Lactobacillus strain and application thereof in preparation of exopolysaccharide protective agent

Abstract

The application discloses a lactic acid bacterial strain and application thereof in preparation of an extracellular polysaccharide protective agent, the strain is classified and named as Lactiplantibacillus plantarum, and is preserved in the China Center for Type Culture Collection on August 31, 2023, with a preservation number of CCTCC M 20231553 and a preservation address of Wuhan University, Wuhan, China. Research results show that the EPS secreted by the strain has good water retention, oil retention and emulsification characteristics, and also has high viscosity physical and chemical characteristics. The strain can effectively improve the stress resistance of a commercial starter strain in low-temperature refrigeration and gastrointestinal digestion, and can also effectively promote the adhesion activity of the commercial starter strain to intestinal cells. The lactic acid bacterial polysaccharide has reliable biological safety as a protective additive of the commercial starter strain, effectively improves the stress resistance and beneficial activity of the strain, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food functional active ingredient processing technology. It relates to a lactic acid bacteria strain and its application in the preparation of extracellular polysaccharide protectants. Specifically, it relates to a lactic acid bacteria strain and its application in the preparation of extracellular polysaccharide protectants that improve the resistance of commercial starter cultures to cold storage, gastrointestinal environment stress, and intestinal adhesion. Background Technology

[0002] Over the past few decades, lactic acid bacteria (LAB) have been widely recognized as safe microorganisms and have been extensively used in fermented foods. Studies have shown that LAB and its metabolites have significant health benefits, including antioxidant, hypoglycemic, anticancer, immunomodulatory, and gut health-regulating effects. Currently, fermented dairy products are considered an effective carrier of lactic acid bacteria. The most common dairy starter cultures on the market are *Streptococcus thermophilus* and *Lactobacillus bulgaricus*, which not only effectively enhance the nutritional value, taste, and quality of fermented dairy products but also possess good gut-regulating activity. In addition, many functional probiotic strains are also widely used in the production of fermented dairy products. However, during the distribution, storage, and retail of probiotic fermented products, they are often affected by temperature and shelf life, leading to a decrease in viable cell counts and thus reducing the effectiveness of beneficial lactic acid bacteria functional activities. Furthermore, the tolerance of these beneficial strains to the human gastrointestinal environment is also a key factor affecting their functional activity expression. Therefore, improving the stability and gastrointestinal resistance of these commercial starter cultures during storage has a positive impact on the development of the probiotic dairy product industry.

[0003] Extracellular polysaccharides (EPS) secreted by lactic acid bacteria are long-chain polymers secreted by bacteria during their growth and metabolism, and are considered safe and reliable natural high-molecular-weight carbohydrates. In recent years, EPS has received widespread research attention due to its excellent bioactivity, such as anti-tumor, pathogen inhibition, antioxidant, and immunomodulatory effects. Studies have shown that EPS plays a crucial role in the hydrophobicity and charge distribution of bacterial surfaces, making significant contributions to the recognition and interaction of biotic and abiotic-specific surfaces. Research has shown that adding exogenous carbohydrates can effectively enhance the stability and survival rate of bacterial cells in fermented milk, the most common including inulin, fructooligosaccharides (FOS), and galactooligosaccharides (GOS). However, research on the impact of natural EPS from lactic acid bacteria on the survival stability of starter cultures in dairy products is relatively limited. *Lactobacillus plantarum* is a highly adaptable beneficial lactobacillus that can thrive in various environments, including dairy products, meat products, fermented vegetable products, and the human and animal gut. Meanwhile, *Lactobacillus plantarum* EPS has attracted widespread attention due to its rich bioactivity. However, there are no reported cases of its safe and reliable application as a protective additive for the survival of commercial fermentation strains. Therefore, developing functional commercial starter culture strain protectants using plant lactobacillus polysaccharide resources, which are green, safe and reliable, can not only effectively enhance the resistance of commercial starter cultures, but also provide a safe and reliable solution for the synergistic development and application of plant lactobacillus produced by EPS and commercial starter cultures in dairy products. Summary of the Invention

[0004] The purpose of this invention is to provide a lactic acid bacteria strain and its application in the production of an extracellular polysaccharide protectant that can improve the cold storage, gastrointestinal environment resistance and intestinal adhesion ability of the starter culture strain.

