CN110878328A - Sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof - Google Patents
Sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof Download PDFInfo
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Abstract
The invention provides a sea buckthorn seed meal non-soluble protein with antioxidant activity and a preparation method thereof, and the preparation method mainly comprises the following steps: (1) pulverizing semen Hippophae; (2) performing alcohol extraction pretreatment on the sea buckthorn seed meal, wherein the liquid-material ratio is 20:1, the volume fraction of alcohol is 70%, the pretreatment time is 30min, the pretreatment temperature is 55 ℃, and the pretreatment is performed for 3 times, filtering is performed to obtain the alcohol extraction residues of the sea buckthorn seed meal, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use; (3) mixing ethanol residue of fructus Hippophae, protease and alkaline aqueous solution at a certain proportion, extracting in constant temperature water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and antioxidant capacity, and drying to obtain fructus Hippophae seed meal insoluble protein. The sea-buckthorn seed meal non-soluble protein provided by the invention has strong oxidation resistance, and can be used as an antioxidant preparation and/or an additive of a product in the industries of food, medicine and cosmetics.
Description
Technical Field
The invention relates to a sea buckthorn seed meal non-soluble protein with antioxidant activity and a preparation method thereof.
Background
Hippophae rhamnoides (Hippophae rhamnoides L.), also known as Hippophae Rhamnoides, Hippophae xanthate, Hippophae nigra, and Hippophae rhamnoides (Elaeagnaceae), is a plant used as both medicine and food. Sea-buckthorn originates from Qinghai-Tibet plateau, and is widely distributed in temperate zones, cold temperate zones and subtropical plateau mountain areas of Asia-European continents due to its extremely strong ecological adaptability. China is the country with the largest amount of sea buckthorn resources, and a large amount of sea buckthorn is cultivated in the three north area, and the sea buckthorn forest accounts for 140 ten thousand hectares and accounts for 95 percent of the total area of the sea buckthorn in the world. The seabuckthorn seed meal is solid waste obtained by extracting oil from seabuckthorn seeds, but the seed meal still contains a large amount of protein and can be used as a protein extraction raw material, and the seabuckthorn industry in China generates about 26.3 million tons of seed meal every year, so that the seabuckthorn seed meal has good market prospect and ecological value for high-value utilization of the part of biological resources.
At present, methods for preparing protein from seabuckthorn seed meal comprise an alcohol washing method, an alkali extraction and acid precipitation method, an enzymolysis method, a water extraction method and the like, have mature industrial foundations, mainly focus on soluble protein of the seabuckthorn seed meal, and hardly pay attention to non-soluble protein in alcohol extraction residues of the seabuckthorn seed meal. Therefore, the invention provides a high-efficiency preparation method of the non-soluble protein of the seabuckthorn seed meal and provides the application of the non-soluble protein in the antioxidant activity product by using the seabuckthorn seed meal as a raw material, extracting the protein by adopting an alcohol extraction pretreatment method, an enzymolysis-alkali extraction combined method, measuring the protein content by a biuret colorimetric method and calculating the yield.
Disclosure of Invention
The invention provides a sea buckthorn seed meal non-soluble protein with antioxidant activity and a preparation method thereof, and the preparation method mainly comprises the following steps:
(1) pulverizing semen Hippophae;
(2) performing alcohol extraction pretreatment on the sea buckthorn seed meal, wherein the liquid-material ratio is 20:1, the volume fraction of alcohol is 70%, the pretreatment time is 30min, the pretreatment temperature is 55 ℃, and the pretreatment is performed for 3 times, filtering is performed to obtain the alcohol extraction residues of the sea buckthorn seed meal, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use;
(3) mixing ethanol residue of fructus Hippophae, protease and alkaline aqueous solution at a certain proportion, extracting in constant temperature water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and antioxidant capacity, and drying to obtain fructus Hippophae seed meal insoluble protein.
Further, as described in step (3), the alcohol extraction residue of hippophae rhamnoides: the ratio of alkaline aqueous solution to alkaline aqueous solution is 30: 1-50: 1, preferably 44: 1.
Further, as described in step (3), the protease is added in an amount of 0.5% to 1.5%, preferably the protease is added in an amount of 1.4%.
