CN111234047A

CN111234047A - Exopolysaccharide rich in fucose and preparation method and application thereof

Info

Publication number: CN111234047A
Application number: CN202010234516.8A
Authority: CN
Inventors: 邹祥; 李姗姗; 夏海钦; 谢爱卿
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-06-05
Anticipated expiration: 2040-03-30
Also published as: CN111234047B

Abstract

The invention discloses fucose-rich exopolysaccharide and a preparation method and application thereof, the fucose-rich exopolysaccharide is composed of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid units, the molar ratio is 2.03:1.00:1.18:0.64:0.67, Mw is 3.65 x 10⁵Da is prepared by fermenting Kosakonia sp.CCTCC M2018092 strain, acid hydrolysis extracellular polysaccharide has reducibility and can be used as a reducing agent and a stabilizing agent, nano silver with uniform size is prepared by a green method, and further the nano silver is preparedThe EPS/nano-silver antibacterial film has an antibacterial effect and has good application potential in the aspect of biodegradable antibacterial films.

Description

Exopolysaccharide rich in fucose and preparation method and application thereof

Technical Field

The invention relates to exopolysaccharides, in particular to exopolysaccharides rich in fucose, and also relates to a preparation method and application of the exopolysaccharides.

Background

Natural polysaccharides are a ubiquitous high molecular weight biopolymer secreted by plants or microorganisms during their growth. Recently, microbial Extracellular Polysaccharides (EPS) having high productivity and various properties have been extensively studied. EPS secreted by bacteria, yeasts, fungi or moulds is very different in composition, comprising one or more monosaccharide residues, linked by linear or branched chains. Researches show that different EPS has different application potentials in the fields of pharmacy, nutrition and health care, functional food and cosmetics due to different components. For example, xanthan gum has been used in the food industry, personal care products and pharmaceutical industries as a thickener, stabilizer and emulsifier. Gellan gum is widely used as a food ingredient and its specific gelling properties make it a multifunctional additive for controlled drug release formulations and tissue engineering applications. In addition to its use as a product ingredient, many EPS exhibit significant immunostimulatory, immunomodulatory, antitumor, antiviral, anti-inflammatory, and antioxidant activity. Despite the wide variety of EPS currently available, there is still growing research interest in exploring new biopolymers for multiple uses.

Among the various EPS, fucose-containing exopolysaccharides have received particular attention due to the unique function of fucose. Fucose is a rare L-configuration 6-deoxyhexose, commonly found in microbial exopolysaccharides, brown algae, and mammals. Fucose modifications have been identified as being associated with a number of biological functions, including immunomodulation and cancer. Sulfate-containing fucoidans rich in brown algae have anticoagulant, antithrombotic, immunomodulatory, anticancer, and antiproliferative activities. However, the yield of fucoidan in plants or algae is low, and its composition varies with climate and seasonal variations. The fucose-rich exopolysaccharides produced by the microorganisms are considered to be a better alternative in view of the advantages of microorganisms having higher growth rates and easier control of production conditions. In recent decades, other fucose-containing exopolysaccharides with the highest yield have been reported to be produced by Enterobacter (Enterobacter A47) at a yield of 13.23 g/L. The various physicochemical properties of the fucose-rich exopolysaccharide, such as rheological properties, adhesive properties, emulsifying capacity, and the use of biodegradable films, show important commercial value.

In previous studies, we isolated a Kosakonia strain and identified it as Kosakonia sp.cctcc M2018092, elucidating the genome-wide sequence and genetic characteristics of this strain (Complete genome sequence of Kosakonia sp.strain CCTCC 2018092, a fusase-Rich exopolysporac producer. singfengfengfeng Niu, 2019). However, no intensive structural and application studies have been carried out on the extracellular polysaccharide produced by Kosakonia sp.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an exopolysaccharide rich in fucose; the second purpose of the invention is to provide a preparation method of the fucose-rich exopolysaccharide; the invention also aims to provide the application of the exopolysaccharide rich in fucose in preparing degradable antibacterial materials; the fourth purpose of the invention is to provide a nano-silver antibacterial film containing the fucose-rich exopolysaccharide; the fifth purpose of the invention is to provide a preparation method of the nano-silver antibacterial film; the sixth purpose of the invention is to provide the application of the nano-silver antibacterial film in preparing antibacterial materials.

