A preparation method and application of polysaccharide in radish seeds

Abstract

The present invention relates to a preparation method and application of polysaccharides in radish seeds, and belongs to the technical field of polysaccharide preparation. The preparation method of the present invention comprises extracting with petroleum ether to remove fat-soluble substances, then using a shabby acid solution produced by fermentation of Aspergillus niger and a complex enzyme system for enzymolysis and extraction to obtain polysaccharides with high purity, and then using enzymolysis to obtain small molecular polysaccharides, and obtaining target polysaccharides after purification with a dextran gel column. The radish seed polysaccharide obtained by the present invention has good biological activity, has good protective effect on the skin, can improve the antioxidant activity of skin tissue, reduce skin damage caused by ultraviolet irradiation, and improve the water content of the skin after irradiation.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide preparation, and relates to a preparation method of polysaccharide in radish seeds and application thereof. Background Art

[0002] Radish seeds are the dried mature seeds of the cruciferous plant radish, which have the effects of digestion, relieving bloating, and reducing qi and phlegm. Radish seeds have a wide range of plant sources and abundant resources, and are produced all over the country. As one of the traditional medicinal and edible items, radish seeds have a wide range of applications. They can not only be used for oil extraction, but also have a long history of medicinal use.

[0003] The main components of radish seeds include glucosinolates, water-soluble alkaloids, volatile oils and fatty acids, flavonoids and polysaccharides. The prior art has disclosed that the use of radish seed polysaccharide coating has a good preservation effect and can reduce the occurrence of fruit rot and water loss. In the radish seed polysaccharide extraction and preparation method disclosed in the prior art, the extraction rate and purity of the polysaccharide are low, and there are few studies on the pharmacological activity of radish seed polysaccharides. Summary of the invention

[0004] The main purpose of the present invention is to provide a radish seed polysaccharide and a preparation method thereof. The radish seed polysaccharide has a protective effect on the skin and can prevent the skin from being damaged by ultraviolet radiation.

[0005] The present invention adopts the following technical solutions to achieve the above purpose:

[0006] A method for preparing polysaccharides from radish seeds mainly comprises the following steps:

[0007] Step 1, after crushing the radish seeds, heat them at 90-100° C. for 20-30 minutes, cool them to room temperature, add petroleum ether and soak them for 30-40 minutes, filter them, evaporate the filter residue I to remove the residual petroleum ether, and set them aside;

[0008] Step 2, inoculating the Aspergillus niger suspension I into a liquid culture medium I containing 35-38% wheat bran, 8-10% soybean meal, and 1-1.5% ammonium nitrate, fermenting and culturing at 28-30° C. for 70-72 hours, filtering, and obtaining a filtrate I for later use;

[0009] Step 3, inoculating the Aspergillus niger suspension II into a liquid culture medium II containing 20-25%, 30-40% corn cob powder, and 1.5-2% potassium nitrate, fermenting and culturing at 30-35° C. for 72-75 hours, filtering to obtain a filtrate II, and drying the filtrate II to obtain a crude enzyme mixture;

[0010] Step 4, after the filter residue I and the filtrate I are mixed at a solid-liquid ratio of 1:(8-10) kg / L, the crude enzyme mixture is added, and enzymolysis is performed at 35-40° C. for 45-48 hours, and then the temperature is raised to 70-80° C., and reflux extraction is performed to obtain an extract for use;

[0011] Step 5, adding xylanase to the extract, performing enzymolysis at 35-40°C for 6-8h, heating to inactivate the enzyme, adjusting the alcohol content of the enzymolysis solution to 50% and 70% in sequence, standing for 1-1.5h after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0012] Step 6, dissolving the precipitate and eluting with Sephadex G-20 dextran gel, retaining the eluate containing polysaccharides with a molecular weight of 30,000-80,000 Da, combining the eluates, and drying to obtain the target radish seed polysaccharide.

[0013] Further, the number of spores in the Aspergillus niger suspension I in step 2 is 5-10×10 8 Pieces / mL.

[0014] Furthermore, in the above step 2, the inoculation amount of the Aspergillus niger suspension I is 5-8 mL / 100 mL.

[0015] Furthermore, the content of citric acid in the filtrate I of step 2 is 0.5-0.8 g / L.

