Pediococcus acidilactici strain MY-5, exopolysaccharide produced by pediococcus acidilactici strain MY-5 and application

Abstract

The invention discloses pediococcus acidilactici MY-5, and the preservation number of the pediococcus acidilactici MY-5 is CCTCC (China Center for Type Culture Collection) M20241066. The invention also discloses an exopolysaccharide produced by the pediococcus acidilactici MY-5 and an application of the exopolysaccharide, the exopolysaccharide comprises MY-5-cEPS and a purified component MY-5-EPS2, the molecular weight distribution of the MY-5-cEPS is 63847 Da (29.29%) and 5189 Da (70.71%) respectively, and the molecular weight distribution of the MY-5-EPS2 is 47870 Da. The extracellular polysaccharide produced by the pediococcus acidilactici MY-5 disclosed by the invention has good antioxidant activity and thermal stability, can reduce intestinal cytotoxicity caused by cadmium exposure, relieve inflammatory response and protect intestinal barriers, and has the potential of being developed into a novel heavy metal biological chelating agent.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial polysaccharides, and in particular relates to a Pediococcus acidilactici strain MY-5, the extracellular polysaccharide produced by the strain and application thereof. Background Art

[0002] Lactic acid bacteria extracellular polysaccharides (LAB EPS) are high molecular weight carbohydrate compounds produced by lactic acid bacteria during their growth. Lactic acid bacteria extracellular polysaccharides are safe and non-toxic, and they are widely available, which can effectively reduce the preparation cost of polysaccharides, making the research and development of related products feasible from an economic perspective, and thus have broad market application prospects.

[0003] Therefore, developing more extracellular polysaccharides with new functions has very important research significance and economic value. As one of the most important products of lactic acid bacteria metabolites, LAB EPS has been widely used in food processing as a food thickener and emulsifier due to its good biological activity.

[0004] However, there are many types of exopolysaccharides with complex structures. Currently, only some exopolysaccharides are used in clinical trials such as anti-tumor, anti-virus, and immunity enhancement. In terms of exploring the relationship between their structure and function, many exopolysaccharides have not been studied in depth. In addition, exopolysaccharides have a certain ability to adsorb heavy metals due to their rich functional groups such as -OH, -COOH, -SH, and -NH2, as well as a three-dimensional network structure, which can protect organisms from heavy metal toxicity.

[0005] At present, polysaccharides are still one of the research hotspots in the field of natural chemistry at home and abroad, and have broad application prospects. Pediococcus acidilactici is a known beneficial bacterium. CN118000408A discloses a method for preparing a fat substitute using Pediococcus acidilactici exopolysaccharide. CN117903987A discloses a method for preparing Pediococcus acidilactici and its multifunctional exopolysaccharide and its application. The exopolysaccharide has a high binding rate of sodium cholate, sodium taurocholate and sodium deoxycholate after simulated gastrointestinal digestion; it has a high clearance rate for hydroxyl radicals, DPPH radicals and superoxide anions, can improve the gel properties of minced meat products, give the product functional properties, and can be applied to low-fat meat products and low-salt meat products. CN116218930A discloses a Pediococcus acidilactici exopolysaccharide with antioxidant, antibacterial and antitumor activities, which can be used to prepare drugs, foods, health products, cosmetics and microecological preparations with antioxidant effects. However, there are few studies on the exopolysaccharides of P. acidilactici in China, and the development and utilization of P. acidilactici polysaccharides are not comprehensive enough. In addition, the research on P. acidilactici mainly focuses on the role of strains, while there are few studies on the characterization of polysaccharide structure and its activity. Summary of the invention

[0006] The inventors of the present application isolated a strain of Pediococcus acidilactici MY-5 from sourdough, and through studying the in vitro physiological activity of its extracellular polysaccharide, found that the extracellular polysaccharide of Pediococcus acidilactici has good antioxidant activity and thermal stability, and also has a certain heavy metal adsorption capacity, which can protect the toxic effects of cadmium exposure on intestinal cells.

[0007] Therefore, the first object of the present invention is to provide a Pediococcus acidilactici MY-5, whose preservation number is CCTCC M 20241066.

[0008] The second object of the present invention is to provide the extracellular polysaccharide produced by the Pediococcus acidilactici MY-5.

[0009] According to the present invention, the extracellular polysaccharide includes MY-5-cEPS and its purified component MY-5-EPS2, wherein the molecular weight distribution of the MY-5-cEPS is 63847Da (29.29%) and 5189Da (70.71%), and the molecular weight distribution of the MY-5-EPS2 is 47870Da.

[0010] Furthermore, the monosaccharide composition of the MY-5-cEPS is: arabinose, galactose, glucose, xylose, mannose and galacturonic acid, and the molar ratio of the six monosaccharides is:

[0011] Arabinose: galactose: glucose: xylose: mannose: galacturonic acid = 0.23: 1.35: 1: 0.34: 1.76: 0.19.

[0012] Furthermore, the monosaccharide composition of MY-5-EPS2 is: arabinose, galactose, glucose, xylose and mannose, and the molar ratio of the five monosaccharides is:

[0013] Arabinose: galactose: glucose: xylose: mannose = 0.38: 0.83: 1: 0.58: 2.2.

[0014] Furthermore, the MY-5-EPS2 includes 7 glycosidic bonds: →3,6)-α-Manp-(1→, →5)-α-Araf-(1→, →4)-α-Glcp-(1→, →2,6)-α-Galp-(1→, →4)-α-Manp-(1→, →4)-β-Glcp-(1→, and β-T-Glcp-(1→.

[0015] The third aspect of the present invention provides the use of the extracellular polysaccharide of Pediococcus acidilactici MY-5 for preparing cosmetics or health products with antioxidant activity.

