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

Abstract

The application discloses a Pediococcus pentosaceus, modified exopolysaccharide, a preparation method of the modified exopolysaccharide and application of the modified exopolysaccharide in preparation of an antioxidant, and has the characteristics that the bacteria are classified and named as Pediococcus pentosaceus YY-13 strain, and the preservation number is CGMCC No.29541; and the steps for preparing the Pediococcus pentosaceus source modified exopolysaccharide through the bacteria are as follows: 1) using an ethanol precipitation method to extract crude exopolysaccharide of the Pediococcus pentosaceus YY-13; 2) using a DEAE-52 cellulose ion exchange column and a Sepharose CL-6B gel column to separate and purify the crude exopolysaccharide, so as to obtain high-purity exopolysaccharide; and 3) using an ultrasonic wave assisted sulfation modification technology to prepare polysaccharide from the high-purity exopolysaccharide, so as to obtain Pediococcus pentosaceus source modified exopolysaccharide with good antioxidant activity, and the Pediococcus pentosaceus source modified exopolysaccharide has the advantages of high antioxidant capacity, high exopolysaccharide yield and anti-aging function.

Description

TECHNICAL FIELD

[0001] The present application relates to a Pediococcus pentosaceus, in particular to a Pediococcus pentosaceus, modified exopolysaccharide and preparation method and application in antioxidant preparation. BACKGROUND

[0002] Lactic acid bacteria (LAB) is a kind of probiotics widely used in food processing, medicine and other fields. Exopolysaccharides (EPS) is a kind of saccharide compounds secreted to the outside of cell wall during the growth and metabolism of strains, often permeating the culture medium, which is often used as emulsifying agent, gelling agent and other applications in food processing industry. Extensive studies have shown that LAB-EPS has biological activities such as antioxidant, antitumor, immunomodulatory, among which antioxidant is the basis of other functions, and has important research value and significance.

[0003] Oxidative stress leads to imbalance between oxidation and antioxidant states, which can accelerate aging, cause neurodegenerative diseases, cancer, atherosclerosis and other diseases, and endanger human health. When cells encounter oxidative stress, EPS plays a good protective role in a physical wrapping way, and enhances antioxidant activity 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 significant influencing factors. In addition to screening natural EPS and changing fermentation conditions, modification methods can effectively change the structure of EPS, which are usually divided into physical, chemical and biological molecular methods. Ultrasonic treatment is a green and economical physical modification, which mainly causes changes in structure and molecular weight. Compared with natural EPS, modified EPS has better antioxidant capacity and stronger ferrous reducing capacity. However, physical modification is relatively mild, and chemical modification is more violent. Sulfation modification is the most common but effective chemical method, which introduces sulfate groups to increase the biological activity of EPS. However, the contact of the reaction system is limited during the reaction, so there are certain limitations in single modification method. SUMMARY

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

[0005] The technical scheme adopted by the present application to solve the above technical problems is: a Pediococcus pentosaceus, which is classified and named as Pediococcus pentosaceus YY-13 strain, was preserved in the China General Microbiological Culture Collection Center on January 8, 2024, and the preservation number is CGMCC No.29541.

[0006] The present application also provides a modified exopolysaccharide from Pediococcus pentosaceus, and the structure of the polysaccharide is as follows:

[0007]

[0008] The present application also provides a preparation method of the modified exopolysaccharide from Pediococcus pentosaceus, which comprises the following steps:

[0009] (1) The Pediococcus pentosaceus (Pediococcus pentosaceus) YY-13 with the preservation number of CGMCC No.29541 is used to extract the crude exopolysaccharide by ethanol precipitation method;

[0010] (2) The crude exopolysaccharide obtained in step (1) is separated and purified by DEAE-52 cellulose ion exchange column and Sepharose CL-6B gel column to obtain high-purity exopolysaccharide;

[0011] (3) The high-purity exopolysaccharide obtained in step (2) is modified by ultrasonic-assisted sulfation technology to prepare polysaccharide, and the modified exopolysaccharide from Pediococcus pentosaceus with good antioxidant activity is obtained.

[0012] Further, the specific steps of the extraction process in step (1) are as follows: after the strain Pediococcus pentosaceus YY-13 is activated, it is inoculated into modified MRS liquid medium at an inoculation amount of 2vt%, and after being cultured at 37℃ for 26.5h, the supernatant is collected by centrifugation and boiled for 10min, then cooled and added with TCA to a final concentration of 4wt%, and placed at 4℃ for 8h, then centrifuged to take the supernatant, and then added with 3 times the volume of anhydrous ethanol in the supernatant and stirred overnight, then centrifuged to take the precipitate part, dissolved in 60℃ water and centrifuged for 10min, and then the supernatant was taken; the supernatant was concentrated by rotary evaporation, then loaded into an 8000Da dialysis bag, dialyzed in an ultrapure water environment at 4℃ for 2 days, and the water was changed every 4h; the dialyzed solution was concentrated by rotary evaporation again, loaded into a centrifuge tube and pre-frozen in a-80℃ refrigerator for 4h, and finally freeze-dried to obtain the crude exopolysaccharide.

[0013] Further, the improved MRS liquid medium preparation method is as follows: 10 g of protein 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, 10 mL of Tween 80, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder are dissolved in 1 L of water.

[0014] Further, the specific steps of the step (2) purification process are as follows: the crude exopolysaccharide obtained in the step (1) is dissolved in water to prepare a solution with a concentration of 20 mg / mL, filtered through a 0.22 μm water phase filter membrane, and then loaded with a loading amount of 5 mL; a DEAE-52 cellulose ion exchange column pre-equilibrated with deionized water at pH 6.0 is used for elution with deionized water as an eluent; the eluate is collected, and each corresponding sugar peak eluate is combined and concentrated by rotary evaporation, and then desalted by dialysis to obtain neutral sugar; the neutral sugar is eluted by a Sepharose CL-6B gel column with distilled water as an eluent, the eluate is concentrated by rotary evaporation, and then freeze-dried to obtain high-purity exopolysaccharide with a purity of more than 90%.

