CN114634960B - Preparation method of edible thymus peptide - Google Patents
Preparation method of edible thymus peptide Download PDFInfo
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- CN114634960B CN114634960B CN202011476914.7A CN202011476914A CN114634960B CN 114634960 B CN114634960 B CN 114634960B CN 202011476914 A CN202011476914 A CN 202011476914A CN 114634960 B CN114634960 B CN 114634960B
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- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims abstract description 12
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
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- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
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- 229960004231 thymalfasin Drugs 0.000 description 2
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- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000002662 enteric coated tablet Substances 0.000 description 1
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Classifications
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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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention relates to a preparation method of edible thymus peptide, S1, taking calf thymus to remove throat and fat, adding purified water, grinding into homogenate by a colloid mill, and taking the homogenate as raw materials for standby; s2, adjusting the pH value to be acidic by using acid, and adding phospholipase for enzymolysis; then adding collagenase and neutral protease for enzymolysis, heating to deactivate enzyme, cooling, and removing oil and solid in the soup by a three-phase centrifuge to obtain clarified enzymolysis liquid; s3, adding active carbon into the enzymolysis liquid, adjusting the temperature for adsorption, and removing the active carbon by a horizontal tank centrifuge to obtain clear extract; s4, adjusting the extracting solution to be neutral by using an alkaline solution, and filtering to obtain a peptide solution with the molecular weight of 0-6000 daltons; s5, removing sodium ions from the peptide solution through ion exchange resin to obtain a low-sodium peptide solution, and concentrating the low-sodium peptide solution by using a three-effect low-temperature concentrator to obtain a concentrated solution; s6, adding auxiliary materials into the concentrated solution, and drying by using a spray drying tower.
Description
Technical Field
The invention belongs to the technical field of generation and preparation, and particularly relates to a preparation method of edible thymus peptide.
Background
The thymosin is a group of protein and polypeptide hormone which is differentiated from thymosin component 5 and used as injection for medicines, and the preparation process of the thymosin with the molecular weight smaller than 10KD generally comprises the processes of tissue cell disruption, centrifugal separation, ultrafiltration, concentration and the like. Generally comprising the following methods: (1) Homogenizing fresh thymus by a homogenizer or a colloid mill, (2) heating to 80-90 ℃ and keeping for 10-30 min to obtain a denatured liquid; repeatedly freezing and thawing; (3) Centrifuging or filtering to remove solid impurities such as tissue cell walls; (4) Clarifying and filtering, collecting filtrate, ultrafiltering with ultrafiltration systems with different molecular weight cut-off, concentrating after twice ultrafiltration, freeze preserving, thawing, ultrafiltering with ultrafiltration system with molecular weight cut-off of 10KD, and collecting filtrate to obtain thymus peptide solution. The operation is complex, three times of ultrafiltration are needed, and the obtained solution can not be directly eaten, so that the cost is high.
The thymus peptide products which are circulated in the market at present are peptide medicines and are mainly used in the clinical medicine field; the main function is to serve as: a. continuously inducing each stage of T cell differentiation and development; b. maintaining the immune balance of the body and enhancing the reaction of T cells to antigens; c. thereby improving the disease resistance of the organism; such as thymus peptide enteric coated tablet, thymus peptide injection, etc. The main active component is thymus peptide alpha 1 (T alpha 1) composed of 28 amino acids. One such product is prepared by a method using protein isoelectric chromatography; the purity of the product is high, the variety of the peptide is single, the waste of other peptide products is caused, the recovery rate of the thymic peptide is reduced, the extraction rate is 0.6-1%, and the production cost is high. The other is to use amino acid as raw material and to use chemical reagent to make artificial grafting synthesis. The method improves the purity and yield of the product to a certain extent and reduces the production cost of the product. However, because a large amount of chemical reagents such as TFA (trifluoroacetic acid), acetic acid and the like are used in the process, certain residues exist in the product, certain toxic and side effects and adverse reactions exist in the use process, and the obtained product is used for injection, cannot be directly eaten, has high cost and high price, and limits the clinical application of the product.
