Pediococcus pentosaceus, modified exopolysaccharide, preparation method of modified exopolysaccharide and application of modified exopolysaccharide in preparation of antioxidant

Abstract

The invention discloses pediococcus pentosaceus, modified extracellular polysaccharide, a preparation method of the modified extracellular polysaccharide and application of the modified extracellular polysaccharide in preparation of an antioxidant, and is characterized in that the pediococcus pentosaceus is classified and named as pediococcus pentosaceus YY-13 strain, the preservation number is CGMCC No.29541, and the modified extracellular polysaccharide is applied to preparation of the antioxidant. The method for preparing the pediococcus pentosaceus source modified extracellular polysaccharide through the pediococcus pentosaceus source modified extracellular polysaccharide comprises the following steps: 1) extracting an extracellular polysaccharide crude product from pediococcus pentosaceus YY-13 by adopting an ethanol precipitation method; (2) carrying out separation and purification on the exopolysaccharide crude product by adopting a DEAE-52 cellulose ion exchange column and a Sepharose CL-6B gel column, so as to obtain high-purity exopolysaccharide; and 3) preparing polysaccharide from the high-purity exopolysaccharide by adopting an ultrasonic-assisted sulfation modification technology to obtain the pediococcus pentosaceus source modified exopolysaccharide with good antioxidant activity, and the pediococcus pentosaceus source modified exopolysaccharide has the advantages of strong antioxidant ability, high yield of the exopolysaccharide and anti-aging function.

Description

Technical Field

[0001] The invention relates to Pediococcus pentosaceus, in particular to Pediococcus pentosaceus, modified extracellular polysaccharide and a preparation method thereof and application thereof in the preparation of an antioxidant. Background Art

[0002] Lactic acid bacteria (LAB) is a kind of probiotic widely used in food processing, medicine and other fields. Its secondary metabolites, exopolysaccharides (EPS), are a type of sugar compound secreted outside the cell wall during the growth and metabolism of the strain and often permeate into the culture medium. They are often used as emulsifiers and gelling agents in the food processing industry. Extensive studies have shown that LAB-EPS has biological activities such as antioxidant, anti-tumor, and immunomodulatory activities, among which antioxidant activity is the basis of other functions and has important research value and significance.

[0003] Oxidative stress leads to an imbalance in the body's oxidative and antioxidant states, which accelerates aging, causes neurodegenerative diseases, cancer, atherosclerosis and other diseases, and endangers human health. When cells encounter oxidative stress, EPS plays a good protective role in the form of physical wrapping, and at the same time enhances antioxidant properties by regulating antioxidant enzyme activity and activating signal pathways. The structural diversity of LAB-EPS leads to differences in antioxidant activity, and the composition, functional groups, and molecular weight of EPS are all significant influencing factors. In addition to screening natural EPS and changing fermentation conditions, modification methods can effectively change the EPS structure, which are usually divided into physical, chemical, and biomolecular methods. Ultrasonic treatment is a green and economical physical modification that mainly causes changes in structure and molecular weight. Compared with natural EPS, modified EPS has better antioxidant capacity and stronger ferrous reduction ability. However, physical modification is relatively mild. In contrast, chemical modification reactions are more intense. Sulfation modification is the most common but most effective chemical method, which introduces sulfate groups to increase the biological activity of EPS. However, as the reaction proceeds, the contact of the reaction system is limited. Therefore, a single modification method has certain limitations. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Pediococcus pentosaceus with strong antioxidant ability and high extracellular polysaccharide yield, a modified extracellular polysaccharide and a preparation method thereof, and an application thereof in the preparation of an antioxidant. The preparation method of the modified extracellular polysaccharide adopts an ultrasonic-assisted sulfation modification method, which can further promote the violent reaction of sulfate modification and improve the antioxidant activity of the product.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a Pediococcus pentosaceus, which is classified and named Pediococcus pentosaceus YY-13 strain, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 8, 2024, with a deposit number of CGMCC No.29541.

[0006] The present invention also provides a modified exopolysaccharide derived from Pediococcus pentosaceus, wherein the polysaccharide structure is as follows:

[0007]

[0008] The present invention also provides a method for preparing modified exopolysaccharide derived from Pediococcus pentosaceus, comprising the following steps:

[0009] (1) Using ethanol precipitation method to extract crude exopolysaccharide from Pediococcus pentosaceus YY-13 with a deposit number of CGMCC No. 29541;

[0010] (2) separating and purifying the crude exopolysaccharide obtained in step (1) using a DEAE-52 cellulose ion exchange column and a SepharoseCL-6B gel column to obtain high-purity exopolysaccharide;

[0011] (3) The high-purity extracellular polysaccharide obtained in step (2) is prepared by ultrasound-assisted sulfation modification technology to obtain modified extracellular polysaccharide derived from Pediococcus pentosaceus with good antioxidant activity.

[0012] Furthermore, the specific steps of the extraction process in step (1) are as follows: after activating the strain Pediococcus pentosaceus YY-13, inoculate it into a modified MRS liquid culture medium at an inoculum size of 2vt%, culture it at 37°C for 26.5h, centrifuge the bacterial liquid to collect the supernatant and boil it for 10min, add TCA to a final concentration of 4wt% after cooling, let it stand at 4°C for 8h, centrifuge to obtain the supernatant, add 3 times the volume of anhydrous ethanol to the supernatant and stir it overnight, then centrifuge to obtain the precipitate, dissolve it in 60°C water and centrifuge it for 10min, and obtain the supernatant; concentrate the supernatant by rotary evaporation and put it into an 8000Da dialysis bag, dialyze it in an ultrapure water environment at 4°C for 2 days, and change the water every 4h; concentrate the solution after dialysis by rotary evaporation again, put it into a centrifuge tube and put it into a -80°C refrigerator for pre-freezing for 4h, and finally freeze-dry it to obtain a crude extracellular polysaccharide.

[0013] Furthermore, the modified MRS liquid culture medium is prepared as follows: 10 grams of peptone, 10 grams of beef extract, 5 grams of yeast extract, 2 grams of diammonium citrate, 5 grams of sodium acetate, 35 grams of maltose, 80 milliliters of Tween, 0.5 grams of magnesium sulfate, 0.25 grams of manganese sulfate and 15 grams of agar powder are dissolved in 1L of water.

[0014] Furthermore, the specific steps of the purification process of step (2) are as follows: the crude extracellular polysaccharide obtained in step (1) is dissolved in water to prepare a solution with a concentration of 20 mg / mL, filtered through a 0.22 μm aqueous phase filter membrane and then loaded with a sample of 5 mL, using a DEAE-52 cellulose ion exchange column pre-equilibrated with deionized water at pH 6.0, eluting with deionized water as an eluent, collecting the eluate, combining the eluates of each corresponding sugar peak respectively, concentrating by rotary evaporation, and desalting by dialysis to obtain neutral sugar; the neutral sugar is eluted using a Sepharose CL-6B gel column with distilled water as an eluent, collecting the eluate, concentrating the eluate by rotary evaporation, and freeze-drying to obtain a high-purity extracellular polysaccharide with a purity of more than 90%.