In order to achieve the purpose, the invention provides the following technical scheme:

1. exopolysaccharide rich in fucose, characterized in that: the Mw of the extracellular polysaccharide is 3.65 x 10⁵Da consists of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid according to the molar ratio of 2.03:1.00:1.18:0.64: 0.67. Preferably, the exopolysaccharide is prepared by fermentation of Kosakonia sp.cctcc M2018092.

Preferably, the exopolysaccharide has the following structural formula:

2. the preparation method of the fucose-rich exopolysaccharide is prepared by fermenting Kosakonia sp.CCTCC M2018092 strain.

Preferably, the pH of fermentation liquor of Kosakonia sp.CCTCC M2018092 strain is adjusted to 1-5 by sulfuric acid, the fermentation liquor is hydrolyzed to 0-10 hours at 50-80 ℃, thalli and calcium sulfate are removed by filtration, small molecules are removed by filtration by an ultrafiltration membrane with the cut-off quantity of 10kDa, after protein is removed, dialysis is carried out in deionized water by the cut-off molecular weight of 8000-14000Mw, and the extracellular polysaccharide rich in fucose is obtained by freeze-drying;

or adjusting the pH of the fermentation broth of the Kosakonia sp.CCTCC M2018092 strain to 1-5 by using sulfuric acid, then centrifuging to remove thalli and calcium sulfate, removing proteins from the supernatant by a Sevage method, dialyzing in deionized water by using molecular weight cut-off of 8000-14000Mw, and freeze-drying to obtain the fucose-rich extracellular polysaccharide.

3. The fucose-rich exopolysaccharide is applied to the preparation of degradable antibacterial materials.

4. The nano-silver antibacterial film containing the fucose-rich exopolysaccharide.

5. The preparation method of the nano-silver antibacterial film comprises the steps of mixing exopolysaccharide rich in fucose and AgNO₃Mixing the solutions, synthesizing nano-silver particles under ultraviolet excitation, and adding an extracellular polysaccharide solution to form a film to obtain the nano-silver antibacterial film.

Preferably, the concentration of the fucose-rich exopolysaccharide is 0.01-0.50 mg/mL, and the AgNO is₃The concentration is 2 mM; the ultraviolet excitation is carried out for 12 minutes under an ultraviolet lamp with the wavelength of 354 nm.

6. The use of the nano-silver antibacterial film of claim 7 in the preparation of antibacterial materials.

The invention has the beneficial effects that: the invention discloses a novel fucose-rich EPS, produced by Kosakonia sp.CCCCCC M2018092, the Mw of which is 3.65X 105Da, and the Mw of separated AH-EPS is 3.47X 104Da, the AH-EPS mainly comprises L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid, and the molar ratio is about 2.03:1.00:1.18:0.64: 0.67; the main linkage structure between sugar residues is elucidated by chemical analysis and NMR analysis and the three-dimensional structure of AH-EPS is determined to be a triple helix chain conformation, and these structural features of AH-EPS may provide key data for studying its potential applications. The purified AH-EPS can further be used as a reducing agent and stabilizer for the preparation of uniform silver nanoparticles (15-30 nm) without any further solvents and reagents. The EPS also has film-forming property, establishes an EPS film containing AH-EPS @ Ag NPs, and shows stronger antibacterial activity to streptococcus aureus. The antibacterial film only contains silver nanoparticles and polysaccharide, so that the antibacterial film has strong application potential in the aspect of developing novel biodegradable antibacterial materials.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a GPC chart of virgin EPS and AH-EPS.

FIG. 2 shows the complete acid hydrolysis product analysis of AH-EPS (A: HPLC of the complete acid hydrolysis product of AH-EPS; B: GC-MS total ion chromatogram of the complete acid hydrolysis product of AH-EPS).

FIG. 3 shows AH-EPS in D₂In O¹H NMR spectrum and¹³c NMR spectrum (a:¹h NMR spectrum; b:¹³c NMR spectrum).

FIG. 4 shows two-dimensional nuclear magnetic spectrum analysis (A: 2D) of AH-EPS¹H/¹³C HSQC spectrum; b: 2D¹H/¹³C HMBC spectra).

