Preparation method of gleditsia sinensis polysaccharide
The method of preparing saponin polysaccharides by combining Ganoderma lucidum fermentation with millet husk has solved the problems of low extraction rate and insufficient biological activity, and achieved efficient preservation and antioxidant properties for fruits and vegetables.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING GUAR SCI&TRADING
- Filing Date
- 2022-10-21
- Publication Date
- 2026-06-16
AI Technical Summary
Existing technologies have low extraction rates and insufficient biological activity of saponin polysaccharides, and their preservation effects on fruits and vegetables are poor. Therefore, it is necessary to improve their biological activity and extraction efficiency.
Saponin polysaccharide was prepared by fermenting Ganoderma lucidum with millet husk as an synergistic ingredient, and then through high-pressure sterilization, enzymatic hydrolysis, alcohol precipitation, and macroporous resin treatment. The specific steps included inoculation with Ganoderma lucidum, enzymatic hydrolysis, filtration, alcohol precipitation, and pigment removal.
The prepared saponin polysaccharides have good preservation and antioxidant effects on fruits and vegetables, and their biological activity is significantly improved.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant polysaccharide technology, specifically relating to a method for preparing saponin polysaccharide. Background Technology
[0002] Soapberry (Gleditsia sinensis) is a tree species endemic to my country, belonging to the genus Gleditsia of the Caesalpiniaceae family. Its seeds are long and flat, with an uneven surface, reddish-brown or purplish-red in color, covered with a grayish-white powdery bloom that leaves a glossy sheen after rubbing. They are slightly pointed at both ends, hard in texture, and rich in polysaccharides and proteins. Soapberry also contains various saponins, as well as fisetin, chalcogenin, betulinic acid, betulin, and senna ketones. Soapberry can be used as a traditional Chinese medicine ingredient, possessing the effects of clearing the orifices and resolving phlegm, dispersing nodules and reducing swelling, moistening dryness and promoting bowel movements; it is classified as an emetic.
[0003] Extracting saponins, triterpenoids, and saponin flavonoids from soapberry is currently a hot research topic. However, there are few reports on the extraction and utilization of polysaccharides from soapberry. Soapberry is hard, and its polysaccharides are mostly stored inside the hard interior. Traditional crushing and chemical extraction methods yield very low polysaccharide yields. The functions of soapberry polysaccharides are also rarely studied. Yu Ming et al. published a study on the preservation of sweet persimmons using a soapberry polysaccharide coating. Under low-temperature conditions, coating with a 1% soapberry polysaccharide solution and PE film packaging significantly delayed the decline in fruit firmness, maintaining a fruit firmness greater than 8 kg / m² after 60 days of storage. 2 During storage, the carotenoid content of the fruit increases. Although saponin polysaccharides have a good preservation effect on sweet persimmons, their effect on other fruits and vegetables is not good, and further improvement of the bioactivity of saponin polysaccharide extract is needed. Whether saponin polysaccharides have other biological activities is also rarely reported. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing saponin polysaccharides.
[0005] A method for preparing saponin polysaccharide, comprising the following steps:
[0006] (1) Take mature and dried soapberry, crush it, add crushed millet husk, sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate and water to prepare raw material culture medium; sterilize the raw material culture medium by high pressure.
[0007] (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 5-10 days;
[0008] (3) Take the fermentation product, beat it into a pulp, and add a compound enzyme for enzymatic hydrolysis;
[0009] (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 1000-3000 rpm, take the supernatant, and remove the protein from the supernatant.
[0010] (5) The supernatant after removing the protein was subjected to alcohol precipitation and freeze-drying to obtain crude saponin polysaccharide;
[0011] (6) Remove pigments from crude soapberry polysaccharide by passing it through macroporous resin to obtain soapberry polysaccharide.
[0012] The weight ratio of the components in the raw material culture medium is as follows: 25-35% soapberry, 10-20% millet husk, 1-5% sucrose, 1-3% calcium carbonate, 0.01-0.05% potassium dihydrogen phosphate, 0.01-0.05% magnesium sulfate, and the balance is water.
[0013] The autoclaving temperature is 121℃ and the time is 20 minutes.
[0014] The inoculation amount of the Ganoderma lucidum strain is 0.1-0.5% of the mass of the raw material culture medium.
[0015] The compound enzyme is a protease and a cellulase, and the amount added is 0.5-1.5% of the weight of the fermentation product, respectively.
[0016] The steps for removing proteins from the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 20-40 minutes, centrifuge at 1000-3000 rpm, and remove the lower organic layer and the intermediate protein layer of the mixed solution.
[0017] In the alcohol precipitation step, the volume of anhydrous ethanol added is 30-70% of the volume of the supernatant.
