Method capable of remarkably improving anti-inflammatory effect of peanut meal polysaccharide

Abstract

The invention belongs to the technical field of peanut meal processing, and particularly relates to a method capable of remarkably improving the anti-inflammatory effect of peanut meal polysaccharide. The invention provides a method capable of remarkably improving the anti-inflammatory effect of peanut meal polysaccharide. The method comprises the following steps: extracting the peanut meal polysaccharide; vc and H2O2 are used for degrading the IL-6, and experiments are respectively carried out aiming at the anti-inflammatory activities of the IL-6, the TNF-alpha and the IL-4, so that the optimal degradation time is obtained. The method has the beneficial effects that the peanut meal polysaccharide is degraded, the technical problem that the peanut meal polysaccharide is difficult to be directly utilized by a human body is solved, and a result shows that the anti-inflammatory activity of the peanut meal polysaccharide degraded for 1.25 hours is the strongest, and the IL-6 level can be reduced by 37.82%; the peanut meal polysaccharide degraded for 2.5 hours has the greatest influence on the expression level of RAW 264.7 cell IL-4, the effect of inhibiting release of IL-4 is the best, the level of IL-4 can be reduced by 39.68%, and the anti-inflammatory activity is remarkably enhanced.

Description

Technical Field

[0001] The invention belongs to the technical field of peanut meal processing, and particularly relates to a method capable of significantly improving the anti-inflammatory effect of peanut meal polysaccharides. Background Art

[0002] Peanut meal polysaccharides have antibacterial, lipid-lowering, immunomodulatory, and anti-tumor effects. There are few literatures on the specific research on the anti-inflammatory effect of peanut meal polysaccharides. Some literature reports that peanut polysaccharides have anti-inflammatory effects, but no detailed experimental research has been disclosed on the specific anti-inflammatory activity.

[0003] If peanut meal is used as raw material, polysaccharides are extracted, and their anti-inflammatory activity is deeply studied, and corresponding products are further developed, it will be of positive significance for improving the comprehensive utilization rate of peanut by-products and increasing the added value of peanut meal.

[0004] Polysaccharides have good biological activity, but their macromolecular structure is difficult to be directly utilized by the human body. Similarly, peanut polysaccharides also have the above-mentioned problems. Therefore, how to moderately degrade polysaccharides to improve solubility is also a difficult technical problem to solve.

[0005] At present, there is still a lack of research on the functions of peanut meal polysaccharides. The complex structure of polysaccharides is also the reason why its structure-activity relationship and functional mechanism are difficult to further study, which limits the industrialization process of peanut meal polysaccharides. Therefore, the technical problem to be solved by this application is to analyze the chemical structure of peanut polysaccharides, moderately degrade the polysaccharides to improve their solubility, and obtain the relationship between the degradation time and the anti-inflammatory effect, so as to achieve the effective utilization of polysaccharides by biological organisms. Summary of the invention

[0006] In order to solve the above-mentioned technical problems, the present invention provides a method that can significantly improve the anti-inflammatory effect of peanut meal polysaccharides. The method uses peanut cake after oil extraction as raw material, extracts it with hot water to obtain peanut meal polysaccharides, and then degrades it to improve its solubility, so as to achieve its high-value utilization.

[0007] The method provided by the present invention for significantly improving the anti-inflammatory effect of peanut meal polysaccharides comprises the following steps:

[0008] (1) Extraction of peanut meal polysaccharides:

[0009] Take peanut meal and extract peanut meal polysaccharide by hot water extraction method;

[0010] (2) Degradation of peanut meal polysaccharides:

[0011] Take the peanut polysaccharide obtained in (1), add pure water to dissolve, add lactobacillus powder at 35-40°C, the amount of which is 0.5-1% of the weight of the peanut polysaccharide, ferment for 1-3 hours, then add Vc and H2O2 solution, and finally add pure water to make the H2O2 concentration 75mM and the Vc concentration 20mM, mix well, and let stand;

[0012] (3) Add manganese dioxide to the mixed solution in (2) to terminate the reaction, centrifuge, separate the precipitate, obtain the supernatant, add anhydrous ethanol, precipitate, centrifuge after the precipitation, separate the supernatant, spread the precipitate on a clean culture dish, dry, grind, collect the dry powder, and obtain peanut meal polysaccharide with enhanced anti-inflammatory effect.

[0013] Preferably, in (2), 2.50 g of peanut meal polysaccharide is accurately weighed and placed in a 500 mL wire-mouth reagent bottle, an appropriate amount of pure water is added and shaken to dissolve, and then Vc and H2O2 solutions are added, and the pure water is added to 250 mL, so that the H2O2 concentration in the solution is 75 mM and the Vc concentration is 20 mM. Mix well, and place the reagent bottle in a water bath shaker at 25-30°C for 1-2.5 h.

[0014] Preferably, in (3), manganese dioxide is added to the mixed solution in (2) at a ratio of 0.05 g / mL to terminate the reaction, and the mixture is centrifuged at 10,000 rpm for 15 min to separate the precipitate and obtain the supernatant. Anhydrous ethanol is added to the supernatant at a volume ratio of 1:4 to alcohol-to-liquid ratio, and the mixture is placed at 4°C for alcohol precipitation for 6 to 12 hours. After the alcohol precipitation is completed, the mixture is centrifuged at 10,000 rpm for 15 min to separate the supernatant. The precipitate is spread on a clean culture dish, placed in an oven, dried at 55°C, ground, and the dry powder is collected and stored in a sealed container at 4°C in the dark.