Further, as described in the step (3), the pH of the alkaline aqueous solution is 12.0-13.0, preferably the pH of the alkaline aqueous solution is 12.3.
Further, as described in step (3), the protease is chymotrypsin or trypsin, preferably trypsin.
Further, as described in step (3), the solute of the alkaline aqueous solution is sodium hydroxide, potassium hydroxide or triethylamine, preferably the solute is sodium hydroxide or/and triethylamine.
Further, the extraction yield of the non-soluble protein of the seabuckthorn seed meal is as high as 41.69-50.53%.
Furthermore, the sea-buckthorn seed meal non-soluble protein has good oxidation resistance, and the capacity of scavenging free radicals reaches 50.4% -73.6%.
Protein content determination:
adopting agricultural industry standard NYT1678-2008 ' determination of protein in milk and dairy products biuret colorimetry ' of the people's republic of China, dividing 18 test tubes into 3 groups, adding 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of casein standard solution of 10 mg/mL in sequence into 6 test tubes in each group, supplementing the test tubes with water to 1.0 mL, adding 4.0 mL of biuret reagent into each tube respectively, fully and uniformly mixing, standing for 30min at room temperature (20-25 ℃), determining the light absorption value under the wavelength of 540 nm, taking the content of casein standard products as a horizontal coordinate and A540 nm as a vertical coordinate, drawing a standard curve, wherein the regression equation is as follows: y =0.0391x +0.0016, R =0.9990, x being the casein concentration (mg/mL), y being the absorbance at 540 nm wavelength, linear range: 2.0-10.0 mg/mL; and (3) sample determination: taking 1.0 mL of solution to be detected, adding 4.0 mL of biuret reagent, substituting the absorbance value of the detected sample at 540 nm into the regression equation by the method, and calculating the protein extraction yield according to the following formula:
the protein extraction yield = [ protein content (g) in supernatant/mass of seabuckthorn seed meal (g) ] x 100%.
Determination of antioxidant Capacity
The antioxidant capacity is determined after adaptation of the method provided in reference W, Brand-Williams, M.E. Cuvelier, C. Berset, Use of a free radial to ocular antioxidant activity, LWT-Food Science and Technology, Volume 28, Issue 1,1995, Pages 25-30, as follows: (1) preparing a DPPH ethanol solution, accurately weighing 2.5 mg of DPPH, sufficiently dissolving the DPPH solution in 50mL of absolute ethanol in a dark state, wherein the concentration of the DPPH solution is 0.05 mg/mL for later use, (2) preparing a sample solution, preparing the sample into a 100mg/mL stock solution by using ethanol, and diluting the stock solution by 200 times to obtain the antioxidant capacity to be detected of a solution to be detected with the concentration of 500 mu g/mL; (3) the test step, 10 mul of test sample is respectively added into a 96-well plate, equal amount of absolute ethyl alcohol is added into a blank well, the concentration of each sample is repeated for 6 wells, the samples are marked in groups, 190 mul of DPPH ethanol solution is added into each well, the reaction is carried out for 30min in a room temperature and dark place, the absorbance value of each well is measured at the wavelength of 517nm, the DPPH clearance rate is calculated according to the following formula, and the oxidation resistance is expressed by the clearance rate of free radical DPPH:
clearance (%) = (a 0-a 1)/(Ac 0-Ac 1) × 100%; in the formula: a0: sample well 0min light absorption value; a1: sample well 30min light absorption value; ac 0: blank control wells; 0min light absorption value; ac 1: blank control wells absorb light for 30 min.
Generally, the invention takes the seabuckthorn seed meal alcohol extraction residues as the raw materials for the first time, creatively adopts an enzymolysis-alkali extraction combined method to prepare the seabuckthorn seed meal protein, and the seabuckthorn seed meal protein has good antioxidant activity.
Compared with the prior art, the sea buckthorn seed meal non-soluble protein and the preparation method thereof provided by the invention have the following advantages:
(1) the invention takes alcohol extraction as the impurity removal step for preparing the non-soluble protein of the seabuckthorn seed meal, and discovers that the alcohol extraction residue of the seabuckthorn seed meal has rich non-soluble protein for the first time;
(2) the invention creatively adopts an enzymolysis-alkali extraction combined method to prepare and purify the non-soluble protein in the alcohol extraction residues of the seabuckthorn seed meal, and the extraction yield of the non-soluble protein is as high as 41.69-50.53%;
(3) the sea-buckthorn seed meal non-soluble protein prepared by the invention has good antioxidant activity, and the free radical scavenging capacity reaches 50.4% -73.6%.