FIG. 5 shows two-dimensional nuclear magnetic spectrum analysis (A: 2D) of AH-EPS¹H/¹H COYY spectrum; b: 2D¹H/¹H NOESY spectrum).

FIG. 6 shows Fourier infrared spectroscopy and differential scanning calorimetry analysis (A: FT-IR spectrum of AH-EPS; B: DSC curve).

FIG. 7 is a graph of the maximum peak absorption change for Congo Red, Congo Red + AH-EPS and Congo Red + dextran complexes in various concentrations of sodium hydroxide solution.

FIG. 8 shows the main structure of AH-EPS.

FIG. 9 shows the preparation of EPS/nanosilver films and the antibacterial tests (A: UV-visible spectra of AH-EPS-synthesized silver nanoparticle solutions of different concentrations; B-C: TEM images of AH-EPS @ Ag NPs at different magnifications; D: size distribution of AH-EPS @ Ag NPs according to TEM analysis; E: pictures of EPS films and EPS/nanosilver films; F: growth inhibition zone for Staphylococcus aureus produced by EPS/nanosilver films, containing 0.89% (a), 0.44% (B), 0.22% (C) and 0% (D) AH-EPS @ Ag NPs).

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 preparation of microbial Extracellular Polysaccharide (EPS)

EPS is produced by fermentation of Kosakonia sp.CCTCC M2018092 strain under the condition of fed-batch, and the specific steps are as follows: culturing Kosakonia sp.CCTCC M2018092 strain in 30mL 250mL shake flask containing 30mL culture medium at 30 deg.C and 200rpm for 20 hr, transferring 30mL bacterial culture solution into 15L fermentation tank, and performing pre-growth culture at 30 deg.C and 300rpm for 13 hr (aeration amount of 1.5M)³H). Thereafter, 3L of the pre-grown bacterial broth was transferred to a 50L fermentor (containing 30L of medium) for fed-batch fermentation. A200 g/L glucose solution was fed in portions starting at a rate of 0.9-3.8rpm 13h after the start of the fermentation using a peristaltic pump according to the residual sugar amount. The ventilation of the fermentation tank is 1.5m³And/h, controlling the dissolved oxygen concentration to be more than 10% by automatically adjusting the rotating speed (300-550rpm) through the linkage of the rotating speed. The pH of the 50L fermenter was controlled at 7.0 by feeding sodium hydroxide and the temperature was controlled at 30 ℃. The composition of each medium during the cultivation is shown in Table 1.

TABLE 1 culture medium composition for extracellular polysaccharide production by Kosakonia sp.CCTCC M2018092 fermentation

And (3) extraction of extracellular polysaccharide:

the method comprises the following steps: the first route is that after the fermentation is finished, the pH value of the fermentation liquor is adjusted to 2.0 by using sulfuric acid, and then the fermentation liquor is centrifuged at 12000rpm for 20min to remove thalli and calcium sulfate; after the protein of the supernatant is removed by a Sevage method, deionized water is dialyzed (the cut-off molecular weight is 8000-14000Mw) and then freeze-dried to obtain the original fermentation polysaccharide (EPS), and the yield is 13.5 g/L. However, the small size of the cells requires high-speed centrifugation, which is not favorable for the industrial large-scale preparation of the polysaccharide.

The method 2 comprises the following steps: the extraction route is that after the fermentation is finished, the pH value of the fermentation liquor is adjusted to 2.0 by sulfuric acid, and the fermentation liquor is hydrolyzed for 4 hours at the temperature of 80 ℃. Then filtering through a 0.22 mu m ceramic membrane to remove thalli and calcium sulfate, and filtering through an ultrafiltration membrane with 10kDa cut-off quantity to remove micromolecules such as pigments and the like. After filtration and protein removal, dialysis (molecular weight cut-off 8000- < 14000 > Mw) and freeze-drying are carried out to obtain the partially hydrolyzed polysaccharide (AH-EPS) with the yield of 12.6 g/L.

