JPH0286741A - Capsule produced by using modified protein as base material - Google Patents

Abstract

PURPOSE:To obtain the subject capsule having excellent elasticity, heat- resistance, solvent resistance, etc., by using a protein modified with transglutaminase as a base material. CONSTITUTION:An aqueous solution or emulsion of a vegetable protein of e.g., soybean or an animal protein of fish, animal meat, etc., having a concentration of 1-30% is added with a preferably 10-100ug-protein of a transglutaminase (TGase) produced by a microorganism such as Streptoverticillium griseocarneum IFO 12776 and reacted at 5-90 deg.C for 1sec-24hr to obtain an aqueous solution, etc., of a protein added with TGase. The solution is added with a seasoning, oil and fat, a plasticizer, etc., injected into a layer of an oil (e.g., corn oil) in the case of the preparation of a single capsule, optionally incubated at 5-60 deg.C for 30sec-24hr while keeping the form of the granular droplet, maintained at 90 deg.C for 10min and separated from the oil layer to obtain the objective capsule.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to capsules based on transglutaminase-denatured proteins.

(Prior art and its problems) Protein-based encapsulation is known, but in this case, conventionally only gelatin was used as the protein, and it is not necessarily suitable for a wide range of purposes. Ta. Furthermore, even when gelatin is used as a base material, chemical cross-linking agents and ultraviolet rays are used to impart heat resistance, water resistance, oil resistance, and freeze resistance, so it is not preferred from a safety perspective for food and pharmaceutical applications. , and the manufacturing temperature is approximately 30℃.
It is limited as follows.

In particular, the protein-based capsule contains transglutaminase (hereinafter referred to as T).
It is sometimes abbreviated as "Gase". ) There are no examples of using denatured proteins.

(Means for solving the problems and their effects) As a result of intensive research to solve the above problems, the present inventors have discovered that various proteins can be produced by utilizing the cross-linking polymerization ability and gelation ability of transglutaminase. The present invention was completed by discovering that the above problems can be solved by modification.

According to the present invention, it is possible to easily encapsulate various proteins other than piratin, which was previously used as a base protein, and the production temperature can also be expanded to a wider range than before, that is, from about 5 to 90°C. . Furthermore, since it can be prepared without using a chemical crosslinking agent, it can be used as food and medicine from the viewpoint of safety. The capsule m of the present invention can be used in a wide range of conventional chemical products.

Hereinafter, the Kabubiru of the present invention and its WIJ manufacturing method will be explained in detail.

The capsules of the present invention can be single capsules, double capsules, etc. Its size depends on its use in food, medicine, chemical products, etc. In addition, it also differs depending on the type of food. (Takashi Kondo [Development of new soft capsule foods and their applications 1 Food and development Vo I, 23. N
13 (Showa 63)). For example, when considering use in foods, the length is 1 to 10 seconds for fish roe-like capsules, and 5 to 30 meters for vitamin-containing capsules.

As the protein to be used, any protein-containing protein can be used, such as plant-based products such as soybean, wheat, cottonseed, and corn, and animal-based products such as fish meat, livestock meat, krill, and milk. These proteins are denatured and used by the action of transglutamine.

Transglutaminose can be produced regardless of its origin, for example, from guinea pig liver (hereinafter referred to as MTG).
(sometimes abbreviated as ase), and those produced by microorganisms (hereinafter sometimes abbreviated as BTG asc). The former Jin MTGase is
For example, Tokukai Akira! It can be prepared by the method described in No. 18-14964. The latter BTGase is a new enzyme, which is related to an invention (Japanese Patent Application No. 62-165067) in which the present inventors were partially involved as inventors, and the enzyme properties, manufacturing method, etc. are described in a separate section.

By applying TGase to the above protein, the winner's FJl!
For example, in order to use the 1i molecularization ability and gelation ability to denature proteins that cannot be encapsulated into capsules using conventional kabubuilization technology and use them as a base material for kabuvir, the following procedure can be performed.

First, an aqueous protein solution or emulsion is prepared.

Its concentration can be between 1 and 30%.

Next, TGaSe is added to this aqueous solution or emulsion at a concentration of 1 to 1,000 u/g protein, preferably 10 to 1
Add 00 u/min of protein and stir. At a lower IS of 1 degree, it cannot be differentiated from the case without additives, and at higher concentrations, gelation progresses too much and the product with good physical properties becomes 1 degree.
Sometimes it gets stuck in the capsule manufacturing machine. Reaction Iff is 5 to 90°C, preferably 20 to 6
The reaction time is 1 second to 24 hours, preferably 5 seconds to 30 minutes.

