JPH02128648A - Solid fat and production thereof - Google Patents

Abstract

PURPOSE:To obtain solid fats useful as a substitute or for increasing the quantity of cattle, pig fat meat, etc., by mixing fats and oils with a water-soluble protein, water and a transglutaminase (TGase). CONSTITUTION:For example, a water-soluble protein is added to water so as to provide 5-30wt.% concentration and then dissolved by warming. Fats and oils are then added thereto so as to afford 0.4-5 pts.wt. amount thereof based on 1 pt.wt. aqueous solution of the water-soluble protein. An aqueous solution of a TGase prepared by dissolving 1 U TGase in 1-10mol water is subsequently added to the resultant emulsion so as to provide 1-500 U TGase based on 1g water soluble protein. The transglutaminase reaction is carried out while stirring the obtained mixture at 30-60 deg.C for 10min-2hr. Thereby, the objective solid fats are obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application 1] The present invention relates to a solid fat containing oil or fat, water-soluble protein, water and transglutaminase (TGase), and a method for producing the same.

[Conventional technology 1] In recent years, as a method for producing components that are extremely similar in appearance, texture, etc. to the fat part found in natural meat, so-called fat, protein IIN, textured protein, etc. are added to fats and oils. Method of producing and thermocoagulable protein, chilled 1
A method of manufacturing by adding coagulating proteins to fats and oils has been proposed (Japanese Patent Publication No. 47-20377, Japanese Patent Publication No. 52-1
56959>.

E Problems to be Solved by the Invention] However, the artificial fat obtained by the conventional method has poor flavor in the products obtained using it, and the products using it are prone to oil spills at the time of manufacture and immediately. There were problems such as the large amount could impair the appearance and texture of the product, and it was difficult to adjust the consistency appropriately.

[Means for Solving the Problems 1] The present invention has been made to develop a solid fat that can solve the drawbacks of the artificial fats conventionally used.

The present inventors have discovered that DingGa5e, one of the acyltransferases,
We focused on the effect of forming crosslinks between glutamine residues and lysine residues, which are abundantly contained in food proteins, and as a result of research, we found that by mixing TGase with fats, oils, water-soluble proteins, and water and causing a reaction. The solid side is easily obtained, and when the solid side thus obtained is mixed and used in a meat paste product, a product exhibiting a preferable flavor and texture can be obtained. It was discovered that the type of fat can be freely determined, and that a solid side with the required hardness can be obtained, and that the solid side can be used for various purposes as an artificial fat. There's only one thing to do.

In other words, the present invention uses fats and oils, water-soluble proteins, water, and TGas.
The present invention relates to a solid side containing e and a method for producing the same.

The fat used in the present invention may be animal fat or vegetable fat, and may be liquid fat or solid fat. Animal fats and oils and 1) include beef tallow, pork fat,
Chicken fat, fish oil, etc., and vegetable oils include soybean oil, corn oil, rice bran oil, peanut oil, rapeseed oil, cottonseed oil, palm oil, olive oil, sesame oil, etc. Also, hydrogenated soybean oil,
Hydrogenated vegetable oils such as hydrogenated cottonseed oil can also be used.

The water-soluble protein used in the present invention is not particularly limited as long as it can be dissolved in water by a normal method, and examples thereof include gelatin, casein, soybean protein, ovalbumin, and serum albumin. be done.

The origin of rGase used in the present invention is not particularly limited, and it forms a crosslink between the glutamine residue and lysine residue contained in the water-soluble protein, and solidifies the heated and dissolved solid fat and liquid fat. Anything that can be placed on the side can be used. Specifically, for example, T G a S e (M
TG a S e ).

Furthermore, patent application No. 62-165067 filed by a portion of the applicant
For example, novel TGaSe derived from microorganisms produced by microorganisms of the genus S.
(BTGase) has been disclosed (new BTG
The method for producing ase, the characteristics of the enzyme, etc. will be described later). In the present invention, such [3-LQ a
Of course, s e can also be used.

The present invention has been developed using 1
The use of Qase is characterized by the fact that it is easy to obtain a solid product whose hardness and oil flow rate are adjusted to any desired range depending on the application.

The solid side of the present invention can be manufactured, for example, as follows.

After heating and dissolving water-soluble protein in water to a concentration of 1-60% by weight, preferably 5-30% by weight, add oil to this, if it is a liquid oil, add it as is, if it is a solid oil, add it. 01 to 10 per disintegration part of 1 water-soluble protein aqueous solution after heating and dissolving.
Add to the stirrer a little at a time so that there are 04 to 5 layers, preferably 04 to 5 layers.

