A method for preparing and applying fused Weissella extracellular polysaccharides

Abstract

This invention belongs to the field of microbial technology, specifically relating to a method for preparing and applying a fused Weissella extracellular polysaccharide. The extracellular polysaccharide of this invention is composed of glucose monosaccharide, and the glucose linkages include terminal glucose t-Glc(p), 1→4 linked glucose, 1→4,6 linked glucose, 1→3,6 linked glucose, and 1→2,6 linked glucose, with a relative molar ratio of (8~8.5):(1~1.2):(81~82):(7~7.5):(1~1.2):(0.5~1). It possesses excellent water-holding capacity, oil-holding capacity, emulsifying properties, and emulsion stability, and can be used as a highly efficient moisturizer, emulsifier, emulsion stabilizer, cosmetic additive, pharmaceutical additive, and food additive, showing broad application prospects in the food, cosmetic, and pharmaceutical fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method for preparing and applying a fused Weissella extracellular polysaccharide. Background Technology

[0002] Weissella spp. Weissella As an important member of the lactic acid bacteria family, Weissella has received increasing attention from academia and industry in recent years due to its unique physiological and biochemical characteristics, as well as its wide distribution and application in fermented foods such as kimchi, sourdough, and fermented meat products. In addition to its excellent fermentation performance and probiotic potential, one of the core functional characteristics of Weissella is its ability to synthesize and secrete extracellular polysaccharides (EPS).

[0003] Extracellular polysaccharides (EPS) are high-molecular-weight carbohydrate polymers secreted outside the cell wall during the growth and metabolism of microorganisms. Microbial-derived EPS exhibits broad application prospects in food, pharmaceuticals, cosmetics, and biomaterials due to its diverse structure, excellent biocompatibility, high safety, unique rheological properties, and potential biological activities—such as antioxidant, immunomodulatory, antitumor, cholesterol-lowering, and prebiotic effects. In the food industry, EPS, as a natural thickener, stabilizer, emulsifier, and gelling agent, can significantly improve product texture, taste, water retention, and sensory quality, precisely meeting consumers' growing demand for "clean labels" and natural additives.

[0004] While research on EPS (extracellular polymeric substances) from some lactic acid bacteria, such as Lactobacillus and Streptococcus, is relatively in-depth, systematic studies on EPS from the genus *Weissella* remain relatively scarce and scattered. The yield, chemical composition (monosaccharide composition, molecular weight), fine structure (glycosidic bond linkage, branching morphology), and their resulting functional properties (such as rheological properties, emulsifying ability, and bioactivity) of *Weissella* EPS exhibit significant differences between species and even at the strain level. Furthermore, a unified standard for efficient and economical extraction and purification methods for *Weissella* EPS has not yet been established, which to some extent restricts the accurate structural analysis and in-depth evaluation of its functional properties. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing and applying a fused Weissella extracellular polysaccharide.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an extracellular polysaccharide, wherein the constituent monosaccharide of the extracellular polysaccharide is glucose, and the glucose linkage includes terminal glucose t-Glc(p), 1→4 linked glucose, 1→4,6 linked glucose, 1→3,6 linked glucose and 1→2,6 linked glucose, with a molar ratio of (8~8.5):(1~1.2):(81~82):(7~7.5):(1~1.2):(0.5~1).

[0008] A second aspect of the present invention provides a method for preparing the extracellular polysaccharide described in the first aspect, comprising the following steps: preparing the fusion strain of *Westernella* described in the second aspect (… Weissella confusa Inoculate the culture medium and incubate at 28-42℃ for 12-120 h to obtain a fermentation broth containing fused Weissella extracellular polysaccharides; extract the fermentation broth with a eutectic solvent to obtain a crude extract of extracellular polysaccharides; purify the crude extract of extracellular polysaccharides to obtain the final product.

[0009] A third aspect of the present invention provides the application of the above-described preparation method in the preparation of extracellular polysaccharides.

[0010] A fourth aspect of the present invention provides the use of the above-described extracellular polysaccharide in the preparation of cosmetics or food.

[0011] A fifth aspect of the present invention provides humectants, emulsifiers, emulsion stabilizers, cosmetic additives, pharmaceutical additives, and food additives containing the above-mentioned extracellular polysaccharides.

[0012] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention provides a method for preparing extracellular polysaccharides, based on *Fusion Weissella* (… Weissella confusa MFO1 was developed using single-factor and orthogonal experiments to optimize fermentation conditions and determine the optimal fermentation process, resulting in the acquisition of Weissella extracellular polysaccharides. The water-holding capacity of the fused Weissella EPS was 371.52 mL / g, and its oil-holding capacity for corn oil was 32.85 g / g, exceeding 32 times its own weight. The oil-holding capacity of Weissella was 4.2 times that of casein and 5.7 times that of xanthan gum, demonstrating superior oil adsorption capacity. It has the ability to replace 50%–60% of fat, effectively reducing product calories, and thus can be considered a promising fat substitute food additive. Simultaneously, the fused Weissella EPS exhibited emulsifying properties and emulsion stability of 70.33% and 99.82%, respectively, demonstrating good emulsifying properties and superior emulsion stability. It can serve as a highly efficient emulsifier and emulsion stabilizer, especially showing broad application prospects in food, cosmetic, or pharmaceutical emulsion systems requiring long shelf life. Attached Figure Description

[0013] Figure 1This is a schematic diagram showing the EPS content of *Westernella fusionis* under different carbon source conditions in the embodiments of the present invention.

[0014] Figure 2 This is a schematic diagram showing the EPS content of *Westernella fusionis* under different sucrose dosages in embodiments of the present invention.