[0015] Further, the specific steps of the step (3) modification process are as follows: the high-purity exopolysaccharide is dissolved in anhydrous dimethylformamide DMF to obtain a solution with a concentration of 5 mg / mL, and then subjected to ultrasonic oscillation under the condition of a frequency of 20 kHz and a power of 500 W for 55 minutes, and then continuously oscillated for 30 minutes; then 100 mg of sulfur trioxide-pyridine complex is added and stirred at 80℃ for 3 hours; after the reaction is completed, the mixture is cooled to room temperature in an ice bath, neutralized with a 4M NaOH solution, and then dialyzed with ultrapure water at 4℃ for 2 days to obtain the modified exopolysaccharide from Pediococcus pentosaceus, wherein the mass ratio of the high-purity exopolysaccharide to the sulfur trioxide-pyridine complex is 2:1.

[0016] The application also provides the use of the above-mentioned modified exopolysaccharide from Pediococcus pentosaceus in the preparation of an in vitro antioxidant agent.

[0017] The application also provides the use of the above-mentioned modified exopolysaccharide from Pediococcus pentosaceus in the preparation of an in vivo antioxidant agent.

[0018] The application also provides the use of the above-mentioned modified exopolysaccharide from Pediococcus pentosaceus in the preparation of an anti-aging drug.

[0019] Compared with the prior art, the advantages of the present application are that the screened Pediococcus pentosaceus has high yield of exopolysaccharide and excellent antioxidant performance; the modified exopolysaccharide obtained by using the Pediococcus pentosaceus is subjected to ultrasonic-assisted sulfation technology to introduce sulfate groups and change the EPS structure, 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 that 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 beneficial to the enhancement of biological activity, thereby optimizing the in-vitro antioxidant performance of the EPS, enhancing the DPPH and ABTS free radical scavenging capacity, and having strong ferrous ion reducing capacity. In in-vivo antioxidant, the S-EPS can effectively improve the activities of antioxidant enzymes SOD, GSH-Px and CAT in the liver of mice, reduce the content of MDA in the liver of mice and the contents of pro-inflammatory factors IL-6, IL-1β and TNF-α in serum, increase the contents of antioxidant master regulator Nrf and antioxidant enzyme HO-1, and improve the loose arrangement of liver cells, unclear boundaries, a large amount of fat accumulation leading to vacuolation and severe inflammatory infiltration, which proves that the S-EPS has excellent antioxidant capacity in vivo and can relieve the oxidative stress pressure caused by the intake of excessive D-galactose.

[0020] The above-mentioned Pediococcus pentosaceus is named Pediococcus pentosaceus YY-13 strain, the preservation number is CGMCC No.29541, and it is preserved in the China General Microbiological Culture Collection Center on January 8, 2024, and the preservation address is No.3, Beichen West Road, Haidian District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Effect of fermentation time on EPS yield;

[0022] Figure 2 Effect of inoculum amount on EPS yield;

[0023] Figure 3 Effect of carbon source on EPS yield;

[0024] Figure 4 Effect of maltose concentration on EPS yield;

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

[0026] Figure 6Figures for the apparent morphology of EPS and S-EPS under scanning electron microscope, wherein (a) EPS (200x); (b) S-EPS (200x); (c) EPS (10000x); (d) S-EPS (10000x), EPS: exopolysaccharide; S-EPS: ultrasonic-assisted sulfated exopolysaccharide;

[0027] Figure 7 Figures for the infrared spectra of EPS and S-EPS, wherein (a) EPS: exopolysaccharide; (b) S-EPS: ultrasonic-assisted sulfated exopolysaccharide;

[0028] Figure 8 Figures for the HPGPC chromatograms of EPS and S-EPS, wherein (a) EPS: exopolysaccharide; (b) S-EPS: ultrasonic-assisted sulfated exopolysaccharide;

[0029] Figure 9 Figures for the PMP pre-column derivatization high performance liquid chromatograms of monosaccharide standard and S-EPS, wherein (a) monosaccharide standard; (b) modified exopolysaccharide S-EPS;

[0030] Figure 10 Figures for the nuclear magnetic resonance structure analysis of modified exopolysaccharide, wherein (a) H spectrum; (b) C spectrum; (c) DEPT spectrum; (d) H-H COSY spectrum; (e) HSQC spectrum; (f) HMBC spectrum; (g) NOESY spectrum;

[0031] Figure 11 Figures for the in vitro antioxidant capacity of S-EPS of different dosages, wherein (a) ABTS free radical scavenging capacity, (b) DPPH free radical scavenging capacity, (c) ferrous ion reducing capacity;

[0032] Figure 12 Figures for the determination results of the contents of IL-6, TNF-α and IL-1β in the serum of the aging model mice under different treatment modes, wherein (a) is IL-6, (b) is TNF-α, and (c) is IL-1β;

[0033] Figure 13 Figures for the determination results of the contents of HO-1 and Nrf2 in the serum of the aging model mice under different treatment modes, wherein (a) is HO-1, and (b) is Nrf2;

[0034] Figure 14 Figure for the determination results of the total antioxidant capacity of the aging model mice under different treatment modes;

[0035] Figure 15 Figure for the determination results of the MDA content in the liver of the aging model mice under different treatment modes;

[0036] Figure 16The pathological changes of the liver of the aging model mice treated in different ways (HE staining, 100x), wherein (a) a CG blank group; (b) an aging model NC group; (c) a positive control VC group; (d) an EPS low-dose LD group; (e) an EPS medium-dose MD group; and (f) an EPS high-dose HLD group. DETAILED DESCRIPTION

[0037] The application will be further described in detail below with reference to the embodiments and the accompanying drawings. Embodiment I

[0039] Strain screening of the application

[0040] 1. Screening by taking EPS yield as the preliminary screening index

[0041] 25 strains of lactic acid bacteria were selected from the strain library of the Laboratory of Animal Products Processing, College of Food Science and Engineering, Ningbo University, activated for 3 times and subjected to experiments. Solid streak culture was performed, and the strains were cultured at 37°C for 48h. 11 strains capable of drawing out filaments were selected. EPS was extracted by using an ethanol precipitation method, and the specific steps were as follows:

[0042] After the strains were activated, the strains were inoculated into MRS liquid medium at a volume ratio of 2%, and cultured at 37°C for 26.5h. The bacterial liquid was centrifuged (4°C, 5000rpm, 20min), and the supernatant was collected and boiled for 10min. After cooling, trichloroacetic acid (TCA) was added to a final concentration of 4%, and the mixture was placed at 4°C for 8h. The supernatant was obtained by centrifugation (4°C, 10000rpm, 10min), and 3 times the volume of anhydrous ethanol was added to the supernatant and stirred overnight by using a magnetic stirrer. The precipitate was obtained by centrifugation (4°C, 10000rpm, 10min), dissolved in water at 60°C and centrifuged for 10min, and the supernatant was collected. The supernatant was concentrated by rotary evaporation, loaded into an 8000Da dialysis bag, dialyzed in an ultrapure water environment at 4°C for 2 days, and the water was changed every 4h. The dialyzed solution was concentrated by rotary evaporation again, loaded into a centrifuge tube and pre-frozen in a-80°C refrigerator for 4h, and finally freeze-dried to obtain the EPS crude product.