[0015] Furthermore, the specific steps of the modification process of step (3) are as follows: dissolving the high-purity extracellular polysaccharide in anhydrous dimethylformamide DMF to obtain a solution with a concentration of 5 mg / mL, oscillating under ultrasonic conditions of a frequency of 20 kHz and a power of 500 W for 55 minutes, and continuing to oscillate for 30 minutes after the ultrasonic oscillation ends, and then adding 100 mg of sulfur trioxide-pyridine complex and stirring at 80°C for 3 hours; after the reaction is completed, the mixture is cooled to room temperature in an ice bath, neutralized with 4M NaOH solution, and then dialyzed with ultrapure water at 4°C for 2 days to obtain modified extracellular polysaccharide derived from Pediococcus pentosaceus, wherein the mass ratio of the high-purity extracellular polysaccharide to the sulfur trioxide-pyridine complex is 2:1.

[0016] The present invention also provides the use of the modified exopolysaccharide derived from Pediococcus pentosaceus in the preparation of an in vitro antioxidant, characterized in that the in vitro antioxidant comprises a DPPH free radical scavenger, an ABTS free radical scavenger and a ferrous ion reducing agent.

[0017] The present invention also provides the use of the modified extracellular polysaccharide derived from Pediococcus pentosaceus in the preparation of an in vivo antioxidant.

[0018] The present invention also provides the use of the modified extracellular polysaccharide derived from Pediococcus pentosaceus in the preparation of anti-aging drugs.

[0019] Compared with the prior art, the invention has the advantages that: the invention discloses a Pediococcus pentosaceus, a modified exopolysaccharide, a preparation method thereof, and an application thereof in the preparation of an antioxidant, wherein the screened Pediococcus pentosaceus exopolysaccharide has a high yield and excellent antioxidant performance; the modified exopolysaccharide obtained by using the Pediococcus pentosaceus is introduced into the EPS structure by using ultrasonic-assisted sulfation technology, and the S-EPS has a small molecular weight, and the low molecular weight S-EPS exposes more hemiacetal hydroxyl groups than the high molecular weight EPS, so the antioxidant capacity is strong. In addition, the S-EPS has a high degree of branching, and the structures such as 1→4 glycosidic bonds are conducive to the enhancement of biological activity, thereby optimizing the in vitro antioxidant performance of EPS, enhancing the DPPH and ABTS free radical scavenging ability, and having a strong ferrous ion reduction ability. In the in vivo antioxidant effect, it can effectively increase the activity of antioxidant enzymes SOD, GSH-Px and CAT in the mouse liver, reduce the MDA content in the mouse liver and the pro-inflammatory factors IL-6, IL-1β and TNF-α in the serum, increase the antioxidant master regulator Nrf and antioxidant enzyme HO-1, and improve the loose arrangement of liver cells, unclear boundaries, cavitation and severe inflammatory infiltration caused by large amounts of fat accumulation, which proves that S-EPS has excellent antioxidant ability in the body and can alleviate the oxidative stress caused by excessive intake of D-galactose.

[0020] The above-mentioned Pediococcus pentosaceus is classified and named as Pediococcus pentosaceus YY-13 strain, with a preservation number of CGMCC No.29541. It was deposited in the General Microbiology Center of China National Culture Collection Administration on January 8, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of fermentation time on EPS production;

[0022] Figure 2 The effect of inoculum amount on EPS production;

[0023] Figure 3 The effect of carbon source on EPS production;

[0024] Figure 4 is the effect of maltose concentration on EPS yield;

[0025] Figure 5 The elution curves of DEAE-52 cellulose ion exchange column and Sepharose CL-6B gel column, wherein (a) DEAE-52 cellulose ion exchange column; (b) Sepharose CL-6B gel column;

[0026] Figure 6The apparent morphology of EPS and S-EPS under scanning electron microscopy, Note: (a) EPS (200×); (b) S-EPS (200×); (c) EPS (10000×); (d) S-EPS (10000×), EPS: extracellular polysaccharide; S-EPS: ultrasound-assisted sulfated extracellular polysaccharide;

[0027] Figure 7 Infrared spectra of EPS and S-EPS, where (a) EPS: extracellular polysaccharide; (b) S-EPS: ultrasound-assisted sulfated modified extracellular polysaccharide;

[0028] Figure 8 HPGPC chromatograms of EPS and S-EPS, where (a) EPS: exopolysaccharide; (b) S-EPS: ultrasound-assisted sulfated exopolysaccharide;

[0029] Fig. 9 The PMP pre-column derivatization HPLC chromatograms of monosaccharide standards and S-EPS, where (a) monosaccharide standards; (b) modified extracellular polysaccharide S-EPS;

[0030] Fig.10 This is the NMR structure analysis of the modified extracellular polysaccharide, including (a) H spectrum; (b) C spectrum; (c) DEPT spectrum; (d) HH COSY spectrum; (e) HSQC spectrum; (f) HMBC spectrum; (g) NOESY spectrum;

[0031] Fig.11 The in vitro antioxidant capacity of S-EPS at different doses, including (a) ABTS free radical scavenging capacity, (b) DPPH free radical scavenging capacity, and (c) ferrous ion reducing capacity;

[0032] Fig.12 The results of determination of IL-6, TNF-α, and IL-1β levels in the serum of aging model mice under different treatments; (a) is IL-6, (b) is TNF-α, and (c) is IL-1β;

[0033] Fig.13 The results of the determination of HO-1 and Nrf2 levels in the serum of aging model mice under different treatments, where (a) is HO-1 and (b) is Nrf2;

[0034] Fig.14 The results are for the determination of total antioxidant capacity of aging model mice under different treatments;

[0035] Fig.15 The results of determination of MDA content in liver of aging model mice under different treatments;

[0036] Fig.16Figure 3 Pathological changes in the liver of aging model mice after different treatments (HE staining, 100×), including (a) CG blank group; (b) aging model NC group; (c) positive control VC group; (d) EPS low-dose LD group; (e) EPS medium-dose MD group; (f) EPS high-dose HLD group. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below with reference to the accompanying drawings. Specific embodiment 1

[0039] Screening of strains of the present invention

[0040] 1. Screening based on EPS production as the primary screening indicator

[0041] 25 lactic acid bacteria strains were selected from the strain bank of the Animal Products Processing Laboratory of the School of Food Science and Engineering of Ningbo University and activated three times for experiments. Solid streak culture was performed at 37°C for 48 hours, and 11 strains that could pull out filaments were selected. EPS was extracted using the ethanol precipitation method. The specific steps are as follows:

[0042] After the strain is activated, it is inoculated into MRS liquid culture medium at a volume ratio of 2%. After culturing at 37°C for 26.5h, the bacterial liquid is centrifuged (4°C, 5000rpm, 20min), the supernatant is collected and boiled for 10min, trichloroacetic acid TCA is added to a final concentration of 4% after cooling, and it is allowed to stand at 4°C for 8h. Centrifuge to obtain the supernatant (4°C, 10000rpm, 10min), and add 3 times the volume of the supernatant to the supernatant with anhydrous ethanol and stir overnight with a magnetic stirrer. Centrifuge (4°C, 10000rpm, 10min) to obtain the precipitate, dissolve it in 60°C water and centrifuge for 10min, and obtain the supernatant. The supernatant is concentrated by rotary evaporation and placed in an 8000Da dialysis bag, and dialyzed in an ultrapure water environment at 4°C for 2 days, with the water changed every 4h. The dialysis solution was concentrated by rotary evaporation again, put into a centrifuge tube and placed in a -80°C refrigerator for pre-freezing for 4 hours, and finally freeze-dried to obtain the crude EPS.