[0015] Figure 3 This is a schematic diagram showing the EPS content of *Westernella fusionis* under different inoculation amounts in an embodiment of the present invention.

[0016] Figure 4 This is a schematic diagram showing the EPS content of *Westernella fusionis* at different culture temperatures in an embodiment of the present invention.

[0017] Figure 5 This is a schematic diagram showing the EPS content of *Westernella fusionis* under different culture times in an embodiment of the present invention.

[0018] Figure 6 This is a schematic diagram showing the EPS content of *Westernella* fusion at different feed-to-liquid ratios in an embodiment of the present invention.

[0019] Figure 7 This is a schematic diagram showing the EPS content of *Westernella fusionis* at different extraction temperatures in an embodiment of the present invention.

[0020] Figure 8 This is a schematic diagram showing the EPS content of *Westernella fusionis* under different culture times in an embodiment of the present invention.

[0021] Figure 9 This is a schematic diagram of the results of EPS separation by DEAE-5 cellulose anion exchange column in an embodiment of the present invention.

[0022] Figure 10 This is a schematic diagram of the EPS separation results using dextran G-100 gel column in an embodiment of the present invention.

[0023] Figure 11 This is a standard ion chromatogram from an embodiment of the present invention.

[0024] Figure 12 This is a chromatogram of EPS ions fused with Weissl bacillus in an embodiment of the present invention.

[0025] Figure 13 This is a molecular configuration diagram of the fused Weissella EPS in an embodiment of the present invention.

[0026] Figure 14 The EPS electron microscope used in this embodiment of the invention is shown in Figure A (100 X), Figure B (2000 X), and Figure C (5.00 KX).

[0027] Figure 15 The image shown is an infrared spectrum of EPS fused with Weissella in an embodiment of the present invention.

[0028] Figure 16 The results of the Congo Red experiment are shown in the embodiments of the present invention.

[0029] Figure 17 This is a schematic diagram showing the hygroscopic properties of the fused Weissella EPS in an embodiment of the present invention.

[0030] Figure 18 This is a schematic diagram illustrating the moisturizing effect of the fused Weissella EPS in an embodiment of the present invention. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0032] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0033] In a typical embodiment of the present invention, an extracellular polysaccharide is provided, wherein the constituent monosaccharide of the extracellular polysaccharide is glucose, and the glucose linkage includes terminal glucose t-Glc(p), 1→4 linked glucose, 1→4,6 linked glucose, 1→3,6 linked glucose and 1→2,6 linked glucose, with a relative molar ratio of (8~8.5):(1~1.2):(81~82):(7~7.5):(1~1.2):(0.5~1), preferably (8.4~8.5):(1~1.1):(81~81.5):(7.1~7.5):(1.1~1.2):(0.5~0.8), more preferably 8.41:1.05:81.31:7.3:1.17:0.76.

[0034] In some embodiments, the extracellular polysaccharide has a molecular weight of 16374.659 kDa.

[0035] In another typical embodiment of the present invention, a method for preparing the extracellular polysaccharide is provided, comprising the following steps: preparing the fusion strain of *Westernella* (… Weissella confusa Inoculate the culture medium and incubate at 28-42℃ for 12-120 h to obtain a fermentation broth containing fused Weissella extracellular polysaccharides; extract the fermentation broth with a eutectic solvent to obtain a crude extract of extracellular polysaccharides; purify the crude extract of extracellular polysaccharides to obtain the final product.

[0036] In some embodiments, the fused Weissella is a fused Weissella ( Weissella confusa MF01, In some embodiments, the fermentation medium is an MRS medium containing 20-100 g / L sucrose. Preferably, it is an MRS medium containing 35 g / L sucrose.

[0037] In some embodiments, the amount of Weissella inoculated is 1% to 5%, preferably 2.5%.

[0038] In some embodiments, the culture temperature is 28~35°C, preferably 30°C; and the culture time is 36 h.

[0039] In some embodiments, the volume ratio of the fermentation broth to the eutectic solvent is (1~5):1, preferably 2:1.

[0040] In some embodiments, the eutectic solvent is composed of choline chloride and oxalic acid; preferably, the molar ratio of choline chloride to oxalic acid is 2:1.

[0041] In some embodiments, the extraction time is 30-90 min, preferably 50-60 min, and more preferably 55 min.

[0042] In some embodiments, the extraction temperature is 40~80°C, preferably 60~70°C, and more preferably 65°C.

[0043] In some embodiments, the extraction of the fermentation broth with a eutectic solvent further includes an alcohol extraction step, specifically: the fermentation broth is mixed with the eutectic solvent for extraction, centrifuged after standing, and a supernatant is obtained; three volumes of 95% ethanol are added to the supernatant, the mixture is allowed to stand overnight at 4°C, centrifuged, the precipitate is collected, distilled water is added to dissolve the precipitate, a 10% trichloroacetic acid aqueous solution is added, and the mixture is placed in a shaker and shaken at 4°C for 4 hours; the sample solution is centrifuged, the supernatant is collected, three volumes of 95% ethanol are added, the mixture is allowed to stand overnight at 4°C, centrifuged, the precipitate is dissolved in distilled water, dialyzed, and a crude extracellular polysaccharide extract, i.e., a crude EPS aqueous solution, is obtained.

[0044] In some embodiments, the purification steps include: the crude extracellular polysaccharide extract is sequentially separated by DEAE-52 cellulose chromatography and dextran G-100 gel chromatography.

