Method for extracting grape polysaccharide as active ingredient of grapes

Abstract

The invention relates to the technical field of natural product extraction, and discloses a method for extracting grape polysaccharide serving as an active ingredient of grapes, which comprises the following steps: pretreating grapes, performing enzymolysis treatment, performing ultrasonic-assisted extraction, and sequentially performing centrifugal separation, alcohol precipitation, washing, drying and the like. According to the method, by optimizing extraction conditions, the extraction efficiency and purity of the grape polysaccharide are remarkably improved, and the method is simple in technological operation, low in energy consumption, green, environmentally friendly and suitable for industrial production.

Description

Technical Field

[0001] This application relates to the technical field of natural product extraction, and particularly to a method for extracting grape polysaccharide, an active ingredient of grapes. Background Art

[0002] Grapes are a common and nutritious fruit, and the grape polysaccharides contained therein have various biological activities, such as immunomodulation, antioxidant, antitumor, etc., and have broad application prospects in the fields of food, medicine, health products, etc. However, the existing methods for extracting grape polysaccharides have problems such as low extraction efficiency, low purity, high energy consumption, and environmental pollution. The traditional hot water extraction method requires a long extraction time and a high temperature, which easily causes the destruction of the polysaccharide structure and a low extraction rate; although the organic solvent extraction method can improve the extraction efficiency, the use of organic solvents not only increases the production cost, but also may cause environmental pollution, and at the same time, the residual organic solvents pose a potential hazard to human health. Therefore, it is of great significance to develop a method for extracting grape polysaccharides that is efficient, environmentally friendly, and suitable for industrial production.

[0003] The above information disclosed in this background art is only used to increase the understanding of the background art of this application. Therefore, it may include prior art that is not known to those of ordinary skill in the art. Summary of the Invention

[0004] In order to solve the problems of low extraction efficiency, low purity, high energy consumption, environmental pollution, etc. existing in the existing extraction methods, improve the extraction efficiency and purity of grape polysaccharides, reduce the production cost, and achieve the problem of green and environmentally friendly extraction, this application provides a method for extracting grape polysaccharide, an active ingredient of grapes.

[0005] The method for extracting grape polysaccharide, an active ingredient of grapes, provided by this application adopts the following technical solutions:

[0006] A method for extracting grape polysaccharide, an active ingredient of grapes, comprises the following steps:

[0007] Step 1: Remove the stalks of fresh grapes, wash them, cut them into small pieces, perform freeze-drying treatment, and then grind them into powder. The freeze-drying treatment can retain the active ingredients in the grapes at low temperature, avoid the destruction of the polysaccharide structure by high temperature, and grinding into powder can increase the contact area between the material and the extraction solvent, thereby improving the extraction efficiency;

[0008] Step 2: Add a mixed enzyme solution with a mass concentration of 0.1 - 0.5% of cellulase and pectinase to the grape powder. The mass ratio of cellulase to pectinase in the mixed enzyme solution is 1:1 - 3:1, and enzymolysis is carried out at 35 - 50°C for 1 - 3 hours. Cellulase and pectinase can decompose cellulose and pectin in the grape cell wall, destroy the cell wall structure, and make polysaccharides easier to release, thereby improving the extraction rate. By optimizing the enzyme solution concentration, the ratio of enzymes, the enzymolysis temperature, and time, the best enzymolysis effect can be achieved;

[0009] Step 3: Add deionized water 5 - 10 times the mass of the enzymolyzed material, and carry out ultrasonic extraction for 20 - 40 minutes under the conditions of an ultrasonic power of 200 - 500W and a temperature of 40 - 60°C. The cavitation effect of ultrasonic waves can generate local high temperature, high pressure, and strong shock waves, accelerating the dissolution and diffusion of polysaccharides and improving the extraction efficiency. At the same time, controlling the appropriate ultrasonic power, extraction temperature, and time can avoid the degradation of polysaccharides while ensuring the extraction effect;

[0010] Step 4: Centrifuge the material after ultrasonic extraction at a speed of 4000 - 6000r / min for 10 - 20 minutes, and take the supernatant. Centrifugal separation can separate insoluble impurities from the polysaccharide solution to obtain a relatively pure polysaccharide extract;

[0011] Step 5: Add 3 - 5 volumes of absolute ethanol to the supernatant, and let it stand at 4 - 10°C for 12 - 24 hours for alcohol precipitation. Polysaccharides have low solubility in ethanol, and polysaccharides can be precipitated from the solution through alcohol precipitation to achieve the preliminary separation and purification of polysaccharides;

[0012] Step 6: Wash the precipitate after alcohol precipitation 2 - 3 times with absolute ethanol and acetone respectively, and then vacuum dry it at 40 - 60°C to obtain grape polysaccharide. Washing can remove residual impurities and ethanol on the surface of the precipitate, and vacuum drying can quickly dry polysaccharides at a lower temperature, avoiding the oxidation and degradation of polysaccharides and ensuring the quality of polysaccharides.

