JPH0581218B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing reinforced protein membranes.

In polymer science, there is a concept called cast film.
This is a casting process in which a liquid substance is poured into a mold, solidified, and the solvent is evaporated to form a film.
This is called casting, and the resulting film is called cast film. Yuba, which is a traditional Japanese food in the food field, is produced by air-drying a gel-like film obtained by heating soy milk, and is considered to fall under the category of cast film. Reports on the formation of cast films using food proteins often involve extreme conditions such as adding 20-30% glycerol or sorbitol to a protein solution, or adding aldehydes or heavy metals to form a film. Many products are manufactured in
There are problems with biodegradability and safety.

The present inventors have carried out various studies in an attempt to compensate for the above-mentioned drawbacks and develop a method for manufacturing a strong and highly safe protein membrane that can be obtained by casting natural materials under mild conditions. As a result, they discovered that a tough and stable protein film could be produced by applying transglutaminase to a solution containing a high concentration of protein and forming a cast film, thereby completing the present invention.

That is, in the present invention, an appropriate amount of acyltransferase transglutaminase (EC2, 3, 2, 13, hereinafter abbreviated as "TGase") is added to a protein-containing solution with a protein concentration of 2% by weight or more, preferably 1 g of protein.
Add at least 1 unit, pour into the formwork,
This method of producing a protein film is characterized by drying at 60° until the moisture content is 20% or less.

The transglutaminase that can be used in the present invention may not only be extracted and separated from natural products, but also be accumulated outside or inside the cells of microorganisms that produce transglutaminase by culturing them. Alternatively, it may be obtained using bioengineering techniques such as genetic engineering techniques and cell engineering techniques.

The membrane-forming protein used in the present invention is not limited by its origin, and any protein such as vegetable protein or animal protein can be used. Examples of vegetable proteins include defatted oilseed products (defatted soybeans, etc.) and proteins separated from them. Furthermore, examples of animal proteins include milk protein, gelatin, and collagen. A protein-containing solution containing 2% by weight or more of these proteins is prepared. It is desirable that the concentration of the protein-containing solution is relatively high, usually 2% by weight or more, preferably 5% by weight.
The amount may be between 15% and 15% by weight.

In this highly concentrated protein-containing solution, JP-A-58-149645
Add at least 1 unit/g of TGase to 1 g of protein, prepared by the method described in the above issue, immediately pour into a flat mold (for example, made of methacrylic resin), and heat at 10 to 60°C. ,Moisture percentage
Until it becomes 20% or less (usually 3 to 5 hours).
Air drying or blast drying yields a tough protein film that is easily peeled off from the mold. When the protein concentration is less than 2% by weight, when the amount of TGase is less than 1U per 1g of substrate protein, the drying temperature is less than 10℃ or 60℃.
If the temperature is above .degree. C., the releasability is poor and a protein film having the characteristics of the present invention cannot be obtained.

The protein membrane obtained as above is
Compared to protein films obtained by casting under similar conditions without the action of TGase, which easily dissolve in water and salt solutions, it is stable and insoluble in water and salt solutions, and has approximately twice the tensile strength. It has elongation. Furthermore, protein membranes formed by TGase can be formed in water by 22
It is a protein film that exhibits a water absorption capacity comparable to that of a ml/g protein film, and has a molecular sieve effect in organic solvents. It is also stable in boiling water and insoluble in all pH ranges. The reason why a protein film produced by TGase has such properties is that it is a protein polymer based on the formation of ε-(γ-glutamyl)lysine crosslinks by the catalytic action of TGase. 1) It must be stable against various protein denaturants (2-mercaptoethanol, sodium dodecyl sulfate, guanidine hydrochloride, urea, etc.). 2)
Even if the Lys residue, which is the reaction site of TGase, is made into a cast film using a completely acetylated protein, it is soluble in water. 3) During protein film formation by TGase, polymerized proteins are
Detected by SDS-polyacrylamide gel electrophoresis. It is proven from etc. Also, this
When a protein membrane is treated with protease using TGase, it is hydrolyzed and becomes a solution. Therefore, since it is biodegradable and the binding agent is an enzyme, the protein membrane obtained by the present invention has little effect on living organisms.

By utilizing the above properties, it can be used not only as an edible film but also as a packaging material, biodegradable membrane,
Medical polymer membrane materials, immobilized enzyme membrane substrates, etc. can be manufactured.

The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 A 5% by weight solution of α _s1 -casein was added to 0.1M Tris-
It was prepared with a hydrochloric acid buffer (5mM CaCl ₂ , 20mM dithiothreitol, 0.1% glycerol, pH 8.0). Add 0.002 units of TGase per 1 mg of α _s1 -casein to this, stir vigorously, and immediately transfer to a methacrylic resin plate (or hard vinyl plate).
Pour into the 20 x 50 x 1.5 mm mold above and heat at 40℃ for 5 minutes.
After drying with air for a period of time, an α _s1 -casein film was obtained. The obtained α _s1 -casein film had a moisture content of 11.2% and a film thickness of
47μm, tensile strength 104.7g/cm ² , elongation 82%,
It became a water-insoluble protein. Compared to that,
When α _s1 -casein membrane was prepared under the same conditions without using TGase, the moisture content was 10.5% and the membrane thickness was
A protein having a diameter of 49 μm, a tensile strength of 40.6 g/cm ² and an elongation of 38% was obtained. Tensile strength and elongation are TGase
Not only is it less than 0.5 times that when
The film was easily soluble in water.