[0043] The EPS content in the crude EPS was further determined by the phenol-sulfuric acid method. The phenol-sulfuric acid method standard curve was prepared using glucose as the standard. The established regression equation was y = 0.0322x + 0.0491, R 2 =0.9963. The production and source of extracellular polysaccharide EPS are shown in Table 1. Three strains of P. pentosaceus YY-13, L. plantarum FR-1 and L. plantarum B-1-42 were initially screened.

[0044] Table 1 Source and yield of experimental strains

[0045]

[0046] 2. Screening based on antioxidant capacity as a secondary screening indicator

[0047] Based on the EPS production, three lactic acid bacteria with better production were preliminarily screened out, namely P. pentosaceus YY-13, L. plantarum FR-1 and L. plantarum B-1-42. Combined with their antioxidant properties, they were rescreened. The DPPH free radical scavenging ability of P. pentosaceus YY-13 was 94.95%, L. plantarum FR-1 was 88.32%, and L. plantarum B-1-42 was 68.37%. Combined with the production and its DPPH free radical scavenging ability, P. pentosaceus YY-13 was determined as the experimental target strain.

[0048] 3. Target strain identification

[0049] Identification process: The target strain was streaked several times on the MRS solid plate, and the colonies with a single morphology were picked out and activated and placed in a modified MRS liquid medium (the formula of the modified MRS liquid medium is 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 35 g of maltose, 80 ml of Tween, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder, dissolved in 1L of distilled water, sterilized at 121°C for 15 min) and cultured overnight at 37°C for 24 h, centrifuged at 5000 rpm for 10 min, and the supernatant was taken. The 16S rDNA gene of the target strain was extracted by the bacterial genomic DNA kit for 16S rRNA sequence analysis. Universal primers 1492R and 27F were used for amplification. The PCR fragment (1,500 bp) purified using the rapid PCR purification kit was tested by Youkang Biotechnology Co., Ltd. MEGA software (https: / / www.megasoftware.net / ) was used to compare the 16S rDNA genes of the selected members in GenBank. Because we found that YY-13 had a high homology with Pediococcus pentosaceus, we confirmed that the strain was classified as Pediococcus pentosaceus again and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 8, 2024, with the deposit number CGMCC No. 29541.

[0050] 4. Optimization of fermentation conditions for target strains

[0051] (1) Single factor experiment:

[0052] The initial conditions controlled in this experiment were 1% (v / v) inoculum dosage, 37°C fermentation temperature, glucose as the only carbon source, and EPS yield as an indicator to study the effects of fermentation time (16h, 20h, 24h, 28h, 30h), inoculum dosage (1%, 2%, 3%, 4%, 5% by volume) and carbon source (glucose, mannose, galactose, maltose, sucrose) on the EPS yield of the strain.

[0053] Effect of fermentation time on EPS production Figure 1 As shown in the figure, when the inoculum amount and other factors such as carbon source were kept the same, the EPS production first increased and then decreased with the fermentation time. When the fermentation time was 20 h, the maximum production was 92.41 mg / L.

[0054] Effect of inoculation amount on EPS production Figure 2 As shown, when the fermentation time was controlled at 20 h, glucose was used as the only carbon source, and the inoculum amount was 2 vt%, the yield was the highest. With the increase of the inoculum amount, the overall trend showed an increase first and then a decrease.

[0055] Maltose, galactose, sucrose, mannose and glucose were selected as the sole carbon sources to explore the effects of different carbon sources on the yield of P. pentosaceus YY-13. Figure 3 As shown in the figure, different carbon sources have a significant effect on EPS production, especially when maltose is used as its carbon source, the EPS production can reach 284.66 mg / L, which is significantly higher than other groups. Therefore, maltose was selected as the carbon source of the culture medium.

[0056] Effect of maltose addition concentration on polysaccharide yield Figure 4 As shown. When the inoculation amount was controlled at 2vt% and the fermentation time was 20h, different concentrations of maltose were added as the carbon source. With the increase of maltose concentration, the polysaccharide yield showed a trend of first increasing and then decreasing. The yield increase was most obvious when it increased from 25g / L to 30g / L, and reached a maximum value of 308.66mg / L at 30g / L, and then showed a downward trend. The increase in maltose concentration is beneficial to the supply of nutrients to the bacteria, promotes bacterial growth and metabolic activities, and increases EPS production. When the maltose concentration is too high, the bacteria may not be able to maintain an appropriate carbon-nitrogen balance, thereby affecting EPS synthesis.

[0057] (2) Determination of optimal fermentation process conditions

[0058] The single factor experiment was used to determine the three optimal levels of maltose concentration, fermentation time and inoculum amount, which were maltose concentration (25g / L, 30g / L and 35g / L), fermentation time (20h, 24h and 28h) and inoculum amount (1vt%, 2vt% and 3vt%). A three-factor and three-level response surface optimization experiment was carried out on maltose concentration, fermentation time and inoculum amount, and the quadratic regression equation obtained was Y=297.556+65.769*A-28.428*B+36.828*C+39.513*AB-27.817*AC-15.723*BC-15.719*A 2 -38.690*B 2 -85.830*C 2 The optimal fermentation process conditions were analyzed as follows: inoculation amount 2vt%, maltose concentration 35g / L, and fermentation time 26.5h. After optimization experiments, EPS production increased from 160.98mg / L to 336.804mg / L, more than doubling the yield. Specific embodiment 2

[0060] The modified exopolysaccharide was prepared by Pediococcus pentosaceus YY-13 screened in Specific Example 1, and the specific steps are as follows:

[0061] Step 1: Preparation of Pediococcus pentosaceus YY-13 exopolysaccharide

[0062] EPS of Pediococcus pentosaceus YY-13 was prepared by ethanol precipitation method, the method of which is shown in Specific Example 1, and the MRS liquid culture medium was replaced by a modified MRS liquid culture medium.