[0043] The EPS content in the EPS crude product was further determined by the phenol-sulfuric acid method. Glucose was used as a standard to prepare a standard curve of the phenol-sulfuric acid method, and the regression equation was y=0.0322x+0.0491, R 2 =0.9963. The yield and source of the exopolysaccharide EPS are shown in Table 1. The three strains of P.pentosaceus YY-13, L.plantarum FR-1 and L.plantarum B-1-42 were preliminarily screened.

[0044] Table 1. Source and yield of the experimental strains

[0045]

[0046] 2. Screening was conducted using antioxidant capacity as a secondary screening indicator.

[0047] Based on EPS yield, three lactic acid bacteria strains with good yields were initially screened: P. pentosaceus YY-13, L. plantarum FR-1, and L. plantarum B-1-42. Further screening was conducted based on their antioxidant properties. P. pentosaceus YY-13 showed a DPPH free radical scavenging capacity of 94.95%, L. plantarum FR-1 showed 88.32%, and L. plantarum B-1-42 showed 68.37%. Based on yield and DPPH free radical scavenging capacity, P. pentosaceus YY-13 was selected as the target strain for the experiment.

[0048] 3. Identification of target strains

[0049] Identification process: The target strain was streaked several times on MRS solid plates. Uniform colonies were selected, activated, and then placed in modified MRS liquid medium (the modified MRS liquid medium formula is: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 5g sodium acetate, 35g maltose, 80ml Tween, 0.5g magnesium sulfate, 0.25g manganese sulfate, and 15g agar powder, dissolved in 1L distilled water and sterilized at 121℃ for 15min) and incubated overnight at 37℃ for 24h. The supernatant was collected by centrifugation at 5000rpm for 10min. The 16S rDNA gene of the target strain was extracted using a bacterial genomic DNA kit for 16S rRNA sequence analysis. Amplification was performed using universal primers 1492R and 27F. The PCR fragment (1,500bp) purified using a rapid PCR purification kit was detected by Youkang Biotechnology Co., Ltd. The 16S rDNA genes of selected members in GenBank were compared using MEGA software (https: / / www.megasoftware.net / ). Because we found that YY-13 showed high homology with *Pediococcus pentosaceus*, this strain was again classified as *Pediococcus pentosaceus* and deposited on January 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29541.

[0050] 4. Optimization of fermentation conditions for the target strain

[0051] (1) Single-factor experiment:

[0052] The initial conditions controlled in the experiment were 1% (v / v) inoculation amount, 37°C fermentation temperature, and glucose as the sole carbon source. The effects of fermentation time (16 h, 20 h, 24 h, 28 h, 30 h), inoculation amount (volume ratio 1%, 2%, 3%, 4%, 5%), and carbon source (glucose, mannose, galactose, maltose, sucrose) on the EPS yield of the strain were studied with the EPS yield as the index.

[0053] The effect of fermentation time on the EPS yield is shown in Figure 1 The EPS yield increased first and then decreased with the increase of fermentation time, and the yield was the highest (92.41 mg / L) when the fermentation time was 20 h.

[0054] The effect of inoculation amount on the EPS yield is shown in Figure 2 The fermentation time was controlled to be 20 h, and glucose was used as the sole carbon source. The yield was the highest when the inoculation amount was 2vt%. The yield increased first and then decreased with the increase of inoculation amount.

[0055] Maltose, galactose, sucrose, mannose, and glucose were used as the sole carbon source, respectively, to explore the effect of different carbon sources on the yield of P. pentosaceus YY-13. As shown in Figure 3 The different carbon sources had a significant effect on the EPS yield, especially when maltose was used as the carbon source, the EPS yield could be as high as 284.66 mg / L, which was significantly higher than that of other groups. Therefore, maltose was selected as the carbon source of the culture medium.

[0056] The effect of maltose addition concentration on the polysaccharide yield is shown in Figure 4 The inoculation amount was controlled to be 2vt%, and the fermentation time was controlled to be 20 h. Different concentrations of maltose were added as the carbon source. The polysaccharide yield increased first and then decreased with the increase of maltose concentration. The yield increased most obviously when the concentration increased from 25 g / L to 30 g / L, and the yield reached the highest value (308.66 mg / L) at 30 g / L, and then decreased. The increase of maltose concentration was beneficial to the supply of nutrients to the bacterial cells, promoted the growth and metabolic activity of the bacterial cells, and increased the EPS yield. However, when the maltose concentration was too high, the bacteria might not be able to maintain proper carbon-nitrogen balance, thereby affecting the synthesis of EPS.

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

[0058] The three levels of maltose concentration, fermentation time and inoculum size were determined by single factor experiment, and the three-factor and three-level response surface optimization experiment was carried out on maltose concentration, fermentation time and inoculum size. The quadratic regression equation obtained is 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 obtained by analysis: inoculum size 2vt%, maltose concentration 35g / L, and fermentation time 26.5h. After the optimization experiment, the EPS yield increased from 160.98mg / L to 336.804mg / L, which was more than doubled. Specific embodiment two

[0060] The modified exopolysaccharide was prepared by Pediococcus pentosaceus YY-13 screened in the specific embodiment one, and the specific steps were as follows:

[0061] Step 1, preparation of Pediococcus pentosaceus YY-13 exopolysaccharide

[0062] The EPS of Pediococcus pentosaceus YY-13 was prepared by ethanol precipitation method, and the method was shown in specific embodiment one, and the MRS liquid medium was replaced by modified MRS liquid medium.

