Method for extracting cactus polysaccharide by using compound enzyme

Abstract

The invention belongs to the technical field of cactus polysaccharide, and discloses a method for extracting cactus polysaccharide by compound enzyme, which comprises the following steps: S1, pretreating fresh cactus to obtain cactus powder, sealing and storing; s2, adding a compound enzyme into the cactus powder, and carrying out polysaccharide extraction by utilizing ultrasonic waves; wherein the compound enzyme is any two or three of papain, cellulase and pectinase; s3, after ultrasonic extraction, enzyme deactivation and centrifugation are conducted, an upper-layer crude extracting solution is taken and concentrated, ethanol is added for precipitation, standing and centrifugation are conducted, precipitates continue to be washed with absolute ethyl alcohol, acetone and diethyl ether respectively, drying is conducted, and crude cactus polysaccharide is obtained. According to the method, the cactus polysaccharide is extracted by a compound enzyme enzyme method mainly comprising natural plant enzymes, so that the cost is reduced, the extraction purity is improved, and the cactus polysaccharide can be efficiently extracted.

Description

Technical Field

[0001] The present invention relates to the technical field of cactus polysaccharides, and specifically to a method for extracting cactus polysaccharides with a composite enzyme. Background Art

[0002] Cacti are succulent plants belonging to the family Cactaceae. They are widely distributed in arid and semi-arid regions and are also cultivated as ornamental plants or cash crops in dry areas around the world due to their strong adaptability. Cacti not only have important value in horticulture but also have extensive applications in fields such as food and medicine. Cacti contain not only rich natural medicinal ingredients such as polysaccharides, alkaloids, and flavonoid compounds but also more than 10 kinds of mineral trace elements, 17 kinds of amino acids essential for the human body, and various vitamins, and have numerous health care functions. Cactus polysaccharides (ODPs) are natural polysaccharide substances extracted from cacti. These polysaccharides are mainly composed of monosaccharides such as glucose, galactose, and arabinose and have various biological activities. Compared with many precious traditional Chinese medicines such as wolfberry and angelica, the polysaccharide yield in cactus dry powder is as high as over 4%, which is 70 times that of wolfberry polysaccharide, 40 times that of trichosanthin polysaccharide, 20 times that of astragalus polysaccharide, and 60 times that of angelica polysaccharide. Research shows that cactus polysaccharides have significant pharmacological effects such as anti-tumor, anti-inflammatory and antibacterial, hypoglycemic and lipid-lowering, and immune-enhancing. Some studies point out that it may be helpful for the prevention and treatment of certain diseases (such as diabetes and cardiovascular diseases). Liu Ting's research shows that cactus polysaccharides can significantly increase the weight of mice, reduce the pathological damage of liver tissue, and improve the physiological and biochemical indexes in the peripheral blood of mice, showing a good therapeutic effect on cadmium-induced liver injury. In addition, due to its moisturizing properties, it is often added to cosmetics and skin care products. Therefore, cacti and their extract - cactus polysaccharides, have broad application prospects in multiple fields. With the continuous advancement of in-depth research on the biological characteristics and components of cacti, more uses of it may be discovered in the future.

[0003] The main methods for separating and extracting active polysaccharides include: water extraction method, alkali extraction method, enzyme extraction method, salt extraction method, acid extraction method, alcohol extraction method, etc. The extraction rate of polysaccharides is affected by many factors, such as extraction temperature, extraction time, type of precipitant, liquid-to-solid ratio, etc. Different separation and purification methods result in different polysaccharide components and different activities. Water extraction is the most traditional method for extracting polysaccharides, but the yield is generally less than 1%. And the crude polysaccharides of cactus extracted by water extraction and alcohol precipitation often contain impurities such as proteins, pigments, and some low-molecular organic compounds, which must be removed separately. Among them, the removal of proteins can be achieved by the Sevag method, trifluorotrichloroethane method, and trichloroacetic acid method. Pigments can be adsorbed and removed by weak basic resin DEAE-cellulose, and the process is relatively complex. Although the acid extraction method improves the polysaccharide yield, many scholars believe that the extraction of cactus polysaccharides under acidic conditions will cause glycosidic bond cleavage and reduce the biological activity of polysaccharides. Using microwave or ultrasonic assisted extraction methods has the advantages of high extraction efficiency, less time consumption, and low cost. Using the enzyme method to extract cactus polysaccharides is gentle, time-consuming, can effectively decompose the plant cell wall to improve the polysaccharide yield and maintain the biological activity of polysaccharides. However, currently, the cactus polysaccharides are generally extracted by the enzyme method, and the enzymes used mainly come from microbial culture, with unstable components, large enzyme dosage, high cost, and low efficiency. Summary of the Invention