[0015] Preferably, in (2), after the peanut meal polysaccharide is degraded for 1.25 to 2.5 hours, the final product obtained contains: 1.287 to 1.329% mannose, 5.626 to 5.744% ribose, 7.819 to 9.121% rhamnose, 10.738% to 11.148% glucose, 19.567 to 21.356% galactose, 1.282 to 1.397% xylose, 17.485 to 18.689% arabinose and 6.991 to 7.536% fucose.

[0016] (3), for inhibiting the secretion of cellular IL-6 or inhibiting the release of TNF-α, the degradation time is controlled to be 1.25h; for inhibiting the secretion of cellular IL-4, the degradation time is controlled to be 2.5h.

[0017] The beneficial effects of the present invention are:

[0018] (1) In the present application, peanut meal polysaccharides were degraded to solve the technical problem that peanut meal polysaccharides are difficult to be directly utilized by the human body. The results showed that peanut meal polysaccharides degraded for 1.25 hours had the strongest anti-inflammatory activity and could reduce the IL-6 level by 37.82%; peanut meal polysaccharides degraded for 2.5 hours had the greatest effect on the IL-4 expression level of RAW 264.7 cells and had the best effect on inhibiting IL-4 release, which could reduce the IL-4 level by 39.68%, and significantly enhanced the anti-inflammatory activity;

[0019] (2) For inhibiting the secretion of IL-6, IL-4, and TNF-α, the present application obtained the corresponding degradation time through experiments, which showed that the longer the degradation time, the better the anti-inflammatory effect on cells;

[0020] (3) In the process of extracting peanut meal polysaccharides in this application, hot water extraction is used, which has the advantages of avoiding the breakage of glycosidic bonds and the destruction of polysaccharide structure in the sample to the greatest extent, and the test conditions are mild and the equipment requirements are low, which provides good conditions for subsequent degradation. In addition, in the process of degrading peanut meal polysaccharides, microbial degradation, VC and H2O2 degradation methods are used in combination to further improve the degradation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 This is the GC spectrum of the undegraded derivative of peanut meal polysaccharide;

[0022] Attached Figure 2 This is the GC spectrum of the derivatives of peanut meal polysaccharide degradation for 1.25h;

[0023] Attached Figure 3 This is the GC spectrum of the derivatives of peanut meal polysaccharide degradation for 2.5 hours;

[0024] Attached Figure 4 The infrared spectra of peanut meal polysaccharides at different degradation times;

[0025] Attached Figure 5 The effect of peanut meal polysaccharide on the expression level of IL-6 in RAW 264.7 cells;

[0026] Attached Figure 6 The effect of peanut meal polysaccharide on the expression level of IL-4 in RAW 264.7 cells;

[0027] Attached Figure 7 This is the effect of peanut meal polysaccharides on the expression level of TNF-α in RAW 264.7 cells. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0029] Example 1 Extraction of peanut meal polysaccharides and determination of yield and purity

[0030] 1.1 Extraction of peanut meal polysaccharides

[0031] In the process of extracting peanut meal polysaccharides and determining their yield and purity, the main reagents used include:

[0032] Table 1 Main reagents and materials

[0033] Reagents or materials Specifications or parameters Manufacturer <![CDATA[Iodine (I2)]]> Analytical grade Tianjin Guangcheng Chemical Reagent Co., Ltd. Potassium iodide (KI) Analytical grade Sinopharm Chemical Reagent Co., Ltd. Thermoamylase Food Grade Beijing Solable Technology Co., Ltd. D-anhydrous glucose 99％ Shanghai MacLean Biochemical Technology Co., Ltd. 3,5-Dinitrosalicylic acid (DNS) Analytical grade Shanghai Yuanye Biotechnology Co., Ltd. Anhydrous ethanol Analytical grade Tianjin Funing Fine Chemical Co., Ltd. sulfuric acid Analytical grade Sinopharm Chemical Reagent Co., Ltd. phenol Analytical grade Sinopharm Chemical Reagent Co., Ltd. MSC80003 ultrafiltration membrane Molecular weight cut-off 3kDa Shanghai Mosu Scientific Instruments Co., Ltd.

[0034] The following instruments are mainly used: electronic balance, digital display overhead electronic stirrer, vertical pressure steam sterilizer, low-temperature refrigerated centrifuge, thermal collector constant temperature heating magnetic stirrer, etc. The above equipment are common instruments available on the market and will not be described in detail here.

[0035] Take 100g of peanut meal and place it in a beaker. Add pure water at a solid-liquid ratio of 1:10, stir for 20 minutes to make it evenly dispersed. Seal the kraft paper and place it in an autoclave (121℃, 30min). Cool to room temperature after extraction. Separate the precipitate by centrifugation (5000rpm, 15min), obtain the supernatant, adjust its pH to 6.0, and measure the volume. Heat in a 90℃ water bath, add high-temperature α-amylase at a ratio of 10U / mL, until the supernatant does not change color when it encounters iodine reagent (2% KI and 1% I2) and the content of reducing sugar produced no longer increases (samples taken at 20min, 30min, and 40min are tested respectively). After the enzymolysis is completed, separate the precipitate by centrifugation (9000rpm, 10min), obtain the supernatant, remove impurities by ultrafiltration with a 3k ultrafiltration membrane, and obtain nearly 200mL of polysaccharide concentrate by rotary evaporation (60℃). Add anhydrous ethanol at a ratio of 1:4 and place it at 4℃ for alcohol precipitation overnight. After the alcohol precipitation is completed, the supernatant is separated by centrifugation (4000 rpm, 10 min), the precipitate is spread on a clean culture dish, placed in an oven, dried at 40-50°C, ground, the dry powder is collected, and stored in a sealed container at 4°C away from light.

