Lactobacillus plantarum exopolysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum exopolysaccharide as well as a preparation method and application thereof. The brand-new exopolysaccharide is separated from lactobacillus plantarum PIAS240228, has a brand-new structure, is heteropolysaccharide composed of monosaccharides in a specific proportion, is uniform in component, has high antioxidant activity and soothing and repairing effects, and is suitable for the field of cosmetics.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to an exopolysaccharide of Lactobacillus plantarum and a preparation method and application thereof. Background Art

[0002] Lactobacillus Plantarum, as an important member of the lactobacillus family, has been listed as a food-grade probiotic recognized as safe by international food regulations. During the growth and metabolism process, the strain synthesizes a variety of bioactive substances through primary and secondary metabolic pathways, including structural polysaccharides such as homopolysaccharides and heteropolysaccharides, as well as functional metabolites such as lactic acid and essential amino acids. Among them, a part of the polysaccharides produced are structural polysaccharides of the bacteria, namely capsular polysaccharides, which are mainly composed of monosaccharide units such as glucose, galactose and rhamnose connected by β-(1→3) and β-(1→6) glycosidic bonds. This type of polysaccharide participates in maintaining the morphology of the bacteria as an important component of the cell wall. On the other hand, the extracellular polysaccharides (EPS) produced by its metabolism have a more complex spatial configuration, and the molecular weight is mostly between 10 4 Da-10 6 Da, with a highly branched repeating unit structure, can capture water through hydrogen bonds to stabilize the microenvironment around the bacteria.

[0003] Microbial exopolysaccharides possess multiple biological activities, including antioxidant activity, immune regulation, and intestinal flora balance, and are largely non-cytotoxic, offering broad application prospects. Currently, the development and utilization of microbial exopolysaccharides primarily focuses on the screening of high-yield strains and the optimization of production conditions, with less research into their structural characteristics, such as crystal morphology, monosaccharide composition, glycosidic bond type, and molecular weight distribution. However, the application of different exopolysaccharides in food, pharmaceuticals, and cosmetics, as well as their industrial production, are of great significance.

[0004] Therefore, it is necessary to further explore the newly isolated exopolysaccharides of Lactobacillus plantarum. Summary of the Invention

[0005] Based on this, an embodiment of the present application provides a Lactobacillus plantarum exopolysaccharide and a preparation method and application thereof.

[0006] On the one hand, the present application provides an extracellular polysaccharide of Lactobacillus plantarum, comprising: sugar residue A, sugar residue B, sugar residue, sugar residue C, sugar residue D, sugar residue E, sugar residue F, sugar residue G, sugar residue H, sugar residue I and sugar residue J connected in sequence.

[0007] The sugar residue A is α-D-Glcp-(1→); the sugar residue B is →4)-α-D-Manp-(1→); the sugar residue C is →3)-α-D-Glcp-(1→); the sugar residue D is →2,3)-α-D-Glcp-(1→); the sugar residue E is α-D-Galp-(1→); the sugar residue F is α-D-Manp-(1→); the sugar residue G is →4,6)-α-D-Manp-(1→); the sugar residue H is →2,6)-α-D-Glcp-(1→); the sugar residue I is →4)-β-D-Galp-(1→); and the sugar residue J is →5)-α-L-Araf-(1→).

[0008] In one embodiment, the Lactobacillus plantarum exopolysaccharide is a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose and galacturonic acid;

[0009] In one embodiment, the molar ratio of the fucose, the arabinose, the rhamnose, the galactose, the glucose, the xylose, the mannose and the galacturonic acid is 0.94:4.62:2.39:14.82:46.10:75:28.46:0.

[0010] In one embodiment, the molecular weight of the Lactobacillus plantarum exopolysaccharide is 550 kDa-560 kDa;

[0011] In one embodiment, the average molecular weight of the Lactobacillus plantarum exopolysaccharide is 554.919 kDa.

[0012] In one embodiment, the Lactobacillus plantarum exopolysaccharide is composed of repeating structural units represented by Formula I:

[0013]

[0014] On the other hand, the present application provides a method for preparing the above-mentioned Lactobacillus plantarum exopolysaccharide, comprising:

[0015] Lactobacillus plantarum PIAS240228 is fermented and cultured to obtain a fermentation broth, and the Lactobacillus plantarum exopolysaccharide is separated from the fermentation broth.

[0016] In one embodiment, the culture medium used for fermentation culture includes: 15g / L-25g / L glucose; 2g / L-3g / L dipotassium hydrogen phosphate trihydrate; 1g / L-3g / L diammonium hydrogen citrate; 4g / L-6g / L sodium acetate; 0.08g / L-0.12g / L magnesium sulfate; 0.08v / v%-0.12v / v% Tween-80; 8g / L-12g / L peptone; 8g / L-12g / L beef extract; 4g / L-6g / L yeast powder FM601;

[0017] In one embodiment, the fermentation culture conditions include an initial pH of 6.2-6.4, a temperature of 36° C.-38° C., and a time of 22 h-26 h; and

[0018] In one embodiment, the number of viable inoculated bacteria of Lactobacillus plantarum PIAS240228 was 2.0×10 9 CFU / mL-4.0×10 9 CFU / mL, the inoculation amount is 1v / v%-3v / v%.