The TGase-added protein liquid aqueous emulsion prepared in this way is then treated so that the generated denatured protein becomes the Kabubil base material, which can be done using conventionally well-known techniques. There it is.

TGase-added protein trade solution or emulsion contains:
Appropriate additives as necessary, such as sugar, salt, extracts, spices, seasonings F+ such as flavors, polysaccharides, fats and oils,
It is also possible to add emulsifiers, plasticizers, glycerin, vitamins, minerals, etc. in a soluble or insoluble state, for example, to the extent that they do not inhibit enzyme reactions.

7 with or without additives prepared in this way
[C using an aqueous solution or emulsion of protein added with ase
To produce Kabuvir, when Kabuvir is a single capsule, the aqueous solution is injected into an oil layer in the form of droplets, but a method is used in which a suitable stirring operation is added to maintain the droplet shape in the oil layer.

As the oil layer, various vegetable oils such as corn oil and soybean oil can be used. Other droplet production conditions can be easily set by those skilled in the art depending on the type of aqueous solution or emulsion, protein concentration, Vf concentration, etc. It should be set to a size that can occur, for example, from 0, 01 to 30111111. Incubation is carried out as necessary, for example, at 5 to 60°C for 30 seconds to 24 hours. Incubation is preferable from the viewpoint of giving +1 to various properties such as shape retention and heat resistance.

In the case of a double capsule, for example, a capsule containing oil yj as the core liquid and denatured protein as the membrane base, the so-called dropping method may be used. That is, the core liquid flows out from the inner nozzle of the double nozzle, and the aqueous solution or emulsion liquid, which is the capsule coating liquid, flows out from the outer nozzle at a constant speed,
These two layers of liquid flow are cut at regular intervals to form droplets in the oil layer. :Eject.

The properties of the film are determined by the shape and size of the nozzle, the injection m and speed, the composition of the protein aqueous solution or emulsion, and the TGase
Although it depends on the type and state of the oil layer, the incubation conditions, etc., those skilled in the art can easily obtain a film with appropriate properties by taking these factors into account.

The turnips produced in the oil layer may be heated as they are, for example, to 90° C. and held for 10 minutes, and then separated and recovered from the oil layer, or conversely, they may be separated and recovered from the oil layer and then heated.

Treatments such as drying may be carried out as needed by methods well known to those skilled in the art.

(Novel transglutaminase BTGase) (1) Transglutaminase and its derived transglutaminase (hereinafter sometimes abbreviated as TGase) performs the acyl transfer reaction of the γ-carbo-4-diamide group of glutamic acid J3 in the peptide chain. It is a catalytic enzyme. In this -rGase, when the ε-amino group of the lysine residue in the protein acts as an acyl acceptor, ε-(γ-Glu)-'cys crosslinks are formed within and between molecules. In addition, when water functions as an acyl acceptor, it is an enzyme that promotes the reaction in which glutamine residues undergo tax amidation to become glutamic acid residues.

Due to these properties of TGase, TGasC can be used to gel a protein-containing solution or slurry.

Until now, TGase was derived from guinea pig liver (MT
However, animal-derived products are inexpensive and difficult to obtain for humans, and when gelling proteins, it is necessary to It is necessary to increase both concentrations, and since it is Ca2+ dependent, its applications are limited by υ1.

Novel 1-Lance Glutaminase (B) that can be used in the present invention
TGase) is produced by microorganisms, for example, bacteria of the genus Stosib-1-Verbusillium, but there are currently no reports on TGasc derived from microorganisms.

BTGase derived from microorganisms that can be used in the present invention is available at low cost and is easy to produce, so it is highly practical. Also, is it possible to use BTG-ase in the absence of calcium or in the presence of calcium? + (B
It has the advantage that a gelled product of excellent quality can be produced only at very low degrees of TGasc)iIIn and substrate concentrations.

■Production of BTGase The microorganism that produces BTGase is, for example, Streptoverticillium griseocalneum (Streptoberticillium griseocalneum).
overticillium griseocarn
eum) I F Q1277G, 3treptoverticillium cinn
amoneuI! 1 sub sp.

cinnamoneum) I F O12852, Streptoverticillium Φmobalaens (S trep
toverticillium mobaraense)
Examples include IF 013819.