[0016] According to a preferred embodiment of the present invention, the antioxidant activity is selected from the group consisting of: scavenging DPPH free radicals, scavenging ABTS free radicals, and scavenging OH free radicals.

[0017] The fourth aspect of the present invention provides another application of the exopolysaccharide of Pediococcus acidilactici MY-5. Both Pediococcus acidilactici and its exopolysaccharide have heavy metal adsorption capacity and can be used to prepare heavy metal biochelator products.

[0018] According to a preferred embodiment of the present invention, the heavy metal biochelator is used to protect cells exposed to cadmium, including: increasing the activity of cadmium-exposed cells, reducing apoptosis of cadmium-exposed cells, reducing the levels of reactive oxygen and lipid peroxides in cadmium-exposed cells, alleviating inflammatory responses in cadmium-exposed cells, increasing tight junction protein expression in cadmium-exposed cells, and protecting cell barriers.

[0019] The present invention has the following beneficial effects:

[0020] 1. The present invention obtains a new Pediococcus acidilactici MY-5.

[0021] 2. The extracellular polysaccharide produced by Pediococcus acidilactici MY-5 of the present invention has good antioxidant activity and thermal stability, and can also reduce the intestinal cell toxicity caused by cadmium exposure, relieve inflammatory response, and protect the intestinal barrier, and has the potential to be developed into a new heavy metal biochelator. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the apparent properties of the extracellular polysaccharide of Pediococcus acidilactici MY-5 after freeze-drying.

[0023] Figure 2 Figure 1 shows the separation and purification results of the extracellular polysaccharide of Pediococcus acidilactici MY-5, where A is the elution curve of MY-5-cEPS by DEAE-Cellulose-52 ion exchange column chromatography, and B is the elution curve of the second polysaccharide peak (EPS-2) in Figure A by Sepharose CL-6B gel column chromatography.

[0024] Figure 3 These are the gel chromatography elution results of the extracellular polysaccharides MY-5-cEPS and MY-5-EPS2.

[0025] Figure 4 These are the results of high-performance ion exchange chromatography detection of the monosaccharide composition of the extracellular polysaccharide of Pediococcus acidilactici MY-5, where A is MY-5-cEPS and B is MY-5-EPS2.

[0026] Figure 5 Fourier transform infrared spectra of Pediococcus acidilactici MY-5 exopolysaccharides MY-5-cEPS and MY-5-EPS2.

[0027] Figure 6 The EI-MS spectrum of MY-5-EPS2 obtained by GC-MS was compared with the standard PMAA spectrum, showing the total ion current of methylated product PMAAs.

[0028] Figures 7A-7D They are the NMR images of MY-5-EPS2, where 7A and 7B are one-dimensional images. 1 H spectrum, 13 C spectrum, 7C and 7D are two-dimensional 1 H- 1 H COSY spectrum, 1 H- 13 C HSQC spectrum.

[0029] Figure 8 This is the circular dichroism spectrum of MY-5-EPS2.

[0030] Fig. 9 The Congo red test results of MY-5-EPS2.

[0031] Fig.10 These are the scanning electron microscopy results of MY-5-cEPS and MY-5-EPS2, where the right image is a partial enlargement of the left image.

[0032] Fig.11 These are the thermogravimetric analysis results of MY-5-cEPS and MY-5-EPS2, where A corresponds to MY-5-cEPS and B corresponds to MY-5-EPS2.

[0033] Fig.12 are the antioxidant activities of MY-5-cEPS and MY-5-EPS2, where A is the DPPH free radical scavenging activity, B is the ABTS free radical scavenging activity, C is the OH free radical scavenging activity, and D is the total reducing power level.

[0034] Fig.13 This is the grouping and treatment method for the experiment in which the extracellular polysaccharide of Pediococcus acidilactici MY-5 alleviates cellular cadmium toxicity.

[0035] Fig.14The effect of MY-5-cEPS on the activity of Caco-2 cells exposed to cadmium is shown, where A and B show the treatment group and the prevention group, respectively.

[0036] Fig.15 Shown are the effects of MY-5-cEPS on the morphology of Caco-2 cells exposed to cadmium.

[0037] Fig.16 The effect of MY-5-cEPS on apoptosis of Caco-2 cells exposed to cadmium is shown.

[0038] Fig.17 The effect of MY-5-cEPS on the level of ROS in Caco-2 cells exposed to cadmium is shown, among which 17A is the result of ROS detection by laser confocal microscopy. Fig.17 B and 17C show the results of further quantitative analysis of intracellular ROS levels using a microplate reader, where B is the treatment group and C is the prevention group.

[0039] Fig.18 The effect of MY-5-cEPS on MDA levels in Caco-2 cells exposed to cadmium is shown.

[0040] Fig.19 The effects of MY-5-cEPS on the relative expression of inflammatory factors in Caco-2 cells exposed to cadmium are shown, where A, B, C, D, and E correspond to the cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10, respectively.

[0041] Fig. 20 The effect of MY-5-cEPS on the relative expression of tight junction proteins in Caco-2 cells exposed to cadmium is shown, where A, B, and C correspond to the cytokines Claudin-1, Occludin-1, and ZO-1, respectively.

[0042] Preservation matters

[0043] The Pediococcus acidilactici MY-5 of the present invention has been deposited in the China Center for Type Culture Collection (CCTCC), the address of the depository is: Wuhan University, Wuhan, China, the deposit date is: May 27, 2024, and the deposit number is: CCTCC M 20241066. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0045] Example 1. Preparation method of Pediococcus acidilactici MY-5 exopolysaccharide (MY-5-cEPS)

[0046] Take a glycerol cryopreservation tube of the MY-5 strain of Pediococcus acidilactici, inoculate it into 10 mL of MRS liquid culture medium at an inoculum volume of 2% (v / v), and culture it at 37°C and 200rpm for 16 hours. Take a small amount of culture solution, streak it on MRS solid culture medium, culture it in a constant temperature incubator at 37°C for 48 hours, select a single colony and inoculate it into MRS liquid culture medium, and culture it in a shaking table at 37°C and 200rpm for 16 hours to obtain the strain activation solution.