[0019] In one embodiment, the separation step includes crude extraction, ion exchange purification, and gel purification.

[0020] In one embodiment, the crude extraction comprises the steps of centrifuging the fermentation broth, collecting the supernatant, filtering with a ceramic membrane, concentrating with a spiral membrane osmosis, and settling the concentrate.

[0021] In one embodiment, the centrifugation parameters include: a rotation speed of 10,000×g-12,000×g and a time of 15 min-25 min.

[0022] In one embodiment, the particle size of the ceramic membrane is 1000 nm-1400 nm.

[0023] In one embodiment, the molecular weight cut-off of the spiral membrane is 4500 Da to 5500 Da.

[0024] In one embodiment, the precipitation of the concentrate comprises subjecting the concentrate to trichloroacetic acid precipitation treatment and ethanol precipitation treatment in sequence.

[0025] In one embodiment, the ion exchange purification comprises treatment with a DEAEseplifeFF anion column.

[0026] In one embodiment, the coacervation purification comprises treatment with a Sephacryl S-400HR gel column.

[0027] On the other hand, the present application provides the use of the above-mentioned Lactobacillus plantarum exopolysaccharide in the preparation of products with soothing and repairing functions.

[0028] Another aspect of the present application is a product with soothing and repairing functions, which includes the above-mentioned Lactobacillus plantarum exopolysaccharide or the Lactobacillus plantarum exopolysaccharide prepared by the above-mentioned preparation method of Lactobacillus plantarum exopolysaccharide.

[0029] In one embodiment, the product comprises a cosmetic.

[0030] The present application isolates a new exopolysaccharide from Lactobacillus plantarum PIAS240228, which has a new structure and is a heteropolysaccharide composed of monosaccharides in a specific proportion with uniform components. In addition, the exopolysaccharide has high antioxidant activity and soothing and repairing effects, and is suitable for the cosmetics field. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0032] Figure 1 For the implementation of EPS in this application, the phenol-sulfuric acid method was used to detect OD490 and draw the ion purification elution curve;

[0033] Figure 2 For the first implementation of this application, EPS1 was tested using the phenol-sulfuric acid method for OD490 and the gel purification elution curve was drawn;

[0034] Figure 3 This is a high-performance gel filtration chromatogram of the exopolysaccharide EPS-11 of Lactobacillus plantarum PIAS240228 in one embodiment of the present application;

[0035] Figure 4 For the monosaccharide composition analysis chromatographic detection in one embodiment of the present application, Figure 4 A in the figure is the chromatogram of the monosaccharide standard. Figure 4 B in the figure is the monosaccharide composition chromatogram of EPS-11;

[0036] Figure 5 This is the infrared spectrum result of EPS-11, an embodiment of the present application;

[0037] Figure 6 Graph analysis for an embodiment of this application 1 H NMR results;

[0038] Figure 7 Graph analysis for an embodiment of this application 13 C NMR results;

[0039] Figure 8 This is the COSY result of the spectrum analysis of an embodiment of the present application;

[0040] Figure 9 This is the NOESY result of the spectrum analysis in one embodiment of the present application;

[0041] Figure 10 This is the HSQC result of the spectrum analysis of an embodiment of the present application;

[0042] Figure 11 This is the HMBC result of the spectrum analysis in one embodiment of the present application;

[0043] Figure 12 The results of DEPT-135 chromatographic analysis in one embodiment of this application are as follows;

[0044] Figure 13 This is the TOCSY result of a spectrum analysis in one embodiment of the present application. DETAILED DESCRIPTION

[0045] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0047] the term

[0048] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0049] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0050] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0051] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0052] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0053] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0054] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0055] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0056] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0057] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0058] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0059] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0060] The present application provides an exopolysaccharide of a newly isolated strain of Lactobacillus plantarum and a method for extracting and preparing the same. The exopolysaccharide is comprehensively analyzed to determine its molecular weight, monosaccharide composition, and structure, and its soothing and repairing effects are studied. The exopolysaccharide has antioxidant, soothing, and repairing effects and can be applied in the cosmetics field.

[0061] On the one hand, the present application provides a Lactobacillus plantarum exopolysaccharide, comprising: sugar residue A-sugar residue B-sugar residue-sugar residue C-sugar residue D-sugar residue E-sugar residue F-sugar residue G-sugar residue H-sugar residue I-sugar residue J connected in sequence;

[0062] Sugar residue A is α-D-Glcp-(1→); sugar residue B is →4)-α-D-Manp-(1→); sugar residue C is →3)-α-D-Glcp-(1→); sugar residue D is →2,3)-α-D-Glcp-(1→); sugar residue E is α-D-Galp-(1→); sugar residue F is α-D-Manp-(1→); sugar residue G is →4,6)-α-D-Manp-(1→); sugar residue H is →2,6)-α-D-Glcp-(1→); sugar residue I is →4)-β-D-Galp-(1→); sugar residue J is →5)-α-L-Araf-(1→).

[0063] In some embodiments, the Lactobacillus plantarum exopolysaccharide is a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose and galacturonic acid.

