Preparation method of peanut skin residue resveratrol

Abstract

The present invention relates to the technical field of nutrient extraction, and more particularly to a method for preparing resveratrol from peanut hulls. This method uses peanut hulls (peanut shells, peanut stalks, and peanut roots), which are residual byproducts after peanut processing, as raw materials after washing. The method then undergoes blanching, drying, crushing, ethanol soaking, subcritical water treatment, centrifugation, pH adjustment, enzymatic hydrolysis with the addition of a complex enzyme, enzyme inactivation, purification, vacuum concentration, and freeze-drying to obtain highly active resveratrol. This method increases the content and purity of resveratrol in the peanut hulls and enhances the functionality of the peanut resveratrol. It also provides a new approach for the comprehensive utilization of peanut byproducts, enabling the intensive processing of agricultural and sideline products and reducing resource waste.

Description

Technical Field

[0001] The invention relates to the technical field of nutrient component extraction, and in particular to a method for preparing resveratrol from peanut skin residue. Background Art

[0002] Peanuts, originally known as groundnuts, are a widely consumed and abundant nut in my country. They promote growth and development, improve brain function, slow aging, moisten the lungs and relieve coughs, lower cholesterol, and prevent cancer. Peanut shells, stems, and roots are rich in crude protein, crude fiber, and resveratrol. They also contain elements such as iron, manganese, and zinc, as well as various vitamins, and 15 common amino acids, including glutamic acid and glycine. Peanut processing produces a large number of byproducts, such as shells, stems, and roots. Currently, these byproducts are not well utilized, contributing to environmental pollution and wasting resources. Therefore, achieving a tiered and rational, comprehensive utilization of peanut processing byproducts to minimize environmental pollution is a pressing issue.

[0003] Resveratrol, a polyphenol compound in peanuts, is an antitoxin produced by plants under stress. Its main functions include: antioxidant, antibacterial, reducing blood viscosity, inhibiting platelet aggregation, maintaining vascular comfort, anti-atherosclerosis, coronary heart disease, ischemic heart disease, lowering blood lipids, estrogen-like effects, preventing a series of diseases caused by insufficient estrogen secretion in women after menopause, and improving immunity.

[0004] At present, the main methods for extracting resveratrol at home and abroad include organic solvent extraction, enzymatic hydrolysis, microwave extraction, ultrasonic method, supercritical CO2 extraction, membrane separation, aqueous two-phase extraction and fermentation. These methods are single and independent, lacking effective combination, resulting in low extraction rate, low product activity, or too complicated process, which is not conducive to industrialization. Therefore, there is an urgent need for a method that can efficiently extract highly active resveratrol.

[0005] Currently, there are few reports in China on extracting resveratrol from plant sources. Patent publication number CN110066834A discloses a resveratrol extraction method that uses high-resveratrol-content Japanese knotweed as the raw material to increase the resveratrol extraction rate from the source. The extraction method utilizes a combination of microbial fermentation, enzymatic hydrolysis, and ultrasonic extraction to maximize the biological activity of resveratrol and achieve a yield of over 1.8% of natural resveratrol (98%). Patent CN109896927A discloses a method for extracting resveratrol from mulberries. Ultrasonic homogenization is used for extraction, and the filtrate is chromatographed on a silica gel column containing Al2O3 and eluted with anhydrous ethanol solution. The eluate is rotary evaporated, the ethanol is recovered, and the residue is dissolved in methanol. The resveratrol is filtered through an organic filter membrane to obtain resveratrol. The resveratrol is freeze-dried to obtain resveratrol powder. Patent CN109896929A discloses an enzymatic extraction method for resveratrol. The Japanese knotweed is washed, dried to constant weight, crushed, ethanol is added, ultrasonicated, propanol is added, heated under reflux, the extract is concentrated, a complex enzyme is added for enzymolysis, the enzymolysis product liquid is filtered, recrystallized with ethanol and freeze-dried to obtain a finished product. The above methods all have the disadvantages of low extraction rate and low product activity. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a method for preparing resveratrol from peanut skin residue.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing resveratrol from peanut skin residue comprises the following steps:

[0009] S1. Pretreatment of raw materials: blanching the peanut shell residue to be processed with hot water (to inactivate PPO enzyme activity and inhibit enzymatic browning), drying, crushing, and sieving in sequence, and mixing the obtained powder with ethanol in a certain proportion to obtain a mixed material;

[0010] S2, subcritical water extraction: Place the mixed material obtained in step S1 in an appropriate amount of water, and perform subcritical water extraction treatment at 100-300kPa for 5-30 minutes, then centrifuge to obtain a supernatant and a precipitate; water is kept in a liquid state by applying a pressure 22.1MPa lower than the critical pressure in the range of 100°C to the critical temperature of 374.2°C. Water in this state is called subcritical water, and the dielectric constant is significantly reduced. Subcritical water has good solubility for hydrophobic substances, and still has sufficient solubility for substances with strong polarity and large relative molecular mass. Based on this, the high temperature and high pressure environment of subcritical water may significantly improve the solubility of resveratrol, which is beneficial to the enrichment of resveratrol;

[0011] S3, complex enzyme hydrolysis: the supernatant obtained in step S2 is adjusted to a pH of 2.5-5.5 with HCl solution, an appropriate amount of complex enzyme (cellulase, xylanase, pectinase) is added, and the mixture is placed in a constant temperature water bath shaker at 40-80°C at a rotation speed of 100-300 r / min. After enzymatic hydrolysis for 10-80 min, the enzymes are inactivated to obtain an enzyme-inactivated solution; the main functions of cellulase, xylanase, and pectinase are to degrade substances such as cellulose, hemicellulose, and pectin in plant cell walls, causing the cell walls to swell and transform, degrading cellulose substances, destroying the plant cell walls, and dissolving the cell contents to release nutrients;

[0012] S4, purification and concentration: the enzyme-inactivated solution obtained in step S3 is separated and purified by silica gel column chromatography, and the clarified liquid is vacuum concentrated to obtain a concentrated solution;

[0013] S5. Drying treatment: The obtained concentrated solution is placed in a vacuum freeze drying box for drying to obtain resveratrol.

[0014] Furthermore, in step S1, the peanut skin residue is obtained by washing the remaining by-products after peanut processing (including peanut shells, peanut stalks and peanut roots), and the blanching temperature is 85-100°C for 1-5 minutes. After draining the blanched peanut skin residue, it is placed in an electric constant temperature blast drying oven at 50-80°C and dried for 24-48 hours, and its moisture content is 4-10%. It is crushed to 20-100 mesh to obtain pretreated peanut skin residue powder, and the mixing ratio of powder to ethanol is 1g:1mL-5mL, and the ethanol concentration is 60-90% (v / v).

[0015] Furthermore, in step S2, the temperature of the subcritical water extraction is 100-150°C; the centrifugal speed is 4000-5000 r / min, and the centrifugal time is 10-40 min.

[0016] Furthermore, in step S3, the enzymatic activity of cellulase is 300U / mL to 400U / mL; the enzymatic activity of xylanase is 1000U / g to 10000U / g; and the enzymatic activity of pectinase is 20,000 to 100,000U / g; wherein the enzymatic activity of the xylanase is defined as: the amount of enzyme required to hydrolyze xylan to form 1μmol of xylose per minute is 1 enzyme activity unit, expressed in U / g; the enzymatic activity of the pectinase is defined as: the amount of enzyme required to decompose pectin to produce 1μg of galacturonic acid under optimal conditions of 1g pectinase powder or 1mL pectinase solution is 1 enzyme activity unit, expressed in U / g.

[0017] Furthermore, in step S3, the pH value is adjusted to 2.5-5.5 with a 0.4 mol / L HCl solution; the amount of cellulase added is 1-6% of the mass of the supernatant, the amount of xylanase added is 1-8% of the mass of the supernatant, and the amount of pectinase added is 0.3-5% of the mass of the supernatant.

[0018] Furthermore, in step S4, the enzyme-inactivated solution is separated and purified by silica gel column chromatography, and the ratio of the enzyme-inactivated solution to silica gel is 1:1 to 1:8 (m / m).

