CN114773445A - Silk fibroin freeze-drying technology - Google Patents
Silk fibroin freeze-drying technology Download PDFInfo
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- CN114773445A CN114773445A CN202210512101.1A CN202210512101A CN114773445A CN 114773445 A CN114773445 A CN 114773445A CN 202210512101 A CN202210512101 A CN 202210512101A CN 114773445 A CN114773445 A CN 114773445A
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- silk fibroin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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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
Abstract
The invention relates to a silk fibroin freeze-drying technology, which comprises the steps of adding a polyethylene glycol solution into a silk fibroin solution, and then carrying out reverse dialysis to obtain a high-concentration silk fibroin solution; adding a protease solution into the mixed solution dropwise for enzymolysis; putting the solution in an environment of 75-90 ℃ for high-temperature enzyme inactivation; and (3) dropwise adding a calcium chloride solution into the obtained enzymolysis silk fibroin solution, and freeze-drying to obtain the silk fibroin freeze-dried powder. After the silk fibroin is prepared into the silk fibroin freeze-dried powder, groups existing on the end chains are easily combined with water vapor in the air to form combined water due to the properties of the silk fibroin freeze-dried powder; in the preparation process, a calcium chloride solution is dripped, free calcium ions can react with groups on the end chains in a chemical bond manner, and the free calcium ions have larger space volume, so that after combination, the influence of space resistance can be reduced, the combination of the vacant end chains and water vapor in the air to form combined water can be reduced, in addition, the calcium ions are necessary substances for human bodies, and the biodegradability is better.
Description
Technical Field
The invention relates to a silk fibroin processing method, in particular to a silk fibroin freeze-drying technology.
Background
Silk fibroin, also called as fibroin, is a natural polymer fibrin extracted from silk, has a content of about 70-80% of silk, and contains various amino acids, wherein glycine (Gly), alanine (Ala), and serine (Ser) account for about more than 80% of the total composition; since silk fibroin has good mechanical properties and physicochemical properties such as good flexibility, tensile strength, air and moisture permeability, slow release property and the like, and different forms such as fibers, solutions, powders, films, gels and the like can be obtained through different treatments, silk fibroin has been well applied to various fields in recent years, such as the field of biomedicine.
The silk fibroin has stable property and can exist in various forms, such as silk fibroin solution, silk fibroin gel and the like, while the silk fibroin lyophilized powder is more extensive in the actual production and use process, because compared with the silk fibroin solution and the silk fibroin gel, the silk fibroin lyophilized powder has light weight, good transportation performance and high content of active ingredients; however, silk fibroin powder has its own disadvantages, and since there are many hydrophilic groups in the peripheral end chains of silk fibroin, it is easily condensed with water vapor in the air to form a moisture regain phenomenon.
Disclosure of Invention
The invention aims to provide a silk fibroin freeze-drying technology.
The technical purpose of the invention is realized by the following technical scheme: a silk fibroin freeze-drying technology comprises the following preparation steps:
step 1: extracting silk fibroin to obtain a clean silk fibroin solution;
step 2: adding a polyethylene glycol solution into the silk fibroin solution obtained in the step 1, and performing reverse dialysis to obtain a high-concentration silk fibroin solution;
and step 3: adjusting the pH value of the mixed solution in the step 2 to 7-9, dropwise adding a protease solution into the mixed solution, and placing the mixed solution in an environment of 40-48 ℃ for enzymolysis for 1-3 hours;
and 4, step 4: putting the solution in the step 3 in an environment of 75-90 ℃ for high-temperature enzyme inactivation;
and 5: dropwise adding a calcium chloride solution into the enzymolysis silk fibroin solution obtained in the step 4, and placing the solution in an environment at the temperature of 75-90 ℃ for 5-40 minutes;
step 6: and (5) concentrating the mixed liquid in the step (5), and freeze-drying to obtain the silk fibroin freeze-dried powder.