[0064] In some embodiments, the molar ratio of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose and galacturonic acid is 0.94:4.62:2.39:14.82:46.10:1.75:28.46:1.0.

[0065] In some embodiments, the molecular weight of the Lactobacillus plantarum exopolysaccharide is 550 kDa-560 kDa; for example, 550 kDa, 551 kDa, 552 kDa, 553 kDa, 554 kDa, 555 kDa, 556 kDa, 557 kDa, 558 kDa, 559 kDa or 560 kDa and any values ​​therebetween.

[0066] In some embodiments, the average molecular weight of the Lactobacillus plantarum exopolysaccharide is 554.919 KDa.

[0067] In some embodiments, the Lactobacillus plantarum exopolysaccharide is composed of repeating structural units represented by Formula I:

[0068]

[0069] On the other hand, the present application provides a method for preparing the Lactobacillus plantarum exopolysaccharide, comprising:

[0070] Lactobacillus plantarum PIAS240228 was fermented to obtain a fermentation broth, and Lactobacillus plantarum exopolysaccharide was isolated from the fermentation broth.

[0071] The strain used in this application is Lactobacillus plantarum PIAS240228, which was deposited in the China Center for Type Culture Collection on April 16, 2024, with the deposit number GDMCC NO: M2024647, and the deposit address is Wuhan University, China.

[0072] In some embodiments, the culture medium for fermentation culture includes: 15g / L-25g / L glucose; 2g / L-3g / L dipotassium hydrogen phosphate trihydrate; 1g / L-3g / L diammonium hydrogen citrate; 4g / L-6g / L sodium acetate; 0.08g / L-0.12g / L magnesium sulfate; 0.08v / v%-0.12v / v% Tween-80; 8g / L-12g / L peptone; 8g / L-12g / L beef extract; and 4g / L-6g / L yeast powder FM601.

[0073] For example, the concentration of glucose is 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L or 25 g / L and any values ​​therebetween.

[0074] For example, the concentration of dipotassium hydrogen phosphate trihydrate is 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L or 3.0 g / L and any value therebetween.

[0075] For example, the concentration of diammonium hydrogen citrate is 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, 2.0 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L or 3.0 g / L and any value therebetween.

[0076] For example, the concentration of sodium acetate is 4.0 g / L, 4.2 g / L, 4.4 g / L, 4.6 g / L, 4.8 g / L, 5.0 g / L, 5.2 g / L, 5.4 g / L, 5.6 g / L, 5.8 g / L or 6.0 g / L and any value in between.

[0077] For example, the concentration of magnesium sulfate is 0.08 g / L, 0.09 g / L, 0.10 g / L, 0.11 g / L or 0.12 g / L and any value therebetween.

[0078] For example, the concentration of Tween-80 is 0.08 v / v%, 0.09 v / v%, 0.10 v / v%, 0.11 v / v% or 0.12 v / v% and any values ​​therebetween.

[0079] For example, the concentration of peptone is 8 g / L, 9 g / L, 10 g / L, 11 g / L or 12 g / L and any value therebetween.

[0080] For example, the concentration of beef extract is 8 g / L, 9 g / L, 10 g / L, 11 g / L or 12 g / L and any value in between.

[0081] For example, the concentration of yeast powder FM601 is 4 g / L, 5 g / L or 6 g / L and any value in between.

[0082] In some embodiments, the culture conditions include an initial pH of 6.2-6.4, a temperature of 36° C.-38° C., and a culture time of 22 h-26 h. For example, the initial pH is 6.2, 6.3, or 6.4, and any values ​​therebetween; the temperature is 36° C., 37° C., or 38° C., and any values ​​therebetween; and the culture time is 22 h, 23 h, 24 h, 25 h, or 26 h, and any values ​​therebetween.

[0083] In some embodiments, the number of viable inoculated bacteria of Lactobacillus plantarum PIAS240228 is 2.0×10 9 CFU / mL-4.0×10 9 CFU / mL, the inoculation amount is 1v / v%-3v / v%.

[0084] For example, the number of viable bacteria inoculated with Lactobacillus plantarum PIAS240228 is 2.0×10 9 CFU / mL, 3.0×10 9 CFU / mL or 4.0×10 9 CFU / mL and any values ​​in between. For example, the inoculum size is 1%, 2%, or 3%, and any values ​​in between.

[0085] In some embodiments, isolating Lactobacillus plantarum exopolysaccharide from the fermentation broth comprises the steps of crude extraction, ion exchange purification, and gel purification.

[0086] In some embodiments, the crude extraction comprises the steps of centrifuging the fermentation broth, collecting the supernatant, filtering with a ceramic membrane, concentrating with a spiral membrane osmosis, and settling the concentrate.

[0087] In some embodiments, the centrifugation parameters include: a rotation speed of 10,000×g-12,000×g and a time of 15 min-25 min.

[0088] For example, the rotational speed is 10000×g, 10100×g, 10200×g, 10300×g, 10400×g, 10500×g, 10600×g, 10700×g, 10800×g, 10900×g, 11000×g, 11100×g, 11200×g, 11300×g, 11400×g, 11500×g, 11600×g, 11700×g, 11800×g, 11900×g or 12000×g and any value in between.

