Preparation method of selenium polysaccharide in cardamine violifolia

Abstract

The invention relates to the technical field of biological extraction, in particular to a preparation method of selenium polysaccharide in cardamine violifolia, which comprises the following steps: extracting selenium polysaccharide in cardamine violifolia by using a deep eutectic solvent as an extraction solvent and adopting an ultrasonic method, and removing protein in the selenium polysaccharide by using an isoelectric precipitation method and an enzymolysis method, thereby obtaining the selenium polysaccharide in cardamine violifolia. Removing water, enzymolysis product amino acid, inorganic selenium and other impurities by a membrane separation concentration system to obtain cardamine violifolia selenium polysaccharide; wherein the eutectic solvent comprises choline chloride and malonic acid in a molar ratio of 1: 1-1: 3, and the water content is 40-60% (mass percent); the compound enzyme is prepared from bromelain and papain in a mass ratio of 1: 1. Protein in the selenium polysaccharide is removed by adopting deep eutectic solvent ultrasonic extraction and an isoelectric precipitation method combined with an enzymolysis method, enzymolysis product amino acid, inorganic selenium and other impurities are removed by adopting an ultrafiltration membrane separation and concentration system, the structure of the polysaccharide is not damaged, and the high-purity cardamine violifolia selenium polysaccharide is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological extraction, in particular to a preparation method of selenium polysaccharide in Pteroxygonum giraldii. BACKGROUND

[0002] Selenium polysaccharide is an organic selenium compound linked by chemical bonds between selenium and polysaccharide, which has the biological functions of selenium and polysaccharide, and has the effects of anti-tumor, reducing blood lipids and blood sugar, antioxidant and enhancing immunity. Plant organic selenium source has the advantages of green, safety, high activity, easy absorption and utilization by human body, etc., and is one of the best-selling selenium raw materials on the market, and is often used for the fortification of selenium nutritional elements in food. Pteroxygonum giraldii is a super selenium plant with rich nutrition, and its high selenium organic conversion rate makes it the best carrier of plant organic selenium source, so the development of safe, green and efficient organic selenium source has high economic value.

[0003] In the prior art, polysaccharide is generally extracted by hot extraction, acid-base extraction, ultrasonic extraction, enzymatic hydrolysis and the like, which has low extraction efficiency, long time consumption and high production cost. The methods for removing protein include organic solvent sedimentation, isoelectric point sedimentation and enzymatic hydrolysis, and a single method cannot safely remove protein. Therefore, the yield and purity of selenium polysaccharide are not high in the prior art. SUMMARY

[0004] In view of the above problems in the prior art, the present application aims to provide a preparation method of selenium polysaccharide in Pteroxygonum giraldii, which can improve the extraction rate of selenium polysaccharide, remove macromolecular protein and small molecular impurities, and improve the yield and purity of selenium polysaccharide.

[0005] In order to achieve the above-mentioned application purposes, the technical scheme adopted by the present application is as follows:

[0006] A preparation method of selenium polysaccharide in Pteroxygonum giraldii is provided, which comprises the following steps:

[0007] The low eutectic solvent is used as the extraction solvent, and the ultrasonic wave method is used to extract the selenium polysaccharide in Pteroxygonum giraldii, the isoelectric point precipitation method is combined with the enzymatic hydrolysis method to remove the protein in the selenium polysaccharide, and the membrane separation and concentration system is used to remove water, enzymatic hydrolysis product amino acid, inorganic selenium and other impurities, so as to obtain the selenium polysaccharide in Pteroxygonum giraldii; wherein the low eutectic solvent is choline chloride and malonic acid with a molar ratio of 1:1-1:3, and the water content is 40wt%-60wt%; the complex enzyme is a mixture of bromelain and papain with a mass ratio of 1:1.