[0045] In some embodiments, the extracellular polysaccharide is separated by a DEAE-5 cellulose anion exchange column, and three fractions are obtained by elution with ultrapure water, 0.1 mol / L NaCl solution, and 0.2 mol / L NaCl solution, respectively. The fraction of the first peak eluted by ultrapure water is selected for subsequent separation experiments.

[0046] In some embodiments, the sample is passed through a dextran G-100 gel column and eluted with ultrapure water, 0.1 mol / L NaCl solution, and 0.2 mol / L NaCl solution to obtain three components. The component of the first peak eluted with ultrapure water is selected for subsequent experiments.

[0047] In another typical embodiment of the present invention, the application of the above-mentioned Weissella bacteria or preparation method in the preparation of extracellular polysaccharides is provided.

[0048] In another typical embodiment of the present invention, the application of the above-mentioned extracellular polysaccharide in the preparation of cosmetics or food is provided.

[0049] In another typical embodiment of the present invention, a humectant, emulsifier, emulsion stabilizer, cosmetic additive, pharmaceutical additive, and food additive containing the above-mentioned extracellular polysaccharide are provided.

[0050] In this invention, the water-holding capacity of the fused Weissella EPS is 371.52 mL / g, and the oil-holding capacity of corn oil is 32.85 g / g, reaching more than 32 times its own weight. The oil-holding capacity of Weissella is 4.2 and 5.7 times that of casein and xanthan gum, respectively, demonstrating superior oil adsorption capacity. It has the ability to replace 50%~60% of fat and can effectively reduce the calories of products. Therefore, it can be used as a fat substitute food additive with promising application prospects. At the same time, the emulsifying properties and emulsifying stability of the fused Weissella EPS are 70.33% and 99.82%, respectively, showing good emulsifying properties and superior emulsifying stability. It can be used as a highly efficient emulsifier and emulsion stabilizer, especially in food, cosmetic or pharmaceutical systems that require long shelf life, with broad application prospects.

[0051] The strain used in the following examples is *Westernella fusionis* (…). Weissella confusa MF01, this strain is disclosed in patent application number "202311254729.7", and is a fusion of Weissella ( Weissella confusa MF01 was deposited at the China Center for Type Culture Collection (CCTCC) on October 21, 2022, with accession number CCTCC NO:M 20221635, at Wuhan University, Wuhan, China.

[0052] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] Example 1: Optimization of fermentation conditions for the preparation of extracellular polysaccharides MRS liquid medium was used as the basal medium. The initial fermentation conditions were 2.0% inoculum, 33℃ fermentation temperature, and 24 h fermentation time.

[0054] MRS medium formulation (g / L): glucose 2 g, yeast extract 4.0 g, beef extract 8.0 g, peptone 10.0 g, K₂HPO₄ 2.0 g, C₆H₂O 14 N2O7 2.0 g, CH3COONa 5.0 g, MnSO4 0.04 g, MgSO4 0.2 g, Tween-80 1.0 g, pH 5.7±0.2.

[0055] Extraction and determination method of EPS from *Westernella fusionis*: The fermentation broth was centrifuged at 9450 r / min, 4℃ for 20 min, and three volumes of 95% ethanol were added. The mixture was then allowed to stand overnight at 4℃. The fermentation broth was centrifuged again at 9450 r / min for 20 min, the precipitate was collected, and 10 mL of distilled water was added to dissolve the precipitate. Then, 10 mL of 10% trichloroacetic acid aqueous solution was added, and the mixture was placed in a shaker and shaken at 4℃ for 4 h. The sample solution was centrifuged at 9450 r / min for 20 min, the supernatant was collected, and three volumes of 95% ethanol were added. The mixture was allowed to stand overnight at 4℃. The sample solution was centrifuged again at 9450 r / min for 20 min, the precipitate was dissolved in 10 mL of distilled water, and dialyzed for 2 days, changing the water every 12 h to obtain a crude EPS aqueous solution. The EPS content was determined using the phenol-sulfuric acid method. The absorbance of each reaction solution was measured at a wavelength of 490 nm. The EPS content was calculated based on the standard curve, and the average value of three tests was taken.

[0056] (1) Single-factor experimental design: Five variables were selected for single-factor experiments: the dosage of three carbon sources (sucrose, glucose, and fructooligosaccharides) (20 g / L, 40 g / L, 60 g / L, 80 g / L, 100 g / L), the culture temperature (28℃, 33℃, 36℃, 39℃, 42℃), the inoculum size (1.0%, 2.0%, 3.0%, 4.0%, 5.0%), and the culture time (12 h, 24 h, 36 h, 72 h, 120 h). EPS was extracted and the EPS yield of the strain was measured. The optimal carbon source, carbon source dosage, culture temperature, inoculum size, and culture time were determined based on the EPS yield. Each experiment was repeated three times, and the final result was the average value.

[0057] like Figure 1 As shown, among the three tested carbon sources, sucrose exhibited the highest EPS production capacity. Under single carbon source conditions, the EPS production of *Westernella* fusion strains was ranked as follows: sucrose > glucose > fructooligosaccharides. The fructooligosaccharide group had the lowest EPS production at 197.95 mg / L, while the sucrose group had the highest production at 1209.50 mg / L. This may indicate that sucrose plays a more significant regulatory role in EPS synthesis than glucose and fructooligosaccharides. pSucrose (<0.05%) can more effectively drive the carbon source to EPS synthesis, thereby significantly increasing EPS yield. Therefore, sucrose is the optimal carbon source for EPS biosynthesis by *Westernella* fusion.