[0016] Further, the number of spores in the Aspergillus niger suspension II in step 3 is 3-5×10 9 Pieces / mL.

[0017] Furthermore, in the above step 3, the inoculation amount of the Aspergillus niger suspension II is 10-12 mL / 100 mL.

[0018] Furthermore, in the above step 3, in the crude enzyme mixture, the enzyme activity of the acid protease is 20000-25000 U / g, and the enzyme activity of the cellulase is 32000-35000 U / g.

[0019] Furthermore, in the above step 4, the amount of crude enzyme mixture added is 10-15 g / L.

[0020] Furthermore, in the above step 5, the amount of xylanase added is 10-12 g / L.

[0021] Furthermore, in the above step 5, the enzyme activity of xylanase is 100,000 U / g.

[0022] Furthermore, in the above step 6, the eluent is a mixed solution of methanol and water.

[0023] Furthermore, the volume ratio of methanol to water in the above eluent is (0.1-0.15):1.

[0024] The present invention also provides a specific use of the radish seed polysaccharide obtained above, that is, the radish seed polysaccharide obtained has a protective effect on the skin and can resist damage caused by ultraviolet radiation. Based on this effect, the radish seed polysaccharide can be used in the preparation of cosmetics.

[0025] The present invention has the following beneficial effects:

[0026] 1. In the preparation process of radish seed polysaccharide of the present invention, the radish seed is heated to remove part of the volatile components, and then the fat and other components are removed by petroleum ether extraction to avoid the presence of fat and other components affecting the extraction of polysaccharide components; secondly, the acid and enzyme production characteristics of Aspergillus niger fermentation are used, and the crude enzyme containing acid protease and cellulase obtained by Aspergillus niger fermentation is used to enzymolyze the radish seed; the fermentation liquid containing citric acid obtained by Aspergillus niger fermentation is used as the extraction solvent, and the complex enzyme system produced by Aspergillus niger fermentation is used, in which the acid protease enzymatically releases protein, and the protein is precipitated under acidic conditions; then, active enzymes such as cellulase and pectinase and other enzymes are used for enzymolysis to destroy the cell structure and release polysaccharides, and polysaccharides that are not easily soluble under acidic conditions are precipitated, and then reflux extraction is performed to improve the extraction rate of crude polysaccharides; the crude polysaccharides are further enzymolyzed to obtain small molecular weight polysaccharides, and then ethanol precipitation and gel column purification are performed to obtain the target polysaccharide of the present invention.

[0027] 2. Pharmacological experiments have confirmed that the radish seed polysaccharide obtained in the present invention has a good skin protection effect. It has good moisturizing properties, increases the water content of the skin after ultraviolet irradiation, increases the antioxidant activity of skin tissue, reduces inflammatory reactions, and maintains collagen activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Macroscopic evaluation of mouse dorsal skin;

[0029] Figure 2 : HE staining of mouse skin tissue, where A is the blank group mice, B is the model group mice, C is the test group 1 mice, and D is the test group 4 mice. DETAILED DESCRIPTION

[0030] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art all fall within the scope of protection of the claims of this application. The Aspergillus niger used in the following embodiments was purchased from the General Microbiological Center of the China Microbiological Culture Collection Administration Committee. It is a commercial strain and is used after laboratory expansion culture; the single hydrolases (xylanase, cellulase) used are all existing commercially available products, which can be purchased through online or offline channels.

[0031] Example 1

[0032] Step 1, taking dried radish seeds and crushing them, passing them through a 150-mesh sieve, placing the radish seed powder in a steamer, heating them at 90° C. for 30 minutes, cooling them to room temperature, adding petroleum ether at a material-liquid ratio of 1:8, kg / L, soaking them for 40 minutes, filtering them, taking the filter residue I, spreading them out to evaporate naturally to remove the residual petroleum ether and setting them aside;

[0033] Step 2: prepare liquid culture medium I according to the ratio of 38% wheat bran, 8% soybean meal and 1% ammonium nitrate, adjust the pH to 4, and sterilize; inoculate Aspergillus niger suspension I into the culture medium at an inoculum volume of 8 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC 3.15297 is activated and expanded according to the instructions, and then prepared into a spore number of 10×10 8 niger suspension of 1 g / mL), fermented and cultured at 30°C for 70 h, filtered, and obtained filtrate I containing 0.6 g / L citric acid for later use;