[0089] For example, the time is 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min or 25 min and any value in between.

[0090] In some embodiments, the particle size of the ceramic membrane is 1000 nm-1400 nm, for example, the particle size of the ceramic membrane is 1000 nm, 1100 nm, 1200 nm, 1300 nm or 1400 nm and any value therebetween.

[0091] In some embodiments, the molecular weight of the spiral membrane is 4500Da-5500Da, for example, the molecular weight of the spiral membrane is 4500Da, 4600Da, 4700Da, 4800Da, 4900Da, 5000Da, 5100Da, 5200Da, 5300Da, 5400Da or 5500Da and any values ​​therebetween.

[0092] The fermentation broth is centrifuged to remove large particles such as bacteria and cell debris, and the supernatant is collected. This step can initially reduce turbidity, but may retain small impurities such as colloids and soluble proteins.

[0093] Ceramic membrane filtration: A ceramic membrane with a pore size of approximately 1200 nm (microfiltration grade) is used for further clarification, reducing the Brix value from 4.7% to 4%. This stage primarily removes colloids, large proteins, and some suspended solids, while retaining soluble sugars (Brix reflects total solids content).

[0094] Spiral wound membrane osmotic concentration: Concentration is performed using a spiral wound ultrafiltration membrane with a molecular weight cutoff of approximately 5000 Da, increasing the Brix value from 4% to 6.3%, ultimately yielding 1600 mL of concentrated solution. This step achieves concentration by selectively retaining small solutes such as salts and small proteins or by evaporating water.

[0095] In some embodiments, the precipitation of the concentrate comprises sequentially subjecting the concentrate to trichloroacetic acid precipitation and ethanol precipitation.

[0096] In some embodiments, the ion exchange purification comprises a DEAE Seplife FF anion column with a gradient NaCl elution. DEAE Seplife FF is a weak anion exchange chromatography medium based on a highly cross-linked agarose matrix with a ligand of diethylaminoethyl (DEAE + ), suitable for separating negatively charged biomolecules, such as acidic polysaccharides, nucleic acids, and some proteins.

[0097] In some embodiments, the coacervation purification includes passage through a Sephacryl S-400HR gel column, elution with pure water, and impurity removal. Sephacryl S-400HR is a dextran-crosslinked acrylamide-based gel filtration medium with a pore size range of 50 kDa-150 kDa, suitable for the fine purification of large molecular proteins, viral particles, and polysaccharides.

[0098] On the other hand, the present application provides the use of the Lactobacillus plantarum exopolysaccharide in the preparation of products with soothing and repairing functions.

[0099] On the other hand, the present application provides a product with soothing and repairing functions, which includes the above-mentioned Lactobacillus plantarum exopolysaccharide or the Lactobacillus plantarum exopolysaccharide prepared by the above-mentioned preparation method of Lactobacillus plantarum exopolysaccharide.

[0100] In some embodiments, the product includes cosmetics having antioxidant, soothing, repairing and other related effects.

[0101] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0102] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0103] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0104] Example 1

[0105] 1. Extraction, purification and preparation of exopolysaccharide from Lactobacillus plantarum PIAS240228

[0106] 1. Preparation of crude polysaccharides

[0107] The fermentation medium formula (based on 1 L of culture medium) is as follows: 20 g glucose; 2.6 g dipotassium phosphate trihydrate; 2 g diammonium citrate; 5 g sodium acetate; 0.1 g magnesium sulfate; 1 mL Tween-80; 10 g peptone; 10 g beef extract; and 5 g yeast extract FM601. The medium has an initial pH of 6.2-6.4 and is sterilized.

[0108] Lactobacillus plantarum PIAS240228 bacterial solution (OD600 = 0.823, viable cell count 2.48 × 10 9 CFU / mL) was inoculated into the culture medium at an inoculum size of 2% and fermented at 37°C for 24 h. The pH value was 3.69 when the tank was filled and the number of viable bacteria was 10 9 CFU / mL or above.

[0109] The fermentation broth was centrifuged at 11,000 × g for 20 min, and the supernatant was collected. The supernatant was filtered through a 1200 nm ceramic membrane to remove impurities (Brix = 4.7% to 4%), and then concentrated through a 5000 Da spiral membrane to obtain 1600 mL (Brix = 6.3%) of concentrated solution.

[0110] Add 4% (m / m) trichloroacetic acid and allow to precipitate overnight at 4°C. Centrifuge at 11,000 × g for 20 minutes, and collect the supernatant. Add 3 volumes of 95% ethanol to 1600 mL of the supernatant, allow to precipitate overnight at 4°C, centrifuge at 11,000 × g for 20 minutes, and collect the precipitate. Wash the precipitate two to three times with 95% ethanol. Redissolve the precipitate in 160 mL of water and freeze-dry to prepare crude polysaccharide (EPS).

[0111] 2. Ion exchange purification

[0112] The EPS obtained above was purified using a DEAE seplife FF anion exchange chromatography column. Approximately 5 g of crude polysaccharide sample was dissolved in 200 mL of pure water and centrifuged at 10,000 g for 10 minutes. The supernatant was then purified by an ion exchange column at a flow rate of 4 mL / min. Elution was performed using a gradient of pure water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L sodium chloride aqueous solutions, with each 15 mL of eluate collected in one tube. OD490 was measured using the phenol-sulfuric acid method, and an ion purification elution curve was plotted.

