CN107267584B - Method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme - Google Patents
Method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme Download PDFInfo
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- CN107267584B CN107267584B CN201710537028.2A CN201710537028A CN107267584B CN 107267584 B CN107267584 B CN 107267584B CN 201710537028 A CN201710537028 A CN 201710537028A CN 107267584 B CN107267584 B CN 107267584B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
Abstract
The invention discloses a method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme, the method takes papaya seed gum as a carrier and trivalent chromium salt as a cross-linking agent to immobilize biological enzyme, the immobilization method is simple and high in efficiency, the obtained immobilized enzyme is physical gel and has good stability, and the immobilized enzyme does not need a high-cost carrier, so that the cost is greatly reduced. The immobilized enzyme is adopted to carry out enzymolysis on the shinyleaf yellowhorn protein to extract the polypeptide, the process is simple, the operation is convenient, the enzyme can be recycled, and the waste of resources is greatly reduced.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to an immobilized enzyme prepared by taking pawpaw seed gum as a carrier, and a method for preparing shinyleaf yellowhorn polypeptide by carrying out enzymolysis on shinyleaf yellowhorn protein by using the immobilized enzyme.
Background
Xanthoceras sorbifolia Bunge (Xanthoceras sorbifolia Bunge) is a deciduous tree or shrub of Xanthoceras in Sapindaceae, is native to loess plateau, is widely distributed in northern areas of China, has strong adaptability, is cold-resistant, drought-resistant and barren-resistant, and can be widely planted in barren hilly lands. Is a unique rare woody oil tree species in China, and is also a water and soil conservation tree species and a rare ornamental tree which have development prospect in northern areas of China. As a natural shinyleaf yellowhorn country, shinyleaf yellowhorn resource development economic benefits and application prospects are immeasurable. The shinyleaf yellowhorn seed not only can be eaten, but also can be developed into health care products, the shinyleaf yellowhorn seed kernel contains a large amount of grease and rich protein, the protein content is 25.75%, 18 amino acids are contained, the types of the necessary amino acids are complete, the amino acid score is high, and the shinyleaf yellowhorn seed kernel has high nutritional value. However, some undesirable properties of vegetable proteins, such as their toxic or anti-nutritional content, their solubility, etc., do not reach the level required for food processing, limiting their use in the food processing industry. In view of this, in recent years, many studies have been made by scientists on the modification of food proteins, and among them, enzymatic hydrolysis of proteins is a method with good effect. The polypeptide obtained after the protein degradation is a protein segment formed by connecting two or more amino acids through peptide bonds, is a basic composition of body tissue cells, and has various biological activities: has effects in regulating enzyme in organism, improving mineral substance transportation and absorption, resisting bacteria and virus, enhancing immunity, resisting oxidation, resisting aging, and resisting hypertension and hyperlipemia. Since the importance of bioactive peptides in food and health products is recognized, research on extraction of bioactive peptides from plant resources by enzymatic methods is also becoming more widespread. The xanthoceras sorbifolia protein peptide has important physiological functions, can eliminate free radicals in vivo, and achieves the effects of resisting oxidation and prolonging the service life of cells. Meanwhile, the nutrient supplement has good digestion and absorption and high food safety, so the nutrient supplement can be applied to infant and child formula food, weight-losing food, athlete food and medical food as a nutrient supplement, and is considered as a new nutrient health-care source and a functional factor with great development potential.