The cultivation method, purification method, etc. for culturing these microorganisms and obtaining transglutaminase are as follows.

Although both liquid culture and solid culture are possible as culture formats, it is advantageous industrially to perform deep aeration agitation culture. In addition, the culture sources used include carbon rays, nitrogen sources, and other sources commonly used for microbial culture. and other micronutrient sources, any nutrient source that can be used by microorganisms belonging to the genus Streptoverticillium can be used. v of medium! In addition to glucose, sucrose, lastagen, glycerin, dextrin, starch, etc., fatty acids,
Fats and oils, Th1fi acids, etc. may be used alone or in combination. As the nitrogen source, either an inorganic nitrogen source or an organic nitrogen source can be used, and examples of the inorganic nitrogen source include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, and ammonium chloride. In addition, soybeans are used as nitrogen sources,
Examples include flours such as rice, corn, and wheat, bran, and defatted lees, as well as cornstarch liquor, peptone, meat extract, casein, amino acids, and yeast extract. Inorganic substances and micronutrients include salts such as phosphoric acid, magnesium, potassium, iron, calcium, and zinc, as well as vitamins, nonionic surfactants, antifoaming agents, etc.
Any substance that promotes the production of can be used as necessary.

The culture is carried out under aerobic conditions, and the culture temperature is set to allow the bacteria to grow.
It may be within the range that e can be produced, preferably 25 to 35
It is ℃. The opening during culture varies depending on the conditions, but BTGa
It is sufficient to culture until the time when se is produced the most, usually 2
~4 days.

In liquid culture, BTGase is dissolved in the culture solution, and after the completion of culture, it is collected from the culture filtrate after removing the solid content from the culture solution.

To purify BTGase from culture filtrate, any method commonly used for enzyme purification can be used.

For example, ethanol, acetone, isopropyl alcohol.
Salting out with ammonium sulfate, common salt, etc.
Methods such as dialysis, ultrafiltration, ion exchange, adsorption chromatography, gel filtration, adsorbents, and isoelectric point fractionation can be used. In addition, by appropriately combining these methods, it is possible to create a skewer that increases the F5 production quality of BTGaSe by appropriately combining them. Enzymes obtained by these methods contain various salts, sugars,
Liquid or solid [3TGaSC
can be obtained.

[To measure the activity of 3TGasc, a reaction was performed with benzyloxycarbonyl-glutaminylglycine and human glycylamine as V substance in the absence of Ca2+, and the resulting human D-4J was reacted with trichloro1 An iron complex is formed, absorption at 525 nm is measured, and the activity of hydroxamic acid is determined from a calibration curve.

RTGase activity was measured by the method described below unless otherwise specified.

Activity measurement method> Reagent A 0.2M Tris-HCl buffer (1) 86.0)
0.1M Hydroxylamine 0.01M Reduced Glutabuone 003M Benzyloxycarbonyl Levonyl-Glutaminylglycine Reagent 8 3N-Hydrochloric acid 12%-tric[co[1 Acetic acid 5%F ccj! 3GH20(0,INl) dissolved in C!
> A 1:1:1 mixture of the above solutions is referred to as Reagent B.

Add 〇 and 5d to the reagent to 0.05m of the enzyme solution and mix.
Complete the reaction at 7℃ for 10 minutes, then add test QB to stop the reaction and F.
e After the formation of the complex, the absorbance at 525 nm is measured. As a control, use an enzyme solution that has been heat-inactivated in advance, and measure the absorbance of the same reaction solution to determine the difference in absorbance from the enzyme solution. Separately, a calibration curve was created using L-glutamic acid γ-monohydroxamic acid instead of the enzyme solution, and the amount of human 1]chizamic acid produced was determined from the absorbance difference.
Enzyme activity that produced 1 μmol of hydroxamic acid per minute was defined as 1 unit.

(3) BTGase (7) Enzyme properties Purified BTGas obtained as above, namely transglutaminase of Strebutvedicillium t-balance IF013819 (named BTG-1) , Streptoverbucilium glileocarnelum I F O1277
The enzyme chemical properties of transglutaminaro of 0 (named BTG-2), Streptoberbusillium cinnamoneum sub sp. cinnamoneum IF○ 12852 (named BTG-3) are as follows: .

a) Optimum pH: When benzyloxycarbonyl-glutaminylglycine or hydroxylamine is used as HH, 37
After reacting at ℃ for 10 minutes, the optimum pH of 13TG-1 is around 6-7, the optimum pH of BTG-2 is around 6-7, and the optimum pH of BTG-2 is around 6-7.
The optimum pH of TG-3 is around 6-7.

b) Optimum temperature: When benzyloxycarbonyl-glutaminylglycine and hydroxylamine are used as additives, pi
-16.The optimum temperature for BTG-1 is 55℃ for 10 minutes reaction.
The optimal temperature for BTG-2 is around 45°C, and the optimal temperature for BTG-3 is around 45m.