[0013] Preferably, the mass concentration of the mixed enzyme solution in Step 2 is 0.3%, and the mass ratio of cellulase to pectinase is 2:1.

[0014] Preferably, the enzymolysis temperature in Step 2 is 45°C, and the enzymolysis time is 2 hours.

[0015] Preferably, the ultrasonic power in Step 3 is 350W, the ultrasonic extraction temperature is 50°C, and the ultrasonic extraction time is 30 minutes.

[0016] Preferably, in step four, the centrifugation speed is 5000 r / min and the centrifugation time is 15 minutes.

[0017] Preferably, in step five, 4 times the volume of absolute ethanol is added, the standing temperature is 6°C, and the standing time is 18 hours.

[0018] Preferably, in step six, the vacuum drying temperature is 50°C.

[0019] In summary, the present application includes the following beneficial technical effects:

[0020] The present invention adopts a method combining enzymatic hydrolysis and ultrasonic-assisted extraction. Enzymatic hydrolysis can destroy the grape cell wall structure, making it easier to release polysaccharides. Ultrasonic-assisted extraction can accelerate the dissolution and diffusion of polysaccharides. The synergistic effect of the two significantly improves the extraction efficiency of grape polysaccharides. Compared with traditional extraction methods, the extraction rate is increased by 30% - 50%.

[0021] By optimizing the extraction conditions, such as enzyme solution concentration, enzyme ratio, enzymatic hydrolysis temperature and time, ultrasonic power, extraction temperature and time, etc., as well as subsequent steps such as centrifugal separation, alcohol precipitation, washing and drying, impurities can be effectively removed, and the purity of grape polysaccharides can be improved. The purity of the obtained grape polysaccharides can reach more than 85%.

[0022] In the extraction process of the present invention, green and environmentally friendly solvents such as water, ethanol, and acetone are mainly used, avoiding the large use of organic solvents, reducing environmental pollution, and at the same time reducing production costs, having good economic and environmental benefits.

[0023] The extraction method of the present invention has simple process operation, is easy to control, and is suitable for large-scale industrial production. Detailed Description of the Invention

[0024] The technical solutions of the present invention will be described in detail below through specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0025] Example 1

[0026] 1. Remove the stalks of fresh grapes, wash them, cut them into small pieces, perform freeze-drying treatment and then grind them into powder.

[0027] 2. Add a mixed enzyme solution of cellulase and pectinase with a mass concentration of 0.1% (the mass ratio of cellulase to pectinase is 1:1) to the grape powder, and perform enzymatic hydrolysis at 35°C for 1 hour.

[0028] 3. Add 5 times the mass of deionized water to the enzymatically hydrolyzed material, and perform ultrasonic extraction at an ultrasonic power of 200 W and a temperature of 40°C for 20 minutes.

[0029] 4. Centrifuge the material after ultrasonic extraction at a speed of 4000 r / min for 10 minutes, and take the supernatant.

[0030] 5. Add 3 volumes of absolute ethanol to the supernatant and let it stand at 4 °C for 12 hours for ethanol precipitation.

[0031] 6. Wash the precipitate after ethanol precipitation twice with absolute ethanol and acetone respectively, and then vacuum dry it at 40 °C to obtain grape polysaccharide. The extraction rate is 65% and the purity is 82%.

[0032] Example 2

[0033] 1. Remove the stalks from fresh grapes, wash them, cut them into small pieces, perform freeze-drying treatment and then grind them into powder.

[0034] 2. Add a mixed enzyme solution of cellulase and pectinase with a mass concentration of 0.3% (the mass ratio of cellulase to pectinase is 2:1) to the grape powder and enzymatically hydrolyze it at 45 °C for 2 hours.

[0035] 3. Add 8 times the mass of deionized water to the material after enzymatic hydrolysis and perform ultrasonic extraction at an ultrasonic power of 350 W and a temperature of 50 °C for 30 minutes.

[0036] 4. Centrifuge the material after ultrasonic extraction at a speed of 5000 r / min for 15 minutes, and take the supernatant.

[0037] 5. Add 4 volumes of absolute ethanol to the supernatant and let it stand at 6 °C for 18 hours for ethanol precipitation.

[0038] 6. Wash the precipitate after ethanol precipitation three times with absolute ethanol and acetone respectively, and then vacuum dry it at 50 °C to obtain grape polysaccharide. The extraction rate is 85% and the purity is 88%.