[0063] Step 2, obtaining purified exopolysaccharide

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

[0065] The exopolysaccharide crude product obtained in step 1 was dissolved in water to prepare a solution with a concentration of 20mg / mL. After filtration through a 0.22μm water phase filter, the sample was loaded with a loading amount of 5mL. DEAE-52 cellulose ion exchange column was used for elution with deionized water as eluent at a flow rate of 1mL / min. The eluate was collected every 5mL, and the sugar content was detected by phenol sulfuric acid method, and the elution curve was drawn. The corresponding sugar peak eluate was combined and concentrated by rotary evaporation, and then desalted by dialysis to obtain neutral sugar. The neutral sugar was eluted by Sepharose CL-6B gel column with distilled water as eluent. The eluate was concentrated by rotary evaporation, and then freeze-dried to obtain high-purity exopolysaccharide EPS with a purity of more than 90%.

[0066] After anion exchange column chromatography, *P. pentasiaceus* YY-13 was separated into two types of polysaccharides—neutral and acidic—based on their ionic charges. Further separation of polysaccharides of different molecular weights was achieved using Sepharose CL-6B gel permeation column chromatography, which utilizes gel particles as the stationary phase, allowing larger molecules to elute first and smaller molecules to elute later. The elution characteristics of DEAE-52 cellulose ion exchange column chromatography are shown below. Figure 5 a. Elution status of gel permeation column chromatography as follows Figure 5 As shown in b. The EPS content was calculated based on the phenol-sulfuric acid standard curve, yielding an EPS purity of 90.265%; Coomassie Brilliant Blue Standard Curve: y = 3.6682x + 0.4168, R 2 =0.9986, and the protein content is calculated to be 0.94% based on the standard curve.

[0067] Step 3: Preparation of modified extracellular polysaccharides

[0068] Preparation of ultrasound-assisted sulfated polysaccharide: 50 mg of high-purity extracellular polysaccharide was dissolved in 10 mL of anhydrous dimethylformamide (DMF). The mixture was vibrated for 55 minutes under ultrasonic conditions at a frequency of 20 kHz and a power of 500 W, followed by continuous vibration at room temperature for 30 minutes. Then, 100 mg of sulfur trioxide-pyridine complex was added and stirred at 80 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature in an ice bath. It was neutralized with 4 M NaOH solution and then dialyzed against ultrapure water at 4 °C for 2 days to obtain the ultrasound-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), and the mixture was shaken continuously at room temperature for 30 minutes. Then, 100 mg of sulfur trioxide-pyridine complex was added, and the mixture was stirred at 80 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature in an ice bath. It was neutralized with 4 M NaOH solution and then dialyzed against ultrapure water at 4 °C for 2 days to obtain sulfated modified polysaccharides. Specific Implementation Example 3

[0071] Performance Comparison Analysis of EPS Before and After Modification

[0072] 1. Determination of sulfate content in 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 can be seen from Table 1, the neutral sugar does not contain sulfate, the introduction of sulfate groups after sulfation treatment significantly increases the sulfate content, and the sulfate content and antioxidant capacity are further improved after ultrasonic treatment. The S% of ultrasonic-sulfation treatment is 1.95 times that of only sulfation treatment, the DPPH free radical scavenging capacity is increased by more than 20%, and the total oxidation capacity is increased by 31%.

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

[0079] An appropriate amount of polysaccharide sample was taken on the conductive carbon tape, gold spraying treatment was carried out, and then it was placed in the scanning electron microscope for observation and photography. The working voltage was set to 10.0 kV, the solid morphology of the sample was observed under different magnifications, and the corresponding clarity adjustment was carried out until the ideal observation field was obtained. The appropriate field was selected for photography and recording.

[0080] The surface microstructure of EPS was observed by scanning electron microscope, as shown in Figure 6 (a), the unmodified exopolysaccharide (EPS) presented irregular blocks of three-dimensional structure and the sheet corner was sharp, and there were also some uniform rod-shaped blocks; as shown in Figure 6 (b), the ultrasonic-assisted sulfation modified polysaccharide (S-EPS) presented flaky, and there were also some broken and broken, which may be caused by ultrasonic oscillation, severe reaction conditions and the introduction of chemical groups. By further enlarging the structure, it can be found that, as shown in Figure 6 (c), there were characteristic wrinkles and banding on the surface of EPS; as shown in Figure 6 (d), the surface of the polysaccharide treated by ultrasonic-assisted sulfation became rough, and there were radial texture and many small pores, indicating that ultrasonic-assisted sulfation treatment significantly changed the polysaccharide structure, and its energy was enough to destroy the glycosidic bond and other covalent bonds of EPS, promoting the separation of polysaccharide molecules.

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

[0082] The type and type of sugar ring in the polysaccharide structure were identified by infrared spectrum scanning, which provided information of glycosidic bond configuration and identified characteristic groups on the sugar chain, and provided part of the basis for the analysis of polysaccharide structure. 1-2 mg of each dried polysaccharide sample EPS in step 2 and S-EPS in step 3 of embodiment two was weighed in a mortar, 200 mg of KBr powder was added and ground uniformly, and then pressed into a tablet. Fourier transform infrared spectrometer was used for infrared scanning of the sample, and the wavelength range was 4000-400 cm -1 . The infrared spectrum was recorded.

[0083] The infrared spectrum of EPS is as follows Figure 7 As shown in (a), the sample exhibits a characteristic absorption peak for polysaccharides: 3445.46 cm⁻¹. -1 A strong and broad absorption peak exists at 2923.89 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity peaks are absorption peaks of the CH stretching vibrations of methyl (-CH3) and methine (-CH2); 1648.50 cm⁻¹ -1 The absorption peak at 1450-1200 cm⁻¹ is due to the C=O stretching vibration of the carbonyl group or the formation of water of crystallization. -1 The absorption peak is at 1461.53 cm⁻¹. -1 1424.46cm -1 1372.95cm -1 The peak at 1159-1110 cm⁻¹ represents the angular vibration absorption of CH, which, along with the stretching vibration of CH, constitutes the characteristic absorption of the sugar ring; 1159-1110 cm⁻¹ represents the stretching vibration of pyranose COC; 1150-1010 cm⁻¹ -1 The three strong absorption peaks between 1081.85 cm⁻¹ -1 1018.85cm -1 and 993.72cm -1 The presence of pyranoside was further confirmed at these three sites, and the absorption at these three sites was due to the bending vibration of the CO bond in the COH or COC structure; 927.50 cm⁻¹ -1 The peak at 846.10 cm⁻¹ shows the asymmetric ring stretching vibration of the pyran ring. -1 The peak represents the CH-angle vibration of the diastereomer of the α-terminal group of pyranose; 759.26 cm⁻¹ -1 The peaks of symmetrical ring stretching vibrations of the pyran ring are shown.