[0004] The present invention aims to provide a method for extracting cactus polysaccharides with a composite enzyme, which uses a composite enzyme mainly composed of natural plant enzymes to extract cactus polysaccharides, reduces costs, improves extraction purity, and can efficiently extract cactus polysaccharides.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for extracting cactus polysaccharides with a composite enzyme, comprising the following steps:

[0007] S1. Take fresh cactus for pretreatment to obtain cactus powder, and store it sealed;

[0008] S2. Add a composite enzyme preparation to the cactus powder and use ultrasonic waves for polysaccharide extraction; wherein, the composite enzyme is any two or three of papain, cellulase, and pectinase;

[0009] S3. After ultrasonic extraction, inactivate the enzyme, centrifuge, take the upper layer of the crude extract and concentrate it, add ethanol for precipitation, let it stand, centrifuge, and continue to wash the precipitate with anhydrous ethanol, acetone, and ether respectively, and dry it to obtain crude cactus polysaccharides.

[0010] Further, in S1, the steps of pretreatment are: take fresh cactus, remove thorns, wash, slice, dry, pulverize, sieve, add ethanol at a liquid-to-solid ratio of 1:2 (g:mL), reflux, dry, and store it sealed.

[0011] Further, in S2, the ratio of cactus powder to the complex enzyme preparation is 1:20 - 30 (g:mL); the extraction temperature is 45°C - 60°C, and the extraction time is 60 min - 90 min.

[0012] Further, in S2, the complex enzyme preparation is 0.1% - 0.3% cellulase and 0.1% - 0.5% pectinase.

[0013] Further, in S2, the complex enzyme preparation is 0.1% - 0.3% cellulase, 0.1% - 0.5% pectinase and 0.1% - 0.3% papain.

[0014] Further, in S3, after ultrasonic extraction, the enzyme is inactivated, centrifuged, the upper-layer crude extract is taken and concentrated, and ethanol is added for precipitation and standing, including: after ultrasonic extraction, the enzyme is inactivated, centrifuged, the upper-layer crude extract is taken and concentrated to 1 / 3 - 1 / 2, and 2 volumes of 95% ethanol are added and allowed to stand for 12 h - 16 h.

[0015] The beneficial effects of the technical solution are:

[0016] A method for extracting cactus polysaccharides with a composite enzyme provided by the present invention uses a composite enzyme enzymatic method mainly composed of natural plant proteases to extract cactus polysaccharides, which can reduce costs, improve purity, and efficiently extract cactus polysaccharides, further maturing the enzymatic polysaccharide extraction technology. Moreover, the composite enzyme method centered on papain, cellulase, and pectinase significantly improves the purity and bioactivity retention of cactus polysaccharides by directionally decomposing cellulose, pectin, and proteins in the cell wall. In addition, the enzymatic method is carried out under mild conditions, avoiding the breakage of polysaccharide molecular chains or the destruction of functional groups (such as sulfate groups and carboxyl groups) caused by high temperatures, and retaining the biological activity of polysaccharides. Specifically, cactus polysaccharides (such as complex polysaccharides composed of glucose, galactose, etc.) are tightly bound to proteins, cellulose, and pectin, and these components together form a physical barrier that hinders the release of polysaccharides. However, cellulase, pectinase, and papain can cooperate to destroy the cell wall structure and release polysaccharides. For example, cellulase can destroy the "skeleton" of the cell wall, loosen the cell wall structure, and expose polysaccharides; pectinase can decompose the polysaccharide-protein crosslinking network of pectin, reduce the binding of polysaccharides to other components, and promote the release of polysaccharides; papain can decompose the proteins in the cell wall, destroy the physical barrier, and separate polysaccharides from non-carbohydrate components. It can also decompose the extracted proteins, omitting the protein removal process, which not only simplifies the extraction process but also improves the purity of polysaccharides. Moreover, the extraction conditions are mild, avoiding the destruction of the polysaccharide structure by high temperatures. Therefore, by extracting cactus polysaccharides with a plant composite enzyme, by directionally decomposing cellulose, pectin, and proteins, releasing polysaccharides bound to non-carbohydrate components, the enzyme has high specificity for the substrate, reducing the dissolution of non-target components (such as pigments and lipids), with fewer impurities in the extract, and improving the product purity. Combining ultrasonic-assisted treatment can accelerate the enzymatic reaction, with high extraction efficiency, no need for high-temperature long-term heating, reducing energy consumption. In addition, plant-derived enzymes have a wide source, stable components, low costs, and the molecular weight and monosaccharide composition of polysaccharides obtained by the same method do not differ significantly. Detailed implementation mode

