Exopolysaccharide of lactobacillus fermentum as well as extraction method and application

Abstract

The invention belongs to but not limited to the technical field of biology, and discloses extracellular polysaccharide of lactobacillus fermentum and an extraction method and application thereof. The average molecular weight of the EPS-W0310 is 2.94 kDa, and the EPS-W0310 is mainly prepared from mannose, galactose, glucose and glucosamine, and the molar percentages of the mannose, the galactose, the glucose and the glucosamine are 38.67%, 29.93%, 26.90% and 4.48% respectively. The main chain structure of EPS-W0310 may be composed of a mannan structural domain and a furan galactan structural domain, the surface is smooth and convex, the shape is irregular, but the structure is compact and firm. And the EPS-W0310 has very high thermal stability at the temperature of 314.16 DEG C. The EPS of the lactobacillus fermentum W03 has better oxidation resistance, has great potential in the aspect of food application, and also has a certain anti-inflammatory effect and an inflammation improving effect.

Description

Technical Field

[0001] The invention belongs to but is not limited to the field of biotechnology, and in particular relates to an extracellular polysaccharide of fermented lactobacillus and an extraction method and application thereof. Background Art

[0002] Lactobacillus (LAB) is a common probiotic, which is generally considered safe (GRAS) and has been widely used in commerce. Lactobacillus fermentum is a species of lactic acid bacteria with extremely high safety. Studies have found that Lactobacillus fermentum strains are often used as natural additives in food production, such as yogurt, sauerkraut, ham, and soy products, while there are relatively few studies on the application of extracellular polysaccharides produced by Lactobacillus fermentum metabolites.

[0003] Extracellular polysaccharide (EPS) is a biopolymer secreted outside the cell, which forms capsules in the form of mucus or combined with the surface of bacterial cells. EPS is divided into homopolymeric polysaccharides (HoPS, composed of one monosaccharide) and heteropolymeric polysaccharides (HePS, composed of two or more monosaccharides). EPS has some important physical and chemical properties and has potential application prospects in the food industry as a stabilizer, gelling agent, emulsifier, flocculant, absorbent and heavy metal absorbent. At the same time, it also plays an important role in various industries such as biology, pharmaceuticals, medicine and cosmetics. Lactic acid bacteria EPS has a variety of physical and chemical properties and biological activities, which is related to the diversity of its chemical structure. The structure of EPS is closely related to the composition of the culture medium, culture conditions and sugar-producing strains.

[0004] So far, there are relatively few studies on the application of extracellular polysaccharides of Lactobacillus fermentum. The key aspect of EPS is to determine the absolute configuration of the carbohydrate part and its derivatives. For polysaccharides of unknown structure, the combination of analytical methods such as Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy is an excellent tool for determining its structure. These techniques have proven their importance in determining the absolute configuration of EPS sugar residues. In this study, the extracellular polysaccharides of the strain were isolated from Lactobacillus fermentum W03, and its physicochemical properties were analyzed, purified, and its structure was resolved. At the same time, its in vitro antioxidant activity was evaluated, which can provide more new research for the study of extracellular polysaccharides of Lactobacillus fermentum, explore its potential applications in more fields, and contribute to human development. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides an extracellular polysaccharide of fermented lactobacillus and an extraction method and application thereof.

[0006] The present invention is achieved by providing an extracellular polysaccharide of fermented lactobacillus. The main chain structure of the extracellular polysaccharide EPS-W0310 of fermented lactobacillus W03 may be composed of two parts, one part is a mannan domain composed of →2)-α-D-Manp-(1→ sugar residues and →2,6)-α-D-Manp-(1→ sugar residues, and the other part is a mannan domain composed of →2,6)-β-D-Galf-(1→ sugar residues and →6)-β-D-Ga The furanose galactan domain is composed of lf-(1→ sugar residues, and its branched structure mainly includes α-D-Manp-(1→ and α-D-Glcp-(1→ sugar residues, and may also contain →4)-β-D-GlcpNAc-(1→, →3)-β-D-Glcp-(1→, α-D-Manp-(1→3)-α-D-Manp-(1→ and other sugar residue fragments. Furthermore, EPS-W0310 at 1681.6 cm -1 There is a polysaccharide functional group at the site, which is the fingerprint region of carbohydrates.

[0007] Furthermore, EPS-W0310 is composed of mannose, galactose, glucose and glucosamine.

