Extraction and purification method of polysaccharide from hawthorn seeds

Abstract

The invention relates to a method for extracting and purifying polysaccharides from hawthorn seeds, and belongs to the technical field of polysaccharide extraction and purification. The method of the invention comprises microbial fermentation, enzymolysis, adsorbent impurity removal, alcohol precipitation to obtain crude polysaccharides, and then gel column separation and purification to obtain target hawthorn seed polysaccharides. The hawthorn seed polysaccharides prepared by the method of the invention have a high yield and high purity, and the obtained hawthorn seed polysaccharides have good antioxidant effects.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction and purification, and relates to a method for extracting and purifying polysaccharides in hawthorn seeds. Background Art

[0002] Hawthorn belongs to the genus Crataegus of the subfamily Apple of the Rosaceae family. It is an important plant in the Rosaceae family and a specialty fruit tree in my country. Hawthorn is famous for its unique flavor, rich nutrition, and dual use as medicine and food. my country's hawthorn products mainly include hawthorn pectin foods such as hawthorn cake and fruit danpi, hawthorn whole fruit products such as hawthorn canned food and hawthorn preserves, as well as hawthorn wine and hawthorn beverages. Hawthorn processing by-products mainly include hawthorn seeds and hawthorn residues. Hawthorn seeds are the seeds of hawthorn, accounting for 1 / 3 of the total weight of hawthorn. Preliminary studies have shown that the ingredients in hawthorn seeds are similar to those in the flesh. Hawthorn seeds are also rich in active substances such as flavonoids, and are high-quality resources for the development of flavonoids. Although the development and utilization of hawthorn seeds are becoming more and more popular, there has been no systematic analysis of the ingredients in hawthorn seeds, especially the polysaccharide components in hawthorn seeds, and no relevant reports have been seen. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for extracting and purifying polysaccharides from hawthorn seeds. The polysaccharides prepared by the method have high yield and purity and good antioxidant activity.

[0004] The present invention adopts the following technical solutions to achieve the above purpose:

[0005] The method for extracting and purifying polysaccharides from hawthorn seeds mainly comprises the following steps:

[0006] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder, adding β-sitosterol and mixing, soaking in a 10-12% H2O2 solution for 1.5-2h, then washing with deionized water, heating at 90-100°C for 40-60min, cooling to room temperature, drying to obtain a preliminary material for later use;

[0007] Step B, adjusting the water content of the initial material to 40-50%, adjusting the pH to 7.0-7.2, inoculating the bacterial solution of the wood porphyra, and fermenting at 25-30° C. for 1.5-2 days; after the fermentation is completed, heating at 90-100° C. for 20-30 minutes, cooling to room temperature, adjusting the pH to 6.0-6.5, inoculating the Pichia pastoris bacterial solution, fermenting again at 25-30° C. for 1-1.5 days, and after the second fermentation is completed, heating at 90-100° C. for 20-30 minutes again, cooling to room temperature, and obtaining the fermented product for standby use;

[0008] Step C, mixing the fermented product with a 10-12% ethanol solution at a material-liquid ratio of 1:(8-10) kg / L, extracting under reflux at 60-65°C, cooling to room temperature, adding 0.08-0.1 kg / L of adsorbent, standing for 6-8 hours, centrifuging, and reserving the supernatant for later use;

[0009] Step D, adjusting the pH of the supernatant to 5.0-5.5, adding 10-12 g / L mannanase and 6-8 g / L β-glucanase and mixing, performing enzymolysis at 35-40° C. for 3-4 hours, heating to 70-80° C. to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30-40 minutes after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0010] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a methanol-water mixed solution with a molecular weight cutoff of 2×10 6 ~5×10 7 The eluates of Da were combined, methanol was removed, and the eluates were dried to obtain the target hawthorn seed polysaccharide.

[0011] As a preferred embodiment, in the above step A, the amount of β-sitosterol added is 10-15% by weight of the hawthorn seed powder.

[0012] As a preferred method, in the above step B, the number of colonies in the fungus solution is 8×10 7 / mL, the number of colonies in the Pichia pastoris solution is 3×10 7 Pieces / mL.

[0013] As a preferred embodiment, in the above step B, the inoculation amount of the seed solution of F. oxysporum is 3-5% of the mass of the initial material, and the inoculation amount of the seed solution of Pichia pastoris is 8-10% of the mass of the initial material.

[0014] As a preferred embodiment, the adsorbent in the above step C is modified brewer's grains microspheres.