[0005] Another objective of this invention is to provide a lactic acid bacteria extracellular polysaccharide and its applications. The preparation method of this lactic acid bacteria extracellular polysaccharide is simple and efficient, exhibiting high water-holding and oil-holding capacity, excellent emulsifying properties, and the ability to improve the tolerance of starter cultures to cold storage and gastrointestinal environments, while promoting intestinal adhesion of starter cultures.

[0006] Another objective of this invention is to provide a lactic acid bacteria extracellular polysaccharide protectant that enhances the stress resistance and intestinal adhesion ability of fermentation strains. This lactic acid bacteria extracellular polysaccharide protectant is a safe and reliable lactic acid bacteria-derived biopolymer with high viscosity and strong water-holding capacity, and can be used to improve the stress resistance of commercial strains.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention seeks protection for a strain of lactic acid bacteria that produces extracellular polysaccharides, classified as *Lactiplantibacillus plantarum*, which was deposited on August 31, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20231553, located at Wuhan University, Wuhan, China.

[0009] Secondly, the present invention seeks protection for the use of the above-mentioned lactic acid bacteria strains in the production of lactic acid bacteria extracellular polysaccharides that can improve the stress resistance and / or intestinal adhesion rate of the starter culture strains.

[0010] Thirdly, the present invention seeks to protect the lactic acid bacteria extracellular polysaccharides secreted by the aforementioned lactic acid bacteria strains.

[0011] Fourthly, the present invention seeks protection for the use of the extracellular polysaccharides of lactic acid bacteria secreted by the above-mentioned lactic acid bacteria strains in the following (1) or (2):

[0012] (1) Improve the survival rate of lactic acid bacteria fermentation strains under cold storage or prepare products that improve the survival rate of lactic acid bacteria fermentation strains under cold storage.

[0013] (2) Promote intestinal adhesion of strains or prepare prebiotic additives that promote intestinal adhesion of strains.

[0014] Furthermore, the lactic acid bacteria extracellular polysaccharides mentioned above are obtained by fermentation culture of the aforementioned lactic acid bacteria strain. Even further, the lactic acid bacteria extracellular polysaccharides are prepared by fermentation culture of the aforementioned lactic acid bacteria strain, followed by separation and purification of the extracellular polysaccharides in the fermentation broth.

[0015] Furthermore, the separation and purification process is as follows:

[0016] (1) Centrifuge the fermentation broth and collect the supernatant; treat the supernatant with 4% (v / v) trichloroacetic acid solution, centrifuge to remove protein; then add anhydrous ethanol for treatment and centrifuge to obtain crude polysaccharide precipitate;

[0017] (2) The crude polysaccharide precipitate was dissolved in deionized water and then transferred into a dialysis bag with a molecular weight cutoff of 8,000-14,000 Da for water dialysis. The dialysis solution was freeze-dried to obtain crude extracellular polysaccharide.

[0018] (3) The crude extracellular polysaccharide was further purified using a DEAE-52 anion exchange column. A gradient elution was performed using deionized water, 0.1M and 0.3M NaCl solutions at a flow rate of 1 mL / min to collect the purified polysaccharide fraction. The sugar content was determined by the phenol-sulfuric acid method. Finally, the main fraction was dialyzed with pure water and freeze-dried to obtain the purified lactic acid bacteria extracellular polysaccharide. The main fraction is the component obtained after purification with deionized water.

[0019] Fifthly, the present invention claims protection for a protective agent that enhances the stress resistance and intestinal adhesion ability of a fermentation agent strain, the protective agent containing lactic acid bacteria extracellular polysaccharide secreted by the lactic acid bacteria strain.

[0020] The fermentation agent strains mentioned are commercial fermentation agent strains, but are not limited to them.

[0021] This invention screened a lactic acid bacteria strain that produces extracellular polysaccharides. This strain was isolated from kefir grains in Aksu, Xinjiang. 16S rDNA sequencing analysis identified it as *Lactiplantibacillus plantarum*, and it was named *Lactiplantibacillus plantarum*. During the research, technicians first extracted, isolated, and purified the extracellular polysaccharides from the lactic acid bacteria. The purified extracellular polysaccharides from the lactic acid bacteria were then studied as follows:

[0022] (1) Evaluation of water-holding capacity, oil-holding capacity and emulsifying properties;

[0023] (2) Evaluation of solution microrheological properties;

[0024] (3) Evaluation of the protective activity of commercial fermentation agent strains after 21 days of low-temperature storage;

[0025] (4) Evaluation of the stress resistance of commercial fermentation strains in the gastrointestinal fluid environment;

[0026] (5) Comprehensive evaluation of the effect of prebiotic additive on promoting intestinal adhesion of commercial fermentation strains.