Disclosure of Invention
First, the technical problem to be solved
In order to solve the problems in the prior art, the invention provides a preparation method of edible thymus peptide, which can effectively extract the edible thymus peptide, is simple to operate, has no toxic or side effect and can be directly eaten.
(II) technical scheme
In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:
a method for preparing edible thymus peptide, which comprises the following steps:
s1, taking calf thymus to remove throats and fat, adding purified water, and grinding into homogenate by a colloid mill to prepare raw materials for later use;
s2, adjusting the pH value to be acidic by using acid, and adding phospholipase for enzymolysis; then adding collagenase and neutral protease for enzymolysis, heating to deactivate enzyme, cooling, and removing oil and solid in the soup by a three-phase centrifuge to obtain clarified enzymolysis liquid;
s3, adding active carbon into the enzymolysis liquid, adjusting the temperature for adsorption, and removing the active carbon by a horizontal tank centrifuge to obtain clear extract;
s4, adjusting the extracting solution to be neutral by using an alkaline solution, and filtering to obtain a peptide solution with the molecular weight of 0-6000 daltons;
s5, removing sodium ions from the peptide solution through ion exchange resin to obtain a low-sodium peptide solution, and concentrating the low-sodium peptide solution by using a three-effect low-temperature concentrator to obtain a concentrated solution;
s6, adding auxiliary materials into the concentrated solution, and drying by using a spray drying tower.
In the preparation method, preferably, in the step S1, the dosage ratio of thymus of the calf to purified water is 1:1-1:1.5.
In the above-mentioned production method, in step S2, the pH is preferably adjusted to be acidic with an acid such as hydrochloric acid, and the pH is preferably adjusted to be 5.5 to 6.2, and the phospholipase is preferably enzymatically hydrolyzed at a temperature of 40 to 48 ℃.
In the preparation method, preferably, in the step S2, the adding amount of the phospholipase is 0.0002 to 0.001 per mill of the calf thymus, and the enzymolysis time of the phospholipase is 30 to 60 minutes; the adding amount of the collagenase is 0.0005-0.001 per mill of the calf thymus, the adding amount of the neutral protease is 0.001-0.01 per mill of the calf thymus, and the enzymolysis time of the neutral protease is 60-150 min.
In the preparation method, preferably, in the step S2, the temperature is raised to 93-100 ℃, and the enzyme is inactivated for 1-5 min; cooling to 70-80 ℃ and centrifuging.
A large number of experimental researches show that the enzyme can be effectively killed when the temperature is raised to 93-100 ℃. In the preparation method, preferably, in the step S3, the addition amount of the activated carbon is 0.01-0.03% of the calf thymus, the temperature is adjusted to 65-80 ℃, and the adsorption is performed for 40-90 min.
In the preparation method as described above, preferably, in step S4, the alkaline solution is 10% -20% sodium hydroxide solution, and the filtration is performed by using a filtration membrane with pore size=0.6 nm, and the filtrate contains thymosin less than 6000 daltons.
The filter membrane can retain proteins with molecular weights greater than 6000 daltons.
In the above-described production method, preferably, in step S5, the solid content of the concentrated solution is 13% to 20%.
In the preparation method, preferably, in the step S6, the auxiliary materials are skim milk and sucrose, the mass ratio of the skim milk to the sucrose is 1-3:1, the mass ratio of the auxiliary materials is 2-5% of the mass of the concentrated solution, and the temperature of the spray drying tower is 75-85 ℃.
(III) beneficial effects
The beneficial effects of the invention are as follows:
the invention provides a preparation method of edible thymus peptide, which is beneficial to thymus cells exposure by decomposing fat and the like through enzymolysis, effectively shortens the decomposition time of neutral protease, ensures that the cells can be completely broken by the collagenase, and the neutral protease decomposes protein into 6000 daltons of thymus peptide.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments for better explaining the present invention.
Example 1
A preparation process of edible thymus peptide comprises the following steps:
step a, taking 10Kg of frozen fresh calf thymus, removing package, thawing to remove throat and fat, adding 10Kg of purified water, grinding into meat paste by a colloid mill, adding 20Kg of purified water, grinding into homogenate, and taking the homogenate as raw material for standby.