Example 2 structural characterization of AH-EPS

The weight average molecular weights (Mw) of EPS and AH-EPS were determined by Gel Permeation Chromatography (GPC). A PL-GPC50 GPC integrated system (Agilent) equipped with a PLAquagel-OH mixed-H8 μm chromatographic column and a differential detector was used. Using 0.1M NaNO at 30 deg.C₃And 500ppm NaN₃The sample with the appropriate concentration is separated as eluent. The results show that EPS produced by Kosakonia sp.CCTCC M2018092 is a heterogeneous high molecular weight polysaccharide with Mw of about 3.65X 10⁵Da (Mw/Mn ═ 1.7). AH-EPS is homogeneous EPS with an average mass of 3.47X 10⁴Da (Mw/Mn ═ 1.2). Due to the advantages of large-scale production and homogeneity of AH-EPS, comprehensive structural characterization of AH-EPS is carried out.

1. High Performance Liquid Chromatography (HPLC) analysis of glycosyl composition

The monomer composition of AH-EPS was determined after complete acid hydrolysis. The purified AH-EPS (5mg/ml) was dissolved in 5ml of purified water, hydrolyzed at 120 ℃ for 2h after addition of 0.1ml of trifluoroacetic acid, and after removal of the trifluoroacetic acid, the sample was dissolved in 10mM sulfuric acid and analyzed by HPLC. The analysis conditions of High Performance Liquid Chromatography (HPLC) are as follows: xtimate (welch) Sugar-H column (7.8mm × 300mm,5 μm); column temperature: 40 ℃; flow rate: 0.5 ml/min; mobile phase: 10Mm sulfuric acid; a detector: and a difference detector (RI-201H). The monomer composition was determined by comparing the retention time with that of each monomer standard, and the results are shown as a in fig. 2. The results show that AH-EPS consists of fucose, glucose, glucuronic acid and galactose.

2. Gas chromatography-mass spectrometry (GC-MS) analysis of glycosyl composition

The above experimental results were further confirmed by thiol-acetate derivatization after complete acid hydrolysis and analysis using GC-MS. That is, 14.6mg of fucoidan was accurately weighed and dissolved in 0.5ml of xylose solution (8g/L), 3ml of TFA (2mol/L) was added, and the resulting solution was covered, heated in an oil bath at 120 ℃ for 2 hours, and dried with nitrogen at 55 ℃.2ml of ethanethiol and 1ml of trifluoroacetic acid were added and stirred magnetically for 25min in a water bath at 25 ℃. Blowing to dry with nitrogen at 55 ℃, then adding 4ml acetic anhydride-pyridine mixture (1: 1, V/V), magnetically stirring for 5 hours in a water bath at 55 ℃, then blowing to dry with 0.5ml nitrogen, redissolving in methanol, and injecting for GC-MS analysis.

The GC-MS conditions were: shimadzu (GCMS-QP2010, Japan), Rtx-5 capillary column (0.25mm × 30m), vaporization chamber temperature of 280 deg.C, high purity helium as carrier gas, and flow rate of 1 ml/min; the sample amount was 0.3 ul. Column temperature program: the initial temperature is 80 deg.C, holding for 2min, heating to 200 deg.C at a rate of 15 deg.C/min, heating to 210 deg.C at a rate of 1 deg.C, heating to 280 deg.C at a rate of 25 deg.C/min, and holding for 6 min. The result is shown as B in FIG. 2. The results showed that AH-EPS was composed of fucose, glucose, glucuronic acid and galactose, the glucuronic acid content was 14.62%, the absolute configuration of monosaccharides in AH-EPS was determined by GC-MS analysis of trimethylsilyl (-) -2-butylglucoside, and it was also found that AH-EPS was composed of L-fucose, D-glucose, D-galactose and D-glucuronic acid.

Acetylation of uronic acid was determined by GC-MS measurement of retention time and ion fragment in the molecule, and the results are shown in table 1, showing that the molar ratio of fucose, glucose, galactose and glucuronic acid in AH-EPS was 2.03:1.00:1.18: 0.64.

TABLE 1 acetylation of uronic acids

3. Pyruvic acid analysis

Pyruvic acid in AH-EPS was quantitatively analyzed at 215nm using an ultraviolet detector (SPD-16) on a Shimadzu (LC-16) HPLC system. AH-EPS (5.1mg) was dissolved in 3M TFA (4mL) and hydrolyzed at 120 ℃ for 2h, the sample was dried under nitrogen at 55 ℃ and redissolved in 100mL mobile phase, and after filtration, the sample was assayed. Detection conditions are as follows: wondasil C18 column (250X 4.6mm, 5 μm) and was washed with 98% K at 30 ℃₂HPO₄-H₃PO₄(0.1M K₂HPO₄-H₃PO₄pH 2.9) and 2% MeOH at a flow rate of 0.25 mL/min. The analysis result showed that the average content of pyruvic acid was 6.82%.