[0008] Further, the specific process of the low eutectic solvent ultrasonic extraction is as follows:

[0009] The super-micro powder of Pteroxygonum giraldii is added into an extraction container, and choline chloride and malonic acid are used as solvents, the solid-liquid ratio is 1:10 g / ml-1:20 g / ml, the ultrasonic power is 500 W, the extraction temperature is 40-60 DEG C, and the extraction time is 30-60 min, then filtration and centrifugation are carried out, and centrifugal liquid 1 is obtained.

[0010] Further, the specific process of removing the protein in the selenium polysaccharide by the isoelectric point precipitation method combined with the enzymatic method is as follows:

[0011] The pH of the centrifugal liquid 1 is adjusted to 8.0-10.0 by using a sodium hydroxide solution, and then standing and sedimentation are carried out for 2 h, centrifugation is carried out, and centrifugal liquid 2 is obtained; the pH of the centrifugal liquid 2 is adjusted to 6.0-8.0 by using acetic acid, and then bromelain and papain are added, the enzymatic temperature is 45-60 DEG C, the enzymatic time is 1-3 h, and centrifugation is carried out, and centrifugal liquid 3 is obtained.

[0012] Further, the specific process of membrane separation and concentration is as follows:

[0013] The centrifugal liquid 3 is subjected to ultrafiltration membrane, one-half volume of pure water of the extraction liquid is added for washing, the concentration is carried out to one-sixth volume of the extraction liquid, and the interception liquid is collected; the interception liquid is subjected to spray drying, and Pteroxygonum giraldii selenium polysaccharide powder is obtained.

[0014] Further, the ultrafiltration membrane is a 3000 Da molecular weight hollow fiber membrane, the membrane pore size is 0.4 nm, and the membrane pressure is 0.4-0.6 MPa.

[0015] Further, the dosage of the compound enzyme is 0.05-0.3 g / L, and the enzyme activity of the bromelain and the papain is 1.2 million u / g and 2 million u / g respectively.

[0016] Further, the inlet air temperature of the spray drying is 170-190 DEG C, and the outlet air temperature is 60-80 DEG C.

[0017] The Pteroxygonum giraldii selenium polysaccharide powder has the advantages that:

[0018] The extraction process of the Pteroxygonum giraldii selenium polysaccharide is established, the low-eutectic solvent ultrasonic extraction method is adopted, the isoelectric point precipitation combined with the enzymatic method is used to remove the protein in the extraction liquid, the membrane separation and concentration system is used to remove the enzymatic product amino acid, inorganic selenium and other impurities, and the structure of the polysaccharide is not damaged, and the high-purity Pteroxygonum giraldii selenium polysaccharide is obtained. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described below to facilitate the understanding of those skilled in the art of the present application, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions using the concept of the present application are within the scope of protection.

[0020] Example 1

[0021] 1. 500g of super-fine powder of Pteridium aquilinum (selenium content 1000mg / kg) was added with 5L of deep eutectic solvent (choline chloride: propylene glycol = 1:3, molar ratio, water content 40wt%), extracted by ultrasonic method, extraction temperature was 40℃, extraction time was 60min, filtered, centrifuged, and centrifugal liquid 1 was obtained.

[0022] 2. The pH of centrifugal liquid 1 was adjusted to 8.5 by sodium hydroxide solution, and it was allowed to stand and settle for 2h, then centrifuged to obtain centrifugal liquid 2. The pH of centrifugal liquid 2 was adjusted to 7 by acetic acid, and 0.05g / L of complex enzyme (papain: papain = 1:1, mass ratio) was added, the enzymolysis temperature was 45℃, the enzymolysis time was 1h, and centrifugation was performed to obtain centrifugal liquid 3.

[0023] 3. Centrifugal liquid 3 was passed through an ultrafiltration membrane, and the volume of pure water added for washing was half of the volume of the extract, and it was concentrated to one sixth of the volume of the extract, and the retentate was collected. The retentate was spray dried (inlet air temperature 180℃, outlet air temperature 70℃) to obtain Pteridium aquilinum selenium polysaccharide powder.