[0027] The results show that this invention isolated and purified an extracellular polysaccharide (EPS) from a lactic acid bacteria strain that produces high levels of EPS, with a molecular weight of 1.41 × 10⁻⁶. 6 Da, composed of galactose and glucose in a molar ratio of 1.21:1.00, exhibits high water and oil retention capacities (621.34% and 834.56%, respectively) and superior emulsifying properties compared to guar gum at a concentration of 4 mg / mL (90.21%, 30 min; 79.02%, 60 min). Rheological analysis shows that when the concentration of this lactic acid bacteria extracellular polysaccharide reaches 40 mg / mL, the solid-liquid equilibrium is below 0.5, exhibiting gel-like properties similar to solids. Furthermore, as a protective additive, EPS-T1 significantly improves the survival rate of three commercial lactic acid bacteria strains (Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus rhamnosus) during a 21-day storage period, while effectively enhancing their adhesion activity to CaCo-2 cells.

[0028] Compared with the prior art, the advantages and effects of the present invention are as follows:

[0029] 1) The polysaccharide prepared by this invention uses naturally sourced lactic acid bacteria as the parent material, has good biocompatibility and is renewable, inexpensive and green.

[0030] 2) The lactic acid bacteria polysaccharide prepared by this invention has excellent water-holding capacity, oil-holding capacity and viscosity characteristics.

[0031] 3) The lactic acid bacteria polysaccharide prepared by this invention has good protective activity against commercial fermentation strains and good intestinal adhesion ability.

[0032] The foregoing summary of certain objectives of the invention is not exhaustive and does not represent all objectives of the invention. Other objectives and advantages of the invention will become clearer from the following description, which, together with the accompanying drawings, illustrations, and embodiments, and specific details of the invention, forms part of this specification. This includes exemplary embodiments of the invention, illustrating various objectives and features of the invention. Attached Figure Description

[0033] Figure 1 This refers to the extraction and purification steps of lactic acid bacteria extracellular polysaccharides in Example 1.

[0034] Figure 2 This is a high-performance liquid chromatography gel permeation chromatogram of lactic acid bacteria extracellular polysaccharides.

[0035] Figure 3 This is a chromatogram showing the monosaccharide composition of extracellular polysaccharides from lactic acid bacteria.

[0036] Figure 4 Physicochemical properties of lactic acid bacteria extracellular polysaccharides (A: water-holding capacity and oil-holding capacity; B: emulsifying properties (30 min); C: emulsifying properties (60 min)).

[0037] Figure 5 Microrheological properties of lactic acid bacteria extracellular polysaccharides.

[0038] Figure 6 The study investigated the cryopreservation properties of extracellular polysaccharides from lactic acid bacteria against commercial starter cultures. (AC: the cell viability protection of different polysaccharide concentrations against *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Lactobacillus rhamnosus* LGG over 20 days; DF: the number of viable cells of the polysaccharide (4 mg / mL) against *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Lactobacillus rhamnosus* LGG over 20 days).

[0039] Figure 7 Extracellular polysaccharides of lactic acid bacteria protect the cell resistance of commercial starter cultures during gastrointestinal digestion. (AC: Polysaccharide protection against the survival of *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Lactobacillus rhamnosus* LGG during gastric digestion; DF: Polysaccharide protection against the survival of *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Lactobacillus rhamnosus* LGG during small intestinal digestion).

[0040] Figure 8 Extracellular polysaccharides of lactic acid bacteria were used as additives to promote intestinal adhesion activity of commercial fermentation strains. (A: Adhesion rate of bacterial cells to CaCo-2 cells; B: Effect of bacterial and cell adhesion). Detailed Implementation

[0041] The method of the present invention will be described below through specific embodiments, but the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0042] Example 1: Screening of lactic acid bacteria strains

[0043] This invention screened a strain of lactic acid bacteria that produces extracellular polysaccharides. This strain was isolated from kefir grains in Aksu, Xinjiang. The strain was identified as *Lactiplantibacillus plantarum* T1 by 16S sequencing analysis and was deposited at the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with accession number CCTCC M 20231553, located at Wuhan University, Wuhan, China.