[0018] The above-prepared saponin polysaccharides are used in the preservation of fruits or vegetables.
[0019] The application of the saponin polysaccharide prepared above in the preparation of antioxidant and lipid-lowering drugs.
[0020] The beneficial effects of the present invention are as follows: The saponin polysaccharide prepared by the present invention has good preservation and antioxidant effects on fruits or vegetables. The preparation process adopts the method of fermenting raw materials with Ganoderma lucidum and adds millet husk as an synergistic material, which makes the prepared saponin polysaccharide more bioactive. Detailed Implementation
[0021] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.
[0022] Example 1
[0023] A method for preparing saponin polysaccharide, comprising the following steps:
[0024] (1) Take mature and dried soapberry, crush it, add crushed millet bran, then add sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate and water to prepare a raw material culture medium; autoclave the raw material culture medium; the weight ratio of the components in the raw material culture medium is: soapberry 30%, millet bran 15%, sucrose 3%, calcium carbonate 2%, potassium dihydrogen phosphate 0.03%, magnesium sulfate 0.03%, and the remainder is water; the autoclave temperature is 121℃ and the time is 20min;
[0025] (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 8 days; the inoculation amount of the Ganoderma lucidum slant culture is 0.3% of the mass of the raw material culture medium;
[0026] (3) Take the fermentation product, beat it, and add a compound enzyme for enzymatic hydrolysis; the compound enzyme is a protease and a cellulase, and the amount added is 1% of the weight of the fermentation product, respectively.
[0027] (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 2000 rpm, take the supernatant, and remove the protein in the supernatant; the operation steps for removing the protein in the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 30 min, centrifuge at 2000 rpm, and remove the lower organic layer and the middle protein layer of the mixed solution.
[0028] (5) The supernatant from which the protein has been removed is subjected to alcohol precipitation and freeze-drying to obtain crude saponin polysaccharide; the volume of anhydrous ethanol added in the alcohol precipitation step is 50% of the volume of the supernatant.
[0029] (6) Remove pigments from crude saponin polysaccharide through macroporous resin to obtain saponin polysaccharide; the specific operation is as follows: soak and clean the macroporous resin with anhydrous ethanol, clean and swell the macroporous resin with distilled water; mix the crude saponin polysaccharide with the macroporous resin to form a mixed solution, statically adsorb for 14 hours, filter and concentrate the mixed solution to remove pigments.
[0030] Example 2
[0031] A method for preparing saponin polysaccharide, comprising the following steps:
[0032] (1) Take mature and dried soapberry, crush it, add crushed millet husk, sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate and water to prepare raw material culture medium; autoclave the raw material culture medium; the weight ratio of the components in the raw material culture medium is: soapberry 25%, millet husk 10%, sucrose 2%, calcium carbonate 1%, potassium dihydrogen phosphate 0.02%, magnesium sulfate 0.02%, and the remainder is water; the autoclave temperature is 121℃ and the time is 20min;
[0033] (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 5-10 days; the inoculation amount of the Ganoderma lucidum slant culture is 0.1% of the mass of the raw material culture medium;
[0034] (3) Take the fermentation product, beat it into a pulp, and add a compound enzyme for enzymatic hydrolysis; the compound enzyme is a protease and a cellulase, and the amount added is 0.5% of the weight of the fermentation product, respectively.
[0035] (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 1000 rpm, take the supernatant, and remove the protein in the supernatant; the operation steps for removing the protein in the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 20 min, centrifuge at 1000 rpm, and remove the lower organic layer and the middle protein layer of the mixed solution.
[0036] (5) The supernatant from which the protein has been removed is subjected to alcohol precipitation and freeze-drying to obtain crude saponin polysaccharide; the volume of anhydrous ethanol added in the alcohol precipitation step is 30% of the volume of the supernatant.
[0037] (6) Remove pigments from crude saponin polysaccharide through macroporous resin to obtain saponin polysaccharide; the specific operation is as follows: soak and clean the macroporous resin with anhydrous ethanol, clean and swell the macroporous resin with distilled water; mix the crude saponin polysaccharide with the macroporous resin to form a mixed solution, statically adsorb for 14 hours, filter and concentrate the mixed solution to remove pigments.
[0038] Example 3
[0039] A method for preparing saponin polysaccharide, comprising the following steps:
[0040] (1) Take mature and dried soapberry, crush it, add crushed millet bran, then add sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate and water to prepare a raw material culture medium; autoclave the raw material culture medium; the weight ratio of the components in the raw material culture medium is: soapberry 35%, millet bran 20%, sucrose 5%, calcium carbonate 3%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.05%, and the remainder is water; the autoclave temperature is 121℃ and the time is 20min;
[0041] (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 10 days; the inoculation amount of the Ganoderma lucidum slant culture is 0.5% of the mass of the raw material culture medium;
[0042] (3) Take the fermentation product, beat it into a pulp, and add a compound enzyme for enzymatic hydrolysis; the compound enzyme is a protease and a cellulase, and the amount added is 1.5% of the weight of the fermentation product, respectively.