[0047] The activated bacterial liquid was inoculated into MRS liquid culture medium at an inoculum size of 2% (v / v), cultured at 30°C for 30 hours, and then centrifuged (8000rpm, 20min, 4°C) to obtain the fermentation supernatant. 80% (m / v) trichloroacetic acid solution was added to the supernatant to a final concentration of 4% (m / v), refrigerated at 4°C for 10 hours, and then centrifuged (8000rpm, 20min, 4°C) to remove protein. The supernatant was then concentrated by rotary evaporation, and 3 times the volume of 95% ethanol solution was slowly added to the concentrate. The concentrate was allowed to stand at 4°C overnight, and centrifuged (8000rpm, 20min, 4°C) to obtain the precipitate. After the precipitate was fully dissolved in deionized water, it was transferred to a 7kDa dialysis bag and dialyzed for 2 days. The water was changed every 6 hours. After the dialysis, it was freeze-dried to obtain the crude exopolysaccharide (MY-5-cEPS) of lactic acid bacteria MY-5, and its appearance was as follows Figure 1 shown.

[0048] Example 2: Isolation and purification of Pediococcus acidilactici MY-5-cEPS

[0049] 2.1. DEAE-Cellulose-52 ion exchange column chromatography purification

[0050] 5 ml of the crude polysaccharide MY-5-cEPS (20 mg / mL) obtained in Example 1 was dissolved in ultrapure water, filtered through a 0.22 μm filter membrane, and loaded onto a DEAE-Cellulose-52 ion exchange column. Gradient elution was performed using different concentrations of NaCl solution (0 M, 0.1 M, 0.2 M, and 0.5 M). 5 mL of the eluate was collected in each tube, and the polysaccharide content in the tube was detected by the phenol-sulfuric acid method. The results are as follows: Figure 2 As shown in A, the EPS-2 component accounts for a relatively high proportion, and the eluted products of the component peak EPS-2 are collected and concentrated by rotary evaporation, dialyzed, and freeze-dried for the next step of purification.

[0051] 2.2. Sepharose CL-6B gel column chromatography purification

[0052] Dissolve the freeze-dried polysaccharide EPS-2 (5 mg / mL) obtained in 2.1 in 5 mL ultrapure water, filter through a 0.22 μm filter membrane, and load onto a Sepharose CL-6B gel column. Elute with a 0.9% (w / v) NaCl solution. Collect 5 mL of the eluate in each tube, and use the phenol-sulfuric acid method to detect the polysaccharide content in the tube. Combine and collect the elution products of the component peak EPS2 ( Figure 2 B), rotary evaporation concentration, dialysis and freeze-drying were performed to obtain the purified extracellular polysaccharide MY-5-EPS2.

[0053] Example 3: Molecular weight distribution of Pediococcus acidilactici exopolysaccharide

[0054] Standard samples were prepared with dextran of different molecular weights (180Da, 10kDa, 40kDa, 70kDa, 150kDa, 270kDa and 410kDa). The above series of standard polysaccharides and the extracellular polysaccharides MY-5-cEPS and MY-5-EPS2 obtained in Example 2 were dissolved in the mobile phase (0.1mol / LNaNO3 solution) to obtain a 1mg / mL solution, which was filtered through a 0.22μm filter membrane and analyzed using a Waters 2695 high performance liquid chromatograph. Based on Mw (weight average molecular weight) and retention time (RT), the 1gMw-RT standard curve and regression equation were obtained. Regression equation: y=-0.5391x+13.93(R 2 =0.993).

[0055] The gel chromatography elution results of the exopolysaccharides MY-5-cEPS and MY-5-EPS2 of Pediococcus acidilactici MY-5 are as follows Figure 3 The molecular weight of the sample was calculated according to the regression equation, and the results are shown in Table 1 below.

[0056] Table 1: Molecular weight determination results of MY-5-cEPS and MY-5-EPS2

[0057]

[0058] From the results in Table 1, it can be seen that the molecular weight distributions of the extracellular polysaccharide MY-5-cEPS of the Pediococcus acidilactici strains are 63847 Da (29.29%) and 5189 Da (70.71%), and the molecular weight distribution of MY-5-EPS2 is 47870 Da.

[0059] Example 4: Determination of Monosaccharide Composition of Pediococcus acidilactici Exopolysaccharide

[0060] 5 mg of polysaccharide was dissolved in 2 mL of trifluoroacetic acid (TFA) solution (2 mol / L) and hydrolyzed in an oven at 120 °C for 2 h. The hydrolyzate was blown dry using a nitrogen blower, and then 3 mL of methanol solution was added and blown dry. The above operation was repeated at least three times to completely remove TFA. Ultrapure water was added to dissolve the hydrolyzate and diluted to an appropriate concentration. After filtering through a 0.22 μm filter membrane, the sample was loaded for detection. Instrument configuration and detection conditions: Thermo Fisher ICS-6000 system; sample volume: 25 μL; Chromeleon7.0 chromatography workstation, DionexTM CarboPacTM PA20 anion exchange analytical column (150 mm×3 mm); elution procedure: first eluted with 2 mM NaOH solution, then gradient eluted with 0.05-0.2 M NaAc solution, flow rate: 0.45 mL / min, analysis time: 40 min. Monosaccharide standards: trehalose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, glucosamine, galacturonic acid and glucuronic acid. Figure 4 shown.