[0063] Step 2: Obtaining purified exopolysaccharides

[0064] DEAE-52 cellulose ion exchange column and Sepharose CL-6B gel column were used for separation and purification.

[0065] The crude exopolysaccharide obtained in step 1 is dissolved in water to prepare a solution with a concentration of 20 mg / mL, and the sample is loaded after filtering through a 0.22 μm aqueous phase filter membrane, with a sample volume of 5 mL, and a DEAE-52 cellulose ion exchange column pre-equilibrated with deionized water at pH 6.0 is used, and deionized water is used as an eluent, and the eluate is eluted at a flow rate of 1 mL / min, and the eluate is collected, 5 mL per tube, and the sugar content is detected by a phenol-sulfuric acid method, and an elution curve is drawn. The eluates of each corresponding sugar peak are respectively combined, concentrated by rotary evaporation, and dialyzed and desalted to obtain neutral sugar; the neutral sugar is eluted using a Sepharose CL-6B gel column with distilled water as an eluent, and the eluate is collected, and the eluate is concentrated by rotary evaporation, and freeze-dried to obtain a high-purity exopolysaccharide EPS with a purity of more than 90%.

[0066] After P.pentasiaceus YY-13 was chromatographed on an anion exchange column, it was divided into two neutral sugars and acidic sugars according to the ionic charge it carried. Then, polysaccharides of different molecular weights were separated by Sepharose CL-6B gel permeation column chromatography. It uses gel particles as the stationary phase. Larger molecules will come out of the column first, and smaller molecules will come out later. Figure 5 a. Elution of gel permeation column chromatography Figure 5 b. The EPS content was calculated according to the phenol-sulfuric acid method standard curve, and the EPS purity was 90.265%; Coomassie brilliant blue standard curve: y = 3.6682x + 0.4168, R 2 =0.9986, and the protein content was calculated based on the standard curve to be 0.94%.

[0067] Step 3: Preparation of modified exopolysaccharide

[0068] Preparation of ultrasonic-assisted sulfated polysaccharide: 50 mg of high-purity extracellular polysaccharide was dissolved in 10 ml of anhydrous dimethylformamide DMF, and then oscillated for 55 minutes under ultrasonic conditions of 20 kHz and 500 W, and then continued to oscillate for 30 minutes at room temperature, and then 100 mg of sulfur trioxide-pyridine complex was added and stirred at 80 ° C for 3 hours; after the reaction, the mixture was cooled to room temperature in an ice bath. Neutralized with 4M NaOH solution, and then dialyzed with ultrapure water at 4 ° C for 2 days to obtain ultrasonic-assisted sulfated polysaccharide S-EPS.

[0069] Preparation of sulfated modified polysaccharides: 50 mg of purified high-purity EPS was dissolved in 10 ml of anhydrous dimethylformamide DMF, shaken continuously at room temperature for 30 minutes, and then 100 mg of sulfur trioxide-pyridine complex was added and stirred at 80°C for 3 hours. After the reaction, the mixture was cooled to room temperature in an ice bath. It was neutralized with 4M NaOH solution and then dialyzed with ultrapure water at 4°C for 2 days to obtain sulfated modified polysaccharides. Specific embodiment three

[0071] Comparative analysis of performance before and after EPS modification

[0072] 1. Determination of sulfate content of S-EPS:

[0073] The sulfate content of S-EPS was determined by the barium chloride gelatin method, and the calculation results are shown in Table 1.

[0074] Table 1 Sulfate content and antioxidant activity of EPS under different treatments

[0075]

[0076] Note: S%: sulfate content; DS: degree of polymerization, used to express the degree of modification.

[0077] As shown in Table 1, it was determined that the neutral sugar did not contain sulfate. After sulfation treatment, sulfate groups were introduced, which significantly increased the sulfate content. After ultrasonic treatment, the sulfate content and antioxidant capacity were further increased. The S% of the ultrasonic-sulfation treatment was 1.95 times that of the sulfation treatment alone, the DPPH free radical scavenging capacity was increased by more than 20%, and the total oxidation capacity was increased by 31%.

[0078] 2. Scanning electron microscopy observation of EPS and S-EPS

[0079] Take an appropriate amount of polysaccharide sample on a conductive carbon tape, spray gold, and place it in a scanning electron microscope for observation and photography. Set the working voltage to 10.0 kV, observe the solid morphology of the sample at different magnifications, and perform corresponding clarity adjustments until the ideal observation field of view is obtained. Select an appropriate field of view for photography and record.

[0080] The microscopic morphology of EPS surface was observed by scanning electron microscopy. Figure 6 As shown in (a), the unmodified extracellular polysaccharide (EPS) presents irregular blocks with sharp corners in a three-dimensional structure, and there are also some uniform rod-shaped blocks; Figure 6 As shown in (b), the ultrasound-assisted sulfated polysaccharide (S-EPS) is in the form of thin sheets and is broken, which may be caused by ultrasonic oscillation, intense reaction conditions, and the introduction of chemical groups. By further magnifying the structure, it can be found that Figure 6 As shown in (c), the surface of EPS has characteristic wrinkles and textures; Figure 6 As shown in (d), the surface of the polysaccharide after ultrasonic-assisted sulfation treatment became rough, and radial textures and many tiny pores appeared, indicating that ultrasonic-assisted sulfation treatment significantly changed the polysaccharide structure, and its energy was sufficient to destroy covalent bonds such as EPS glycosidic bonds and promote the separation of polysaccharide molecules.

[0081] 3. Infrared spectroscopy analysis of EPS and S-EPS

[0082] Infrared spectroscopy is used to identify the type of sugar and sugar ring in the polysaccharide structure, provide information on the configuration of glycosidic bonds, and identify characteristic groups on the sugar chain, providing partial basis for the analysis of the polysaccharide structure. Weigh 1-2 mg of the polysaccharide sample EPS in step 2 and the polysaccharide sample S-EPS in step 3 of each dried specific embodiment 2 in a mortar, add 200 mg of KBr powder respectively, grind evenly, press into tablets, and use a Fourier transform micro-infrared spectrometer to perform infrared scanning on the samples, with a wavelength range of 4000-400 cm -1 , record the infrared spectrum.

[0083] The infrared spectrum of EPS is as follows Figure 7 As shown in (a), the sample has a characteristic absorption peak of polysaccharide: 3445.46 cm -1 There is a strong and broad absorption peak at 2923.89cm, which is the strong absorption peak of OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The medium-intensity peaks nearby are the CH stretching vibration absorption peaks of methyl (-CH3) and methine (-CH2); 1648.50cm -1 The absorption peak at 1450-1200cm is the stretching vibration of C=O of carbonyl or the formation of crystal water; -1 The absorption peak is 1461.53cm -1 ,1424.46cm -1 ,1372.95cm -1 is the angle-variable vibration absorption peak of CH, which together with the stretching vibration of CH constitutes the characteristic absorption of the sugar ring; 1159.29 is the stretching vibration of pyranose COC; 1150-1010cm -1 There are three strong absorption peaks between 1081.85cm -1 、1018.85cm -1 and 993.72cm -1 The presence of pyranoside was confirmed again at 927.50 cm-1. The absorption at these three locations is the bending vibration of the CO bond in the COH or COC structure; -1 The peak of asymmetric ring stretching vibration of pyran ring is shown at 846.10 cm -1 It is the CH angle vibration peak of the α-anomer of pyranose; 759.26cm -1 Showing the symmetric ring stretching vibration peaks of the pyran ring.

