸均匀的纳米银，进一步制备EPS/纳米银抗菌薄膜，制得的薄膜具有抗菌作用，在可生物降解抗菌膜方面应用；胞外多糖水解的岩藻寡糖具有促进碳水化合物代谢、糖的生物合成与代谢、降低膜运输功能、增强消化系统功能或降低疾病感染的功能，促进肠道内短链脂肪酸产生，改善肠道微生物菌群水平，可以用于制备肠道益生元，用于人体或畜禽动物，具有广泛的应用前景。

Description

富含岩藻糖的胞外多糖及其制备方法和应用 技术领域
本发明涉及胞外多糖，具体涉及富含岩藻糖的胞外多糖，还涉及胞外多糖的制备方法和应用。
背景技术
天然多糖是由植物或者微生物生长过程中分泌的一种普遍存在的高分子量生物聚合物。近来，具有高产能力和性质各异的微生物胞外多糖(EPS)已被广泛研究。由细菌、酵母菌、真菌或者霉菌分泌的EPS在成分上差异很大，EPS中包含一种或者多种单糖残基，并通过直链或者支链的形式链接起来。研究表明不同的组成成分使得不同的EPS在制药、营养保健、功能食品和化妆品领域有着不同的应用潜力。例如，黄原胶已经作为增稠剂、稳定剂和乳化剂应用于食品工业、个人护理产品和制药行业。结冷胶被广泛用作食品成分，其特殊的胶凝特性使得结冷胶成为用于药物控释制剂和组织工程应用的多功能添加剂。除了用作产品成分外，许多EPS还表现出了显著的免疫刺激、免疫调节、抗肿瘤、抗病毒、抗炎和抗氧化活性。尽管目前可利用的EPS种类繁多，但对于探索多用途的新型生物聚合物的研究兴趣仍在增长。
在各种各样的EPS中，由于岩藻糖的独特功能，含岩藻糖的胞外多糖受到了特别关注。岩藻糖是一种罕见的L-构型的6-脱氧己糖，通常发现于微生物胞外多糖、褐藻和哺乳动物中。岩藻糖修饰被确定与许多生物学功能有关，其中包括免疫调节和癌症。褐藻中富含的含硫酸盐的岩藻多糖(fucoidans)具有抗凝血、抗血栓形成、免疫调节、抗癌和抗增殖活性。然而，植物或藻类中岩藻多糖的产量较低、其组成随气候和季节变化而变化。鉴于微生物具有更高的生长速率和更易于控制生产条件的优点，其生产的富含岩藻糖的胞外多糖被认为是一种更好的替代品。在最近的几十年中，其他报道中产量最高的含岩藻糖胞外多糖是由肠杆菌(Enterobacter A47)产生，产量为13.23g/L。这种富含岩藻糖胞外多糖的多种理化特性，如流变性、粘合特性、乳化能力，以及生物可降解性薄膜的应用，显示出了其重要的商业价值。
前期研究中，我们分离出了一株Kosakonia菌株，并将其鉴定为Kosakonia sp.CCTCC M2018092，阐明了该菌株的全基因组序列和遗传特性(Complete Genome Sequence of Kosakonia sp.Strain CCTCC 2018092,a Fucose-Rich Exopolysaccharide Producer.Songfeng Niu,2019)。但是并未对Kosakonia sp.产生的荚膜胞外多糖进行深入的结构及应用研究。
发明内容
有鉴于此，本发明的目的之一在于提供一种富含岩藻糖的胞外多糖；本发明的目的之二在于提供所述富含岩藻糖的胞外多糖的制备方法；本发明的目的之三在于提供富含岩藻糖的胞外多糖在制备可降解抗菌材料中的应用；本发明的目的之四在于提供含有所述富含岩藻糖的胞外多糖的纳米银抗菌薄膜；本发明的目的之五在于提供纳米银抗菌薄膜的制备方法；本发明的目的之六在于提供所述纳米银抗菌薄膜在制备抗菌材料中的应用；本发明的目的之七在于提供基于所述富含岩藻糖的胞外多糖水解的岩藻寡糖；本发明的目的之八在于提供所述岩藻寡糖在制备肠道益生元中的应用。
为达到上述目的，本发明提供如下技术方案：
1、富含岩藻糖的胞外多糖，所述胞外多糖Mw为3.65×10 5Da，由L-岩藻糖、D-葡萄糖、D-半乳糖、D-葡萄糖醛酸和丙酮酸按摩尔比为2.03:1.00:1.18:0.64:0.67组成。
优选的，所述胞外多糖由Kosakonia sp.CCTCC M2018092发酵制得。
优选的，所述胞外多糖结构式如下：
Figure PCTCN2020123082-appb-000001
2、所述富含岩藻糖的胞外多糖的制备方法，由Kosakonia sp.CCTCC M2018092菌株发酵制得。
优选的，将Kosakonia sp.CCTCC M2018092菌株的发酵液用硫酸将pH调节至1～5，在50～80℃条件下水解至0～10小时，过滤去除菌体与硫酸钙，再用截流量10kDa的超滤膜过滤去除小分子，去除蛋白后，在去离子水中用截留分子量8000-14000Mw进行透析，冻干得富含岩藻糖的胞外多糖；
或将Kosakonia sp.CCTCC M2018092菌株的发酵液用硫酸将发酵液pH调至1～5，然后离心去除菌体和硫酸钙，上清通过Sevage法去除蛋白后，在去离子水中用截留分子量8000-14000Mw进行透析，冻干得富含岩藻糖的胞外多糖。
3、所述富含岩藻糖的胞外多糖在制备可降解抗菌材料中的应用。
4、含有所述富含岩藻糖的胞外多糖的纳米银抗菌薄膜。
5、所述的纳米银抗菌薄膜的制备方法，将富含岩藻糖的胞外多糖与AgNO 3溶液混合，在紫外激发合成纳米银颗粒，然后加入胞外多糖溶液成膜，制得纳米银抗菌薄膜。
优选的，所述富含岩藻糖的胞外多糖的浓度为0.01～0.50mg/mL，所述AgNO 3浓度为2mM；所述紫外激发为在354nm紫外灯下照射12分钟。
6、所述纳米银抗菌薄膜在制备抗菌材料中的应用。
7、基于所述富含岩藻糖的胞外多糖水解的岩藻寡糖。
优选的，所述岩藻寡糖由Kosakonia sp.CCTCC M2018092发酵产生的含岩藻糖的胞外多糖经三氟乙酸水解制得。
优选的，所述三氟乙酸水解为将质量分数为5％的胞外多糖加入三氟乙酸使三氟乙酸终浓度为0.1M，100℃水解至少1小时，然后用200Da纳滤膜除去三氟乙酸，获得岩藻寡糖。
8、所述岩藻寡糖在制备肠道益生元中的应用。
优选的，所述岩藻寡糖在制备促进碳水化合物代谢、糖的生物合成与代谢、降低膜运输功能、增强消化系统功能或降低疾病感染的益生元中的应用。
优选的，所述岩藻寡糖在促进肠道内短链脂肪酸产生中的应用。
优选的，所述短链脂肪酸为乙酸或丙酸。
优选的，所述岩藻寡糖在改善肠道微生物菌群水平中的应用。
优选的，所述改善肠道微生物菌群水平为增加拟杆菌门水平，降低厚壁菌门和变形菌门水平。