[0084] The infrared spectrum of S-EPS is as follows: Figure 7 As shown in (b), the sample exhibits a characteristic absorption peak for polysaccharides: 3431.74 cm⁻¹. -1 A strong and broad absorption peak exists at 2919.63 cm⁻¹, which is the strong absorption peak of the OH stretching vibration of hydrogen bonds between or within polysaccharide molecules; -1 The nearby moderate-intensity peaks are absorption peaks of the CH stretching vibrations of methyl (-CH3) and methine (-CH2) groups; 1639.99 cm⁻¹ -1 The absorption peak at 1254 cm⁻¹ is due to the C=O stretching vibration of the carbonyl group or the formation of water of crystallization. Compared with unsulfated polysaccharides, the peak at 1254 cm⁻¹ is... -1 The absorption peaks appearing on the left and right are characteristic peaks of S=O, at 855.82 cm⁻¹. -1 The absorption peaks appearing on the left and right are due to the stretching vibration of the COS bond. These two peaks are characteristic absorption peaks of sulfate esters, indicating that the polysaccharide sulfation was successful.

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

[0086] (1) Establishment of the molecular weight correction curve

[0087] The dextran standard of different molecular weight (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analysis standard) was precisely weighed, 0.05M NaCl solution was added to prepare a standard solution of 5mg / mL dextran, and the solution was filtered with a 0.22μm microporous filter for use. The HPGPC method was used, and a high-performance gel permeation chromatography tandem column was used for detection. The logarithmic value of the relative molecular mass of the standard was used as the vertical coordinate, and the retention time of the corresponding chromatographic peak was used as the horizontal coordinate for linear regression to obtain the correction curve.

[0088] (2) Preparation of the test sample solution

[0089] 5mg of the sample was precisely weighed, 1mL of 0.05M NaCl solution was added to the sample to prepare a test sample solution of 5mg / mL, and the solution was centrifuged at 8000rpm for 10min. The supernatant was filtered with a 0.22μm microporous filter, and then the sample was transferred to a 2mL sample bottle for use.

[0090] (3) Chromatographic method

[0091] The HPGPC method was used, and a high-performance gel permeation chromatography tandem column was used for detection. The instrument was a Waters high-performance liquid chromatograph, the detector was a Waters differential detector, the chromatographic column was a polymer matrix water-soluble SEC (GFC) chromatographic column (8x300mm) with 3 columns in series, the mobile phase was 0.05M NaCl solution, the flow rate was 0.65mL / min, the column temperature was 40℃, and the injection volume was 30μL.

[0092] (4) Analysis of the results

[0093] The logarithmic value of the relative molecular mass of the standard was used as the vertical coordinate, and the retention time (T) of the corresponding chromatographic peak was used as the horizontal coordinate for linear regression to obtain the correction curve equation: Log Mw=-0.1761T+11.0299, R 2 =0.9927. According to the HPGPC chromatogram of EPS (a) and S-EPS (b) in Figure 8

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

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

[0096] (1) Standard preparation

[0097] Take 5 mg of each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride monosaccharides, and 10 mg of fucose, dissolve and dilute to 10 mL with a volumetric flask to prepare a standard stock solution. Further dilute to the following gradient and load into a sample bottle after passing through a 0.22 micron microporous filter.

[0098] (2) Preparation of test sample solution

[0099] Take a clean chromatographic flask and accurately weigh 5 mg (±0.05 mg) of the polysaccharide sample. Add 1 mL of 2M TFA acid solution and heat at 121°C for 2 hours. Blow dry with nitrogen. Add 3 mL of methanol for washing, and then blow dry. Repeat the methanol washing 2-3 times. Dissolve in 5 mL of sterile water and transfer into a chromatographic flask for testing.

[0100] (3) PMP derivatization

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

[0102] (4) Chromatographic method

[0103] Thermo U3000 liquid chromatography system, chromatographic column is ZORBAX Eclipse XDB-C18, mobile phase is acetonitrile: phosphate buffer (12 g / L potassium dihydrogen phosphate, 2M NaOH to adjust PH to 6.8) gradient elution, the volume ratio of acetonitrile and 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) Result analysis

[0105] According to Table 2 and Figure 9 (a) monosaccharide standards and, Figure 9(b) The PMP column pre-derivative high performance liquid chromatography of S-EPS, by calculation, S-EPS is composed of glucose and mannose, and the main component is glucose. From the monosaccharide composition results, the polysaccharide is composed of only two monosaccharides, of which the proportion of glucose is 98.562%, and the proportion of mannose is about 1.438%, so the polysaccharide can be attributed to glucan.

[0106] Table 2 summary of calibration information

[0107]

[0108]

[0109] 6, methylation analysis results of S-EPS

[0110] The S-EPS sample prepared in step 3 of the specific embodiment is derivatized: 1-3 mg of the sample to be tested is weighed and dissolved in 500 μL of DMSO. 1 mg of NaOH is added and incubated for 30 min. 50 μL of iodomethane solution is added and reacted for 1 h; 1 mL of water and 2 mL of dichloromethane are added, vortexed, centrifuged, and the water phase is discarded. Water washing is repeated 3 times; the lower dichloromethane phase is taken and evaporated; 100 μL of 2M TFA is added, reacted at 121°C for 90 min, and evaporated at 30°C; 50 μL of 2M ammonia water and 50 μL of 1M NaBD4 are added, mixed, and reacted at room temperature for 2.5 h; 20 μL of acetic acid is added to terminate the reaction, nitrogen is blown dry, 250 μL of methanol is washed twice, and nitrogen is blown dry; 250 μL of acetic anhydride is added, vortexed, and reacted at 100°C for 2.5 h; 1 mL of water is added and left for 10 min; 500 μL of dichloromethane is added, vortexed, centrifuged, and the water phase is discarded. Water washing is repeated 3 times; the lower dichloromethane phase is taken and detected by GC-MS.