[0084] The infrared spectrum of S-EPS is shown in Figure 7 As shown in (b), the sample has a characteristic absorption peak of polysaccharide: 3431.74 cm -1 There is a strong and broad absorption peak at 2919.63cm, which is the strong absorption peak of OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The medium-intensity peaks nearby are the CH stretching vibration absorption peaks of methyl (-CH3) and methine (-CH2); 1639.99cm -1 The absorption peak at 1254 cm is the C=O stretching vibration of the carbonyl group or the formation of crystal water. -1 The absorption peaks on the left and right are the characteristic peaks of S=O, 855.82cm -1 The absorption peaks on the left and right are the stretching vibrations of the COS bonds. These two peaks are characteristic absorption peaks of sulfate esters, indicating that the polysaccharide has been successfully sulfated.

[0085] 4. Determination of molecular weight of EPS and S-EPS

[0086] (1) Establishment of molecular weight calibration curve

[0087] Dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards) were accurately weighed, and 0.05M NaCl solution was added to prepare 5 mg / mL dextran standard solution, which was filtered through a 0.22 μm microporous filter membrane for later use. The HPGPC method was used with high-performance gel permeation chromatography tandem columns for detection, and linear regression was performed with the logarithm of the relative molecular mass of the standard as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain a calibration curve.

[0088] (2) Preparation of test sample solution

[0089] Accurately weigh 5 mg of the sample, add 1 mL of 0.05 M NaCl solution to the sample to prepare a 5 mg / mL test sample solution, centrifuge at 8000 rpm for 10 min, take the supernatant and filter it with a 0.22 μm microporous filter membrane, and then transfer the sample to a 2 mL injection bottle for use.

[0090] (3) Chromatographic method

[0091] HPGPC method was used, and high-performance gel permeation chromatography series columns were used for detection. Instrument: Waters high-performance liquid chromatograph; Detector: Waters differential detector; Chromatographic column: 3 polymer-based water-soluble SEC (GFC) chromatographic columns (8×300mm) connected in series; Mobile phase: 0.05M NaCl solution; Flow rate: 0.65mL / min; Column temperature: 40℃; Injection volume: 30μL.

[0092] (4) Results analysis

[0093] Linear regression was performed with the logarithmic value of the relative molecular mass of the standard as the ordinate and the retention time (T) of the corresponding chromatographic peak as the abscissa to obtain the calibration curve equation: Log Mw = -0.1761T + 11.0299, R 2 =0.9927. Figure 8 From the HPGPC chromatograms of EPS (a) and S-EPS (b), the molecular weight of EPS was calculated to be 5990Da; that of S-EPS was 11808Da, which was more than doubled. In addition, small peaks with different molecular weights appeared. However, according to their elution time, the monosaccharide components were consistent.

[0094] 5. Analysis of monosaccharide composition of S-EPS

[0095] After pre-column derivatization, the monosaccharide composition was determined by high performance liquid chromatography (HPLC).

[0096] (1) Standard product configuration

[0097] Weigh 5 mg of each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucosamine hydrochloride, and galactosamine hydrochloride monosaccharides, and 10 mg of fucose in sequence, dissolve them in a volumetric flask to 10 mL, and prepare the standard stock solution. Dilute them to the following gradient dilutions, filter through a 0.22 μm microporous membrane, and place them in a sample injection bottle.

[0098] (2) Preparation of test sample solution

[0099] Take a clean chromatographic bottle, accurately weigh 5 mg (± 0.05 mg) of polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add 3 mL of methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add 5 mL of sterile water to dissolve, and transfer to the chromatographic bottle for testing.

[0100] (3) PMP derivatization

[0101] Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolyzate into a stoppered conical bottom centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP methanol solution, vortex mix well and react in a 70°C water bath for 1 h. After the reaction is completed, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the added sodium hydroxide, add 1 mL of chloroform and vortex extract 3 times to remove excess PMP, discard the chloroform layer, take 0.3 mL and add water to make up to 1 mL.

[0102] (4) Chromatographic method

[0103] ThermoU3000 liquid chromatography system, the chromatographic column is ZORBAX EclipseXDB-C18, the mobile phase is acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, 2M NaOH to adjust the pH to 6.8) gradient elution, the volume ratio of acetonitrile to phosphate buffer is 17:83, the flow rate is 0.8 mL / min, the column temperature is 30°C, the detection wavelength is 250 nm, and the injection volume is 10 μL.

[0104] (5) Results analysis

[0105] According to Table 2 and Fig. 9 (a) Monosaccharide standards and, Fig. 9(b) PMP pre-column derivatization HPLC of S-EPS. It was calculated that S-EPS is composed of glucose and mannose, with glucose being the main component. From the results of monosaccharide composition, the polysaccharide is composed of only two monosaccharides, of which glucose accounts for the largest proportion, accounting for 98.562% by molar ratio, followed by mannose, accounting for about 1.438%, so the polysaccharide can be classified as dextran.

[0106] Table 2 Summary of the information of the marked song

[0107]

[0108]

[0109] 6. Methylation analysis results of S-EPS

[0110] The S-EPS sample prepared in step 3 of specific embodiment 2 was derivatized: 1-3 mg of the sample to be tested was weighed and dissolved in 500 μL DMSO. 1 mg NaOH was added and incubated for 30 min. 50 μL iodomethane solution was added and reacted for 1 h; 1 mL water and 2 mL dichloromethane were added, vortexed and mixed, centrifuged, and the aqueous phase was discarded. Repeat the water washing for 3 times; absorb the lower dichloromethane phase and evaporate to dryness; add 100μL 2M TFA, react at 121℃ for 90min, evaporate to dryness at 30℃; add 50μL 2M ammonia, 50μL 1M NaBD4, mix well, react at room temperature for 2.5h; add 20μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250μL methanol, blow dry with nitrogen; add 250μL acetic anhydride, vortex mix well, react at 100℃ for 2.5h; add 1mL water and let stand for 10min; add 500μL dichloromethane, vortex mix well, centrifuge, and discard the water phase. Repeat the water washing for 3 times; take the lower dichloromethane phase and detect on the machine.