By adopting the technical scheme, after the silk fibroin is prepared into the silk fibroin freeze-dried powder, groups existing on the end chains are easy to combine with water vapor in the air to form combined water due to the properties of the silk fibroin freeze-dried powder, namely the silk fibroin freeze-dried powder is often damp; in the preparation process, the calcium chloride solution is dripped, free calcium ions can react with groups on the end chains in a chemical bond mode, and the free calcium ions are large in space volume and are affected by space resistance after combination, so that the phenomenon that the vacant end chains are combined with water vapor in the air to form combined water can be reduced, in addition, the calcium ions are substances necessary for a human body, and the biodegradability is good.
Preferably, the method further comprises the following steps:
step 7.1: placing the silk fibroin freeze-dried powder prepared in the step 6 in an environment at the temperature of lower than 10 ℃, and introducing clean air at the temperature of higher than 10 ℃ for 1-3 minutes;
step 7.2: repeating the above steps for multiple times;
step 7.3: and (3) freeze-drying the silk fibroin freeze-dried powder in the step (2) again.
Preferably, the freezing temperature in the step 6 is-1 ℃ to 2 ℃.
Preferably, the mixed liquid in the step 5 is concentrated to 8% -15% of the original solution in the step 6.
Preferably, the step 6 is performed by reverse dialysis concentration or reduced pressure concentration.
Preferably, the air in step 7.1 comprises inert gas, hydrogen gas and water vapor.
Preferably, the temperature of the clean air in step 7.1 is 15-25 ℃.
Preferably, the moisture content is less than 5%.
Preferably, the inert gas is one or more of nitrogen, nitrogen dioxide, helium and argon.
In conclusion, the invention has the following beneficial effects:
1. after the silk fibroin is prepared into the silk fibroin freeze-dried powder, due to the nature of the silk fibroin freeze-dried powder, groups existing on a terminal chain are easy to combine with water vapor in the air to form combined water, namely the combined water is frequently moistened; in the preparation process, the calcium chloride solution is dripped, free calcium ions can react with groups on the end chains in a chemical bond mode, and the free calcium ions are large in space volume and are affected by space resistance after combination, so that the phenomenon that the vacant end chains are combined with water vapor in the air to form combined water can be reduced, in addition, the calcium ions are substances necessary for a human body, and the biodegradability is good.
Detailed Description
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Example 1:
a silk fibroin freeze-drying technology comprises the following preparation steps:
step 1: extracting silk fibroin to obtain a clean silk fibroin solution;
and 2, step: adding a polyethylene glycol solution into the silk fibroin solution obtained in the step (1) and then carrying out reverse dialysis to obtain a high-concentration silk fibroin solution;
and step 3: after the pH value of the mixed solution in the step 2 is adjusted to be 7-9, adding a protease solution into the mixed solution dropwise, and placing the mixed solution in an environment of 40-48 ℃ for enzymolysis for 1-3 hours; in the embodiment, the pH value is adjusted to 7, and the mixture is placed in an environment of 47 ℃ for enzymolysis for 1.5 hours;
and 4, step 4: placing the solution in the step 3 in an environment of 75-90 ℃ for high-temperature enzyme deactivation;
and 5: dropwise adding a calcium chloride solution into the enzymolysis silk fibroin solution obtained in the step (4), and placing in an environment of 75-90 ℃ for 5-40 minutes;
step 6: and (5) concentrating the mixed liquid in the step (5), and freeze-drying to obtain the silk fibroin freeze-dried powder.
Further comprising the steps of:
step 7.1: placing the silk fibroin freeze-dried powder prepared in the step 6 in an environment at the temperature of lower than 10 ℃, and introducing clean air at the temperature of higher than 10 ℃ for 1-3 minutes;
and 7.2: repeating the steps for many times, wherein the temperature of the clean air is 18 ℃, and the duration time of each time is 1 minute and lasts for three times;
step 7.3: and (3) freeze-drying the silk fibroin freeze-dried powder in the step (2) again.