And b, adjusting the PH to be 6.0 by using hydrochloric acid with the mass fraction of 5%, wherein the temperature is 45 ℃, and adding 2g of phospholipase for enzymolysis for 50min. Then adding 10g of collagenase and 20g of neutral protease for enzymolysis for 120min, heating to 100 ℃, sterilizing for 5min, cooling to 75 ℃, and removing oil and solids in the soup by using a three-phase centrifuge to obtain a clear enzymolysis liquid.
And c, adding 150g of active carbon into the enzymolysis liquid, adjusting the temperature to 75 ℃, adsorbing for 60min, and removing the active carbon by a horizontal tank centrifuge to obtain a clear extract.
And d, adjusting the pH value of the extracting solution to be 7.0 by using 10% sodium hydroxide solution, filtering by using a filtering membrane with the pore diameter of 0.6nm, and intercepting proteins with the molecular weight of more than 6000 daltons to obtain peptide solution with the molecular weight of 0-6000 daltons. And e, removing sodium ions from the peptide solution through ion exchange resin to obtain a low-sodium peptide solution, and concentrating the low-sodium peptide solution by using a three-effect low-temperature concentrator to obtain a concentrated solution with 15% of solid content.
Step f, adding 3% of the concentrated solution into the concentrated solution, wherein the mass ratio is 2:1 and sucrose, and drying the mixture at 80 ℃ in a spray drying tower.
The content of the thymus peptide is 16.21mg/ml and the content of the thymus peptide alpha 1 is 3.2 percent by the conventional method. The activity of the thymus peptide is measured by adopting a T cell activity measuring method-an E-removing receptor method, the number of cells formed by E rosettes is counted to be combined with 3 or more sheep red blood cells, the sample is diluted to 0.002mg/ml, and the activity is increased by 37.5%.
Example 2
A preparation process of edible thymus peptide comprises the following steps:
step a, taking 10Kg of frozen fresh calf thymus, removing package, thawing to remove throat and fat, adding 10KG of purified water, grinding into meat paste by a colloid mill, adding 50Kg of purified water, grinding into homogenate, and taking as raw materials for standby.
And b, adjusting pH=5.5 by using 8% hydrochloric acid with the temperature=48 ℃, and adding 10g of phospholipase for enzymolysis for 30min. Then 8g of collagenase and 25g of neutral proteinase are added for enzymolysis for 100min, the temperature is raised to 95 ℃, sterilization is carried out for 3min, the temperature is lowered to 70 ℃, and the oil and solid matters in the soup are removed by a three-phase centrifuge, thus obtaining clarified enzymolysis liquid.
And c, adding 200g of active carbon into the enzymolysis liquid, adjusting the temperature to be 70 ℃, adsorbing for 40min, and removing the active carbon by a horizontal tank centrifuge to obtain a clear extract.
And d, adjusting the pH value of the extracting solution to be 7.0 by using 15% sodium hydroxide solution, filtering by using a filtering membrane with the pore diameter of 0.6nm, and intercepting proteins with the molecular weight of more than 6000 daltons to obtain peptide solution with the molecular weight of 0-6000 daltons.
And e, removing sodium ions from the peptide solution through ion exchange resin to obtain a low-sodium peptide solution, and concentrating the low-sodium peptide solution by using a three-effect low-temperature concentrator to obtain a concentrated solution with 19% of solid content.
Step f, adding 5% of the mass of the concentrated solution into the concentrated solution, wherein the mass ratio is 1:1 and sucrose, and then drying the mixture at a spray drying tower operating temperature of 85 ℃.
The content of the thymus peptide is 18.03mg/ml and the content of the thymus peptide alpha 1 is 3.7 percent by the conventional method. The activity of thymus peptide is measured by adopting a T cell activity measuring method-an E-removing receptor method, the number of cells formed by E rosettes is counted to be combined with 3 or more sheep red blood cells, and a sample is diluted to 0.002mg/ml, and the activity is 36.4%.