4. Methylation analysis

Methylation analysis of AH-EPS was performed using conventional methods with some modifications. Prior to methylation, pyruvate was removed by heating AH-EPS (5mg/ml AH-EPS solution in 1mM oxalic acid, 0.1M sodium chloride, pH 3.0) for 2h at 95 ℃ as per the Holzwarth and Ogletree studies. The solution was then neutralized with NaOH, dialyzed against deionized water and freeze-dried. In addition, the uronic acid should be reduced prior to methylation, and the uronic acid reduced AH-EPS prepared by reducing pyruvate-free AH-EPS with EDC, specifically AH-EPS (5mg) was added to 2mL of 75% THF-0.1mol/L MES solution and the pH adjusted to 4.75 using 10% Et3N, followed by EDC (20mg) addition and stirring at ambient temperature for 1 h. The reaction was then quenched with 2M acetic acid solution. The reaction solution was dialyzed 24 using a dialysis bag with a cut-off of 3.5kDa, and then lyophilized. And (3) re-dissolving the freeze-dried sample in 1.0ml of water, adding 0.5ml of 10% acetic acid-methanol solution, drying by nitrogen to remove boric acid generated in the reduction process, continuously adding 1.0ml of 10% acetic acid-methanol solution, drying by nitrogen, and repeating for 3-4 times. And finally, adding 0.5ml of methanol, blowing the mixture by using nitrogen, repeating the blowing for 3 times to ensure that the boric acid is completely removed, obtaining an uronic acid reduction sample, and drying the sample at 60 ℃ for 5 hours for methylation analysis.

AH-EPS (10mg) without pyruvic acid and uronic acid was dissolved in 0.1mL of water, and the solution was transferred to 3mL of DMMSO and mixed well. The water was then absorbed by 2g of 3A molecular sieve for 24 hours. After removing the molecular sieve, the sample was treated with CH₃I methylation and use of NaOH as in DMSOA catalyst. The methylated product was then hydrolyzed in 3M TFA at 120 ℃ for 2h and treated with NaBH at 25 ℃₄And reducing for 12 h. The sample was finally acetylated with acetic anhydride-pyridine (1: 1, v/v) at 55 ℃ for 5h and then analyzed by GC-MS, the results are shown in Table 2.

TABLE 2 results of methylation analysis

^aPartially methylated alditolacetate.

^bRelative to the 1,4-linked-fucose residue.

The results show that AH-EPS consists mainly of 1, 4-linked fucose, 1, 3-linked glucose, 1, 3-linked galactose and terminal galactose in a molar ratio of 1: 1.02: 1.63: 0.33: 0.68, and the AH-EPS chain consists of a unique branch point at fucose residue C3. Further based on previous studies, pyruvate was deduced to be linked to a terminal galactose.

5. Periodic acid oxidation and Smith degradation

AH-EPS (56mg) was dissolved in 50ml of a sodium periodate solution (0.015M) and stored in a refrigerator at 4 ℃. 0.2ml of the solution was taken at 12h intervals and made up to 50ml with purified water and the absorbance of the diluted solution at 233 nm. After the absorbance stabilized for 126h, the reaction was stopped by adding 4ml of ethylene glycol and a small amount of the aqueous solution was analyzed for formic acid by HPLC. The remaining reaction was dialyzed against purified water (cut-off 8000MW) and lyophilized. To the lyophilized product was added 3ml of sodium borohydride solution (26g/L) and reduced at room temperature for 22 h. The reduced product was hydrolyzed in an oil bath at 120 ℃ for 2h with 2ml TFA (3M), dried with nitrogen and redissolved in the mobile phase for HPLC analysis.

High Performance Liquid Chromatography (HPLC) analysis conditions: xtimate (welch) Sugar-H column (7.8mm × 300mm,5 μm); column temperature: 40 ℃; flow rate: 0.5 ml/min; mobile phase: 10Mm sulfuric acid; a detector: and a difference detector (RI-201H). Sample introduction volume: 15 μ L.