[0024] Example 2

[0025] 1. 500g of super-fine powder of Pteridium aquilinum (selenium content 1000mg / kg) was added with 9L of deep eutectic solvent (choline chloride: propylene glycol = 1:3, molar ratio, water content 50wt%), extracted by ultrasonic method, extraction temperature was 50℃, extraction time was 50min, filtered, centrifuged, and centrifugal liquid 1 was obtained.

[0026] 2. The pH of centrifugal liquid 1 was adjusted to 9.5 by sodium hydroxide solution, and it was allowed to stand and settle for 2h, then centrifuged to obtain centrifugal liquid 2. The pH of centrifugal liquid 2 was adjusted to 7 by acetic acid, and 0.2g / L of complex enzyme (papain: papain = 1:1, mass ratio) was added, the enzymolysis temperature was 50℃, the enzymolysis time was 2h, and centrifugation was performed to obtain centrifugal liquid 3.

[0027] 3. Centrifugal liquid 3 was passed through an ultrafiltration membrane, and the volume of pure water added for washing was half of the volume of the extract, and it was concentrated to one sixth of the volume of the extract, and the retentate was collected. The retentate was spray dried (inlet air temperature 185℃, outlet air temperature 80℃) to obtain Pteridium aquilinum selenium polysaccharide powder.

[0028] Example 3

[0029] 1. 500 g of super-micro powder of Pteridium aquilinum (selenium content 1000 mg / kg) was added with 10 L of deep eutectic solvent (choline chloride: propylene glycol = 1:3, molar ratio, water content 50 wt%), extracted by ultrasonic method, extraction temperature was 60℃, extraction time was 30 min, filtered, centrifuged, and centrifugal liquid 1 was obtained.

[0030] 2. The pH of centrifugal liquid 1 was adjusted to 8 by sodium hydroxide solution, and it was allowed to stand and settle for 2 h, then centrifuged to obtain centrifugal liquid 2. The pH of centrifugal liquid 2 was adjusted to 6 by acetic acid, and 0.1 g / L of complex enzyme (papain: papain = 1:1, mass ratio) was added, the enzymolysis temperature was 60℃, the enzymolysis time was 3 h, and centrifugation was performed to obtain centrifugal liquid 3.

[0031] 3. Centrifugal liquid 3 was passed through an ultrafiltration membrane, and the volume of pure water added for washing was half of the volume of the extract, and the concentration was to one sixth of the volume of the extract, and the retentate was collected. The retentate was spray dried (inlet air temperature 185℃, outlet air temperature 80℃) to obtain Pteridium aquilinum selenium polysaccharide powder.

[0032] Example 4

[0033] 1. 500 g of super-micro powder of Pteridium aquilinum (selenium content 1000 mg / kg) was added with 7.5 L of deep eutectic solvent (choline chloride: propylene glycol = 1:3, molar ratio, water content 50 wt%), extracted by ultrasonic method, extraction temperature was 50℃, extraction time was 40 min, filtered, centrifuged, and centrifugal liquid 1 was obtained.

[0034] 2. The pH of centrifugal liquid 1 was adjusted to 8.5 by sodium hydroxide solution, and it was allowed to stand and settle for 2 h, then centrifuged to obtain centrifugal liquid 2. The pH of centrifugal liquid 2 was adjusted to 7.5 by acetic acid, and 0.3 g / L of complex enzyme (papain: papain = 1:1, mass ratio) was added, the enzymolysis temperature was 50℃, the enzymolysis time was 2 h, and centrifugation was performed to obtain centrifugal liquid 3.

[0035] 3. Centrifugal liquid 3 was passed through an ultrafiltration membrane, and the volume of pure water added for washing was half of the volume of the extract, and the concentration was to one sixth of the volume of the extract, and the retentate was collected. The retentate was spray dried (inlet air temperature 185℃, outlet air temperature 80℃) to obtain Pteridium aquilinum selenium polysaccharide powder.

[0036] Example 5

[0037] The difference between this example and Example 2 is that in step (1), “choline chloride: propylene glycol = 1:3 (molar ratio)” is replaced with “choline chloride: propylene glycol = 1:1 (molar ratio)”, and the others are the same as Example 2.