[0034] Step 3: prepare liquid culture medium II according to the ratio of 25% rice bran, 30% corn cob powder and 2% potassium nitrate, adjust the pH to 5.5, and sterilize; inoculate Aspergillus niger suspension II into the culture medium at an inoculum volume of 10 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC3.11455 is activated and expanded according to the instructions, and then prepared into a spore number of 5×10 9 niger suspension of 100 g / mL), fermented and cultured at 35°C for 72 hours, filtered to obtain filtrate II, and dried to obtain a crude enzyme mixture containing 25000 U / g acid protease and 32000 U / g cellulase;

[0035] Step 4, after the filter residue I and the filtrate I are mixed at a solid-liquid ratio of 1:10, kg / L, 15 g / L of the crude enzyme mixture is added, and enzymolysis is performed at 35° C. for 48 hours, and then the temperature is raised to 80° C., reflux extraction is performed, and the extract is obtained for standby use;

[0036] Step 5, adding 12 g / L xylanase with an enzyme activity of 100,000 U / g to the extract, performing enzymolysis at 40°C for 6 h, heating to 90°C to inactivate the enzyme, cooling to room temperature, and adjusting the alcohol content of the enzymolysis solution to 50% and 70% with anhydrous ethanol in sequence, standing for 1 h after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0037] Step 6, dissolve the precipitate in an appropriate amount of water, load the sample on Sephadex G-20 dextran gel, elute with a methanol-water mixed solution with a volume ratio of 0.15:1, collect the eluate, detect, retain the eluate containing polysaccharides with a molecular weight of 30000-80000Da, combine the eluates, and dry to obtain the target radish seed polysaccharide.

[0038] Example 2

[0039] Step 1, taking dried radish seeds and crushing them, passing them through a 150-mesh sieve, placing the radish seed powder in a steamer, heating them at 100° C. for 20 minutes, cooling them to room temperature, adding petroleum ether at a material-liquid ratio of 1:10, kg / L, soaking them for 30 minutes, filtering them, taking the filter residue I, spreading them out to evaporate naturally to remove the residual petroleum ether and setting them aside;

[0040] Step 2: prepare liquid culture medium I according to the ratio of 35% wheat bran, 10% soybean meal and 1.5% ammonium nitrate, adjust the pH to 3, and sterilize; inoculate Aspergillus niger suspension I into the culture medium at an inoculum volume of 5 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC 3.15297 is activated and expanded according to the instructions, and then prepared into a spore number of 5×10 8 niger suspension of 1 g / mL), fermented and cultured at 28°C for 72 h, filtered, and the filtrate I containing 0.5 g / L citric acid was obtained for later use;

[0041] Step 3: Prepare liquid culture medium II according to the ratio of 20% rice bran, 40% corncob powder and 1.5% potassium nitrate, adjust the pH to 4.5, and sterilize; inoculate Aspergillus niger suspension II into the culture medium at an inoculum volume of 12 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC3.11455 is activated and expanded according to the instructions, and then prepared into a spore number of 3×10 9 The mixture was mixed with an Aspergillus niger suspension having an activity of 10000 U / g and a cellulase having an activity of 34000 U / g, and then fermented and cultured for 75 hours at 30°C, filtered to obtain a filtrate II, and dried to obtain a crude enzyme mixture containing an acid protease having an activity of 20000 U / g and a cellulase having an activity of 34000 U / g;

[0042] Step 4, after the filter residue I and the filtrate I are mixed at a solid-liquid ratio of 1:8, kg / L, 10 g / L of the crude enzyme mixture is added, and enzymolysis is performed at 40° C. for 45 hours, and then the temperature is raised to 70° C., and reflux extraction is performed to obtain an extract for standby use;

[0043] Step 5, adding 10 g / L xylanase with an enzyme activity of 100,000 U / g to the extract, performing enzymolysis at 35° C. for 8 h, heating to 80° C. to inactivate the enzyme, cooling to room temperature, and adjusting the alcohol content of the enzymolysis solution to 50% and 70% with anhydrous ethanol in sequence, standing for 1.5 h after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0044] Step 6, dissolve the precipitate in an appropriate amount of water, load the sample on Sephadex G-20 dextran gel, elute with a methanol-water mixed solution with a volume ratio of 0.1:1, collect the eluate, detect, retain the eluate containing polysaccharides with a molecular weight of 30000-80000Da, combine the eluates, and dry to obtain the target radish seed polysaccharide.