[0044] (1) Screening method:

[0045] Kefir grains were sourced from dairy products made by residents in Aksu, Xinjiang. After sampling in sterile containers, the samples were sent back to the laboratory under low-temperature conditions for the isolation and purification of lactic acid bacteria. First, MRS liquid and solid culture media were prepared for the growth and plating of lactic acid bacteria colonies. Then, a piece of kefir grain was selected in a laminar flow hood and immersed in MRS liquid culture medium, followed by thorough vortexing. After 24 hours of incubation, the culture medium containing the bacterial solution was serially diluted with 0.85% (g / 100ml) sterile physiological saline, and then the colonies were plated. After culturing at 37℃ for 30 hours, the colony morphology on the solid plates was observed. Single colonies with differences in size, color, and fibrillation were selected for streak purification and labeled. The streak purification process was repeated 3-4 times. Single colonies were then selected for Gram staining and microscopic examination to ensure that the selected strains were Gram-positive and catalase-negative, and were non-spore-forming. The purified strains were selected through the above operations, and then activated by liquid culture. The cultured single bacteria were then sent to a biotechnology company for 16S rDNA sequencing. The sequencing results were compared with the bioinformatics data in the GenBank database to determine that strain T1 was *Lactobacillus plantarum*.

[0046] Example 2: Extraction and physicochemical composition analysis of extracellular polysaccharides from lactic acid bacteria

[0047] (1) Extraction of extracellular polysaccharides from lactic acid bacteria

[0048] A glycerol-preserved lactic acid bacteria strain (Lactiplantibacillus plantarum T1) was inoculated into MRS liquid medium and activated twice at 30°C for 20 h each time. Subsequently, it was transferred to a large flask of MRS medium at a ratio of 4% (v / v) and incubated at 30°C for 28 h. The fermented medium was centrifuged at 12000 rpm for 15 min to obtain a supernatant containing extracellular polysaccharides (EPS). The supernatant was then treated with 4% (v / v) trichloroacetic acid (TCA) solution, allowed to stand at 4°C for 6 h, and centrifuged to remove proteins. Then, three volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight at 4°C to precipitate crude polysaccharides. The crude extracellular polysaccharide precipitate was then obtained by centrifugation and dissolved in deionized water. The precipitate was transferred to a dialysis bag (MD34, 8000-14000 Da) and dialyzed against running water for 72 h. The dialysate was freeze-dried to obtain a dried crude extracellular polysaccharide sample. The extracellular polysaccharide was further purified using a DEAE-52 anion exchange column (2.6 × 30 cm). Gradient elution was performed using deionized water, 0.1 M, and 0.3 M NaCl solutions at a flow rate of 1 mL / min. Twenty tubes (10 mL each) were collected under each elution condition. The sugar content in each tube was determined by the phenol-sulfuric acid method. The fractions obtained primarily through deionized water purification were collected, dialyzed against pure water, and freeze-dried to obtain the purified lactic acid bacteria extracellular polysaccharide EPS-T1 for further research (see [link to study]). Figure 1 ).

[0049] (2) Physicochemical composition analysis of polysaccharides

[0050] High-performance liquid chromatography-gel permeation chromatography (HPGPC) was used to determine the homogeneity and relative molecular mass of polysaccharides. The selected columns were an Ultrahydrogel™ guard column (6 mm × 40 mm) and an Ultrahydrogel™ linear gel column (7.8 mm × 300 mm). The mobile phase was 0.02% (w / v, g / 100 mL) sodium azide, the flow rate was 0.6 mL / min, and the detectors were a differential detection detector (RID) and a UV detector, with the detection wavelength set at 280 nm. The column temperature was 35 °C, the injection volume was 20 μL, and the data acquisition time was 30 min.