[0015] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0016] Step 1, after drying the brewer's grains, soak them in a phosphoric acid solution with a mass fraction of 30-40% for 6-8 hours, filter them, and dry them; calcine them in an oxygen-free environment at 400-500° C. for 1-1.5 hours, and cool them to room temperature to obtain a primary product for use;

[0017] Step 2, soaking the primary product in a potassium permanganate solution with a mass fraction of 10-12% for 2-4 hours, filtering, and drying; adding oyster shell powder and mixing, calcining in an oxygen-free environment at 700-800° C. for 2.5-3 hours, cooling to room temperature, washing with deionized water until neutral, and drying to obtain a secondary product for later use;

[0018] Step 3, adding the by-product, succinic anhydride, FeCl3, and polyethyl methacrylate to molten chitosan, placing it on a magnetic stirrer and stirring it for 1-1.5 hours to obtain a mixed solution; while stirring, adding 2 mol / L NaOH solution to the mixed solution, letting it stand for 6-8 hours, and washing it with deionized water to obtain modified brewer's grains microspheres.

[0019] Furthermore, in the above step 2, the mass ratio of the primary product to the oyster shell powder is 1:(0.3-0.5).

[0020] Furthermore, in the above step 3, the mass ratio of the by-product, succinic anhydride, FeCl3, polyethyl methacrylate, and chitosan is 1:(0.08-0.12):(0.1-0.15):(0.2-0.3):(1.5-1.8).

[0021] Furthermore, in the above step 3, the amount of NaOH solution added is 8-10 times the volume of the mixed solution.

[0022] As a preferred embodiment, the volume ratio of methanol to water in the methanol-water mixed solution in the above step E is (0.1-0.12):1.

[0023] The present invention has the following beneficial effects:

[0024] The hawthorn seed polysaccharide prepared by the invention has high purity and good antioxidant effect; hawthorn seeds contain a large amount of lignin components, and in the preparation process, the hawthorn seeds are soaked in hydrogen peroxide and heated at high temperature to change the structure of lignin, and the hawthorn seeds are activated, and beta-sitosterol is added as a stabilizer to avoid the influence of high temperature on the polysaccharide components; the hawthorn seeds that have been initially activated are fermented with phyllopodiaceae and heated at high temperature to further change the lignin structure, and then the lignin is further modified by yeast fermentation and high temperature heating to improve the solubility, and the lignin is decomposed into monosaccharides through microbial fermentation; the modified hawthorn seeds can significantly improve the extraction rate of polysaccharides when reflux extraction is performed; after extraction, the components in the extract are relatively complex, and there are multiple components such as polysaccharides, flavonoids, proteins, etc. After the adsorbent prepared by the invention is added, impurities can be effectively removed. In the preparation process of the adsorbent, brewer's grains are calcined at high temperature to form biochar. During the calcination process, oyster shell powder is added. The oyster shell powder is calcined to produce carbon dioxide gas, which etches the pores of the brewer's grains to obtain biochar with small and abundant pore sizes. When prepared into microspheres with other ingredients, it can effectively adsorb ingredients such as proteins without affecting polysaccharides, reduce the occurrence of undesired adsorption, and effectively remove impurities. The obtained hawthorn seed crude polysaccharide is enzymatically hydrolyzed and purified using a dextran gel column to obtain the target hawthorn seed polysaccharide. Experimental measurements have shown that it has high antioxidant activity. DETAILED DESCRIPTION

[0025] 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. In the following embodiments, the inoculated microbial bacterial liquid is obtained by culturing and dilution in the laboratory. The strains of CGMCC 5.1083 Pyricularia oxysporum and CGMCC 2.5688 Pichia pastoris purchased from the China General Microbiological Culture Collection Administration Center are activated and expanded to seed liquid according to the instructions, and the seed liquid is diluted and prepared into a colony count of 8×10 7 / mL of the fungus solution, the colony count was 3×10 7 The activity of the mannanase (CAS No.: 37288-54-3) and the activity of the β-glucanase (CAS No.: 9025-70-1) used was 10000u / g and 40u / mg, respectively, both of which are commercially available products.