[0045] Example 3

[0046] Step 1, taking dried radish seeds and crushing them, passing them through a 150-mesh sieve, placing the radish seed powder in a steamer, heating them at 100° C. for 25 minutes, cooling them to room temperature, adding petroleum ether at a material-liquid ratio of 1:9, kg / L, soaking them for 35 minutes, filtering them, taking the filter residue I, spreading them out to evaporate naturally to remove the residual petroleum ether and setting them aside;

[0047] Step 2: Prepare liquid culture medium I according to the ratio of 37% wheat bran, 9% soybean meal, and 1.3% ammonium nitrate, adjust the pH to 4.0, and sterilize; inoculate Aspergillus niger suspension I into the culture medium at an inoculum volume of 7 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC 3.15297 is activated and expanded according to the instructions, and then prepared into a spore number of 8×10 8 niger suspension of 1 g / mL), fermented and cultured at 30°C for 72 h, filtered, and the filtrate I containing 0.8 g / L citric acid was obtained for later use;

[0048] Step 3: Prepare liquid culture medium II according to the ratio of 23% rice bran, 35% corn cob powder and 1.8% potassium nitrate, adjust the pH to 4.5, and sterilize; inoculate Aspergillus niger suspension II into the culture medium at an inoculum volume of 11 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC3.11455 is activated and expanded according to the instructions, and then prepared into a spore number of 4×10 9 The mixture was mixed with an Aspergillus niger suspension of 100 g / mL, and fermented at 30°C for 75 hours, filtered to obtain a filtrate II, and dried to obtain a crude enzyme mixture containing an acid protease with an activity of 23400 U / g and a cellulase with an activity of 34800 U / g.

[0049] Step 4, after the filter residue I and the filtrate I are mixed at a solid-liquid ratio of 1:9, kg / L, 13 g / L of the crude enzyme mixture is added, and enzymolysis is performed at 40° C. for 45 hours, and then the temperature is raised to 80° C., and reflux extraction is performed to obtain an extract for standby use;

[0050] Step 5, adding 11 g / L xylanase with an enzyme activity of 100,000 U / g to the extract, performing enzymolysis at 40°C for 7 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the alcohol content of the enzymolysis solution to 50% and 70% with anhydrous ethanol in sequence, standing for 1.5 h after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0051] Step 6, dissolve the precipitate in an appropriate amount of water, load the sample on Sephadex G-20 dextran gel, elute with a methanol-water mixed solution with a volume ratio of 0.13:1, collect the eluate, detect, retain the eluate containing polysaccharides with a molecular weight of 30000-80000Da, combine the eluates, and dry to obtain the target radish seed polysaccharide.

[0052] Comparative Example 1

[0053] The dried radish seeds were crushed, passed through a 150-mesh sieve, placed in a Soxhlet extractor, petroleum ether was added, refluxed for degreasing twice, each time for 2 hours, and the petroleum ether extract was discarded; refluxed with 80% ethanol for extraction for 1 hour, and the extract was discarded; after the residual ethanol was evaporated and removed, refluxed with distilled water for extraction twice, each time for 2 hours, filtered under reduced pressure, the filtrates were combined, concentrated, and dried to obtain radish seed polysaccharides.

[0054] Comparative Example 2

[0055] Step 1, taking dried radish seeds and crushing them, passing them through a 150-mesh sieve, placing the radish seed powder in a steamer, heating them at 100° C. for 25 minutes, cooling them to room temperature, adding petroleum ether at a material-liquid ratio of 1:9, kg / L, soaking them for 35 minutes, filtering, taking the filter residue, spreading them out for natural volatilization to remove the residual petroleum ether and setting them aside;

[0056] Step 2: prepare a liquid culture medium according to the ratio of 37% wheat bran, 9% soybean meal, and 1.3% ammonium nitrate, adjust the pH to 4.0, and sterilize; inoculate the culture medium with an Aspergillus niger suspension at an inoculum volume of 7 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC3.15297 is activated and expanded according to the instructions, and then prepared into a spore number of 8×10 8 niger suspension of 1 g / mL), fermented and cultured at 30°C for 72 h, filtered, and the fermented liquid containing 0.8 g / L citric acid was obtained for later use;