[0036] 1.2 Determination of the yield and purity of peanut polysaccharides

[0037] The yield and purity were determined as follows:

[0038]

[0039] The purity was determined by the phenol-sulfuric acid method. Reference: Research on steam explosion-assisted extraction of Brasenia schrenkiana polysaccharides and determination of their polysaccharide content by phenol-sulfuric acid method [J]. Grain and Oils, 2022, 35(05): 116-120. Author: Jin Jin, Liu Yanqi, Qin Lingxiang.

[0040] Calculation formula:

[0041] In Example 1, a total of 100 g of peanut meal was used to extract 5.62 g of peanut meal polysaccharide. According to the above formula, the yield of peanut meal polysaccharide was 5.62%.

[0042] The purity was calculated to be 48.83%.

[0043] Example 2 Degradation of peanut meal polysaccharides

[0044] The peanut meal polysaccharide prepared in Example 1.1 was degraded, and the specific degradation method was as follows:

[0045] 2.1 Degradation of peanut meal polysaccharides

[0046] Weigh 2.50g peanut meal polysaccharide accurately, place it in a 500mL wire-mouthed reagent bottle, add appropriate amount of pure water to shake and dissolve, heat the aqueous solution of peanut meal polysaccharide to 35-40℃, then add lactobacillus powder, the amount added is about 0.8% of the weight of peanut polysaccharide, ferment for 2h, add appropriate amount of Vc and H2O2 solution to the solution, add pure water to 250mL, make the H2O2 concentration in the solution to 75mM, Vc concentration to 20mM, mix well. Place the reagent bottle in a water bath shaker at 25℃ for 1.25h; repeat the above steps for one group and treat for 2.5h.

[0047] After the treatment, add manganese dioxide (MnO2) at a ratio of 0.05g / mL to terminate the reaction, separate the precipitate (MnO2) by centrifugation (10000rpm, 15min), and obtain the supernatant. Add anhydrous ethanol at a ratio of 1:4 and place it at 4℃ for alcohol precipitation overnight. After the alcohol precipitation is completed, separate the supernatant by centrifugation (10000rpm, 15min), spread the precipitate on a clean culture dish, place it in an oven, dry it at 55℃, grind it, collect the dry powder, and store it in a sealed container at 4℃ away from light.

[0048] 2.2 Degradation rate of peanut meal polysaccharides

[0049] Take the undegraded, 1.25h degraded, and 2.5h degraded peanut meal polysaccharide dry powders and place them in a clean beaker, add appropriate amount of pure water, stir and dissolve, and prepare a solution of a certain concentration. Take out the Ubbelohde viscometer, inject the first group of sample solutions into ball B along tube 2, fix the instrument vertically in a 25℃ constant temperature water bath, make the water bath liquid level higher than ball C, and let it stand for 15 minutes. Connect the clamped latex tube to the tube mouth 1, use the ear washing bulb to draw air at the tube mouth 3, and slowly raise the liquid level of the solution in the tube to the middle of ball C. Release the tube mouth 1 and the tube mouth 3 in turn, let the sample solution fall naturally in the tube, start timing at the scale line m1, and stop timing at the scale line m2.

[0050] Repeat the measurement twice, the difference between the two measured values ​​should not exceed 0.1 second, and the average value is taken as the outflow time of the sample solution. Take pure water as the control group, repeat the above steps and measure the data of the remaining two groups.

[0051] Calculate intrinsic viscosity:

[0052] Where:

[0053] η sp (Specific viscosity) = η r -1

[0054] t n : Sample measurement time

[0055] t0: Pure water measurement time

[0056] C: Sample concentration

[0057] The pure water outflow time was 1:39:59 as measured by an Ubbelohde viscometer.

[0058] Table 2 Viscosity of polysaccharide solutions at different degradation times

[0059] Solution Grouping Undegraded group Degradation 1.25h group Degradation 2.5h group time 1:59:49 1:51:10 1:45:94 <![CDATA[η r ]]> 1.200 1.116 1.064 <![CDATA[η sp ]]> 0.200 0.116 0.064 [η] 0.5719 0.4253 0.3212

[0060] According to the above formula, the viscosity of peanut meal polysaccharide solution at different degradation times is shown in Table 6-2. The degradation rate of peanut meal polysaccharide is 25.63% when it is degraded for 1.25h, and the degradation rate is 43.84% when it is degraded for 2.5h.