The freezing temperature in step 6 was-1 ℃ to 2 ℃ and in this example was 2 ℃.
And 6, concentrating the mixed liquid in the step 5 to 8-15% of the original solution.
And step 6, concentration by reverse dialysis or reduced pressure concentration is adopted.
The air in the step 7.1 comprises inert gas, hydrogen and water vapor, wherein the content of the inert gas accounts for 80%, the content of the hydrogen accounts for 15%, and the content of the hydrogen dioxide accounts for 5%.
The clean air temperature in step 7.1 is 15-25 ℃.
The moisture content is less than 5%.
The inert gas is one or more of nitrogen, nitrogen dioxide, helium and argon.
The working principle is as follows:
after the silk fibroin is prepared into the silk fibroin freeze-dried powder, due to the nature of the silk fibroin freeze-dried powder, groups existing on a terminal chain are easy to combine with water vapor in the air to form combined water, namely the combined water is frequently moistened; in the preparation process, the calcium chloride solution is dripped, free calcium ions can react with groups on the end chains in a chemical bond mode, and the free calcium ions are large in space volume and are affected by space resistance after combination, so that the phenomenon that the vacant end chains are combined with water vapor in the air to form combined water can be reduced, in addition, the calcium ions are substances necessary for a human body, and the biodegradability is good.
Example 2:
a silk fibroin freeze-drying technology comprises the following preparation steps:
step 1: extracting silk fibroin to obtain a clean silk fibroin solution;
and 2, step: adding a polyethylene glycol solution into the silk fibroin solution obtained in the step (1) and then carrying out reverse dialysis to obtain a high-concentration silk fibroin solution;
and 3, step 3: after the pH value of the mixed solution in the step 2 is adjusted to be 7-9, adding a protease solution into the mixed solution dropwise, and placing the mixed solution in an environment of 40-48 ℃ for enzymolysis for 1-3 hours; in the embodiment, the pH value is adjusted to be 9, and the mixture is placed in an environment of 47 ℃ for enzymolysis for 3 hours;
and 4, step 4: placing the solution in the step 3 in an environment of 75-90 ℃ for high-temperature enzyme deactivation;
and 5: dropwise adding a calcium chloride solution into the enzymolysis silk fibroin solution obtained in the step (4), and placing in an environment of 75-90 ℃ for 5-40 minutes;
step 6: and (5) concentrating the mixed liquid in the step (5), and freeze-drying to obtain the silk fibroin freeze-dried powder.
Further comprising the steps of:
step 7.1: placing the silk fibroin freeze-dried powder prepared in the step 6 in an environment at the temperature of lower than 10 ℃, and introducing clean air at the temperature of higher than 10 ℃ for 1-3 minutes;
and 7.2: repeating the steps for many times, wherein the temperature of the clean air is 25 ℃, and the duration time of each time is 1 minute and lasts for three times;
step 7.3: and (3) freeze-drying the silk fibroin freeze-dried powder in the step (2) again.
The freezing temperature in step 6 was-1 ℃ to 2 ℃ and-1 ℃ in this example.
And 6, concentrating the mixed liquid in the step 5 to 8-15% of the original solution.
In step 6, concentration by reverse dialysis or concentration under reduced pressure is adopted.
The air in the step 7.1 comprises inert gas, hydrogen and water vapor, wherein the content of the inert gas accounts for 80%, the content of the hydrogen accounts for 15%, and the content of the hydrogen dioxide accounts for 5%.
The clean air temperature in step 7.1 is 15-25 ℃.
The moisture content is less than 5%.
The inert gas is one or more of nitrogen, nitrogen dioxide, helium and argon.