[0113] like Figure 1As shown, after gradient elution with NaCl, the crude EPS was primarily separated into four components. The eluates from each collection tube corresponding to the same elution peak were combined and concentrated to 1 / 5 of the original volume by rotary evaporation. The samples were then dialyzed for 24-48 hours using a 3000 Da dialysis bag to remove salts, yielding EPS1, EPS2, EPS3, and EPS4, respectively. The polysaccharide purity of the desalted samples was determined using the phenol-sulfuric acid method, and the DPPH free radical scavenging activity of the four polysaccharides at a 2% concentration was compared. The results are shown in Table 1 below. EPS1 exhibited the largest sample size and the highest DPPH scavenging activity, so subsequent gel chromatography purification focused on the neutral polysaccharide EPS1.

[0114] Table 1: DPPH scavenging activity of EPS components

[0115]

[0116]

[0117] 3. Gel purification

[0118] The neutral exopolysaccharide EPS1 was isolated and purified using a Sephacryl S-400HR gel purification column. 1.5 column volumes of pure water were used for elution, with 10 mL collected in one tube as the eluent at a rate of 1 mL / min. OD490 values ​​were determined using the phenol-sulfuric acid method, and the gel purification elution curve was plotted.

[0119] like Figure 2 As shown, the elution peak was selected and confirmed, and the eluates from each collection tube corresponding to the same elution peak were combined and concentrated by rotary evaporation to 1 / 5 of the original volume. The sample was dialyzed for 24-48 hours using a 3000Da dialysis bag to remove small molecules, and then freeze-dried to obtain the highly pure extracellular neutral polysaccharide EPS11. The polysaccharide purity of the gel-purified sample was tested using the phenol-sulfuric acid method. The test results are shown in Table 1 below. Subsequent composition analysis and functional verification focused on EPS11.

[0120] Verification results:

[0121] 1. Molecular weight determination

[0122] 1. The EPS-11 obtained in Example 1 was dissolved in a 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL and filtered through a filter with a pore size of 0.45 μm before testing on an instrument.

[0123] 2. Inject the filtrate (μL) obtained in step 1 into gel exclusion chromatography columns Ohpak SB-805H (300×8 mm) and Ohpak SB-805HQ (300×8 mm) connected in series, use 0.02% NaN3, 0.1 M NaNO3 as the mobile phase, elute at a flow rate of 0.6 mL / min, and set the column temperature at 45°C to obtain the response value data.

[0124] 3. Use the software ASTRA6.1 to collect and process the chromatographic data, and calculate the molecular weight of each component according to the Mark-Houwink equation.

[0125] 4. Figure 3 This is the high-performance gel filtration chromatogram of the exopolysaccharide EPS-11 of Lactobacillus plantarum PIAS240228. The average molecular weight of EPS-11 is 554.919 KDa.

[0126] 2. Monosaccharide composition analysis

[0127] 1. Preparation of standards: Accurately weigh the required standard substances, fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid, and add deionized water to prepare a 10 mg / mL standard stock solution. Then, take an appropriate amount of the standard stock solution and mix them to prepare a standard mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL.

[0128] 2. Sample pretreatment: Take an appropriate amount of EPS-11 sample obtained in Example 1, add 1 mL of 2M TFA solution, heat at 121°C for 2 h, purge with nitrogen, blow dry, and then wash with 99.99% methanol to remove trifluoroacetic acid. Repeat 2-3 times, add sterile water to dissolve, and transfer to a chromatographic bottle for testing.

[0129] 3. Instrument parameters:

[0130] The chromatographic system used was Thermo ICS 5000 + Ion Chromatography System (ICS 5000 + , Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector.

[0131] Using Dionex TM CarboPac TMPA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; column temperature 30°C; elution gradient: 0 min phase A / phase B / phase C (95:5:0, v / v), 26 min phase A / phase B / phase C (85:5:10, v / v), 42 min phase A / phase B / phase C (85:5:10, v / v), 42.1 min phase A / phase B / phase C (60:0:40, v / v), 52 min phase A / phase B / phase C (60:40:0, v / v), 52.1 min phase A / phase B / phase C (95:5:0, v / v), 60 min phase A / phase B / phase C (95:5:0, v / v).

[0132] 4. The determination results were compared and calculated with the standard. The results showed that EPS-11 is a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose, and galacturonic acid with a molar ratio of 0.94:4.62:2.39:14.82:46.10:1.75:28.46:1.0, among which the content of mannose and glucose is relatively high. Figure 4 A in the figure is the chromatogram of the monosaccharide standard. Figure 4 B in FIG is a chromatogram of the monosaccharide composition of EPS-11.

[0133] 3. Infrared spectroscopy analysis

[0134] 1. Using the potassium bromide tableting method, a small amount of the EPS-11 sample obtained in Example 1 was weighed, 200 mg of potassium bromide powder was added and mixed, and then a tablet press was used to form a 1 mm thick sheet. The infrared spectrum was scanned in the range of 4000-450 cm-1 using a Fourier transform infrared spectrometer (Nicolet iZ-10) and the data was recorded (32 scans).

