CN111662555A

CN111662555A - Method for preparing silk protein film

Info

Publication number: CN111662555A
Application number: CN201910170715.4A
Authority: CN
Inventors: 李松泰; 林楠凯; 蔡国龙
Original assignee: Daileng Technology Co ltd; Taichuang Biotechnology Co ltd; Southern Taiwan University of Science and Technology
Current assignee: Daileng Technology Co ltd; Taichuang Biotechnology Co ltd; Southern Taiwan University of Science and Technology; Univacco Tech Inc
Priority date: 2019-03-07
Filing date: 2019-03-07
Publication date: 2020-09-15

Abstract

A method for preparing fibroin membrane comprises film forming step. In the film forming step, a heating process is applied to the mixture provided in the atmosphere. The mixture comprises a fibroin aqueous solution and a polyhydroxy compound, wherein the fibroin aqueous solution comprises fibroin and water, and the polyhydroxy compound accounts for 20 wt% to 60 wt% of the total amount of the fibroin and the polyhydroxy compound being 100 wt%. In the heat treatment, the temperature is controlled to 60 ℃ to 170 ℃, and the relative humidity of the environment is controlled to be below 30% during the heat treatment. By controlling the temperature to 60 ℃ to 170 ℃ and the relative humidity to be less than 30%, the method for manufacturing the fibroin film has the characteristic of short film forming time, and particularly can obtain the fibroin film within 30 seconds to 30 minutes, so that the production efficiency can be improved.

Description

Method for preparing silk protein film

Technical Field

The invention relates to a method for manufacturing a membrane, in particular to a method for manufacturing a fibroin membrane.

Background

Plastic products, such as plastic wrap, plastic cup, plastic bag, or plastic tube, etc., are widely used by people, but due to the extensive abuse, the plastic products pose serious garbage problems, especially the plastic products which are not incineratable or biologically and difficultly decomposed, and the environmental harm degree is quite large.

With the rising of environmental awareness, natural polymer films, such as silk fibroin films, have superior physical properties and easily biodegradable properties compared with plastic films, and can be used for replacing plastic products, thereby solving the problem that the plastic products are difficult to decompose or generate toxic substances such as dioxin during burning.

The preparation method of the silk fibroin membrane comprises the steps (a) to (c). In the step (a), silk is sequentially subjected to sericin removal treatment, dissolution treatment and dialysis treatment to obtain a silk fibroin aqueous solution. In the step (b), the silk fibroin aqueous solution is coated on a carrier and is allowed to evaporate naturally for 12 to 16 hours to form a water-soluble silk fibroin film on the carrier. In the step (c), a conversion treatment is applied to the water-soluble silk fibroin film to convert the water-soluble silk fibroin film into a water-insoluble silk fibroin film. There are two ways to this conversion process. In one embodiment, the water-soluble silk fibroin film is placed in a vacuum dryer comprising a water tray containing water. Then, an air extractor connected to the vacuum dryer is started to extract air from the inside of the vacuum dryer to generate steam, and at this time, the water-soluble silk fibroin film is subjected to a one-day moistening (annealing) treatment with steam, so that the configuration of the silk fibroin in the water-soluble silk fibroin film is converted from random coil (random coil) to a stable silk I structure [ including α -helix (α -helix) and β -turn (β -turn) ], thereby obtaining the water-insoluble silk fibroin film. In another embodiment, the water-soluble silk fibroin film is immersed in a solution containing an alcohol compound such as methanol or ethanol at a concentration of 95 wt% or more for one day to convert random coils into β -sheets (β -sheets).

Although the silk fibroin film preparation method can obtain the water-soluble silk fibroin film and the water-insoluble silk fibroin film, the film forming time of the water-soluble silk fibroin film and the water-insoluble silk fibroin film is at least over 12 hours, and the problem of poor production efficiency exists. Furthermore, the water-insoluble fibroin films obtained by any of the above-described conversion treatments have problems of being wrinkled, having high surface hardness and poor flexibility, and having low ductility and being easily broken by stretching. The water-insoluble silk fibroin film obtained by any of the above-described conversion treatments is stored in water, and is flexible, but is more hardened after drying. Therefore, the water-insoluble fibroin films obtained by any of the above-mentioned conversion treatments have problems of poor flexibility and ductility when used as a wrap film, and are not easily attached to the surface of an object to be wrapped (for example, tableware such as bowls and dishes). In addition, the silk fibroin film is prepared by coating or spraying aqueous solution of silk fibroin on paper carrier to form water-soluble silk fibroin film, and can not be used as waterproof film on paper product (such as paper tableware, paper cup, or paper straw). The water-soluble silk fibroin film on the outer layer or the inner layer of the paper product is moistened by water vapor or by soaking in a solution containing a high concentration of alcohol compounds, so that the paper product is deformed due to water absorption.