The enzyme immobilization technology is a new technology which is rapidly developed in the later stage of the sixties of the twentieth century, and mainly uses a carrier to bind or limit enzymes in a certain region by means of adsorption, embedding, crosslinking, covalent bonding and the like, so that enzyme molecules can perform active catalytic action in the specific region and can be recovered and reused for multiple times. Compared with the traditional free enzyme, the immobilized enzyme technology can realize the repeated utilization of the enzyme, improve the stability of the enzyme, ensure the continuous reaction, realize the effective separation of a substrate and a product, improve the use efficiency of the enzyme and reduce the cost. In recent years, with the development of immobilized enzyme technology and the continuous expansion of application range, immobilized enzymes become one of active research fields, and the research on the immobilization of the enzymes is more and more at home and abroad, but the immobilized enzymes which are really put into industrial application are few, and the main reasons are high immobilization cost, low efficiency and poor stability.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for preparing shinyleaf yellowhorn polypeptide by using immobilized enzyme enzymolysis of shinyleaf yellowhorn protein by using natural pawpaw seed gum as a carrier.
The technical scheme adopted for solving the technical problems comprises the following steps:
1. respectively dissolving biological enzyme, pawpaw seed gum and trivalent chromium soluble salt in a phosphate buffer solution to prepare a biological enzyme solution with the mass concentration of 0.5-6%, a pawpaw seed gum solution with the mass concentration of 1.5-5% and a trivalent chromium soluble salt solution with the mass concentration of 1-4%; then uniformly mixing 1.5-5% of pawpaw seed gum solution, 0.5-6% of biological enzyme solution and 1-4% of trivalent chromium soluble salt solution according to the volume ratio of 1 (0.5-1) to (1-3), standing for 1-12 hours at 20-50 ℃, centrifuging and drying to obtain the pawpaw seed gum immobilized enzyme.
2. Adjusting the pH value of a shinyleaf yellowhorn egg white aqueous solution with the mass concentration of 2% -5% to 6-10, adding papaya seed gum immobilized enzyme according to the proportion of 2000-8000U of biological enzyme/g shinyleaf yellowhorn protein, carrying out enzymolysis for 30-210 minutes at 40-60 ℃, carrying out centrifugal separation, and carrying out freeze drying on the supernatant to obtain the shinyleaf yellowhorn polypeptide.
In the step 1, a papaya seed gum solution with a mass concentration of 2.5-4%, a biological enzyme solution with a mass concentration of 1-2.5% and a trivalent chromium soluble salt solution with a mass concentration of 2.5-4% are preferably uniformly mixed according to a volume ratio of 1 (0.5-1) to (1-3), and the mixture is stood at a temperature of 20-50 ℃ for 1-12 hours, centrifuged and dried to obtain the papaya seed gum immobilized enzyme.
In the step 1, the mixture is preferably allowed to stand at 40 to 50 ℃ for 2 to 9 hours.
In the step 2, preferably, the pH value of the shinyleaf yellowhorn protein aqueous solution with the mass concentration of 2-5% is adjusted to 6-10 by using phosphate, and then papaya seed gum immobilized enzyme is added according to the proportion of 3000-5000U biological enzyme/g shinyleaf yellowhorn protein.
In the step 2, enzymolysis is preferably performed at 50-55 ℃ for 60-120 minutes.
The above biological enzyme is at least one of alkaline protease, neutral protease, trypsin and papain, and the trivalent chromium soluble salt is chromium acetate or chromium propionate.
The immobilized biological enzyme takes the pawpaw seed gum as a carrier and the trivalent chromium salt as a cross-linking agent, wherein the pawpaw seed gum belongs to a natural product, is environment-friendly, healthy, cheap and easily available, has good mechanical property, stable chemical property and biodegradability, the immobilized enzyme has high efficiency, the immobilization method is simple, and the obtained immobilized enzyme is physical gel and has good stability. The immobilized enzyme does not need a carrier with high cost, so the cost is greatly reduced. The immobilized enzyme is adopted to carry out enzymolysis on the shinyleaf yellowhorn protein to extract the polypeptide, the process is simple, the operation is convenient, the enzyme can be recycled, and the waste of resources is greatly reduced.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1
1. Respectively dissolving 1g of papain, 3g of pawpaw seed gum and 3.8g of chromium acetate in 100g of phosphate buffer solution with the pH value of 6.5 to prepare a papain solution with the mass concentration of 1%, a pawpaw seed gum solution with the mass concentration of 3% and a chromium acetate solution with the mass concentration of 3.8%; uniformly mixing 20mL of papaya seed gum solution with the mass concentration of 3% and 10mL of papain solution with the mass concentration of 1%, adding 60mL of chromium acetate solution with the mass concentration of 3.8%, standing at 40 ℃ for 6 hours, then performing centrifugal separation at the rotating speed of 5000rpm, and performing freeze drying on the precipitate to obtain the papaya seed gum immobilized enzyme.