[0057] Step 3, after mixing the filter residue obtained in step 1 with the fermentation liquid at a solid-liquid ratio of 1:9, kg / L, adding 13 g / L of cellulase with an enzyme activity of 50,000 U / g, performing enzymatic hydrolysis at 40° C. for 45 hours, then heating to 80° C., reflux extraction, and obtaining an extract for standby use;

[0058] Step 4, adding 11 g / L xylanase with an enzyme activity of 100,000 U / g to the extract, performing enzymolysis at 40°C for 7 hours, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the alcohol content of the enzymolysis solution to 50% and 70% with anhydrous ethanol in sequence. After each adjustment, standing for 1.5 hours, centrifuging to obtain a precipitate, namely, radish seed polysaccharide.

[0059] Comparative Example 3

[0060] Step 1: prepare a liquid culture medium according to the ratio of 23% rice bran, 35% corn cob powder, and 1.8% potassium nitrate, adjust the pH to 4.5, and sterilize; inoculate the culture medium with an Aspergillus niger suspension at an inoculum volume of 11 mL / 100 mL (the Aspergillus niger strain with the deposit number of CGMCC3.11455 is activated and expanded according to the instructions, and then prepared into a spore number of 4×10 9 The mixture was mixed with an Aspergillus niger suspension of 100 g / mL, and fermented at 30°C for 75 hours, filtered to obtain a filtrate II, and dried to obtain a crude enzyme mixture containing an acid protease with an activity of 22,600 U / g and a cellulase with an activity of 315,000 U / g;

[0061] Step 2, taking the dried radish seeds and crushing them, passing them through a 150-mesh sieve, mixing them with water at a solid-liquid ratio of 1:9, kg / L, adding 13 g / L of the crude enzyme mixture, performing enzymolysis at 40°C for 45 hours, then heating the temperature to 80°C, reflux extraction, obtaining an extract, adjusting the alcohol content of the enzymolysis solution to 80% with anhydrous ethanol, standing for 1.5 hours, centrifuging, and obtaining a precipitate for standby use;

[0062] Step 3, dissolving the precipitate with an appropriate amount of water, loading the precipitate onto Sephadex G-20 dextran gel, eluting the precipitate with a methanol-water mixed solution having a volume ratio of 0.13:1, collecting the eluate, combining the eluate and drying the eluate to obtain radish seed polysaccharide.

[0063] Comparative Example 4

[0064] Step 1, taking dried radish seeds and crushing them, passing them through a 150-mesh sieve, placing the radish seed powder in a steamer, heating them at 100° C. for 25 minutes, cooling them to room temperature, adding petroleum ether at a material-liquid ratio of 1:9, kg / L, soaking them for 35 minutes, filtering, taking the filter residue, spreading them out for natural volatilization to remove the residual petroleum ether and setting them aside;

[0065] Step 2, after the filter residue is mixed with water at a solid-liquid ratio of 1:9, kg / L, citric acid is added to adjust the pH to 5.0, 10 g / L of cellulase with an enzyme activity of 50,000 U / g and 11 g / L of xylanase with an enzyme activity of 100,000 U / g are added, enzymolysis is carried out at 40°C for 45 hours, and then the temperature is raised to 80°C, reflux extraction is performed to obtain an extract, and the alcohol content of the enzymolysis solution is adjusted to 50% and 70% with anhydrous ethanol in sequence, and the solution is allowed to stand for 1.5 hours after each adjustment, centrifuged, and a precipitate is obtained for standby use;

[0066] Step 3, dissolving the precipitate with an appropriate amount of water, loading the precipitate onto Sephadex G-20 dextran gel, eluting the precipitate with a methanol-water mixed solution having a volume ratio of 0.13:1, collecting the eluate, combining the eluate and drying the eluate to obtain radish seed polysaccharide.

[0067] Experimental test of the protective and repair effects of radish seed polysaccharide on photodamaged skin

[0068] 1. Test materials and process

[0069] 1.1 Test materials

[0070] Experimental animals: SPF KM mice, 7 weeks old, weight 25±2 g, license number: SYXK(Lu) 2024 0016, adaptive feeding for 10 days.

