Vinasse polysaccharide OIR with immune regulation function and preparation method thereof

Abstract

The invention belongs to the technical field of functional food, and particularly relates to a preparation method of vinasse polysaccharide OIR with immunomodulatory activity. The OIR in the invention is extracted by an ultrasonic-assisted water extraction method, deproteinized by a Sevage method, decolored by D101 macroporous adsorption resin, and separated and purified by DEAE-52 ion exchange column and Sephadex G-100 gel column chromatography. The OIR is mainly composed of 12 monosaccharides such as glucose, mannose, galactose, arabinose and xylose, wherein the content of glucose in the OIR is as high as 94.38%; an infrared spectrum result shows that the OIR contains an alpha-configuration glucosidic bond, which indicates that the OIR is alpha-glucan. The vinasse polysaccharide OIR provided by the invention has an immunomodulatory effect, and can improve the proliferative activity and the phagocytic activity of RAW264.7 macrophages, promote the NO release of the RAW264.7 macrophages, promote the RAW264.7 macrophages to secrete cell factors TNF-alpha, IL-6 and IL-1beta, and improve the immunomodulatory activity mediated by the macrophages.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional foods, and particularly relates to a preparation method of distiller's grains polysaccharide OIR with immunomodulatory activity. Background Art

[0002] Distiller's grains are solid mixtures composed of fermented grain residues and are the main by-products of the liquor industry. Distiller's grains are the products remaining after most of the starch in grains is utilized, including crude protein, starch, organic acids, small peptides, soluble sugars, and various functional substances produced during fermentation. At present, the main treatment methods for distiller's grains are as livestock feed, fertilizer, biogas production, etc. The economic value generated by these methods is low, the demand is small and unstable. Therefore, distiller's grains are often directly discarded, burned, or landfilled, but such treatment methods often cause environmental pollution.

[0003] Distiller's grains contain rich nutritional components such as protein, dietary fiber, trace elements, and vitamins, among which the content of natural active polysaccharides is relatively high. However, there are still some blanks in the research on the functional activities of distiller's grains polysaccharides at home and abroad. Therefore, in summary, it is necessary to further study the biological effects of distiller's grains polysaccharides and explore more efficient preparation methods of distiller's grains polysaccharides to promote the effective utilization of distiller's grains polysaccharide resources and their applications in food and medicine. Summary of the Invention

[0004] The object of the present invention is to provide a preparation method of distiller's grains polysaccharide with immunomodulatory activity in view of the deficiencies in the existing research on distiller's grains polysaccharides and the low utilization rate of distiller's grains resources.

[0005] The specific technical solutions are as follows:

[0006] A distiller's grains polysaccharide OIR with immunomodulatory function, the distiller's grains polysaccharide OIR contains the following components: guluronic acid, mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose; preferably, the molar ratio of each monosaccharide component is 0.51:15.89:0.77:0.74:1.68:1.32:0.14:776.25:10.31:5.43:7.04:0.19.

[0007] A preparation method of distiller's grains polysaccharide OIR, the method comprises the following steps:

[0008] (1) Extraction: Dry and crush the distiller's grains, add ultrapure water, extract for 2 h, and perform ultrasonic-assisted extraction for 30 min; centrifuge, take the supernatant, rotary evaporate and concentrate the extract at 50 °C, then add 3 volumes of absolute ethanol, and keep it at 4 °C for 12 h to precipitate the polysaccharide, centrifuge to obtain the precipitate;

[0009] (2) Deproteinization: After the precipitate obtained in step (1) is redissolved with ultrapure water, ethanol is removed by rotary evaporation at 45°C, and then it is mixed evenly with Sevage reagent in a volume ratio of 1:3, magnetically stirred for 1 h, centrifuged, the organic layer and the middle layer are discarded, and the aqueous layer is collected; then 3 times the volume of the aqueous layer of Sevage reagent is added to the aqueous layer, and the above operation is repeated for extraction 2 times until there is no middle layer, and the aqueous layers are collected and combined; then dialysis is carried out, and freeze-drying is performed to obtain polysaccharide;

[0010] (3) Decoloration: The polysaccharide obtained in step (2) is subjected to column chromatography on a D101 macroporous adsorption resin column, and the water-washing part is collected, and freeze-dried to obtain crude distiller's grains polysaccharide DYT.