[0061] Figure 4 The results showed that the monosaccharide composition of MY-5-cEPS of P. acidilactici was arabinose, galactose, glucose, xylose, mannose and galacturonic acid, and the molar ratio of the six monosaccharides was arabinose: galactose: glucose: xylose: mannose: galacturonic acid = 0.23: 1.35: 1: 0.34: 1.76: 0.19. The monosaccharide composition of the purified polysaccharide MY-5-EPS2 was arabinose, galactose, glucose, xylose, mannose, and the molar ratio of the five monosaccharides was arabinose: galactose: glucose: xylose: mannose = 0.38: 0.83: 1: 0.58: 2.2.

[0062] Example 5: Fourier transform infrared spectroscopy determination of Pediococcus acidilactici exopolysaccharide

[0063] 2 mg of polysaccharide was mixed with 100 mg of KBr powder and ground in a mortar to make the mixture uniform. The mixture was put into a tablet press to make thin slices and then subjected to infrared spectrometer at 4000-400 cm -1 The spectrum scan within the range is shown in the following figure. Figure 5 shown.

[0064] Figure 5 The results showed that polysaccharides at 3300 cm –1 The absorption peak at 2930 cm is caused by the stretching vibration of OH. -1 The absorption peaks on the left and right are caused by CH stretching vibration. These two absorption peaks are characteristic peaks of polysaccharides. -1The absorption peaks appearing around 1419 cm are caused by the stretching vibration of the C=O bond. -1 The absorption peak of polysaccharide comes from the deformation vibration of CH. -1 ~1200cm -1 The absorption peak at 805cm is produced by COC or CO stretching. -1 and 808cm -1 The weak signal peak appearing at indicates that mannose may exist in the polysaccharide, which is consistent with the analysis of the monosaccharide composition.

[0065] Example 6: Determination of the connection mode of Pediococcus acidilactici exopolysaccharide

[0066] 6.1 Methylation analysis

[0067] Pretreatment of reagents and samples (drying and dehydration): Use 4A° molecular sieves to dry the reaction reagents (such as DMSO, etc.) in anhydrous form. Dry the polysaccharide and sodium hydroxide in a desiccator overnight.

[0068] CH3I methylation: Add 2-3 mg of anhydrous polysaccharide sample to the reaction bottle, add 1 mL of anhydrous DMSO, fill with nitrogen, and sonicate at 45°C until the polysaccharide sample is dissolved. Add 20 mg of dry sodium hydroxide powder, fill with nitrogen, and sonicate at room temperature for 3 hours. Slowly add 500 μL of iodomethane in an ice bath, fill with nitrogen, sonicate for 1 hour, and add 1 mL of deionized water to terminate the methylation reaction. Add 1 mL of chloroform, vortex, and let stand for a few minutes to allow it to separate, collect the chloroform phase and repeat this operation 3 times. Add a small amount of anhydrous sodium sulfate to the chloroform phase and mix thoroughly to remove residual moisture. Evaporate the chloroform phase under reduced pressure, test the infrared absorption spectrum of the product, and the infrared spectrum of the methylated product is between 3100 and 3500 cm -1 If the hydroxyl peak at the position disappears, it indicates that the degree of methylation is very high, otherwise the second methylation needs to be repeated.

[0069] TFA acid hydrolysis: The methylated sample was transferred to a pear-shaped bottle, 1 mL of 2 mol / L TFA was added, nitrogen was introduced, and the sample was hydrolyzed in an oven at 120°C for 2 h. The hydrolyzate was blown dry using a nitrogen blower, and 3 mL of methanol solution was added. The above operation was repeated at least three times to completely remove TFA.

[0070] NaBD4 reduction: Add 0.5 mL of freshly prepared 10 mg / mL NaBD4 (50 mmol / L NaOH) solution to the reaction bottle, stir and react overnight at room temperature. Add 500 μL of 10% acetic acid methanol solution, blow dry with a nitrogen blower, and repeat the above operation at least three times to completely remove the boric acid to obtain the reduced monosaccharide.

[0071] Acetyl anhydride acetylation: After the reduced monosaccharide is dried in a desiccator overnight, 500 μL of ethyl acetate, 1.5 mL of acetic anhydride, and 50 μL of perchloric acid are added in sequence, ultrasonicated for 5 min, and allowed to stand for 15 min to complete the reaction. 5 mL of deionized water is added and vortexed to remove excess acetic anhydride. 2 mL of dichloromethane is added, vortexed, and the dichloromethane layer is collected by centrifugation. The above operation is repeated three times. The dichloromethane layer is washed with 2 mL of deionized water, the water layer is removed, and the above operation is repeated three times. Rotary evaporate and redissolve the sample in dichloromethane for GC–MS analysis.

[0072] GC-MS analysis: GC-MS conditions: chromatographic column Agilent HP5-ms (30m×0.32mm, 0.25μm), carrier gas N2, flow rate 1.0ml / min, column temperature conditions: initial temperature 50℃, maintained for 1min, 50℃ / min to 130℃, 3℃ / min to 230℃, maintained for 2min, MS detector, EI ionization 70eV, mass spectrum scanning range 40~500m / z. Injection port temperature 250℃, injection volume 1μL.

[0073] Data analysis: Figure 6 As shown, the EI-MS spectrum obtained by GC-MS was compared with the standard PMAA spectrum. Combined with the results of monosaccharide composition, it can be determined that MY-5-EPS2 contains eight sugar residues: 2,3,5-Me3-Araf, 2,3-Me2-Araf, 2,3,4,6-Me4-Glcp, 2,3,6-Me3-Galp, 2,3,6-Me3-Manp, 2,4-Me2-Manp, 2,3,6-Me3-Glcp, and 3,4-Me2-Galp. The specific results are shown in Table 2.