[0111] S-EPS is a glycosidic bond connected sugar chain, which is acid hydrolyzed to obtain a series of intermediate products, and finally obtains monosaccharide. Methylation analysis is a means for determining the connection mode of various monosaccharide residues in S-EPS. First, the free hydroxyl groups in each monosaccharide residue in the polysaccharide chain are methylated, then the glycosidic bond is hydrolyzed, and finally the product is analyzed by GC-MS. According to the relative retention time and mass spectrum of each chromatographic peak, qualitative analysis is carried out by comparing the ion fragment mass spectrum of the literature data and the database of the Complex Carbohydrate Research Center of the University of Georgia, and the type of sugar residue corresponding to each chromatographic peak is analyzed. Then, the content of each is calculated by using the corresponding sugar residue type chromatographic peak area. The results are shown in Table 3.

[0112] Table 3 summary of glycosidic bond configuration of S-EPS

[0113]

[0114] From the methylation test results of Table 3, four kinds of residual sugars composed of glucose were known, which basically coincided with the monosaccharide composition results, among which the largest proportion was →4)-Glcp-(1→ residual sugar, which constituted the main chain of glucan, followed by about 25% of glucose end sugar, confirming that the polysaccharide had a relatively high degree of branching. Combined with the main →4,6)-Glcp-(1→ residual sugar, it was speculated that the branching might be formed by the 6 position of →4,6)-Glcp-(1→ residual sugar. The relatively small amount of →6)-Glcp-(1→ residual sugar might be a component of the branching. Through GPC molecular weight detection, the weight average molecular weight of glucan was about 5990, and combined with the monosaccharide composition calculation, it was found that the average intramolecular structure of the polysaccharide contained 37 residual sugars.

[0115] 7. NMR technology analysis of S-EPS structure

[0116] 100 mg of polysaccharide sample S-EPS was dissolved in D2O (heavy water) and exchanged 3 times by freeze-drying, so that the active hydrogen was replaced by deuterium. After being dissolved in 0.5 mL of D2O, it was placed in a nuclear magnetic tube, and one-dimensional nuclear magnetic resonance spectrum of the polysaccharide sample was analyzed by using a nuclear magnetic resonance spectrometer. 1 H-NMR, 13 C-NMR and two-dimensional nuclear magnetic COSY, HSQC, HMBC, NOESY analysis.

[0117] As shown in Figure 10 (a), in the anomeric region of the nuclear magnetic hydrogen spectrum 1 H NMR, a plurality of anomeric hydrogen signals were found, and the main peak positions were identified as 5.30, 5.26, 5.12, 4.92, 4.87, and 4.54 ppm, confirming that the polysaccharide was composed of multiple residual sugars, which basically echoed the methylation results. In the 3.0-4.2 ppm interval of the hydrogen spectrum, there were typical anomeric hydrogen signals outside the residual sugar ring, and the signals in this region were more and mixed, which was consistent with the general characteristics of polysaccharide hydrogen spectrum.

[0118] As shown in Figure 10 (b), in the nuclear magnetic carbon spectrum 13 C NMR, no obvious uronic acid carbonyl carbon signal was found in 170-210 ppm, confirming that the polysaccharide was neutral. In the anomeric region (90-110 ppm) of the carbon spectrum, a plurality of main anomeric carbon signals were identified, which were 99.70, 98.54, 95.86, and 92.04 ppm, respectively. The multiple anomeric carbon signals echoed the multiple anomeric hydrogen signals; and in the range of 55-85 ppm, there were multiple typical polysaccharide residual sugar signals except anomeric carbon, among which the signals at 60.69 and 63.42 ppm were attributed to the methylene-CH2- secondary carbon outside the residual sugar ring, as shown in Figure 10(c) shown, DEPT135 spectrum confirmed the signal attribution, it is worth noting that the weak negative peak signal of 67.06ppm in DEPT spectrum, suggesting that the place may belong to the methylene-CH2- C6 forming glycosidic bond; multiple signals in 74-82ppm represent the carbon information of glucose C2-C5, and the weak out-of-peak in the range of 75-82ppm belongs to C4 forming glycosidic bond.

[0119] The structure of the polysaccharide was further analyzed by two-dimensional nuclear magnetic spectrum. As shown in Figure 10 (e) shown, in the HSQC two-dimensional nuclear magnetic spectrum reflecting the hydrogen-carbon correlation of the connected, a group of weak signals of H1 / C1 correlation in the anomeric region were identified, respectively 5.29 / 99.57, 5.12 / 91.82, 4.92 / 98.39, 4.85 / 97.93, 4.53 / 95.81ppm. Combined with the methylation results, the integral intensity of the anomeric hydrogen, and the out-of-peak signal of the literature, it was comprehensively judged that 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→, 4.92 / 98.39 was the H1 / C1 signal of α→6)-Glcp-(1→.

[0120] The carbon and hydrogen signal positions and structural information of various residual sugars in the glucan structure were further attributed by combining multiple two-dimensional nuclear magnetic spectra. It was determined that the anomeric signal 5.29ppm belonged to the anomeric H1 in the residual sugar α→4)-Glcp-(1→. By identifying the hydrogen-hydrogen correlated COSY two-dimensional spectrum Figure 10 (d)) the strongly correlated signal was found to be 3.47ppm, confirming that H1 and H2 were at 5.29 and 3.47ppm; combined with the HSQC spectrum, the signal of H2 / C2 was found to be 3.47 / 71.41; according to the COSY information, the signal of H2 and H3 was found to be 3.75, confirming that the hydrogen spectrum position of C3 was at 3.75ppm, and the signal of 3.75 / 70.94 was found in the HSQC, confirming that the position of C3 was 70.94ppm. The position information of H4 / C4, H5 / C5, and H6 / C6 was determined by the same method to be 3.55 / 76.57, 3.87 / 73.05, 3.67, 3.76 / 60.42. Other main position residual sugars were found by the same method, and the following table 4 was formed:

[0121] Table 4 Main residual sugar attribution table of S-EPS

[0122]