[0111] S-EPS is a sugar chain connected by glycosidic bonds. After acid hydrolysis, a series of intermediate products are obtained, and finally monosaccharides are obtained. Methylation analysis is a means to determine the connection mode of various monosaccharide residues in S-EPS. First, the free hydroxyl group in each monosaccharide residue in the polysaccharide chain is methylated, and then the glycosidic bonds therein are hydrolyzed. Finally, the product is subjected to GC-MS analysis. According to the relative retention time and mass spectrum of each chromatographic peak, the literature data and the ion fragment mass spectrum of the database of the Center for Complex Carbohydrate Research at the University of Georgia in the United States are compared for qualitative analysis. The type of sugar residue corresponding to each chromatographic peak is analyzed, and then the content of each sugar residue type is calculated using the chromatographic peak area of ​​the corresponding sugar residue type. The results are shown in Table 3.

[0112] Table 3 Summary of glycosidic bond configurations of S-EPS

[0113]

[0114] From the methylation test results in Table 3, it can be seen that there are four kinds of residual sugars constituting glucose, which is basically consistent with the monosaccharide composition results. The largest proportion is →4)-Glcp-(1→ residual sugar, which constitutes the main chain of glucan, followed by glucose terminal sugar accounting for about 25%, which confirms that the polysaccharide has a high degree of branching. Combined with the main →4,6)-Glcp-(1→ residual sugar, it is speculated that the branch may be formed by the 6th position of →4,6)-Glcp-(1→ residual sugar. The →6)-Glcp-(1→ residual sugar with a smaller content may be a component of the branch. Through GPC molecular weight detection, the weight average molecular weight of glucan was obtained to be about 5990. Combined with the monosaccharide composition calculation, it was obtained that the polysaccharide structure contained an average of 37 residual sugars in the molecule.

[0115] 7. Analysis of S-EPS structure using nuclear magnetic resonance technology

[0116] Dissolve 100 mg of polysaccharide sample S-EPS in D2O (heavy water) and exchange it three times by freeze drying to replace active hydrogen with deuterium. Dissolve it in 0.5 mL of D20 and place it in a nuclear magnetic resonance tube. Perform one-dimensional nuclear magnetic resonance on the polysaccharide sample using a nuclear magnetic resonance spectrometer. 1 H-NMR, 13 C-NMR and two-dimensional NMR COSY, HSQC, HMBC, NOESY analysis.

[0117] like Fig.10 As shown in (a), in the H NMR spectrum 1 In the anomeric region of H NMR (4.3-5.5ppm), multiple groups of anomeric hydrogen signals can be found. After identification, the main peak positions are 5.30, 5.26, 5.12, 4.92, 4.87, and 4.54ppm, confirming that the polysaccharide is composed of multiple residual sugars, which basically echoes the methylation results. In the 3.0-4.2ppm range of the hydrogen spectrum, there are typical proton signals outside the anomeric hydrogen of the residual sugar ring. The signals in this region are numerous and complex, which is consistent with the general characteristics of the hydrogen spectrum of polysaccharides.

[0118] like Fig.10 (b) shows the NMR carbon spectrum 13 In C NMR, no obvious uronic acid carbonyl carbon signal was found within 170-210ppm, confirming that the polysaccharide is neutral. In the anomeric region of the carbon spectrum (90-110ppm), multiple groups of main anomeric carbon signals can be identified, which are 99.70, 98.54, 95.86, and 92.04ppm, respectively. Multiple anomeric carbon signals correspond to multiple anomeric hydrogen signals; in the range of 55-85ppm, there are multiple typical polysaccharide residual sugars excluding anomeric carbons. The signals at 60.69 and 63.42ppm are attributed to the residual sugar ring exomethylene -CH2- secondary carbon, such as Fig.10As shown in (c), the DEPT135 spectrum confirms the signal attribution at this location. It is worth noting that the weaker negative peak signal at 67.06 ppm in the DEPT spectrum suggests that this location may belong to C6 of the methylene -CH2- that forms the glycosidic bond; the multiple signals at 74-82 ppm represent the carbon information of glucose C2-C5, among which the weak peak in the range of 75-82 ppm belongs to C4 that forms the glycosidic bond.

[0119] The structure of the polysaccharide was further analyzed by two-dimensional NMR spectroscopy. Fig.10 As shown in (e), in the HSQC two-dimensional NMR spectrum reflecting the connected hydrogen-carbon correlation, multiple groups of H1 / C1 related weak signals in the anomeric region were identified, which were 5.29 / 99.57, 5.12 / 91.82, 4.92 / 98.39, 4.85 / 97.93, and 4.53 / 95.81 ppm, respectively. Combining the methylation results, the integrated intensity of the anomeric hydrogen in the hydrogen spectrum, and referring to the peak signals in the literature, a comprehensive judgment was made: 5.29 / 99.57 was the H1 / C1 signal of α→4)-Glcp-(1→, 5.26 / 99.89 was the H1 / C1 signal of α→4,6)-Glcp-(1→, 4.85 / 97.93 was the H1 / C1 signal of α→t)-Glcp-(1→, and 4.92 / 98.39 was the H1 / C1 signal of α→6)-Glcp-(1→.

[0120] By combining multiple two-dimensional NMR spectra, the positions and structural correspondence information of the carbon-hydrogen signals of various residual sugars in the glucan structure were assigned in turn. The anomeric signal 5.29 ppm was determined to be assigned to the anomeric H1 in the residual sugar α→4)-Glcp-(1→ Fig.10 (d)) A strongly correlated signal of 3.47ppm was found, confirming the positions of H1 and H2 at 5.29 and 3.47ppm; combined with the HSQC spectrum, the signal of H2 / C2 was confirmed to be 3.47 / 71.41; based on the COSY information, the signal related to H2 and H3 was found to be 3.75, confirming the hydrogen spectrum position of C3 at 3.75ppm, and combined with HSQC to find 3.75 / 70.94, confirming the position of C3 at 70.94ppm, and the position information of H4 / C4, H5 / C5, and H6 / C6 was determined to be 3.55 / 76.57, 3.87 / 73.05, 3.67, and 3.76 / 60.42 by the same method. Based on this method, other main positions of residual sugars were found and the same signal positions were assigned, forming the following Table 4:

[0121] Table 4 Main residual sugars in S-EPS

[0122]