Disclosure of Invention

The invention aims to provide a method for manufacturing a fibroin film, which has short film forming time and improves the production efficiency. The silk protein film prepared by the method has good flexibility and ductility.

The method for manufacturing the fibroin film comprises a film forming step. In the film forming step, a mixture disposed in an environment is subjected to a heating treatment, wherein the mixture includes an aqueous fibroin solution and a polyol, and the aqueous fibroin solution includes fibroin and water, and the amount of the polyol is in the range of 20 wt% to 60 wt% with the total amount of the fibroin and the polyol being 100 wt%. In the heat treatment, the temperature is controlled to 60 ℃ to 170 ℃, and the relative humidity of the environment is controlled to be below 30% during the heat treatment.

The method for producing a fibroin film of the present invention comprises a step of preparing a fibroin film.

The method for manufacturing the fibroin film of the present invention is such that the amount of the polyhydroxy compound is in the range of 20 wt% to 50 wt% based on 100 wt% of the total amount of the fibroin and the polyhydroxy compound.

The method for manufacturing the fibroin film of the present invention is such that the amount of the polyhydroxy compound is in the range of 25 wt% to 40 wt% based on 100 wt% of the total amount of the fibroin and the polyhydroxy compound.

The method for producing a fibroin film of the present invention is characterized in that the polyol is selected from the group consisting of glycerol, monosaccharides, disaccharides, and combinations thereof.

The temperature of the silk protein film manufacturing method is controlled to be 100-120 ℃.

The relative humidity of the silk protein film manufacturing method is controlled to be below 10%.

The method for producing a fibroin film of the present invention further comprises a pretreatment step before the film formation step, wherein the mixture is applied to a support in the pretreatment step.

The method for producing a fibroin film of the present invention further comprises a pretreatment step before the film formation step, wherein the mixture is applied onto a support by spraying.

The invention has the beneficial effects that: by controlling the temperature to 60 ℃ to 170 ℃ and the relative humidity to be less than 30%, the method for manufacturing the fibroin film has the characteristic of short film forming time, and can especially obtain the fibroin film within 30 seconds to 30 minutes, thereby improving the production efficiency. And the silk protein film obtained by the silk protein film manufacturing method contains the polyhydroxy compound through the existence of the polyhydroxy compound in the film forming step, thereby having good flexibility and ductility.

Detailed Description

The present invention will be described in detail below.

[ mixture ]

< aqueous fibroin solution >

The silk protein comprises silk fibroin. Such silk fibroin is not limited to random coil (random coil) type silk fibroin, or alpha-helix (alpha-helix) type silk fibroin, and the like.

< polyhydroxy Compound >

When the silk fibroin is a random-crimp type silk fibroin, the use of the polyol helps to transform the configuration of the silk fibroin from random-crimp to α -helix (α -helix) or β -sheet (β -sheet) to obtain a water-insoluble silk fibroin film. In addition, by the presence of the polyhydroxy compound at the film forming step, the process and time required for the conversion treatment in the past can be eliminated, thereby enabling the productivity to be improved. The polyol has the following characteristics: can be mutually soluble and difficult volatile with this fibroin aqueous solution, and this polyol's hydroxyl can increase the probability of fibroin's the inside hydrogen bond that forms of peptide chain to fold into the configuration of alpha-spiral or beta-folded plate easily, moreover, can increase the torsion space between the fibroin molecule, and regard as plasticizer (plastizer), in order to promote the pliability and the ductility of fibroin membrane.