[0061] 2.3 Analysis of monosaccharide composition of peanut meal before and after degradation

[0062] The monosaccharide composition was determined as follows:

[0063] For the obtained peanut meal polysaccharide, ion exchange chromatography was used to determine the monosaccharide composition of the sample. The chromatographic column conditions are as follows: the monosaccharide composition analysis was performed using a Dion ICS-3000 ion chromatograph equipped with a chromatographic column (3×150mm); the chromatographic column was Xtimate C18 (4.6×200mm, 5μm), the injection volume was 20μL; the mobile phase was ultrapure water, 0.02mol / L sodium hydroxide (NaOH) solution and 0.5mol / L sodium acetate (NaAc) solution. A gradient elution method was used, the elution program is shown in Table 4-3, the flow rate was 1.0mL / min, and the column temperature was 30°C.

[0064] Control solution: Accurately weigh appropriate amounts of mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), N-acetyl-glucosamine (GlcNAc), glucose (Glc), N-acetyl-galactosamine (GalNAc), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucose (Rha), dissolve in water and dilute to a mixed control solution containing 50 μg of each in 1 mL.

[0065] Hydrolysis of the sample to be tested: Accurately weigh an appropriate amount of the sample into a 10mL ampoule, add 3.0mL of 2mol / L trifluoroacetic acid (TFA) into the ampoule, fill with nitrogen, seal the tube, and perform acid hydrolysis at 120℃ for 4h. After the acid hydrolysis is completed, transfer 1.0mL and add an appropriate amount of methanol, and repeatedly blow nitrogen to dry to remove residual acid. Finally, add 1.0mL of water to re-dissolve, filter with a 0.22μm microporous filter head and set aside.

[0066] Table 3 Monosaccharide composition analysis ion exchange chromatography elution procedure

[0067] Elution time / min 0 15 15.01 30 Ultrapure water (%) 90 90 70 70 0.02mol / L NaOH(%) 10 10 10 10 0.5mol / L NaAc(%) 0 0 20 20

[0068] (1) Attachment Figure 1 Table 4 is the GC spectrum analysis results of the undegraded derivatives of peanut meal polysaccharides:

[0069] Table 4 GC spectrum analysis results of undegraded derivatives of peanut meal polysaccharides

[0070]

[0071]

[0072] Combination Figure 1As shown in Table 4, the elution order of the eight standard monosaccharide derivatives in the capillary column; among them, the elution time of ribose and rhamnose is close, and the elution time of galactose, xylose and arabinose is relatively close; the baselines of all monosaccharides have achieved good separation. As shown in Table 6-3, the main monosaccharide composition of the peanut meal polysaccharide in the undegraded group is mannose (0.860%), ribose (5.632%), rhamnose (1.605%), glucose (55.156%), galactose (5.970%), xylose (0.697%), arabinose (8.629%) and fucose (4.716%), among which the glucose content is the highest.

[0073] (2) Analysis of monosaccharide composition after 1.25 h degradation

[0074] Attached Figure 2 The monosaccharide analysis of the peanut meal polysaccharide in Example 1 after degradation for 1.25 hours using the method in Example 2 is shown in Table 5 as the GC spectrum analysis results of the derivatives of the peanut meal polysaccharide degradation for 1.25 hours:

[0075] Table 5 GC spectrum analysis results of peanut meal polysaccharide degradation 1.25h derivatives

[0076] Peak order Monosaccharide name <![CDATA[Retention time (T R / min)]]> Composition percentage (%) 1 Mannose (Man) 14.079 1.287 2 Ribose 18.041 5.626 3 Rhamnose (Rha) 19.169 9.121 4 Glucose (Glc) 29.820 10.738 5 Galactose (Gal) 34.073 21.356 6 Xylose 35.775 1.282 7 Arabinose (Ara) 37.154 18.689 8 Fucose (Rha) 41.978 6.991

[0077] Combination Figure 2 As shown in Table 5, the elution order of the derivatives of eight standard monosaccharides in the capillary column; among them, the elution time of ribose and rhamnose is close, and the elution time of galactose, xylose and arabinose is relatively close; the baselines of all monosaccharides have achieved good separation. As shown in Table 6-4, the main monosaccharide composition of peanut meal polysaccharide degraded for 1.25h is mannose (1.287%), ribose (5.626%), rhamnose (9.121%), glucose (10.738%), galactose (21.356%), xylose (1.282%), arabinose (18.689%) and fucose (6.991%), among which the galactose content is the highest.

[0078] (3) Analysis of monosaccharide composition after 2.5 h degradation

[0079] Attached Figure 3 The monosaccharide analysis of the peanut meal polysaccharide in Example 1 after degradation for 1.25 hours using the method in Example 2 is shown in Table 6 as the GC spectrum analysis results of the derivatives of the peanut meal polysaccharide degradation for 1.25 hours:

[0080] Table 6 GC spectrum analysis results of peanut meal polysaccharide degradation 2.5h derivatives

[0081] Peak order Monosaccharide name <![CDATA[Retention time (T R / min)]]> Composition percentage (%) 1 Mannose (Man) 14.085 1.329 2 Ribose 18.069 5.744 3 Rhamnose (Rha) 19.178 7.819 4 Glucose (Glc) 29.860 11.148 5 Galactose (Gal) 34.100 19.567 6 Xylose 35.797 1.397 7 Arabinose (Ara) 37.190 17.485 8 Fucose (Rha) 42.000 7.536

[0082] Combination Figure 3As shown in Table 6, the elution order of the derivatives of eight standard monosaccharides in the capillary column; among them, the elution time of ribose and rhamnose is close, and the elution time of galactose, xylose and arabinose is relatively close; the baselines of all monosaccharides have achieved good separation. As shown in Table 6-4, the main monosaccharide composition of peanut meal polysaccharide degraded for 1.25h is mannose (1.329%), ribose (5.744%), rhamnose (7.819%), glucose (11.148%), galactose (19.567%), xylose (1.397%), arabinose (17.485%) and fucose (7.536%), among which the galactose content is the highest.