The results indicate the presence of glucose, galactose and fucose, and that glucose, galactose and fucose have 1 → 3 linkages. The formation of ethylene glycol and erythritol indicates the presence of a 4-substituted sugar group. The presence of 1,2, 3-butanetriol indicates the presence of a 1, 4-disubstituted fucose residue in AH-EPS.

6. Nuclear magnetic resonance spectroscopy

AH-EPS

Dissolution of the sample in D₂O and charged into a 5mm nuclear magnetic tube for NMR analysis.¹H and¹³CNMR spectra, two-dimensional nuclear magnetic spectra (including 2D)¹H-¹H COSY, HSQC, HMBC, NOESY, and TOCSY) were determined using a Bruker AvanceIII 600MHz NMR spectrometer to determine the sequence of sugar residues. The results are shown in FIGS. 3 to 5 and Table 3.¹The signal peak at 1.27ppm in the H NMR spectrum (FIG. 3, A) is typically the CH of the 6-deoxy sugar (fucose here)₃The signal at the group, δ 1.45, is attributed to CH of the acetonyl substituent₃. The integration data showed that the ratio between acetonyl and fucose residues was 1: 3, this is consistent with the results of the HPLC analysis. Thus, AH-EPS has a composition of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid in a molar ratio of 2.03:1.00:1.18:0.64: 0.67.¹³the C NMR spectrum (FIG. 3, B) shows two CH groups of fucose₃Signals (. delta.15.32 and 15.49ppm), one CH of the acetone substituent₃Signal (. delta.25.04 ppm) and two C ═ O group signals for the acetone substituent and the glucuronic acid substituent (. delta. 175.99 and 176.36ppm), at

The free hydroxyl C-6 signal of glucose and galactose was observed.

TABLE 3 AH-EPS in D₂In O¹H NMR spectrum and¹³c NMR spectra

¹H/¹³Six of the C HSQC spectra (FIG. 4, A)¹H/¹³The C signal (A-F, delta 5.40/99.35, 5.35/93.11, 5.35/99.25, 5.17/93.91, 4.99/101.00, 4.50/102.51ppm) anomeric protons (H-1) and anomeric carbons (C-1) due to sugar residues.signals above delta 4.9ppm are due to α -anomeric protons, while signals between delta 4.9-4.4ppm are due to β -anomeric protons.coupling constant analysis of bound anomeric signals determined that residues A-E have the α -configuration, residue F has the β -configuration, residue G represents an acetone substituent¹H/¹³In the C HMBC spectrum (FIG. 4, B), the methyl proton signal of pyruvate (1.45ppm) is at 102.04ppm¹³C signal coupling (O-C-O group of pyruvate). The results indicate that pyruvic acid participates in six-membered cyclic ketal formation including the O-4 and O-6 positions.

By passing¹H/¹H COSY，¹H/¹H NOESY，¹H/¹³C HSQC and¹H/¹h TOCSY experiments completed the A-G residues¹H and¹³assignment of C chemical shifts (Table 3). Low field shifts of the C-4(78.78) and C-6(69.31) carbon signals of residue B indicate that it is 1,4,6- α -D-Galp¹H/¹³In the C HMBC spectrum (FIG. 4, B), the methyl proton of fucose shows triple bond coupling with fucose C-4 at 1.27ppm (δ 79.80ppm) and two bond coupling with fucose C-5 (δ 67.41 ppm). C-5 of fucose is further coupled with anomeric protons of residue C (δ 5.35ppm) and residue E (δ 4.99ppm), indicating that residues C and E are fucose residues (FIG. 5, A). C-4 of fucose is coupled with anomeric protons of residue E in residue C, indicating that residue C is 1,4- α -L-Fucp, i.e., presence → 4) - α -L-Fucp- (1 → 4) - α -L-Fucp → 1 → a link, similarly, the anomeric proton coupling of residue E C-4 with residue F, i.e., presence → 3) - β -D-Glcp- (1 → 4) - α -L-Fucp- (1-Fucp → 7-Fucp → 1-3H → 7-3H → 1H → 3H → 1H → 3H → 1H → 3H → 1H → 3H → 1H →Residue D is α -D-GlcpA, and the last residue, residue B, may be linked only to residue D.