[0011] (4) Purification: The crude polysaccharide DYT obtained in step (3) is subjected to column chromatography on a DEAE-52 column, eluted with 0.1 mol / L NaCl solution to obtain OI, and purified by Superdex G-100 gel to obtain the distiller's grains polysaccharide OIR.

[0012] Preferably, in step (1), the distiller's grains powder is less than 40 mesh; the specific extraction method is: adding ultrapure water according to the mass-to-volume ratio of material to liquid of 1:15, and extracting at 65°C for 2 hours.

[0013] Preferably, in step (2), the conditions for dialysis are: the dialysis molecular weight is 3500 Da, the temperature is 4°C, the time is 48 h, and the water is changed every 4 - 10 h.

[0014] Preferably, in step (3), the conditions for column chromatography elution using a D101 macroporous adsorption resin column are: the eluent is 50% ethanol, the diameter and length of the column are 1.6 cm × 40 cm, the elution speed is 1 mL / min, and 10 mL / tube.

[0015] Preferably, in step (4), the conditions for column chromatography elution using a DEAE-52 column and a Superdex G-100 gel column are: the diameter and length of the column are 1.6 cm × 40 cm, the elution speed is 1 mL / min, and 10 mL / tube.

[0016] The application of the distiller's grains polysaccharide OIR disclosed by the present invention in the preparation of health products or drugs for improving immunity.

[0017] The total sugar content of OIR involved in the present invention is 94.99 ± 1.05%, and the relative molecular mass is 1.65×10 5Da. OIR is composed of guluronic acid, mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose and fucose. The molar ratio of each monosaccharide component calculated according to the peak area is 0.51:15.89:0.77:0.74:1.68:1.32:0.14:776.25:10.31:5.43:7.04:0.19. Among them, glucose has the highest proportion, which is 94.38%, indicating that OIR is glucan. And OIR contains α-configured glycosidic bonds, indicating that OIR is α-glucan; and OIR has good thermal stability.

[0018] The distiller's grains homogeneous polysaccharide prepared by the above method can be used as one of the active ingredients or the only active ingredient in the application of immunomodulation. The immunomodulation includes significantly promoting the proliferation of mouse macrophages RAW264.7, enhancing the cell phagocytosis ability, inducing NO production and the secretion of TNF-α, IL-6 and IL-1β.

[0019] Advantages of the present invention:

[0020] (1) The technical solution of the present invention can obtain a distiller's grains polysaccharide with excellent immunological effects by separating the crude extract of distiller's grains through DEAE-52 column chromatography. The polysaccharide yield is relatively high, which is beneficial to improving the added value of distiller's grains, realizing the transformation of waste into treasure, and reducing environmental pollution.

[0021] (2) The present invention proves that the distiller's grains polysaccharide OIR has good in vitro immunomodulatory effects, can significantly promote the proliferation of mouse macrophages RAW264.7, enhance the cell phagocytosis ability, and induce the production of NO and the secretion of TNF-α, IL-6 and IL-1β.

[0022] (3) The distiller's grains polysaccharide OIR provided by the present invention can be used in immunopotentiating products, including but not limited to vaccine adjuvants, functional foods, health products or drugs for immunological uses. Description of the drawings

[0023] Figure 1 a is the elution curve of the crude polysaccharide DYT of distiller's grains on cellulose DEAE-52 column chromatography; Figure 1 b is the elution curve of the polysaccharide component OI on Sephadex G-100 column chromatography;

[0024] Figure 2 is the glucose standard curve;

[0025] Figure 3 a is the standard curve drawn according to the logarithm of the weight-average molecular weight and the retention time of dextrans with different weight-average molecular weights; Figure 3 b is the GPC chromatogram of OIR;

[0026] Figure 4 a is the liquid chromatography of monosaccharide standard; Figure 4 b is the liquid chromatography of OIR;

