CN105624248B - Preparation method of alfalfa bioactive peptide - Google Patents
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
A preparation method of alfalfa biological active peptide takes alfalfa fresh grass or hay as raw material, and obtains raw material liquid by screening, removing impurities, cleaning, draining, crushing, adding water and homogenizing; adding trypsin into the raw material solution for enzymolysis; carrying out enzyme deactivation treatment after the enzymolysis is finished, and centrifuging or filtering the mixed solution after enzyme deactivation to obtain supernatant or filtrate; vacuum concentrating the supernatant or filtrate to obtain herba Medicaginis bioactive peptide paste; the alfalfa bioactive peptide paste is subjected to freeze drying treatment to obtain alfalfa bioactive peptide powder. Compared with the prior art, the method directly uses trypsin to carry out enzymolysis on the alfalfa, has simple and convenient process, low production cost, high peptide yield and hydrolysis degree reaching 32.56 percent, mainly concentrates the molecular weight of peptide segments below 1000Da, and prepares the alfalfa biological active peptide with stronger diphenyl bitter hydrazine radical free radical (DPPH & ltcndot & gt) scavenging capability, and can be popularized and applied in the fields of food, health care products, cosmetics, feed processing and the like.
Description
Technical Field
The invention belongs to the field of preparation of plant-derived bioactive peptides, and particularly relates to a preparation method of alfalfa bioactive peptides.
Background
Alfalfa is a leguminous plant, belongs to perennial herbaceous plants, is the most widely planted and earliest cultivated pasture in the world, has the characteristics of high yield, good palatability, rich nutrition and the like, is known as the king of pasture, and is generally planted in northeast, northwest and northwest regions of China. The alfalfa has complete and balanced amino acid types and high nutritive value, and is an excellent vegetable protein resource.
At present, the researches on alfalfa at home and abroad mainly focus on breeding, planting, screening, extraction of flavonoid compounds and leaf protein and the like, and the researches on alfalfa bioactive peptides in the prior art are few. Wherein Zhang Yong and the like research the optimal conditions for preparing the oligopeptide by hydrolyzing the alfalfa leaf protein with alkaline protease, the hydrolysis degree under the optimal conditions is 27.50 percent, and the average peptide chain length of the oligopeptide is 3.64 (Chinese feed, 2007, 18: 15-17); the invention discloses a preparation method of alfalfa polypeptide in Chinese patent application publication specification CN104195204A, which comprises the steps of obtaining crude alfalfa protein by a heating method, leaching, decoloring and deodorizing by ethanol, carrying out enzymolysis on the crude alfalfa protein by Protamex enzyme and Flavourzyme1000L enzyme, and finally carrying out spray drying to obtain the alfalfa polypeptide, wherein the content of the polypeptide reaches 70-75%, and the obtained peptide powder has no bitter and astringent feeling and can be directly taken. However, the two methods are both prepared by extracting leaf protein from alfalfa and then carrying out enzymolysis on the leaf protein to prepare polypeptide, the extraction rate of the leaf protein is low, the energy consumption is high, and the two methods have the defects of complex process and high production cost.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of alfalfa bioactive peptide, which has the advantages of simple and convenient process, low production cost and stronger antioxidant activity of a finished product, aiming at the defects of the prior art.
The technical problem to be solved by the present invention is achieved by the following technical means. The invention relates to a preparation method of alfalfa bioactive peptide, which is characterized by comprising the following steps:
(1) raw material treatment: selecting fresh alfalfa or hay, removing impurities, cleaning, draining, crushing in a crusher, adding water according to a material-water mass ratio of 1: 9-1: 11, and homogenizing to obtain a raw material solution;
(2) enzymolysis: pumping the raw material liquid into an enzymolysis tank, adjusting the pH value by using a sodium hydroxide solution or a hydrochloric acid solution, continuously stirring, adding trypsin into the raw material liquid according to the enzyme addition amount of 800-1200U/g substrate protein for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 3-5.5 h, the enzymolysis temperature is 45-60 ℃, and the enzymolysis pH is 6-7.5;
(3) enzyme deactivation: heating to above 90 ℃ after enzymolysis, keeping for 10min, inactivating enzyme, cooling, and centrifuging the mixture after enzyme inactivation to obtain supernatant, or filtering the mixture after enzyme inactivation to obtain filtrate;
(4) and (3) vacuum concentration: and pumping the clear liquid or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 50-65 ℃, and concentrating until the final water content is 40-60%, thus obtaining the alfalfa bioactive peptide paste.