[0074] Table 2: MY-5-EPS2 methylation analysis results

[0075]

[0076] 6.2. Nuclear Magnetic Resonance Spectroscopy Analysis

[0077] 30 mg of MY-5-EPS2 polysaccharide sample was dissolved in 0.55 mL of D2O and the one-dimensional structure of the sample was analyzed by 400 MHz NMR spectrometer. 1 H spectrum, 13 C spectrum and two-dimensional 1 H- 1 H COSY spectrum, 1 H- 13 The C HSQC spectrum was tested and the results were as follows: Fig. 7A , 7B , 7C and 7D.

[0078] according to Fig. 7A , 7B , 7C and 7D, and the carbon-hydrogen signals of MY-5-EPS2 were assigned. The results are shown in Table 3. It can be seen that the exopolysaccharide of Pediococcus acidilactici MY-5-EPS2 mainly includes seven glycosidic bonds: →3,6)-α-Manp-(1→、→5)-α-Araf-(1→、→4)-α-Glcp-(1→、→2,6)-α-Galp-(1→、→4)-α-Manp-(1→、→4)-β-Glcp-(1→、β-T-Glcp-(1→).

[0079] Table 3: Attribution of carbon and hydrogen signals of MY-5-EPS2 NMR

[0080]

[0081]

[0082] Example 7: Determination of the Higher-Level Structure of Pediococcus acidilactici Exopolysaccharide

[0083] 7.1 Circular dichroism analysis

[0084] A 2 mg / mL MY-5-EPS2 polysaccharide solution was prepared and the circular dichroism of the polysaccharide solution in the range of 190 to 300 nm was measured using a circular dichroism spectrometer. Figure 8 shown.

[0085] Figure 8 The results showed that MY-5-EPS2 exhibited a large negative peak near 200 nm, indicating that the polysaccharide had an asymmetric structure.

[0086] 7.2 Congo red test

[0087] Take 1mL of 2mg / mL MY-5-EPS2 polysaccharide solution and 1mL of 80μmol / L Congo red solution and mix them evenly. Add NaOH solution to make the final concentrations 0, 0.1, 0.2, 0.3, 0.4, and 0.5mol / L, respectively. Let it stand for 5min to stabilize the system. Use an ELISA reader to scan the maximum absorption wavelength λmax of the mixed solution in the range of 400-800nm ​​and record the results. Fig. 9 shown.

[0088] Depend on Fig. 9 The results show that when the NaOH concentration is 0.1 mol / L, the complex formed by extracellular polysaccharide and Congo red shows a red shift phenomenon, indicating that there may be a triple helix structure in the polysaccharide. After that, as the NaOH concentration increases, the absorbance value gradually decreases, indicating that the triple helix structure in the polysaccharide is destroyed by strong alkali.

[0089] Example 8: Scanning electron microscopy determination of Pediococcus acidilactici exopolysaccharide

[0090] The dried polysaccharide samples of MY-5-cEPS and MY-5-EPS2 were pasted on a copper stage with a conductive double-sided black adhesive, sprayed with gold for 1 min, and their surface morphology was observed using a scanning electron microscope. The results are as follows: Fig.10 shown.

[0091] Depend on Fig.10 The results showed that the extracellular polysaccharide was in irregular flakes and loose network structure. After increasing the magnification, it was found that its surface was smooth and had a porous structure and smaller spherical fragments.

[0092] Example 9: Determination of thermal stability of Pediococcus acidilactici exopolysaccharide

[0093] 5 mg of MY-5-cEPS and MY-5-EPS2 polysaccharide samples were weighed and placed in a crucible. The samples were heated from room temperature to 800 °C at a heating rate of 10 °C / min under N2 atmosphere using a thermogravimetric analyzer. Fig.11 As shown in A and 11B, A corresponds to MY-5-cEPS and B corresponds to MY-5-EPS2.

[0094] Depend on Fig.11 The thermogravimetric analysis results of A and 11B show that the final weight of the extracellular polysaccharide is MY-5-cEPS (37.53%) > MY-5-EPS2 (23.03%). In the DTG curve, the maximum decomposition rates of MY-5-cEPS and MY-5-EPS2 are 0.46% / min and 1.66% / min, respectively. The temperature corresponding to the maximum decomposition rate is MY-5-cEPS (295.1℃) > MY-5-EPS2 (274.86℃). The maximum decomposition rate of MY-5-cEPS occurs at a higher temperature, which shows good thermal stability.

[0095] Example 10: Determination of the antioxidant activity of Pediococcus acidilactici exopolysaccharide

[0096] 10.1. DPPH free radical scavenging activity

[0097] Take 100 μL of polysaccharide solutions of different concentrations of MY-5-cEPS and MY-5-EPS2 and 100 μL of 0.1 mmol / L DPPH-anhydrous ethanol solution, mix them thoroughly, react at 37°C in the dark for 30 minutes, and measure the absorbance at 517 nm. At the same time, Vc solution was used as a positive control. The DPPH free radical scavenging rate of polysaccharides was calculated by the following formula:

[0098]

[0099] Among them: A SIndicates the absorbance of DPPH solution and polysaccharide solution; A c Indicates the absorbance of ethanol solution and polysaccharide solution; A b Indicates the absorbance value of DPPH solution and ultrapure water.

[0100] Fig.12 A shows the results of DPPH free radical scavenging rate, which shows that the scavenging ability of MY-5-cEPS and its purified component MY-5-EPS2 on DPPH free radicals is positively correlated with the polysaccharide concentration. When the polysaccharide concentration is 5 mg / mL, MY-5-cEPS and its purified component MY-5-EPS2 have the best DPPH free radical scavenging efficiency, which are 84.51% and 77.18%, respectively, slightly lower than the positive control Vc.