[0017] The following further elaborates on the present invention in combination with the implementation mode:

[0018] Example 1

[0019] A method for extracting cactus polysaccharides with a composite enzyme includes the following steps:

[0020] S1. Take fresh cactus, remove thorns, wash, slice, dry, crush, pass through a 20-mesh sieve, add ethanol at a material-liquid ratio of 1:2 (g:mL), reflux for 2 h, dry to obtain cactus powder, and store it sealed;

[0021] S2. Take 10 g of cactus powder, add 0.3% cellulase, 0.2% pectinase and 0.3% papain enzyme preparation, with a material-liquid ratio of 1:30 (g:mL), and use ultrasonic waves for polysaccharide extraction at an extraction temperature of 45 °C and an extraction time of 90 min;

[0022] S3. After ultrasonic extraction, inactivate the enzyme, centrifuge, take the upper-layer crude extract and concentrate it to one-third, add 2 volumes of 95% ethanol for precipitation, let it stand for 12 h and then centrifuge, wash the precipitate with anhydrous ethanol, acetone and ether for 3 times respectively, and dry to obtain crude cactus polysaccharide. The yield of crude cactus polysaccharide is 10.7%, and the purity measured by the phenol-sulfuric acid method is 76.5%.

[0023] Example 2

[0024] A method for extracting cactus polysaccharide with a composite enzyme, comprising the following steps:

[0025] S1. Take fresh cactus, remove thorns, wash, slice, dry, crush, pass through a 20-mesh sieve, add ethanol at a material-liquid ratio of 1:2 (g:mL), reflux for 2 h, dry to obtain cactus powder, and store it sealed;

[0026] S2. Take 10 g of cactus powder, add 0.2% cellulase and 0.5% pectinase enzyme preparation, with a material-liquid ratio of 1:30 (g:mL), and use ultrasonic waves for polysaccharide extraction at an extraction temperature of 60 °C and an extraction time of 75 min;

[0027] S3. After ultrasonic extraction, inactivate the enzyme, centrifuge, take the upper-layer crude extract and concentrate it to one-third, add 2 volumes of 95% ethanol for precipitation, let it stand for 12 h and then centrifuge, wash the precipitate with anhydrous ethanol, acetone and ether for 3 times respectively, and dry to obtain crude cactus polysaccharide. The yield of crude cactus polysaccharide is 11.9%, and the purity is 64.7%.

[0028] Example 3

[0029] A method for extracting cactus polysaccharide with a composite enzyme, comprising the following steps:

[0030] S1. Take fresh cactus, remove thorns, wash, slice, dry, crush, pass through a 20-mesh sieve, add ethanol at a material-liquid ratio of 1:2 (g:mL), reflux for 2 h, dry to obtain cactus powder, and store it sealed;

[0031] S2. Take 10 g of cactus powder, add 0.1% cellulase and 0.1% pectinase enzyme preparation, with a material-liquid ratio of 1:20 (g:mL), and use ultrasonic waves for polysaccharide extraction at an extraction temperature of 50 °C and an extraction time of 60 min;

[0032] S3. After ultrasonic extraction, inactivate the enzymes, centrifuge, take the upper-layer crude extract and concentrate it to one-half, add 2 volumes of 95% ethanol for precipitation, let it stand for 12 h and then centrifuge, wash the precipitate with absolute ethanol, acetone, and ether for 3 times respectively, and dry to obtain crude cactus polysaccharide. The yield of crude cactus polysaccharide is 3.54%, and the purity is 73.2%.