2. After the pH value of shinyleaf yellowhorn egg white aqueous solution with the mass concentration of 2% is adjusted to 6.5 by phosphate, papaya seed gum immobilized enzyme is added according to the proportion of 3300U of papain/g of shinyleaf yellowhorn protein, enzymolysis is carried out for 60 minutes at 55 ℃, after the enzymolysis is finished, centrifugal separation is carried out at the centrifugal rotation speed of 5000rpm, the papaya seed gum immobilized enzyme is recovered, and the obtained supernatant is freeze-dried to obtain shinyleaf yellowhorn polypeptide, wherein the hydrolysis degree of the shinyleaf yellowhorn protein is 17.78%. The immobilized papaya seed gum enzyme can be reused for 6 times, and the enzyme activity can still be kept above 50%.
Example 2
1. Dissolving 1.5g of protease (a mixture of trypsin and neutral protease at an activity ratio of 1: 1), 2.5g of papaya seed gum and 3g of chromium propionate in 100g of phosphate buffer with a pH of 8.0 respectively to prepare a protease solution with a mass concentration of 1.5%, a papaya seed gum solution with a mass concentration of 2.5% and a chromium propionate solution with a mass concentration of 3%; uniformly mixing 20mL of 2.5 mass percent papaya seed gum solution and 20mL of 1.5 mass percent protease solution, adding 40mL of 3 mass percent chromium propionate solution, standing at 50 ℃ for 3 hours, then performing centrifugal separation at the rotating speed of 5000rpm, and freeze-drying the precipitate to obtain the papaya seed gum immobilized enzyme.
2. After the pH value of shinyleaf yellowhorn egg white aqueous solution with the mass concentration of 4.5% is adjusted to 8.0 by phosphate, adding papaya seed gum immobilized enzyme according to the proportion of 3300U protease/g shinyleaf yellowhorn protein, carrying out enzymolysis for 180 minutes at 50 ℃, carrying out centrifugal separation at the centrifugal rotation speed of 4500rpm after the enzymolysis is finished, recovering the papaya seed gum immobilized enzyme, and carrying out freeze drying on the obtained supernatant to obtain shinyleaf yellowhorn polypeptide, wherein the hydrolysis degree of the shinyleaf yellowhorn protein is 16.98%. The immobilized papaya seed gum enzyme can be reused for 8 times, and the enzyme activity can still be maintained above 50%.
Example 3
1. Respectively dissolving 2.5g of alkaline protease, 4g of pawpaw seed gum and 2.5g of chromium acetate in 100g of phosphate buffer solution with the pH value of 9.0 to prepare an alkaline protease solution with the mass concentration of 2.5%, a pawpaw seed gum solution with the mass concentration of 4% and a chromium acetate solution with the mass concentration of 2.5%; uniformly mixing 20mL of a 4% pawpaw seed gum solution and 10mL of a 2.5% alkaline protease solution, adding 24mL of a 2.5% chromium acetate solution, standing at 45 ℃ for 2.5 hours, then carrying out centrifugal separation at a rotation speed of 5000rpm, and carrying out freeze drying on the precipitate to obtain the pawpaw seed gum immobilized enzyme.