[0083] 2.4 Analysis of monosaccharide and uronic acid composition at different degradation times

[0084] Table 7 Percentage of monosaccharide and uronic acid composition at different degradation times

[0085]

[0086] As shown in Table 7, the glucose content of peanut meal polysaccharides decreased significantly during the degradation process, while the contents of galactose and arabinose increased; the glucuronic acid content decreased, while the galacturonic acid content increased.

[0087] 2.5 Infrared spectroscopic determination results of peanut meal polysaccharides at different degradation times

[0088] Infrared spectrum scanning

[0089] The peanut meal polysaccharide sample and potassium bromide (KBr) were dried in an oven at 105°C for 4 hours, and then placed in a dryer to cool for 2 hours. 4 mg of sample and 80 mg of KBr were accurately weighed and fully ground in an agate mortar until mixed evenly. The ground sample and KBr mixture were transferred to a tablet pressing mold and subjected to a pressure of 20 MPa for 20 seconds. The pressed sample potassium bromide tablets were dried for 30 minutes and scanned using a Fourier infrared spectrometer with a scanning range of 4000-400 cm -1 , resolution 4cm -1 .

[0090] according to Figure 4 It can be seen that the three peanut meal polysaccharides all have characteristic peaks of carbohydrate compounds. -1 The broad peak at 2937.8-2962.9cm is the absorption peak caused by the stretching vibration of a large number of -OH and OH in the polysaccharide molecules. -1 and 1400.5~1416.4cm -1 The absorption peaks are caused by CH stretching vibration and bending vibration, which are characteristic absorption peaks of sugar compounds.

[0091] Figure 4 Medium, 1651.7~1663.2cm -1 The absorption peak at 1238.3~1245.1cm is caused by the C=O stretching vibration of the -COOH functional group. -1 The CO stretching vibration absorption peak is 1048.7~1082.9cm -1 The absorption peak is the ether bond COC in the pyranose ring.

[0092] The undegraded group was at 1539.8 cm -1 There is an absorption peak at 795.1cm, while the absorption peaks of the two groups of degradation are less obvious. This is caused by the vibration of the benzene ring skeleton and is one of the most important peaks for determining the presence of benzene rings in polysaccharides. -1 There is a small peak at each of the two groups, which can be inferred that the benzene ring moves during the degradation process, resulting in ortho-meta-substitution. The two groups of degraded polysaccharides have an absorption peak at 1315.9 cm-1, which is the characteristic absorption peak of -CH3. It can be inferred that the degradation process causes the glycosidic bond to break, thereby producing more -CH3.

[0093] In summary, except for the substitution of the benzene ring at 1539.8 cm-1 by the ortho-meta-position, the other major functional groups did not change significantly, but there were changes in quantity, indicating that the degradation process led to the breakage of the glycosidic bond and the generation of more free groups.

[0094] 2.6 Verification of the anti-inflammatory effect of peanut meal polysaccharides

[0095] 2.6.1(1) Reagent preparation

[0096] Cell culture medium: sterilize the wire-top reagent bottle in advance, take out 20 mL of fetal bovine serum (FBS) from the sterile biological cabinet and thaw it, filter it with a sterilized 0.22 μm filter membrane, make the volume of DMEM high-glucose culture medium to 200 mL, add 2 mL of double antibody (penicillin-streptomycin 100X), put it into the treated wire-top reagent bottle, and store it at 4°C for later use.

[0097] High-glucose DMEM complete medium: DMEM high-glucose medium + 10% FBS.

[0098] Preparation of LPS solution: Accurately weigh 1.0 mg of LPS, dissolve it in 4 mL of high-glucose DMEM by ultrasonication, prepare a 0.25 mg / mL solution, and store it in aliquots at -20°C.

[0099] Cell freezing solution: Pipette 3 mL FBS and 1.2 mL DMSO into a 15 mL centrifuge tube, and add high-glucose DMEM to make up to 15 mL (72% DMEM high-glucose medium + 20% FBS + 8% DMSO). Prepare it before use.

[0100] (2) Cell culture

[0101] Cell recovery: Take the RAW 264.7 cell line out of the -80℃ refrigerator, quickly put it into a water bath (37℃) to thaw, and then transfer it to the clean bench. Pipette 5mL of complete culture medium into a 15mL sterile centrifuge tube, and then transfer the dissolved cell suspension into the centrifuge tube. Take 1mL of complete culture medium from the centrifuge tube and gently blow the cell cryopreservation tube, wash it once, wash the remaining attached cells and transfer them into the centrifuge tube, discard the supernatant by centrifugation (1000rpm, 5min), replace with new complete culture medium, and gently blow and mix to suspend and evenly distribute the cells. Mark well and place in an incubator (37℃, 5% CO2) for culture.

[0102] Cell culture medium replacement: Cultivate cells according to the above conditions, observe their status and replace the culture medium, usually 1 to 2 days. When replacing the culture medium, wash it twice with PBS buffer in advance. Select cells with good growth status for the test.