[0058] like Figure 2 As shown, sucrose, as the main carbon source, significantly affected the biosynthesis of extracellular polysaccharides (EPS) in *Westernella* (p < 0.05). A sucrose concentration of 40 g / L yielded the highest EPS yield of 2.96 g / L. Previous studies have also found that an appropriate concentration provides a balanced carbon source supply and osmotic pressure environment for cell growth and EPS synthesis, thereby maximizing polysaccharide synthesis. Above this concentration, EPS content decreased, indicating that sucrose concentrations exceeding the optimal level are detrimental to *Westernella* secretion of extracellular polysaccharides. Therefore, a sucrose concentration of 4% (by mass) is considered the optimal sucrose addition level.

[0059] like Figure 3 As shown, the growth and metabolism of *Westernella* are affected by the inoculum size. The inoculum size can influence the initial cell content in the bacterial culture, thereby affecting cell growth and metabolism. With increasing inoculum size, the EPS content increases. At a 3% inoculum size, the extracellular polysaccharide content of *Westernella* reaches its highest level, reaching 2.18 g / L. Further increases in inoculum size do not significantly alter the EPS content compared to 3% (p > 0.05). When the inoculum size is appropriate, the cells can grow and multiply rapidly, thus forming sufficient biomass to synthesize EPS in a short time. However, when the inoculum size is too high, the excessive number of cells leads to a large consumption of nutrients to maintain basic cell metabolism, thereby inhibiting EPS synthesis. Therefore, a 3% inoculum size is chosen as the appropriate inoculum size.

[0060] like Figure 4 As shown, EPS production peaked at 2.48 g / L (p < 0.05) at a culture temperature of 33℃. Overall, EPS synthesis was higher at culture temperatures below 33℃. The optimal growth temperature for *Westernella* is around 30℃, at which temperature the strain grows vigorously, accumulating large cell volumes, which in turn promotes increased EPS synthesis. However, when the temperature is further increased to 39℃, the excessively high temperature accelerates the synthesis rate of cell cells and cell walls, a condition unfavorable for EPS production, ultimately leading to a significant decrease in extracellular polysaccharide production. Based on these results, 33℃ was determined to be the suitable culture temperature for EPS synthesis by *Westernella* fusion strain.

[0061] like Figure 5As shown, in the early stage of cultivation, EPS yield was positively correlated with time, reaching a maximum of 2.01 g / kg at 36 h (p < 0.05). After cultivation time exceeded 36 h, EPS content began to decrease. There are likely two main reasons for this yield decline: firstly, the degradation of EPS by accumulated organic acids during fermentation; and secondly, the decomposition and utilization of EPS by the microbial cells under carbon source limitation in the later stages. Therefore, 36 h was selected as the optimal cultivation time.

[0062] (2) Orthogonal experimental design: Using initial pH, inoculum size, and culture time as variables, a 4-factor, 3-level L9 (3 3 An orthogonal experiment was conducted to determine the yield of EPS from *Westernella fusionis* under different fermentation conditions, to determine the influence of each factor on the results, and to screen out the optimal fermentation parameters. The experimental design is shown in Table 1.

[0063] Table 1. Factor Level Design for Orthogonal Experiments

[0064] The sucrose content of the culture medium, culture temperature, culture time, and inoculum size were selected for a 4-factor, 3-level L9 (3) experiment. 3 An orthogonal experiment was conducted to determine the EPS yield of *Westernella fusionis* under different fermentation conditions, identify the influence of each factor on the results, and screen the optimal fermentation parameters. The orthogonal experiment analysis is shown in Table 2. The results showed that the order of influence of the factors affecting fermentation was D>A>B>C, with inoculum size having the greatest impact, followed by sucrose dosage and culture temperature, while culture time had the smallest range. Based on the orthogonal experiment results, the optimal fermentation condition combination was determined to be A1B1C2D1, i.e., a sucrose dosage of 35 g·L⁻¹. -1 The culture temperature was 30℃, the culture time was 36 h, and the inoculum size was 2.5%.

[0065] Table 2. Results and Analysis of Orthogonal Experiments

[0066] The validation experiment was conducted under the optimal conditions: the sucrose content in the MRS medium was 35 g / L, the inoculum size was 2.5%, the culture time was 36 h, and the culture temperature was 30℃. The experiment was conducted in triplicate. The yield of EPS from the fusion Weissella after the optimization of the fermentation process was 5.87±0.07 g / L, which was 385.12% higher than that before optimization.

[0067] Example 2: Optimization of extraction conditions for Weissella extracellular polysaccharides MRS liquid medium was used as the basal medium. The fermentation conditions were: sucrose content of 35 g / L in MRS medium, inoculum size of 2.5%, culture time of 36 h, and culture temperature of 30℃.

[0068] The method for determining EPS of *Westernella fusionis* is the same as in Example 1, except that the extraction steps are as follows: the fermentation broth is first mixed with a eutectic solvent for extraction, and after standing, it is centrifuged to obtain the supernatant. The supernatant is then used to continue the extraction steps of Example 1.

[0069] Preparation method of eutectic reagent: Weigh choline chloride and oxalic acid in a molar ratio of 2:1, put them into a constant temperature water bath at 80℃, and stir magnetically for 2 h until the solvent becomes clear and homogeneous.

[0070] (1) Single-factor experimental design: Three variables were selected for single-factor experiments: eutectic solvent: fermentation broth (1:1, 1:2, 1:3, 1:4, 1:5), extraction temperature (40℃, 50℃, 60℃, 70℃, 80℃), and extraction time (30 min, 45 min, 60 min, 75 min, 90 min). EPS was extracted and the EPS yield of the strain was measured. The optimal material-to-liquid ratio, extraction temperature, and extraction time were determined based on the EPS yield. Each experiment was repeated three times, and the final result was the average value.

