CN114621992B - Preparation method of quinoa antioxidant polypeptide - Google Patents
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- C12P21/00—Preparation of peptides or proteins
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses a preparation method of quinoa antioxidant polypeptide, which comprises the steps of soaking quinoa into a mixed salt solution of sodium bicarbonate and sodium chloride for dissolving, centrifuging to obtain quinoa protein extract, respectively carrying out ultrasonic treatment and enzymolysis treatment by pepsin and trypsin, ultrafiltering to remove impurities, and freeze-drying to obtain quinoa antioxidant polypeptide. The invention has the advantages that the DPPH free radical clearance rate of the quinoa antioxidant polypeptide reaches more than 65%, the hydroxyl free radical clearance rate reaches more than 40%, and the superoxide anion free radical clearance rate reaches more than 50%.
Description
Technical Field
The invention belongs to the technical field of polypeptide preparation, and particularly relates to a method for extracting and preparing antioxidant polypeptide from quinoa.
Background
Quinoa is native to the andes mountain in south america, is an annual dicotyledonous amaranthaceae quinoa plant, and is a pseudocereal crop with cereal crop characteristics. At present, quinoa is planted in large scale in mountain western, gansu, qinghai, tibet and other places in China.
The content of the complete protein in quinoa reaches more than 16 percent, which is higher than that of barley, rice and corn. Quinoa protein contains all natural amino acids and is rich in all essential amino acids of human body, and especially the lysine content is far higher than that of common grains. In addition, quinoa contains rich dietary fibers, mineral elements, unsaturated fatty acids and the like. The quinoa can enhance the immunity of the organism and promote health after being eaten for a long time, and is used for preventing and assisting in treating diseases such as obesity, cardiovascular diseases, diabetes, cancer and the like. Therefore, quinoa is one of ideal food choices for astronauts to engage in long-term space tasks as a high-quality food with complete protein, low fat, low calorie and comprehensive nutrition, and is also considered as the only one single plant which can meet the basic nutritional requirements of human bodies.
Free radicals are a class of harmful compounds that are produced by the body during normal metabolism and can cause protein damage, lipid peroxidation, DNA fragmentation. The production of excess free radicals is closely linked to a variety of diseases, such as aging, neurodegenerative diseases, cardiovascular diseases, and tumors. Studies prove that the quinoa extract can inhibit lipid peroxidation of liver microsomes and has good antioxidant activity. The antioxidant activity of quinoa is mainly dependent on the antioxidant substances such as polyphenol and flavone contained in quinoa, but the contents of the polyphenol and flavone in quinoa are low, the extraction difficulty is high, the production cost is high, and the industrial production is difficult to realize.
The antioxidant polypeptide is used as a small molecular bioactive peptide capable of effectively reducing in vivo active oxygen free radicals, achieves the antioxidant purpose by removing excessive in vivo free radicals, improving antioxidant enzyme activity, inhibiting lipid peroxidation, chelating metal ions and the like, has the advantages of high stability, strong activity, easy absorption, safety, no toxicity and the like, and has wide application prospect in the aspects of medicine, functional foods, cosmetics and the like.
The quinoa has rich protein content and balanced amino acid ratio, and reports about preparing antioxidant polypeptide by deep processing of quinoa protein are less.
The enzymolysis method is a biological synthesis method for preparing the polypeptide by degrading the protein by using biological enzyme, however, the enzymolysis method adopted at present has obvious defects of large investment, low yield, poor uniformity of enzymolysis product quality, high separation and purification difficulty and the like. How to prepare high-activity antioxidant polypeptide rapidly and safely with low cost, enriches the deep processing technology of quinoa products, increases the economic added value of quinoa products, meets the requirements of the market on safe, healthy and efficient natural antioxidants, and is a problem which needs to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a preparation method of quinoa antioxidant polypeptide, which is characterized in that quinoa protein is purified by a modern separation technology, and the quinoa protein is hydrolyzed by a specific double-enzyme method, so that the quinoa active polypeptide with antioxidant activity is obtained under mild reaction conditions and high selectivity.