[0051] The monosaccharide composition of polysaccharides was determined using high-performance anion exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). First, 5 mg of the polysaccharide sample was weighed into a glass tube, and 0.5 mL of 12 M H₂SO₄ was added. A stir bar was added to the glass tube, and the mixture was magnetically stirred for 30 min in an ice bath. After the reaction, deionized water was added to dilute the H₂SO₄ concentration to 2 M, and then the mixture was transferred to a 120 °C oil bath for hydrolysis for 2 h. The hydrolysate was cooled and diluted 100-fold, then filtered through a 0.22 μm pinhole filter before analysis. Different monosaccharide standards and their standard curves were established using the same procedure, mainly including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid. Chromatographic conditions were performed using a Dionex ICS-5000 ion-exchange chromatography system, equipped with a pulsed amperometric detector, a CarboPac™ PA20 guard column, and a CarboPac™ PA20 analytical column (4 mm × 250 mm). Mobile phase A was ultrapure water, mobile phase B was 250 mmol / L NaOH solution, and mobile phase C was 1 M NaOAc solution. Separation was achieved using gradient elution. The flow rate was set at 0.5 mL / min, and the elution program is detailed in Table 1.

[0052] Table 1 Mobile phase elution program for Dionex chromatography system

[0053]

[0054] The results showed that the purified extracellular polysaccharide had a single and symmetrical chromatographic peak, indicating the homogeneity of the polysaccharide component. The molecular weight (Mw) of the polysaccharide was determined to be 1.41 × 10⁻⁶. 6 Da is composed of galactose and glucose in a molar ratio of 1.21:1.00. Figure 2 , Figure 3 ).

[0055] Example 3 Physicochemical properties of lactic acid bacteria extracellular polysaccharide solution

[0056] 1. Analysis of the water-holding, oil-holding and emulsifying properties of polysaccharide protectants

[0057] First, accurately weigh 50 mg of the polysaccharide sample and completely dissolve it in 1 mL of distilled water. Then, centrifuge the completely dissolved polysaccharide solution at 20,000 × g for 30 minutes to remove unbound water. Record the mass of the polysaccharide after complete water absorption. The WHC of EPS-T1 is calculated using the following formula:

[0058] WHC (%) = [Total weight of sample after water absorption] / [Total weight of dry sample] × 100

[0059] Furthermore, the procedure for determining oil holding capacity (OHC) was the same as that for water holding capacity, and sunflower oil was used in the experiment.

[0060] Different concentrations of EPS solutions (0.5, 1.0, 2.0, and 4.0 mg / mL) were prepared using phosphate-buffered saline (PBS, pH 6.8). Then, 1 mL of the polysaccharide solution was mixed with 0.25 mL of hexadecyl alkane and mixed for 1 min. The absorbance was measured at 550 nm, and the absorbance before and after shaking (A0) was recorded. The absorbance values ​​after incubation at room temperature for 30 and 60 min (A1) were also recorded. PBS solution was used as a negative control. Guar gum and xanthan gum were used as control groups. Emulsifying activity was calculated using the following formula:

[0061] Emulsifying activity (%) = (A1 / A0) × 100

[0062] The results showed that EPS-T1 possessed excellent hydration and oil retention capabilities (621.34% and 834.56%, respectively). Figure 4 (A) This may be attributed to its higher molecular weight. Comparative analysis with commercial polysaccharides xanthan gum and guar gum showed that the emulsifying properties of EPS-T1 increased with increasing concentration. At a concentration of 4 mg / mL, EPS-T1 maintained emulsifying activity of 96.78% and 94.10% after 30 min and 60 min, respectively, significantly higher than guar gum (A). Figure 4 B,C).

[0063] 2. Microrheological properties of polysaccharide protectants

[0064] Microrheological analysis of EPS-T1 was performed using a commercial multispot diffusion spectroscopy system (MS-DWS, Rheolaser Lab, Formulaction, France). Briefly, EPS-T1 solutions of different concentrations (5, 10, 20, and 40 mg / mL) were prepared using deionized water. Then, 20 mL of the polysaccharide solution was transferred to a specially designed glass vial and then to the measurement chamber. The measurement temperature was set to 25 °C. Initial data collection and analysis were performed using the instrument's software. All polysaccharide samples were fully hydrated before measurement.