[0043] (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 3000 rpm, take the supernatant, and remove the protein in the supernatant; the operation steps for removing the protein in the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 40 min, centrifuge at 3000 rpm, and remove the lower organic layer and the middle protein layer of the mixed solution.
[0044] (5) The supernatant from which the protein has been removed is subjected to alcohol precipitation and freeze-drying to obtain crude saponin polysaccharide; the volume of anhydrous ethanol added in the alcohol precipitation step is 60% of the volume of the supernatant.
[0045] (6) Remove pigments from crude saponin polysaccharide through macroporous resin to obtain saponin polysaccharide; the specific operation is as follows: soak and clean the macroporous resin with anhydrous ethanol, clean and swell the macroporous resin with distilled water; mix the crude saponin polysaccharide with the macroporous resin to form a mixed solution, statically adsorb for 14 hours, filter and concentrate the mixed solution to remove pigments.
[0046] Comparative Example 1
[0047] A method for preparing saponin polysaccharide, comprising the following steps:
[0048] (1) Take mature and dried soapberry, crush it, add sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate and water to prepare a raw material culture medium; autoclave the raw material culture medium; the weight ratio of the components in the raw material culture medium is: soapberry 45%, sucrose 3%, calcium carbonate 2%, potassium dihydrogen phosphate 0.03%, magnesium sulfate 0.03%, and the remainder is water; the autoclave temperature is 121℃ and the time is 20min;
[0049] (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 8 days; the inoculation amount of the Ganoderma lucidum slant culture is 0.3% of the mass of the raw material culture medium;
[0050] (3) Take the fermentation product, beat it, and add a compound enzyme for enzymatic hydrolysis; the compound enzyme is a protease and a cellulase, and the amount added is 1% of the weight of the fermentation product, respectively.
[0051] (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 2000 rpm, take the supernatant, and remove the protein in the supernatant; the operation steps for removing the protein in the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 30 min, centrifuge at 2000 rpm, and remove the lower organic layer and the middle protein layer of the mixed solution.
[0052] (5) The supernatant from which the protein has been removed is subjected to alcohol precipitation and freeze-drying to obtain crude saponin polysaccharide; the volume of anhydrous ethanol added in the alcohol precipitation step is 50% of the volume of the supernatant.
[0053] (6) Remove pigments from crude saponin polysaccharide through macroporous resin to obtain saponin polysaccharide; the specific operation is as follows: soak and clean the macroporous resin with anhydrous ethanol, clean and swell the macroporous resin with distilled water; mix the crude saponin polysaccharide with the macroporous resin to form a mixed solution, statically adsorb for 14 hours, filter and concentrate the mixed solution to remove pigments.
[0054] Comparative Example 2
[0055] A method for preparing millet polysaccharide, comprising the following steps:
[0056] (1) Take millet husks, crush them, and then add sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate, and water to prepare a raw material culture medium; autoclave the raw material culture medium; the weight ratio of the components in the raw material culture medium is: millet husks 45%, sucrose 3%, calcium carbonate 2%, potassium dihydrogen phosphate 0.03%, magnesium sulfate 0.03%, and the remainder is water; the autoclaving temperature is 121℃ and the time is 20min;
[0057] (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 8 days; the inoculation amount of the Ganoderma lucidum slant culture is 0.3% of the mass of the raw material culture medium;
[0058] (3) Take the fermentation product, beat it, and add a compound enzyme for enzymatic hydrolysis; the compound enzyme is a protease and a cellulase, and the amount added is 1% of the weight of the fermentation product, respectively.
[0059] (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 2000 rpm, take the supernatant, and remove the protein in the supernatant; the operation steps for removing the protein in the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 30 min, centrifuge at 2000 rpm, and remove the lower organic layer and the middle protein layer of the mixed solution.
[0060] (5) The supernatant from which the protein has been removed is subjected to alcohol precipitation and freeze-drying to obtain millet bark polysaccharide; the volume of anhydrous ethanol added in the alcohol precipitation step is 50% of the volume of the supernatant.
[0061] (6) Remove pigments from the crude polysaccharide of millet husk through macroporous resin to obtain millet husk polysaccharide; the specific operation is as follows: soak and clean the macroporous resin with anhydrous ethanol, and clean and swell the macroporous resin with distilled water; mix the crude polysaccharide of millet husk with the macroporous resin to form a mixed solution, statically adsorb for 14 hours, filter and concentrate the mixed solution to remove pigments.
