Cardamine selenium polysaccharide and preparation method thereof

Abstract

The present invention relates to the technical field of polysaccharide preparation, and in particular, to Cardamine selenopolysaccharide and a preparation method thereof. The preparation method includes: Cardamine selenopolysaccharide and hydroxide aqueous solution are mixed for extraction to form an extract; The extract is mixed with a composite protease for enzymolysis; wherein the concentration of the hydroxide aqueous solution is 0.028-0.032 mol / L, and 20-30 L hydroxide aqueous solution is used for each kilogram of Cardamine selenopolysaccharide; The hydroxide is selected from sodium hydroxide, and the composite protease is composed of alkaline protease, neutral protease and papain; The amount of alkaline protease is 0.18-0.22 g / L, the amount of papain is 0.18-0.22 g / L, and the amount of neutral protease is 0.7-0.9 g / L. The method can improve the yield of selenopolysaccharide, and can further improve the content of polysaccharide, reduce protein content, and then can reduce the production cost of Cardamine selenopolysaccharide.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide preparation, and in particular to Cardamine selenium polysaccharide and a preparation method thereof. Background Art

[0002] Cardamine occulta Hornem. is an annual herb of the genus Cardamine in the family Cruciferae. Also known as Caiziqi and Banbiancai, it is a common folk medicine. Cardamine has super selenium-rich ability and high organic selenium content, among which selenium polysaccharide is an important occurrence form of its organic selenium. Selenium polysaccharide has obvious antioxidant and immune activity, can increase the activity of glutathione peroxidase in whole blood and liver homogenate, enhance the phagocytic function of macrophages in immune cells, promote lymphocyte transformation and promote antibody production, thereby improving immune function in many aspects. In addition, studies have found that selenium-rich Cardamine polysaccharide has a significant inhibitory effect on the cell activity, proliferation ability, migration ability, and invasion ability of human lung cancer A549, and can promote cancer cell apoptosis.

[0003] Although selenium polysaccharides have many beneficial effects, they are rarely used in practice. The main reason is that the existing reported extraction technology of Cardamine selenium polysaccharides generally has the disadvantages of low extraction rate, long time consumption, high energy consumption, etc., which leads to high production cost of Cardamine selenium polysaccharides. Therefore, it is urgent to develop a preparation method that can improve the difficulty of extraction, separation and purification to improve the production quality and production efficiency of selenium polysaccharides.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide Cardamine selenopolysaccharide and a preparation method thereof. The embodiment of the present invention provides a novel preparation method of Cardamine selenopolysaccharide, which can improve the yield of selenopolysaccharide, further improve the content of polysaccharide, reduce the protein content, and then reduce the production cost of Cardamine selenopolysaccharide.

[0006] The present invention is achieved in that: In a first aspect, the present invention provides a method for preparing Cardamine selenium polysaccharide, comprising: mixing Cardamine and a hydroxide aqueous solution for extraction to form an extract; The extract is mixed with a composite protease for enzymolysis; The concentration of the hydroxide aqueous solution is 0.028-0.032 mol / L, and 20-30 L of the hydroxide aqueous solution is used for every kilogram of the Broken Rice Mustard; the hydroxide is selected from sodium hydroxide; The composite protease consists of alkaline protease, neutral protease and papain; The dosage of the alkaline protease is 0.18-0.22 g / L, the dosage of the papain is 0.18-0.22 g / L, and the dosage of the neutral protease is 0.7-0.9 g / L; The enzyme activity of the alkaline protease is 150,000-250,000 U / g; the enzyme activity of the neutral protease is 30,000-70,000 U / g; the enzyme activity of the papain is 150,000-250,000 U / g; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis system is subjected to membrane separation, wherein the membrane separation collects the interception portion with a molecular weight greater than 1000.

[0007] In an optional embodiment, the extraction conditions include: extraction temperature of 100-120° C., extraction time of 1.5-3 h, and extraction times of 1-3 times.

[0008] In an optional embodiment, the conditions for enzymatic hydrolysis include: a temperature of 43-47° C. and a time of 95-120 min.

[0009] In an optional embodiment, the method comprises: cooling the extract to an enzymatic hydrolysis temperature, and then performing enzymatic hydrolysis.

[0010] In an optional embodiment, the method comprises: performing membrane separation on the enzymatic hydrolysis system and then sequentially performing concentration, standing, centrifugation and drying.

[0011] In an optional embodiment, the centrifugal conditions include: a rotation speed of 4500-7500 rpm and a time of 5-15 minutes.