7. Fourier Infrared Spectroscopy and differential scanning calorimetry analysis

The FT-IR spectrum and DSC curve of AH-EPS were tested on a Shimadzu IRPresting-21 spectrometer and a TA Q200 thermal analyzer, respectively. FT-IR at 4000cm^-1To 400cm^-1Is performed in an atmosphere of air at a heating rate of 10 DEG K/min under an environment of 30 ℃ to 400 ℃. FT-IR spectrum of AH-EPS (FIG. 6, A) at 3429cm^-1Shows a strong peak due to the stretching vibration of O — H. 1650 and 1575cm^-1The peak at (a) belongs to the stretching vibration of group C ═ O. At 2928 and 2858cm^-1The peak at (a) is a characteristic peak of the sugar, particularly due to the stretching vibration of C-H. 1097cm^–1The peak at (b) reflects the tensile vibration of C-O-H and C-O-C, and 885cm^–1The DSC curve of AH-EPS (FIG. 6, B) shows a broad endothermic peak at 94.5 ℃ as a result of the dehydration process, further increasing the temperature, an exothermic band is observed up to 356.5 ℃ which is probably due to the change in three-dimensional structure and oxidation of AH-EPS.

Methylation analysis and NMR analysis identified a possible structural organization of AH-EPS by Smith degradation experiments, as shown in figure 8.

8. Congo Red test

A4 mg/mL AH-EPS solution (2mL) was mixed with 80. mu.M Congo red (2mL) and reacted at different NaOH concentrations (0.00M, 0.05M, 0.10M, 0.15M, 0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M and 0.50M) for 10 minutes. The maximum absorbance was measured by a UV-Vis spectrophotometer in the range of 400nm to 800 nm. In addition, a dextran (Mw ═ 40,000Da) solution to which congo red was added and a congo red solution without any polysaccharide were used as controls.

Polysaccharide chains exhibit different three-dimensional structures, such as triple helix chains, single random coil chains and random coil chains. Congo red can form a specific complex with triple helical polysaccharides in alkaline solutions. Maximum absorption wavelength (. lamda.) with increasing NaOH concentration_max) A red shift will occur. As shown in fig. 7Lambda of Congo Red at NaOH concentrations close to 0.2M_maxIncreasing to a maximum value. While congo red lambda with or without dextran_maxThe decrease with increasing NaOH concentration was gradual until a constant value was reached, which indicated that AH-EPS had a triple helix conformation.

Example 3 preparation of EPS/Nanosilver film and antibacterial testing

Different concentrations (0.01mg/mL, 0.03mg/mL, 0.05mg/mL, 0.10mg/mL, 0.20mg/mL, 0.50mg/mL) of AH-EPS solution and 2mM AgNO₃The solution was mixed into 5mL of water and shaken for 30 minutes. Thereafter, the solution was irradiated under a 354nm UV lamp for 12 minutes to prepare silver nanoparticles, and the nano silver solutions were respectively added (0mL, 1.5mL, 3.0mL, 6.0mL) to 25mL eps (1.5 wt%) solutions, and then the solutions were poured into a plastic plate (d ═ 6.5cm), and dried at 50 ℃ for 12 hours to form a thin film. The film was peeled off and cut into a sheet shape (d ═ 0.5cm) for antibacterial testing using the Kirby-Bauer method, using gram-positive staphylococcus aureus (ATCC29213) as an experimental bacterium. The absorption spectra of silver nanoparticle solutions prepared with different concentrations of AH-EPS are shown as a in fig. 9. The results show that the UV-vis spectrum of the solution shows the strongest absorption peak of silver nanoparticles at 423nm when the concentration of AH-EPS is 0.05 mg/mL. Therefore, 0.05mg/mL AH-EPS was used to prepare silver nanoparticles for further characterization and application. This phenomenon is caused by the fact that AH-EPS contains abundant-OH groups, which can convert Ag to⁺Reduction to Ag⁰. The ratio of reducing agent and stabilizer in the reaction has a significant effect on the morphology and size distribution of the silver nanoparticles.