[0033] Example 4

[0034] A method for extracting cactus polysaccharide with a composite enzyme, comprising the following steps:

[0035] S1. Take fresh cactus, remove thorns, wash, slice, dry, pulverize, pass through a 20-mesh sieve, add ethanol at a material-liquid ratio of 1:2 (g:mL), reflux for 2 h, dry to obtain cactus powder, and store it sealed;

[0036] S2. Take 10 g of cactus powder, add 0.3% cellulase, 0.4% pectinase, and 0.3% papain enzyme preparation, with a material-liquid ratio of 1:20 (g:mL), and use ultrasonic waves to extract polysaccharides at an extraction temperature of 60 °C and an extraction time of 90 min;

[0037] S3. After ultrasonic extraction, inactivate the enzymes, centrifuge, take the upper-layer crude extract and concentrate it to one-half, add 2 volumes of 95% ethanol for precipitation, let it stand for 12 h and then centrifuge, wash the precipitate with absolute ethanol, acetone, and ether for 3 times respectively, and dry to obtain crude cactus polysaccharide. The yield of crude cactus polysaccharide is 9.4%, and the purity is 67.5%.

[0038] Example 5

[0039] A method for extracting cactus polysaccharide with a composite enzyme, comprising the following steps:

[0040] S1. Take fresh cactus, remove thorns, wash, slice, dry, pulverize, pass through a 20-mesh sieve, add ethanol at a material-liquid ratio of 1:2 (g:mL), reflux for 2 h, dry to obtain cactus powder, and store it sealed;

[0041] S2. Take 10 g of cactus powder, add 0.15% cellulase, 0.3% pectinase, and 0.2% papain enzyme preparation, with a material-liquid ratio of 1:25 (g:mL), and use ultrasonic waves to extract polysaccharides at an extraction temperature of 50 °C and an extraction time of 75 min;

[0042] S3. After ultrasonic extraction, inactivate the enzymes, centrifuge, take the upper-layer crude extract and concentrate it to one-half, add 2 volumes of 95% ethanol for precipitation, let it stand for 12 h and then centrifuge, wash the precipitate with absolute ethanol, acetone, and ether for 3 times respectively, and dry to obtain crude cactus polysaccharide. The yield of crude cactus polysaccharide is 10.2%, and the purity is 77.6%.

[0043] Example 6

[0044] A method for extracting cactus polysaccharide with a composite enzyme, comprising the following steps:

[0045] S1. Take fresh cactus, remove thorns, wash, slice, dry, crush, pass through a 20-mesh sieve, add ethanol at a solid-liquid ratio of 1:2 (g:mL), reflux for 2 h, dry to obtain cactus powder, and store it sealed;

[0046] S2. Take 10 g of cactus powder, add 0.2% cellulase, 0.3% pectinase and 0.3% papain enzyme preparation, with a solid-liquid ratio of 1:30 (g:mL), and use ultrasonic waves for polysaccharide extraction, with an extraction temperature of 55 °C and an extraction time of 70 min;

[0047] S3. After ultrasonic extraction, inactivate the enzyme, centrifuge, take the upper-layer crude extract and concentrate it to one-half, add 2 volumes of 95% ethanol for precipitation, let it stand for 12 h and then centrifuge, wash the precipitate with absolute ethanol, acetone, and ether for 3 times respectively, and dry to obtain crude cactus polysaccharide. The yield of crude cactus polysaccharide is 12.7%, and the purity is 69.8%.