[0026] Example 1 Preparation and purification of hawthorn seed polysaccharide

[0027] First, prepare the adsorbent (modified brewer's grains microspheres):

[0028] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0029] Step 1, after drying 10 kg of brewer's grains, soak them in a 40% by mass phosphoric acid solution for 6 hours, filter and dry them; calcine them in an oxygen-free environment at 500° C. for 1 hour, cool them to room temperature, and obtain a primary product for use;

[0030] Step 2, taking 10 kg of the prepared primary product and placing it in a 10% potassium permanganate solution by mass fraction and soaking it for 4 hours, filtering it, and drying it; adding 5 kg of oyster shell powder and mixing it evenly, placing it in an oxygen-free environment at 700° C. and calcining it for 3 hours, cooling it to room temperature, washing it with deionized water until it is neutral, and drying it to obtain a secondary product for use;

[0031] Step 3, take 10kg of the prepared by-product, 1.2kg of succinic anhydride, 1.5kg of FeCl3, and 2kg of polyethyl methacrylate, add them to 18kg of molten chitosan, place it on a magnetic stirrer and stir for 1.5h to obtain a mixed solution; under stirring, add 10 times the volume of 2mol / L NaOH solution to the mixed solution, let it stand for 8h, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0032] Secondly, prepare hawthorn seed polysaccharide:

[0033] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1 kg of β-sitosterol and mixing, placing in a 12% by mass H2O2 solution and soaking for 1.5 hours, then washing with deionized water, heating at 90°C for 60 minutes, cooling to room temperature, and drying to obtain a preliminary material for use;

[0034] Step B, taking 10 kg of the prepared initial material, adjusting the water content to 50%, adjusting the pH to 7.0, inoculating 0.5 kg of the fungus solution, and fermenting at 30° C. for 1.5 days; after the fermentation is completed, heating at 100° C. for 20 minutes, cooling to room temperature, adjusting the pH to 6.5, inoculating 0.8 kg of the Pichia pastoris solution, fermenting again at 30° C. for 1 day, and after the second fermentation is completed, heating at 100° C. for 20 minutes again, cooling to room temperature, and obtaining the fermented product for standby use;

[0035] Step C, taking 1 kg of the fermented product and mixing it with 8 L of 12% ethanol solution by mass fraction, reflux extraction was carried out at 65° C., cooled to room temperature, and 0.1 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 6 hours, centrifuged, and the supernatant was reserved for use;

[0036] Step D, adjusting the pH of the supernatant to 5.5, adding 12 g / L mannanase and 6 g / L β-glucanase and mixing, performing enzymolysis at 40°C for 3 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30 min each time, centrifuging, and obtaining a precipitate for standby use;

[0037] Step E: After the precipitate is dissolved in water, the sample is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.1:1 by volume, and the molecular weight cut-off is 2×10 6 ~5×10 7 The eluates of Da were combined, methanol was removed, and the eluates were dried to obtain the target hawthorn seed polysaccharide.

[0038] Example 2

[0039] First, prepare the adsorbent (modified brewer's grains microspheres):

[0040] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0041] Step 1, after drying 10 kg of brewer's grains, soak them in a 30% by mass phosphoric acid solution for 8 hours, filter and dry them; calcine them in an oxygen-free environment at 400-500° C. for 1.5 hours, cool them to room temperature, and obtain a primary product for use;

[0042] Step 2, taking 10 kg of the prepared primary product and placing it in a potassium permanganate solution with a mass fraction of 12% for 2 hours, filtering and drying; adding 3 kg of oyster shell powder and mixing, placing it in an oxygen-free environment at 800° C. and calcining it for 2.5 hours, cooling it to room temperature, washing it with deionized water until it is neutral, and drying it to obtain a secondary product for use;

[0043] Step 3, take 10kg of the prepared by-product, 0.8kg of succinic anhydride, 1kg of FeCl3, and 3kg of polyethyl methacrylate, add them to 15kg of molten chitosan, place it on a magnetic stirrer and stir for 1h to obtain a mixed solution; under stirring, add 8 times the volume of 2mol / L NaOH solution to the mixed solution, let it stand for 6h, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0044] Secondly, prepare hawthorn seed polysaccharide:

[0045] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1.5 kg of β-sitosterol and mixing, placing in a 10% by mass H2O2 solution and soaking for 2 hours, then washing with deionized water, heating at 100° C. for 40 minutes, cooling to room temperature, and drying to obtain a preliminary material for later use;

[0046] Step B, taking 10 kg of the prepared initial material, adjusting the water content to 40%, adjusting the pH to 7.2, inoculating 0.3 kg of the wood hoof porphyra liquid, and fermenting at 25° C. for 2 days; after the fermentation is completed, heating at 90° C. for 30 minutes, cooling to room temperature, adjusting the pH to 6.0, inoculating 1 kg of Pichia pastoris liquid, fermenting again at 25° C. for 1.5 days, and after the second fermentation is completed, heating at 90° C. for 30 minutes again, cooling to room temperature, and obtaining the fermented product for standby use;

[0047] Step C, taking 1 kg of the fermented product and mixing it with 10 L of 10% ethanol solution by mass fraction, reflux extraction was carried out at 60° C., cooled to room temperature, and 0.08 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 8 hours, centrifuged, and the supernatant was reserved for later use;

[0048] Step D, adjusting the pH of the supernatant to 5.0-5.5, adding 10-12 g / L mannanase and 6-8 g / L β-glucanase and mixing, performing enzymolysis at 35-40° C. for 3-4 hours, heating to 70-80° C. to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30-40 minutes after each adjustment, centrifuging, and obtaining a precipitate for later use;

[0049] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.12:1 by volume, and the molecular weight cut-off is 2×10 6 ~5×10 7 The eluates of Da were combined, methanol was removed, and the eluates were dried to obtain the target hawthorn seed polysaccharide.