[0027] Figure 5 is the Fourier transform infrared spectrum of OIR;

[0028] Figure 6 are the TGA and DTG curves of OIR pyrolysis;

[0029] Figure 7 is the proliferation effect of OIR on mouse macrophage RAW264.7;

[0030] Figure 8 is the effect of OIR on the phagocytic ability of macrophage RAW264.7;

[0031] Figure 9 is the effect of OIR on the release of nitric oxide by RAW264.7 macrophages;

[0032] Figure 10 is the effect of OIR on the release of tumor necrosis factor (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β) by macrophages. Detailed implementation mode

[0033] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] Example 1: Preparation method of distiller's grains polysaccharide OIR

[0035] (1) First, place the distiller's grains in an oven at 60 °C to constant weight, crush them, and pass through a 40-mesh sieve to obtain distiller's grains powder. Weigh 800 g of the distiller's grains powder, add ultrapure water according to a solid-liquid ratio of 1:15 (g / mL), extract at 65 °C for 2 h, and perform ultrasonic-assisted extraction for 30 min. Centrifuge and take the supernatant. Rotate and evaporate to concentrate the extract at 50 °C, add three volumes of absolute ethanol to make the final ethanol concentration 75%, and keep it at 4 °C for 12 h to precipitate the polysaccharide. Centrifuge to obtain the precipitate, redissolve it with ultrapure water, and rotate and evaporate to remove ethanol at 45 °C. Remove proteins by the Sevage method, dialyze (dialysis conditions: dialysis molecular weight is 3500 Da, temperature is 4 °C, time is 48 h, change water every 4 - 10 h), and then freeze-dry. Then, perform chromatography elution and decolorization using D101 macroporous adsorption resin. The eluent is 50% ethanol. The diameter and length of the column are 1.6 cm × 40 cm, the elution speed is 1 mL / min, 10 mL / tube. Collect the water-washed part and freeze-dry to obtain crude polysaccharide DYT from distiller's grains.

[0036] (2) Then, prepare a solution of crude polysaccharide DYT at 10 mg / mL, filter it through a 0.45-μm filter membrane, load 10 mL each time, perform DEAE-52 column chromatography, and elute with 0.1 mol / L NaCl to obtain OI. The elution curve is as Figure 1 shown in a. After freeze-drying, prepare a solution of OI at 10 mg / mL, filter it through a 0.45-μm filter membrane, load 10 mL each time, and then perform purification using Superdex G-100 gel. The elution curve is as Figure 1 shown in b. The conditions for chromatography elution using DEAE-52 column and Superdex G-100 gel column are: the diameter and length of the column are 1.6 cm × 40 cm, the elution speed is 1 mL / min, 10 mL / tube. After freeze-drying, obtain homogeneous polysaccharide OIR from distiller's grains.

[0037] Example 2: Identification of homogeneous polysaccharide OIR from distiller's grains

[0038] (1) Determination of total sugar content and relative molecular mass

[0039] The total sugar content was determined with a slight modification of the phenol-sulfuric acid method. The standard regression equation was fitted to obtain y = 5.6724x + 0.0696, R 2 = 0.996 ( Figure 2 as shown). Absorb the sample solution and measure the absorbance in the same way. Calculate the total sugar content in the polysaccharide sample OIR according to the standard curve.

[0040] Using a Shimadzu high-performance liquid chromatography system combined with a differential refractive index detector, Waters UltrahydrogeTM120 (7.8 mm × 300 mm), Waters Ultrahydroge TM250 (7.8 mm × 300 mm) and WatersUltrahydroge TM500 (7.8 mm × 300 mm) chromatographic columns were connected in series, and the column temperature was 40 °C. The mobile phase was 0.1 M NaNO3, the flow rate was 1 mL / min, and the homogeneity and molecular weight of OIR were determined.