[0019] Furthermore, in step S4, the clarified liquid is concentrated to 1 / 5 to 1 / 15 of the original volume by rotary evaporation at 40 to 70° C. and 0.08 to 0.1 MPa to obtain a concentrated solution.

[0020] Furthermore, in step S5, the vacuum freeze drying is performed at a temperature of -10 to -70°C, for 10 to 30 hours, and at a vacuum degree of 10 to 100 Pa.

[0021] The present invention has the following beneficial effects:

[0022] Resveratrol is extracted from peanut hulls by coupling subcritical water extraction with enzymatic hydrolysis. After subcritical water extraction and enzymatic hydrolysis, the extraction rate of resveratrol is improved, and the antioxidant and antibacterial properties of resveratrol are improved, so that the obtained high-activity resveratrol product has better physiological effects in reducing blood viscosity, inhibiting platelet aggregation, maintaining vascular comfort, resisting atherosclerosis, coronary heart disease, ischemic heart disease, and lowering blood lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a process flow chart of the method for preparing resveratrol from peanut skin residue according to an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0025] Example 1

[0026] S1. Pretreatment of raw materials:

[0027] The peanut skin residue obtained by washing the residual by-products after peanut processing (peanut shells, peanut stalks, and peanut roots) was blanched at 90° C. for 2 minutes. After the blanching, the peanut skin residue was drained and placed in a 60° C. electric constant temperature forced air drying oven to dry for 24 hours until the moisture content was 5%. The peanut skin residue was crushed to 70 mesh and mixed with 75% (v / v) ethanol at a mixing ratio of 1 g:3 mL to obtain a mixture;

[0028] S2. Subcritical water extraction:

[0029] The obtained mixture was placed in an appropriate amount of water, subjected to subcritical water extraction at 120°C and 250 kPa for 15 minutes, and then centrifuged at 4000 r / min for 15 minutes to obtain a supernatant;

[0030] S3, compound enzyme hydrolysis:

[0031] The resulting supernatant was adjusted to a pH of 5.0 with a 0.4 mol / L HCl solution, and an appropriate amount of complex enzyme (cellulase, xylanase, and pectinase) was added. The mixture was placed in a 60°C constant temperature water bath shaker at a speed of 150 r / min, subjected to enzymatic hydrolysis for 30 min, and inactivated at 95°C for 3 min. The mixture was cooled to room temperature to obtain an enzyme-inactivated solution. The amount of cellulase added was 3% of the mass of the supernatant, the amount of xylanase added was 4% of the mass of the supernatant, and the amount of pectinase added was 2.5% of the mass of the supernatant.

[0032] S4. Purification:

[0033] The obtained enzyme-inactivated solution was separated and purified by silica gel column chromatography, and then concentrated to 1 / 10 of the original volume by rotary evaporation at 50° C. and 0.085 MPa to obtain a concentrated solution, wherein the ratio of the enzyme-inactivated solution to the silica gel was 1:4 (m / m).

[0034] S5. Drying treatment:

[0035] The concentrated solution was placed in a vacuum freeze drying oven and dried at -40°C for 15 h at a vacuum degree of 30 Pa.

[0036] Under these conditions, the extraction rate of resveratrol was 3.02% and the purity was 99.1%. · and DPPH scavenging capacity IC 50 They are 4.16 mg / mL, 4.09 mg / mL and 7.90 mg / mL respectively.

[0037] Example 2

[0038] S1. Pretreatment of raw materials:

[0039] The peanut skin residue obtained by washing the residual by-products after peanut processing (peanut shells, peanut stalks and peanut roots) was blanched at 95° C. for 1.5 min. After the blanching, the peanut skin residue was drained and placed in a 70° C. electric constant temperature forced air drying oven to dry for 20 h to a moisture content of 4.5%. The peanut skin residue was then crushed to 80 mesh and mixed with 80% (v / v) ethanol at a mixing ratio of 1 g:4 mL to obtain a mixture;

[0040] S2. Subcritical water extraction:

[0041] The obtained mixture was placed in an appropriate amount of water, subjected to subcritical water extraction at 130°C and 200 kPa for 25 minutes, and then centrifuged at 4200 r / min for 18 minutes to obtain a supernatant;

[0042] S3, compound enzyme hydrolysis:

[0043] The obtained supernatant was adjusted to a pH of 5.5 with a 0.4 mol / L HCl solution, and an appropriate amount of complex enzyme (cellulase, xylanase, and pectinase) was added. The mixture was placed in a 65°C constant temperature water bath shaker at a speed of 200 r / min, subjected to enzymatic hydrolysis for 25 minutes, and inactivated at 95°C for 3 minutes. The mixture was cooled to room temperature to obtain an enzyme-inactivated solution. The amount of cellulase added was 3% of the mass of the supernatant, the amount of xylanase added was 3.5% of the mass of the supernatant, and the amount of pectinase added was 3.0% of the mass of the supernatant.

[0044] S4. Purification:

[0045] The obtained enzyme-inactivated solution was separated and purified by silica gel column chromatography, and then concentrated to 1 / 12 of the original volume by rotary evaporation at 60° C. and a vacuum degree of 0.09 MPa to obtain a concentrated solution, wherein the ratio of the enzyme-inactivated solution to the silica gel was 1:3 (m / m);

[0046] S5. Drying treatment:

[0047] The concentrated solution was placed in a vacuum freeze drying oven and dried at -50°C for 12 h at a vacuum degree of 35 Pa.

[0048] Under these conditions, the extraction rate of resveratrol was 3.11% and the purity was 99.3%. · and DPPH scavenging capacity IC 50 They are 5.02 mg / mL, 4.52 mg / mL and 8.10 mg / mL respectively.

[0049] Example 3

[0050] S1. Pretreatment of raw materials:

[0051] The peanut skin residue obtained by washing the residual by-products after peanut processing (peanut shells, peanut stalks, and peanut roots) was blanched at 95° C. for 2.5 min. After the blanching, the peanut skin residue was drained and placed in a 65° C. electric constant temperature forced air drying oven to dry for 25 h until the moisture content was 4.0%. The peanut skin residue was crushed to 90 mesh and mixed with 85% (v / v) ethanol at a mixing ratio of 1 g:4.5 mL to obtain a mixture;

[0052] S2. Subcritical water extraction:

[0053] The obtained mixture was placed in an appropriate amount of water, subjected to subcritical water extraction at 135°C and 300 kPa for 20 minutes, and then centrifuged at 4500 rpm for 15 minutes to obtain a supernatant.

[0054] S3, compound enzyme hydrolysis:

[0055] The obtained supernatant was adjusted to a pH of 5.0 with a 0.4 mol / L HCl solution, and an appropriate amount of complex enzyme (cellulase, xylanase, pectinase) was added. The mixture was placed in a 70°C constant temperature water bath shaker at a speed of 200 r / min. After enzymatic hydrolysis for 28 minutes, the enzymes were inactivated at 95°C for 3 minutes, and the mixture was cooled to room temperature to obtain an enzyme-inactivated solution. The amount of cellulase added was 3.5% of the mass of the supernatant, the amount of xylanase added was 4.5% of the mass of the supernatant, and the amount of pectinase added was 2.0% of the mass of the supernatant.

[0056] S4. Purification: The obtained enzyme-inactivated solution was separated and purified by silica gel column chromatography, and then concentrated to 1 / 8 of the original volume by rotary evaporation at 65° C. and 0.09 MPa to obtain a concentrated solution, wherein the ratio of the enzyme-inactivated solution to the silica gel was 1:5 (m / m).

[0057] S5. Drying treatment:

[0058] The concentrated solution was placed in a vacuum freeze drying oven and dried at a temperature of -55°C for 20 h and a vacuum degree of 30 Pa.

[0059] Under these conditions, the extraction rate of resveratrol was 3.08% and the purity was 99.0%. · and DPPH scavenging capacity IC 50 They are 4.85 mg / mL, 4.36 mg / mL and 7.95 mg / mL respectively.

[0060] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. The embodiments of this application and the features in the embodiments may be combined with each other in any manner unless there is a conflict.