(5) And (3) freeze drying: and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of minus 60 to minus 40 ℃ until the final water content is below 10%, thus obtaining the alfalfa bioactive peptide powder.
In the preparation method of the alfalfa bioactive peptide, a further preferable technical scheme or technical characteristics is as follows:
1. in step (1): the mass ratio of the raw materials to water is 1:10 during homogenization.
2. In step (2): adding trypsin into the raw material solution according to the enzyme adding amount of 900U/g substrate protein for enzyme, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 4h, the enzymolysis temperature is 55 ℃, and the enzymolysis pH is 6.5.
3. In step (4): and pumping the supernatant or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 55 ℃, and concentrating until the final water content is 50% to obtain the alfalfa bioactive peptide paste.
4. In step (5): and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of-50 ℃ until the final water content is 8%, thus obtaining the alfalfa bioactive peptide powder.
Compared with the prior art, the method directly uses trypsin to carry out enzymolysis on the alfalfa, the peptide yield is high, the hydrolysis degree can reach 32.56%, the molecular weight of a peptide segment is mainly concentrated below 1000Da, and the prepared alfalfa biological active peptide has stronger diphenyl bitter hydrazine radical free radical (DPPH & gt) scavenging capacity; the steps of extracting crude protein and decoloring and deodorizing are omitted in the preparation process, so that the production process is simplified, the production cost is further reduced, and the use titer of the product is improved. The alfalfa biological active peptide obtained by the invention is a plant natural extract, is light green, has light green grass fragrance, has good flavor and strong antioxidant activity, can be popularized and applied in the fields of food, health care products, cosmetics, feed processing and the like, has simple and convenient process and low production cost, and is suitable for large-scale industrial production.
Drawings
FIG. 1 shows the results of measurement of DPPH.RTM.of ascorbic acid (Vc);
FIG. 2 shows the DPPH-scavenging ability of alfalfa bioactive peptide powder prepared by the method of the present invention in example 4.
Detailed Description
The embodiments of the present invention will be further described with reference to the accompanying drawings so as to facilitate the further understanding of the present invention by those skilled in the art, and do not limit the right thereto.
Example 1, a method for preparing a bioactive peptide of alfalfa, comprising the steps of:
(1) raw material treatment: selecting fresh alfalfa or hay, removing impurities, cleaning, draining, crushing in a crusher, adding water according to a material-water mass ratio of 1:9, and homogenizing to obtain a raw material solution;
(2) enzymolysis: pumping the raw material liquid into an enzymolysis tank, adjusting the pH value by using a sodium hydroxide solution or a hydrochloric acid solution, continuously stirring, adding trypsin into the raw material liquid according to the enzyme adding amount of 800U/g substrate protein for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 3h, the enzymolysis temperature is 45 ℃, and the enzymolysis pH is 6;
(3) enzyme deactivation: heating to above 90 ℃ after enzymolysis, keeping for 10min, inactivating enzyme, cooling, and centrifuging the mixture after enzyme inactivation to obtain supernatant, or filtering the mixture after enzyme inactivation to obtain filtrate;
(4) and (3) vacuum concentration: and pumping the clear liquid or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 50 ℃, and concentrating until the final water content is 40% to obtain the alfalfa bioactive peptide paste.
(5) And (3) freeze drying: and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of-60 ℃ until the final water content is 10%, thus obtaining the alfalfa bioactive peptide powder.
Example 2, a method for preparing a bioactive peptide of alfalfa, comprising the steps of:
(1) raw material treatment: selecting fresh alfalfa or hay, removing impurities, cleaning, draining, crushing in a crusher, adding water according to a material-water mass ratio of 1:11, and homogenizing to obtain a raw material solution;
(2) enzymolysis: pumping the raw material liquid into an enzymolysis tank, adjusting the pH value by using a sodium hydroxide solution or a hydrochloric acid solution, continuously stirring, adding trypsin into the raw material liquid according to the enzyme adding amount of 1200U/g substrate protein for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 5.5h, the enzymolysis temperature is 60 ℃, and the enzymolysis pH is 7.5;
(3) enzyme deactivation: heating to above 90 ℃ after enzymolysis, keeping for 10min, inactivating enzyme, cooling, and centrifuging the mixture after enzyme inactivation to obtain supernatant, or filtering the mixture after enzyme inactivation to obtain filtrate;
(4) and (3) vacuum concentration: and pumping the clear liquid or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 65 ℃, and concentrating until the final water content is 60% to obtain the alfalfa bioactive peptide paste.