[0101] 10.2. ABTS free radical scavenging activity

[0102] Mix 10mL 7mmol / L ABT solution and 10mL 2.45mmol / L K2S2O8 solution, and react in the dark for 16h to produce ABTS free radicals. Dilute the above solution with PBS (pH=7.4) to an absorbance of 0.7±0.02 at a wavelength of 734nm to prepare ABTS solution. Take 100μL of polysaccharide solutions of MY-5-cEPS and MY-5-EPS2 of different concentrations and 750μL ABTS solution, mix them and react in the dark for 6min, and immediately detect the absorbance of OD734nm. At the same time, use Vc solution as a positive control. Calculate the ABTS free radical scavenging rate of polysaccharides by the following formula:

[0103]

[0104] Wherein: Ac represents the absorbance value of ABTS solution and ultrapure water; As represents the absorbance value of ABTS solution and polysaccharide solution.

[0105] Fig.12 B shows the results of ABTS free radical scavenging rate. It can be seen that when the polysaccharide concentration is 5 mg / mL, MY-5-cEPS and its purified component MY-5-EPS2 have the best ABTS free radical scavenging efficiency, which are 87.13% and 83.13%, respectively, which are slightly lower than the positive control Vc.

[0106] 10.3. OH Radical Scavenging Activity

[0107] 100 μL of polysaccharide solutions of MY-5-cEPS and MY-5-EPS2 with different concentrations were mixed with 100 μL of 6 mmol / L FeSO4 solution, 100 μL of 9 mmol / L salicylic acid-ethanol solution, and 100 μL of 3% H2O2 solution, and reacted for 30 min in a 37°C water bath, and the absorbance at OD517nm was determined. At the same time, Vc solution was used as a positive control. The OH radical scavenging rate of the polysaccharide was calculated by the following formula:

[0108]

[0109] Among them: A S Indicates the absorbance value of polysaccharide solution and FeSO4, salicylic acid-ethanol, and H2O2 solution; A C Indicates the absorbance of polysaccharide solution with FeSO4, salicylic acid-ethanol, and ultrapure water solution; A b It indicates the absorbance value of the mixture of ultrapure water and FeSO4, salicylic acid-ethanol, and H2O2 solution.

[0110] Fig.12 C shows the results of OH radical scavenging rate. It can be seen that when the concentration of extracellular polysaccharide is 5 mg / mL, MY-5-cEPS and its purified component MY-5-EPS2 have the best OH radical scavenging rates, which are 28.81% and 11.65%, respectively, but both are significantly lower than the hydroxyl radical scavenging rate of the control group Vc.

[0111] 10.4. Reduction Level

[0112] Take 100 μL of polysaccharide solutions of MY-5-cEPS and MY-5-EPS2 of different concentrations, mix them with 100 μL of phosphate buffer (pH=6.6) and 100L of 1% potassium ferricyanide solution, react at 50°C for 30 minutes, add 100 μL of 10% TCA solution, mix and centrifuge at 4500rpm for 15 minutes. Take 200 μL of supernatant and 100 μL of 0.1% ferric chloride solution and 500 μL of ultrapure water, mix them thoroughly, react for 10 minutes in a 37°C water bath, and measure the absorbance at OD700nm. At the same time, use Vc solution as a positive control. The size of the absorbance value indicates the strength of the sample's reducing ability.

[0113] Fig.12 D shows the results of total reducing capacity. It can be seen that when the polysaccharide concentration is 5 mg / mL, the total reducing capacity of MY-5-cEPS and its purified component MY-5-EPS2 reaches the highest level of 0.23% and 0.215%.

[0114] The above results showed that MY-5-cEPS from Pediococcus acidilactici and its purified component MY-5-EPS2 had certain DPPH, ABTS, and OH free radical scavenging abilities and had good antioxidant activity.

[0115] Example 11: Effect of Pediococcus acidilactici exopolysaccharide on alleviating cadmium toxicity in intestinal cells

[0116] This example uses a Cd-induced Caco-2 cell injury model to evaluate the protective effect of exopolysaccharides produced by Pediococcus acidilactici MY-5 on cadmium-exposed intestinal cell injury. The specific implementation plan is as follows:

[0117] The whole cell experiment was divided into two groups: treatment and prevention. Each group was further divided into different treatment groups. Specifically, they included: blank control group (Control), heavy metal exposure modeling group (Cd Only), polysaccharide only group (MY-5-EPSOnly) and cadmium + polysaccharide group (MY-5-EPS+Cd). Fig.13 As shown, "no treatment" means adding complete cell culture medium; "Cd treatment" means adding cell culture medium containing cadmium chloride; "MY-5-cEPS treatment" means adding cell culture medium containing polysaccharide. For both the treatment group and the prevention group, the treatment time was 48 hours. After the cadmium treatment of the cells, the cells were repeatedly washed three times with PBS solution to remove cadmium ions in the cells.

[0118] 11.1 Cell morphology and viability detection

[0119] Approximately 2 × 10 cells were added to each well of a 96-well cell culture plate. 4 Caco-2 cells were cultured. After the cells were treated with cadmium ions and extracellular polysaccharide MY-5-cEPS, the culture medium was removed and the cells were washed twice with PBS. 10 μL CCK8 solution and 100 μL MEM complete culture medium were added to each well, and the reaction was carried out in a 37°C incubator for 2 hours. The absorbance at 450 nm was measured with an ELISA reader. After completing the cell morphology and cell viability tests, subsequent studies were carried out.

[0120]

[0121] Wherein: A represents the absorbance value of the culture medium, cells, CCK8 solution, cadmium ions and extracellular polysaccharide treatment group, A0 represents the absorbance value of the culture medium and CCK8 solution group, and A1 represents the absorbance value of the culture medium, cells, CCK8 solution, no cadmium ions and extracellular polysaccharide treatment group.

[0122] Fig.14A and B show the cell viability test results of the treatment group and the cell group, respectively. It can be seen that MY-5-cEPS in the treatment group (EPS was given after the cadmium stimulation) significantly restored the cell survival rate (p < 0.05), and the prevention group (EPS was given first and then cadmium stimulation) also restored cell viability, but there was no significant difference.