The present invention is described in further detail with reference to the following embodiments, but the present invention is not limited thereto, and various other modifications, substitutions and alterations can be made without departing from the basic technical idea of the present invention based on the above-mentioned contents of the present invention and common technical knowledge and conventional means in the art.
Detailed Description
Example 1: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:44, adding protease 1.4%, extracting with sodium hydroxide aqueous solution at pH 12.3 in constant temperature water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein A. Through test determination, the extraction yield of the protein A of the seabuckthorn seed meal is 50.53%, and the antioxidant capacity is 73.6%.
Example 2: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:30, adding protease 1.0%, extracting with sodium hydroxide aqueous solution pH of 13.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein B. Through test determination, the extraction yield of the protein B of the seabuckthorn seed meal is 41.69 percent, and the oxidation resistance is 50.4 percent.
Example 3: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:50, adding protease 0.5%, extracting with sodium hydroxide aqueous solution pH of 12.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein C. Through test determination, the extraction yield of the protein C of the seabuckthorn seed meal is 42.98%, and the oxidation resistance is 53.6%.
Example 4: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:50, adding protease 1.5%, extracting with sodium hydroxide aqueous solution pH of 12.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein D. Through test determination, the extraction yield of the protein D of the seabuckthorn seed meal is 48.72 percent, and the oxidation resistance is 65.3 percent.
Comparative example 1: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:30, adding protease 1.0%, extracting with sodium hydroxide aqueous solution pH of 11.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein E. The test shows that the extraction yield of the protein E of the seabuckthorn seed meal is 15.69 percent, and the oxidation resistance is 10.4 percent.
Comparative example 2: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:50, adding protease 1.0%, extracting with sodium hydroxide aqueous solution pH of 11.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein F. Through test determination, the extraction yield of the protein F of the sea buckthorn seed meal is 23.58 percent, and the oxidation resistance is 13.7 percent.
Comparative example 3: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:40, adding protease 0.5%, extracting with sodium hydroxide aqueous solution pH of 11.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein G. Through test determination, the extraction yield of the protein G of the sea buckthorn seed meal is 19.22 percent, and the oxidation resistance is 11.5 percent.
Comparative example 4: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) weighing 90g of sea buckthorn seed meal, performing alcohol extraction pretreatment with the liquid-material ratio of 20:1, the alcohol volume fraction of 70%, the pretreatment time of 30min, the pretreatment temperature of 55 ℃ and 3 times, filtering to obtain sea buckthorn seed meal alcohol extraction residues, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use. (3) Mixing the ethanol residue of fructus Hippophae, protease and sodium hydroxide aqueous solution at a ratio of 1:40, adding protease 1.5%, extracting with sodium hydroxide aqueous solution pH of 11.0 in thermostatic water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein H. Through test determination, the extraction yield of the protein H of the sea buckthorn seed meal is 24.74%, and the antioxidant capacity is 18.1%.
Comparative example 5: sea-buckthorn seed meal non-soluble protein with antioxidant activity and preparation method thereof
(1) Crushing 100g of sea buckthorn seed meal for later use; (2) mixing fructus Hippophae seed meal, protease and sodium hydroxide aqueous solution at a ratio of 1:44, adding protease 1.4%, extracting with sodium hydroxide aqueous solution pH of 12.0 in constant temperature water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and oxidation resistance, and drying to obtain fructus Hippophae seed meal protein I. Through test determination, the extraction yield of the seabuckthorn seed meal protein I is 18.56%, and the antioxidant capacity is 10.37%.