[0135] 2. Measurement results: 3306.15cm -1 It is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. -1 The absorption peak at 1023.07 cm is attributed to CH stretching vibration. -1 There is an absorption peak at , which is attributed to the stretching vibration of CO. Figure 5 This is the infrared spectrum result of EPS-11.

[0136] 4. Determination of the exopolysaccharide structure of Lactobacillus plantarum PIAS240228

[0137] 1. Methylation analysis

[0138] 1) Sample Pretreatment: Dissolve a small amount (2-3 mg) of the EPS-11 sample from Example 1 in 500 μL of DMSO until completely dissolved. Add 1 mL of NaOH and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Then, add 1 mL of water and 2 mL of dichloromethane, vortex to mix, and centrifuge to discard the aqueous phase. Repeat the water wash three times. Aspirate the lower dichloromethane phase and dry it with nitrogen. Add 100 μL of 2M TFA, react at 121°C for 90 min, and evaporate to dryness at 30°C. Add 50 μL of 2M ammonia and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h. Terminate the reaction by adding 20 μL of acetic acid, dry it with nitrogen, wash twice with 250 μL of methanol, and dry it with nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100°C for 2.5 h. Add 1 mL of water and let it stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the water wash three times. Remove the dichloromethane phase and analyze it on a GC-MS machine.

[0139] 2) GC-MS Conditions: The instrument was an Agilent gas chromatography system (Agilent 6890A; Agilent Technologies, USA), using a TG-200 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was high-purity helium at a flow rate of 1.5 mL / min. The column oven temperature was initially set at 150°C for 2.0 min, then programmed to 210°C at 2°C / min, held for 3 min, and then to 240°C at 2°C / min, held for 5 min. The mass spectrometry system used was an Agilent 5977B quadrupole mass spectrometry detection system (Agilent 5977B, Agilent Technologies, USA). The mass spectrometry ion source was an electron impact ionization source (EI), the ion source temperature was 200°C, the MS quadrupole temperature was 110°C, the ionization energy was 50 EV, the transfer line temperature was 210°C, and the mass scan range (m / z) was 50-350.

[0140] 3) Data Analysis: The EI-MS spectrum and methylation analysis of EPS-11 are shown in Table 2 below.

[0141] Table 2: EI-MS spectrum and methylation analysis of EPS-11

[0142] Linkage mode Derivative name RT Relative molar ratio (%) t-Ara(f) 1,4-di-O-acetyl-2,3,5-tri-O-methyl arabinitol 7.737 1.27 t-Xyl(p) 1,5-di-O-acetyl-2,3,4-tri-O-methyl xylitol 9.189 1.02 t-Fuc(p) 1,5-di-O-acetyl-6-deoxy-2,3,4-tri-O-methyl fucitol 9.581 0.61 t-Man(p) 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol 11.889 8.02 t-Glc(p) 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol 11.988 15.69 2-Rha(p) 1,2,5-tri-O-acetyl-6-deoxy-3,4-di-O-methyl rhamnitol 12.739 1.05 t-Gal(p) 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol 12.864 8.20 5-Ara(f) 1,4,5-tri-O-acetyl-2,3-di-O-methyl arabinitol 13.450 1.30 3-Glc(p) 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl glucitol 16.024 11.30 2-Gal(p) 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl galactitol 16.748 3.18 4-Man(p) 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl mannitol 17.019 12.35 2,4-Rha(p) 1,2,4,5-tetra-O-acetyl-6-deoxy-3-O-methyl rhamnitol 17.211 1.02 4-Gal(p) 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol 17.576 3.86 4-Glc(p) 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol 17.867 2.37 6-Glc(p) A1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol 18.220 2.08 3,4-Man(p) 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl mannitol 20.074 1.55 3,4-Gal(p) 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl galactitol 21.079 0.96 2,3-Glc(p) 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl glucitol 21.598 11.12 4,6-Man(p) 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl mannitol 22.598 6.82 2,6-Glc(p) 1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl glucitol 24.263 5.42 3,4,6-Glc(p) 1,3,4,5,6-penta-O-acetyl-2-O-methyl glucitol 25.735 0.82

[0143] 2. Nuclear Magnetic Resonance Analysis

[0144] 1) Sample pretreatment: Take an appropriate amount of the EPS-11 sample obtained in Example 1 and fully dissolve it in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. After dissolution, transfer it to an NMR tube with an addition amount of 0.5 mL. Place the NMR tube in an NMR spectrometer to scan the one-dimensional 1H spectrum, 13C spectrum, DEPT-135, and two-dimensional COSY, HSQC, HMBC, NOESY, and TOCSY spectra.

[0145] 2) NMR Parameters: Chromatographic analyses were performed on a 500 MHz NMR instrument (Bruker, Germany) at a scanning temperature of 25°C. The liquid probe was a QXI 1H / 31P / 13C / 15N 5 mm quadruple resonance reverse detection probe (Z-gradient, ATMAcc). Technical parameters: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating). The BBFO 1H-19F, 31P-15N, 1H decoupling / observe multinuclear forward detection probe (Z-gradient, ATM) had the following technical parameters: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1.