[0065] The results showed that the MVI of the EPS-T1 solution increased rapidly with increasing concentration, indicating the gradual expansion of the EPS-T1 polysaccharide molecular chains and the high degree of polymerization in the aqueous solution. Figure 5 A). Meanwhile, the EI gradually increases with increasing concentration, indicating that the polysaccharide solution mainly exhibits solid-phase behavior in the dispersion system and forms a gel-like structure. Figure 5B). In addition, the solid-liquid balance (SLB) directly reflects the time preference of the sample for solid or liquid state. When 0 < SLB < 0.5, the sample exhibits elastic modulus and tends to be solid; while when 0.5 < SLB < 1, it exhibits viscous modulus and tends to be liquid. As the concentration increases, the SLB value of EPS-T1 gradually decreases. At a concentration of 40 mg / mL, due to the aggregation of polysaccharide molecules, the sample exhibits colloidal properties tending to solid state, and the elastic modulus reaches the maximum value ( Figure 5 C). Monitoring the flow index (FI) can effectively reflect the movement rate of EPS-T1 molecules. This parameter clarifies the movement of microparticles in fermented milk samples. When the FI value exceeds 10 Hz, the molecules exhibit high mobility, making the overall solution present liquid characteristics; on the contrary, when the flow index drops to 10-2 Hz, it indicates that the fluidity of the sample decreases, the intermolecular resistance increases, and the sample presents solid characteristics. The gradual increase in the concentration of EPS-T1 leads to a corresponding decrease in its FI value, which is consistent with the analysis results of MVI and SLB ( Figure 5 D).

[0066] Example 4 Evaluation on the improvement of stress resistance of commercial starter strains by lactic acid bacteria exopolysaccharide

[0067] 1. Protection of polysaccharide protective agent on cell survival rate of commercial starter strains during cold storage

[0068] Three strains (Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus LGG) were cultured in MRS medium for 20 h. Bacterial cells were collected by centrifugation (5000 rpm / min) and washed twice with sterile normal saline (0.9% w / v, g / 100 ml, NaCl solution). Subsequently, the bacterial cells were suspended in 2 mL of EPS-T1 solutions with different concentrations (0, 1, 2, 4 mg / mL). These suspensions were stored at 4 °C for 21 days, with sterile normal saline used as the control group. Galacto-oligosaccharides (GOS) and inulin were used as positive controls. The agar plate counting method was used to count the bacterial survival rate on days 0, 3, 6, 9, 15 and 21 to evaluate the survival status.

[0069] The results showed that: different concentrations of EPS-T1 treatment groups significantly improved the survival rate of the three lactic acid bacteria strains under low temperature storage conditions ( Figure 6 A-C). Specifically, when the concentration was 4 mg / mL, the survival rates of Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus LGG reached 85.13%, 71.62% and 83.34% respectively. Comparative analysis of EPS-T1 and two prebiotics (inulin and fructooligosaccharides) showed that after 20 days of cold storage, EPS-T1 exhibited a good protective effect on Lactobacillus bulgaricus, with the number of viable bacteria exceeding 10 8 CFU / mL ( Figure 6D), similar to the inulin and fructooligosaccharide group. Furthermore, EPS-T1 showed slightly better protection against Streptococcus thermophilus than the two commercial prebiotics, maintaining a microbial count of 10 on day 20. 7 CFU / mL or higher ( Figure 6 E). Under the protection of EPS-T1, inulin, and fructooligosaccharides, the number of surviving *Lactobacillus rhamnosus* LGG cells significantly exceeded 10 at the end of storage. 7 CFU / mL Figure 6 F).

[0070] 2. Protection of cell survival rate of commercial fermentation strains during gastrointestinal digestion by polysaccharide protectants

[0071] Simulated gastric buffer solution: Weigh 2.8g of sodium chloride, 514.4mg of potassium chloride, 2.1g of sodium bicarbonate, 225.0mg of potassium dihydrogen phosphate, 20.3mg of magnesium chloride hexahydrate, and 78.6mg of ammonium carbonate, dissolve them in 1,000mL of distilled water, and adjust the pH to 2.5 with 0.1M HCl.

[0072] Simulated intestinal buffer solution: Weigh 2.2g of sodium chloride, 507.0mg of potassium chloride, 7.1g of sodium bicarbonate, 108.9mg of potassium dihydrogen phosphate, and 67.1mg of magnesium chloride hexahydrate, dissolve them in 1,000mL of distilled water, and adjust the pH to 7.5.