优选的，所述改善肠道微生物菌群属水平为增加副拟杆菌属、降结肠拟杆菌和普雷沃菌属水平，降低乳酸菌属和拟杆菌属水平。
本发明的有益效果在于：本发明公开了一种新型的富含岩藻糖的EPS，由Kosakonia sp.CCCCC M2018092生产，其Mw为3.65×105Da，而分离出AH-EPS的Mw为3.47×104Da，AH-EPS主要由L-岩藻糖，D-葡萄糖，D-半乳糖，D-葡萄糖醛酸和丙酮酸组成，摩尔比约为2.03：1.00：1.18：0.64：0.67；通过化学分析和NMR分析阐明了糖残基之间的主要链接结构，并且确定AH-EPS的三维结构为三螺旋链构象，AH-EPS的这些结构特征可能为研究其潜在应用提供关键数据。纯化的AH-EPS可以进一步用作还原剂和稳定剂，用于制备均匀的银纳米颗粒(15–30nm)，而无需任何其他溶剂和试剂。EPS还具有成膜性能，建立了含有AH-EPS@Ag NPs的EPS膜，并对金黄色链球菌表现出较强的抗菌活性。这种抗菌膜仅包含银纳米颗粒和多糖，使其在开发新型生物可降解抗菌材料方面具有强大的应用潜力。
本发明的富含岩藻糖的EPS水解后获得岩藻寡糖，岩藻寡糖具有益生功能，首次公开岩藻寡糖具有促进碳水化合物代谢、糖的生物合成与代谢、降低膜运输功能、增强消化系统功能或降低疾病感染的功能，能够促进肠道内短链脂肪酸产生，改善肠道微生物菌群水平，因此能够制备肠道益生元，可以应用到人体健康及畜禽动物养殖等领域。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为原始EPS和AH-EPS的GPC图。
图2为AH-EPS完全酸水解产物分析(A：AH-EPS的完全酸水解产物的HPLC；B：AH-EPS完全酸水解产物的GC-MS总离子流色谱图)。
图3为AH-EPS在D 2O中的 1H NMR谱和 13C NMR谱(A： 1H NMR谱；B： 13C NMR谱)。
图4为AH-EPS的二维核磁波谱分析(A：2D  1H/ 13C HSQC谱；B：2D  1H/ 13C HMBC谱)。
图5为AH-EPS的二维核磁波谱分析(A：2D  1H/ 1H COYY谱；B：2D  1H/ 1H NOESY谱)。
图6为傅立叶红外光谱和差示扫描量热法分析(A：AH-EPS的FT-IR光谱；B：DSC曲线)。
图7为在各种浓度的氢氧化钠溶液中，刚果红，刚果红+AH-EPS和刚果红+葡聚糖络合物的最大吸峰的变化。
图8为AH-EPS的主体结构。
图9为EPS/纳米银薄膜的制备和抗菌测试(A：不同浓度的AH-EPS合成的银纳米颗粒溶液的紫外-可见光谱；B-C：AH-EPS@Ag NP在不同放大倍数下的TEM图像；D：根据TEM分析得出AH-EPS@Ag NP的大小分布；E：EPS薄膜和EPS/纳米银薄膜的图片；F：由EPS/纳米银薄膜产生的对金黄色葡萄球菌的生长抑制区，其中含有0.89％(a)，0.44％(b)，0.22％(c)和0％(d)AH-EPS@Ag NPs)。
图10为单糖标准品的GC-MS总离子流色谱图。
图11为寡糖完全酸水解产物的GC-MS总离子流色谱图。
图12为岩藻寡糖对发酵液pH的影响。
图13为岩藻寡糖对短链脂肪酸的影响。
图14为试管发酵肠道菌群门水平(SGCON为对照组，SGFOP为添加岩藻寡糖组)。
图15为试管发酵肠道菌群属水平(SGCON为对照组，SGFOP为添加岩藻寡糖组)。
图16为粘膜小球的制备。
图17为粘膜小球模拟结果(A：内腔；B：粘膜；S、H、J分辨代表升结肠、横结肠、降结肠，后面的数字代表发酵天数，w代表停止添加样品)。
图18为模拟肠道结果(A：肠道门水平；B：肠道属水平；C：粘膜门水平；D：肠道粘膜属水平；S为升结肠，H为横结肠，J为降结肠；0,3,w分别代表：对照组，添加岩藻寡糖第3天，停止添加岩藻寡糖)。
图19为岩藻寡糖对肠道菌群代谢活动的影响(左边柱状图为该代谢途径在所有二级代谢途径中占的比例，右边为两组比较的显著性值，蓝色代表对照组强的代谢途径，橘色代表0.1％岩藻寡糖实验组代谢强的途径，代谢途径按照显著性值从小到大依次排列)。
图20为岩藻寡糖安全性评价结果(A：岩藻寡糖对重量的影响；B：岩藻寡糖对脑和心脏的影响；C：岩藻寡糖对结肠长度的影响；D：岩藻寡糖对肝重量的影响；E：岩藻寡糖对肝功能和肾功能的影响)。
图21为小鼠粪便中短链脂肪酸测定结果(A：HPLC；B：GC；C：GC-MS；D：短链脂肪酸含量统计结果)。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好的理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1、微生物胞外多糖(EPS)的制备
EPS由Kosakonia sp.CCTCC M2018092菌株在分批补料的条件下发酵生产，具体步骤如下：取Kosakonia sp.CCTCC M2018092菌株在含30mL培养基的250mL摇瓶中于30℃、200rpm转速下培养20小时，然后30ml细菌培养液转移到15L大小的发酵罐中于30℃、300rpm条件进行预成长培养13小时(通气量1.5m3/h)。之后，取3L预生长的细菌液转移到50L发酵罐中(含30L培养基)进行分批补料发酵。根据残糖量使用蠕动泵在发酵开始13h后以0.9-3.8rpm的速率开始分批补入200g/L的葡萄糖溶液。发酵罐通气量为1.5m3/h，通过转速联动自动调节转速(300-550rpm)将溶氧浓度控制在10％以上。50L发酵罐的pH通过补入氢氧化钠控制在7.0，温度控制在30℃。培养过程中的各培养基组成见表1。
表1.Kosakonia sp.CCTCC M2018092发酵产生胞外多糖的培养基成分
Figure PCTCN2020123082-appb-000002
胞外多糖的提取：
方法1：第一条路线为发酵结束后，用硫酸将发酵液pH调至2.0，然后12000rpm高速离心20min去除菌体和硫酸钙；上清通过Sevage法去除蛋白后，去离子水进行透析(截留分子量8000-14000Mw)后冻干得原始发酵多糖(EPS)，得率为13.5g/L。但由于菌体体积小，需要高速离心去除，不利于多糖的工业化大规模制备。