[0123] like Fig.10 As shown in (f), the HMBC of the two-dimensional NMR spectrum plays a greater role in determining the hydrogen-carbon correlation between 2-3 chemical bonds, and has a certain significance in determining the connection of glycosidic bonds in the analysis of polysaccharides. For example, in the 1,4-Glc(p) residual sugar with a large content, the anomeric carbon-related signal is found in the HMBC spectrum: 5.31 / 72.58, which confirms that the anomeric hydrogen of the residual sugar is related to H4 of the same residual sugar, confirming the existence of 1→4 connected glycosidic bonds of the same residual sugar, and forming the main chain glycosidic bonds in the glucan; the correlation signal of 5.30 / 76.80 confirms that the main residual sugars 1,4-Glc(p) and 1,4,6-Glc(p) have 1→4 connected glycosidic bonds. In addition, the correlation signal of 5.26 / 72.58 inferred that 1,4,6-Glc(p) and 1,4-Glc(p) had a 1→4 glycosidic bond connection, while the anomeric signal of 4.87 / 67.65 inferred that t-Glc(p) and 1,6-Glc(p) had a 1→6 glycosidic bond connection, the signal of 4.88 / 66.98 inferred that t-Glc(p) and 1,4,6-Glc(p) had a 1→6 glycosidic bond connection, and the signal of 4.92 / 67.07 inferred that 1,6 -Glc(p) and 1,4,6-Glc(p) are connected by a 1→6 glycosidic bond; the overall HMBC signal indicates that the glucan is connected by 1,4-Glc(p) and 1,4,6-Glc(p) to form a main chain, with the 6 position of 1,4,6-Glc(p) forming a branch chain, 1,6-Glc(p) is a component of the branch chain, and the terminal sugar t-Glc(p) may be capped with the 6 position of 1,6-Glc(p) and 1,4,6-Glc(p).

[0124] like Fig.10 As shown in (g), the existence of glycosidic bonds was verified by NOESY spectra. The signal of 5.30 / 3.55 confirmed that 1,4-Glc(p) had glycosidic bonds with the same residual sugar 1→4; the signal of 5.29 / 3.87 confirmed that the main residual sugar 1,4-Glc(p) and 1,4,6-Glc(p) had glycosidic bonds with 1→4; the signal of 4.87 / 3.80 of the anomeric part speculated that t-Glc(p) and 1,6-Glc(p) had glycosidic bonds with 1→6; the signal of 4.92 / 4.12 of the anomeric part speculated that 1,6-Glc(p) and 1,4,6-Glc(p) had glycosidic bonds with 1→6, and the signal of 4.86 / 4.22 of the anomeric part speculated that t-Glc(p) and 1,4,6-Glc(p) had glycosidic bonds with 1→6. The above main signals confirmed the correctness of the residual sugar connection.

[0125] Therefore, combined with the above analysis, the main structure of the modified extracellular polysaccharide is as follows:

[0126]

[0127] 8. Results of in vitro antioxidant capacity assay

[0128] Depend on Fig.11 (a)-(c) show that S-EPS has good antioxidant capacity, excellent ability to scavenge DPPH and ABTS free radicals, and strong ability to reduce ferrous ions. It shows a dose-dependent relationship with the increase of S-EPS concentration, and the antioxidant capacity increases with the increase of concentration. Among them, the DPPH free radical scavenging ability is the strongest. At a concentration of 8 mg / mL, it is equivalent to the same dose of vitamin C, and the free radical scavenging ability can reach 95.89%. Specific embodiment 4

[0130] Study on the in vivo antioxidant activity of S-EPS prepared in Example 2

[0131] 1. Experimental materials: SPF-grade healthy male ICR mice, weighing approximately 20.00±2g / mouse, provided by the Experimental Animal Center of Ningbo University.

[0132] 2. Experimental plan: Construction of D-galactose subacute aging model: Mice were randomly divided into 6 groups after one week of adaptation: blank control group (CG), aging model group (NC), positive control group (VC), S-EPS gradient dose feeding group, low dose group (LD), medium dose group (MD), and high dose group (HLD). From the day after grouping, 0.2 mL of normal saline was injected into the CG group at a relatively fixed time every day, and 0.2 mL of 5% D-galactose was injected into the subcutaneous part of the neck of the other groups at a dose of 800 mg / kg. 4 hours after each subcutaneous injection of D-galactose, 0.2 mL of vitamin C was gavaged into the CG group and the NC group, 0.2 mL of vitamin C was gavaged into the VC group at 200 mg / kg, and 200 mg / kg, 400 mg / kg, and 800 mg / kg were gavaged into the LD, MD, and HLD groups, respectively. The drug was administered continuously for 40 days, and the food intake, mental state, and activity of the mice were observed daily.

[0133] 3. Experimental results

[0134] (1) Results of cytokine content determination in mouse serum

[0135] Highly reactive free radicals covalently bind to macromolecules, inducing changes in gene expression in hepatocytes and leading to the release of large amounts of cytokines (IL-6, TNF-α, and IL-1β). Therefore, the level of pro-inflammatory factors can also be used to measure inflammation and oxidative stress. Fig.12As shown in (a)-(c), the level of pro-inflammatory factors in the NC group was lower than that in the CG group, and that in the VC and all experimental groups were significantly higher than that in the CG group. The effect in the VC group was the most significant. The levels of cytokines in the LD, MD, and HLD groups increased with the increase of the dose, showing a dose-dependent manner.

[0136] Nrf2 is the main regulator of antioxidant response. It is a transcription factor sensitive to ROS and can drive the upregulation of various antioxidant genes. Nrf2 can also downregulate IL-1β secretion. Nrf / HO-1 is also a common antioxidant pathway. HO-1 is also an important antioxidant enzyme that can help the kidneys resist oxidative stress damage. Under stress conditions, redox-sensitive transcription factors bind to stress response elements upstream of the promoter to induce the expression of the HO-1 gene (HMOX1). The Nrf2 and HO-1 levels in the NC group were significantly lower than those in the CG group, and the VC group had the highest content. The S-EPS gavage groups of different doses increased HO-1 to varying degrees ( Fig.13 (a)) and Nrf2( Fig.13 (b)) content, and the effect showed a dose-dependent relationship.

[0137] (2) Results of the total antioxidant capacity test of mouse liver

[0138] Total antioxidant capacity (T-AOC) represents the overall ability of a system to resist oxidative stress. It mainly includes the contribution of various antioxidant substances, such as vitamin C, vitamin E, glutathione, and related antioxidant enzymes. It represents the overall effect of substances on the body's antioxidant efficacy. The effect of S-EPS on the galactose subacute aging model is shown in Figure 2. Fig.14 As shown. The CG group represents the normal level without treatment, and the total antioxidant capacity is the strongest. The NC group is the weakest compared with it, with significant differences (P<0.01). The experimental groups gavaged with different doses of S-EPS and the VC group all enhanced their antioxidant capacity to varying degrees. Among them, the antioxidant capacity of the VC group was the most significantly enhanced, close to the CG group, followed by the HLD group. The antioxidant capacity of the MD and LD groups also showed a certain degree of enhancement. The antioxidant molecular mechanism of S-EPS is that these biomacromolecules exposed to an acidic environment will cause the biomacromolecules to hydrolyze and produce a large number of active hemiacetal hydroxyl groups. Electrons are transported to the reaction system, thereby promoting the conversion of free radicals into stable molecules, resulting in a decrease in free radical concentration.