[0041] Results: The total sugar content of OIR was 94.99 ± 1.05%; the relative molecular mass was 1.65×10 5 Da, (the results are as Figure 3 shown in Figure 3 Figures a and

[0042] (2) Determination of monosaccharide composition

[0043] The monosaccharide composition of OIR was determined with a slightly modified PMP pre-column derivatization method. Weigh 15 mg of OIR into a 10 mL hydrolysis tube, add 5 mL of 2 mol / L trifluoroacetic acid (TFA), fill with N 2 seal the tube (10 L / min, 1 min), and hydrolyze in an oven at 110 °C for 2 h; after cooling, take out 1 mL of the solution, add 1 mL of methanol, and blow dry with N 2 at 70 °C in a water bath. Repeat adding methanol and blowing dry with N 2 twice to remove TFA; add 1 mL of 0.3 mol / L NaOH solution to fully dissolve the residue to obtain a polysaccharide hydrolysis solution, which was diluted and then derivatized for determination. Accurately transfer 400 μL of the polysaccharide hydrolysis solution into a 5 mL stoppered test tube, add 400 μL of PMP methanol solution, and mix well by vortexing; react in a water bath at 70 °C for 2 h; take out and let it cool to room temperature; add 400 μL of 0.3 mol / L HCl for neutralization (pH 6 - 7), add 1200 μL of water, then add an equal volume of chloroform, mix well by vortexing and shaking, let it stand, and discard the chloroform phase. Extract twice in this way. Filter the aqueous phase through a 0.45 μm microporous membrane (water system) for HPLC injection analysis. The monosaccharide mixed standard solution was treated with the same derivatization method, and the results are as Figure 4 shown in

[0044] HPLC chromatographic detection: C18 chromatographic column (250 mm × 4.6 mm, 5 μm), column temperature 30 °C, flow rate 1 mL / min; mobile phase A: 100 mM sodium phosphate buffer (pH = 6.7), mobile phase B: acetonitrile; injection volume was 5 μL, and the detection wavelength was 250 nm.

[0045] Results: As Figure 4As shown in Figure b, OIR is composed of guluronic acid, mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose and fucose. The molar ratio of each monosaccharide component calculated based on the peak area is 0.51:15.89:0.77:0.74:1.68:1.32:0.14:776.25:10.31:5.43:7.04:0.19. Among them, glucose has the highest proportion in OIR, which is 94.38%, indicating that OIR is dextran.

[0046] (3) Infrared spectroscopy analysis

[0047] Weigh 2 mg of the dried OIR and mix it with anhydrous KBr powder, grind it, and press it into a tablet. Use the KBr tablet as a blank control, and scan it in the wavenumber range of 4000 - 400 cm -1 using an infrared spectroscopy scanner, and record the infrared spectrum.

[0048] Result: The Fourier transform infrared spectrum of OIR is as shown in Figure 5 Figure, and it has characteristic absorption peaks of polysaccharides between 4000 - 400 cm -1 . The broad and strong stretching peak at 3373.49 cm -1 is the stretching vibration of O - H. The stretching peak at 2926.48 cm -1 is the stretching vibration and bending vibration of C - H of alkyl groups; there is a strong absorption band near 1640 cm -1 which is the absorption band of bound water. Since polysaccharides are prone to absorb water, a relatively large absorption peak appears here; the relatively weak absorption peak around 1384 cm -1 may be caused by the C - H deformation vibration; the absorption peak near 1155 cm -1 is caused by the stretching vibrations of two C - O (C - O - C and C - O - H); the relatively strong absorption peaks at 1023.60 cm -1 and 1022.95 cm -1 are the characteristic absorption peaks of pyranoside, further verifying that OIR is dextran. The absorption peak at 855.99 cm -1 is the characteristic absorption peak of α - glycosidic bond, and OIR is α - dextran.

[0049] (4) Thermogravimetric analysis

[0050] Precisely weigh 3 mg of the sample OIR, place it in a crucible, compact it, and under the protection of nitrogen, increase the temperature from 30 °C to 600 °C at a heating rate of 10 °C / min to detect the mass change and thermal stability of the sample during the heating process.