方法2：提取路线为发酵结束后，用硫酸将发酵液pH调至2.0，在80℃条件下水解4h。然后通过0.22μm陶瓷膜过滤去除菌体与硫酸钙，再用10kDa截流量的超滤膜过滤去除色素等小分子。过滤浓相除蛋白后，透析(截留分子量8000-14000Mw)并冻干得部分水解多糖(AH-EPS)，得率为12.6g/L。
实施例2、AH-EPS的结构鉴定
EPS和AH-EPS的重均分子量(Mw)通过凝胶渗透色谱(GPC)进行测定。使用配备了PL aquagel-OH mixed-H 8μm色谱柱和示差检测器的PL-GPC50GPC集成系统(安捷伦)。在30℃下用0.1M NaNO 3和500ppm NaN 3作为洗脱液分离具有适当浓度的样品，结果如图1所示。结果显示，产自Kosakonia sp.CCTCC M2018092的EPS是一种异质高分子量多糖，Mw约为3.65×10 5Da(Mw/Mn＝1.7)。AH-EPS是均质的EPS，平均质量为3.47×10 4Da(Mw/Mn＝1.2)。由于AH-EPS具有能大规模生产和具有同质性的优越之处，因此对AH-EPS进行了全面的结构表征。
1、高效液相色谱(HPLC)分析糖基组成
将AH-EPS完全酸水解后测定其单体组成。纯化后的AH-EPS(5mg/ml)溶于5ml纯化水中，加入0.1ml三氟乙酸后于120℃水解2h，除去三氟乙酸后，将样品溶解于10mM的硫酸中，进行HPLC分析。高效液相色谱(HPLC)分析条件为：Xtimate(Welch)Sugar-H column(7.8mm×300mm,5μm)色谱柱；柱温：40℃；流速：0.5ml/min；流动相：10Mm硫酸；检测器：示差检测器(RI-201H)。通过将保留时间与各个单体标准品的保留时间进行比较来确定单体组成，结果如图2中A所示。结果显示，AH-EPS由岩藻糖，葡萄糖，葡萄糖醛酸和半乳糖组成。
2、气相色谱-质谱(GC-MS)分析糖基组成
完全酸水解后进行缩硫醇-乙酸酯衍生化并使用GC-MS分析进一步证实了上述实验结果。即，精确称取14.6mg岩藻多糖溶解于0.5ml木糖溶液(8g/L)中，再加入3ml TFA(2mol/L)，压盖后在120℃油浴中加热2小时，55℃条件下氮气吹干。加入2ml乙硫醇和1ml三氟乙酸，于25℃水浴中磁力搅拌25min。再于55℃条件下氮气吹干，然后加入4ml醋酸酐-吡啶混合物(1：1，V/V)，于55℃水浴中磁力搅拌5小时后取0.5ml氮气吹干后复溶于甲醇中，进样进行GC-MS分析。
GC-MS条件为：Shimadzu(GCMS-QP2010，日本)，Rtx-5毛细管柱(0.25mm×30m)，汽化室温度为280℃，以高纯氦气为载气，流速为1ml/min；进样量为0.3ul。柱温升温程序：初始温度为80℃，保持2min，以15℃/min的速率升温到200℃，再以1℃的速率升温至210℃，以25℃/min的速率升温到280℃，保持6min。结果如图2中B所示。结果显示，AH-EPS由岩藻糖，葡萄糖，葡萄糖醛酸和半乳糖组成，葡萄糖醛酸含量为14.62％，AH-EPS中单糖的绝对构型经GC-MS分析三甲基甲硅烷基化的(-)-2-丁基糖苷经确定，同样发现AH-EPS由L-岩藻糖，D-葡萄糖，D-半乳糖和D-葡萄糖醛酸组成。
通过GC-MS测定保留时间和分子中的离子碎片来确定糖醛酸的乙酰化，结果如表1所示，结果显示，AH-EPS中的岩藻糖，葡萄糖，半乳糖和葡萄糖醛酸的摩尔比为2.03：1.00：1.18：0.64。
表1、糖醛酸的乙酰化
Figure PCTCN2020123082-appb-000003
3、丙酮酸分析
在Shimadzu(LC-16)HPLC系统上使用紫外检测器(SPD-16)在215nm波长下对AH-EPS中的丙酮酸进行定量分析。将AH-EPS(5.1mg)溶于3M TFA(4mL)中，并在120℃水解2h，样品在55℃的氮 气中干燥，并重新溶于100mL流动相中，过滤后，将进样检测。检测条件：WondaSil C18柱(250×4.6mm，5μm)，并在30℃下用含98％K 2HPO 4-H 3PO 4(0.1M K 2HPO 4-H 3PO 4，pH 2.9)和2％MeOH的缓冲液洗脱，流速为0.25mL/min。分析结果显示，丙酮酸的平均含量为6.82％。
4、甲基化分析
AH-EPS的甲基化分析是使用传统方法并进行了一些修改。在甲基化之前，按照Holzwarth和Ogletree的研究，在95℃下加热AH-EPS(5mg/ml的AH-EPS溶液在1mM草酸，0.1M氯化钠，pH 3.0条件下)2h除去丙酮酸。然后将溶液用NaOH中和，用去离子水透析并冷冻干燥。此外，在甲基化之前应先将糖醛酸还原，通过用EDC还原不含丙酮酸的AH-EPS来制备还原了糖醛酸的AH-EPS，具体是将AH-EPS(5mg)添加到2mL 75％THF-0.1mol/L MES溶液中，并使用10％Et3N将pH值调节至4.75，再添加EDC(20mg)后于环境温度搅拌1h。随后用2M醋酸溶液终止反应。反应液使用截留量3.5kDa的透析袋透析24后，冷冻干燥。冻干样品复溶于1.0ml水中，加入0.5ml 10％醋酸-甲醇溶液，氮气吹干以除去还原过程产生的硼酸，继续加入1.0ml 10％醋酸-甲醇溶液，氮气吹干，重复3-4次。最后加入0.5ml甲醇，氮气吹干，重复3次，以保证硼酸除尽，得到糖醛酸还原样品后，60℃干燥5h用于甲基化分析。
将不含丙酮酸和糖醛酸的AH-EPS(10mg)溶于0.1mL水中，然后将溶液转移至3mL DMSO中进行充分混合。然后水被2g 3A分子筛吸收24小时。除去分子筛后，将样品用CH 3I甲基化，并使用NaOH作为DMSO中的催化剂。然后将甲基化的产物在3M TFA中120℃水解2h，并在25℃下用NaBH 4还原12h。样品最后在55℃下用醋酸酐-吡啶(1：1，v/v)乙酰化5h，然后通过GC-MS分析，结果如表2所示。
表2、甲基化分析结果
Figure PCTCN2020123082-appb-000004
a Partially methylated alditol acetate. b Relative to the 1,4-linked-fucose residue.