[0008] Furthermore, EPS-W0310 has a smooth and glossy surface and a tight structure.

[0009] Another object of the present invention is to provide a method for extracting extracellular polysaccharides of fermented lactobacillus, and obtain crude polysaccharide of fermented lactobacillus W03 through fermentation, deproteinization, ethanol precipitation, dialysis and other processes; first, the crude EPS is purified by an anion exchange chromatography column; the fractions from the main peak are collected, named EPS-W031, and further separated using a gel chromatography column to obtain a single peak, named EPS-W0310.

[0010] The method for preparing the extracellular polysaccharide of Lactobacillus fermentum W03 is also within the scope of protection of the present invention. Specifically, the extracellular polysaccharide of Lactobacillus fermentum W03 is prepared by fermentation using the strain CCTCC NO: M20242010 of Lactobacillus fermentum W03.

[0011] Wherein, the fermented lactobacillus W03 exopolysaccharide is produced by fermenting the fermented lactobacillus W03 (Limosilactobacillusfermentum W03) strain inoculated in MRS medium. The strain is deposited with CCTCC NO: M 20242010, deposited on September 20, 2024, and deposited in the China Center for Type Culture Collection, referred to as CCTCC, classified and named: Limosilactobacillus fermentum fermented lactobacillus W03, deposited at: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072, Tel: 027-68752319, Email: cctcc@whu.edu.cn.

[0012] Another object of the present invention is to provide an application of extracellular polysaccharide of fermented lactobacillus in the field of food.

[0013] Another object of the present invention is to provide an application of extracellular polysaccharide of fermented lactobacillus in the preparation of anti-inflammatory drugs.

[0014] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0015] First, the present invention extracts EPS from fermented Lactobacillus W03, purifies it with an anion exchange chromatography column (EPS-W031), and further elutes it with a gel chromatography column (EPS-W0310). The solubility, water holding capacity and thermal stability of EPS-W031 and EPS-W0310 were measured, and the results showed that the physicochemical properties of EPS-W0310 were better than those of EPS-W031. The particle sizes of EPS-W031 and EPS-W0310 were 723±1.23nm and 349.1±1.28nm, respectively. The zeta potentials of EPS-W031 and EPS-W031 were -25.51±2.29mV and -18.36±1.62mV, respectively. In addition, both EPS-W031 and EPS-W0310 exhibited strong antioxidant activity. The scavenging rates of EPS-W0310 and EPS-W0310 for DPPH were 66.78% and 81.08%, respectively, while the scavenging rates for ABTS were 58.09% and 59.20%, respectively. The above data show that EPS-W0310 exhibits better physicochemical and antioxidant properties, and its structure will be further characterized. The average molecular weight of EPS-W0310 is 2.94 kDa, and it is mainly composed of mannose, galactose, glucose and glucosamine, with molar percentages of 38.67%, 29.93%, 26.90% and 4.48%, respectively. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that the surface of EPS-W0310 is smooth and convex, with an irregular shape, but a compact and strong structure. It was inferred that the structure of EPS-W0310 may be composed of two parts, one of which is the mannan domain composed of →2)-α-D-Manp-(1→ sugar residues and →2,6)-α-D-Manp-(1→ sugar residues, and the other is the furanose galactan domain composed of →2,6)-β-D-Galf-(1→ sugar residues and →6)-β-D-Galf-(1→ sugar residues. Its branched structure mainly contains α-D-Manp-(1→ and α-D-Glcp-(1→ sugar residues, and may also contain →4)-β-D-GlcpNAc-(1→, →3)-β-D-Glcp-(1→, α-D-Manp-(1→3)-α-D-Manp-(1→ and other sugar residue fragments. In short, EPS-W0310 has great potential in food applications.

[0016] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0017] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0018] The extracellular polysaccharide of Lactobacillus fermentum W03 prepared by the present invention has excellent in vitro antioxidant activity and can be used to develop natural, non-toxic and side-effect-free functional foods or drugs for preventing or alleviating inflammatory diseases.