And (3) knotting:
(1) the research of the invention finds that the alcohol extraction residue of the seabuckthorn seed meal contains rich non-soluble protein components, and if the alcohol extraction pretreatment is not carried out, the enzymolysis-alkali extraction combined method can not achieve the high extraction yield of the non-soluble protein, and the high extraction yield is often covered or interfered by the soluble protein of the seabuckthorn seed meal;
(2) the research of the invention finds that the enzymolysis-alkali extraction combined method is influenced by various factors, particularly the feed-liquid ratio, the adding amount of protease and the pH value of an aqueous solution, as shown in the examples or the comparative examples, and is not obvious;
(3) the sea-buckthorn seed meal non-soluble protein provided by the invention has strong oxidation resistance, and can be used as an antioxidant preparation and/or an additive of a product in the industries of food, medicine and cosmetics.
Claims (10)
1. A sea-buckthorn seed meal non-soluble protein with antioxidant activity is prepared by the following main steps:
(1) pulverizing semen Hippophae;
(2) performing alcohol extraction pretreatment on the sea buckthorn seed meal, wherein the liquid-material ratio is 20:1, the volume fraction of alcohol is 70%, the pretreatment time is 30min, the pretreatment temperature is 55 ℃, and the pretreatment is performed for 3 times, filtering is performed to obtain the alcohol extraction residues of the sea buckthorn seed meal, drying at 50 ℃, crushing, and sieving with a 50-mesh sieve for later use;
(3) mixing ethanol residue of fructus Hippophae, protease and alkaline aqueous solution at a certain proportion, extracting in constant temperature water bath at 60 deg.C for 30min, centrifuging to obtain supernatant, measuring protein content and antioxidant capacity, and drying to obtain fructus Hippophae seed meal insoluble protein.
2. The preparation method according to claim 1, wherein the ethanol residue of seabuckthorn in the step (3): the ratio of alkaline aqueous solution to alkaline aqueous solution is 30: 1-50: 1, preferably 44: 1.
3. The method of claim 1, wherein the protease is added in the amount of 0.5% to 1.5% in step (3), preferably the protease is added in an amount of 1.4%.
4. The method according to claim 1, wherein the pH of the basic aqueous solution is 12.0 to 13.0, preferably 12.3.
5. The protease according to claim 3, characterized in that the protease is chymotrypsin or trypsin, preferably trypsin.
6. The aqueous alkaline solution according to claim 1, 2 or 4, characterized in that the solute is sodium hydroxide, potassium hydroxide, triethylamine, preferably the solute is sodium hydroxide or/and triethylamine.
7. The preparation method of claim 1, wherein the extraction yield of the non-soluble protein of the seabuckthorn seed meal is 41.69% -50.53%.
8. The sea buckthorn seed meal insoluble protein according to claim 1, wherein the sea buckthorn seed meal insoluble protein has good oxidation resistance and free radical scavenging capacity of 50.4% -73.6%.
9. The seabuckthorn seed meal non-soluble protein according to claim 8, which can be used as an antioxidant product, an antioxidant preparation product and/or a raw material.
10. The seabuckthorn seed meal insoluble protein according to claim 9, which can be used in food, medicine or cosmetics for scavenging free radicals.
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Cited By (2)
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| CN114344444A (en) * | 2022-01-14 | 2022-04-15 | 完美（广东）日用品有限公司 | Application of sea buckthorn seed meal protein peptide in preparation of product for preventing and treating anxiety and depression |
| CN114404560A (en) * | 2022-01-14 | 2022-04-29 | 完美（广东）日用品有限公司 | Application of sea-buckthorn seed meal protein peptide in preparation of product for improving liver function |
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| CN114344444A (en) * | 2022-01-14 | 2022-04-15 | 完美（广东）日用品有限公司 | Application of sea buckthorn seed meal protein peptide in preparation of product for preventing and treating anxiety and depression |
| CN114404560A (en) * | 2022-01-14 | 2022-04-29 | 完美（广东）日用品有限公司 | Application of sea-buckthorn seed meal protein peptide in preparation of product for improving liver function |
| CN114404560B (en) * | 2022-01-14 | 2023-08-18 | 完美（广东）日用品有限公司 | Application of sea buckthorn seed meal protein peptide in preparation of products for improving liver function |
| CN114344444B (en) * | 2022-01-14 | 2023-08-29 | 完美（广东）日用品有限公司 | Application of sea buckthorn seed meal protein peptide in preparation of products for preventing and treating anxiety and depression |
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Application publication date: 20200313 |