[0146] 3) Spectral data analysis: 1H NMR ( Figure 6 )、13C NMR( Figure 7 )、COSY( Figure 8 )、NOESY( Figure 9 )、HSQC( Figure 10 )、HMBC( Figure 11 )、DEPT-135( Figure 12 )、TOCSY( Figure 13 The sample's hydrogen spectrum signals are mainly concentrated between δ3.0ppm and δ5.5ppm. Multiple coupled signal peaks are identified in the anomeric signal region of δ4.3ppm-δ5.4ppm, indicating that this sample contains a variety of sugar residues. The chemical shifts of the corresponding anomeric hydrogens are δ4.59ppm, δ4.81ppm, δ4.83ppm, δ4.9ppm, δ4.95ppm, δ4.99ppm, δ5.04ppm, δ5.06ppm, δ5.09ppm, δ5.24ppm, etc. The non-anomeric hydrogen signals are mainly concentrated in the δ3.1ppm-δ4.2ppm region. Due to the serious overlap of some individual signals, it is necessary to combine COSY and TOCSY spectra to assign the H2-H6 chemical shifts of each sugar residue separately.

[0147] The strong signal peak near δ4.71 ppm is a solvent peak. Multiple signal peaks were identified in the anomeric carbon region of the sample. Combining the cross-peaks in the anomeric region of the 13C NM spectrum and the HSQC spectrum, the anomeric signals present in this sample were determined to be: δ4.9 / 102.37, 5.24 / 100.48, 4.83 / 101.54, 4.81 / 101.82, 5.09 / 102.09, 5.06 / 98.17, 4.99 / 101.99, 4.95 / 102.14, 4.59 / 104.32, and 5.04 / 107.51 ppm, which were respectively labeled as sugar residues A, B, C, D, E, F, G, H, I, and J.

[0148] Combining the DEPT-135 and 13C NMR spectra, signals of methylene (δ60.77 ppm, δ60.73 ppm, δ60.9 ppm, δ60.82 ppm, δ61.11 ppm, δ60.78 ppm, δ66.89 ppm, δ66.98 ppm, δ61.06 ppm, δ66.83 ppm) can be obtained.

[0149] 3. Polysaccharide structure analysis

[0150] Combined with the sample bonding structure (methylation) information, anomeric signals and literature reports, sugar residue A was determined to be α-D-Glcp-(1→), sugar residue B was →4)-α-D-Manp-(1→, sugar residue C was →3)-α-D-Glcp-(1→, sugar residue D was →2,3)-α-D-Glcp-(1→, sugar residue E was α-D-Galp-(1→, sugar residue F was α-D-Manp-(1→), sugar residue G was →4,6)-α-D-Manp-(1→, sugar residue H was →2,6)-α-D-Glcp-(1→, sugar residue I was →4)-β-D-Galp-(1→, sugar residue J was →5)-α-L-Araf-(1→, and their 1H and 13C chemical shifts were assigned.

[0151] The structure of EPS-11 is shown in Formula 1:

[0152]

[0153] 5. Determination of the soothing and repairing efficacy of Lactobacillus plantarum PIAS240228 exopolysaccharide

[0154] 1. Human VEGF and VEGF R2 Binding Inhibitory Activity Assay (Bepsix VEGFA [Biotinylated]: VEGFR2 Inhibitor Screening ELISA Kit, Catalog No. EP-141). This inhibitor screening ELISA kit is used to identify new VEGFA pathway inhibitors. It uses a colorimetric ELISA platform to detect the binding status of biotinylated VEGF to immobilized human VEGF R2. The main steps are as follows:

[0155] 1) Coat the plate with human VEGF R2 and incubate at 4°C overnight or for 16 hours.

[0156] 2) The coated plate was washed three times and incubated with blocking buffer at 37° C. for 1.5 h. The plate was washed again and 1% crude extracellular polysaccharide of Lactobacillus plantarum was added.

[0157] 3) Add VEGF-Biotin to bind to the coated VEGF R2.

[0158] 4) Repeat washing the coated plate 3 times, add streptavidin-HRP, and then add TMB or other colorimetric HRP substrate.

[0159] 5) Finally, the inhibitory activity of Lactobacillus plantarum extracellular crude polysaccharide on the binding of VEGF and VEGF R2 was determined by comparing the OD450 readings between different experimental groups. The test results are shown in Table 3 below.

[0160] 2. Human TNF-α and TNFR1 binding inhibitory activity assay (Biopsy TNF-alpha [Biotinylated]: TNFR1 Inhibitor Screening ELISA Kit, Catalog No. EP-143). This inhibitor screening ELISA kit is used to identify TNF-α pathway inhibitors. It uses a colorimetric ELISA platform to detect the binding status of biotinylated human TNF-α to immobilized human TNFR1. The main steps are as follows:

[0161] 1) Coat the plate with human TNFR1 and incubate at 4°C overnight or for 16 hours.

[0162] 2) The coated plate was washed three times and incubated with blocking buffer at 37° C. for 1.5 h. The plate was washed again and 1% crude extracellular polysaccharide of Lactobacillus plantarum was added.