(5) And (3) freeze drying: and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of-40 ℃ until the final water content is 6%, thus obtaining the alfalfa bioactive peptide powder.
Example 3, a method for preparing a bioactive peptide of alfalfa, comprising the steps of:
(1) raw material treatment: selecting fresh alfalfa or hay, removing impurities, cleaning, draining, crushing in a crusher, adding water according to a material-water mass ratio of 1:10, and homogenizing to obtain a raw material solution;
(2) enzymolysis: pumping the raw material liquid into an enzymolysis tank, adjusting the pH value by using a sodium hydroxide solution or a hydrochloric acid solution, continuously stirring, adding trypsin into the raw material liquid according to the addition of 1000U/g substrate protein for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 4.5h, the enzymolysis temperature is 55 ℃, and the enzymolysis pH is 7;
(3) enzyme deactivation: heating to above 90 ℃ after enzymolysis, keeping for 10min, inactivating enzyme, cooling, and centrifuging the mixture after enzyme inactivation to obtain supernatant, or filtering the mixture after enzyme inactivation to obtain filtrate;
(4) and (3) vacuum concentration: and pumping the clear liquid or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 60 ℃, and concentrating until the final water content is 50% to obtain the alfalfa bioactive peptide paste.
(5) And (3) freeze drying: and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of-50 ℃ until the final water content is 6%, thus obtaining the alfalfa bioactive peptide powder.
Example 4, a method for preparing a bioactive peptide of alfalfa, comprising the steps of:
(1) raw material treatment: selecting fresh alfalfa or hay, removing impurities, cleaning, draining, crushing in a crusher, adding water according to a material-water mass ratio of 1:10, and homogenizing to obtain a raw material solution;
(2) enzymolysis: pumping the raw material liquid into an enzymolysis tank, adjusting the pH value by using a sodium hydroxide solution or a hydrochloric acid solution, continuously stirring, adding trypsin into the raw material liquid according to the substrate protein with the enzyme adding amount of 900U/g for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 4 hours, the enzymolysis temperature is 55 ℃, and the enzymolysis pH is 6.5;
(3) enzyme deactivation: heating to above 90 ℃ after enzymolysis, keeping for 10min, inactivating enzyme, cooling, and centrifuging the mixture after enzyme inactivation to obtain supernatant, or filtering the mixture after enzyme inactivation to obtain filtrate;
(4) and (3) vacuum concentration: and pumping the supernatant or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 55 ℃, and concentrating until the final water content is 50% to obtain the alfalfa bioactive peptide paste.
(5) And (3) freeze drying: and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of-50 ℃ until the final water content is 8%, thus obtaining the alfalfa bioactive peptide powder.
The alfalfa bioactive peptide powder prepared by the method in the embodiment is light green, has light green grass fragrance and good flavor, the molecular weight and the removing effect on diphenyl bitter hydrazine radical free radical (DPPH. DEG) are measured, and the measuring method and the result are as follows:
1. determination of molecular weight
The molecular weight of the alfalfa bioactive peptide is detected by a sephadex (G-15) column chromatography, and the result is shown in table 1.
TABLE 1 molecular weight test results of alfalfa bioactive peptides.
The result shows that the molecular weight of the alfalfa bioactive peptide is mainly concentrated in 443-509 Da, the content is about 73.6%, and the obtained small molecular peptide is easier to be absorbed by organisms.
2. Measurement of DPPH.removing Effect.
The antioxidant activity of the alfalfa bioactive peptide is evaluated by a DPPH method. Dissolving the alfalfa bioactive peptide powder with water, and concentrating the supernatant to obtain concentrated solution of alfalfa bioactive peptide for use. Using ascorbic acid (Vc) as a positive control, and preparing the Vc and alfalfa bioactive peptide concentrated solutions into to-be-detected solutions with different concentrations by using absolute ethyl alcohol respectively; DPPH is prepared with absolute ethanol to the concentration of 1 × 10
-3And storing the mother liquor of mol/L at 0-4 ℃. For the experiment, the mother liquor was diluted to 1X 10
-4And after mol/L, adding 2mL of the solution into a 10mL colorimetric tube, adding the prepared solution to be detected, fully mixing, and standing at room temperature for 30 min. Absolute ethanol was used as a blank control at a maximum wavelength of 517nm to measure the absorbance (A), and each absorbance was measured in parallel 3 times to obtain an average value.