[0123] As Figure 10 (f) The HMBC spectrum is important for determining the hydrogen-carbon correlation of 2-3 chemical bonds apart, and is useful for determining the linkage of glycosidic bond in polysaccharide analysis. In the 1,4-Glc(p) residue with a relatively high content, the anomeric carbon correlation signals were found in the HMBC spectrum: 5.31 / 72.58, confirming that the anomeric hydrogen of the residue is correlated with H4 of the same residue, confirming the presence of the 1→4 linked glycosidic bond of the same residue, and constituting the main chain glycosidic bond in the glucan; the correlation signal of 5.30 / 76.80 confirms that the main residue 1,4-Glc(p) and 1,4,6-Glc(p) have a 1→4 linked glycosidic bond. In addition, the 1,4,6-Glc(p) and 1,4-Glc(p) are speculated to have a 1→4 glycosidic bond by the correlation signal of 5.26 / 72.58, and the t-Glc(p) and 1,6-Glc(p) are speculated to have a 1→6 glycosidic bond by the anomeric 4.87 / 67.65 signal, the t-Glc(p) and 1,4,6-Glc(p) are speculated to have a 1→6 glycosidic bond by the 4.88 / 66.98 signal, and the 1,6-Glc(p) and 1,4,6-Glc(p) are speculated to have a 1→6 glycosidic bond by the 4.92 / 67.07 signal; the signals of HMBC as a whole determine that the glucan is connected by 1,4-Glc(p) and 1,4,6-Glc(p) to form the main chain, and the 6 position of 1,4,6-Glc(p) forms the branch chain, 1,6-Glc(p) is a part of the branch chain, and the terminal sugar t-Glc(p) may form a cap with the 6 position of 1,6-Glc(p) and 1,4,6-Glc(p).

[0124] As Figure 10 (g) The presence of glycosidic bond was verified by the NOESY spectrum. The signal of 5.30 / 3.55 confirms that 1,4-Glc(p) has a 1→4 linked glycosidic bond of the same residue; the signal of 5.29 / 3.87 confirms that the main residue 1,4-Glc(p) and 1,4,6-Glc(p) have a 1→4 linked glycosidic bond; the anomeric 4.87 / 3.80 signal suggests that t-Glc(p) and 1,6-Glc(p) have a 1→6 glycosidic bond; the anomeric 4.92 / 4.12 signal suggests that 1,6-Glc(p) and 1,4,6-Glc(p) have a 1→6 glycosidic bond, and the anomeric 4.86 / 4.22 signal suggests that t-Glc(p) and 1,4,6-Glc(p) have a 1→6 glycosidic bond, and the above main signals confirm the correctness of the residue linkage.

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

[0126]

[0127] 8. In vitro antioxidant capacity determination results

[0128] From Figure 11 (a)-(c), it can be seen that S-EPS has good antioxidant capacity, has excellent DPPH and ABTS free radical scavenging capacity, and has strong ferrous ion reducing capacity. With the increase of S-EPS concentration, the antioxidant capacity shows a dose-dependent relationship, and the antioxidant capacity increases with the increase of concentration. Among them, the DPPH free radical scavenging capacity is the strongest, and at a concentration of 8 mg / mL, it is equivalent to the effect of the same dose of vitamin C, and the free radical scavenging capacity can reach 95.89%. Specific embodiment four

[0130] In vivo antioxidant activity study of S-EPS prepared in specific embodiment two

[0131] 1. Experimental materials: SPF healthy, male ICR mice, weighing about 20.00±2 g per mouse. Provided by the Experimental Animal Center of Ningbo University.

[0132] 2. Experimental scheme: Construct a D-galactose subacute aging model: take the mice after one week of adaptation, and randomly divide them into 6 groups: 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 next day of grouping, 0.2 mL of normal saline was injected into the CG group at a relatively fixed time every day, and the rest of the groups were injected with 5% D-galactose at a dose of 800 mg / kg, 0.2 mL subcutaneously in the neck. 4 hours after each subcutaneous injection of D-galactose, the CG and NC groups were given vitamin C 0.2 mL by gavage, the VC group was given vitamin C 0.2 mL by gavage at a dose of 200 mg / kg, and the LD, MD, and HLD groups were given by gavage at doses of 200 mg / kg, 400 mg / kg, and 800 mg / kg, respectively. Continuous administration for 40 days and daily observation of the mice's food intake, mental state, and activity ability, etc.

[0133] 3. Experimental results

[0134] (1) Determination results of cytokine content in mouse serum

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

[0136] Nrf2 is the main regulator of antioxidant response, which is a transcription factor sensitive to ROS, and can drive the up-regulation of various antioxidant genes. Nrf2 can also down-regulate IL-1β secretion. Nrf / HO-1 is also a common antioxidant pathway, and HO-1 is an important antioxidant enzyme that helps the kidney resist oxidative stress damage. Under stress conditions, the redox-sensitive transcription factor binds to the stress response element upstream of the promoter, inducing the expression of the HO-1 gene (HMOX1). The Nrf2 and HO-1 contents of the NC group were significantly lower than those of the CG group, and the VC group had the highest content. The S-EPS groups of different dose groups increased the HO-1 Figure 13 (a)) and Nrf2 Figure 13 (b)) contents to different degrees, and the effect showed a dose-dependent relationship.

[0137] (2) Determination results of total antioxidant capacity of mouse liver

[0138] Total antioxidant capacity (T-AOC) represents the overall ability of a system to resist oxidative stress, which mainly includes the contribution of various antioxidant substances such as vitamin C, vitamin E, glutathione, related antioxidant enzymes, etc. The overall effect of the substance on the antioxidant efficiency of the body, and the effect of S-EPS on the galactose subacute aging model is shown in Figure 14 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 the CG group, and the difference is significant (P<0.01). The S-EPS experimental groups of different doses and the VC group all enhance the antioxidant capacity to different degrees, among which the antioxidant capacity of the VC group is the most significant, close to the CG group, followed by the HLD group, and the antioxidant capacity of the MD and LD groups also shows a certain degree of enhancement. The antioxidant molecular mechanism of S-EPS is that the exposure of these biological macromolecules in the acidic environment will lead to the hydrolysis of biological macromolecules and produce a large number of active hemiacetal hydroxyl groups. Electrons are transported to the reaction system, thereby promoting the conversion of free radicals to stable molecules, resulting in a decrease in free radical concentration.