TEM image of 0.05mg/mL AH-EPS preparation of silver nanoparticles it was confirmed that the silver nanoparticles covered with AH-EPS (AH-EPS @ Ag NPs) were spherical with an average diameter of 20nm (FIG. 9, B-D). EPS films and EPS/nanosilver films were prepared containing varying amounts of AH-EPS @ Ag NPs (fig. 9, E). The antibacterial activity of the EPS/nano-silver film is analyzed by a disc diffusion method, and an obvious inhibition zone appears around the EPS/nano-silver film, which shows that AH-EPS @ Ag NPs effectively inhibit the growth of staphylococcus aureus (figure 9, F). The inhibition zones of 0mm, 12.3mm, 14.1mm and 16.5mm for the growth of staphylococcus aureus of the EPS/nano-silver film containing 0, 0.22%, 0.44% and 0.89% AH-EPS @ Ag NP are respectively. The antibacterial ability showed dose dependence. And after 12 hours of plating, the EPS-based antibacterial film disappeared and fused into the medium. Therefore, the novel polysaccharides (EPS and AH-EPS) found in this study have potential application prospects in the development of novel water-soluble and biodegradable antibacterial materials.

It was found from the above studies that Kosakonia sp.CCCCCC M2018092 produces a novel fucose-rich EPS having Mw of 3.65X 10⁵Da, while the Mw of the isolated AH-EPS is 3.47X 10⁴Da. AH-EPS consists essentially of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid in a molar ratio of about 2.03:1.00:1.18:0.64: 0.67. the main linkage structure between sugar residues is also elucidated by chemical analysis and NMR analysis, as shown in fig. 8, and the three-dimensional structure of AH-EPS is a triple helix chain conformation. The purified AH-EPS can further be used as a reducing agent and stabilizer for the preparation of uniform silver nanoparticles (15-30 nm) without any further solvents and reagents. Based on the EPS film-forming property, an EPS film containing AH-EPS @ Ag NPs is established, and has stronger antibacterial activity on the streptococcus aureus. The antibacterial film only contains silver nanoparticles and polysaccharide, so that the antibacterial film has strong application potential in the aspect of developing novel biodegradable antibacterial materials.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. Exopolysaccharide rich in fucose, characterized in that: the Mw of the extracellular polysaccharide is 3.65 x 10⁵Da consists of L-fucose, D-glucose, D-galactose, D-glucuronic acid and pyruvic acid according to the molar ratio of 2.03:1.00:1.18:0.64: 0.67.

2. Fucose-rich exopolysaccharide according to claim 1, characterized in that: the exopolysaccharide is prepared by fermentation of Kosakonia sp.CCTCC M2018092.

3. Fucose-rich exopolysaccharide according to claim 1 or 2, characterized in that: the structural formula of the exopolysaccharide is as follows:

4. the method for preparing fucose-rich exopolysaccharide of any one of claims 1 to 3, wherein: prepared by fermentation of Kosakonia sp.CCTCC M2018092 strain.

5. The method for preparing fucose-rich exopolysaccharides as claimed in claim 4, wherein: adjusting the pH of fermentation liquor of Kosakoniasp.CCTCC M2018092 strain to 1-5 by using sulfuric acid, hydrolyzing for 0-10 hours at 50-80 ℃, filtering to remove thalli and calcium sulfate, filtering to remove small molecules by using an ultrafiltration membrane with the cut-off value of 10kDa, removing proteins, dialyzing by using the cut-off molecular weight of 8000-14000Mw in deionized water, and freeze-drying to obtain extracellular polysaccharide rich in fucose;

6. Use of the fucose-rich exopolysaccharide of any one of claims 1 to 3 in the preparation of degradable antibacterial materials.

7. A nanosilver antimicrobial film comprising the fucose-rich exopolysaccharide of claim 6.

8. The method of claim 7 for preparing nano silver antibacterial filmThe preparation method is characterized by comprising the following steps: mixing fucose-rich exopolysaccharide with AgNO₃Mixing the solutions, synthesizing nano-silver particles under ultraviolet excitation, and adding an extracellular polysaccharide solution to form a film to obtain the nano-silver antibacterial film.

9. The method for preparing a nano-silver antibacterial film according to claim 8, characterized in that: the concentration of the fucose-rich extracellular polysaccharide is 0.01-0.50 mg/mL, and the AgNO is₃The concentration is 2 mM; the ultraviolet excitation is carried out for 12 minutes under an ultraviolet lamp with the wavelength of 354 nm.

10. The use of the nano-silver antibacterial film of claim 7 in the preparation of antibacterial materials.