[0012] In a second aspect, the present invention provides a Cardamine selenopolysaccharide, which is prepared by the preparation method of Cardamine selenopolysaccharide described in the aforementioned embodiment.

[0013] The present invention has the following beneficial effects: the embodiment of the present invention uses a hydroxide aqueous solution to extract Cardamine, and then performs enzymatic hydrolysis and membrane separation, and limits the extraction conditions and the amount of enzyme used in the enzymatic hydrolysis, thereby improving the yield of selenopolysaccharides, increasing the content of polysaccharides in selenopolysaccharides, reducing the content of protein in selenopolysaccharides, and further improving the quality of the obtained selenopolysaccharides. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0015] In a first aspect, the present invention provides a method for preparing Cardamine selenium polysaccharide, comprising: S1, extraction; The cardamine is dried and then crushed appropriately. The crushing is to process the cardamine with a crusher to reduce the volume of the extracted cardamine and improve the extraction effect. The degree of crushing can be crushed according to actual production needs.

[0016] Then, Cardamine and the hydroxide aqueous solution are mixed, specifically, 20-30 L of the hydroxide aqueous solution is used for every kilogram of Cardamine. If the amount of water used is too low, the yield of selenium polysaccharide may be reduced.

[0017] It should be noted that the ratio between Broccoli and the hydroxide aqueous solution is only an example of the embodiment of the present invention, and the ratio can be adjusted according to the conversion of the weight and volume units used in production.

[0018] Further, the hydroxide is selected from sodium hydroxide, and the concentration of the hydroxide aqueous solution is 0.028-0.032 mol / L, for example, 0.028 mol / L, 0.029 mol / L, 0.030 mol / L, 0.031 mol / L and 0.032 mol / L, any value between 0.028-0.032 mol / L or a range value between any two values.

[0019] The use of the hydroxide concentration within the above range can ensure the yield of selenopolysaccharides, increase the content of polysaccharides in selenopolysaccharides, and reduce the content of protein in selenopolysaccharides. If the concentration of the hydroxide is changed, for example, the concentration of the hydroxide is reduced, the yield of selenopolysaccharides will be reduced. If the concentration of the hydroxide is increased, the content of polysaccharides in selenopolysaccharides may be reduced, while the protein content may be increased, which is not conducive to improving the quality of the obtained selenopolysaccharides.

[0020] It should be noted that the selenium polysaccharide recorded in the embodiments of the present invention refers to a crude selenium polysaccharide or a selenium polysaccharide sample containing protein and polysaccharide, and the polysaccharide content refers to the content of polysaccharides in the above-mentioned selenium polysaccharide (crude product or sample), and the protein content refers to the content of protein in the above-mentioned selenium polysaccharide (crude product or sample).

[0021] The embodiment of the present invention not only improves the yield of selenium polysaccharides, but also improves the content of polysaccharides in selenium polysaccharides. If the yield of selenium polysaccharides is high, but the polysaccharide content is low, then the actual content of polysaccharides that can exert the effect is not high, which also leads to poor quality of selenium polysaccharides (crude products). For example, the yield of selenium polysaccharide A reaches 5%, and the polysaccharide content therein is only 50%. The yield of selenium polysaccharide B is 3%, but its polysaccharide content is 90%. Obviously, the quality of selenium polysaccharide B is better than that of selenium polysaccharide A.

[0022] The extraction is performed at a temperature of 100-120°C for 1.5-3 hours and 1-3 times. Specifically, the extraction temperature is any value between 100-120°C, such as 100°C, 105°C, 110°C, 115°C and 120°C, or a range value between any two values. The extraction time is any value between 1.5-3 hours, such as 1.5 hours, 2 hours, 2.5 hours and 3 hours, or a range value between any two values.

[0023] S2, enzymatic hydrolysis; Then the extracts formed by each extraction are mixed and cooled to the enzymatic hydrolysis temperature.

[0024] The mixed extract is mixed with a composite protease for enzymatic hydrolysis; The complex protease includes alkaline protease, neutral protease and papain; The dosage of the alkaline protease is 0.18-0.22 g / L, the dosage of the papain is 0.18-0.22 g / L, and the dosage of the neutral protease is 0.7-0.9 g / L.

[0025] The above enzyme dosage refers to the number of grams (g) of enzyme contained in the mixed solution formed by the extract and the composite protease per liter (L). It is understood that the dosage unit can be converted to mg / L or g / mL, etc. according to the conversion relationship.

[0026] Specifically, the dosage of alkaline protease is 0.18 g / L, 0.19 g / L, 0.20 g / L, 0.21 g / L and 0.22 g / L, any value between 0.18-0.22 g / L or a range value between any two values.