Claims (9)
1. A silk fibroin freeze-drying technology is characterized by comprising the following preparation steps:
step 1: extracting silk fibroin to obtain a clean silk fibroin solution;
step 2: adding a polyethylene glycol solution into the silk fibroin solution obtained in the step 1, and performing reverse dialysis to obtain a high-concentration silk fibroin solution;
and step 3: after the pH value of the mixed solution in the step 2 is adjusted to be 7-9, adding a protease solution into the mixed solution dropwise, and placing the mixed solution in an environment of 40-48 ℃ for enzymolysis for 1-3 hours;
and 4, step 4: putting the solution in the step 3 in an environment of 75-90 ℃ for high-temperature enzyme inactivation;
and 5: dropwise adding a calcium chloride solution into the enzymolysis silk fibroin solution obtained in the step 4, and placing the solution in an environment at the temperature of 75-90 ℃ for 5-40 minutes;
and 6: and (5) concentrating the mixed liquid in the step (5), and freeze-drying to obtain the silk fibroin freeze-dried powder.
2. The silk fibroin lyophilization technique of claim 1, further comprising the steps of:
step 7.1: placing the silk fibroin freeze-dried powder prepared in the step 6 in an environment lower than 10 ℃, and introducing clean air with the temperature higher than 10 ℃ into the environment for 1-3 minutes;
and 7.2: repeating the above steps for multiple times;
step 7.3: and (3) freeze-drying the silk fibroin freeze-dried powder in the step (2) again.
3. The silk fibroin lyophilization technique of claim 1, wherein: the freezing temperature in the step 6 is-1-2 ℃.
4. The silk fibroin lyophilization technique of claim 1, wherein: and in the step 6, the mixed liquid in the step 5 is concentrated to 8-15% of the original solution.
5. The silk fibroin lyophilization technique of claim 1, wherein: and in the step 6, reverse dialysis concentration or reduced pressure concentration is adopted.
6. The silk fibroin lyophilization technique of claim 2, wherein: the air in step 7.1 comprises inert gas, hydrogen and water vapor.
7. The silk fibroin lyophilization technique of claim 2, wherein: the temperature of the clean air in step 7.1 is 15-25 ℃.
8. The silk fibroin lyophilization technique of claim 6, wherein: the moisture content is less than 5%.
9. The silk fibroin lyophilization technique of claim 6, wherein: the inert gas is one or more of nitrogen, nitrogen dioxide, helium and argon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210512101.1A CN114773445A (en) | 2022-05-12 | 2022-05-12 | Silk fibroin freeze-drying technology |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210512101.1A CN114773445A (en) | 2022-05-12 | 2022-05-12 | Silk fibroin freeze-drying technology |
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| CN202210512101.1A Pending CN114773445A (en) | 2022-05-12 | 2022-05-12 | Silk fibroin freeze-drying technology |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990074787A (en) * | 1998-03-14 | 1999-10-05 | 김강권 | Method for preparing silk powder peptide by enzymatic digestion |
| KR100961470B1 (en) * | 2009-08-11 | 2010-06-08 | 주식회사 브레인가드 | Manufacturing method of silk peptide by enzyme resolve |
| CN103910789A (en) * | 2014-03-27 | 2014-07-09 | 苏州丝美特生物技术有限公司 | Preparation process of high molecular weight silk fibroin freeze-dried powder |
| CN107445191A (en) * | 2017-10-12 | 2017-12-08 | 广西碳酸钙产业化工程院有限公司 | A kind of preparation method of spherical calcium carbonate |
-
2022
- 2022-05-12 CN CN202210512101.1A patent/CN114773445A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990074787A (en) * | 1998-03-14 | 1999-10-05 | 김강권 | Method for preparing silk powder peptide by enzymatic digestion |
| KR100961470B1 (en) * | 2009-08-11 | 2010-06-08 | 주식회사 브레인가드 | Manufacturing method of silk peptide by enzyme resolve |
| CN103910789A (en) * | 2014-03-27 | 2014-07-09 | 苏州丝美特生物技术有限公司 | Preparation process of high molecular weight silk fibroin freeze-dried powder |
| CN107445191A (en) * | 2017-10-12 | 2017-12-08 | 广西碳酸钙产业化工程院有限公司 | A kind of preparation method of spherical calcium carbonate |
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