[0071] Test samples: radish seed polysaccharides obtained in Examples 1 to 3, and radish seed polysaccharides obtained in Comparative Examples 1 to 4.

[0072] 1.2 Experimental process

[0073] The mice were randomly divided into 9 groups, namely blank group, model group, test group 1, test group 2, test group 3, test group 4, test group 5, test group 6, and test group 7, with 10 mice in each group. The back hair of the mice was removed, and the hair removal area was about 2cm×4cm. Except for the blank group mice, the other groups of mice were treated with 200mJ / cm 2 The hair removal area on the back of mice was irradiated with UVB at a distance of about 10 cm, once a day, each irradiation lasted for 30 minutes, and irradiation lasted for 30 consecutive days. 30 minutes before each irradiation, mice in each group were treated as follows:

[0074] Model group: physiological saline was applied to the hairless skin on the back;

[0075] Experimental group 1: The radish seed polysaccharide obtained in Example 1 was applied to the depilated skin on the back;

[0076] Experimental group 2: The radish seed polysaccharide obtained in Example 2 was applied to the depilated skin on the back;

[0077] Experimental group 3: The radish seed polysaccharide obtained in Example 3 was applied to the depilated skin on the back;

[0078] Experimental group 4: applying the radish seed polysaccharide obtained in comparative example 1 to the depilated skin on the back;

[0079] Experimental group 5: The radish seed polysaccharide obtained in comparative example 2 was applied to the depilated skin on the back;

[0080] Experimental group 6: The radish seed polysaccharide obtained in comparative example 3 was applied to the depilated skin on the back;

[0081] Experimental group 7: The radish seed polysaccharide obtained in comparative example 4 was applied to the depilated skin on the back;

[0082] The mice in the blank group were only smeared with saline after hair removal without UVB irradiation. During the experiment, the hair removal area on the back of the mice was kept hairless.

[0083] 1.3 Detection indicators

[0084] After the last irradiation, the skin moisture content of the hair removal area on the back of each group of mice was measured using a skin moisture content tester in the living state, and three areas were selected for measurement and the average value was taken.

[0085] The skin tissue from the depilated area on the back of the mouse was obtained, sliced ​​and stained with HE to observe the state of the skin tissue.

[0086] Skin tissue from the depilated area on the back of mice was obtained to prepare tissue homogenate, and the superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) content, and hydroxyproline (HYP) content in the skin tissue were detected according to the instructions of the detection kit.

[0087] 2. Test results and analysis

[0088] 2.1 Comparison of mouse skin conditions

[0089] According to the relevant evaluation standards in existing literature, the condition of the skin in the hair-depleting area on the back of the mice was observed and scored during the experiment.

[0090] Table 1 Skin condition scoring criteria

[0091]

[0092] The back skin of mice in the blank group was ruddy, smooth, wrinkle-free and elastic; the skin of mice in the model group was dry, rough and hard, with obvious wrinkles, severe erythema and ulceration, showing typical photoaging phenomena; some mice in test groups 1-3 had fine wrinkles, but the overall condition was good; the wrinkles of mice in test groups 4-7 were relatively obvious, with a small amount of erythema, and the skin was rough overall. Figure 1 It can be seen from the evaluation curve of the mouse back skin that the radish seed polysaccharides prepared in Examples 1 to 3 of the present invention have a good improvement effect on mouse skin damage caused by ultraviolet irradiation.

[0093] 2.2 Changes in mouse skin tissue

[0094] Compared with the blank group, the skin moisture content of the mice in the model group was significantly reduced and the skin was dry; although the skin moisture content of the mice in the test groups 1-3 was reduced, there was no significant difference compared with the blank group; the skin moisture content of the mice in the test groups 4-7 was higher than that in the model group, but lower than that in the test groups 1-3.

[0095] Table 2 Skin water content

[0096]

[0097] Note: Compared with the model group, * P<0.05,# P<0.01

[0098] HE staining results showed that the skin structure of the blank group mice was intact, and the epidermal cells were arranged regularly; the skin surface of the model group mice was abnormally thickened, the stratum corneum was shed, the collagen fibers were firm, broken or curled, and the structure was loose and disordered; the skin epidermis of the mice in test groups 1-3 had no abnormal thickening, the stratum corneum was relatively intact, the collagen fibers in the dermis were arranged in a steam-like manner, and the breakage was reduced; the skin of the mice in test groups 4-7 had no abnormal thickening, but the collagen fibers were reduced, the arrangement was slightly disordered, and the density distribution was uneven.