As the stirring device, one that is commonly used for YJ manufacturing of foods can be used. In this case, it is preferable to stir the mixture so as to form a 0/W type 1-mulsion.

This allows the fats and oils in the inner phase to be wrapped in the water-soluble proteins in the outer phase, making it easier for the reaction between the rGase and the water-soluble proteins to be added next. Although an emulsifier is not necessarily required to form Nimalgin, O/W emulsions are used in the production of foods such as resins, sorbitan fatty acid esters, and sucrose fatty acid esters. Emulsifiers may also be used. Furthermore, emulsion stabilizers, thickeners, etc. can also be used.

TGaSe was added to the emulsion obtained as described above.
An appropriate amount of TGase aqueous solution obtained by dissolving 1 unit (Ll) in 1 to 101 dl of water is added to the water-soluble protein. When the transglutaminase reaction is carried out with stirring at 30 to 60°C for 10 minutes to 2 hours, the emulsion solidifies and the solid fat of the present invention is obtained. When using MTGase that is dependent on M T Gase, it is necessary to add a calcium (Ca)
l2, CaCO3, Ca30 2820, etc.
Approximately 1 to 100111M may be added to a5e1tJ. In addition, if necessary, seasonings, coloring agents, flavoring agents, etc. can be added at any stage of the above-mentioned manufacturing process to obtain solid fats having various colors and flavors.

The method for producing solid fats of the present invention is not limited to the above-mentioned production method, and for example, fats and oils are added to a mixture containing water-soluble protein, water, and TGase in the above-mentioned ratios, and a transglutaminase reaction is carried out under stirring. It can also be obtained by doing.

The mixing ratio of water-soluble protein, water, fat and oil, and -rGase contained in the solid fat of the present invention obtained in this way is usually 0.67 to 999 parts water per 1 g of water-soluble protein, preferably 0.67 to 999 parts water. 2.3-19g, fats and oils 0.17-10003, preferably 0.67-500g, TGase 0.1
-1000tJ, preferably 1-500U.

In the present invention, the hardness of the solid fat and the amount of oil flowing out can be freely adjusted by appropriately selecting the types of fat and water-soluble protein, the ratio of fat and water, and the enzyme reaction conditions.

For example, when fats and oils with high melting points such as beef tallow and pig intestines are used, and gelatin is used as water-soluble proteins, harder solid fats are obtained; liquid fats and oils such as vegetable oil are used as fats; When soybean protein is used, a softer solid fat can be obtained. Moreover, by increasing the mixing ratio of water to oil and fat, it is possible to obtain solid fat with less oil spillage.

The solid fat of the present invention can be used as it is after production, or after being frozen if necessary, shredded into pieces of appropriate size using mince, flakers, etc., to be used as a substitute for or as a supplement to beef fat, pork intestines, etc. It is added to meat paste products such as ham, sausage, hamburger steak, shumai, and dumplings. In addition, it can be used as a solid fat with various flavors such as spices and soybeans, as a flavoring agent for food, and in products that have a healthy image, such as in Muslim countries where pork intestines cannot be used for religious reasons. It can also be used for applications such as liver fat substitutes made from vegetable oils for products characterized by.

(Novel transglutaminase B l' used in the present invention
(1) Transglutaminase and its derived transglutaminase (hereinafter sometimes abbreviated as TGase).

) is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxyamide group of the glutamine residue in the peptide chain.

When the ε-amino group of the lysine residue in the protein acts as an acyl acceptor, ε-(γ-Q+u)-Lys-Lys bridge bonds are formed within and between the molecules. When it functions as an acyl acceptor, it is an enzyme that proceeds with the reaction in which glutamine residues undergo amidation to become glutamic acid residues.

Novel transglutaminase (BTG) used in the present invention
Ase) is produced by microorganisms, such as bacteria of the genus Streptoverticillium.

■BTGase+7) Production Signal-1 substances that produce BTGase are, for example, Streptoverticillium griseocalneum (S
treptoverticillius arise
ocarneum) I F 012776, Streptoverticillium cinnamoneum sub.
・S treptover mouth cilli
um cinnamoneua sub sp.

cinnamoneum) l F 012852, Strep verticillium-1 balance (Strep
toverticilliummobaraense3
1 F 013819 etc.