[0123] Fig.15 The cell morphology is shown, with the upper row being the treatment group and the lower row being the prevention group. Fig.15 The results showed that the cell morphology of the treatment group (EPS was given after the cadmium exposure) and the prevention group (EPS was given first and then cadmium exposure) was normal and no significant changes occurred, indicating that the extracellular polysaccharide MY-5-cEPS has a certain protective effect on heavy metal-damaged cells.

[0124] 11.2. Cell apoptosis detection

[0125] Approximately 1 × 10 cells were added to each well of a 6-well cell culture plate. 6 Caco-2 cells were cultured. After the cells were treated with cadmium ions and extracellular polysaccharide MY-5-cEPS, the culture medium was removed and the cells were washed twice with PBS. The cells were digested with 0.25% trypsin-EDTA solution for about 2 to 3 minutes and the cells were collected. Subsequently, the cells were washed twice with PBS to remove residual trypsin. 100 μL of binding solution was added to resuspend the cells, 5 μL of Annexin V-FITC was added, and the mixture was gently mixed. The cells were stained at 37°C in the dark for 30 minutes. Finally, 5 μL of PI solution was added for staining for 5 minutes. After staining, flow cytometry was performed immediately.

[0126] Fig.16 The results of cell apoptosis are shown, with the upper row representing the treatment group and the lower row representing the prevention group. Fig.16 The results showed that after cadmium exposure, the apoptosis rate of Caco-2 cells (18.7% in the treatment group and 27.9% in the prevention group) increased significantly. After treatment with the extracellular polysaccharide MY-5-cEPS, it can be seen that the apoptosis rates of both the treatment group (10.3%) and the prevention group (8.42%) decreased, indicating that the extracellular polysaccharide MY-5-cEPS can effectively alleviate the effect of cadmium exposure on cell toxicity.

[0127] 11.3. Intracellular Reactive Oxygen Species (ROS) Detection

[0128] Approximately 1 × 10 cells were added to each well of a 6-well cell culture plate. 6Caco-2 cells were cultured. After the cells were treated with cadmium ions and extracellular polysaccharide MY-5-cEPS, the medium was removed and the cells were washed twice with PBS. DCFH-DA was diluted with serum-free medium to a final concentration of 10 μM. Subsequently, 1 mL of DCFH-DA was added to each well and placed in a 37°C cell culture incubator for 30 min. After stopping the incubation, the cells were washed three times with serum-free medium. Under the conditions of 488 nm excitation wavelength and 525 nm emission wavelength, laser confocal microscopy and microplate reader were used to detect the changes in cell fluorescence intensity before and after stimulation to evaluate the level of reactive oxygen species.

[0129] Fig.17 A shows the results of ROS detection using a laser confocal microscope, where the upper row is the treatment group and the lower row is the prevention group. It can be seen that cadmium exposure in both the treatment group and the prevention group led to increased cellular ROS levels, while MY-5-cEPS treatment significantly reduced intracellular ROS levels.

[0130] Fig.17 B and 17C show the results of further quantitative analysis of intracellular ROS levels using an ELISA instrument, where B is the treatment group and C is the prevention group. It can be seen that compared with the blank control group, cadmium exposure caused a significant increase in ROS levels in the treatment group (172.66%) and the prevention group (153.08%). In the treatment group and the prevention group, the ROS level of the MY-5-cEPS+Cd group was significantly lower than that of the Cd Only group (p < 0.05), which is consistent with the results of laser confocal microscopy observation.

[0131] 11.4. Detection of intracellular malondialdehyde (MDA) content

[0132] Approximately 1 × 10 cells were added to each well of a 6-well cell culture plate. 6 Caco-2 cells were added. After the cells were treated with cadmium ions and extracellular polysaccharide MY-5-cEPS, the culture medium was removed and the cells were washed twice with PBS. 200 μL of lysis buffer was added to lyse the cells. After lysis, the cells were centrifuged at 4°C and 8000 rpm for 10 min and the supernatant was collected. The MDA content was determined using the MDA content detection kit, and the protein concentration was determined using the BCA protein concentration kit.

[0133] Fig.18 The results of MDA content detection showed that in both the treatment group and the prevention group, the MDA production level of Caco-2 cells was significantly reduced after polysaccharide protection (p < 0.05). On the other hand, it confirmed that MY-5-cEPS has a protective effect on the oxidative stress of cells after cadmium exposure.

[0134] 11.5. Cytokine Content Detection

[0135] Approximately 1 × 10 cells were added to each well of a 6-well cell culture plate.6 Caco-2 cells were cultured in 6-well plates. After the cells were treated with cadmium ions and extracellular polysaccharide MY-5-cEPS, the culture medium was removed and the cells were washed twice with PBS. Cell RNA was extracted using the RNA extraction kit, and then the RNA was reverse transcribed into cDNA using the high-efficiency cDNA first-strand synthesis kit. The cDNA template obtained by reverse transcription was used to configure the PCR system for amplification according to the instructions of RT SuperMix Perfect for qPCR. β-actin was selected as the internal reference gene, and 2 -△△CT The data were calculated and analyzed by the method. The specific primer information is shown in Table 4.