[0139] (3) Results of determination of MDA content in mouse liver

[0140] MDA is a common byproduct of lipid peroxidation and is generally considered to be one of the markers of intracellular oxidative stress. The change in its content can reflect the degree of cell membrane damage and the tendency of body peroxidation. After the galactose subacute aging model mice and mice in each experimental group were killed, the mouse livers were removed to prepare tissue homogenates and the MDA content was detected. Fig.15 As shown. The MDA content of mice in the CG group was the lowest, and that in the NC group was the highest, with significant differences between the CG group and the NC group (P<0.01), indicating that the D-galactose subacute aging model group was successfully established. Comparison of the VC group, the NC group, and the CG group showed that the MDA content could be reduced to a near normal level by the intake of antioxidants, while the MDA content of the LD, MD, and HLD groups decreased to varying degrees, and may be dose-dependent. The HLD group had the lowest MDA content and the strongest antioxidant capacity.

[0141] (4) HE staining results of mouse liver

[0142] Fig.16 (a)-(f) are 100× HE-stained liver pathological sections of mice under different treatment conditions. In normal mice, liver cells should be tightly arranged, with clear boundaries and no fatty lesions. In the NC group, liver cells were loosely arranged, with unclear boundaries, and a large amount of fat accumulation led to cavitation and severe inflammatory infiltration. The EPS gavage group and the VC group were relieved to a certain extent, and there were no obvious pathological changes in the liver sections of each group, indicating that EPS within an appropriate dose has no toxic side effects on the mouse liver.

[0143] In summary, the in vivo antioxidant activity of S-EPS is mainly reflected in enhancing the activity of antioxidant enzymes SOD, GSH-Px and CAT, reducing the MDA content in mouse liver and the proinflammatory factors IL-6, TNF-α and IL-1β, and increasing the content of antioxidant master regulator Nrf and antioxidant enzyme HO-1, indicating that S-EPS achieves the effect of enhancing antioxidant activity through the joint action of enzyme-mediated and non-enzyme-mediated antioxidant systems. On the one hand, it enhances the activity of antioxidant enzymes and total oxidative capacity, and on the other hand, it promotes the expression of important signal factors Nrf and HO-1 in related pathways such as Nrf / HO-1. The MDA content in mouse liver and the proinflammatory factors in serum are significantly reduced, and the loose arrangement of liver cells, unclear boundaries, cavitation and severe inflammatory infiltration caused by a large amount of fat accumulation are improved, which proves that S-EPS has excellent antioxidant capacity in vivo and can relieve the oxidative stress caused by excessive intake of D-galactose.

[0144] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A Pediococcus pentosaceus, characterized in that The bacteria was classified and named Pediococcus pentosaceus YY-13 strain, and was deposited in the General Microbiology Center of China Culture Collection Administration on January 8, 2024, with the deposit number CGMCC No.29541.

2. A modified exopolysaccharide derived from Pediococcus pentosaceus, characterized in that: The polysaccharide structure is as follows:

3. A method for preparing modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 2, characterized in that The following steps are involved: (1) Using ethanol precipitation method to extract crude exopolysaccharide from Pediococcus pentosaceus YY-13 with a deposit number of CGMCC No. 29541; (2) separating and purifying the crude exopolysaccharide obtained in step (1) using a DEAE-52 cellulose ion exchange column and a Sepharose CL-6B gel column to obtain high-purity exopolysaccharide; (3) The high-purity extracellular polysaccharide obtained in step (2) is prepared by ultrasound-assisted sulfation modification technology to obtain modified extracellular polysaccharide derived from Pediococcus pentosaceus with good antioxidant activity.

4. The method for preparing a modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 3, characterized in that The specific steps of the extraction process of step (1) are as follows: Pediococcus pentosaceus YY-13 with a preservation number of CGMCC No. 29541 is activated and inoculated into a modified MRS liquid culture medium at an inoculum amount of 2vt%, and after culturing at 37°C for 26.5h, the bacterial liquid is centrifuged to collect the supernatant and boiled for 10min, TCA is added to the supernatant to a final concentration of 4wt% after cooling, and the supernatant is centrifuged to obtain the supernatant, and anhydrous ethanol 3 times the volume of the supernatant is added to the supernatant and stirred overnight, and then the precipitate is centrifuged to obtain the supernatant, dissolved in 60°C water and centrifuged for 10min, and the supernatant is obtained; the supernatant is concentrated by rotary evaporation and then loaded into an 8000Da dialysis bag, and dialyzed in an ultrapure water environment at 4°C for 2 days, with the water changed every 4h; the solution after the dialysis is concentrated by rotary evaporation again, loaded into a centrifuge tube and placed in a -80°C refrigerator for pre-freezing for 4h, and finally freeze-dried to obtain a crude exopolysaccharide.

5. The method for preparing modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 4, characterized in that The modified MRS liquid culture medium described in step 1 was prepared as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 35 g of maltose, 80 ml of Tween, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder were dissolved in 1 L of water.

6. The method for preparing a modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 3, characterized in that The specific steps of the purification process of step (2) are as follows: the crude extracellular polysaccharide obtained in step (1) is dissolved in water to prepare a solution with a concentration of 20 mg / mL, filtered through a 0.22 μm aqueous phase filter membrane and then loaded with a sample of 5 mL, using a DEAE-52 cellulose ion exchange column pre-equilibrated with deionized water at pH 6.0, eluting with deionized water as an eluent, collecting the eluate, combining the eluates of each corresponding sugar peak respectively, concentrating by rotary evaporation, and desalting by dialysis to obtain neutral sugar; the neutral sugar is eluted using a Sepharose CL-6B gel column with distilled water as an eluent, collecting the eluate, concentrating the eluate by rotary evaporation, and freeze-drying to obtain high-purity extracellular polysaccharide with a purity of more than 90%.

7. The method for preparing modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 3, characterized in that The specific steps of the modification process of step (3) are as follows: dissolving high-purity extracellular polysaccharide in anhydrous dimethylformamide DMF to obtain a solution with a concentration of 5 mg / mL, oscillating under ultrasonic conditions of a frequency of 20 kHz and a power of 500 W for 55 minutes, and continuing to oscillate for 30 minutes after the ultrasonic oscillation ends, and then adding 100 mg of sulfur trioxide-pyridine complex and stirring at 80°C for 3 hours; after the reaction is completed, the mixture is cooled to room temperature in an ice bath, neutralized with 4M NaOH solution, and then dialyzed with ultrapure water at 4°C for 2 days to obtain modified extracellular polysaccharide derived from Pediococcus pentosaceus, wherein the mass ratio of high-purity extracellular polysaccharide to sulfur trioxide-pyridine complex is 2:

1.

8. Use of the modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 2 in the preparation of an in vitro antioxidant, characterized in that: The in vitro antioxidants include DPPH free radical scavenger, ABTS free radical scavenger and ferrous ion reducing agent.

9. Use of the modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 2 in the preparation of an in vivo antioxidant.

10. Use of the modified exopolysaccharide derived from Pediococcus pentosaceus according to claim 2 in the preparation of anti-aging drugs.

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