[0139] (3) Determination results of MDA content in mouse liver

[0140] MDA is a common by-product in lipid peroxidation reaction, and is generally considered as one of the markers of intracellular oxidative stress, and its content change can reflect the degree of cell membrane damage and the trend of body peroxidation. After the mice in the galactose subacute aging model and each experimental group were sacrificed, the mouse liver was taken out to prepare tissue homogenate, and the MDA content was detected, as shown in Table 2. Figure 15 As shown in Table 2, the MDA content of the CG group was the lowest, and the content of the NC group was the highest, and there was a significant difference between the CG group and the NC group (P<0.01), which indicated that the D-galactose subacute aging model group was successfully modeled. By comparing the VC group, the NC group and the CG group, it can be seen that the MDA content can be reduced to near normal level by the intake of antioxidant substances, and the MDA content of the LD, MD and HLD groups is reduced to different degrees, and may have a dose-dependent effect, and the MDA content of the HLD group is the least, and the antioxidant capacity is the strongest.

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

[0142] Figure 16 (a)-(f) are 100x HE staining of liver pathological sections of mice under different treatment conditions. The normal mouse liver cells should be closely arranged, the boundary is clear, and there is no fatty lesion phenomenon. The liver cells of the NC group are loosely arranged, the boundary is not clear, and a large amount of fat accumulation leads to vacuolization and severe inflammatory infiltration. The EPS gavage group and the VC group have a certain degree of relief, and the liver sections of each group have no obvious pathological changes, which indicates that the appropriate dose of EPS has no toxic side effects on the mouse liver.

[0143] In summary, the in vivo antioxidant activity of S-EPS is mainly reflected in the enhancement of the activities of antioxidant enzymes SOD, GSH-Px and CAT, the reduction of MDA content in mouse liver and pro-inflammatory factors IL-6, TNF-α and IL-1β, and the increase of antioxidant master regulator Nrf and antioxidant enzyme HO-1 content, which indicates 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, the activity of antioxidant enzymes and the total oxidation capacity are enhanced, and on the other hand, the expression of important signal factors Nrf and HO-1 in Nrf / HO-1 related pathways is promoted, the MDA content in mouse liver and the pro-inflammatory factors in serum are significantly reduced, and the phenomenon of loose arrangement of liver cells, unclear boundary, a large amount of fat accumulation leading to vacuolization and severe inflammatory infiltration is improved, which proves that S-EPS has excellent antioxidant capacity in vivo, and can relieve the oxidative stress pressure caused by the intake of excessive D-galactose.

[0144] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled in the art within the essential scope of the present application shall also fall within the protection scope of the present application.

Claims

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

2. A method for the preparation of a pentasaccharide Pediococcus -derived modified exopolysaccharide according to claim 1, characterized in that The method comprises the following steps: (1) Pediococcus pentosaceus with accession number CGMCC No. 29541 ( Pediococcus pentosaceus YY-13 was used to extract crude extracellular polysaccharides using the ethanol precipitation method. The specific steps of the extraction process are as follows: The strain... Pediococcus pentosaceus After activation, YY-13 was inoculated into modified MRS medium at a 2 wt% inoculum. After culturing at 37°C for 26.5 h, the supernatant was collected by centrifugation and boiled for 10 min. After cooling, TCA was added to a final concentration of 4 wt%, and the mixture was allowed to stand at 4°C for 8 h. The supernatant was then collected by centrifugation, and three times its volume of anhydrous ethanol was added and stirred overnight. The precipitate was then dissolved in 60°C water and centrifuged for 10 min, and the supernatant was collected. The supernatant was concentrated by rotary evaporation and placed in an 8000 Da dialysis bag. Dialysis was performed in ultrapure water at 4°C for 2 days, with water changed every 4 h. The dialysis solution was concentrated again by rotary evaporation, placed in centrifuge tubes, and pre-frozen at -80°C for 4 days. h, and finally freeze-drying to obtain crude extracellular polysaccharide. The modified MRS medium is prepared as follows: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 5 g sodium acetate, 35 g maltose, 80 ml Tween, 0.5 g magnesium sulfate, 0.25 g manganese sulfate and 15 g agar powder are dissolved in 1 L of water. (2) The crude exopolysaccharide obtained in step (1) is separated and purified by a DEAE-52 cellulose ion exchange column and a Sepharose CL-6B gel column to obtain high-purity exopolysaccharide, specifically as follows: The crude exopolysaccharide 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 water phase filter, and then loaded with a loading amount of 5 mL; a DEAE-52 cellulose ion exchange column pre-equilibrated with deionized water at pH 6.0 is used as an eluent, and deionized water is used as an eluent to elute the sample; the eluate is collected, and the corresponding sugar peaks are combined and concentrated by rotary evaporation, and then desalted by dialysis to obtain neutral sugar; the neutral sugar is eluted by a Sepharose CL-6B gel column with distilled water as an eluent, and the eluate is concentrated by rotary evaporation and freeze-dried to obtain high-purity exopolysaccharide with a purity of more than 90%; (3) The high-purity exopolysaccharide obtained in step (2) is modified by an ultrasonic-assisted sulfation technology to prepare polysaccharide, and pentosaccharide-derived modified exopolysaccharide with good antioxidant activity is obtained, specifically as follows: The high-purity exopolysaccharide is dissolved in anhydrous dimethylformamide DMF to obtain a solution with a concentration of 5 mg / mL, and is oscillated under the condition of an ultrasonic frequency of 20 kHz and a power of 500 W for 55 minutes, and then is continuously oscillated for 30 minutes; then 100 mg of sulfur trioxide-pyridine complex is added and stirred at 80°C for 3 hours; after the reaction is completed, the mixture is cooled to room temperature in an ice bath, neutralized with a 4M NaOH solution, and then dialyzed against ultrapure water at 4°C for 2 days to obtain pentosaccharide-derived modified exopolysaccharide, wherein the mass ratio of the high-purity exopolysaccharide to the sulfur trioxide-pyridine complex is 2:

1.

3. Use of the pentasaccharide Pediococcus -derived modified exopolysaccharide according to claim 2 for the preparation of an in vitro antioxidant agent, characterized by the fact that: The in-vitro antioxidant agent comprises a DPPH free radical scavenger, an ABTS free radical scavenger, and a ferrous ion reducing agent.

4. Use of the pentosaccharide-derived modified exopolysaccharide of claim 2 in the preparation of an in-vivo antioxidant agent.

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