结果表明，AH-EPS主要由1,4-连接的岩藻糖，1,3,4-连接的岩藻糖，1,3-连接的葡萄糖，1,3-连接的半乳糖和末端半乳糖组成，摩尔比为1：1.02：1.63：0.33：0.68，以及AH-EPS链由岩藻糖残基C3处的唯一分支点组成。进一步根据先前的研究，丙酮酸被推导与末端半乳糖连接。
5、高碘酸氧化和Smith降解
AH-EPS(56mg)溶解于50ml高碘酸纳溶液(0.015M)中，并于4℃冰箱中保存。间隔12h取0.2ml溶液用纯化水定容至50ml，并稀释后溶液在233nm处的吸光度。待126h吸光度稳定后，向其中加入4ml乙二醇终止反应，取少量该水溶液用HPLC分析甲酸。剩下的反应物使用纯化水透析(截留量8000MW)后冻干。向冻干产物中加入3ml硼氢化钠溶液(26g/L)室温下还原22h。还原后的产物加入2ml TFA(3M)于120℃油浴中水解2h，氮气吹干后复溶于流动相中，进行HPLC分析。
高效液相色谱(HPLC)分析条件：Xtimate(Welch)Sugar-H column(7.8mm×300mm,5μm)色谱柱；柱温：40℃；流速：0.5ml/min；流动相：10Mm硫酸；检测器：示差检测器(RI-201H)。进样体积：15μL。
结果表明存在葡萄糖，半乳糖和岩藻糖，并且葡萄糖，半乳糖和岩藻糖具有1→3键。乙二醇和赤藓 糖醇的形成表明存在4-取代的糖基。1,2,3-丁三醇的存在表明AH-EPS中存在1,4-二取代岩藻糖残基。
6、核磁共振波谱分析
将AH-EPS
Figure PCTCN2020123082-appb-000005
样品溶于D 2O并装入5mm核磁管中，以进行NMR分析。 1H和 13C NMR谱，二维核磁波谱(包括2D  1H- 1H COSY，HSQC，HMBC，NOESY和TOCSY)使用Bruker Avance III 600MHz NMR光谱仪测定以确定糖残基的序列。结果如图3～5和表3所示。 1H NMR谱(图3，A)在1.27ppm处的信号峰通常是6-脱氧糖(此处为岩藻糖)的CH 3基团，δ1.45处的信号归因为丙酮基取代基的CH 3。积分数据表明，丙酮基和岩藻糖残基之间的比例为1：3，这与HPLC分析的结果一致。因此，AH-EPS的组成为L-岩藻糖，D-葡萄糖，D-半乳糖，D-葡糖醛酸和丙酮酸，摩尔比为2.03：1.00：1.18：0.64：0.67。 13C NMR谱图(图3，B)显示了岩藻糖的两个CH 3信号(δ15.32和15.49ppm)，丙酮取代基的一个CH 3信号(δ25.04ppm)和丙酮取代基和葡萄糖醛酸取代基的两个C＝O基团信号(δ175.99和176.36ppm)，在
Figure PCTCN2020123082-appb-000006
Figure PCTCN2020123082-appb-000007
之间观察了葡萄糖和半乳糖的游离羟基C-6信号。
表3、AH-EPS在D 2O中的 1H NMR谱和 13C NMR谱
Figure PCTCN2020123082-appb-000008
1H/ 13C HSQC谱所示(图4，A)，有六个 1H/ 13C信号(A-F，δ5.40/99.35、5.35/93.11、5.35/99.25、5.17/93.91、4.99/101.00、4.50/102.51ppm)归因于糖残基的异头质子(H-1)和异头碳(C-1)。超过δ4.9ppm的信号归因于α-异头质子，而在δ4.9-4.4ppm之间的信号属于β-异头质子。结合异头信号的耦合常数分析，确定残基A-E具有α-构型，残基F具有β-构型。残基G代表了丙酮取代基。在 1H/ 13C HMBC谱中(图4，B)，丙酮酸的甲基质子信号(1.45ppm)与102.04ppm处的 13C信号耦合(丙酮酸的O–C–O基团)。结果表明，丙酮酸参与了包括O-4和O-6位置在内的六元环状缩酮形成。
通过 1H/ 1H COSY， 1H/ 1H NOESY， 1H/ 13C HSQC和 1H/ 1H TOCSY实验完成了A–G残基的 1H和 13C化学位移的归属(表3)。残基B的C-4(78.78)和C-6(69.31)碳信号的低场移位表明它是1,4,6-α-D-Galp。在 1H/ 13C HMBC谱中(图4，B)，岩藻糖的甲基质子在1.27ppm处显示出与岩藻糖C-4的三键耦合(δ79.80ppm)和岩藻糖C-5的两键耦合(δ67.41ppm)。岩藻糖的C-5进一步与残基C(δ5.35ppm)和残基E(δ4.99ppm)的异头质子偶联，表明残基C和E是岩藻糖残基(图5，A)。残基C中岩藻糖的C-4与残基E的异头质子偶联，这表明残基C为1,4-α-L-Fucp，即存在→4)-α-L-Fucp-(1→4)-α-L-Fucp-(1→链接。类似地，残基E的C-4与残基F的异头质子偶联，即存在→3)-β-D-Glcp-(1→4)-α-L-Fucp-(1→4)-α-L-Fucp-(1→链接。NOESY谱(图5，B)显示在残基F的H-3与残基C的H-1之间的存在残基交叉峰，确定出Fucp-(1→3)-Glcp链接和→3)-β-D-Glcp-(1→4)-α-L-Fucp-(1→4)-α-L-Fucp-(1→为重复的骨架单元。NOESY谱中残基C的H-3，H-5和残基A的H-1之间的信号表明残基A链接在残基C的3号C上；残基A的H-4和残基D的H-1之间的信号表明残基D与残基A连接。结合积分和典型的化学位移归属，确定了残基D为α-D-GlcpA，最后一个残基-残基B可能只与残基D相连。
7.傅立叶红外光谱和差示扫描量热法分析
AH-EPS的FT-IR光谱和DSC曲线分别在Shimadzu IRPresting-21光谱仪和TA Q200热分析仪上测试。FT-IR在4000cm -1至400cm -1的扫描范围内进行，DSC在30℃至400℃的环境下以10°K/min的加热速率在空气氛围中进行。AH-EPS的FT-IR光谱(图6，A)在3429cm -1处显示出强烈的峰，这归因于O-H的拉伸振动。1650和1575cm -1处的峰属于C＝O组的拉伸振动。在2928和2858cm -1处的峰是糖的特征峰，具体归因于C-H的拉伸振动。1097cm -1处的峰反映了C–O–H和C–O–C的拉伸振动，而885cm –1处的峰表明存在β-糖苷键。AH-EPS的DSC曲线(图6，B)显示了在94.5℃处的宽吸热峰，这是脱水过程的结果。进一步升高温度，观察到最大在356.5℃的放热带。这可能是由于三维结构的变化和AH-EPS的氧化引起的。
通过Smith降解实验，甲基化分析和NMR分析鉴定出了AH-EPS可能的结构构成，如图8所示。
8.刚果红实验
将4mg/mL AH-EPS溶液(2mL)与80μM刚果红(2mL)混合后在不同NaOH浓度下(0.00M，0.05M，0.10M，0.15M，0.20M，0.25M，0.30M，0.35M，0.40M，0.45M和0.50M)反应10分钟。通过UV-Vis分光光度计在400nm至800nm的范围内测量最大吸光度。另外，以添加有刚果红的葡聚糖(Mw＝40,000Da)溶液和没有任何多糖的刚果红溶液作为对照。