[0050] Example 3

[0051] First, prepare the adsorbent (modified brewer's grains microspheres):

[0052] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0053] Step 1, after drying 10 kg of brewer's grains, soak them in a 35% by mass phosphoric acid solution for 7 hours, filter and dry them; calcine them in an oxygen-free environment at 500° C. for 1 hour, cool them to room temperature, and obtain a primary product for use;

[0054] Step 2, taking 10 kg of the prepared primary product and placing it in a 10% potassium permanganate solution with a mass fraction for 2 hours, filtering and drying; adding 4 kg of oyster shell powder and mixing, placing it in an oxygen-free environment at 800° C. and calcining it for 2.5 hours, cooling it to room temperature, washing it with deionized water until it is neutral, and drying it to obtain a secondary product for use;

[0055] Step 3, take 10kg of the prepared by-product, 1kg of succinic anhydride, 1.3kg of FeCl3, and 2.5kg of polyethyl methacrylate, add them to 17kg of molten chitosan, place it on a magnetic stirrer and stir for 1.5h to obtain a mixed solution; under stirring, add 9 times the volume of 2mol / L NaOH solution to the mixed solution, let it stand for 7h, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0056] Secondly, prepare hawthorn seed polysaccharide:

[0057] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1.3 kg of β-sitosterol and mixing, placing in a H2O2 solution with a mass fraction of 11% for 2 hours, then washing with deionized water, heating at 100° C. for 40 minutes, cooling to room temperature, and drying to obtain a preliminary material for use;

[0058] Step B, taking 10 kg of the prepared initial material, adjusting the water content to 45%, adjusting the pH to 7.2, inoculating 0.4 kg of the fungus solution, and fermenting at 25° C. for 2 days; after the fermentation is completed, heating at 100° C. for 20 minutes, cooling to room temperature, adjusting the pH to 6.5, inoculating 0.9 kg of the Pichia pastoris solution, fermenting again at 30° C. for 1 day, and after the second fermentation is completed, heating at 100° C. for 20 minutes again, cooling to room temperature, and obtaining the fermented product for standby use;

[0059] Step C, taking 1 kg of the fermented product and mixing it with 9 L of 11% ethanol solution by mass fraction, reflux extraction was carried out at 65° C., cooled to room temperature, and 0.09 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 7 hours, centrifuged, and the supernatant was reserved for use;

[0060] Step D, adjusting the pH of the supernatant to 5.5, adding 11 g / L mannanase and 7 g / L β-glucanase and mixing, performing enzymolysis at 40°C for 3 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30 min each time, centrifuging, and obtaining a precipitate for standby use;

[0061] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.11:1 by volume, and the molecular weight cut-off is 2×10 6 ~5×10 7 The eluates of Da were combined, methanol was removed, and the eluates were dried to obtain the target hawthorn seed polysaccharide.

[0062] Example 4

[0063] First, prepare the adsorbent (modified brewer's grains microspheres):

[0064] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0065] Step 1, after drying 10 kg of brewer's grains, soak them in a 40% by mass phosphoric acid solution for 6 hours, filter and dry them; calcine them in an oxygen-free environment at 400° C. for 1.5 hours, cool them to room temperature, and obtain a primary product for use;

[0066] Step 2, taking 10 kg of the prepared primary product and placing it in a potassium permanganate solution with a mass fraction of 11% and soaking it for 3 hours, filtering it, and drying it; adding 3.5 kg of oyster shell powder and mixing it evenly, placing it in an oxygen-free environment at 700° C. and calcining it for 3 hours, cooling it to room temperature, washing it with deionized water until it is neutral, and drying it to obtain a secondary product for use;

[0067] Step 3, take 10kg of the prepared by-product, 1.1kg of succinic anhydride, 1.1kg of FeCl3, and 2.5kg of polyethyl methacrylate, add them to 17kg of molten chitosan, place it on a magnetic stirrer and stir for 1.5h to obtain a mixed solution; under stirring, add 10 times the volume of 2mol / L NaOH solution to the mixed solution, let it stand for 7h, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0068] Secondly, prepare hawthorn seed polysaccharide:

[0069] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1.2 kg of β-sitosterol and mixing, placing in a H2O2 solution with a mass fraction of 11% for 2 hours, then washing with deionized water, heating at 100° C. for 50 minutes, cooling to room temperature, and drying to obtain a preliminary material for use;

[0070] Step B, taking 10 kg of the prepared initial material, adjusting the water content to 50%, adjusting the pH to 7.0, inoculating 0.35 kg of the phytomycete solution, and fermenting at 30° C. for 1.5 days; after the fermentation is completed, heating at 100° C. for 20 minutes, cooling to room temperature, adjusting the pH to 6.5, inoculating 1 kg of Pichia pastoris solution, fermenting again at 30° C. for 1 day, and after the second fermentation is completed, heating at 100° C. for 20 minutes again, cooling to room temperature, and obtaining a fermented product for standby use;

[0071] Step C, taking 1 kg of the fermented product and mixing it with 10 L of 10% ethanol solution by mass fraction, reflux extraction was carried out at 65° C., cooled to room temperature, and 0.1 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 7 hours, centrifuged, and the supernatant was reserved for use;

[0072] Step D, adjusting the pH of the supernatant to 5.0, adding 11 g / L mannanase and 7 g / L β-glucanase and mixing, performing enzymolysis at 40°C for 3.5 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 40 min each time, centrifuging, and obtaining a precipitate for standby use;

[0073] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.12:1 by volume, and the molecular weight cut-off is 2×10 6 ~5×10 7 The eluates of Da were combined, methanol was removed, and the eluates were dried to obtain the target hawthorn seed polysaccharide.

[0074] Comparative Example 1 Extraction and purification of hawthorn seed polysaccharide

[0075] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1.3 kg of β-sitosterol and mixing, placing in a H2O2 solution with a mass fraction of 11% for 2 hours, then washing with deionized water, heating at 100° C. for 40 minutes, cooling to room temperature, and drying to obtain a preliminary material for use;

[0076] Step B, taking 10 kg of the prepared initial material, adjusting the water content to 45%, adjusting the pH to 7.2, inoculating 0.4 kg of the fungus solution, and fermenting at 25° C. for 2 days; after the fermentation is completed, heating at 100° C. for 20 minutes, cooling to room temperature, adjusting the pH to 6.5, inoculating 0.9 kg of the Pichia pastoris solution, fermenting again at 30° C. for 1 day, and after the second fermentation is completed, heating at 100° C. for 20 minutes again, cooling to room temperature, and obtaining the fermented product for standby use;

[0077] Step C, taking 1 kg of the fermented product and mixing it with 9 L of 11% ethanol solution by mass fraction, reflux extraction was performed at 65° C., cooled to room temperature, centrifuged, and the supernatant was reserved for later use;

[0078] Step D, adjusting the pH of the supernatant to 5.5, adding 11 g / L mannanase and 7 g / L β-glucanase and mixing, performing enzymolysis at 40°C for 3 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30 min each time, centrifuging, and obtaining a precipitate for standby use;

[0079] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.11:1 by volume, and the molecular weight cut-off is 2×106 ~5×10 7 The eluates of Da are combined, methanol is removed, and the eluates are dried to obtain hawthorn seed polysaccharide.

[0080] Comparative Example 2

[0081] First, prepare the adsorbent (modified brewer's grains microspheres):

[0082] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0083] Step 1, after drying 10 kg of brewer's grains, soak them in a 35% by mass phosphoric acid solution for 7 hours, filter and dry them; calcine them in an oxygen-free environment at 500° C. for 1 hour, cool them to room temperature, and obtain a primary product for use;

[0084] Step 2, taking 10 kg of the prepared primary product and placing it in a 10% potassium permanganate solution with a mass fraction for 2 hours, filtering and drying; adding 4 kg of oyster shell powder and mixing, placing it in an oxygen-free environment at 800° C. and calcining it for 2.5 hours, cooling it to room temperature, washing it with deionized water until it is neutral, and drying it to obtain a secondary product for use;

[0085] Step 3, take 10kg of the prepared by-product, 1kg of succinic anhydride, 1.3kg of FeCl3, and 2.5kg of polyethyl methacrylate, add them to 17kg of molten chitosan, place it on a magnetic stirrer and stir for 1.5h to obtain a mixed solution; under stirring, add 9 times the volume of 2mol / L NaOH solution to the mixed solution, let it stand for 7h, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0086] Secondly, prepare hawthorn seed polysaccharide:

[0087] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adjusting the water content to 45%, adjusting the pH to 6.5, inoculating 0.4 kg of phytomycete solution and 0.9 kg of Pichia pastoris solution, fermenting at 25° C. for 3 days, and obtaining a fermented product for use;

[0088] Step B, taking 1 kg of the fermented product and mixing it with 9 L of 11% ethanol solution by mass fraction, reflux extraction was carried out at 65° C., cooled to room temperature, and 0.09 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 7 hours, centrifuged, and the supernatant was reserved for later use;

[0089] Step C, adjusting the pH of the supernatant to 5.5, adding 11 g / L mannanase and 7 g / L β-glucanase and mixing, performing enzymolysis at 40°C for 3 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30 min each time, centrifuging, and obtaining a precipitate for later use;

[0090] Step D: After the precipitate was dissolved in water, the sample was loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.11:1 by volume, and the molecular weight cut-off was 2×10 6 ~5×10 7 The eluates of Da are combined, methanol is removed, and the eluates are dried to obtain hawthorn seed polysaccharide.

[0091] Comparative Example 3

[0092] First, prepare the adsorbent (modified brewer's grains microspheres):

[0093] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0094] Step 1, after drying 10 kg of brewer's grains, calcine them in an oxygen-free environment at 800° C. for 2.5 hours, cool them to room temperature, wash them with deionized water until they are neutral, and dry them to obtain a secondary product for later use;

[0095] Step 3, take 10 kg of the prepared by-product and 17 kg of chitosan, add water, place on a magnetic stirrer and stir for 1.5 hours to obtain a mixed solution; under stirring, add 3 times the volume of 2 mol / L NaOH solution to the mixed solution, let it stand for 7 hours, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0096] Secondly, prepare hawthorn seed polysaccharide:

[0097] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1.3 kg of β-sitosterol and mixing, placing in a H2O2 solution with a mass fraction of 11% for 2 hours, then washing with deionized water, heating at 100° C. for 40 minutes, cooling to room temperature, and drying to obtain a preliminary material for use;

[0098] Step B, taking 10 kg of the prepared initial material, adjusting the water content to 45%, adjusting the pH to 7.2, inoculating 0.4 kg of the fungus solution, and fermenting at 25° C. for 2 days; after the fermentation is completed, heating at 100° C. for 20 minutes, cooling to room temperature, adjusting the pH to 6.5, inoculating 0.9 kg of the Pichia pastoris solution, fermenting again at 30° C. for 1 day, and after the second fermentation is completed, heating at 100° C. for 20 minutes again, cooling to room temperature, and obtaining the fermented product for standby use;

[0099] Step C, taking 1 kg of the fermented product and mixing it with 9 L of 11% ethanol solution by mass fraction, reflux extraction was carried out at 65° C., cooled to room temperature, and 0.09 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 7 hours, centrifuged, and the supernatant was reserved for use;

[0100] Step D, using ethanol solution to adjust the ethanol volume fraction in the supernatant to 10%, 30%, 50%, and 70% in sequence, and each time the solution is adjusted, the solution is allowed to stand for 30 minutes and centrifuged to obtain a precipitate for later use;

[0101] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.11:1 by volume, and the molecular weight cut-off is 2×10 6 ~5×10 7 The eluates of Da are combined, methanol is removed, and the eluates are dried to obtain hawthorn seed polysaccharide.

[0102] Comparative Example 4

[0103] First, prepare the adsorbent (modified brewer's grains microspheres):

[0104] The present invention provides a method for preparing modified brewer's grains microspheres, which specifically comprises the following steps:

[0105] Step 1, after drying 10 kg of brewer's grains, soak them in a 35% by mass phosphoric acid solution for 7 hours, filter and dry them; calcine them in an oxygen-free environment at 500° C. for 1 hour, cool them to room temperature, and obtain a primary product for use;

[0106] Step 2, taking 10 kg of the prepared primary product and placing it in a 10% potassium permanganate solution with a mass fraction for 2 hours, filtering and drying; adding 4 kg of oyster shell powder and mixing, placing it in an oxygen-free environment at 800° C. and calcining it for 2.5 hours, cooling it to room temperature, washing it with deionized water until it is neutral, and drying it to obtain a secondary product for use;

[0107] Step 3, take 10kg of the prepared by-product, 1kg of succinic anhydride, 1.3kg of FeCl3, and 2.5kg of polyethyl methacrylate, add them to 17kg of molten chitosan, place it on a magnetic stirrer and stir for 1.5h to obtain a mixed solution; under stirring, add 9 times the volume of 2mol / L NaOH solution to the mixed solution, let it stand for 7h, and then wash with deionized water to obtain modified brewer's grains microspheres.