The cultivation method, purification method, etc. for culturing these microorganisms and obtaining 1-lansglutami and 1-se are as follows.

Culture formats include liquid culture and solid culture.

However, from an industrial perspective, it is advantageous to perform deep aeration agitation culture. In addition, the culture sources used include carbon sources, nitrogen sources, xiR salts, and other microorganism sources commonly used for microbial culture. In addition to this, any nutrient source that can be used by microorganisms belonging to the genus Streptoverbucilium can be used. Carbon sources for the medium include glucose, sucrose, lastagen, glycerin, dextrin, starch, etc.
Fatty acids, fats and oils, 1fi acids, etc. can be used alone or in combination. As a nitrogen source, both inorganic nitrogen sources and organic nitrogen sources are reusable. [Includes nium etc. In addition, the 14-Nada Nitrogen Source Binding uses soybeans, rice, 1-U-chi, powder, bran, and defatted lees, as well as 1-1-teen staple liquor, peptone, meat extract, casein, amino acids, and fermentation. (iI Kiss, etc.) Inorganic salts and micronutrients include salts such as phosphoric acid, magnesium, potassium, iron, calcium, and zinc, as well as vitamins, nonionic surfactants, antifoaming agents, etc. It can be used as required if it promotes the production of B'TGasc.

The culture is carried out in an aerobic tea scoop, and the culture temperature is such that the bacteria grow and reach BTGas.
It may be within the range that e can be produced, preferably 25 to 35
It is ℃. Culture time (1, varies depending on conditions, but B T
It is sufficient to culture until the time when Gase is produced the most, which is usually about 2 to 411 times.

B-[Qase is dissolved in the culture solution in liquid culture, and is collected from the culture filtrate obtained by removing the solid content from the culture solution after the completion of the culture.

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

For example, treatment with organic solvents such as ethanol, acetone, and isopropyl alcohol, salting out with ammonium sulfate, salt, etc.
Methods such as dialysis, ultrafiltration, ion-exchange chromatography, adsorption O mudography, gel filtration, stone absorbing agents, and isoelectric point fractionation can be used. Also, by appropriately combining these methods, you can create the essence of B'r Gase! If it is all done once, it can be done in combination as appropriate. Enzymes that can be removed by these methods can be processed by ultrafiltration concentration, reverse osmosis acondensation, vacuum drying, with or without the addition of various salts, sugars, fleas, lipids, surfactants, etc. as stabilizers. ,
B T in liquid or solid form by freeze-drying or spray-drying.
G a se can be obtained.

The activity of BT Gas is measured by reacting benzyloxycarbonyl-L-glutaminylglycine with hydroxylamine in the absence of Ca2+ as a substrate, and then using the generated hydroxamic acid to form an iron complex in the presence of trichloroacetic acid. ! The absorption of 525 nl is measured, and the activity of human 1]chizamic acid is determined from the calibration curve.

B T Gase activity was measured by the method described below unless otherwise specified.

<Activity measurement method> Reagent A 0.2M Tris-HCl buffer (1) H6,0)
0.1M hydroxylamine 0.01M reduced glutathione 003M benzyloxycarbonyl monoglutaminylglycine reagent 8 3N-hydrochloric acid 12%-tricloacetic acid 5% FeCj! - 6H20 (dissolved in 0.1N ICl) Reagent B is a 1:1:1 mixture of the above solutions.

0 of the enzyme solution. Reagent △0.05me. 5d was added and mixed, and after reacting at 37°C for 10 minutes, reagent B was added to stop the reaction and form Fe 11 bodies (measure the absorbance at 1525 nm. As a control, an enzyme solution that had been heat-inactivated in advance was used. Measure the absorbance of the same reaction using
P? f Find the absorbance difference with the basic solution. Separately, a calibration curve was created using L-glutamic acid γ-L nohydroxamic acid instead of the enzyme solution, and the amount of hydroxamic acid produced was determined from the difference in absorbance, and 1 μmol of hydroxamic acid was produced per minute.85 This activity was defined as 1 unit.

Enzyme properties of GBTGase Purified Bl-GaSe obtained as above, f1 Transglutaminase (named BTG-1) of Streptoveticillium suprabalance IFOI3819, Streptoveticillium griseolneum I F 0
12776 transglutaminase (BTG
-2), Strebtovertisi.-.