[0019] (2) The technical solution of the present invention solves a technical problem that people have been eager to solve but have never been able to solve successfully:

[0020] The present invention utilizes fermentation of Lactobacillus fermentum W03 to prepare a polysaccharide EPS-W0310 with a molecular weight of 2.94 kDa. The preparation method of the extracellular polysaccharide is simple, the conditions are easy to control, the cost is low and the cycle is short, and large-scale production can be achieved. It is expected to replace traditional high-cost fruiting body polysaccharides, be widely used in the food industry, and can also be used as an anti-inflammatory drug, thereby reducing the side effects of traditional drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The purification curve diagram of the fermented Lactobacillus W03 EPS provided in the embodiment of the present invention is as follows: (A) anion exchange chromatogram; (B) gel chromatography chromatogram; (C) freeze-dried diagram of the purified EPS, a): EPS-W031; b): EPS-W0310.

[0022] Figure 2 1 and 2. The figures are the thermal analysis diagrams of the EPS of Lactobacillus fermentum W03 provided in the embodiments of the present invention, (A) EPS-W031, (B) EPS-W0310; TGA curve (black line), DSC curve (red line), and DTG curve (blue line).

[0023] Figure 3 This is a water holding capacity and solubility analysis diagram of the Lactobacillus fermentum W03 EPS provided in an embodiment of the present invention.

[0024] Figure 4 It is the particle size diagram (A) and potential diagram (B) of the extracellular polysaccharides EPS-W031 and EPS-W0310 of Lactobacillus fermentum W03 provided in the embodiments of the present invention.

[0025] Figure 5 It is a schematic diagram of the absorption wavelength of Congo red and its EPS complex provided in an embodiment of the present invention.

[0026] Figure 6 The in vitro antioxidant activity of EPS of Lactobacillus fermentum W03 provided in the embodiment of the present invention is shown in FIG. EPS uses ascorbic acid (Vc) as a positive control. (A) DPPH free radical scavenging activity, (B) ABTS free radical scavenging activity.

[0027] Figure 7(A) Molecular weight analysis chart of the extracellular polysaccharide EPS-W0310 of Lactobacillus fermentum W03 provided in an embodiment of the present invention; (B) UV spectrum analysis chart; (C) Monosaccharide composition analysis chart.

[0028] Figure 8 This is a graph showing the functional group determination results of the extracellular polysaccharide EPS-W0310 of Lactobacillus fermentum W03 provided in an embodiment of the present invention.

[0029] Fig. 9 The electron microscope scanning images of the extracellular polysaccharide EPS-W0310 of Lactobacillus fermentum W03 provided in the embodiments of the present invention are as follows: (A): scanning electron microscope (SEM); (B): atomic force scanning electron microscope (AFM). DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] The present invention relates to the structure and characteristics of an extracellular polysaccharide (EPS-W0310) of fermented lactobacillus W03. The polysaccharide is secreted by the strain during fermentation and has a diverse sugar residue structure, characterized by a special main chain and branched chain structure. This complex structure may provide a wide range of potential for its application in food, pharmaceuticals and functional materials. The following is a detailed description of the structure of EPS-W0310:

[0032] 1. Main chain structure

[0033] The main chain structure of EPS-W0310 may be composed of two parts, namely the mannan domain and the furangalactan domain. These two parts together give EPS-W0310 unique chemical properties.

[0034] Mannan Domain:

[0035] This part is mainly composed of →2)-α-D-Manp-(1→ sugar residues and →2,6)-α-D-Manp-(1→ sugar residues. α-D-Mannose (Manp) residues are connected to each other through 1,2 and 1,2,6 glycosidic bonds to form the main chain structure of mannan. This structure makes it show high branching degree and good water solubility in the molecule.

[0036] The presence of this structural domain enables EPS-W0310 to form a relatively stable colloidal structure in the solution, which helps to maintain a high viscosity in the solution and is suitable for use as a thickener, stabilizer, etc.

[0037] Galactofuranose Domain:

[0038] This part is composed of →2,6)-β-D-Galf-(1→ sugar residues and →6)-β-D-Galf-(1→ sugar residues, forming a chain structure with β-D-galactose (Galf) as the main body.

[0039] The β-D-furanose form of this domain has higher flexibility, which is conducive to the curling and folding of the molecular chain, thus having good rheological and gel-forming properties in solution. This structure may also affect its biological activities such as antioxidant and immunomodulatory.