[0038] Example 6

[0039] The present example differs from Example 2 in that "chloride choline: propylene glycol = 1:3 (molar ratio)" in step (1) is replaced with "chloride choline: propylene glycol = 1:2 (molar ratio)", and the others are the same as Example 2.

[0040] Example 7

[0041] The present example differs from Example 2 in that "0.05 g / L of complex enzyme (bromelain: papain = 1:1) is added" in step (2) is replaced with "0.05 g / L of bromelain is added", and the others are the same as Example 2.

[0042] Example 8

[0043] The present example differs from Example 2 in that "0.05 g / L of complex enzyme (bromelain: papain = 1:1) is added" in step (2) is replaced with "0.05 g / L of papain is added", and the others are the same as Example 2.

[0044] Example 9

[0045] The present example differs from Example 2 in that "500 g of P. coriacea powder (total selenium content: 1000 mg / kg) is taken" is replaced with "5 kg of P. coriacea powder (total selenium content: 2000 mg / kg) is taken", and the others are the same as Example 2.

[0046] Example 10

[0047] The present example differs from Example 2 in that "5 kg of P. coriacea powder (total selenium content: 1000 mg / kg) is taken" is replaced with "100 kg of P. coriacea powder (total selenium content: 1000 mg / kg) is taken", and the others are the same as Example 2.

[0048] Comparative Example 1

[0049] The present comparative example differs from Example 2 in that P. coriacea stem and leaf with a length of 1-3 cm is used as the extraction raw material, and water is used as the extraction solvent, and the others are the same as Example 2.

[0050] Comparative Example 2

[0051] The present comparative example differs from Example 2 in that the step of adjusting the pH of the centrifuged liquid with acetic acid to precipitate the protein is not added, and the others are the same as Example 2.

[0052] Comparative Example 3

[0053] The present comparative example differs from Example 2 in that bromelain and papain are not added, and the others are the same as Example 2.

[0054] Comparative Example 4

[0055] The comparative example is different from example 2 in that the two steps of acid precipitation and enzymatic hydrolysis of proteins are cancelled, the extract is centrifuged and then passed through an ultrafiltration membrane, one-half volume of pure water is added to the extract to rinse, the extract is concentrated to one-sixth of its original volume, and the retentate is collected. Anhydrous ethanol is added to the retentate to a concentration of 75%, the mixture is allowed to stand for 2 hours, and then centrifuged. The precipitate is collected and mixed with water, and then spray dried (inlet temperature 180°C, outlet temperature 70°C) to obtain a powder of selenium polysaccharides from P. cordata. The rest is the same as example 2.

[0056] Comparative example 5

[0057] 500g of ultra-fine powder of P. cordata (total selenium content: 1000mg / kg) is extracted using the existing ultrasonic extraction method, filtered, centrifuged, concentrated under reduced pressure, and then anhydrous ethanol is added to a concentration of 75%, the mixture is allowed to stand for 2 hours, and then centrifuged. The precipitate is collected, mixed with water, and then spray dried to obtain a powder of selenium polysaccharides.

[0058] The products of selenium polysaccharides from P. cordata obtained in examples 1-10 and comparative examples 1-5 are collected, and the yield of the product selenium polysaccharides (i.e. the percentage of the mass of the product selenium polysaccharides to the mass of the P. cordata powder) is calculated. The contents of selenium, polysaccharides, inorganic selenium, and proteins in the selenium polysaccharides obtained in examples 1-10 and comparative examples 1-5 are detected (the content of selenium is detected using the first method in GB 2009.93-2017, the content of polysaccharides is detected using the phenol-sulfuric acid method, the content of inorganic selenium is detected using the first method in DBS42010-2018, and the content of proteins is detected using the first method in GB5009.5-2016), and the results are shown in Table 1.

[0059] Table 1: Experimental results of examples and comparative examples

[0060]

[0061] From the above results, it can be concluded that the extraction method of the present application can effectively improve the yield of selenium polysaccharides and the contents of total selenium and polysaccharides, while removing inorganic selenium and proteins.