[0103] Cell passaging: Observe the cell growth status under a microscope. Sterilize the test bench, discard the original culture medium, and wash the RAW 264.7 cells at about 80% with PBS buffer. Take an appropriate amount of trypsin to digest the adherent cells, transfer the cell suspension into a 15mL centrifuge tube with 3mL complete culture medium, discard the supernatant by centrifugation (1000rpm, 5min), add 3mL complete culture medium, gently blow and mix, and culture under the above culture conditions.

[0104] Cell freezing: Take out serum from -80℃ refrigerator, take out DMSO from -4℃ refrigerator, and place in water bath (37℃) to thaw. Select RAW 264.7 cells in logarithmic phase, discard the original culture medium, wash once with 5mL PBS buffer, blow gently to blow off the attached cells, and discard the supernatant by centrifugation (1000rpm, 5min). Add cell freezing solution, blow repeatedly to evenly, transfer 1mL per tube into freezing tube, mark it, put it into program freezing box and move it into -80℃ refrigerator.

[0105] (3) Preparation and sterilization of polysaccharide solution

[0106] The concentration of peanut meal polysaccharide intervention cells was 300 μg / mL. Because the polysaccharide has a general solubility, it is impossible to prepare a 10X stock solution. Therefore, the experiment selected full culture medium to prepare a 2X stock solution, that is, a polysaccharide solution with a concentration of 600 μg / mL.

[0107] Preparation of sample solution: In a sterile clean bench, take about 20 mL of complete culture medium into a sterile centrifuge tube, accurately weigh 0.0030 g of undegraded, 1.25 h degraded, and 2.5 h degraded polysaccharide powder, place them in 10 mL centrifuge tubes and add 5 mL of sterile culture medium. After vortexing to dissolve, prepare a solution with a concentration of 600 μg / mL.

[0108] Sterilization: After the centrifuge tube is sterilized with a large amount of alcohol spray, it is placed on the centrifuge tube rack in the clean bench. (Note: At this time, the tube mouth, the inside of the tube and the lid, and the liquid in the tube are still sterile, so be careful to place them separately from the sterile instruments to avoid contamination.) Take 3 sterile centrifuge tubes and place them on another centrifuge tube rack in the clean bench, and open the lid. After tearing open the package of the 0.22um sterile filter, use tweezers to grab the cylindrical tube part of the filter and place it on the sterile centrifuge tube. Use one filter for each sample to avoid cross contamination. Use a sterile syringe with a needle to absorb the prepared polysaccharide solution, take the needle cap to cover the needle, remove the needle with force, insert the syringe into the cylindrical tube of the sterile filter, press down the syringe, and connect the filtrate to a new sterile centrifuge tube. After the filtration is completed, discard the syringe, filter and original centrifuge tube to avoid contamination. Put the sterilized polysaccharide solution at 4℃ for standby use.

[0109] (4) Cell treatment

[0110] A blank control group (RAW 264.7 cells + PBS), an LPS model group (RAW 264.7 cells + LPS), and an experimental group (i.e., peanut meal polysaccharides + LPS groups at different degradation time periods) were set up.

[0111] Observe the cells under a microscope, and select RAW 264.7 cells in the logarithmic growth phase and in good condition for the experiment. Take 500 μL of polysaccharide solution and add it to 500 μL of cell culture medium and mix well. Digest the adherent cells, resuspend them with fetal bovine serum medium, and inoculate them in the above medium. After inoculation, the cells were placed in an incubator (37°C, 5% CO2) for 24 hours, and the original medium was discarded. According to the experimental grouping, LPS with a final concentration of 1 μg / mL was added to each group in turn for 24 hours. After the intervention, the precipitate was separated by centrifugation (1000rpm, 5min), and the supernatant was taken into a sterile centrifuge tube, marked, and placed at 4°C for a short period of time and -20°C for a long period of time.

[0112] (5) ELISA method for determination of cytokines IL-6, IL-4, and TNF-α

[0113] ① Reagent preparation: Take the kit out of the refrigerator in advance and equilibrate it at room temperature for 20 minutes. All liquid components must be shaken thoroughly before use.

[0114] Dilution of 20X Wash Buffer: dilute 1:20 with purified water.

[0115] ② Reagent steps: Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and put them back at 4°C. Set up standard wells and sample wells, and add 50μL of standards of different concentrations to the standard wells. First add 10μL of the sample to be tested to the sample well, and then add 40μL of sample diluent; do not add to the blank well. Except for the blank well, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate in a constant temperature box (37°C) for 1h.

[0116] After the incubation, discard the liquid, pat dry on absorbent paper, fill each well with diluted washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this operation 5 times. After the washing, add 50μL of substrate A and B to each well, and incubate in a constant temperature box (37℃) in the dark for 15 minutes. After the incubation, add 50μL of stop solution to each well, and measure the absorbance of each well at a wavelength of 450nm with an enzyme reader within 15 minutes.

[0117] ③Draw a standard curve

[0118] With the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis, a linear regression curve of the standard was drawn, and the concentration value of each sample was calculated according to the curve equation.

[0119] ④Statistical analysis

[0120] The experimental data were expressed as mean ± standard deviation, and repeated three times. SPSS software was used for data analysis, P < 0.05 was significant, P < 0.01 was extremely significant, and Graphpad Prism 9.5 was used for graphing.