The polyol may be used singly or in combination of plural kinds, and the polyol is, for example, but not limited to, glycerin, a monosaccharide, a polysaccharide or the like. Such as, but not limited to, glucose. Such as but not limited to disaccharide materials. Such as, but not limited to trehalose. In some embodiments of the invention, the polyol is glycerol or trehalose. In order to provide the fibroin film with better flexibility, extensibility, transparency and lack of sticky feel, in some embodiments of the present invention, the amount of the polyol ranges from 20 wt% to 50 wt% based on 100 wt% of the total amount of the fibroin and the polyol. Further, the amount of the polyhydroxy compound ranges from 25 wt% to 40 wt% based on 100 wt% of the total amount of the silk protein and the polyhydroxy compound.

< Environment and Heat treatment >

To make the production efficiency better, in some embodiments of the present invention, the temperature is controlled to be 100 ℃ to 120 ℃ in the heating process. To improve the production efficiency, in some embodiments of the invention, the relative humidity of the environment is controlled to be less than 10%.

The method for manufacturing the fibroin film further comprises a pretreatment step before the film forming step.

< pretreatment step >

In the pretreatment step, the mixture is coated or laminated on a carrier. Examples of the coating method include, but are not limited to, spray coating (spraying coating) and spin coating (spinning coating). The carrier is not limited to, for example, a plastic carrier, a silica gel carrier, a paper carrier, a metal carrier, or the like.

< fibroin Membrane >

The fibroin film has the characteristics of biodegradability, transparency, heat resistance, flexibility, ductility and the like, still has flexibility in the environment of-80 ℃, and can be applied to a freezing environment. In the method for manufacturing the silk protein film, the mixture comprises the silk protein aqueous solution and the polyhydroxy compound, and the formed silk protein film has water insoluble property and flexibility.

The fibroin film can be used as a wrap film or a container (such as a reusable bag) or the like. In addition, the fibroin film can also be used as a waterproof film for an outer layer or an inner layer on paper products (such as paper tableware, paper cups, paper straws and the like) because the fibroin film has water-insoluble characteristics.

The invention will be further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the practice of the invention.

Preparation example 1 aqueous fibroin solution

Sequentially carrying out sericin removal treatment, dissolution treatment, first dialysis treatment and second dialysis treatment on the silkworm cocoons.

In the sericin removal treatment, silkworm cocoons are cut into pieces, and put into a boiling aqueous solution of sodium carbonate (containing water and sodium carbonate, and the concentration of the sodium carbonate is 0.02M) to be boiled for 30 minutes, so that sericin on the fibers of the silkworm cocoons is dissolved in the sodium carbonate solution and separated from the silkworm cocoons, and a mixture is obtained. The mixture comprises a sodium carbonate solution, dissolved sericin and silk fibroin fibers floating in the sodium carbonate solution. And fishing out the silk fibroin fibers from the mixture, washing the silk fibroin fibers for three times by reverse osmosis water, squeezing the silk fibroin fibers, and drying the silk fibroin fibers in an oven with the temperature set at 37 ℃ to form the dried silk fibroin fibers.

In the dissolution treatment, 5 g of the dried silk fibroin fibers were put in a container containing 20 ml of a lithium bromide solution (containing water and lithium bromide, and having a concentration of 9.3M). The vessel was placed in an oven at a temperature of 60 ℃ for 4 hours to dissolve the silk fibroin fibers in the lithium bromide solution by contacting the silk fibroin fibers with the lithium bromide solution, thereby obtaining a silk fibroin-containing solution.

In the first dialysis treatment, 23 ml of the solution containing silk fibroin is injected into a dialysis bag with a dialysis pore size of 3.5kDa and placed in a container with a volume of more than 2 l, a magnet for stirring is arranged in the container, and every 12 ml of the solution containing silk fibroin corresponds to 1 l of deionized water, then the container is placed on a stirrer, the stirrer drives the magnet for stirring in the container to rotate, so that the deionized water in the container flows to perform the first dialysis treatment to remove the lithium bromide, thereby obtaining a first solution of silk fibroin, wherein 48 hours are passed and water is replaced six times in total.

In the second dialysis treatment, a dialysis bag containing the first silk fibroin solution after the first dialysis treatment was put into 1 liter of a 10% (wt/vol) aqueous solution of polyethylene glycol (m.w. 10,000) to be subjected to dialysis treatment for 20 hours, to obtain a second silk fibroin solution in which the silk fibroin concentration was 15 wt%. Then, secondary deionized water was added to adjust the concentration of silk fibroin to 8 wt%, to obtain an aqueous fibroin solution.