[0071] Effect of different solid-liquid ratios on EPS extraction yield: EPS extraction yield showed a trend of first increasing, then decreasing and then stabilizing with the change of solid-liquid ratio. 1:2 was the optimal solid-liquid ratio within the experimental range, which could obtain the highest polysaccharide extraction yield of 7.47 g (p<0.05). After the solid-liquid ratio exceeded 1:4, further increasing the amount of solvent did not have a significant effect on improving the extraction yield, but instead increased the cost of subsequent concentration.

[0072] Effect of different temperature ratios on EPS extraction yield: The extraction yield of extracellular polysaccharides showed a trend of first decreasing, then increasing, then decreasing again, and finally stabilizing with temperature. The optimal extraction temperature was 60℃, at which the extraction yield of extracellular polysaccharides was the highest at 7.17 g (p < 0.05). At temperatures too low (40℃), the polysaccharides may not have been fully released into the extract due to insufficient molecular motion. At temperatures too high (above 60℃), the extraction yield may have decreased because some polysaccharide structures were damaged or denatured. The extraction yields were the same at 70℃ and 80℃, indicating that the stability of polysaccharides did not change significantly within this temperature range.

[0073] Effect of different extraction times on EPS extraction yield: The EPS content of *Westernella fusionis* gradually increased with prolonged extraction time, reaching a peak and then slightly decreasing. 75 min was the optimal incubation time within the experimental range, yielding the highest EPS yield of 6.13 g (p < 0.05). Too short an extraction time would result in incomplete extraction, while too long an extraction time might lead to a slight decrease in yield due to reduced extraction efficiency or EPS loss.

[0074] (2) Orthogonal experimental design: Orthogonal experiments were designed using software orthogonal design assistant. The material-liquid ratio, extraction temperature and extraction time were used as variables to conduct a 3-factor 3-level L9 (33) orthogonal experiment. The yield of fused Weissella EPS produced under different extraction conditions was measured to determine the influence of each factor on the results and to screen out the optimal fermentation parameters. The experimental design is shown in Table 3.

[0075] Table 3. Factor Level Design of Orthogonal Experiment

[0076] The material-to-liquid ratio, extraction temperature, and extraction time were selected for a 3-factor, 3-level L9 (3) analysis. 3 An orthogonal experiment was conducted to determine the EPS yield of *Westernella fusionis* under different extraction conditions, identify the influence of each factor on the results, and screen the optimal extraction parameters. The orthogonal experimental analysis is shown in Table 4. The results showed that the order of influence of the factors affecting extraction was C>A>B, with extraction time having the greatest impact, followed by the solid-liquid ratio, and extraction temperature having the smallest range. Based on the orthogonal experimental results, the optimal fermentation condition combination was determined to be A2B1C1, i.e., a solid-liquid ratio of 1:2, an extraction temperature of 65℃, and an extraction time of 55 min.

[0077] Table 4. Results of Orthogonal Experiment Analysis

[0078] The validation experiment was conducted under the optimal conditions: a material-to-liquid ratio of 1:2, an extraction temperature of 65℃, and an extraction time of 55 min. The experiment was conducted in three parallel trials. The yield of fused Weissella EPS after optimizing the extraction conditions was 8.77±0.03 g / L.

[0079] Example 3 Purification of fused Weissella EPS 1. Separation of EPS by DEAE-52 cellulose chromatography Before regenerating and swelling the DEAE-52 cellulose column packing material, use 3-5 times the packing material volume of 0.1M acetic acid solution to swell for 1 hour. Discard the supernatant, repeat 3 times, and then replace with pure water to equilibrate. After repeatedly rinsing with pure water, pack the column, add buffer solution to the bottom of the column, close the column outlet, and leave a small amount of buffer solution inside the column.

[0080] Take 10 mg of crude EPS extracted under orthogonal optimal conditions (Example 2), add pure water to prepare a 10 mg / mL sample solution, filter the sample solution through a membrane to remove impurities, and then add the filtered sample solution to a DEAE-52 column and elute with different concentrations of sodium chloride (0 mol / L, 0.1 mol / L, 0.2 mol / L) at a flow rate of 1 mL / min, using 200 μL of [unspecified solution]. Evaporate and concentrate the collected fractions, dialyze for 12 h, freeze at -80℃ for 24 h, and then freeze-dry under vacuum for 48 h to obtain a preliminarily purified sample.

[0081] 2. Separation of EPS by dextran G-100 gel chromatography The packing material was placed in deionized water and allowed to swell at room temperature for 48 h. After swelling, the supernatant was removed. The packing material, buffer solution, and other materials were equilibrated to room temperature, and all buffer solutions were filtered and degassed. After packing the column, the peristaltic pump was turned on to allow the buffer solution to flow through at a rate of 1.33 mL / min to stabilize the column bed. The column was equilibrated until the baseline was stable before sample loading.

[0082] Take 10 mg of the sample purified by DEAE-52 cellulose column, add pure water to prepare a 10 mg / mL sample solution, filter the sample solution through a membrane to remove impurities, and then add the filtered sample solution to a dextran G-100 gel column. Elute with different concentrations of sodium chloride (0 mol / L, 0.1 mol / L, 0.2 mol / L) at a flow rate of 1 mL / min. Evaporate and concentrate the collected fractions, dialyze for 12 h, freeze at -80℃ for 24 h, and then freeze-dry under vacuum for 48 h to obtain the purified sample.

[0083] like Figure 9 As shown, the pretreated extracellular polysaccharides were separated by a DEAE-5 cellulose anion exchange column and eluted with ultrapure water, 0.1 mol / L NaCl solution, and 0.2 mol / L NaCl solution to obtain three fractions. The first peak eluted with ultrapure water had the largest molecular weight, and this fraction was selected for subsequent separation experiments.