多糖链表现出不同的三维结构，如三重螺旋链，单个无规则卷曲链和无规则卷曲链。刚果红可以在碱性溶液中与三螺旋多糖形成特殊的复合物。随着NaOH浓度的增加，最大吸收波长(λ max)将发生红移。如图7所示，在NaOH浓度接近0.2M时，刚果红的λ max增大到最大值。而在有或没有葡聚糖的情况下，刚果红的λ max随NaOH浓度的增加而逐渐减小，直至达到恒定值，该结果表明AH-EPS具有三重螺旋链构象。
实施例3、EPS/纳米银薄膜的制备和抗菌测试
将不同浓度(0.01mg/mL、0.03mg/mL、0.05mg/mL、0.10mg/mL、0.20mg/mL、0.50mg/mL)的AH-EPS溶液和2mM AgNO 3溶液混入5mL水中震荡30分钟。之后，将溶液在354nm UV灯下照射12分钟以制备银纳米颗粒，将纳米银溶液分别添加(0mL，1.5mL，3.0mL，6.0mL)到25mL EPS(1.5wt％)溶液中，然后将溶液倒入塑料平板(d＝6.5cm)中，在50℃干燥12h后形成薄膜。使用Kirby-Bauer方法， 将薄膜剥离并切成薄片形状(d＝0.5cm)以进行抗菌测试，使用革兰氏阳性金黄色葡萄球菌(ATCC29213)作为实验细菌。不同浓度的AH-EPS制备的银纳米颗粒溶液的吸收光谱如图9中A所示。结果显示，当AH-EPS的浓度为0.05mg/mL时，溶液的UV-vis光谱在423nm处显示出银纳米颗粒的最强吸收峰。因此，使用0.05mg/mL的AH-EPS制备银纳米颗粒，以用于进一步的表征和应用。形成此现象的原因是AH-EPS含有丰富的–OH基团，可以将Ag +还原为Ag 0。反应中还原剂和稳定剂的比例对银纳米颗粒的形态和尺寸分布具有显著影响。
0.05mg/mL的AH-EPS制备银纳米颗粒TEM图证实，被AH-EPS(AH-EPS@Ag NPs)覆盖的银纳米颗粒呈球形，平均直径为20nm(图9，B～D)。制备了含有不同数量的AH-EPS@Ag NP的EPS膜和EPS/纳米银膜(图9，E)。通过圆盘扩散法分析了EPS/纳米银薄膜的抗菌活性，EPS/纳米银薄膜周围出现明显的抑制区，表明AH-EPS@Ag NPs有效抑制金黄色葡萄球菌的生长(图9，F)。含有0、0.22％，0.44％和0.89％AH-EPS@Ag NP的EPS/纳米银薄膜对金黄色葡萄球菌生长的抑制区分别为0mm，12.3mm，14.1mm和16.5mm。抗菌能力表现出剂量依赖性。并且在平板培养12小时后，基于EPS的抗菌膜消失并融合到培养基中。因此，在这项研究中发现的新型多糖(EPS和AH-EPS)在开发水溶性和生物可降解的新型抗菌材料方面具有潜在的应用前景。
通过上述研究发现，Kosakonia sp.CCCCC M2018092生产了一种新型的富含岩藻糖的EPS，其Mw为3.65×10 5Da，而分离出AH-EPS的Mw为3.47×10 4Da。AH-EPS主要由L-岩藻糖，D-葡萄糖，D-半乳糖，D-葡萄糖醛酸和丙酮酸组成，摩尔比约为2.03：1.00：1.18：0.64：0.67。还通过化学分析和NMR分析阐明了糖残基之间的主要链接结构，如图8所示，并且AH-EPS的三维结构为三螺旋链构象。纯化的AH-EPS可以进一步用作还原剂和稳定剂，用于制备均匀的银纳米颗粒(15–30nm)，而无需任何其他溶剂和试剂。基于EPS的成膜性能，建立了含有AH-EPS@Ag NPs的EPS膜，并对金黄色链球菌表现出较强的抗菌活性。这种抗菌膜仅包含银纳米颗粒和多糖，使其在开发新型生物可降解抗菌材料方面具有强大的应用潜力。
实施例4、制备低分子量岩藻寡糖
将实施例1制得的水解多糖以去离子水溶解配置成质量分数为5％的多糖水溶液，加入三氟乙酸使其终浓度为0.1M，100℃油浴水解0.5h、1h、1.5h、2h、3h，得到的多糖水解液使用200Da纳滤膜除去三氟乙酸，在40℃旋蒸浓缩至小体积，冻干得到寡糖样品，使用高效液相色谱进行分子量测定，色谱柱为G3000PWXL，柱温40℃，流动相为0.1M NaNO 3，进样体积15μl，结果如表4所示。结果显示，使用200Da纳滤膜除三氟乙酸岩藻寡糖提取率在93％，能在除去三氟乙酸的情况下有较高的岩藻寡糖提取率，同时经过硫酸一次水解的岩藻多糖分子量为47916Da，且PDI(聚合物分散度)较高，表明样品分子量处于极不均一状态，而经过三氟乙酸水解0.5h、1h、1.5h、2h、3h后得到岩藻寡糖分子量分别为14813Da、1702Da、2650Da、7528Da、1875Da，在水解1h后分子量不再减小，表明该浓度三氟乙酸能够断裂的多糖分子糖苷键已经达到上限，在水解2h时出现分子量异常上升，表明酸水解多糖具有不稳定性。
表4、三氟乙酸水解多糖
组分名 保留体积(mL) 数均分子量(Mn) 重均分子量(Mw) PDI
一次水解 7.42 3940.89 47916.31 12.16
0.1M TFA,0.5h,100℃ 8.08 10932.02 14813.61 1.36
0.1M TFA,1h,100℃ 8.77 1613.3 1702.95 1.06
0.1M TFA,1.5h,100℃ 8.78 2042.87 2650.55 1.3
0.1M TFA,2h,100℃ 8.73 2600.63 7528.55 2.89
0.1M TFA,3h,100℃ 8.83 1683.18 1875.48 1.11
GC-MS分析岩藻寡糖中单糖组成：
以木糖作为内标物，采用内标法测定岩藻寡糖中岩藻糖、葡萄糖、半乳糖、葡萄糖醛酸的定量校正因子。精确称取14.6mg岩藻寡糖溶解于0.5mL木糖溶液(8g/L)中再加入3mL TFA(2mol/L)，压盖后在120℃油浴中加热2h，55℃条件下氮气吹干。加入2mL乙硫醇和1mL TFA于25℃水浴中磁力搅拌25min。再于55℃条件下氮气吹干，然后加入4mL醋酸酐-吡啶混合物(1:1，V/V)，于55℃水浴中磁力搅拌5h后，取0.5mL样品氮气吹干后，复溶于甲醇中，进样进行GC-MS分析。移取8g/L的岩藻糖、葡萄糖、半乳糖、木糖、葡萄糖醛酸标样各0.5mL于同一玻璃管中混合。氮气吹干后，按上述方法进行衍生化后进样GC-MS分析。GC-MS条件：Shimadzu(GCMS-QP2010，日本)，Rtx-5毛细管柱(0.25mm×30m)，汽化室温度为280℃，以高纯氦气为载气，流速为1mL/min；进样量为1μL。柱温升温程序：初始温度为80℃，保持2min，以15℃/min的速率升温到200℃，再以1℃的速率升温至210℃，以25℃/min的速率升温到280℃，保持6min。接口温度为260℃；正离子电离方式，质量范围：m/z 35–600。标准品GC-MS总离子流色谱图如图10所示，寡糖完全酸水解产物GC-MS总离子流色谱图如图11所示。
以木糖为内标的GC-MS分析表明岩藻寡糖由岩藻糖，葡萄糖，葡萄糖醛酸和半乳糖组成，分析得到岩藻寡糖中的岩藻糖，葡萄糖，半乳糖和葡萄糖醛酸的摩尔比为1.58:1.00:1.00:1.46。
实施例3、试管模拟肠道发酵岩藻寡糖
取发酵培养基1L，培养基组分如下：蛋白胨2g/L；酵母提取物2g/L；NaCl 0.1g/L；K 2HPO 4 0.04g/L；KH 2PO 4 0.04g/L；MgSO 4·7H 2O 0.01g/L；CaCl 2·6H 2O 0.01g/L；NaHCO 3 2g/L；吐温80 2mL/L，灭菌后按0.02g/L加入血红素；10mL/L维生素K 1；0.5g/L胆盐；0.5g/L半胱氨酸盐酸盐，按质量分数0.1％加入岩藻寡糖。