C) flH stability: After treatment at 37°C for 10 minutes, BTG-1 is stable at p1]5-9, BTQ-2 is stable at p15-9, and BTG-3 is stable at p116-9. It is stable.

d) Temperature stability: tlH7TeIOmin1i'j Treatment F Ha, [3TG-1
At +o℃, [1883 talkability remains, and at 50℃, 14%
Activity remains, and BTG~2 has 86% activity remaining at 40°C, and ha! at 50°C. i G % '61 activity remains, BTG-3 remains 80% active at 40"C, 50"C
In this case, 53% activity remains.

e) Substrate specificity: Using each 13TGasc, reactions between various synthetic substrates and hydroxylamine were investigated. None of the BTGases reacts when the synthesized W Y (is benzyloxycarbonyl aspara-1!nylglycine or benzyloxycarbonylglutamine. However, the reactivity is highest when the synthetic group \'[ is benzyloxylponylglutaminylglycine. The concentration of various synthetic antagonists at this time was 5 mM.The results are shown in Table 1.

CBZ in Table 1 is an abbreviation for pendyloxycarbonyl group, Qln is an abbreviation for glutamyl, Gly is an abbreviation for glycyl group, and ASI) is an abbreviation for asparaginyl group. It is.

f) Effect of metal ions: Various metal ions were added to the activity measurement system at a concentration of 1 mM to investigate the effects (results are shown in Table 2). The activity of both BTGases is also inhibited by Cu2+z n24.

g) Influence of inhibitors: Each inhibitor was added at a concentration of 1 mM, left to stand at 25°C for 30 minutes, and activity was measured (results are shown in Table 3).

The activity of any [3TQase is inhibited by L]romercurybenfI acid (abbreviated as PCMB), N-ethylmaleimide (abbreviated as NEM), and monoiodo M acid.

Table 3 In Table 3, PMSF is an abbreviation for phenylmethylsulfonylfluorite.

h) Isoelectric point: As determined by ampholine isoelectric focusing, BT
The isoelectric point plf of G-1 is around 49, the isoelectric point pl of BTO-2 is around 97, and the isoelectric point pt of BTG3 is around 9.
It is around 8.

i) Molecular weight: As determined by SDS disk electrophoresis, BTGl
The molecular weight of BTG-3 is about 38,000, the molecular weight of 13T G2 is about 41,000, and one molecule of BTG-3 is about 41,000.

j) Comparison with MTGase: Next, the properties of BTGaSC and guinea pig liver-derived transglutaminase (MTGase) will be compared. Furthermore, M
TG a se +, Japanese Patent Publication No. 14904/1983
It was prepared by the method described in No. 5.

Table 4 shows a comparison of each enzyme's chemical properties, and Table 5 shows a comparison of each enzyme's chemical properties.
The effect on a2+ activity is shown. Table-4 and Table-5
J, sea urchin, which is more obvious than MT, which has been mainly studied in the past.
GaSe and BTGase derived from actinomycetes have an enzyme chemical +! There are various differences in quality, especially temperature stability,
Differences are seen in molecular jd, isoelectric point, and substrate specificity. Also,
Even in the presence of Ca24 and in the presence of
There is also a clear difference in the way e works. obey,
Each Vf element that succumbs to the novel enzyme [3TGase is MTGaS
It is thought that e is responsible for that property.

Table-4 Table-5 (4) B T G a S e no % J 3i) Example a
) Production of BTG-1 Streptoverticillium mobalaens IF0138
19, medium composition Volibeb] 0.2%, glycose 0.
5%, dipotassium phosphate 02%, magnesium sulfate 0.
A medium (13) 17) consisting of 1% polypeptone, 2.0% lastagene, 0.2% dipotassium phosphate, 2.0% polypeptone, 2.0% lastagen, 0.2% dipotassium phosphate, and 200rnIl was added to the culture medium and cultured at 30°C for 48 hours. 2%, magnesium sulfate 0.1%, ffH
Medium 2ON (1'
1l-17), cultured at 30°C for 3 days, filtered, and obtained a culture solution of 18.5j! It's 49. The activity of this thing is 0.351
1 ml of membrane! It is.