[0062] It will be obvious to a person skilled in the art that, without departing from the spirit or essential characteristics of the application, the present application can be implemented in other specific forms. The present examples are therefore to be considered in all respects as illustrative and not restrictive, the scope of the application being indicated by the appended claims rather than by the foregoing description which is intended to be exemplary only and the intention is that all changes that come within the meaning of the equivalences of the claims are embraced therein.

[0063] Furthermore, it should be understood that although the specification is described in terms of embodiments, not every embodiment includes every feature described. The specification can include implicit combinations of explicitly mentioned features and / or explicit combinations of implicitely mentioned features. Each embodiment depends on the explicit combinations of features and / or the implicit combinations of features made specifically within that embodiment, and each such embodiment can be combined with every other such embodiment to create further embodiments.

Claims

1. A method for preparing selenium polysaccharide from *Violet Leaf Mint*, characterized in that, Includes the following steps: Selenium polysaccharide was extracted from *Corydalis violacea* using an ultrasonic method with a eutectic solvent as the extraction solvent. Proteins in the selenium polysaccharide were removed by a combination of isoelectric point precipitation and enzymatic hydrolysis. Water, amino acids from the enzymatic hydrolysis products, inorganic selenium, and other impurities were removed by a membrane separation and concentration system to obtain *Corydalis violacea* selenium polysaccharide. The eutectic solvent consisted of choline chloride and malonic acid in a molar ratio of 1:1 to 1:3, with a water content of 40 wt% to 60 wt%. The complex enzyme consisted of bromelain and papain in a mass ratio of 1:

1.

2. The method for preparing selenium polysaccharide from *Violet Leaf Mint* according to claim 1, characterized in that, The specific process of ultrasonic extraction of eutectic solvents is as follows: Add the ultrafine powder of violet leaf and water chestnut to the extraction container, use choline chloride and malonic acid as solvents, the material-to-liquid ratio is 1:10g / ml-1:20g / ml, the ultrasonic power is 500W, the extraction temperature is 40℃-60℃, the extraction time is 30min-60min, filter, centrifuge, and obtain centrifuged liquid 1.

3. The method for preparing selenium polysaccharide from *Violet Leaf Mint* according to claim 2, characterized in that, The specific process for removing proteins from selenium polysaccharides using the isoelectric point precipitation method combined with enzymatic hydrolysis is as follows: Adjust the pH of centrifuge solution 1 to 8.0-10.0 with sodium hydroxide solution, let it stand for 2 hours to settle, and then centrifuge to obtain centrifuge solution 2; adjust the pH of centrifuge solution 2 to 6.0-8.0 with acetic acid, add bromelain and papain, enzymatically hydrolyze at 45℃-60℃ for 1-3 hours, and then centrifuge to obtain centrifuge solution 3.

4. The method for preparing selenium polysaccharide from *Violet Leaf Mint* according to claim 3, characterized in that, The specific process of membrane separation and concentration is as follows: The centrifuged liquid was passed through an ultrafiltration membrane, rinsed with half the volume of pure water, concentrated to one-sixth the volume of the extract, and the retentate was collected. The retentate was spray-dried to obtain Viola yedoensis selenium polysaccharide powder.

5. The method for preparing selenium polysaccharide from *Violet Leaf Mint* according to claim 4, characterized in that, The ultrafiltration membrane is a hollow fiber membrane with a molecular weight of 3000 Da, a pore size of 0.4 nm, and a membrane pressure of 0.4 MPa-0.6 MPa.

6. The method for preparing selenium polysaccharide from *Violet Flos* according to claim 1, characterized in that, The dosage of the compound enzyme was 0.05 g / L-0.3 g / L; the enzyme activities of bromelain and papain were 1.2 million u / g and 2 million u / g, respectively.

7. The method for preparing selenium polysaccharide from *Violet Leaf Mint* according to claim 4, characterized in that, The inlet air temperature for spray drying is 170-190℃, and the outlet air temperature is 60-80℃.

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