Claims

1. A method for preparing resveratrol from peanut skin residue, characterized by: The steps include: S1. Pretreatment of raw materials: blanching the peanut shells to be processed with hot water, drying, crushing, and sieving in sequence, and mixing the obtained powder with ethanol in a proportion to obtain a mixed material; S2. Subcritical water extraction: The mixed material obtained in step S1 is placed in an appropriate amount of water, subjected to subcritical water extraction treatment at 100-300 kPa for 5-30 minutes, and then centrifuged to obtain a supernatant and a precipitate; S3, complex enzyme hydrolysis: the supernatant obtained in step S2 is adjusted to a pH of 2.5-5.5 with HCl solution, complex enzyme is added, and the mixture is placed in a constant temperature water bath shaker at 40-80°C and a rotation speed of 100-300 r / min. After enzymatic hydrolysis for 10-80 min, the enzyme is inactivated to obtain an enzyme-inactivated solution; S4, purification and concentration: the enzyme-inactivated solution obtained in step S3 is separated and purified by silica gel column chromatography, and the clarified liquid is vacuum concentrated to obtain a concentrated solution; S5, drying treatment: placing the obtained concentrated solution in a vacuum freeze drying box to dry it, thereby obtaining resveratrol; In step S3, the complex enzyme comprises cellulase, xylanase, and pectinase; the enzymatic activity of the cellulase is 300 U / mL to 400 U / mL; the enzymatic activity of the xylanase is 1000 U / g to 10000 U / g; and the enzymatic activity of the pectinase is 20,000 to 100,000 U / g; wherein the enzymatic activity of the xylanase is defined as: the amount of enzyme required to hydrolyze xylan to form 1 μmol of xylose per minute is 1 enzyme activity unit, expressed in U / g; the enzymatic activity of the pectinase is defined as: the amount of enzyme required to decompose pectin to form 1 μg of galacturonic acid under optimal conditions by 1 g of pectinase powder or 1 mL of pectinase solution is 1 enzyme activity unit, expressed in U / g; In step S3, the pH value is adjusted to 2.5-5.5 using a 0.4 mol / L HCl solution; the amount of cellulase added is 1-6% of the mass of the supernatant, the amount of xylanase added is 1-8% of the mass of the supernatant, and the amount of pectinase added is 0.3-5% of the mass of the supernatant.

2. The method for preparing resveratrol from peanut skin residue according to claim 1, characterized in that: In step S1, the peanut husk residue is a residual by-product after peanut processing, including peanut shells, peanut stalks and peanut roots, which are obtained after washing. The blanching temperature is 85-100° C. and the time is 1-5 minutes. After the blanched peanut husk residue is drained, it is placed in an electric constant temperature blast drying oven at 50-80° C. and dried for 24-48 hours. The water content is 4-10%. The peanut husk residue is crushed to 20-100 mesh to obtain pretreated peanut husk residue powder. The mixing ratio of the powder to ethanol is 1 g: 1 mL-5 mL, and the ethanol concentration is 60-90% v / v.

3. The method for preparing resveratrol from peanut skin residue according to claim 1, characterized in that: In step S2, the temperature of the subcritical water extraction is 100-150° C.; the centrifugal speed is 4000-5000 r / min, and the centrifugal time is 10-40 min.

4. The method for preparing resveratrol from peanut skin residue according to claim 1, wherein: In the step S4, the enzyme-inactivated solution is separated and purified by silica gel column chromatography, and the mass ratio of the enzyme-inactivated solution to the silica gel is 1:1 to 1:

8.

5. The method for preparing resveratrol from peanut skin residue according to claim 1, characterized in that: In the step S4, the clarified liquid is concentrated by rotary evaporation at 40-70° C. and 0.08-0.1 MPa to 1 / 5-1 / 15 of the original volume to obtain a concentrated liquid.

6. The method for preparing resveratrol from peanut skin residue according to claim 1, characterized in that: In step S5, the vacuum freeze drying is performed at a temperature of -10 to -70°C, for 10 to 30 hours, and at a vacuum degree of 10 to 100 Pa.

Images