The preparation method of the quinoa antioxidant polypeptide provided by the invention comprises the following steps:
1) Soaking quinoa in water until the quinoa swells and softens, taking out and drying, and grinding to obtain quinoa flour;
2) Placing quinoa flour into a mixed salt solution of sodium bicarbonate and sodium chloride, stirring and fully dissolving, and centrifuging to obtain quinoa protein extract;
3) Regulating the pH value of the quinoa protein extract to 1.5-3, carrying out enzymolysis treatment by pepsin, regulating the pH value of the quinoa protein extract to 8-9, and carrying out enzymolysis treatment by trypsin to obtain an enzymolysis solution;
4) Filtering the enzymolysis liquid by an ultrafiltration membrane with the maximum permeation molecular weight of 10KD, collecting filtrate, and freeze-drying to obtain the quinoa antioxidant polypeptide.
Wherein, preferably, the enzymolysis treatment is carried out by adding pepsin into 0.5-1.5 ten thousand U/g quinoa protein.
More specifically, after pepsin is added, ultrasonic auxiliary treatment is carried out for 0.5-2 hours, enzymolysis is carried out for 2-4 hours at 45-55 ℃, and then heating is carried out to inactivate enzymes.
Wherein, preferably, 200-600U/g quinoa protein is added with trypsin for enzymolysis treatment.
More specifically, after trypsin is added, ultrasonic auxiliary treatment is carried out for 0.5 to 2 hours, enzymolysis is carried out for 2 to 4 hours at 37 to 47 ℃, and then enzyme is deactivated by heating.
Further, the invention is to centrifuge the enzymolysis liquid for 10-20 min at 4000rpm, and then to take the supernatant for ultrafiltration membrane filtration.
The invention carries out ultrasonic auxiliary treatment before enzymolysis, can promote the dissolution of quinoa protein, exposes more enzymolysis sites and is beneficial to improving the enzymolysis efficiency.
In the invention, the mixed salt solution of sodium bicarbonate and sodium chloride is a solution prepared by mixing 0.033mol/L sodium bicarbonate solution and 0.086mol/L sodium chloride solution according to the volume ratio of (0.5-3) to 1. The mixed salt solution can specifically separate quinoa protein.
Further, the invention is to put the quinoa flour in the mixed salt solution of sodium bicarbonate and sodium chloride according to the feed liquid ratio of 1:10-20.
More specifically, the invention is to soak quinoa in water for 6-24 hours to fully expand and soften the quinoa.
In more detail, the invention grinds the dried quinoa into 80-120 mesh fine powder.
The preparation method of the quinoa antioxidation polypeptide has mild reaction conditions, is simple, convenient and easy to operate, and has high selectivity for extracting the quinoa antioxidation polypeptide.
Amino acid sequencing is carried out on the quinoa antioxidant polypeptide prepared by the invention, and the sequence of the quinoa antioxidant polypeptide comprises amino acid combination sequences such as GTM, KHTC and the like which are different from other antioxidant polypeptides.
The test result of the antioxidation capability of the quinoa antioxidation polypeptide prepared by the invention shows that the quinoa antioxidation capability has remarkable antioxidation capability for DPPH free radical, hydroxyl free radical and superoxide anion free radical with clearance of 30-70%, 25-45% and 30-60% respectively.
Detailed Description
The following describes the present invention in further detail with reference to examples. The following examples are presented only to more clearly illustrate the technical aspects of the present invention so that those skilled in the art can better understand and utilize the present invention without limiting the scope of the present invention.
The experimental methods, production processes, apparatuses and devices involved in the examples and comparative examples of the present invention, the names and abbreviations thereof all belong to the names conventional in the art, and are clearly and clearly understood in the related fields of use, and the skilled person can understand the conventional process steps according to the names and apply the corresponding devices, and perform according to the conventional conditions or the conditions suggested by the manufacturer.
The various raw materials or reagents used in the examples and comparative examples of the present invention are not particularly limited in source, and are conventional products commercially available. The preparation may also be carried out according to conventional methods known to the person skilled in the art.
Example 1.
Weighing 50g of quinoa, rinsing for 2-3 times by tap water, filtering to remove impurities such as scraps, soaking in purified water for 8 hours to fully expand and soften the quinoa, and drying in a drying oven to loosen and facilitate grinding the quinoa.