[0051] Result: As shown in Figure 6They are the TGA and DTG curves of the thermal decomposition of OIR. The first mass loss of OIR (12.90%) occurs between 30.67 °C and 164.18 °C, mainly due to the evaporation of water in the polysaccharide. The second mass loss (72.63%) occurs between 265.31 °C and 448.10 °C, mainly due to the destruction of the chemical bonds of OIR and its thermal decomposition. At the same time, the temperature of the maximum weight loss rate of OIR is 316.30 °C, and it is relatively stable below 282 °C. The results show that OIR has good thermal stability.

[0052] Example 3 Effect of distiller's grains polysaccharide OIR on the immunity of mouse macrophages RAW264.7

[0053] (1) Experimental grouping and cell culture

[0054] Dissolve the polysaccharide with the special medium for RAW 264.7 cells, adjust the concentration of the OIR polysaccharide solution to 25, 50, 100, 200, 400, 800 μg / mL, filter it with a 0.22 μm microporous filter membrane and set aside.

[0055] After taking RAW264.7 macrophages out of liquid nitrogen, quickly thaw them, remove the preservation solution after simple centrifugation, add the special medium for RAW 264.7 cells, mix evenly and add them into a cell culture flask, place them in an incubator for culture, change the medium every 24 h. After the cells grow to 80 - 90% of the area of the culture flask, passage them to keep the cells in the logarithmic growth phase. After three passages, observe that the cell morphology is normal and then proceed to the next experiment.

[0056] (2) Proliferation experiment of RAW264.7 cells

[0057] Use the CCK-8 cell proliferation and toxicity detection kit to determine the effect of distiller's grains polysaccharide on the proliferation of RAW264.7 macrophages. The concentration of the OIR polysaccharide solution is 25, 50, 100, 200, 400, 800 μg / mL. Use LPS (1 μg / mL) as the positive control group. Select the cell culture flasks with RAW264.7 cells in the logarithmic growth phase, count them and prepare a cell suspension with a cell density of 1×10 5 cell / mL with complete medium. Add 100 μl of the cell suspension to each well for cell plating. Place the cells in an incubator at 37 °C and 5% CO 2 for 24 h, aspirate the cell medium, add 100 μL of the culture solution containing different concentrations of distiller's grains polysaccharide and the culture solution containing LPS to each well respectively. After culturing for 24 h, discard the cell culture supernatant containing the drug in each well, add 100 μl of the CCK-8 culture solution containing 10%, incubate in a 37 °C incubator for 4 h, and measure the absorbance value at 450 nm with an enzyme-labeled instrument. The cell survival rate is calculated according to the following formula:

[0058] Cell viability (%) = A 实验组 / A 空白组 × 100%

[0059] Results: To investigate the toxic effect of OIR on RAW264.7 cells, the CCK-8 method was used to detect the effect of OIR polysaccharide from distiller's grains at different concentrations (25, 50, 100, 200, 400, and 800 μg / mL) on the viability of mouse macrophages RAW264.7 after 24 hours of treatment. From Figure 7 the data, it can be seen that compared with the blank control group, OIR polysaccharide from distiller's grains was non-toxic to cells within the six concentration gradients measured. On the contrary, it could significantly increase the growth rate of macrophages, with statistical significance (P < 0.01). This indicates that the concentrations within this range can be selected for subsequent research.

[0060] (3) Phagocytic ability of RAW264.7 cells

[0061] The neutral red phagocytosis assay was used to determine the effect of OIR polysaccharide from distiller's grains on the phagocytic ability of RAW264.7 macrophages. The concentrations of the OIR polysaccharide solution were 25, 50, 100, 200, 400, and 800 μg / mL. LPS (1 μg / mL) was used as the positive control group. Cell culture flasks with RAW264.7 cells in the logarithmic growth phase were selected. After counting, cell suspensions with a density of 1 × 10 5 cell / mL were prepared with complete medium, and 100 μl of the cell suspension was added to each well for cell plating. The cells were placed in an incubator at 37°C and 5% CO2 for 24 h. The cell culture medium was aspirated, and 100 μL of the culture solution containing OIR polysaccharide at different concentrations and the culture solution containing LPS were added to each well. After washing with PBS, 100 μL of 0.1% neutral red solution was added to each well. After incubation in the incubator for 2 h, the cells were taken out, washed 2 or 3 times with PBS, 100 μL of cell lysate was added to each well, and then placed in the incubator for 12 h. The absorbance value at 540 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The relative phagocytosis rate was calculated as follows:

[0062] Relative phagocytosis rate (%) = (A 实验组 - A 空白组 ) / (A 阳性组 - A 空白组 ) × 100%

[0063] Results: As Figure 8As shown, compared with the blank control group, OIR significantly enhanced the phagocytic ability of macrophages (P<0.01), and with the increase of concentration, the phagocytic activity generally showed a trend of first increasing and then decreasing. When the mass concentration of OIR was 100 μg / mL, the phagocytic activity of RAW264.7 reached the maximum, and was significantly higher than that of the positive control LPS group (P<0.01). The results indicated that the distiller's grains polysaccharide OIR had the ability to activate mouse macrophages RAW264.7, and could promote its immune activity by enhancing the phagocytic activity of macrophages.

[0064] (4) Release level of NO from RAW264.7 cells

[0065] The effects of distiller's grains polysaccharide on the secretion of NO by RAW264.7 macrophages were determined using a total nitric oxide detection kit. The concentrations of the OIR polysaccharide solution were 25, 50, 100, 200, 400, and 800 μg / mL. LPS (1 μg / mL) was used as the positive control group. Cell culture flasks with RAW264.7 cells in the logarithmic growth phase were selected. After counting, cell suspensions with a density of 1×10 5 cell / mL were prepared with complete medium, and 100 μl of the cell suspension was added to each well for cell plating. The cells were placed in an incubator at 37°C and 5% CO2 for 24 h. The cell culture medium was aspirated, and 100 μL of the culture medium containing different concentrations of distiller's grains polysaccharide and the culture medium containing LPS were added to each well. After culturing for 24 h, the cell culture supernatants containing different drug concentrations were respectively aspirated into 1.5 mL centrifuge tubes. Centrifugation was carried out at 1200 rpm for 5 min, and the supernatant was collected to measure the release amount of NO according to the kit instructions.

[0066] Results: As Figure 9 shown, OIR could stimulate macrophages to release NO, showing a concentration gradient dependence. When the concentration of OIR was 800 μg / mL, the production amount of NO was 19.64±0.63 μM, which was significantly higher than that of the blank control group. In addition, the release of NO in the OIR treatment group was lower than that in the positive group, indicating that the effect of OIR was milder than that of lipopolysaccharide. These results showed that OIR could significantly promote the production of NO and had strong immunomodulatory activity.

[0067] (5) Determination of cytokine content

[0068] The effects of distiller's grains polysaccharide on the secretion of cytokines by RAW264.7 macrophages were determined using mouse tumor necrosis factor α (TNF-α), mouse interleukin-6 (IL-6) and mouse interleukin-1β (IL-1β) ELISA kits. The concentrations of OIR polysaccharide solution were 25, 50, 100, 200, 400, and 800 μg / mL respectively. LPS (1 μg / mL) was used as the positive control group. A cell culture flask with RAW264.7 cells in the logarithmic growth phase was selected. After counting, a cell suspension with a cell density of 1×10 5 cells / mL was prepared with complete medium, and 100 μl of the cell suspension was added to each well for cell plating. The cells were placed in an incubator at 37 °C and 5% CO2 for 24 h. The cell culture medium was aspirated, and 100 μL of the culture solution containing different concentrations of distiller's grains polysaccharide and the culture solution containing LPS were added to each well respectively. After culturing for 24 h, the cell culture supernatants containing different drug concentrations were respectively aspirated into 1.5 mL centrifuge tubes. Centrifuge at 1200 rpm for 5 min, and collect the supernatant to measure the release amounts of TNF-α, IL-6 and IL-1β respectively according to the kit instructions.