[0048] In summary, the method for extracting cactus polysaccharides provided by the present invention uses a complex enzyme method with natural plant proteases as the main component to extract cactus polysaccharides, which can reduce costs, improve the purification purity, and efficiently extract cactus polysaccharides, further maturing the technology of extracting polysaccharides by the enzyme method. Moreover, the complex enzyme method centered on papain, cellulase, and pectinase significantly improves the purity and bioactivity retention of cactus polysaccharides by directionally decomposing the cellulose, pectin, and proteins in the cell wall. In addition, the enzyme method is carried out under mild conditions, avoiding the breakage of polysaccharide molecular chains or the destruction of functional groups (such as sulfate groups and carboxyl groups) caused by high temperature, and retaining the biological activity of polysaccharides. Specifically, cactus polysaccharides (such as complex polysaccharides composed of glucose, galactose, etc.) are tightly bound to proteins, cellulose, and pectin, and these components together form a physical barrier that hinders the release of polysaccharides. However, cellulase, pectinase, and papain can cooperate to destroy the cell wall structure and release polysaccharides. For example, cellulase can destroy the "skeleton" of the cell wall, making the cell wall structure loose and the polysaccharides exposed; pectinase can decompose the polysaccharide-protein cross-linking network of pectin, reduce the binding of polysaccharides to other components, and promote the release of polysaccharides; papain can decompose the proteins in the cell wall, destroy the physical barrier, and separate the polysaccharides from non-carbohydrate components. Moreover, the extraction conditions are mild, avoiding the destruction of the polysaccharide structure by high temperature. Therefore, by using plant complex enzymes to extract cactus polysaccharides, the polysaccharides bound to non-carbohydrate components are released by directionally decomposing cellulose, pectin, and proteins. The enzyme has high specificity for the substrate, reducing the dissolution of non-target components (such as pigments and lipids), with less impurities in the extract and higher product purity. Combining ultrasonic-assisted treatment can accelerate the enzymatic reaction, with high extraction efficiency, no need for high-temperature long-term heating, and reduced energy consumption. In addition, plant-derived enzymes are widely sourced, with stable components and low costs, and there are no obvious differences in the molecular weight and monosaccharide composition of the polysaccharides obtained by the same method.

[0049] The above are only examples of the present invention, and specific technical solutions or common knowledge such as well-known characteristics in the solution are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A method for extracting cactus polysaccharide with a composite enzyme, characterized in that, It includes the following steps: S1. Take fresh cactus for pretreatment to obtain cactus powder, and store it sealed; S2. Add a complex enzyme preparation to the cactus powder and use ultrasonic waves for polysaccharide extraction; among them, the complex enzyme is any two or three of papain, cellulase, and pectinase; S3. After ultrasonic extraction, inactivate the enzyme, centrifuge, take the upper-layer crude extract and concentrate it, add ethanol for precipitation, let it stand, centrifuge, and continue to wash the precipitate with anhydrous ethanol, acetone, and ether respectively, and dry to obtain crude cactus polysaccharide.

2. The method for extracting cactus polysaccharide with a composite enzyme according to claim 1, characterized in that: In S1, the steps of pretreatment are: take fresh cactus, remove thorns, wash, slice, dry, crush, sieve, add ethanol at a solid-liquid ratio of 1:2 (g:mL), reflux, dry, and store it sealed.

3. The method for extracting cactus polysaccharide by using a composite enzyme according to claim 1, wherein: In S2, the ratio of cactus powder to the complex enzyme preparation is 1:20 - 30 (g:mL); the extraction temperature is 45°C - 60°C, and the extraction time is 60 min - 90 min.

4. The method for extracting cactus polysaccharide by using a composite enzyme according to claim 1, characterized in that: In S2, the complex enzyme preparation is 0.1% - 0.3% cellulase and 0.1% - 0.5% pectinase.

5. A method for extracting cactus polysaccharide with a composite enzyme according to claim 1, characterized in that: In S2, the complex enzyme preparation is 0.1% - 0.3% cellulase, 0.1% - 0.5% pectinase, and 0.1% - 0.3% papain.

6. The method for extracting cactus polysaccharide with a composite enzyme according to claim 1, wherein: In S3, the steps of inactivating the enzyme, centrifuge, taking the upper-layer crude extract and concentrating it, and adding ethanol for precipitation and letting it stand after ultrasonic extraction include: inactivating the enzyme, centrifuge, taking the upper-layer crude extract and concentrating it to 1 / 3 - 1 / 2 after ultrasonic extraction, adding 2 volumes of 95% ethanol and letting it stand for 12 h - 16 h.