Grinding the dried quinoa into powder by a high-speed pulverizer, and filtering by a 80-mesh screen to obtain quinoa powder.
Sodium bicarbonate solution with the concentration of 0.033mol/L is prepared and mixed with sodium chloride solution with the concentration of 0.086mol/L in the volume ratio of 1:1 to obtain salt solution.
50mL of the salt solution is taken, 5g of quinoa flour is added, stirring is carried out at 4 ℃ for overnight to enable the quinoa flour to be fully dissolved, centrifugation is carried out at 12000rpm for 50min, quinoa protein solution is obtained by extraction, and the protein concentration in the solution is measured to be 12.8mg/mL by a BCA method.
Taking 20mL of quinoa protein solution, regulating the pH value to 1.5-2, adding pepsin according to 0.5U/g of quinoa protein, carrying out ultrasonic auxiliary treatment for 1h, carrying out enzymolysis for 2h at 45 ℃, heating for 15min at 80 ℃ to inactivate enzymes, and cooling to room temperature to obtain the pepsin-hydrolyzed quinoa protein solution.
Regulating pH value of the pepsin quinoa protein solution to 8-9, adding trypsin according to 200U/g quinoa protein, performing ultrasonic auxiliary treatment for 1h, performing enzymolysis for 2h at 37 ℃, heating for 15min at 80 ℃ to inactivate enzymes, and cooling to room temperature to obtain the trypsin quinoa protein solution.
The trypsin quinoa protein solution was centrifuged at 4000rpm for 10min and the supernatant was collected and filtered through an ultrafiltration membrane having a maximum permeation molecular weight of 10 KD. And freeze-drying the filtrate to obtain the quinoa antioxidant polypeptide.
Preparing 0.25mg/mL aqueous solution by using quinoa antioxidant polypeptide, respectively mixing and incubating with 0.2mol/L DPPH solution, 9mmol/L Fenton reagent and 25mmol/L pyrogallol solution, and detecting the clearance rate of the quinoa antioxidant polypeptide to DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals.
The results in Table 1 show that the scavenging rates of quinoa antioxidant polypeptide on DPPH free radical, hydroxyl free radical and superoxide anion free radical are respectively 32.8%, 25.7% and 36.5%, and are respectively improved by 212.3%, 56.7% and 77.2% compared with that of non-enzymatic quinoa protein.
Comparative example 1.
According to the method of example 1, only after pepsin and trypsin are added respectively, enzymolysis is directly carried out, ultrasonic auxiliary treatment is not carried out for 1h, and other steps are exactly the same as those of example 1, so that the finally prepared quinoa antioxidant polypeptide is used as an un-ultrasonic treatment group.
The procedure of example 1 was followed except that the pepsin-hydrolyzed quinoa protein solution was used to directly prepare quinoa antioxidant polypeptide without trypsin hydrolysis, and the finally prepared quinoa antioxidant polypeptide was used as a pepsin treatment group in exactly the same manner as in example 1.
The method of example 1 was followed except that pepsin was not used to carry out the enzymolysis, and the quinoa protein solution was directly subjected to trypsin to obtain a trypsin-hydrolyzed quinoa protein solution to prepare quinoa antioxidant polypeptide, and the procedure was otherwise exactly the same as in example 1, except that the finally prepared quinoa antioxidant polypeptide was used as a trypsin treatment group.
Quinoa antioxidant polypeptide prepared in example 1 was used as pepsin/trypsin treatment group.
Taking 20mL of quinoa protein solution prepared in example 1, regulating the pH value to 6-8, adding neutral protease according to 200U/g, carrying out ultrasonic auxiliary treatment for 1h, carrying out enzymolysis at 45 ℃ for 2h, heating at 80 ℃ for 15min to inactivate the enzyme, cooling to room temperature, regulating the pH value to 10-12, adding alkaline protease according to 200U/g, carrying out ultrasonic auxiliary treatment for 1h, carrying out enzymolysis at 50 ℃ for 2h, heating at 80 ℃ for 15min to inactivate the enzyme, cooling to room temperature, centrifuging at 4000rpm for 10min, collecting supernatant, and filtering by an ultrafiltration membrane with the maximum transmission molecular weight of 10 KD. And freeze-drying the filtrate to obtain quinoa antioxidant polypeptide serving as an alkaline protease/neutral protease treatment group.