[0084] like Figure 10 As shown, EPS was separated by a DEAE-5 cellulose anion exchange column and then lyophilized. It was then passed through a dextran G-100 gel column and eluted with ultrapure water, 0.1 mol / L NaCl solution, and 0.2 mol / L NaCl solution to obtain three fractions. The fraction obtained from the first peak eluted with ultrapure water was selected for subsequent experiments.

[0085] Example 4: Extracellular Polysaccharide Structure Analysis 1. Molecular composition (1) Preparation of standard products: After accurately weighing the standard products required for this project, add water to prepare a 10 mg / mL standard solution stock solution single standard. Then take an appropriate amount of standard stock solution single standard and mix them to prepare a standard mixed standard with the highest index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL. Prepare a series of standard products required for the instrument according to the concentration gradient in Table 5.

[0086] Table 5. Monosaccharide mixed standard gradient concentration information

[0087] (2) Sample pretreatment: Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample, add 1 mL of 2 M TFA acid solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Add 99.99% methanol to wash, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic bottle for analysis.

[0088] The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), which used an electrochemical detector to analyze and detect monosaccharide components.

[0089] Utilizing Dionex™ CarboPac™ PA20 (150) 3.0 mm, 10 μm liquid chromatography column; injection volume: 5 μL. Mobile phases A (H₂O), B (0.1 M NaOH), and C (0.1 M NaOH, 0.2 M NaAc) were used at a flow rate of 0.5 mL / min. The column temperature was 30 °C. The elution gradients were as follows: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10, V / V), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), and 60 min A / B / C (95:5:0, V / V). Chromatographic data were processed using Chromeleon software. The figure below shows the ion chromatograms of the standard sample and the sample, with the horizontal axis representing the retention time (Time, min) and the vertical axis representing the ion detection response value (Response, nC).

[0090] 2. Determination of molecular configuration and molecular weight The sample 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 then filtered through a 0.45 μm filter before being analyzed.

[0091] The chromatographic system used was a gel chromatography-differential chromatography-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt Technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt Technology, CA, USA). Specific column and elution conditions were as follows: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) gel size exclusion columns were used in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0092] Chromatographic data were processed using ASTRA 6.1 software. Absolute molecular weight analysis chromatogram: retention time (time, min) was plotted on the x-axis, and molar mass (g / mol) on the y-axis; Molecular configuration chromatogram: molar mass (Molar Mass, g / mol) was plotted on the x-axis, and root mean square radius (RMS Radius, nm) on the y-axis.

[0093] like Figure 11 and Figure 12 As shown, by comparing the ion chromatogram of *Westernella fusionis* EPS with that of the standard, it was found that *Westernella fusionis* EPS is mainly composed of glucose. Plotting log(Molar Mass) on the x-axis and log(RMS Radius) on the y-axis, the slope can be used as a reference for molecular configuration. Generally, a slope of 1 indicates a rod-shaped molecule, a slope of 0.5-0.6 indicates a random coil, and a slope of 1 / 3 indicates a spherical shape. *Westernella fusionis* EPS is spherical.

[0094] Table 6 Molecular Composition

[0095] As shown in Table 6 and Figure 13As shown, the peak molecular weight is significantly higher than other molecular weight parameters, indicating that the component with the highest content in the sample is a high molecular weight component. The polydispersity index (PDI) of 1.400 is a relatively narrow molecular weight distribution, indicating good stability and high molecular homogeneity in the synthesis of extracellular polysaccharides by *Westernella*. The root mean square radii are close to and within the hundreds of nanometers, indicating that the polysaccharide exhibits a relaxed and uniform spatial conformation in solution, generally a linear or slightly branched chain. This conformation is the structural basis for its viscosity and emulsifying effect in solution. The 100% component content indicates high extraction purity of the polysaccharide with no significant impurities interfering.

[0096] 3. Electron microscopy The dried *Westernella fusionis* EPS powder sample was placed on a carbon-coated electrothermal film adhered with double-sided tape. Any sample that was not adhered was blown off with a syringe. Gold was sputtered onto the sample surface using an MC 1000 sputtering coating machine. The gold-sputtered sample was then placed in a scanning electron microscope, and the morphology and structural characteristics of the extracellular polysaccharide were observed at magnifications of 500 and 5000.

[0097] like Figure 14 As shown, the extracellular polysaccharide has a rough surface without obvious breakage or deformation, maintaining its intact morphology. The loose overlap of secondary units and the surface irregularities together create a high specific surface area, providing ample "interaction space" for the adsorption of small molecules or the binding of bioactive targets (such as enzymes and cell receptors), resulting in outstanding adsorption and bioactivity potential. The microscopically rough surface further increases the specific surface area, exposing more active groups such as hydroxyl and carboxyl groups, which not only enhances solubility but also increases the number of binding sites with biomolecules, theoretically leading to higher potential for bioactivity (such as antioxidation and immune regulation).

[0098] 4. Fourier transform infrared spectroscopy The dried polysaccharide sample and potassium bromide (spectral grade) were mixed at a mass ratio of 1:100, ground into powder, pressed into thin sheets, and scanned using a Fourier transform infrared spectroscopy (FTIR) spectrometer. The scanning range was 400–4000 cm⁻¹. -1 , with 4 cm -1 The resolution was scanned 16 times.