[0108] Secondly, prepare hawthorn seed polysaccharide:

[0109] Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder; taking 10 kg of hawthorn seed powder, adding 1.3 kg of β-sitosterol and mixing, placing in a H2O2 solution with a mass fraction of 11% for 2 hours, then washing with deionized water, heating at 100° C. for 40 minutes, cooling to room temperature, and drying to obtain a preliminary material for use;

[0110] Step B, taking 1 kg of the prepared initial material and mixing it with 9 L of 11% ethanol solution by mass fraction, reflux extraction was carried out at 65° C., cooled to room temperature, and 0.09 kg / L of the modified brewer's grains microsphere adsorbent prepared above was added, stirred evenly, allowed to stand for 7 hours, centrifuged, and the supernatant was reserved for later use;

[0111] Step D, adjusting the pH of the supernatant to 5.5, adding 11 g / L mannanase and 7 g / L β-glucanase and mixing, performing enzymolysis at 40°C for 3 h, heating to 80°C to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30 min each time, centrifuging, and obtaining a precipitate for standby use;

[0112] Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a mixed solution of methanol: water = 0.11:1 by volume, and the molecular weight cut-off is 2×10 6 ~5×10 7 The eluates of Da are combined, methanol is removed, and the eluates are dried to obtain hawthorn seed polysaccharide.

[0113] Comparative Example 5

[0114] Take hawthorn seeds, crush them, and sieve them to obtain hawthorn seed powder; add the hawthorn seed powder into a 15% by mass ethanol solution, mix them, reflux extract them at 65°C, and filter them to obtain a filtrate; adjust the alcohol content of the filtrate to 70%, let it stand overnight, and centrifuge it to obtain a precipitate, thereby obtaining hawthorn seed polysaccharide.

[0115] Performance Testing

[0116] 1. Determination of total sugar and protein content in hawthorn seed polysaccharides

[0117] With reference to existing literature, the phenol-sulfuric acid method was used to determine the total sugar content in hawthorn seed polysaccharides, and the Coomassie brilliant blue method was used to determine the protein content.

[0118] 2. Determination of the antioxidant effect of hawthorn seed polysaccharides

[0119] With reference to existing literature, the antioxidant effect of hawthorn seed polysaccharides was determined.

[0120] 2.1 Scavenging ability of hawthorn seed polysaccharide on DPPH free radicals

[0121] The hawthorn seed polysaccharides prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were prepared into a 1.0 mg / mL polysaccharide solution; 1 mL of the polysaccharide solution was mixed evenly with 1 mL of 0.5 mmol / L DPPH ethanol solution, and the mixture was reacted in a dark place for 30 min; anhydrous ethanol was used instead of the DPPH solution as a sample control, distilled water was used instead of the polysaccharide solution as a blank control, VC was used as a positive control, and the absorbance was measured at 517 nm by an ELISA instrument to calculate the clearance rate.

[0122] 2.2 Scavenging ability of hawthorn seed polysaccharide on ABTS free radicals

[0123] The hawthorn seed polysaccharides prepared in Examples 1 to 4 and Comparative Examples 1 to 5 were prepared into a 1.0 mg / mL polysaccharide solution; 1 mL of the polysaccharide solution was mixed evenly with 5 mL of the ABTS solution, and placed in a dark place for 20 min. The absorbance was measured at 734 nm, and distilled water was used instead of the ABTS solution as a sample control, and distilled water was used instead of the polysaccharide solution as a blank control. VC was used as a positive control to calculate the clearance rate.

[0124] The above data were analyzed using SPSS22.0 software.

[0125] Experimental results and analysis

[0126] 1. Hawthorn seed polysaccharide content

[0127] Table 1 Polysaccharide and protein content in hawthorn seed polysaccharide

[0128]

[0129] As can be seen from the results in Table 1, the polysaccharide preparation method provided by the present invention has a relatively high yield of polysaccharides and can ensure the high purity of polysaccharides. The yields of polysaccharides in Comparative Examples 1 and 3 are higher, but the purity of polysaccharides is lower and the protein content is higher, indicating that the presence and different effects of the adsorbent on the purity of the polysaccharide. The total yields of polysaccharides in Comparative Examples 2, 4 and 5 are lower, and the purity of the polysaccharide in Comparative Example 4 is higher.

[0130] Table 2 Antioxidant activity of hawthorn seed polysaccharides

[0131]

[0132] From the results in Table 2, it can be seen that the hawthorn seed polysaccharide prepared by the present invention has good antioxidant properties, and the antioxidant effect is significantly better than the polysaccharides prepared by Comparative Examples 1 to 5. The reason is that the preparation methods are different, and the components of the obtained polysaccharides are different. The polysaccharides prepared by Comparative Examples 1 to 5 contain components or monosaccharides that do not have antioxidant activity.