[0062] Experimental example:
[0063] Zijinguan eggplants of uniform maturity were selected for the experiment. They were washed and placed in preservation bags on the day of harvest. The polysaccharides prepared in Examples 1-3 and Comparative Examples 1-2 were prepared into a 1% aqueous solution and coated onto the eggplants. After manual coating, the eggplants were air-dried. Ten eggplants were treated per group, with three replicates. The eggplants were stored in preservation bags at 4℃ for 15 days. Respiration intensity was measured using a DCY-2 dual-channel atmospheric sampler. The control group consisted of Zijinguan eggplants treated only with preservation bags. The results are shown in Table 1.
[0064] Table 1
[0065]
[0066]
[0067] Note: * indicates P<0.05 compared with the control group, # indicates P<0.05 compared with Example 1.
[0068] The weight change of Zijinguan eggplant before and after storage was determined using an electronic analytical balance. The percentage of weight loss was calculated as: (weight before storage - weight after storage) / weight before storage x 100%. The results are shown in Table 2.
[0069] Table 2
[0070]
[0071] Note: * indicates P<0.05 compared with the control group, # indicates P<0.05 compared with Example 1.
[0072] The polysaccharides prepared in Examples 1-3 and Comparative Examples 1-2 were prepared into 5 mg / mL solutions. 1.2 mg of DPPH powder was weighed and dissolved in 15 mL of anhydrous ethanol, and thoroughly mixed to obtain a 0.2 mmol / L DPPH stock solution, which was stored in the dark. 100 μL of sample was taken, and 100 μL of DPPH stock solution was added. The reaction was carried out in the dark for 30 min, and the absorbance (A1) was measured at 517 nm. The scavenging capacity was calculated using the formula R% = (1 - (A1 - A2) / A0) × 100%. Wherein, A2 is 100 μL of sample and 100 μL of anhydrous ethanol, representing the background group; A0 is 100 μL of DPPH and 100 μL of 50% ethanol, representing the blank control group.
[0073] Table 3
[0074]
[0075]
[0076] Note: * indicates that P < 0.05 compared with Example 1.
[0077] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A method for preparing saponin polysaccharide, characterized in that, Follow these steps: (1) Take mature and dried soapberry, crush it, add crushed millet husk, sucrose, calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate and water to prepare raw material culture medium; sterilize the raw material culture medium by high pressure; the weight ratio of the components in the raw material culture medium is: soapberry 25-35%, millet husk 10-20%, sucrose 1-5%, calcium carbonate 1-3%, potassium dihydrogen phosphate 0.01-0.05%, magnesium sulfate 0.01-0.05%, and the remainder is water; (2) Inoculate with Ganoderma lucidum slant culture, place on a shaker for culture, and culture for 5-10 days; (3) Take the fermentation product, beat it, add a compound enzyme for enzymatic hydrolysis. The compound enzyme is a protease and a cellulase, and the amount added is 0.5-1.5% of the weight of the fermentation product, respectively. (4) Filter the enzymatic hydrolysate to remove large impurities, centrifuge at 1000-3000 rpm, take the supernatant, and remove the protein from the supernatant; (5) The supernatant after removing the protein was subjected to alcohol precipitation and freeze-drying to obtain crude saponin polysaccharide; (6) Remove pigments from crude soapberry polysaccharide by passing it through macroporous resin to obtain soapberry polysaccharide.
2. The method for preparing saponin polysaccharide according to claim 1, characterized in that, The autoclaving temperature is 121℃ and the time is 20 minutes.
3. The method for preparing saponin polysaccharide according to claim 1, characterized in that, The inoculation amount of the Ganoderma lucidum strain is 0.1-0.5% of the mass of the raw material culture medium.
4. The method for preparing saponin polysaccharide according to claim 1, characterized in that, The steps for removing proteins from the supernatant are as follows: mix the supernatant with Sevage reagent at a volume ratio of 4:1 to obtain a mixed solution, shake the mixed solution thoroughly for 20-40 minutes, centrifuge at 1000-3000 rpm, and remove the lower organic layer and the intermediate protein layer of the mixed solution.
5. The method for preparing saponin polysaccharide according to claim 1, characterized in that, In the alcohol precipitation step, the volume of anhydrous ethanol added is 30-70% of the volume of the supernatant.
6. The application of the saponin polysaccharide prepared according to claim 1 in the preservation of fruits or vegetables.
7. The use of the saponin polysaccharide prepared according to claim 1 in the preparation of antioxidant drugs.