[0099] like Figure 15 As shown, at 3430 cm -1 The peak at 2920 cm⁻¹ is a characteristic broad peak representing the OH stretching vibration (hydroxyl group) of polysaccharides, indicating intermolecular / intramolecular hydrogen bonding and representing the core functional group responsible for the hydrophilicity of polysaccharides. -1 The position represents the CH stretching vibration (-CH2- / -CH3), a characteristic vibration of the hydrocarbon backbone of polysaccharides, reflecting the alkyl structure of the sugar chain. At 1416 cm⁻¹ -1 The position represents the CH bending vibration (-CH2-), indicating the in-plane bending vibration of the alkyl chain, which can assist the reaction of the hydrocarbon skeleton. At 1315 cm⁻¹ -1The denoted by COH bending vibration (hydroxyl group) represents the in-plane bending vibration of the hydroxyl group in polysaccharides. At 1158 cm⁻¹ -1 The peak at 10¹³ cm⁻¹ represents the COC stretching vibration (glycosidic bond), a key characteristic peak of the polysaccharide backbone that reflects the presence of glycosidic bonds. -1 The peak at 915 cm⁻¹ represents the vibration of the pyranose ring skeleton and is a typical absorption peak of the pyranose ring fused with *Weisseria gonorrhoeae* EPS. -1 The peak at 890–910 cm⁻¹ is a characteristic peak for β-glycosidic bonds. -1 The peak in the region suggests that this polysaccharide contains β-glycosidic bonds. At 861 cm⁻¹... -1 The out-of-plane bending vibration at 782 cm⁻¹ is a characteristic vibration of the sugar ring and can help verify the sugar ring configuration. -1 The peak at 915.96 cm⁻¹ represents the out-of-plane bending vibration of CH, which is also an auxiliary characteristic peak of the sugar ring structure. This ultimately indicates that this is a typical natural polysaccharide, containing core structures such as hydroxyl groups, glycosidic bonds, and pyranose rings, and is detected through a peak at 915.96 cm⁻¹. -1 The peak indicates that its glycosidic bond is β-type.

[0100] 5. Methylation experiment Take a small sample (2-3 mg) and dissolve it in 500 μL DMSO. Add 1 mg NaOH and incubate for 30 min. Add 50 μL iodomethane solution and react for 1 h. Add 1 mL water and 2 mL dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water 3 times. Take the lower dichloromethane phase and dry it under nitrogen. Add 100 μL 2 M TFA and react at 121 °C for 90 min. Evaporate to dryness at 30 °C. Add 50 μL 2 M ammonia and 50 μL 1 M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL acetic acid to terminate the reaction, dry under nitrogen, wash twice with 250 μL methanol, and dry under nitrogen. Add 250 μL acetic anhydride, vortex to mix, and react at 100 °C for 2.5 h. Add 1 mL water and let stand for 10 min. Add 500 μl of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water three times. Collect the lower dichloromethane phase and analyze it using GC-MS. The results are shown in Table 7. Table 7. Methylation Detection Results

[0101] The above test results show that the monosaccharide of the extracellular polysaccharide of Weissella is glucose, and the detected linkage modes include: terminal glucose t-Glc(p), 1→4 linked glucose, 1→4,6 linked glucose, 1→3,6 linked glucose, and 1→2,6 linked glucose.

[0102] The extracellular polysaccharide backbone of Weissella is based on glucose linked in a 1→4 manner; 81.31% of the backbone glucose forms branches at the C6 position, while a small number of branching sites exist at the C3 (7.3%) and C2 (1.93%) positions; the ends of the branches consist of 8.4% free glucose residues t-Glc(p).

[0103] 6. Congo Red Experiment Prepare 2 mg / mL fusion solutions of *Westernella* EPS and 0.16 mg / mL Congo red. Mix 2 mL of polysaccharide solution with 1 mL of Congo red solution, then add 1 mL of NaOH solution (0, 0.2, 0.4, 0.6, 0.8, 1.0 mol / mL) to the above solution to obtain six test solutions. Replace the polysaccharide solution with water as a blank. After mixing the solutions, let them stand for 30 min, and record the UV absorption peaks in the wavelength range of 400-600 nm.

[0104] like Figure 16 As shown, with increasing alkali concentration, the absorbance of the Congo red-polysaccharide complex did not change significantly, and the maximum absorption wavelength remained essentially unchanged, resulting in a relatively flat curve without a noticeable red shift. The extracellular polysaccharide of *Westernella* does not possess a triple helical structure.

[0105] Example 5: Performance Verification of Extracellular Polysaccharides 1. Hygroscopicity and moisture retention determination A certain amount of EPS sample was dried at 105℃ to constant weight. 50 mg (ml) was accurately weighed and placed in a desiccator with a relative humidity of 81% (saturated ammonium sulfate solution). The EPS sample was weighed every 1 hour at room temperature. t ), with glycerol as a positive control.

[0106] Hygroscopicity (%) = ×100% The moisture-saturated sample (m0) was placed in a desiccator containing modified silica gel. A moisture retention test was conducted at room temperature, with the EPS sample weighed every 1 hour at room temperature (m0). t ), with glycerol as a positive control.

[0107] Moisturizing properties (%) = ×100% Study on the hygroscopicity and moisture retention of EPS The hygroscopicity of *E. fusionis* EPS is lower than that of glycerol, and it can reach hygroscopic equilibrium in about 1 hour and maintain a stable hygroscopic state of 10%. Figure 17 , 18While the hygroscopicity of glycerol increased nearly linearly over time, its moisturizing rate decreased almost linearly. This indicates that although polysaccharides such as EPS have relatively weak hygroscopicity, their longer molecular chains allow them to form a more stable network structure, encapsulating water molecules and thus providing a better moisturizing effect. The core function of EPS is not strong hygroscopicity, but rather its excellent water-locking ability and water-holding stability. Therefore, this polysaccharide can serve as a moisturizing material with potential application value in inhibiting water evaporation and maintaining the humidity stability of the system, especially suitable for food and cosmetic systems that require dynamic moisture balance.