[0073] Three commercial starter cultures—Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus rhamnosus—were selected and cultured in MRS medium to the logarithmic growth phase. Cells were collected by centrifugation and washed with sterile physiological saline (0.9% w / v, g / 100 ml, NaCl solution). The bacterial cells were then resuspended in EPS-T1 solution at a concentration of 6 mg / mL. The EPS-T1-containing suspension was then mixed with equal volumes of simulated gastric and intestinal fluids and incubated at 37°C for 0–3 h. Bacterial viability was assessed at 0, 1.5, and 3 h to evaluate their tolerance to the gastrointestinal environment.

[0074] The results showed that EPS-T1 exhibited excellent gastrointestinal digestive stress protection against three commercial starter cultures. Figure 7 After 3 hours of gastric digestion, the survival rates of *Lactobacillus bulgaricus* SRFM-1, *Streptococcus thermophilus* MB5-1, and *Lactobacillus rhamnosus* LGG in the control group decreased to 18.01%, 16.37%, and 15.07%, respectively. However, EPS-T1 significantly improved their survival rates, reaching 63.35%, 48.10%, and 71.83%, respectively. Figure 7(AC). During the enteric digestion phase, the survival rates of *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Lactobacillus rhamnosus* in the control group after 3 hours of digestion were 16.12%, 24.23%, and 29.12%, respectively. In contrast, EPS-T1 effectively improved their survival rates to 66.69%, 57.08%, and 63.37%, respectively. Figure 7 (DF). In summary, the above results indicate that EPS-T1 can significantly improve the stress resistance of these three commercial starter cultures during gastrointestinal digestion.

[0075] 3. Polysaccharide protectants promote gut adhesion activity of commercial fermentation strains.

[0076] Caco-2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20% ​​fetal bovine serum, 100 U / mL penicillin, and streptomycin. The cell concentration was adjusted to 2.0 × 10⁸ CFU / mL through appropriate dilution, and then aliquoted into 6-well plates containing 1 × 10⁵ cells each. Cells were incubated at 37°C and 5% CO₂ until complete attachment. Subsequently, three test bacterial strains were cultured in logarithmic growth phase for 18 h, followed by centrifugation to collect the bacterial cells. The cells were washed twice with PBS buffer and then resuspended in DMEM to obtain a concentration of 5.0 × 10⁹ CFU / mL for subsequent use. In the control group (blank), the bacterial suspension (2 mL) was co-cultured with Caco-2 cells at 37°C for 2 h. In contrast, the experimental group (sample group) used bacterial suspension and EPS (final concentrations of 0.2%, 0.4%, and 0.6% (w / v, g / 100 ml) 1 mL), which were thoroughly mixed before being added to 6-well culture dishes containing Caco-2 cells and co-cultured at 37°C for 2 hours. After co-culture, the cell monolayer was washed at least three times with PBS buffer and then fixed with 4% (v / v) formaldehyde for 20 minutes. Finally, Gram staining was performed on coverslips in 6-well culture dishes. Subsequently, bacterial attachment on each Caco-2 cell was observed under a microscope, and the contents of each well were collected and plated on MRS agar medium for counting.

[0077] The results showed that EPS-T1 effectively promoted the adhesion of the three commercial fermentation agents to the intestine. Figure 8B). Compared with the control group, the adhesion rates of the three lactic acid bacteria strains to Caco-2 cells after the addition of EPS-T1 extracellular polysaccharide showed a concentration-dependent relationship. At a polysaccharide concentration of 3 mg / mL, the adhesion rate of *Lactobacillus rhamnosus* LGG significantly increased from the initial value of 14.63% to 27.49%, while the adhesion rates of *Streptococcus thermophilus* MB5-1 and *Lactobacillus bulgaricus* SRFM-1 also increased from the initial values ​​of 5.87% and 10.58% to 26.14% and 21.36%, respectively. Figure 8 A). These results indicate that EPS-T1 effectively enhances the adhesion of three commercial lactic acid bacteria strains to Caco-2 cells, facilitating their colonization and adhesion in the gut and further highlighting their beneficial prebiotic properties.