[0121] 2.6.2(1) Effects of polysaccharides on IL-6 expression in RAW 264.7 cells

[0122] according to Figure 5It can be seen that there is a very significant difference between the control group and the LPS group. Compared with the control group, the IL-6 level in the LPS group was significantly increased, which shows that the inflammatory model was successfully established, that is, after LPS induction, RAW 264.7 cells showed significant inflammation; there is no significant difference between the LPS group and the non-degraded group. Compared with the LPS group, the IL-6 level in the non-degraded group did not decrease significantly, indicating that the peanut meal polysaccharide in the non-degraded group cannot effectively inhibit the secretion of IL-6 in the cells; there is a very significant difference between the LPS group and the 1.25h degradation group. Compared with the LPS group, the IL-6 level in the 1.25h degradation group was significantly reduced, indicating that the peanut meal polysaccharide degraded for 1.25h can significantly inhibit the secretion of IL-6 in the cells; there is a significant difference between the LPS group and the 2.5h degradation group. Compared with the LPS group, the IL-6 level in the 2.5h degradation group was reduced to a certain extent, indicating that the peanut meal polysaccharide degraded for 2.5h can inhibit the secretion of IL-6 in the cells, but the inhibitory effect is not as obvious as that of the 1.25h degradation group.

[0123] In summary, the three peanut meal polysaccharides all showed an inhibitory effect on the secretion of IL-6 by RAW 264.7 cells induced by LPS, showing a certain anti-inflammatory activity. Among them, the peanut meal polysaccharide degraded for 1.25 h had the strongest anti-inflammatory activity, which could reduce the IL-6 level by 37.82% (compared with the LPS group). (2) Effect of polysaccharides on the expression level of IL-4 in RAW 264.7 cells

[0124] according to Figure 6 It can be seen that there is a significant difference between the control group and the LPS group. Compared with the control group, the IL-4 level in the LPS group was significantly increased, which indicates that the inflammatory model was successfully established. That is, after LPS induction, RAW 264.7 cells showed significant inflammation; there was no significant difference between the LPS group and the 1.25h degradation group, but compared with the LPS group, the IL-4 level of the 1.25h degradation group was reduced to a certain extent, indicating that the peanut meal polysaccharide degraded for 1.25h can inhibit the secretion of cellular IL-4, but the inhibitory effect is not obvious; the difference between the LPS group and the 2.5h degradation group was extremely significant, compared with the LPS group, the IL-4 level of the 2.5h degradation group was significantly reduced, indicating that the peanut meal polysaccharide degraded for 2.5h can significantly inhibit the secretion of cellular IL-4; there was no significant difference between the LPS group and the non-degraded group, compared with the LPS group, the IL-4 level of the non-degraded group was slightly increased, there may be errors, but the two groups of degraded polysaccharides had a certain inhibitory effect on the secretion of IL-4 by cells. It is speculated that the peanut meal polysaccharide in the non-degraded group can also inhibit the secretion of IL-4 in cells, but the effect is not obvious.

[0125] In summary, the three peanut meal polysaccharides all showed an inhibitory effect on the secretion of IL-4 by RAW 264.7 cells induced by LPS, showing a certain anti-inflammatory activity. Among them, the peanut meal polysaccharide degraded for 2.5 hours had the strongest anti-inflammatory activity, which could reduce the IL-4 level by 39.68% (compared with the LPS group). (3) Effect of polysaccharides on the expression level of TNF-α in RAW 264.7 cells

[0126] according to Figure 7 It can be seen that there is a very significant difference between the control group and the LPS group. Compared with the control group, the TNF-α level in the LPS group was significantly increased, which shows that the inflammatory model was successfully established, that is, after LPS induction, RAW 264.7 cells showed significant inflammation; there is a significant difference between the LPS group and the non-degraded group. Compared with the LPS group, the TNF-α level in the non-degraded group decreased to a certain extent, indicating that the peanut meal polysaccharide in the non-degraded group can inhibit the secretion of TNF-α in cells; there is a very significant difference between the LPS group and the 1.25h degradation group. Compared with the LPS group, the TNF-α level in the 1.25h degradation group was significantly reduced, indicating that the peanut meal polysaccharide degraded for 1.25h can significantly inhibit the secretion of cellular TNF-α; there is a significant difference between the LPS group and the 2.5h degradation group. Compared with the LPS group, the TNF-α level in the 2.5h degradation group was reduced to a certain extent, indicating that the peanut meal polysaccharide degraded for 2.5h can inhibit the secretion of cellular TNF-α, and compared with the non-degraded group, the inhibitory effect is better.

[0127] In summary, the three peanut meal polysaccharides all showed an inhibitory effect on the secretion of TNF-α by RAW 264.7 cells induced by LPS, and exhibited certain anti-inflammatory activity. Among them, the peanut meal polysaccharide that was degraded for 1.25h had the strongest anti-inflammatory activity, which could reduce the TNF-α level by 37.32% (compared with the LPS group). Combined with the figure, it can be seen that after stimulating RAW 264.7 cells with LPS, the levels of IL-6, IL-4 and TNF-α increased significantly from 13.12, 5.20 and 132.89 pg / mL to 17.85, 12.60 and 168.75 pg / mL, respectively. Compared with the LPS group, peanut meal polysaccharides can extremely significantly or significantly inhibit the secretion of these three cytokines. Among them, peanut meal polysaccharides degraded for 1.25 hours had the greatest effect on the expression levels of IL-6 and TNF-α in RAW 264.7 cells, and had the best effect in inhibiting the release of IL-6 and TNF-α. Compared with the LPS group, the levels of IL-6 and TNF-α could be reduced by 37.82% and 37.32%, respectively; peanut meal polysaccharides degraded for 2.5 hours had the greatest effect on the expression level of IL-4 in RAW 264.7 cells, and had the best effect in inhibiting the release of IL-4, which could reduce the IL-4 level by 39.68%.