The clearance of DPPH radicals, hydroxyl radicals and superoxide anion radicals by each group of quinoa antioxidant polypeptides was determined separately as in example 1.
The results in Table 2 above show that the pepsin/trypsin treated groups had clearance rates of 36.5%, 30.1% and 45.3% for DPPH free radical, hydroxyl free radical and superoxide anion free radical, which were improved by 37.2%, 31.4% and 30.5% respectively compared to the non-sonicated groups; compared with pepsin groups, 97.3%, 40.7% and 17.4% are respectively improved; compared with trypsin group, the trypsin group is respectively improved by 78.0%, 91.7% and 75.6%; the improvement was 247.6%, 67.2% and 79.8% compared to the alkaline protease/neutral protease group, respectively.
Example 2.
Weighing 50g of quinoa, rinsing for 2-3 times by tap water, filtering to remove impurities such as scraps, soaking in purified water for 10 hours to fully expand and soften the quinoa, and drying in a drying oven to loosen and facilitate grinding the quinoa.
Grinding the dried quinoa into powder by a high-speed pulverizer, and filtering by a 120-mesh screen to obtain quinoa powder.
Sodium bicarbonate solution with the concentration of 0.033mol/L is prepared and mixed with sodium chloride solution with the concentration of 0.086mol/L in the volume ratio of 1:2 to obtain salt solution.
50mL of the salt solution is taken, 5g of quinoa flour is added, stirring is carried out at 4 ℃ for overnight to enable the quinoa flour to be fully dissolved, centrifugation is carried out at 12000rpm for 30min, quinoa protein solution is obtained by extraction, and protein concentration in the solution is measured to be 10.1mg/mL by a BCA method.
Taking 20mL of quinoa protein solution, regulating the pH value to 1.5-3, adding pepsin according to 0.5U/g of quinoa protein, carrying out ultrasonic auxiliary treatment for 2h, carrying out enzymolysis for 2h at 45 ℃, heating for 15min at 80 ℃ to inactivate enzymes, and cooling to room temperature to obtain the pepsin-hydrolyzed quinoa protein solution.
Regulating pH value of the pepsin quinoa protein solution to 8-9, adding trypsin according to 200U/g quinoa protein, performing ultrasonic auxiliary treatment for 2h, performing enzymolysis for 2h at 37 ℃, heating for 15min at 80 ℃ to inactivate enzymes, and cooling to room temperature to obtain the trypsin quinoa protein solution.
The trypsin quinoa protein solution was centrifuged at 4000rpm for 10min and the supernatant was collected and filtered through an ultrafiltration membrane having a maximum permeation molecular weight of 10 KD. And freeze-drying the filtrate to obtain the quinoa antioxidant polypeptide.
The quinoa antioxidant polypeptide is prepared into 0.75mg/mL aqueous solution, and the aqueous solution is respectively mixed with 0.2mol/L DPPH solution, 9mmol/L Fenton reagent and 25mmol/L pyrogallol solution for incubation, and the clearance rates of the quinoa antioxidant polypeptide on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals are detected to be 67.5%, 43.6% and 52.1% respectively.
Example 3.
Weighing 50g of quinoa, rinsing for 2-3 times by tap water, filtering to remove impurities such as scraps, soaking in purified water for 24 hours to fully expand and soften the quinoa, and drying in a drying oven to loosen and facilitate grinding the quinoa.
Grinding the dried quinoa into powder by a high-speed pulverizer, and filtering by a 80-mesh screen to obtain quinoa powder.
Sodium bicarbonate solution with the concentration of 0.033mol/L is prepared and mixed with sodium chloride solution with the concentration of 0.086mol/L in the volume ratio of 2:1 to obtain salt solution.
50mL of the salt solution is taken, 5g of quinoa flour is added, stirring is carried out at 4 ℃ for overnight to enable the quinoa flour to be fully dissolved, centrifugation is carried out at 12000rpm for 50min, quinoa protein solution is obtained by extraction, and protein concentration in the solution is measured to be 11.3 mg/mL by BCA method.