Preparation example 2

The preparation method of the aqueous fibroin solution of preparation example 2 of the present invention is similar to that of preparation example 1, except that: adding secondary deionized water to the second silk fibroin solution to adjust the concentration of silk fibroin to 10 wt% to obtain an aqueous silk fibroin solution.

Example 1

1 ml of the silk fibroin aqueous solution of preparation example 1 (including 80 mg of silk fibroin and 920 mg of water) having a concentration of 8 wt% was mixed with 27 mg of glycerol to form a mixture, wherein the glycerol was used in an amount of 25.2 wt% based on 100 wt% of the sum of the amounts of the silk fibroin and the glycerol. The mixture is subjected to a pretreatment step and a film formation step in this order. In the pretreatment step, the mixture is spray coated on a polyethylene terephthalate carrier to form a coating layer on the polyethylene terephthalate carrier, thereby obtaining a laminate. In the film forming step, the laminated body is placed in a cavity which has a temperature of 110 ℃ and is connected with an air pump, and then a heating treatment is applied to the laminated body, wherein the temperature of the heating treatment is set to be 110 ℃. During the heat treatment, the air pump is started to control the relative humidity of the chamber to be 10%. After 5 minutes, a heated laminate was formed, wherein the heated laminate comprised the polyethylene terephthalate carrier and a fibroin film formed on the polyethylene terephthalate carrier, wherein the fibroin film had a moisture content of 19%. Then, the heated laminate is taken out from the cavity and cooled to room temperature (25-30 ℃). No water stagnation trace exists in the appearance of the heated laminated body, and no hole is formed after the silk protein film is separated from the polyethylene terephthalate carrier, namely the film forming is finished.

Examples 2 to 7

The preparation method of the fibroin films of examples 2 to 7 of the present invention is similar to that of the fibroin film of example 1, except that: the aqueous fibroin solution of preparation example 2, the amount of glycerin used was 34 mg, and the temperature of the heat treatment in the film formation step were used, see table 1.

Examples 8 to 9

The preparation method of the fibroin films of examples 8 to 9 of the present invention is similar to that of example 3, except that: the amount of glycerol was varied, see tables 1 and 2.

Example 10

The preparation method of the fibroin film of example 10 of the present invention is similar to that of the fibroin film of example 3, except that: trehalose was substituted for glycerol, see table 2.

Example 11

The preparation method of the fibroin film of example 11 of the present invention is similar to that of the fibroin film of this example 8, except that: the pretreatment step is to apply the mixture to a paper sheet as a carrier by spin coating to form a coating layer on the paper sheet to obtain a laminate.

Comparative example 1

The fibroin film of comparative example 1 was prepared in a similar manner to that of example 3, except that: the mixture contained no glycerol, see table 2.

Comparative example 2

The fibroin film of comparative example 2 was prepared in a similar manner to that of example 8, except that: in comparative example 2, the temperature of the heat treatment in the film formation step was 50 ℃.

Comparative example 3

The fibroin film of comparative example 3 was prepared in a similar manner to that of example 8, except that: in comparative example 3, the temperature of the heat treatment in the film formation step was 180 ℃. The fibroin film of comparative example 3 had a problem that the coating was not easily uniform and the thickness was not uniform.

Evaluation item

And (3) transparency measurement: the silk protein films of examples 1 to 10 and comparative examples 1 to 3 were measured for transmittance using a multi-functional spectrometer (brand: cloud technology; model: TRF 2006). The scanning wavelength range is 200nm to 850 nm.

Heat resistance temperature: the fibroin films of examples 1 to 10 and comparative examples 1 to 3 were measured with reference to the heat temperature resistance indication compliance test method (MOHWU0023.02) of plastic-based food appliance container packages and taiwan standard total number 2446 test method (CNS2446, published year: 2017, 11 months and 13 days). When the temperature was measured at 200 ℃ by the heat resistance temperature compliance test method (MOHWU0023.02) for plastic food container packages, the double-sided tape as a fixing material was melted and failed to exert a fixing effect, and therefore the heat resistance temperature was measured up to 185 ℃.