[0040] 2. Branched chain structure

[0041] In addition to the main chain structure, EPS-W0310 also has a relatively complex branched chain structure. These branches include α-D-mannose (Manp) and α-D-glucose (Glcp) residues. Possible branched chain structures include the following:

[0042] α-D-Manp-(1→Sugar residue:

[0043] As a major branching residue, α-D-Manp can further connect with α-D-Manp or β-D-Galf residues on the main chain to form branch points. Its terminal structure is usually exposed on the surface of the polysaccharide molecule and may play a role in the interaction with external molecules.

[0044] α-D-Glcp-(1→Sugar residue:

[0045] α-D-glucose (Glcp), as a branched residue, enhances the water solubility of EPS-W0310 and may bring it additional biological activities, such as antioxidant or intestinal flora balance.

[0046] Other branched structures that may be included:

[0047] The presence of the N-acetylglucosamine (GlcpNAc) residue may endow EPS-W0310 with more biological activities, such as anti-inflammatory and antibacterial activities.

[0048] →3)-β-D-Glcp-(1→: This residue may further extend the length of the side chain, increasing the volume and branching of the molecule.

[0049] α-D-Manp-(1→3)-α-D-Manp-(1→: This unique dismannose residue connection mode helps to improve the stability and branching density of polysaccharides, which is beneficial to their application in biomedicine.

[0050] 3. Chemical and biological properties

[0051] Structural stability: Due to the combination of mannan and furan galactan in EPS-W0310, it has high thermal stability and can still maintain its structural and functional stability under high temperature conditions.

[0052] Water solubility and viscosity: EPS-W0310 has excellent water solubility and high viscosity, and is suitable for use as a thickener or suspending agent in food and medicine. At the same time, these properties give it good application prospects in the fields of colloids, emulsion stabilizers, etc.

[0053] Biological activity: Sugar residues such as mannose, glucose and galactose contained in EPS-W0310 may give it biological activities such as antioxidant, anti-tumor, immunomodulatory and intestinal flora balance.

[0054] Safety and biocompatibility: As a common probiotic species, EPS-W0310 is from a safe source and meets the safety requirements of food-grade additives. It also has certain application potential in intestinal flora balance and anti-inflammation.

[0055] 4. Application potential

[0056] This polysaccharide has the following application potentials:

[0057] Food industry: Due to its good viscosity and water solubility, EPS-W0310 can be used as a thickener, stabilizer, emulsifier, etc., and is widely used in dairy products, beverages, condiments and other fields.

[0058] Medical and health products: In medicine, EPS-W0310, as a product of probiotics, has potential activities of antioxidant, anti-inflammatory and immunomodulatory effects, and is suitable for use in health products to enhance health effects.

[0059] Biomaterials: Due to its good biocompatibility, EPS-W0310 may become a candidate for tissue engineering and drug sustained-release materials, especially in the application of bone, cartilage and other tissue repair.

[0060] In summary, the main chain and branched chain structure of EPS-W0310 make it excellent in functionality and stability, and provide broad prospects for its application in food, medicine, health products and biomaterials.

[0061] The embodiment of the present invention provides an extracellular polysaccharide of fermented lactobacillus. The main chain structure of the extracellular polysaccharide EPS-W0310 of the fermented lactobacillus W03 may be composed of two parts, one part is a mannan domain composed of →2)-α-D-Manp-(1→ sugar residues and →2,6)-α-D-Manp-(1→ sugar residues, and the other part is a furanose galactan domain composed of →2,6)-β-D-Galf-(1→ sugar residues and →6)-β-D-Galf-(1→ sugar residues. Its side chain structure mainly includes α-D-Manp-(1→ and α-D-Glcp-(1→ sugar residues, and may also include →4)-β-D-GlcpNAc-(1→, →3)-β-D-Glcp-(1→, α-D-Manp-(1→3)-α-D-Manp-(1→ and other sugar residue fragments.

[0062] The structural characteristics of EPS-W0310 were analyzed by Fourier transform infrared spectroscopy, HPGPC and scanning electron microscopy. The results of Fourier transform infrared spectroscopy showed that EPS-W0310 had a peak at 1681.6 cm -1 There is a polysaccharide functional group at the center, which is the fingerprint region of carbohydrates. HPGPC shows that EPS-W0310 is composed of mannose, glucose, galactose and glucosamine. Scanning electron microscopy shows that EPS-W0310 has a smooth, shiny surface, spherical shape and a compact structure. Thermogravimetric analysis shows that EPS-W0310 has high thermal stability at 314.16°C. The high thermal stability of EPS is a necessary condition for its industrial application. The antioxidant properties of EPS were analyzed by DPPH and ABTS. The EPS of Lactobacillus fermentum W03 has better antioxidant properties. This work has established a new scientific basis for the structure and physicochemistry of the exopolysaccharide of Lactobacillus fermentum W03, laying the foundation for its application and potential in the food field.