2. Adjusting the pH value of shinyleaf yellowhorn egg white aqueous solution with the mass concentration of 3% to 9.0 by using phosphate, adding papaya seed gum immobilized enzyme according to the proportion of 4000U of alkaline protease/g shinyleaf yellowhorn protein, carrying out enzymolysis for 120 minutes at 55 ℃, carrying out centrifugal separation at the centrifugal rotation speed of 3500rpm after the enzymolysis is finished, recovering the papaya seed gum immobilized enzyme, and carrying out freeze drying on the obtained supernatant to obtain the shinyleaf yellowhorn polypeptide, wherein the hydrolysis degree of shinyleaf yellowhorn substitutional protein is 19.21%. The papaya seed gum immobilized enzyme is reused for 6 times, and the enzyme activity is still kept above 55%.
Claims (6)
1. A method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme is characterized by comprising the following steps:
(1) respectively dissolving biological enzyme, pawpaw seed gum and trivalent chromium soluble salt in a phosphate buffer solution to prepare a biological enzyme solution with the mass concentration of 0.5-6%, a pawpaw seed gum solution with the mass concentration of 1.5-5% and a trivalent chromium soluble salt solution with the mass concentration of 1-4%; then uniformly mixing 1.5-5% of pawpaw seed gum solution, 0.5-6% of biological enzyme solution and 1-4% of trivalent chromium soluble salt solution according to the volume ratio of 1 (0.5-1) to (1-3), standing for 1-12 hours at 20-50 ℃, centrifuging and drying to obtain the pawpaw seed gum immobilized enzyme;
(2) adjusting the pH value of a shinyleaf yellowhorn egg white aqueous solution with the mass concentration of 2-5% to 6-10, adding papaya seed gum immobilized enzyme according to the proportion of 2000-8000U of biological enzyme/g of shinyleaf yellowhorn protein, performing enzymolysis for 30-210 minutes at 40-60 ℃, performing centrifugal separation, and freeze-drying the supernatant to obtain shinyleaf yellowhorn polypeptide;
the biological enzyme is at least one of alkaline protease, neutral protease, trypsin and papain;
the trivalent chromium soluble salt is chromium acetate or chromium propionate.
2. The method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme according to claim 1, which is characterized in that: in the step (1), papaya seed gum immobilized enzyme is obtained by uniformly mixing a papaya seed gum solution with the mass concentration of 2.5-4%, a biological enzyme solution with the mass concentration of 1-2.5% and a trivalent chromium soluble salt solution with the mass concentration of 2.5-4% according to the volume ratio of 1 (0.5-1) to (1-3), standing for 1-12 hours at the temperature of 20-50 ℃, centrifuging and drying.
3. The method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme according to claim 2, which is characterized in that: in the step (1), standing for 2-9 hours at 40-50 ℃.
4. The method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme according to claim 1, which is characterized in that: in the step (2), the pH value of the shinyleaf yellowhorn egg white water solution with the mass concentration of 2% -5% is adjusted to 6-10 by phosphate.
5. The method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme according to claim 1, which is characterized in that: in the step (2), adding the papaya seed gum immobilized enzyme according to the ratio of 3000-5000U of biological enzyme/g of shinyleaf yellowhorn protein.
6. The method for preparing shinyleaf yellowhorn polypeptide based on papaya seed gum immobilized enzyme according to claim 5, which is characterized in that: in the step (2), enzymolysis is carried out for 60-120 minutes at 50-55 ℃.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5577888A (en) * | 1978-12-07 | 1980-06-12 | Takashi Ito | Immobilized gamma-glutamyl-cysteine-synthetase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5577888A (en) * | 1978-12-07 | 1980-06-12 | Takashi Ito | Immobilized gamma-glutamyl-cysteine-synthetase |
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| Title |
|---|
| 文冠果蛋白酶解条件研究;杨永强;《万方数据知识服务平台》;20150701;摘要、第2页第1.3节、第5页第2.2.4节、第11页3.2.1节、第21页表3-4、第25页表3-9、第30页表3-14 * |
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