Rum cinnamonium sub sp. cinnamoneum I
F 0 12852 transglutaminase (BTG
The enzymatic chemical properties of the enzyme (named -3) [actually, the following is true.

a) When benzyloxycarbonyl-glutaminylglycine and hydroxylamine are used as the optimum pH, 37
℃, 10 minutes reaction, optimal p1-1 of BTG-1 is 6-7
The optimum pH of B 1' G-2 is around 6-7, and the optimum pH of B TG-3 is around 6-7.

b) Optimal temperature: When using benzyloxycarbonyl-L to glutaminylglycine and hydroxylamine as substrates, the pH
6. In a 10-minute reaction, the optimal temperature for BTG-1 is around 55°C, the optimal temperature for BTG-2 is around 45°C, and the optimal temperature for BTG-3 is around 45°C. It is in.

c) pH stability: After treatment at 37°C for 10 minutes, BTG-1 had a pH of 5 to 9.
BTG-2 is stable at pH 5-9, and BTG-3 is stable at pH 6-9.

d) Humidity stability: When treated with DH7 for 10 minutes, BTG-1 remained at 88% activity at 40°C, and at 50°C, 14% activity remained (BTG-1).
TG-2 has 86% activity remaining at 40°C, bran has 56% activity remaining at 50°C, and BrG-3 has 80% activity remaining at 40°C.
At 50°C, 53% activity remains.

e) Substrate specificity: Using each BTGase, reactions between various synthetic substrates and hydroxylamine were investigated. Eventually 1', 1・1BT Gas
e also does not react when the synthetic substrate is benzyloxycar-1-nylasparaginylglycine, benzyloxy, □'rubonylglutamine, glycylglutaminylglycine. The reactivity was the highest in the case of .The concentration of various synthetic substrates at this time was 5 g+M.The results are summarized in Table 4-1.

In addition, CBZ in Table 1 is an abbreviation for benzyloxycarbonyl group, Qln is an abbreviation for glutamyl group, and Gly
is an abbreviation for glycyl group, and ASI) is an abbreviation for asparaginyl group.

Table 1: Effects were investigated by adding ions (results are shown in Table 2). The activity of 3rGase is also inhibited by Cu2+7n""tc.

Table-2 r) Metal ion concentration: Various metals were added to the activity measurement system so that the concentration was 11 IMm.
Effect of inhibitors: Add each inhibitor to 1111M and incubate at 25°C for 30
Distribute! 1, to measure the activity (results are shown in Table 3), both B T Gases were parachlorinated [
Commer-t J Riyann Q Froic Acid (abbreviated as P CM 13)
, If-] nibblemaleimide (abbreviated as NEM), -[
)-1-doacetic acid inhibits the activity.

Table = 3 In Table 3, F' M S F is an abbreviation of mono-r-methyl sulfonyl fluoride.

h) Isoelectric point: +3 as determined by ampholine isoelectric focusing
TG-1's 313 points PL is close to 9, BT
The isoelectric point pi of G-2 is around 9.7, and B T G
It is around 8 from the isoelectric point plL of 3.

) Molecular weight: As determined by SDS disk electrophoresis, BTG-
The molecular weight of 1 is about 38.000, and the molecular weight of )3T G2 is about 41.000. OOO'r, and the molecular weight of BTG-3 is approximately 41,000.

(4) Manufacturing example of BTG a) BTG
1 production of Streptoverticillium (Varaens I [0138
19, medium composition: polypeptone 0.2%, green, -]-
Medium consisting of 0.5% sodium chloride, 0.2% dipotassium phosphate, and 0.1% magnesium sulfate (pl'7) 200#lI
! I and Z were inoculated and cultured at 30°C for 48 hours, and the resulting seed culture was mixed with polypeptone 2.0% and lastagen 2.0.
%, dipotassium phosphate 0.2%, ngnesium sulfate 0.
Medium 2 consisting of 1% yeast extract, 0.2% yeast extract, and 0.05% ADEKA NOL (trade name, Asahi Denka product) as an antifoaming agent.
01 (+)H7) and cultured at 30° C. for 3 days, followed by filtration to obtain cultures 118 and 51. The activity of this thing is 0.35
u/mj! It is.

The culture solution was adjusted to pH 6.5 with hydrochloric acid and passed through a column of CG50 (trade name, manufactured by Organo Inc.) which had been equilibrated with 0.05M phosphoric acid W[II solution (DI-16.5).

Transglutaminase was adsorbed by this operation.