In the formula: a. the
i: adding the absorbance value of the DPPH solution to be detected;
A
j: mixing a solvent of the DPPH solution with a solution to be detected to obtain an absorbance value;
A
0: absorbance value of DPPH solution without test solution.
The result shows that the clearance capability of the alfalfa bioactive peptide to DPPH & is increased along with the increase of the mass concentration within a certain mass concentration range, and the clearance capability and the mass concentration have a good logarithmic regression relationship. Will scavenge 50% DPPH & CEffective mass concentration of the required agent is defined as EC
50，EC
50Smaller values indicate greater ability to scavenge free radicals. The EC of ascorbic acid was calculated
50EC of alfalfa bioactive peptide with value of 0.033mg/mL
50The value is 2.22mg/mL, which indicates that the alfalfa bioactive peptide powder has strong DPPH-clearance capability, and the results refer to fig. 1 and fig. 2.
Claims (5)
1. A preparation method of alfalfa bioactive peptide is characterized by comprising the following steps:
(1) raw material treatment: selecting fresh alfalfa or hay, removing impurities, cleaning, draining, crushing in a crusher, adding water according to a material-water mass ratio of 1: 9-1: 11, and homogenizing to obtain a raw material solution;
(2) enzymolysis: pumping the raw material liquid into an enzymolysis tank, adjusting the pH value by using a sodium hydroxide solution or a hydrochloric acid solution, continuously stirring, adding trypsin into the raw material liquid according to the enzyme addition amount of 800-1200U/g substrate protein for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 3-5.5 h, the enzymolysis temperature is 45-60 ℃, and the enzymolysis pH is 6-7.5;
(3) enzyme deactivation: heating to above 90 ℃ after enzymolysis, keeping for 10min, inactivating enzyme, cooling, and centrifuging the mixture after enzyme inactivation to obtain supernatant, or filtering the mixture after enzyme inactivation to obtain filtrate;
(4) and (3) vacuum concentration: pumping the clear liquid or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 50-65 ℃, and concentrating until the final water content is 40-60% to obtain the alfalfa bioactive peptide paste;
(5) and (3) freeze drying: and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of minus 60 to minus 40 ℃ until the final water content is below 10%, thus obtaining the alfalfa bioactive peptide powder.
2. The method for preparing alfalfa bioactive peptides of claim 1, wherein in step (1): the mass ratio of the raw materials to water is 1:10 during homogenization.
3. The method for preparing alfalfa bioactive peptides of claim 1, wherein in step (2): adding trypsin into the raw material solution according to the enzyme adding amount of 900U/g substrate protein for enzymolysis, wherein the enzymolysis process parameters are as follows: the enzymolysis time is 4h, the enzymolysis temperature is 55 ℃, and the enzymolysis pH is 6.5.
4. The method for preparing alfalfa bioactive peptides of claim 1, wherein in step (4): and pumping the supernatant or the filtrate into a concentration tank for concentration treatment, wherein the concentration temperature is 55 ℃, and concentrating until the final water content is 50% to obtain the alfalfa bioactive peptide paste.
5. The method for preparing alfalfa bioactive peptides of claim 1, wherein in step (5): and (3) carrying out freeze drying treatment on the alfalfa bioactive peptide paste at the drying temperature of-50 ℃ until the final water content is 8%, thus obtaining the alfalfa bioactive peptide powder.
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| CN106820113A (en) * | 2016-12-28 | 2017-06-13 | 陕西天宝大豆食品技术研究所 | Full clover peptide nutrient food and preparation method thereof |
| CN107319543A (en) * | 2017-07-18 | 2017-11-07 | 诸城市和生食品有限公司 | A kind of alfalfa cream and preparation method thereof |
| CN107446980B (en) * | 2017-09-26 | 2021-03-19 | 成都中医药大学 | Sea buckthorn leaf polypeptide and preparation method thereof |
| CN112807416B (en) * | 2021-01-18 | 2023-05-05 | 宁波御坊堂生物科技有限公司 | Traditional Chinese medicine bone peptide composition with function of improving bone joint health and preparation method thereof |
| KR102605467B1 (en) * | 2023-05-04 | 2023-11-23 | 한국콜마주식회사 | Cosmetic composition for skin improvement comprising an extract of alfalfa seed-derived glycoprotein |
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| CN104195204A (en) * | 2014-07-30 | 2014-12-10 | 内蒙古天奇生物科技有限公司 | Method for preparing alfalfa polypeptide |
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