[0039] Example 3

[0040] 1. Remove the stalks from fresh grapes, wash them, cut them into small pieces, perform freeze-drying treatment and then grind them into powder.

[0041] 2. Add a mixed enzyme solution of cellulase and pectinase with a mass concentration of 0.5% (the mass ratio of cellulase to pectinase is 3:1) to the grape powder and enzymatically hydrolyze it at 50 °C for 3 hours.

[0042] 3. Add 10 times the mass of deionized water to the material after enzymatic hydrolysis and perform ultrasonic extraction at an ultrasonic power of 500 W and a temperature of 60 °C for 40 minutes.

[0043] 4. Centrifuge the material after ultrasonic extraction at a speed of 6000 r / min for 20 minutes, and take the supernatant.

[0044] 5. Add 5 times the volume of absolute ethanol to the supernatant, and let it stand at 10 °C for 24 hours for ethanol precipitation.

[0045] 6. Wash the precipitate after ethanol precipitation 3 times with absolute ethanol and acetone respectively, and then vacuum dry at 60 °C to obtain grape polysaccharide with an extraction rate of 80% and a purity of 86%.

[0046] It can be seen from the above embodiments that the extraction method of the present invention can effectively improve the extraction efficiency and purity of grape polysaccharide, and has good application prospects.

[0047] Finally, several points should be noted: First, in the description of the present application, it should be noted that unless otherwise specified and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense, which can be mechanical connection or electrical connection, or the communication inside two components, and can be directly connected. "Up", "down", "left", "right", etc. are only used to represent the relative position relationship. When the absolute position of the object being described changes, the relative position relationship may change;

[0048] Second: In the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments are involved. For other structures, reference can be made to the general design. Without conflict, the same embodiment and different embodiments of the present invention can be combined with each other;

[0049] Finally: The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0050] The above are all the preferred embodiments of the present application, and do not limit the protection scope of the present application accordingly. Therefore, all equivalent changes made according to the structure, shape, and principle of the present application shall be covered by the protection scope of the present application.

Claims

1. A method for extracting the active ingredient of grapes, grape polysaccharide, characterized in that It includes the following steps: Step 1: Remove the stalks of fresh grapes, wash them, cut them into small pieces, perform freeze-drying treatment and then grind them into powder; Step 2: Add a mixed enzyme solution of cellulase and pectinase with a mass concentration of 0.1 - 0.5% to the grape powder. The mass ratio of cellulase to pectinase in the mixed enzyme solution is 1:1 - 3:1, and enzymolysis is carried out at 35 - 50°C for 1 - 3 hours; Step 3: Add deionized water with a volume 5 - 10 times the mass of the enzymolyzed material, and perform ultrasonic extraction at an ultrasonic power of 200 - 500W and a temperature of 40 - 60°C for 20 - 40 minutes; Step 4: Centrifuge the material after ultrasonic extraction at a rotational speed of 4000 - 6000r / min for 10 - 20 minutes, and take the supernatant; Step 5: Add anhydrous ethanol with a volume 3 - 5 times that of the supernatant, and let it stand at 4 - 10°C for 12 - 24 hours for alcohol precipitation; Step 6: Wash the precipitate after alcohol precipitation with anhydrous ethanol and acetone 2 - 3 times each, and then perform vacuum drying at 40 - 60°C to obtain grape polysaccharide.

2. The extraction method of grape polysaccharide, an active ingredient of grapes, according to claim 1, is characterized in that: In Step 2, the mass concentration of the mixed enzyme solution is 0.3%, and the mass ratio of cellulase to pectinase is 2:

1.

3. The extraction method of a grape active ingredient, grape polysaccharide, according to claim 1, characterized in that: In Step 2, the enzymolysis temperature is 45°C and the enzymolysis time is 2 hours.

4. The extraction method of grape polysaccharide, an active ingredient of grapes, according to claim 1, is characterized in that: In Step 3, the ultrasonic power is 350W, the ultrasonic extraction temperature is 50°C, and the ultrasonic extraction time is 30 minutes.

5. The extraction method of grape polysaccharide, an active ingredient of grapes, according to claim 1, is characterized in that: In Step 4, the centrifugation rotational speed is 5000r / min and the centrifugation time is 15 minutes.

6. The extraction method of grape polysaccharide, an active ingredient of grapes, according to claim 1, is characterized in that: In Step 5, add anhydrous ethanol with a volume 4 times that of the supernatant, the standing temperature is 6°C, and the standing time is 18 hours.

7. The extraction method of a grape active ingredient, grape polysaccharide, according to claim 1, is characterized in that: In Step 6, the vacuum drying temperature is 50°C.