Example 2 80 mg of α _s1 -casein membrane obtained in Example 1 was
The increase in weight of this membrane was measured over time by placing it in 100 ml of deionized water, and the water absorption amount (gwater/g membrane) was calculated.
Expressed as. The results are shown in FIG. 1, and approximately 22 g of water was absorbed per gram of α _s1 -casein membrane.

Example 3 By the same method as in Example 1, gelatin 10
Gelatin membrane prepared from wt% solution and TGase
10 mg of each gelatin film prepared under exactly the same conditions without adding any additives was placed in a 1 cm optical path length quartz cell, 3 ml of deionized water was added, and an electronic cooling temperature controller (manufactured by Shimadzu Corporation) was used. Heat the temperature from 20° to 35°C every 5°C for 10 minutes using
Absorbance at 280 nm was measured to determine the amount of dissolved protein. The results are shown in Figure 2.
For gelatin membranes that have not been treated with TGase, the absorbance increases rapidly from around 28°C and gradually dissolves. In contrast, the rate of increase in absorbance of the TGase-treated film was slow, suggesting that it was fairly stable against heat. this is,
If TGase treatment is not performed, the film is mainly composed of secondary bonds such as hydrogen bonds, so the bonds will be broken by heating.
If treated with TGase, it will gradually dissolve.
It was thought that ε-(γ-Glu)Lys crosslinking occurred, resulting in a film mainly composed of covalent bonds, making it relatively stable against heat. Furthermore, when both were immersed in boiling water, the TGase-treated membrane did not dissolve, but the TGase-free membrane did.

Example 4 Sen et al.'s method (J.Agric.Food Chem., 29 , 348
(1981)), the digestibility of the α _s1 -casein membrane obtained in Example 1 and the α _s1 -casein powder was compared. That is, 5 mg of each was added to 0.1M phosphate buffer (PH
8.0), 0.1 mg of chymotrypsin was added, and the mixture was incubated at 37°C. Chymotrypsin was inactivated and the reaction was stopped by heating the reaction solution at 100° C. for 3 minutes. The amount of amino groups in this reaction solution was determined by the Fields method (Biochem.J., 124 ,
581 (1971)), and the digestibility at each time was defined as follows.

Digestibility = amount of amino groups at the time of observation/amount of amino groups after 24 hours x 100 (%) The results are shown in Figure 3. After 24 hours, the amounts of amino groups in both were almost the same, and even if they were formed into a membrane, they could be sufficiently digested. However, when comparing the digestibility up to 120 minutes, α _s1 -casein powder is 100% digested, whereas α s1 -casein powder is digested at 100%.
With the cast film, it was approximately 60%, which was quite controlled. Therefore, protein membranes can be digested by TGase, and the rate of degradation can be controlled to some extent.

Example 5 The α _s1 -casein membrane obtained in Example 1 is stable and insoluble in organic solvents. Therefore, we investigated the permeability of this α _s1 -casein membrane in organic solvents using methylene blue (MW.373.90, ν _nax
660nm), Coomassie Brilliant Blue (MW.695.61, ν _nax 590nm), Vitamin B ₁₂
(MW.1355.42, ν _nax 560 nm).
Place the three compounds in each small test tube and add TGase.
The resulting α _s1 -casein cast membrane was sealed and immersed in a large amount of ethanol. If these compounds are released through the cast membrane,
The external solution (ethanol) takes on a color unique to each compound,
By observing the maximum absorption wavelength, it is possible to determine how much light is being transmitted through the film. 15
℃ to 40℃ at a rate of 5℃/min, then 5℃/min.
The operation of cooling from 40°C to 15°C in portions was repeated, and the transmittance of the above three types of compounds released into the external liquid for 60 minutes was determined and is shown in Figure 4. Methylene blue was penetrated relatively quickly after immersion, and completely penetrated after 40 minutes. In comparison, it was observed that about 20% of Coomassie Brilliant Blue penetrated after 60 minutes, and almost no penetration of vitamin _B12 . Therefore, when comparing the transmittance after 60 minutes, the order was methylene blue>Coomassie brilliant blue>vitamin _B12 . That is, the permeability varies depending on the molecular weight. Therefore, it was shown that the larger the molecular weight, the more difficult it is to permeate, and that there is a molecular sieve effect.

[Brief explanation of the drawing]

Figures 1, 2, 3 and 4 are Example 2, respectively.
The experimental results of 3, 4 and 5 are shown. In Figure 1, the horizontal axis is immersion time (minutes), and the vertical axis is water absorption amount (g
water/gcast film). In the diagram of FIG. 2, the horizontal axis shows temperature (°C), and the vertical axis shows absorbance at 280 nm. ● indicates the value for the gelatin membrane with TGase added, and ○ indicates the value for the gelatin membrane without the addition of TGase. In the diagram of FIG. 3, the horizontal axis shows the digestion time (minutes), and the vertical axis shows the digestibility (%).
● indicates the value of TGase-added α _s1 casein membrane, and ○ indicates the value of α _s1 casein powder. In FIG. 4, the horizontal axis represents immersion time (minutes), and the vertical axis represents transmittance (%). △ indicates methylene blue, ● indicates Coomassie brilliant blue, and ○ indicates the value of vitamin _B12 .

Claims (1)

[Claims] 1. Adding 1 unit or more of transglutaminase per 1 g of protein to a protein-containing solution with a protein concentration of 2% by weight or more, pouring into a mold, and drying at 10 to 60°C until the moisture content is 20% or less. protein membrane.