Taking 20mL of quinoa protein solution, regulating the pH value to 1.5-2, adding pepsin according to 1.5U/g of quinoa protein, carrying out ultrasonic auxiliary treatment for 1h, carrying out enzymolysis for 3h at 45 ℃, heating for 15min at 80 ℃ to inactivate enzymes, and cooling to room temperature to obtain the pepsin-hydrolyzed quinoa protein solution.
Regulating pH value of the pepsin quinoa protein solution to 8-9, adding trypsin according to 500U/g quinoa protein, performing ultrasonic auxiliary treatment for 1h, performing enzymolysis for 3h at 37 ℃, heating for 15min at 80 ℃ to inactivate enzymes, and cooling to room temperature to obtain the trypsin quinoa protein solution.
The trypsin quinoa protein solution was centrifuged at 4000rpm for 20min and the supernatant was collected and filtered through an ultrafiltration membrane having a maximum permeation molecular weight of 10 KD. And freeze-drying the filtrate to obtain the quinoa antioxidant polypeptide.
The quinoa antioxidant polypeptide is prepared into 0.5mg/mL aqueous solution, and the aqueous solution is respectively mixed with 0.2mol/L DPPH solution, 9mmol/L Fenton reagent and 25mmol/L pyrogallol solution for incubation, and the clearance rates of the quinoa antioxidant polypeptide on DPPH free radicals, hydroxyl free radicals and superoxide anion free radicals are detected to be 62.6%, 40.5% and 59.5% respectively.
The above embodiments of the invention are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Various changes, modifications, substitutions and alterations may be made by those skilled in the art without departing from the principles and spirit of the invention, and it is intended that the invention encompass all such changes, modifications and alterations as fall within the scope of the invention.
Claims (6)
1. A method of preparing a quinoa antioxidant polypeptide, the method comprising:
1) Soaking quinoa in water until the quinoa swells and softens, taking out and drying, and grinding to obtain quinoa flour;
2) Placing quinoa flour into a mixed salt solution of sodium bicarbonate and sodium chloride, stirring and fully dissolving, and centrifuging to obtain quinoa protein extract;
3) Regulating the pH value of quinoa protein extract to 1.5-3, adding pepsin according to 0.5-1.5U/g quinoa protein for enzymolysis treatment, carrying out ultrasonic auxiliary treatment for 0.5-2 h, carrying out enzymolysis for 2-4 h at 45-55 ℃, heating to inactivate enzymes, regulating the pH value to 8-9, adding trypsin according to 200-600U/g quinoa protein for enzymolysis treatment, carrying out ultrasonic auxiliary treatment for 0.5-2 h, carrying out enzymolysis for 2-4 h at 37-47 ℃, and heating to inactivate enzymes to obtain an enzymolysis solution;
4) Filtering the enzymolysis liquid by an ultrafiltration membrane with the maximum permeation molecular weight of 10KD, collecting filtrate, and freeze-drying to obtain the quinoa antioxidant polypeptide.
2. The method for preparing quinoa antioxidant polypeptide according to claim 1, wherein the enzymatic hydrolysate is centrifuged at 4000rpm for 10-20 min, and the supernatant is filtered by ultrafiltration membrane.
3. The method for preparing quinoa antioxidant polypeptide according to claim 1, wherein the mixed salt solution of sodium bicarbonate and sodium chloride is a solution prepared by mixing 0.033mol/L sodium bicarbonate solution and 0.086mol/L sodium chloride solution according to a volume ratio of (0.5-3) to 1.
4. The method for preparing quinoa antioxidant polypeptide according to claim 1 or 3, wherein quinoa flour is placed in the mixed salt solution of sodium bicarbonate and sodium chloride according to a feed liquid ratio of 1: (10-20).
5. The method for preparing the quinoa antioxidant polypeptide according to claim 1, wherein quinoa is soaked in water for 6-24 hours to be swelled and softened.
6. The method for preparing the quinoa antioxidant polypeptide according to claim 1, wherein the dried quinoa is ground into 80-120 mesh fine powder.
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| The effects of hydrolysis condition on antioxidant activity of protein hydrolyzate from q uinoa;Mina Mahdavi-Yekta et al,;food Science & Nutrition;20191231(第7期);第930–936页 * |
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