先将6支试管接入25ml培养基，接种10％粪便菌液，通入厌氧气体保持5min(85％N 2，10％CO 2，5％H 2)，并用石蜡液封，每12h取样一次，测定发酵液pH及气体，结果如图12所示。结果显示，添加0.1％岩藻寡糖的发酵液pH下降速度高于对照组。
36小时后，取样送至测定16s rRNA，取1mL发酵液测定短链脂肪酸，发酵液中短链脂肪酸测定方法如下：
短链脂肪酸提取：取1ml上清液，6000rpm/min离心10min，吸出上清液，加入100μL浓盐酸和5mL乙醚混匀，常温萃取20分钟后以5000r/min在4℃下离心10分钟，收集上清液。将上清液转移到另一管中，加入500μL的1M NaOH混匀，常温萃取20分钟后以5000r/min在4℃下离心10分钟，收集下层水相。将下层水相转移到另一管中，加入100μL浓盐酸混匀，所得样品用0.22μm滤膜过滤后进行高效液相色谱分析。
色谱条件：采用ZORBAX SB-Aq(4.6×250mm 5-Micron)色谱柱进行分析，流动相为0.025％磷酸水溶液(pH＝2.8)：乙腈＝95：5，以1.0mL/min进行洗脱，每针进样量20μL；检测波长：210nm；柱温：30℃。
短链脂肪酸标曲测定：分别配置浓度为1mL/L的乙酸、丙酸、正丁酸、正戊酸的标准样品溶液，在上述检测条件下进行高效液相色谱分析，确定每个酸的保留时间。根据预实验估算的发酵液中短链脂肪酸浓度，配置乙酸浓度为2mL/L，丙酸浓度为4mL/L，正丁酸浓度为2mL/L，正戊酸浓度为1ml/L的混标溶液。 再按比例用超纯水稀释。在上述检测条件下对混标溶液进行高效液相色谱分析，测定各个浓度下的峰面积，绘制短链脂肪酸标准曲线，结果如表5所示。
表5、短链脂肪酸标准曲线
Figure PCTCN2020123082-appb-000009
计算结果如图13所示。结果显示，添加0.1％的岩藻寡糖后丙酸产量为对照组的2倍，可以初步证实岩藻寡糖可以促进肠道菌产生丙酸而促进人体健康。
试管发酵16s rRNA测序
1)DNA提取与PCR扩增
微生物基因组使用天根试剂盒，提取自人体粪便和发酵后人体粪便基因组。细菌16S rRNA基因的V4-V5区通过PCR进行扩增，扩增引物为：338F 5’-barcode-ACTCCTACGGGAGGCAGCA-3’；806R 5’-GGACTACHVGGGTWTCTAAT-3’其中barcode是每个样本特有的八碱基序列。扩增体系为(20μL)：4μL 5×FastPfu Buffer，2μL of 2.5mM dNTPs，0.8μL of正反引物(5μM)，0.4μL of FastPfu聚合酶，10ng DNA模板；扩增程序如为：95℃预变性2min；95℃变性30s，55℃退火30s；72℃延伸30s；72℃后延伸5min；循环25次。
2)Illumina MiSeq高通量测序
扩增子经2％琼脂糖凝胶电泳后凝胶回收提取，使用AxyPrep DNA凝胶提取试剂盒按照制造商的说明进行纯化，并使用QuantiFluor TM-ST(Promega,U.S.)进行定量。纯化后的扩增子汇集在等分子量的测序池中，在成都罗宁生物科技有限公司Illumina MiSeq上进行配对测序(2×300)。
3)测序数据处理与分析
原始fastq文件经割库后用QIIME(1.17版)按照以下标准对其进行质量过滤：在10个bp滑动窗口的平均质量得分小于20的任何位点上截断250个bp的reads，丢弃短于50bp的截断reads。去除barcode序列、引物中有2个核苷酸错配的序列、含有模糊碱基的reads。仅组装重叠区域大于10bp的reads，无法组装的reads则被丢弃。具有97％相似性的序列通过UPARSE聚类为一个操作单元(OTUs)，并使用UCHIME识别和去除嵌合体序列。利用RDP分类器(http://rdp.cme.msu.edu/)对置信阈值为70％的silva(SSU115)16S rRNA数据库中每个16S rRNA基因序列的系统发育亲缘关系进行分析及注释。
结果如图14和图15所示，结果显示拟杆菌门(Bacteroidetes)(p＜0.05)水平显著增加，而包含多种革兰氏阴性致病或条件致病菌的变形菌门(Proteobacteria)(p＜0.05)水平显著降低。
实施例4、CDMN模拟肠道发酵
配置肠道发酵培养基2L：(g/L)：玉米淀粉8.0g/L；蛋白胨3.0g/L；酵母提取物4.5g/L；胰蛋白胨3.0g/L；粘液素0.5g/L；L-半胱氨酸盐酸盐0.8g/L；3号胆盐0.4g/L；血红素0.05g/L；氯化钠4.5g/L；吐温80 1.0mL/L；氯化钾2.5g/L；磷酸二氢钾0.4g/L；六水氯化镁4.5g/L；六水氯化钙0.2g/L；微量元素2mL/L。
微量元素储备液(g/L)：七水硫酸镁3.0；七水硫酸亚铁0.1；二水氯化钙0.1；四水氯化锰0.32；七水硫酸钴0.18；五水硫酸铜0.01；七水硫酸锌0.18；六水氯化镍0.092。
先将玉米淀粉用蒸馏水在100℃溶解5min，再加入肠道发酵培养基(不含粘液素、血红素、3号胆盐、半胱氨酸、微量元素)，121℃灭菌15min后在超净台加入粘液素、血红素、3号胆盐、半胱氨酸、微量元素。
粘膜小球的制备：称取2g琼脂于100mL蒸馏水中加热溶解，待琼脂溶液澄清透明后，冷却至60±5℃，称取0.5g粘液素趁热溶解，调节pH为6.8左右，转移到在超净工作台中紫外灭菌30min后球形磨具中得直径为7-8mm粘膜凝胶小球并将凝胶小球装袋悬挂于三个发酵罐中以模拟肠道粘膜(图16)，参数如表6所示。
表6、各结肠段参数
参数 升结肠 横结肠 降结肠
发酵体积(ml) 300 400 300
温度(℃) 37 37 37
转速 150 130 170
pH 5.8 6.2 6.8
肠道菌液：收集两名正常青年(24周岁)男性的粪便共20g，溶解至160ml PBS溶液中，使用三层纱布过滤，除去固体不溶物，得到粪便菌液，粪便微生物以10％的接种量分别接入到3个发酵罐中，每个罐子中加入15颗黏膜小球。pH自动控制系统补充0.5mol/L的NaOH溶液和0.5mol/L HCl来调节发酵pH，依靠加热冷凝系统保持发酵温度恒定在37℃。为控制发酵严格的厌氧环境，每日早、中、晚对每个发酵罐通氮气以排尽发酵罐内的空气。接种培养24h之后，为维持微生物的正常生长，每日补给养料和排出300mL，以维持发酵体积不变，每天用3个新的黏膜小球替换罐中的黏膜小球，以模拟黏膜的再生。
结果如图17所示，结果显示在肠腔中岩藻寡糖促进了乙酸和丙酸产生，而在粘膜主要分布的为丁酸和乙酸，岩藻寡糖促进了乙酸的产生，且停止添加样品后，肠道菌产生短链脂肪酸水平变化不大，可以表明岩藻寡糖有类似重塑肠道菌群的效果。
16s rRNA测序：微生物基因组使用天根试剂盒，提取自人体粪便。细菌16S rRNA基因的V4-V5区通过PCR进行扩增，扩增引物为：338F：5’-barcode-ACTCCTACGGGAGGCAGCA-3’；806R：5’-GGACTACHVGGGTWTCTAAT-3’其中barcode是每个样本特有的八碱基序列。扩增体系为(20μL)：4μL 5×FastPfu Buffer，2μL of 2.5mM dNTPs，0.8μL of正反引物(5μM)，0.4μL of FastPfu聚合酶，10ng DNA模板。扩增程序如为：95℃2min；95℃30s；55℃30s；72℃30s；72℃5min；循环25次。