[0027] The dosage of papain is 0.18 g / L, 0.19 g / L, 0.20 g / L, 0.21 g / L and 0.22 g / L, any value between 0.18-0.22 g / L or a range value between any two values.

[0028] The dosage of neutral protease is 0.7 g / L, 0.75 g / L, 0.8 g / L, 0.85 g / L and 0.9 g / L, etc., any value between 0.7-0.9 g / L or a range value between any two values.

[0029] The embodiment of the present invention adopts the above enzymes in combination and limits the dosage of the above enzymes, which can effectively reduce the protein content in selenium polysaccharides and increase the polysaccharide content.

[0030] The enzyme activity of the alkaline protease is 150,000-250,000 U / g; the enzyme activity of the neutral protease is 30,000-70,000 U / g; and the enzyme activity of the papain is 150,000-250,000 U / g.

[0031] The conditions for enzymolysis include: temperature of 43-47°C and time of 95-120 minutes. The above enzymolysis temperature and time can ensure the yield of selenium polysaccharide, while ensuring the content of polysaccharide in selenium polysaccharide and reducing the protein content.

[0032] Specifically, the enzymolysis temperature is any value between 43-47° C., such as 43° C., 44° C., 45° C., 46° C., and 47° C., or a range between any two values. The enzymolysis time is any value between 95-120 min, such as 95 min, 100 min, 105 min, 110 min, 115 min, and 120 min, or a range between any two values.

[0033] S3, post-processing; After enzymatic hydrolysis, membrane separation is performed, and the retained portion with a molecular weight greater than 1000 is taken, and reduced pressure concentration is performed (reduced pressure concentration at 100°C) to remove the solvent. The concentrate is allowed to stand at 0-4°C overnight, and then centrifuged (4500-7500 rpm, time is 5-15 minutes), the supernatant is taken, and spray-dried to obtain Cardamine selenium polysaccharide.

[0034] In a second aspect, the present invention provides a Cardamine selenopolysaccharide, which is prepared by the preparation method of Cardamine selenopolysaccharide described in the aforementioned embodiment.

[0035] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0036] Example 1 The embodiment of the present invention provides a method for preparing Cardamine selenium polysaccharide, comprising: Cardamine, dried, moderately crushed, take 2.0 kg, add 20 times volume (40L) of 0.03 mol / L NaOH aqueous solution, extract twice at 100℃, each extraction for 2 hours, filter, combine the filtrate, cool to 45℃, add composite protease, among which the enzyme activity of alkaline protease is 200,000 U / g; the enzyme activity of neutral protease is 50,000 U / g; the enzyme activity of papain is 200,000 U / g. The dosage of alkaline protease and papain is 0.2 g / L, and the dosage of neutral protease is 0.8 g / L. The enzymolysis temperature is 45℃, and the enzymolysis time is 90 min. After enzymolysis, membrane separation is performed, and the interception part with a molecular weight of compound>1000 is taken, and vacuum concentration (vacuum concentration at 100℃) is concentrated to 3 L. The concentrate was allowed to stand at 4°C overnight, centrifuged (6000 rpm, 10 min), the supernatant was taken, and spray-dried to obtain Cardamine selenium polysaccharide (100 g).

[0037] The polysaccharide content was determined by the sulfuric acid phenol method, and the protein content was determined by the Kjeldahl method.

[0038] It can be seen that the yield of selenium polysaccharides is 4.98%, of which the polysaccharide content is 90.57% and the protein content is 2.37%.

[0039] Process Optimization 1 - Sodium Hydroxide Concentration Selenium polysaccharide was prepared according to the preparation method provided in Example 1. The preparation method was the same as that in Example 1, except that the concentration of sodium hydroxide used was different, for example, 0.01 mol / L, 0.02 mol / L, 0.04 mol / L and 0.05 mol / L, and the other conditions were the same as those in Example 1.

[0040] The results are shown in Table 1. In Table 1, the polysaccharide yield refers to the obtained selenium polysaccharide / Cardamine*100%.

[0041] Table 1 Effect of different sodium hydroxide concentrations

[0042] Process Optimization 2 - Dosage of Sodium Hydroxide Aqueous Solution Selenium polysaccharide was prepared by referring to the preparation method provided in Example 1. The preparation method was the same as that in Example 1, except that the amount of sodium hydroxide aqueous solution was different, specifically 10 times the volume of 20 L, 15 times the volume of 30 L, 25 times the volume of 50 L and 30 times the volume of 60 L, and the other conditions were the same as those in Example 1. The results are shown in Table 2.