[0163] 3) Add TNF-α-Biotin to bind to the coated TNFR1.

[0164] 4) Repeat washing the coated plate 3 times, add streptavidin-HRP, and then add TMB or other colorimetric HRP substrate.

[0165] 5) Finally, the inhibitory activity of Lactobacillus plantarum extracellular crude polysaccharide on the binding of TNF-α and TNFR1 was determined by comparing the OD450 readings between different experimental groups. The test results are shown in Table 3 below.

[0166] Table 3: EPS soothing and repairing activity index data

[0167]

[0168] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A Lactobacillus plantarum exopolysaccharide, characterized in that include: Sugar residue A, sugar residue B, sugar residue, sugar residue C, sugar residue D, sugar residue E, sugar residue F, sugar residue G, sugar residue H, sugar residue I and sugar residue J are connected in sequence; The sugar residue A is α-D-Glcp-(1→); the sugar residue B is →4)-α-D-Manp-(1→); the sugar residue C is →3)-α-D-Glcp-(1→); the sugar residue D is →2,3)-α-D-Glcp-(1→); the sugar residue E is α-D-Galp-(1→); the sugar residue F is α-D-Manp-(1→); the sugar residue G is →4,6)-α-D-Manp-(1→); the sugar residue H is →2,6)-α-D-Glcp-(1→); the sugar residue I is →4)-β-D-Galp-(1→); and the sugar residue J is →5)-α-L-Araf-(1→).

2. The plant lactobacillus exopolysaccharide according to claim 1, wherein The plant lactobacillus exopolysaccharide is a heteropolysaccharide composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, mannose and galacturonic acid; Optionally, the molar ratio of the fucose, the arabinose, the rhamnose, the galactose, the glucose, the xylose, the mannose and the galacturonic acid is 0.94:4.62:2.39:14.82:46.10:1.75:28.46:1.

0.

3. The plant lactobacillus exopolysaccharide according to claim 1, wherein The molecular weight of the Lactobacillus plantarum exopolysaccharide is 550kDa-560kDa; Optionally, the average molecular weight of the Lactobacillus plantarum exopolysaccharide is 554.919 kDa.

4. The plant lactobacillus exopolysaccharide according to claim 1, wherein The Lactobacillus plantarum exopolysaccharide is composed of repeating structural units shown in Formula I:

5. The method for preparing the Lactobacillus plantarum exopolysaccharide according to any one of claims 1 to 4, characterized in that: include: Lactobacillus plantarum PIAS240228 is fermented and cultured to obtain a fermentation broth, and the Lactobacillus plantarum exopolysaccharide is separated from the fermentation broth.

6. The method for preparing the plant lactobacillus exopolysaccharide according to claim 5, It is characterized in that The fermentation culture meets one or more of the following conditions: (1) The culture medium used for fermentation culture includes: 15g / L-25g / L glucose; 2g / L-3g / L dipotassium hydrogen phosphate trihydrate; 1g / L-3g / L diammonium hydrogen citrate; 4g / L-6g / L sodium acetate; 0.08g / L-0.12g / L magnesium sulfate; 0.08v / v%-0.12v / v% Tween-80; 8g / L-12g / L peptone; 8g / L-12g / L beef extract; 4g / L-6g / L yeast powder FM601; (2) fermentation conditions include an initial pH of 6.2-6.4, a temperature of 36°C-38°C, and a fermentation time of 22 hours-26 hours; and (3) The number of viable bacteria inoculated with Lactobacillus plantarum PIAS240228 was 2.0×10 9 CFU / mL-4.0×10 9 CFU / mL, the inoculation amount is 1v / v%-3v / v%.

7. The method for preparing the plant lactobacillus exopolysaccharide according to claim 5, wherein The separation steps include crude extraction, ion exchange purification and gel purification; Optionally, the crude extraction comprises the steps of centrifuging the fermentation broth, collecting the supernatant, filtering with a ceramic membrane, concentrating with a spiral membrane osmosis, and settling the concentrate; Optionally, the centrifugation parameters include: a rotation speed of 10,000×g-12,000×g, and a time of 15 min-25 min; Optionally, the particle size of the ceramic membrane is 1000nm-1400nm; Optionally, the molecular weight cut-off of the spiral membrane is 4500Da-5500Da; Optionally, the precipitation of the concentrated solution comprises subjecting the concentrated solution to trichloroacetic acid precipitation treatment and ethanol precipitation treatment in sequence; Optionally, ion exchange purification includes treatment with a DEAE seplife FF anion column; Optionally, the coacervation purification comprises treatment with a Sephacryl S-400HR gel column.

8. Use of the Lactobacillus plantarum exopolysaccharide according to any one of claims 1 to 4 in the preparation of a product with soothing and repairing functions.

9. A product with soothing and repairing functions, characterized in that: The product comprises the Lactobacillus plantarum exopolysaccharide according to any one of claims 1 to 4 or the Lactobacillus plantarum exopolysaccharide prepared by the preparation method of the Lactobacillus plantarum exopolysaccharide according to any one of claims 5 to 7.

10. The use according to claim 8 or the product with soothing and repairing function according to claim 9, characterized in that: The products include cosmetics.

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