Comparative example 1
The process of example 1 was used, except that trypsin was used in place of collagen, and the final thymosin content was 10.54mg/ml and the thymosin alpha 1 content was 1.4%. The activity of the thymus peptide was measured as above, and the sample was diluted to 0.002mg/ml with 15.5% activity.
Comparative example 2
The procedure of example 2 was used, except that phospholipase was not added, and the final thymosin content was 14.48mg/ml and the thymosin alpha 1 content was 2.1%. The activity of the thymus peptide was measured as above, and the sample was diluted to 0.002mg/ml with an activity of 18.7%.
Comparative example 3
The same procedure as in example 2 was used, except that in step f, spray drying was directly carried out without addition of skim milk and sucrose. The activity of thymus peptide is measured by adopting a T cell activity measuring method-an E-removing receptor method, the number of cells formed by E rosettes is counted to be combined with 3 or more sheep red blood cells, and the sample is diluted to 0.002mg/ml, and the activity is 20.5%.
From the above, the edible thymus peptide prepared by the method has simple operation, no toxic and side substance residue, the product is powdery, can be directly eaten, and the vitality of the product is greatly maintained and is higher than that of the thymus peptide prepared in the prior art.
Claims (6)
1. The preparation method of the edible thymus peptide is characterized by comprising the following steps of:
s1, taking calf thymus to remove throats and fat, adding purified water, and grinding into homogenate by a colloid mill to prepare raw materials for later use;
s2, adjusting the pH value to be acidic by using acid, and adding phospholipase for enzymolysis; then adding collagenase and neutral protease for enzymolysis, heating to deactivate enzyme, cooling, and removing oil and solid in the soup by a three-phase centrifuge to obtain clarified enzymolysis liquid;
s3, adding active carbon into the enzymolysis liquid, adjusting the temperature for adsorption, and removing the active carbon by a horizontal tank centrifuge to obtain clear extract;
s4, adjusting the extracting solution to be neutral by using an alkaline solution, and filtering to obtain a peptide solution with the molecular weight of 0-6000 daltons;
s5, removing sodium ions from the peptide solution through ion exchange resin to obtain a low-sodium peptide solution, and concentrating the low-sodium peptide solution by using a three-effect low-temperature concentrator to obtain a concentrated solution;
s6, adding auxiliary materials into the concentrated solution, and drying by using a spray drying tower;
in the step S2, the pH is adjusted to be acidic by acid, hydrochloric acid is adopted, the pH is adjusted to be 5.5-6.2, and phospholipase enzymolysis is carried out at the temperature of 40-48 ℃;
in the step S4, the alkaline solution is 10% -20% sodium hydroxide solution, the filtering is carried out by adopting a filtering membrane with the pore diameter of=0.6 nm, and the filtrate contains thymus peptide smaller than 6000 daltons;
in the step S2, the adding amount of the phospholipase is 0.0002-0.001 per mill of the thymus of the calf, and the enzymolysis time of the phospholipase is 30-60 min; the adding amount of the collagenase is 0.0005-0.001 per mill of the calf thymus, the adding amount of the neutral protease is 0.001-0.01 per mill of the calf thymus, and the enzymolysis time of the neutral protease is 60-150 min.
2. The method of claim 1, wherein in step S1, the ratio of thymus to purified water is 1:1 to 1:1.5.
3. the preparation method according to claim 1, wherein in step S2, the temperature is raised to 93-100 ℃, and the enzyme is inactivated for 1-5 min; cooling to 70-80 ℃ and centrifuging.
4. The preparation method according to claim 1, wherein in the step S3, the addition amount of the activated carbon is 0.01% -0.03% of the calf thymus, the temperature is adjusted to 65-80 ℃, and the activated carbon is adsorbed for 40-90 min.
5. The method according to claim 1, wherein in step S5, the solid content of the concentrated solution is 13% to 20%.
6. The preparation method of claim 1, wherein in the step S6, the auxiliary materials are skim milk and sucrose, and the mass ratio of the skim milk to the sucrose is 1-3: 1, the dosage of the auxiliary materials is 2-5% of the mass of the concentrated solution, and the temperature of the spray drying tower is 75-85 ℃.
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