[0136] Table 4: Gene and primer information of Caco-2 cells

[0137]

[0138] Fig.19 The effect of MY-5 exopolysaccharide MY-5-cEPS on the relative expression of inflammatory factors in Caco-2 cells exposed to cadmium is shown, among which A, B, C, D and E correspond to cytokines TNF-α, IL-1β, IL-6, IL-8 and IL-10, respectively. It can be seen that cadmium exposure caused a significant increase in the secretion levels of cytokines such as TNF-α and IL-1β in Caco-2 cells, indicating that cadmium exposure may cause an inflammatory response in intestinal cells. In the treatment group and the prevention group, MY-5-cEPS reduced the levels of TNF-α and IL-1β in Caco-2 cells exposed to cadmium (p < 0.05), proving that MY-5-cEPS does have the function of alleviating the inflammatory response caused by cadmium exposure. In addition, MY-5-cEPS also increased the secretion level of IL-10 after cadmium exposure, further indicating that MY-5-cEPS has the effect of inhibiting the inflammatory response of Caco-2 cells caused by cadmium exposure.

[0139] Fig. 20The effect of MY-5 exopolysaccharide of lactic acid bacteria on the relative expression of tight junction proteins in Caco-2 cells exposed to cadmium was shown. A, B, and C correspond to cytokines Claudin-1, Occludin-1, and ZO-1, respectively. It can be seen that cadmium exposure caused a significant decrease in the secretion levels of cytokines such as Claudin-1 (treatment group and prevention group), Occludin-1 (prevention group), and ZO-1 (prevention group) in Caco-2 cells, indicating that cadmium exposure may cause increased intestinal cell permeability and damage to tight junction proteins, thereby increasing cellular absorption of cadmium. In the treatment group and the prevention group, MY-5-cEPS significantly upregulated the level of ZO-1 in Caco-2 cells exposed to cadmium (p < 0.05), while there was no significant change in Claudin-1 and Occludin-1 genes. This shows that MY-5-cEPS protects the integrity of the cell barrier by regulating the expression of tight junction proteins in Caco-2 cells under cadmium exposure conditions.

[0140] Example 12: Ability of Pediococcus acidilactici exopolysaccharide to adsorb heavy metal Cd

[0141] 5 mg of MY-5-cEPS was added to 20 mL of 500 mg / L cadmium chloride solution and shaken for 3 h at 150 rpm and 25 °C in a constant temperature shaker. 500 mg / L cadmium chloride solution was used as a blank control. After the adsorption reaction, EPS was separated by ultrafiltration using an ultrafiltration tube, and the heavy metal concentration in the filtrate was determined using ICP-MS. The equilibrium adsorption amount of EPS for heavy metals was calculated by the following formula:

[0142]

[0143] Where C represents the concentration of cadmium ions at adsorption equilibrium mg / L, C0 represents the initial concentration of cadmium ions mg / L, and C EPS represents the EPS concentration in g / L, and Q represents the EPS adsorption capacity of cadmium ions in mg / g.

[0144] The results of in vitro heavy metal adsorption showed that the extracellular polysaccharide MY-5-cEPS of Pediococcus acidilactici had the ability to adsorb cadmium ions, and the adsorption capacity of MY-5-cEPS for Cd was 100±4.714 mg / g.

[0145] In summary, the exopolysaccharide of Pediococcus acidilactici MY-5 of the present invention has good antioxidant activity and thermal stability. The cell model confirms that Pediococcus acidilactici MY-5-cEPS can reduce the intestinal cell toxicity caused by cadmium exposure, relieve inflammatory response, protect the intestinal barrier, and has the potential to be developed into a new heavy metal biochelator, and has broad market application value.

[0146] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A Pediococcus acidilactici MY-5, characterized in that The preservation number of the Pediococcus acidilactici is CCTCC M20241066.

2. The exopolysaccharide produced by Pediococcus acidilactici MY-5 according to claim 1.

3. The extracellular polysaccharide according to claim 2, characterized in that The extracellular polysaccharide includes MY-5-cEPS and its purified component MY-5-EPS2, wherein the molecular weight distribution of the MY-5-cEPS is 63847Da (29.29%) and 5189Da (70.71%), respectively, and the molecular weight distribution of the MY-5-EPS2 is 47870Da.

4. The extracellular polysaccharide according to claim 3, characterized in that The monosaccharide composition of the MY-5-cEPS is: arabinose, galactose, glucose, xylose, mannose and galacturonic acid, and the molar ratio of the six monosaccharides is: Arabinose: galactose: glucose: xylose: mannose: galacturonic acid = 0.23: 1.35: 1: 0.34: 1.76: 0.

19.

5. The extracellular polysaccharide according to claim 3, characterized in that The monosaccharide composition of MY-5-EPS2 is: arabinose, galactose, glucose, xylose and mannose, and the molar ratio of the five monosaccharides is: Arabinose: galactose: glucose: xylose: mannose = 0.38: 0.83: 1：0.58：2.2。 6. The exopolysaccharide according to claim 3, characterized in that The MY-5-EPS2 contains seven glycosidic bonds: →3,6)-α-Manp-(1→, →5)-α-Araf-(1→, →4)-α-Glcp-(1→, →2,6)-α-Galp-(1→, →4)-α-Manp-(1→, →4)-β-Glcp-(1→, and β-T-Glcp-(1→).

7. Use of the exopolysaccharide of Pediococcus acidilactici MY-5 according to any one of claims 2 to 6, characterized in that: Used for preparing cosmetics or health products with antioxidant activity.

8. The use according to claim 7, characterized in that: The antioxidant activity is selected from the group consisting of: scavenging DPPH free radicals, scavenging ABTS free radicals, and scavenging OH free radicals.

9. Use of the exopolysaccharide of Pediococcus acidilactici MY-5 according to any one of claims 2 to 6, characterized in that: Used to prepare heavy metal biochelator products.

10. The use according to claim 9, characterized in that: The heavy metal biochelator is used to protect cells exposed to cadmium, including: improving the activity of cadmium-exposed cells, reducing apoptosis of cadmium-exposed cells, reducing the levels of reactive oxygen and lipid peroxides in cadmium-exposed cells, alleviating inflammatory responses in cadmium-exposed cells, improving tight junction protein expression in cadmium-exposed cells, and protecting cell barriers.

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