[0128] As shown in the figure, after stimulating RAW 264.7 cells with LPS, the levels of IL-6, IL-4 and TNF-α increased significantly from 13.12, 5.20 and 132.89 pg / mL to 17.85, 12.60 and 168.75 pg / mL, respectively. Compared with the LPS group, peanut meal polysaccharides can significantly or extremely significantly inhibit the secretion of these three cytokines. Among them, the peanut meal polysaccharides degraded for 1.25 hours had the greatest effect on the expression levels of IL-6 and TNF-α in RAW264.7 cells, and had the best effect on inhibiting the release of IL-6 and TNF-α. Compared with the LPS group, the levels of IL-6 and TNF-α were reduced by 37.82% and 37.32%, respectively; the peanut meal polysaccharides degraded for 2.5 hours had the greatest effect on the expression level of IL-4 in RAW264.7 cells, and had the best effect on inhibiting the release of IL-4, which could reduce the level of IL-4 by 39.68%.

[0129] Through the analysis of peanut meal polysaccharides, it was found that the monosaccharides such as arabinose, glucose, xylose, mannose and galactose contained in peanut meal polysaccharides accounted for a relatively high proportion, and these monosaccharides endowed peanut meal polysaccharides with better anti-inflammatory activity.

[0130] According to the experiments of the present application, it can be inferred that the higher the content of mannose and xylose, the higher the anti-inflammatory activity of peanut meal polysaccharides on IL-4; the higher the content of galactose and arabinose, the higher the anti-inflammatory activity of peanut meal polysaccharides on IL-6 and TNF-α.

[0131] Therefore, the present application solves the problem of low solubility of peanut meal polysaccharides and difficulty in effective utilization by degrading peanut meal polysaccharides, and conducts a large number of experiments on the anti-inflammatory activity of IL-6, TNF-α and IL-4 to obtain the optimal degradation time.

Claims

1. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides, characterized in that: The following steps are involved: (1) Extraction of peanut meal polysaccharides: Take peanut meal and extract peanut meal polysaccharide by hot water extraction method; (2) Degradation of peanut meal polysaccharides: Take the peanut polysaccharide obtained in (1), add pure water to dissolve, add lactobacillus powder at 35-40°C, the amount of which is 0.5-1% of the weight of the peanut polysaccharide, ferment for 1-3 hours, then add Vc and H2O2 solution, and finally add pure water to make the H2O2 concentration 75mM and the Vc concentration 20mM, mix well, and let stand; (3) Add manganese dioxide to the mixed solution in (2) to terminate the reaction, centrifuge, separate the precipitate, obtain the supernatant, and then add anhydrous ethanol to precipitate. After the precipitation is completed, centrifuge, separate the supernatant, spread the precipitate on a clean culture dish, dry, grind, and collect the dry powder to obtain peanut meal polysaccharide with enhanced anti-inflammatory effect.

2. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides as claimed in claim 1, characterized in that: (2) Accurately weigh 2.50 g of peanut meal polysaccharide and place it in a 500 mL wire-capped reagent bottle. Add an appropriate amount of pure water and shake to dissolve. Then add Vc and H2O2 solution and make up to 250 mL with pure water so that the H2O2 concentration in the solution is 75 mM and the Vc concentration is 20 mM. Mix well and place the reagent bottle in a water bath shaker at 25 ~30℃ for 1 ~ 2.5 h.

3. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides as claimed in claim 1, characterized in that: (3), add manganese dioxide at a ratio of 0.05 g / mL to the solution mixed in (2) to terminate the reaction, centrifuge at 10,000 rpm for 15 min, separate the precipitate, obtain the supernatant, add anhydrous ethanol to the supernatant at a volume ratio of 1:4, place it at 4°C for alcohol precipitation for 6 to 12 hours, after the alcohol precipitation is completed, centrifuge at 10,000 rpm for 15 min, separate the supernatant, spread the precipitate on a clean culture dish, place it in an oven, dry it at 55°C, grind it, collect the dry powder, and store it in a sealed container at 4°C in the dark.

4. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides as claimed in claim 2, characterized in that: (2) After the peanut meal polysaccharide was degraded for 1.25-2.5 h, the final product contained 1.287-1.329% mannose, 5.626-5.744% ribose, 7.819-9.121% rhamnose, 10.738%-11.148% glucose, 19.567-21.356% galactose, 1.282-1.397% xylose, 17.485-18.689% arabinose and 6.991-7.536% fucose.

5. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides as claimed in claim 2, characterized in that: (3), for inhibiting the secretion of cellular IL-6 or inhibiting the release of TNF-α, the degradation time is controlled to be 1.25h.

6. A method for significantly improving the anti-inflammatory effect of peanut meal polysaccharides as claimed in claim 2, characterized in that: (3) To inhibit the secretion of IL-4 in cells, the degradation time was controlled to be 2.5h.

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