Illumina MiSeq高通量测序：扩增子经2％琼脂糖凝胶电泳后凝胶回收提取，使用AxyPrep DNA凝胶提取试剂剂盒按照制造商的说明进行纯化，并使用QuantiFluor TM-ST(Promega,U.S.)进行定量。纯化后的扩增子汇集在等分子量的测序池中，在成都罗宁生物科技有限公司Illumina MiSeq上进行配对测序(2×300)。
测序数据处理与分析：原始fastq文件经割库后用QIIME(1.17版)按照以下标准对其进行质量过滤：在10个bp滑动窗口的平均质量得分小于20的任何位点上截断250个bp的reads，丢弃短于50bp的截断reads。去除barcode序列、引物中有2个核苷酸错配的序列、含有模糊碱基的reads。仅组装重叠区域大于10bp的reads，无法组装的reads则被丢弃。具有97％相似性的序列通过UPARSE聚类为一个操作单元(OTUs)， 并使用UCHIME识别和去除嵌合体序列。利用RDP分类器(http://rdp.cme.msu.edu/)对置信阈值为70％的silva(SSU115)16S rRNA数据库中每个16S rRNA基因序列的系统发育亲缘关系进行分析及注释。
结果如图18所示，图18中A为肠道门水平，可以看出添加岩藻寡糖三天后，横结肠厚壁菌门(Firmicutes)与变形菌门(Proteobacteria)水平降低，拟杆菌门(Bacteroidetes)增加，且停止添加岩藻寡糖，横结肠与降结肠拟杆菌门(Bacteroidetes)维持在相对稳定的水平。图18中B为肠道属水平，可以看出添加岩藻寡糖三天后，横结肠乳酸菌属(Lactobacillus)水平显著降低，拟杆菌属(Bacteroidetes)略微降低，副拟杆菌属(Parabacteroides)显著增加(p＜0.05)，降结肠拟杆菌(Bacteroides)显著增加(p＜0.05)，副拟杆菌属(Parabacteroides)显著增加(p＜0.05)，且在停止添加岩藻寡糖后横结肠副拟杆菌属(Parabacteroides)水平与未添加岩藻寡糖相比有所增加(p＜0.05)。图18中C为肠道粘膜门水平，可以看出添加岩藻寡糖3天后，升结肠、横结肠、降结肠中拟杆菌门(Bacteroidetes)显著上升，升结肠中拟杆菌门(Bacteroidetes)提升显著，且其中变形菌门(Proteobacteria)水平显著下降，厚壁菌门(Firmicutes)水平下降。停止添加岩藻寡糖后升结肠厚壁菌门(Firmicutes)水平增加，变形菌门(Proteobacteria)水平增加。图18中D为粘膜中添加岩藻寡糖后Prevotella(p＜0.05)属在升结肠中增加最多，横结肠中拟杆菌(Bacteroidetes)(p＜0.05)增加，乳酸菌(Lactobacillus)(p＜0.05)水平在各个肠段均减少；停止添加岩藻寡糖后，升结肠和降结肠粘膜乳酸菌(Lactobacillus)水平回升。
岩藻寡糖对肠道菌群代谢活动的影响，结果如图19所示。结果显示，寡糖具有促进碳水化合物代谢、糖的生物合成与代谢、降低膜运输功能、增强消化系统功能、降低疾病感染等功能。
实施例5、岩藻寡糖体内安全性评价
取8周龄雄性野生型小鼠，随机分为3组，每组6只，分别灌胃生理盐水(0.9％氯化钠)、0.1％岩藻寡糖(79.2mg/d/kg)、0.5％岩藻寡糖(396mg/d/kg)，每周收集一次粪便，1个月后收集小鼠各个器官测定重量，结果如图20中A所示。
粪便采集：固定小鼠，将其尾部提起，用手指轻轻按压小鼠下腹部，收集新鲜粪便于对应编号的带盖塑料管中，立即密封，将小管置于冰盒中保存，将所有样品管置于-80℃低温冰箱中保存备用。
各项生理指标能够初步反映岩藻寡糖对小鼠机体是否有不良效果，1个月后测定小鼠体重、脑、结肠、胰腺、肝重量，以及结肠长度，结果如图20中B～D。
实验结束时，摘眼球取血，血清样本分析一式三份，γ谷氨酰转移酶(GGT)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALP)作为肝脏功能的评价指标，按照试剂盒说明书的方法处理并用全自动酶标仪进行检测，结果如图20中E所示。
摘眼球取血，血清样本分析一式三份，尿素氮(BUN)、肌酸酐(CRE)等作为肾功能的评价指标，按照试剂盒说明书的方法处理并用全自动酶标仪进行检测，结果如图20中E所示。
结果表明，岩藻寡糖处理小鼠生理指标及肝肾功能与对照组比较无显著性差异(P＞0.05)，表明岩藻寡糖是一种较为安全的寡糖，能够用于生物体食用。
岩藻寡糖处理小鼠生理指标及肝肾功能与对照组比较无显著性差异(P＞0.05)，表明岩藻寡糖是一种较为安全的寡糖，能够用于生物体食用。
小鼠粪便中短链脂肪酸测定：
分别取少量小鼠新鲜排出粪便称重，将其溶于含500μL甲醇溶液的EP管中，静置5～10min，振荡混匀，制成粪便悬液。用硫酸调节悬液pH至2～3，静置5min，其间震荡混匀数次。将EP管于5 000r/min 离心20min后，取上清以5 000r/min离心5min后，其上清用于气相色谱－质谱联用分析。分别使用HPLC、GC-MS、GC测定小鼠粪便中短链脂肪酸进行初实验，摸索最佳提取方法，结果如图21所示。
结果显示，上述三种测定方法测定出的图谱表明，GC-MS峰分离及峰形较好，并且根据标准得到上述峰，从左到右依次为乙酸、丙酸、丁酸、戊酸可以用于测定小鼠粪便中的短链脂肪酸。测定结果表明，添加0.1％岩藻寡糖的小鼠产生乙酸及丙酸有所增加，表明我们的岩藻寡糖样品在小鼠肠道内被特殊肠道菌群利用并刺激其产生短链脂肪酸，从而对人体表现出益生效果。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (20)

  1. 富含岩藻糖的胞外多糖，其特征在于：所述胞外多糖Mw为3.65×10 5Da，由L-岩藻糖、D-葡萄糖、D-半乳糖、D-葡萄糖醛酸和丙酮酸按摩尔比为2.03:1.00:1.18:0.64:0.67组成。
  2. 根据权利要求1所述富含岩藻糖的胞外多糖，其特征在于：所述胞外多糖由Kosakonia sp.CCTCC M2018092发酵制得。
  3. 根据权利要求1或2所述富含岩藻糖的胞外多糖，其特征在于：所述胞外多糖结构式如下：
    Figure PCTCN2020123082-appb-100001
  4. 权利要求1～3任一项所述富含岩藻糖的胞外多糖的制备方法，其特征在于：由Kosakonia sp.CCTCC M2018092菌株发酵制得。
  5. 根据权利要求4所述富含岩藻糖的胞外多糖的制备方法，其特征在于：将Kosakonia sp.CCTCC M2018092菌株的发酵液用硫酸将pH调节至1～5，在50～80℃条件下水解至0～10小时，过滤去除菌体与硫酸钙，再用截流量10kDa的超滤膜过滤去除小分子，去除蛋白后，在去离子水中用截留分子量8000-14000Mw进行透析，冻干得富含岩藻糖的胞外多糖；
    或将Kosakonia sp.CCTCC M2018092菌株的发酵液用硫酸将发酵液pH调至1～5，然后离心去除菌体和硫酸钙，上清通过Sevage法去除蛋白后，在去离子水中用截留分子量8000-14000Mw进行透析，冻干得富含岩藻糖的胞外多糖。