[0099] 2.3 Changes in SOD, GSH-Px activity and MDA, HYP content in mouse skin tissue

[0100] Compared with the blank group, the SOD, GSH-Px activity and HYP content in the skin tissue of mice in the model group were significantly decreased, and the MDA content was significantly increased; the SOD, GSH-Px activity and HYP content in the skin tissue of mice in test groups 1-3 were significantly increased, and the MDA content was significantly decreased; the SOD, GSH-Px activity and HYP content in the skin tissue of mice in test groups 4-7 were increased, and the MDA content was decreased, but the overall change level was not as good as that in test groups 1-3.

[0101] Table 3 SOD, GSH-Px activities and MDA, HYP contents in mouse skin tissue

[0102]

[0103] Note: Compared with the model group, * P<0.05, # P<0.01

Claims

1. A method for preparing polysaccharides from radish seeds, characterized in that: The preparation method comprises the following steps: Step 1, after crushing the radish seeds, heat them at 90-100° C. for 20-30 minutes, cool them to room temperature, add petroleum ether and soak them for 30-40 minutes, filter them, evaporate the residue I to remove the residual petroleum ether, and set them aside; Step 2, inoculating Aspergillus niger suspension I into a liquid culture medium I containing 35-38% wheat bran, 8-10% soybean meal, and 1-1.5% ammonium nitrate, fermenting and culturing at 28-30° C. for 70-72 hours, filtering, and obtaining filtrate I for standby use; the Aspergillus niger suspension I is prepared by expanding the culture of Aspergillus niger with a preservation number of CGMCC 3.15297; the content of citric acid in the filtrate I is 0.5-0.8 g / L; Step 3, inoculating Aspergillus niger suspension II into a liquid culture medium II containing 20-25%, 30-40% corn cob powder, and 1.5-2% potassium nitrate, fermenting and culturing at 30-35° C. for 72-75 hours, filtering to obtain filtrate II, and drying the filtrate II to obtain a crude enzyme mixture; the Aspergillus niger suspension II is prepared by expanding the culture of Aspergillus niger with a preservation number of CGMCC 3.11455; Step 4, after the filter residue I and the filtrate I are mixed at a solid-liquid ratio of 1:(8-10) kg / L, a crude enzyme mixture is added, and enzymolysis is performed at 35-40° C. for 45-48 hours, and then the temperature is raised to 70-80° C., and reflux extraction is performed to obtain an extract for standby use; the amount of the crude enzyme mixture added is 10-15 g / L; Step 5, adding xylanase to the extract, performing enzymolysis at 35-40°C for 6-8h, heating to inactivate the enzyme, adjusting the alcohol content of the enzymolysis solution to 50% and 70% in sequence, standing for 1-1.5h after each adjustment, centrifuging, and obtaining a precipitate for later use; Step 6, dissolving the precipitate and eluting with Sephadex G-20 dextran gel, retaining the eluate containing polysaccharides with a molecular weight of 30,000-80,000 Da, combining the eluates, and drying to obtain the target radish seed polysaccharide; the eluate is a mixed solution of methanol and water; the volume ratio of methanol to water is (0.1-0.15):

1.

2. The preparation method according to claim 1, characterized in that The number of spores in the Aspergillus niger suspension I in step 2 is 5-10×10 8 / mL, and the inoculum volume is 5-8mL / 100mL.

3. The preparation method according to claim 1, characterized in that: The number of spores in the Aspergillus niger suspension II in step 3 is 3-5×10 9 / mL, and the inoculum volume is 10-12mL / 100mL.

4. The preparation method according to claim 1, characterized in that: The enzyme activity of the acidic protease in the crude enzyme mixture obtained in step 3 is 20000-25000 U / g, and the enzyme activity of the cellulase is 32000-35000 U / g.

5. The preparation method according to claim 1, characterized in that: In step 5, the amount of xylanase added is 10-12 g / L; the enzyme activity of the xylanase is 100,000 U / g.

6. Use of radish seed polysaccharide prepared by the preparation method according to any one of claims 1 to 5 in cosmetics.

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