The culture solution was adjusted to pH 6.5 with hydrochloric acid and equilibrated with 0.05M phosphate buffer (rlH6.5) in advance.
50 (trade name, Organo Co., Ltd. product) column.

Transglucuminase was adsorbed by this operation.

Furthermore, after washing away impure proteins with the same buffer solution,
．． A concentration range of 0.05 to 0.5 M was prepared for the same buffer solution, and the eluate was fractionated and collected by passing through the solution, and fractions with high specific activity were collected.

After dilution so that the conductivity was less than Ioms, it was passed through a blue sepharose column. Transglutaminase was adsorbed by this operation. Plus 0.05M phosphorus! After washing away impure proteins with a buffer solution (pH 7), a salt concentration gradient of 0 to 1 M was created and the eluate was collected, and both fractions with high specific activity were collected. Concentrated using UF 6000 membrane, 0
．． 0.05M phosphate buffer (pH 7) containing 5M sodium chloride
Equilibration was performed using a buffer solution.

1. The collected stentate was equilibrated in advance with the same FIim solution and passed through a column containing Fadex G-75 (manufactured by Pharmacia Fine Chemical Co., Ltd.), and the same buffer solution was passed therethrough to fractionate the eluate. As a result, the compressible fraction was eluted as the peak A-. The specific activity of this product was 625 times that of the culture medium, and the recovery rate was 41%.

b) Production of +3Tc+, -2 In the same manner as in the case of BTG-1, Strebtoverdicillium griseocalneum [FO12776 was cultured at 30°C for 3 days, filtered, and culture solution 19. (qta.

The activity of this product was 0.28 u/m-('.

The enzyme was purified in the same manner as for BTG-1 and SO
A single enzyme was obtained by S disk electrophoresis.

C) Production of BTG-3 In the same manner as in the case of BTG-1, Streptoberbu-Cirium cinnamoneum zabu sp. sinenaeum IF 012852 was cultured at 30°C for 3 days, filtered, and
8 nutrient solution 18.5β was added to 1!7. The enzyme activity of this thing is 0
．． It was 5u/d.

Purify the enzyme in the same manner as for BTGl and add it to SDS.
A single enzyme was obtained by disk electrophoresis.

Hereinafter, the present invention will be further explained by citing as an example a production example of Chibureru containing the denatured protein 7' of the present invention as an ingredient.

Example 1 15-L' InM Sodium (1'J OW Zc
Alanate 180) 409 manufactured by Airy 3 Oard was placed in a beaker, water 2809 was added thereto, and the mixture was completely dissolved in a Rin bath at 55°C. After that, add 32ff of soybean oil (Ajinomoto ■!I) and use a homogenizer (
KIN[HATICA GmbllLITTAII P
OIVTrtON■, shaft φ20m) was emulsified (12.OOOrpm x 3 minutes).

BTG-1 (specific activity 2.Ou #9) 4 was added to this emulsion.
00IQ was dissolved in 5 d of water and added, and the mixture was stirred using the above-mentioned shaker to make it homogeneous (12.OO
Orpm x 10 seconds). The sample was then kept in a hot bath at 55°C for 10 minutes.

After that, the above emulsion was poured into
An oil layer consisting of soybean oil (55℃) at a rate of 5ml per minute.
dripped into. The oil layer was stirred using a magnetic stirrer so that the stirrer rotated approximately 180 revolutions per minute. After the dripping was allowed to stand for 30 minutes, only the Kabrel-like pseudo-solidified mold emulsion was taken up and heated in hot water at 90° C. for 10 minutes.

The product thus obtained has a white spherical shape (spherical diameter 1-2
At M), he loved the soft and smooth cod roe-like texture.

Example 2 Isolated soybean protein (manufactured by Ajinomoto, Ajibron 182)
BTG-1 Specific activity 2, Ou/rng) 2
5q was dissolved in 1d of water and added, and mixed until uniform.

Next, the above emulsion was passed through four nozzles with a diameter of 2M at a rate of 12mf per minute! An oil layer (55°C
). After leaving the mixture for 10 minutes while continuing to stir roughly, the temperature of the i1+ layer was raised to 90° C. for 10 minutes.

This produces a white spherical (spherical (1~3M) capsule) (
It's 9.

Claims (1)

[Claims] (1) Capsules based on transglutaminase-denatured protein.