  6. 权利要求1～3任一项所述富含岩藻糖的胞外多糖在制备可降解抗菌材料中的应用。
  7. 含有权利要求6所述富含岩藻糖的胞外多糖的纳米银抗菌薄膜。
  8. 根据权利要求7所述的纳米银抗菌薄膜的制备方法，其特征在于：将富含岩藻糖的胞外多糖与AgNO 3溶液混合，在紫外激发合成纳米银颗粒，然后加入胞外多糖溶液成膜，制得纳米银抗菌薄膜。
  9. 根据权利要求8所述的纳米银抗菌薄膜的制备方法，其特征在于：所述富含岩藻糖的胞外多糖的浓度为0.01～0.50mg/mL，所述AgNO 3浓度为2mM；所述紫外激发为在354nm紫外灯下照射12分钟。
  10. 权利要求7所述纳米银抗菌薄膜在制备抗菌材料中的应用。
  11. 基于权利要求1所述富含岩藻糖的胞外多糖水解的岩藻寡糖。
  12. 根据权利要求11所述的岩藻寡糖，其特征在于：所述岩藻寡糖由Kosakonia sp.CCTCC M2018092发酵产生的含岩藻糖的胞外多糖经三氟乙酸水解制得。
  13. 根据权利要求12所述的岩藻寡糖，其特征在于：所述三氟乙酸水解为将质量分数为5％的胞外多糖加入三氟乙酸使三氟乙酸终浓度为0.1M，100℃水解至少1小时，然后用200Da纳滤膜除去三氟乙酸，获得岩藻寡糖。
  14. 权利要求11所述岩藻寡糖在制备肠道益生元中的应用。
  15. 根据权利要求14所述的应用，其特征在于：所述岩藻寡糖在制备促进碳水化合物代谢、糖的生物合成与代谢、降低膜运输功能、增强消化系统功能或降低疾病感染的益生元中的应用。
  16. 权利要求11所述岩藻寡糖在促进肠道内短链脂肪酸产生中的应用。
  17. 根据权利要求16所述的应用，其特征在于：所述短链脂肪酸为乙酸或丙酸。
  18. 权利要求11所述岩藻寡糖在改善肠道微生物菌群水平中的应用。
  19. 根据权利要求18所述的应用，其特征在于：所述改善肠道微生物菌群水平为增加拟杆菌门水平，降低厚壁菌门和变形菌门水平。
  20. 根据权利要求18所述的应用，其特征在于：所述改善肠道微生物菌群属水平为增加副拟杆菌属、降结肠拟杆菌和普雷沃菌属水平，降低乳酸菌属和拟杆菌属水平。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114133464A (zh) * 2022-01-19 2022-03-04 集美大学 一种红藻硫酸低聚糖及其制备方法与应用
CN115058369A (zh) * 2022-07-20 2022-09-16 威海迪普森生物科技有限公司 一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012034996A1 (en) * 2010-09-13 2012-03-22 Inalco S.P.A. Process for production of l-fucose
US20140378556A1 (en) * 2009-12-15 2014-12-25 73100- Setenta E Tres Mil E Cem, Lda. Fucose-containing bacterial biopolymer
CN110106163A (zh) * 2019-06-19 2019-08-09 西南大学 岩藻多糖及其水解寡糖在制备益生菌保护剂中的应用及方法
CN111234047A (zh) * 2020-03-30 2020-06-05 西南大学 富含岩藻糖的胞外多糖及其制备方法和应用
CN111248258A (zh) * 2020-03-30 2020-06-09 西南大学 岩藻糖多糖在制备保鲜剂中的应用及其保鲜涂膜剂
CN111728976A (zh) * 2020-07-15 2020-10-02 西南大学 岩藻寡糖在制备肠道益生元中的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140378556A1 (en) * 2009-12-15 2014-12-25 73100- Setenta E Tres Mil E Cem, Lda. Fucose-containing bacterial biopolymer
WO2012034996A1 (en) * 2010-09-13 2012-03-22 Inalco S.P.A. Process for production of l-fucose
CN110106163A (zh) * 2019-06-19 2019-08-09 西南大学 岩藻多糖及其水解寡糖在制备益生菌保护剂中的应用及方法
CN111234047A (zh) * 2020-03-30 2020-06-05 西南大学 富含岩藻糖的胞外多糖及其制备方法和应用
CN111248258A (zh) * 2020-03-30 2020-06-09 西南大学 岩藻糖多糖在制备保鲜剂中的应用及其保鲜涂膜剂
CN111728976A (zh) * 2020-07-15 2020-10-02 西南大学 岩藻寡糖在制备肠道益生元中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI SHANSHAN; XIA HAIQIN; XIE AIQING; WANG ZHENYU; LING KAIJIAN; ZHANG QIXIONG; ZOU XIANG: "Structure of a fucose-rich polysaccharide derived from EPS produced by Kosakonia sp. CCTCC M2018092 and its application in antibacterial film", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, ELSEVIER BV, NL, vol. 159, 8 May 2020 (2020-05-08), NL, pages 295 - 303, XP086248257, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2020.05.029 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114133464A (zh) * 2022-01-19 2022-03-04 集美大学 一种红藻硫酸低聚糖及其制备方法与应用
CN114133464B (zh) * 2022-01-19 2022-11-08 集美大学 一种红藻硫酸低聚糖及其制备方法与应用
CN115058369A (zh) * 2022-07-20 2022-09-16 威海迪普森生物科技有限公司 一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法
CN115058369B (zh) * 2022-07-20 2024-02-20 威海迪普森生物科技有限公司 一种胞外多糖来源岩藻寡糖发酵型合生元的制备方法

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