Elongation (tensile strength) measurement: in order to clearly describe the measurement process, the fibroin film of example 1 is described below, and the fibroin films of examples 2 to 10 and comparative examples 1 to 3 are measured in this manner. The fibroin film of example 1 was cut into test samples having a length of 12.5 cm and a width of 3 cm, and 3 samples were prepared in total. The sample to be measured was measured by a tensile testing machine (brand name: high and high universal materials testing machine; model number: H35), and the measurement conditions were as follows: the moving speed was 0.2 mm/min. The elongation (%) [ (L2-L1)/L1] × 100%, where L1 represents the original length and L2 represents the stretched length at break of the sample to be measured.

Measuring the water content: the coatings of examples 1 to 10 and comparative examples 1 to 2 were weighed with a precision microbalance (brand: SHIMADZU; model: AX120) to obtain a weight A. Then, the sample was placed in an oven set at 65 ℃ for 16 hours, and then, taken out of the oven and weighed with the precision microbalance to obtain a weight B. The water content was [ (a-B)/a ] × 100%.

TABLE 1

TABLE 2

The silk protein films obtained by the methods for manufacturing silk protein films of examples 1 to 10 of the present invention had excellent elongation, compared to the method for manufacturing silk protein films of comparative example 1 in table 2, in which glycerin was not used, indicating that the method for manufacturing silk protein films of the present invention can indeed obtain silk protein films having good flexibility and ductility. Further, from the experimental results of the methods for manufacturing silk protein films of comparative examples 2 and 3 in table 2, it was found that the method for manufacturing silk protein films could not obtain silk protein films within 30 seconds to 30 minutes and could achieve uniformity of the thickness of the obtained silk protein films, and on the contrary, the methods for manufacturing silk protein films of examples 1 to 11 could obtain silk protein films within 30 seconds to 30 minutes and could achieve uniformity of the thickness of the obtained silk protein films.

In summary, the present invention controls the temperature to 60 ℃ to 170 ℃ and the relative humidity to 30% or less, the method for manufacturing the silk protein film has the characteristic of short film forming time, and particularly, the silk protein film can be formed in 30 seconds to 30 minutes, so that the production efficiency can be improved, and the silk protein film obtained by the method for manufacturing the silk protein film contains the polyhydroxy compound in the presence of the polyhydroxy compound in the film forming step, so that the silk protein film has good flexibility and ductility, and thus the object of the present invention can be achieved.

Claims

1. A method for preparing fibroin membrane; it is characterized by comprising:

a film forming step of applying heat treatment to a mixture disposed in an environment, wherein the mixture includes a fibroin aqueous solution and a polyol, the fibroin aqueous solution includes fibroin and water, and the amount of the polyol ranges from 20 wt% to 60 wt% based on 100 wt% of the total amount of the fibroin and the polyol; in the heat treatment, the temperature is controlled to 60 ℃ to 170 ℃, and the relative humidity of the environment is controlled to be below 30% during the heat treatment.

2. The method for producing a fibroin film according to claim 1, characterized in that: the silk protein comprises silk fibroin.

3. The method for producing a fibroin film according to claim 1, characterized in that: the amount of the polyhydroxy compound ranges from 20 wt% to 50 wt% based on the total amount of the silk protein and the polyhydroxy compound as 100 wt%.

4. The method for producing a fibroin film according to claim 3, characterized in that: the amount of the polyhydroxy compound ranges from 25 wt% to 40 wt% based on the total amount of the silk protein and the polyhydroxy compound as 100 wt%.

5. The method for producing a fibroin film according to claim 1, characterized in that: the polyol is selected from glycerol, monosaccharides, disaccharides, or any combination thereof.

6. The method for producing a fibroin film according to claim 1, characterized in that: the temperature is controlled at 100 ℃ to 120 ℃.

7. The method for producing a fibroin film according to claim 1, characterized in that: the relative humidity was controlled to 10% or less.

8. The method for producing a fibroin film according to claim 1, characterized in that: the method for manufacturing the fibroin film further comprises a pretreatment step before the film forming step, wherein the mixture is coated on a carrier in the pretreatment step.

9. The method for producing a fibroin film according to claim 1, characterized in that: also comprises a pretreatment step before the film forming step, in the pretreatment step, the mixture is coated on a carrier.