Even more same! After washing away impure proteins with lj solution, make a concentration gradient of 0.05 to 0.5 M of the same buffer, pass through the solution, collect the eluate in fractions, and collect the fractions with high specific activity. Ta. The mixture was diluted to have an electrical conductivity of 10 m5 or less, and then passed through a blue sepharose column. Through this operation, transglutaminase was adsorbed. Further, impure proteins were washed away with 0.05M phosphate buffer (pH 7), and then an 11 degree gradient of 0 to 1M sodium chloride was passed through the solution, the eluate was collected, and fractions with high specific activity were collected. It was concentrated using UF 60001J and equilibrated using a buffer containing 0.5M(7)itl, 0.5M phosphate buffer (pH 7).

The obtained concentrate was pre-equilibrated with the same buffer solution.
The eluate was fractionated by passing the same buffer through a column containing No7dex G-75 (manufactured by Pharmacia Fine Chemicals). As a result, the active fraction was eluted as a single peak. The specific activity of this product is 625 against culture filtrate.
The recovery rate was 47%.

b) [Production of 3TG-2 In the same manner as in the case of BTG-1, Streptoberbusillium griseoluneum IF012776 was cultured at 30°C for 3 days and filtered, and culture solution 19j! I got it.

The activity of this product was 0.28 u/d.

The enzyme was purified in the same manner as for 8TG-1, and the SD
A single enzyme was obtained by S disk electrophoresis.

C) Production of t3 T G-3 In the same manner as in the case of BTG-1, Strebtoverticilli cum cinnamoneum subsp. cinnamoneum IF 012852 was cultured at 30°C for 3 days! I'm late,
Culture solution 18.5j! I got it. The enzyme activity of this product is 0.
It was 5u/d.

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

Examples of the present invention will be described below.

Example 1 Three-layered gelatin was added to 16-layered water to swell and then heated to dissolve. Next, 80 parts of purified lard, which had been separately heated and dissolved, was added little by little while stirring and mixing to obtain an emulsion. A solution prepared by dissolving B'rGaSe-10,02 heavy part (18 U per 1 g of water-soluble protein) in 1 part by weight of water was added to this, and the mixture was kept at 50°C for 1 hour to obtain a solid fat. Even when the solid fat obtained is heated above the melting point of lard, all the oil does not melt and flow out, leaving solids as residue, and the properties are extremely similar to natural pork fat. there were.

11 pieces of this solid product was frozen, thawed, minced, and used to prepare a hamburger steak with the following composition. The resulting hamburger steak was comparable in taste, flavor, and texture to those obtained using natural pork fillet.

<Hamburger Steak BTGase-1 Containing Solid II 15.0il
lfiffi minced meat 45
．． 0 onions 20.0 eggs

6.9 Milk bread crumbs 1i! Example 2-3 Water, purified food, and gelatin mineralization were changed as shown in Table 1.
) different solid fats were obtained between the oil spills.

Example 4 31 parts of Na-caseine-1 was dissolved in 16 parts of water, and 8 parts of refined soybean oil was added thereto a little at a time while stirring and mixing to obtain white maltene. Ta.

B T G a S e -1 in this -L multi-column 1
0.04-fold dissolved in 1 small bottle of water is 1 part protein.
7 to 35 U and kept at 50°C for 21 hours until it became solid (obtained fat).

1q of the obtained solid fat was solid at room temperature, and like the one obtained in Example 1, even when heated, the oil did not flow out at the ribbed.

Example 5-12 Types of fats and oils and water-soluble proteins, fats and oils, water-soluble proteins, water, r
Each solid fat was produced 1:1 in the same manner as the production method A described in Example 1, except that the a content of the gas was changed to the one shown in Table 2. Table 2 shows the hardness of the obtained solid fat and the oil spilled product when heated to 95°C.

[Effects of the Invention] When the solid product 2 of the present invention is mixed and used in meat paste products, oil spillage during heating can be freely controlled, resulting in a pleasant flavor and good taste! Y
You can make 1 meat paste product that is 8 to [I2]. Historically, according to the method for producing solid fat of the present invention, the types of fats and oils and water-soluble proteins, the n-combination ratio of fats and oils, water-soluble proteins, and fermented water, or the acid (5) reaction conditions may be adjusted as appropriate. A solid fat with a consistency comparable to that of 500ml is obtained.

(Representative)”: Masao Shimokoshi

Claims (2)

[Claims] (1) A solid fat containing oil, water-soluble protein, water, and transglutaminase. (2) A method for producing solid fat, which comprises mixing fats and oils, water-soluble protein, water, and transglutaminase.