[0063] 1. Extraction and purification of EPS

[0064] The crude polysaccharide of lactobacillus W03 was obtained by fermentation, deproteinization, ethanol precipitation, dialysis and other processes. The crude EPS was first purified by an anion exchange column, and a main peak and two secondary peaks were observed ( Figure 1 A), the main peak was named EPS-W031, and fractions from the main peak were collected and further separated using a gel chromatography column. Figure 1 As shown in B, a single peak was obtained, named EPS-W0310. Finally, the fractions of EPS-W031 and EPS-W0310 were collected and freeze-dried to obtain the purified polysaccharide, as shown in Figure 1 C, wherein a) is the polysaccharide component EPS-W031 obtained by preliminary purification and elution through cellulose filler; b) is the polysaccharide component EPS-W0310 obtained by secondary purification and elution through gel chromatography column. Figure 1It can be seen from C that the polysaccharides obtained after two elutions are all flocculent, and from the color point of view, the polysaccharide eluted for the second time is lighter than the second time, indicating that a small amount of pigment can be washed away during the elution process.

[0065] 2. Physical and chemical properties of EPS-W0310

[0066] 2.1 Thermal stability analysis

[0067] Thermal analysis of EPS-W031 and EPS-W0310 Figure 2 As shown in the TGA curve, the extracellular polysaccharide of Lactobacillus fermentum W03 mainly experienced two stages of weight loss. The first stage was the weight loss at 35°C to 105°C (10-15%). The weight loss in this stage was due to the large amount of carboxyl groups in EPS, which led to the elimination of polymer moisture as the temperature increased, including physical absorption and structural water. The second stage was the weight loss above 200°C, which was due to the depolymerization of EPS, including chemical reactions and changes in functional groups. As the temperature rises, EPS will form polynuclear aromatic carbon and graphite carbon structures. At the same time, the decomposition caused by the breaking of C=C and CO bonds leads to the generation of CO, CO2 and H2O. These two are the main reasons for the weight loss of EPS.

[0068] from Figure 2 From the DTG curves in Figure 1, EPS-W031 and EPS-W0310 have two obvious peaks, one of which is around 250℃~300℃, which is related to the bound water in EPS. The peak values ​​of EPS-W031 and EPS-W0310 are 315.16℃ and 314.16℃ respectively, which are the degradation temperatures (Td) at which the weight of EPS decreases fastest. These temperatures are higher than the results of EPS production by Lactobacillus acidophilus LA5 (266.7℃).

[0069] The DSC curve is used to evaluate the heat absorption and heat release of EPS as the temperature rises. From the curve change results, it can be seen that EPS-W031 and EPS-W0310 both show two peaks. The first endothermic peak is 38.83℃ and 38.66℃, which is related to the evaporation of water and the melting of the crystal structure formed by the long fat chain of EPS molecules. The second endothermic peak (melting temperature T m ) are 191.83℃ and 348.83℃, respectively, which is related to the dehydration and depolymerization of EPS. The results of the corresponding TGA and DTG curves show that EPS-W0310 has broad application prospects in the fields of food, pharmaceuticals, and chemicals. m T higher than that of EPS produced by Lactobacillus acidophilus LA5 m (257.4℃), which reflects that EPS-W0310 can be used in heat treatment process.

[0070] 2.2 Water holding capacity and solubility analysis

[0071] The solubility (WSI) and water holding capacity (WHC) of EPS are as follows: Figure 3 As shown. EPS-W0310 showed a WSI of 18.03±1.45% and a WHC of 122.56±3.79%. The WSI and WHC of EPS-W031 were 22.46±2.30% and 120.97±3.45%, respectively. In this study, EPS had high water solubility and good water holding capacity. These results indicate that EPS has permeable polymer chains that can retain a large amount of water through hydrogen bonds and has potential application value as a water binder.