[0078] 7. Discussion:

[0079] This study investigated the emulsifying properties and solution rheological behavior of viscous EPS-T1 secreted by *Lactobacillus plantarum* T1. Furthermore, the potential of these properties as protectants to enhance the extracellular resistance of commercial fermentation strains (*Lactobacillus bulgaricus*, *Streptococcus thermophilus*, and *Streptococcus lactis*) was explored, as well as their adhesion activity to CaCo-2 cells. EPS-T1 exhibited high water and oil holding capacities (621.34%, 834.56%, respectively) and superior emulsifying properties compared to guar gum (90.21%, 30 min; 79.02%, 60 min). Rheological analysis showed that the macroscopic viscosity index gradually increased with increasing EPS-T1 concentration. Moreover, at a concentration of 40 mg / mL, the solid-liquid equilibrium value was below 0.5, exhibiting solid-gel-like characteristics. As a protective agent, EPS-T1 significantly improved the cell survival rate of three lactic acid bacteria strains—*Lactobacillus bulgaricus* SRFM, *Streptococcus thermophilus* MB5-1, and *Lactobacillus rhamnosus* LGG—during 20 days of low-temperature storage, and enhanced their resistance to gastrointestinal digestion as a bacterial encapsulation matrix. Furthermore, EPS-T1 effectively enhanced the adhesion of *Lactobacillus bulgaricus* SRFM, *Streptococcus thermophilus* MB5-1, and *Lactobacillus rhamnosus* LGG to CaCo-2 cells. Our findings have research value in the development and utilization of EPS-T1 derived from *Lactobacillus plantarum* T1 as a potential food emulsifier, thickener, and prebiotic protective delivery material.

[0080] sequence list

[0081] Lactiplantibacillus plantarum T1 strain 16S rDNA

[0082] TTGATCACTTAGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAA

[0083] CTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCAT

[0084] GCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATC

[0085] CGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTA

[0086] CCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCA

[0087] TCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCT

[0088] GGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACA

[0089] CGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAAT

[0090] CTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTA

[0091] AACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTG

[0092] CGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGA

[0093] AACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCT

[0094] GTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCG

[0095] CCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTC

[0096] CTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCT

[0097] TTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAA

[0098] CGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGG

[0099] TTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAG

[0100] TTTTACGAGCCGAAACCCCTTCTTCACTCACGCGCGGTGGCTCATCAGACTTTCGTTC

[0101] CATGTGAGATCCCCTACTGCTGCCTCCGGTAAGAGTTTGGTCCTGTTCTCAGTCCTAA

[0102] TGTGGTCGAATGACCTCCTCAGGATCGACTTACGGTATTA。

Claims

1. Application of lactic acid bacteria extracellular polysaccharides secreted by lactic acid bacteria strains in the following (1) or (2): (1) Improve the survival rate of lactic acid bacteria fermentation strains under cold storage or prepare products that improve the survival rate of lactic acid bacteria fermentation strains under cold storage; (2) Preparation of prebiotic additives that promote intestinal adhesion of lactic acid bacteria fermentation strains; The lactic acid bacteria strain described is classified as *Lactobacillus plantarum* (Lactobacillus plantarum). Lactiplantibacillus plantarum The lactic acid bacteria extracellular polysaccharide was deposited at the China Center for Type Culture Collection on August 31, 2023, with accession number CCTCC M 20231553, and the deposit address is Wuhan University, Wuhan, China. The lactic acid bacteria extracellular polysaccharide was obtained by fermentation culture of the lactic acid bacteria strain.

2. The application according to claim 1, characterized in that, The lactic acid bacteria extracellular polysaccharide is prepared by fermenting the lactic acid bacteria strain and then separating and purifying the extracellular polysaccharide in the fermentation broth.

3. The application according to claim 2, characterized in that, The separation and purification process is as follows: (1) Centrifuge the fermentation broth and collect the supernatant; treat the supernatant with 4% trichloroacetic acid solution, centrifuge to remove protein; then add anhydrous ethanol for treatment and centrifuge to obtain crude polysaccharide precipitate; (2) The crude polysaccharide precipitate was dissolved in deionized water and then transferred to a dialysis bag with a molecular weight cutoff of 8,000-14,000 Da for water dialysis. The dialysis solution was freeze-dried to obtain crude extracellular polysaccharide. (3) The crude extracellular polysaccharide was further purified using a DEAE-52 anion exchange column. The purified polysaccharide fraction was collected by gradient elution using deionized water, 0.1 M and 0.3 M NaCl solutions at a flow rate of 1 mL / min. The sugar content was detected by the phenol-sulfuric acid method. Finally, the main fraction was dialyzed with pure water and freeze-dried to obtain the purified lactic acid bacteria extracellular polysaccharide.

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