Claims

1. A method for extracting and purifying polysaccharides from hawthorn seeds, characterized in that: The following steps are involved: Step A, crushing hawthorn seeds, sieving to obtain hawthorn seed powder, adding β-sitosterol and mixing, soaking in a 10-12% H2O2 solution for 1.5-2h, then washing with deionized water, heating at 90-100°C for 40-60min, cooling to room temperature, drying to obtain a preliminary material for later use; Step B, adjusting the water content of the initial material to 40-50%, adjusting the pH to 7.0-7.2, inoculating the bacterial solution of the wood porphyra, and fermenting at 25-30° C. for 1.5-2 days; after the fermentation is completed, heating at 90-100° C. for 20-30 minutes, cooling to room temperature, adjusting the pH to 6.0-6.5, inoculating the Pichia pastoris bacterial solution, fermenting again at 25-30° C. for 1-1.5 days, and after the second fermentation is completed, heating at 90-100° C. for 20-30 minutes again, cooling to room temperature, and obtaining the fermented product for standby use; Step C, mixing the fermented product with a 10-12% ethanol solution at a material-liquid ratio of 1:(8-10) kg / L, extracting under reflux at 60-65°C, cooling to room temperature, adding 0.08-0.1 kg / L of adsorbent, standing for 6-8 hours, centrifuging, and reserving the supernatant for later use; Step D, adjusting the pH of the supernatant to 5.0-5.5, adding 10-12 g / L mannanase and 6-8 g / L β-glucanase and mixing, performing enzymolysis at 35-40° C. for 3-4 hours, heating to 70-80° C. to inactivate the enzyme, cooling to room temperature, and adjusting the ethanol volume fraction in the enzymolysis solution to 10%, 30%, 50%, and 70% with ethanol solution in sequence, standing for 30-40 minutes after each adjustment, centrifuging, and obtaining a precipitate for later use; Step E: After the precipitate is dissolved in water, it is loaded on Sephadex G type dextran gel and eluted with a methanol-water mixed solution with a molecular weight cutoff of 2×10 6 ~5×10 7 The polysaccharide eluate of Da was combined, the methanol was removed, and the eluate was dried to obtain the target hawthorn seed polysaccharide; Wherein, the adsorbent in step C is modified brewer's grains microspheres; the preparation method of the modified brewer's grains microspheres comprises the following steps: Step 1, after drying the brewer's grains, soak them in a phosphoric acid solution with a mass fraction of 30-40% for 6-8 hours, filter them, and dry them; calcine them in an oxygen-free environment at 400-500° C. for 1-1.5 hours, and cool them to room temperature to obtain a primary product for use; Step 2, soaking the primary product in a potassium permanganate solution with a mass fraction of 10-12% for 2-4 hours, filtering, and drying; adding oyster shell powder and mixing, calcining in an oxygen-free environment at 700-800° C. for 2.5-3 hours, cooling to room temperature, washing with deionized water until neutral, and drying to obtain a secondary product for standby use; wherein the mass ratio of the primary product to the oyster shell powder is 1:(0.3-0.5); Step 3, adding the by-product, succinic anhydride, FeCl3, and polyethyl methacrylate to molten chitosan, placing it on a magnetic stirrer and stirring for 1-1.5 hours to obtain a mixed solution; under stirring, adding 8-10 times the volume of 2 mol / L NaOH solution to the mixed solution, letting it stand for 6-8 hours, and washing with deionized water to obtain modified brewer's grains microspheres; wherein the mass ratio of the by-product, succinic anhydride, FeCl3, polyethyl methacrylate, and chitosan is 1:(0.08-0.12):(0.1-0.15):(0.2-0.3):(1.5-1.8).

2. The method according to claim 1, characterized in that In the step A, the amount of β-sitosterol added is 10-15% of the mass of the hawthorn seed powder.

3. The method according to claim 1, characterized in that In step B, the colony count in the fungus solution is 8×10 7 / mL, the number of colonies in the Pichia pastoris solution is 3×10 7 Pieces / mL.

4. The method according to claim 1, characterized in that: In the step B, the inoculation amount of the seed solution of the phytomycetes is 3-5% of the mass of the initial material, and the inoculation amount of the seed solution of the Pichia pastoris is 8-10% of the mass of the initial material.

5. The method according to claim 1, characterized in that The volume ratio of methanol to water in the methanol-water mixed solution in step E is (0.1-0.12):1.

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