[0043] Table 2 Effect of different dosages of sodium hydroxide aqueous solution

[0044] Process Optimization 3 - Enzymatic Hydrolysis Temperature Selenium polysaccharide was prepared by referring to the preparation method provided in Example 1. The preparation method was the same as that in Example 1, except that the enzymatic hydrolysis temperature was different, specifically, 35°C, 40°C, 50°C and 55°C, and the other conditions were the same as those in Example 1.

[0045] See Table 3 for the results.

[0046] Table 3 Effect of different enzymatic hydrolysis temperatures

[0047] Process Optimization 4 - Enzymatic Hydrolysis Time Selenium polysaccharide was prepared by referring to the preparation method provided in Example 1. The preparation method was the same as that in Example 1, except that the enzymatic hydrolysis time was different, specifically 60 min, 75 min, 90 min, 105 min and 120 min, and the other conditions were the same as those in Example 1.

[0048] The results are shown in Table 4.

[0049] Table 4 Effect of different enzymatic hydrolysis time

[0050] Process Optimization 5 - Combination of Different Proteases Selenium polysaccharide was prepared by referring to the preparation method provided in Example 1. The preparation method was the same as that in Example 1, except that the protease or composite protease was different, and the other conditions were the same as those in Example 1. The results are shown in Table 5.

[0051] Table 5 Effects of different compositions of proteases

[0052] Process Optimization6 Selenium polysaccharide was prepared by referring to the preparation method provided in Example 1. The preparation method was the same as that in Example 1, except that the amount of the complex enzyme used was different, and the other conditions were the same as those in Example 1. The results are shown in Table 6.

[0053] Table 6 Effect of different enzyme dosages

[0054] In summary, according to Tables 1 to 6 above, if the conditions specified in the embodiments of the present invention are changed, for example, the amount or combination of the complex enzyme, the enzymatic hydrolysis temperature, the amount of sodium hydroxide, and the amount of water are changed, the yield of selenium polysaccharides may be reduced, or the content of polysaccharides in selenium polysaccharides may be reduced, or the content of protein in selenium polysaccharides may be increased. The preparation method provided in the embodiments of the present invention can ensure the yield of selenium polysaccharides, while ensuring the content of polysaccharides in selenium polysaccharides and reducing the content of protein in selenium polysaccharides.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing Cardamine selenium polysaccharide, characterized in that: include: Mixing Cardamine and a hydroxide aqueous solution to extract and form an extract; The extract is mixed with a composite protease for enzymolysis; The concentration of the hydroxide aqueous solution is 0.028-0.032 mol / L, and 20-30 L of the hydroxide aqueous solution is used for every kilogram of the Broken Rice Mustard; the hydroxide is selected from sodium hydroxide; The composite protease consists of alkaline protease, neutral protease and papain; The dosage of the alkaline protease is 0.18-0.22 g / L, the dosage of the papain is 0.18-0.22 g / L, and the dosage of the neutral protease is 0.7-0.9 g / L; The enzyme activity of the alkaline protease is 150,000-250,000 U / g; the enzyme activity of the neutral protease is 30,000-70,000 U / g; the enzyme activity of the papain is 150,000-250,000 U / g; After the enzymatic hydrolysis is completed, the enzymatic hydrolysis system is subjected to membrane separation, wherein the membrane separation collects the interception portion with a molecular weight greater than 1000.

2. The method for preparing Cardamine selenium polysaccharide according to claim 1, characterized in that: The extraction conditions include: extraction temperature of 100-120° C., extraction time of 1.5-3 h, and extraction times of 1-3 times.

3. The method for preparing Cardamine selenium polysaccharide according to claim 1, characterized in that: The conditions for enzymatic hydrolysis include: temperature of 43-47°C and time of 95-120 min.

4. The method for preparing Cardamine selenium polysaccharide according to claim 1, characterized in that: include: The extract is cooled to an enzymolysis temperature and then enzymolyzed.

5. The method for preparing Cardamine selenium polysaccharide according to any one of claims 1 to 4, characterized in that: include: The enzymatic hydrolysis system is subjected to membrane separation and then concentrated, allowed to stand, centrifuged and dried in sequence.

6. The method for preparing Cardamine selenium polysaccharide according to claim 5, characterized in that: The centrifugal conditions include: a rotation speed of 4500-7500 rpm and a time of 5-15 minutes.

7. A Cardamine selenium polysaccharide, characterized in that: The polysaccharide is prepared by the method for preparing Cardamine selenopolysaccharide according to claim 1.

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