[0072] 2.3 Particle size and Zeta potential analysis

[0073] The particle size of nanoparticles is one of the important factors affecting the delivery efficiency of nanoparticles. The dynamic light scattering method was used to determine the effect of EPS-W031 and EPS-W0310 on the particle size at a concentration of 2 mg / ml ( Figure 4 A). The particle size of EPS-W031 is 123.1nm, and the particle size of EPS-W031 is 223.8nm.

[0074] Zeta potential is a key indicator used to characterize the mutual attraction and repulsion between polymers and is used to evaluate the stability of polymers. When the absolute value of Zeta potential is large, the solution system has a higher stability. Figure 4 B) It can be seen that all EPS have negative charges, which is related to the presence of sulfate / aldehyde groups in the functional groups. The negative charge intensity of EPS-W0310 is -18.36±1.62mV, and the negative charge intensity of EPS-W031 is -25.51±2.29mV. The negative charge in EPS may be caused by functional groups, thus affecting biological activity. Existing studies have shown that negatively charged EPS has higher biological activity. Generally speaking, the larger the absolute value of Zeta potential, the smaller the particle size of the polysaccharide molecule, the easier it is for the polysaccharide molecule to dissolve or disperse in the aqueous solution system, and the better its solubility and water retention.

[0075] 2.4 Congo red test

[0076] Figure 5 is the maximum absorption wavelength of Congo red and its EPS complex (λ max ). In different concentrations of NaOH solution, Congo red was compared with the purified polysaccharide fractions WPS-W031 and EPS-W0310 of Lactobacillus fermentum W03 exopolysaccharide, and no λ was observed with the increase of NaOH concentration. maxThe obvious red shift phenomenon indicates that WPS-W031 and EPS-W0310 do not have a regular triple helical structure and may exist in an irregular helix rather than a triple helix conformation. max The pattern of first decreasing and then increasing shows that it may contain an irregular triple helical structure. In addition, the λ max There is a gradually weakening trend with the increase of NaOH concentration, which further indicates that EPS-W0310 has a triple helix structure and its structure changes in a higher concentration alkaline environment.

[0077] 3. Analysis of the antioxidant activity of EPS-W0310

[0078] The antioxidant activity of EPS extracts was evaluated by scavenging DPPH free radicals and scavenging ABTS free radicals. The DPPH experiment showed that all EPS samples had significant antioxidant capacity. Among them, EPS-W0310 had the highest scavenging capacity (81.08%), followed by EPS-W031 (66.78%) ( Figure 6 A). In addition, the scavenging effects of EPS-W031 and EPS-W0310 on ABTS free radicals are shown in Figure 6 As shown in Figure B, at a concentration of 4 mg / mL, the clearance rates of EPS-W031 and EPS-W0310 were 58.09% and 59.20%, respectively.

[0079] 4. Characterization of EPS-W0310

[0080] 4.1 Molecular weight and monosaccharide composition

[0081] In the HPGPC analysis, EPS-W0310 showed a relatively symmetrical sharp peak, which indicated that it was homogeneous ( Figure 7 A). The Mw of EPS-W0310 is 2.94 kDa. The molecular weight of LAB EPS varies depending on the specific strain and culture conditions. The difference in molecular weight will affect the solubility and antioxidant properties of LAB EPS. In addition, the UV-visible spectrum of EPS-W0310 has no obvious peaks at 280 or 260 nm, further confirming that proteins and nucleic acids have been completely removed ( Figure 7 B).

[0082] It is well known that the properties of EPS are affected by its monosaccharide content. HPGPC analysis showed that EPS-W0310 is composed of mannose, galactose, glucose and glucosamine ( Figure 7 C), the molar percentages are 38.67%, 29.93%, 26.90% and 4.48% respectively.

[0083] 4.2 Fourier transform infrared spectroscopy analysis

[0084] The main functional groups and chemical bonds of EPS-W0310 were analyzed by FT-IR spectroscopy. Figure 8 As shown. At 3353.6cm -1 There is a strong and broad peak at 2948.6 cm-1, which corresponds to the stretching vibration of hydroxyl (OH). -1 The absorption peak at 1681.6cm belongs to the CH stretching vibration of alkanes, while the absorption peak at 1681.6cm -1 The absorption peak at 1103.0 cm -1 A peak was found at 727.0 cm, which may be the ring vibration overlapping the stretching vibration of the COH group, indicating that the polysaccharide is in the pyranose form. -1 The characteristic signal at 619.0 cm reflects the α-glycoside peak. -1 The characteristic signals nearby reflect the α-configuration of the mannose unit.