[0108] 2. Determination of water-holding and oil-holding properties Take 200 mg EPS (m0) and place it in a 25 mL expansion tube. Add 10 mL of water and let it stand for 24 hours after the EPS is completely wetted. Discard the supernatant and read the volume (V) of the mixture.

[0109] Water holding capacity (mL / g) = ×100% Weigh a 50 mL centrifuge tube (m0), then place 200 mg of EPS (mL) into the tube. Weigh 10 mL of corn oil and pour it into the tube. Stir magnetically at 100 rpm for 30 min to mix thoroughly. Centrifuge at 7000 rpm for 20 min, discard the supernatant, and weigh the total weight (m2). Casein and xanthan gum are used as positive controls.

[0110] Oil retention (g / g) = ×100% Table 8. Comparison of oil-holding properties of Fusion Weissella EPS with casein and xanthan gum

[0111] As shown in Table 8, the water-holding capacity of the fused *Weissella asiatica* EPS is 3.71 mL / g, indicating a certain level of water retention. Its oil-holding capacity for corn oil is 32.85 g / g, exceeding 32 times its own weight. The oil-holding capacity of *Weissella asiatica* is 4.2 and 5.7 times that of casein and xanthan gum, respectively, demonstrating excellent oil adsorption capacity. Observations revealed that the gel structure of this polysaccharide remained stable after adsorbing oil, with no sugar-oil separation, indicating its applicability in fatty food systems such as mayonnaise and cheese, improving product texture and processing performance. *Weissella asiatica* EPS can replace 50%–60% of fat, effectively reducing product calories. Therefore, it can be considered a promising fat substitute food additive, similar to inulin as a fat substitute in appetizers, improving food texture, forming a smooth and delicate gel, thus giving food an excellent fat texture and a fat-like mouthfeel.

[0112] 3. Determination of emulsifying ability and emulsification stability Weigh 200 mg EPS and dissolve it completely in 10.0 mL of distilled water. Add 5.0 mL of corn oil, shake well, and centrifuge at 2500 r / min for 10 min. Record the volume of the emulsion layer (V1).

[0113] Emulsifying properties (%) = ×100% The emulsion was heated in an 80 °C water bath for 30 min, then centrifuged at 2500 r / min for 10 min, and the volume of the emulsion layer (V2) was recorded. Soy lecithin was selected as a positive control.

[0114] Emulsion stability (%) = ×100% Table 9 Comparison of emulsifying properties and emulsifying stability between *Westernella fusca* EPS and soybean lecithin

[0115] As shown in Table 9, the emulsifying properties and emulsifying stability of *Westernella fusion-based* EPS were 70.33% and 99.82%, respectively, while those of the emulsifier soybean lecithin were 88.67% and 91.26%, respectively. EPS exhibited worse emulsifying properties than soybean lecithin, but better emulsifying stability. This indicates that *Westernella fusion-based* extracellular polysaccharides possess better emulsifying properties and superior emulsifying stability. Previous reports indicated that the emulsifying properties of plant polysaccharides such as mung bean polysaccharides were 15%, and their emulsifying stability was 47%, both significantly lower than those of *Westernella fusion-based* EPS. This suggests that *Westernella fusion-based* EPS, as a natural and safe biopolymer, can serve as a highly efficient emulsifier and emulsion stabilizer, especially in food, cosmetic, or pharmaceutical systems requiring long shelf lives, where it has broad application prospects.

[0116] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An extracellular polysaccharide, characterized in that, The extracellular polysaccharide is composed of glucose monosaccharide, and the glucose linkage includes terminal glucose t-Glc(p), 1→4 linked glucose, 1→4,6 linked glucose, 1→3,6 linked glucose and 1→2,6 linked glucose, with a molar ratio of (8~8.5):(1~1.2):(81~82):(7~7.5):(1~1.2):(0.5~1).

2. The extracellular polysaccharide as described in claim 1, characterized in that, The extracellular polysaccharide has a molecular weight of 16374.659 kDa.

3. A method for preparing the extracellular polysaccharide according to any one of claims 1 to 2, characterized in that, The steps include: merging Weissella (… Weissella confusa Inoculate the culture medium and incubate at 28-42℃ for 12-120 h to obtain a fermentation broth containing fused Weissella extracellular polysaccharides; extract the fermentation broth with a eutectic solvent to obtain a crude extract of extracellular polysaccharides; purify the crude extract of extracellular polysaccharides to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The fermentation medium is an MRS medium containing 20-100 g / L sucrose.

5. The preparation method according to claim 3, characterized in that, The amount of *Westernella fusionis* inoculated is 1% to 5%.

6. The preparation method according to claim 3, characterized in that, The volume ratio of the fermentation broth to the eutectic solvent is (1~5):

1.

7. The preparation method according to claim 6, characterized in that, The eutectic solvent is composed of choline chloride and oxalic acid; preferably, the molar ratio of choline chloride to oxalic acid is 2:

1.

8. The application of the preparation method according to claims 3 to 7 in the preparation of extracellular polysaccharides.

9. The use of the extracellular polysaccharide according to claim 1 or 2 in the preparation of cosmetics or food.

10. Moisturizers, emulsifiers, emulsion stabilizers, cosmetic additives, pharmaceutical additives, and food additives containing the extracellular polysaccharides of claim 1 or 2.

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