[0069] Results: As Figure 10 shown, compared with the blank control group, after treatment with different concentrations of OIR, the contents of TNF-α, IL-6 and IL-1β increased significantly (P<0.01), and showed a concentration gradient dependence. When the OIR concentration was 800 μg / mL, the contents of TNF-α, IL-6 and IL-1β reached 981.70±8.63 ng / L, 158.54±1.14 ng / L and 96.73±0.51 ng / L respectively. It indicates that OIR can significantly promote the secretion of TNF-α, IL-6 and IL-1β by RAW264.7 macrophages.

[0070] In summary, it can be seen that the novel distiller's grains homogeneous polysaccharide OIR isolated and purified from white wine distiller's grains of the present invention can be used as a good in vitro immunomodulatory substance to reduce LPS-induced phagocyte immune imbalance. Therefore, it is expected to be used as the main or only active ingredient to prepare health foods or drugs for immunomodulation. Compared with the prior art, it has made significant progress and is of great significance for the research, development and utilization of distiller's grains resources.

Claims

1. A distiller's grains polysaccharide OIR with immunomodulatory function, characterized in that, The distiller's grains polysaccharide OIR contains the following components: guluronic acid, mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose.

2. The lees polysaccharide OIR with immunomodulatory function according to claim 1, characterized in that, The molar ratio of guluronic acid, mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose is 0.51:15.89:0.77:0.74:1.68:1.32:0.14:776.25:10.31:5.43:7.04:0.

19.

3. A preparation method of the distiller's grains polysaccharide OIR according to any one of claims 1-2, characterized in that, The preparation method comprises the following steps: (1) Extraction: Dry and crush the distiller's grains, add ultrapure water, extract, and then perform ultrasonic-assisted extraction for 30 min; centrifuge, take the supernatant, concentrate the extract at 50 °C, add 3 volumes of absolute ethanol, and keep it at 4 °C for 12 h, then centrifuge to obtain the precipitate; (2) Deproteinization: After redissolving the precipitate obtained in step (1) with ultrapure water, remove ethanol by rotary evaporation at 45 °C, then add it to Sevage reagent in a volume ratio of 1:3, mix evenly, stir and centrifuge, discard the organic layer and the middle layer, and collect the aqueous layer; add 3 times the volume of Sevage reagent to the aqueous layer, repeat the above operation for extraction 2 times until there is no middle layer, collect and combine the aqueous layers; then dialyze the combined aqueous layer and freeze-dry to obtain the polysaccharide; (3) Decolorization: Subject the polysaccharide obtained in step (2) to macroporous adsorption resin column chromatography, collect the water-washing part, and freeze-dry to obtain the crude distiller's grains polysaccharide DYT; (4) Purification: Subject the crude polysaccharide DYT obtained in step (3) to DEAE-52 column chromatography, elute with 0.1 mol / L NaCl solution to obtain OI, and purify it through Superdex G-100 gel to obtain the distiller's grains polysaccharide OIR.

4. The preparation method according to claim 3, characterized in that, In step (1), the distiller's grains powder is less than 40 mesh; the specific extraction method is: add ultrapure water according to the mass-volume ratio of the material to the liquid of 1:15, and extract at 65 °C for 2 h.

5. The preparation method according to claim 3, characterized in that, In step (2), the dialysis conditions are: the dialysis molecular weight is 3500 Da, the dialysis temperature is 4 °C, the dialysis time is 48 h, and the water is changed every 4 - 10 h.

6. The preparation method according to claim 3, characterized in that, In step (3), a D101 macroporous adsorption resin column is selected for column chromatography elution, and the specific conditions are: the eluent is 50% ethanol, the diameter and length of the column are 1.6 cm × 40 cm, the elution speed is 1 mL / min, and 10 mL / tube.

7. The preparation method according to claim 3, characterized in that, In step (4), the conditions for column chromatography elution using a DEAE-52 column and a Superdex G-100 gel column are: the diameter and length of the column are 1.6 cm × 40 cm, the elution speed is 1 mL / min, and 10 mL / tube.

8. Use of the distiller's grains polysaccharide OIR according to any one of claims 1 - 2 or the distiller's grains polysaccharide OIR obtained by the preparation method according to any one of claims 3 - 7 in the preparation of health products or drugs for enhancing immunity.

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