[0085] 4.3 Atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis

[0086] The SEM image of EPS-W0310 is shown in Fig. 9 As shown in A and B, the images show smooth, irregular flake structures and smooth structures, indicating that EPS-W0310 can be used to make flexible film materials. Fig. 9 B) It was observed that the surface structure of EPS is smoother, which also shows that EPS-W0310 can change the rheology of food, increase viscosity and water retention capacity. In previous reported studies, similar structures were found in EPS produced by Lactobacillus plantarum KF5 and Mesenteric bacteria XR1. A large number of studies have shown that this type of EPS can be used as a thickener and emulsifier in food, and has the ability to retain moisture in food and produce plastic films. A small number of EPS-W0310 present spherical structures of different diameters, indicating that the sugar chains of EPS have not been fully unfolded and are entangled with each other to form agglomerates, which is related to the hydrogen bonding within the EPS molecules and its internal triple helix structure.

[0087] AFM is a tool for further understanding of the morphological characteristics of EPS. It is a tool that can quantify and characterize the surface protrusions of macromolecular polymers at the nanoscale and is an effective tool that can be directly applied to observe the conformation of polysaccharides. EPS-W0310 image presented by AFM ( Fig. 9 C, D), the size is 20μm×20μm, the height is 100μm, and the images show that the surface of EPS-W0310 is rough, with protrusions of different heights and some irregular substances (such as Fig. 9C). EPS has a rough surface with good water retention and biocompatibility, and has broad application prospects in the medical field. The protrusions of different heights may be due to certain regions or side chains of the polysaccharide molecular chain, which may have specific functions, such as interaction with other molecules to regulate the stability and solubility of EPS. The protrusions will affect the morphology and stability of EPS, thereby affecting its function and application in the body. The irregular shape may be a microstructure formed by the self-assembly of EPS molecules, or it may be a complex formed by the interaction of EPS with other molecules, such as proteins and phospholipids.

[0088] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. An exopolysaccharide of fermented lactobacillus, characterized in that: The main chain structure of the extracellular polysaccharide EPS-W0310 includes: a part of a mannan domain composed of →2)-α-D-Manp-(1→ sugar residues and →2,6)-α-D-Manp-(1→ sugar residues; the other part is a furanose galactan domain composed of →2,6)-β-D-Galf-(1→ sugar residues and →6)-β-D-Galf-(1→ sugar residues, and its side chain structure contains α-D-Manp-(1→ and α-D-Glcp-(1→ sugar residues, and may contain →4)-β-D-GlcpNAc-(1→, →3)-β-D-Glcp-(1→, α-D-Manp-(1→3)-α-D-Manp-(1→ and other sugar residue fragments.

2. The exopolysaccharide of Lactobacillus fermentum W03 according to claim 1, characterized in that The mannan domain of the extracellular polysaccharide gives it good water solubility and a stable colloidal structure, and the furan galactan domain provides flexibility and folding ability of the molecular chain, thereby improving its application potential in food, medicine and biomaterials.

3. The exopolysaccharide of Lactobacillus fermentum according to claim 1, characterized in that EPS-W0310 at 1681.6cm -1 There is a polysaccharide functional group at the site, which is the fingerprint region of carbohydrates.

4. The exopolysaccharide of Lactobacillus fermentum according to claim 1, characterized in that EPS-W0310 is composed of mannose, glucose, galactose and glucosamine.

5. The exopolysaccharide of Lactobacillus fermentum according to claim 1, characterized in that EPS-W0310 has a smooth and shiny surface, a spherical shape and a tight structure.

6. A method for extracting extracellular polysaccharides of Lactobacillus fermentum according to any one of claims 1 to 5, characterized in that: The crude polysaccharide of fermented lactobacillus W03 was obtained through fermentation, deproteinization, ethanol precipitation and dialysis processes; the crude EPS was first purified using an anion exchange chromatography column; the fractions from the main peak were collected and further separated using a gel chromatography column to obtain a single peak named EPS-W0310.

7. Use of the extracellular polysaccharide of Lactobacillus fermentum according to any one of claims 1 to 5 in the food field.

8. Use of the extracellular polysaccharide of a fermented lactobacillus according to any one of claims 1 to 5 in the preparation of anti-inflammatory drugs.

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