JPH0269146A - Production of protein-textured product - Google Patents

Abstract

PURPOSE:To obtain a protein textured product favorable in both flavor and color tone by adding transglutaminase when a protein-contg. raw material is processed with an extruder to enable said processing under relatively milder conditions than those for conventional extruder processing. CONSTITUTION:A protein-contg. raw material is incorporated with transglutaminase (hereafter, referred to as TGase) followed by making an extruder processing. The TGase is of any origin, e.g., separated from the liver of a guinea pig, produced by microorganisms. The amount of the TGase to be added, though somewhat different depending on protein-contg. raw materials, is, in general, ca.1-100u/g protein. The protein-contg. raw material is any one containing protein, e.g., vegetable protein stemmed from soybeans, wheat flour or cotton seeds; animal protein stemmed from albumen, meat, fish meat, casein or defatted powdered milk; or a combination of at least two kinds of them.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a method for producing a protein tissue by subjecting a protein-containing material to an extruder treatment, in which transglutaminase (hereinafter referred to as TGase) and (sometimes abbreviated)), it relates to a method for providing a protein assembly of excellent quality.

(Prior Art and its Problems) Conventionally, structured protein products have been produced by, for example, extruder treatment, based on melting (and molding and pressurizing) protein-containing materials at high temperatures and high pressures.

However, because the protein is processed under high temperature and pressure, the resulting protein texture is inferior in color, taste, flavor, and juiciness, and when used in deli dishes such as hamburgers, shumai, gyoza, and korotsuku, it absorbs water and has a hard texture. Even if it is possible to use bottski, the quality of the product is lacking in juiciness, and the taste and flavor are a concern, especially when using vegetable protein materials as raw materials.For example, soybean protein materials Does it have a strong smell of roasted beans? The amount used in these foods has been limited (for example, 5% or more on a dry matter basis). Therefore, a texturing method that utilizes shear force at lower temperatures is desired.

However, there have been no examples of using TGase during T-tastruder treatment to produce a protein structure from a protein-containing material.

(Means for Solving the Problems) The present invention (changes protein-containing T-cords into protein tissue-[
The use of Qase is defined as 1!r iM, and by doing so, it is possible to perform Iv and rd at a lower temperature than before during the Ex1-ruder treatment, which makes it relatively less expensive. It is possible to organize the protein at a suitable temperature, and it also prevents browning even when processed at the same temperature as before, making it possible to produce a white structured product without a roasted odor. ..

The protein structure produced by the method of the present invention can also be used for livestock meat,
1. Foods that have a high affinity with surimi, etc., and do not become sticky, have a jicky feel, and are difficult to swallow.
You can do it.

The method of the present invention will be explained in detail below.

The outline of the method of the present invention is to add water (if necessary) and transglutaminase to the protein-containing material.
What is involved in Ruda treatment to form a protein structure?
Since the method for producing a protein tissue by Ex1 to Ruda treatment of a protein-containing material without using transglutaminase is well known, it is necessary to use Transglucumi Inu-12 and this method when implementing the method of the present invention. For songs that have some restrictions in use, the above-mentioned well-known extruder processing rules can be used as is or with necessary modifications.

First, the raw materials will be explained.

The protein-containing material may be any protein-containing material, for example,
Examples include vegetable proteins derived from cabbage, wheat, cottonseed seeds, etc., egg white, livestock meat, fish meat, casein, '+h physical proteins derived from skim milk powder, and combinations of two or more of these.

Water (additionally, if necessary, such as when the protein-containing material is dry), but usually 0.1
Add ~2 parts.

Transglutami canine, regardless of its origin, can be isolated, for example, from the liver of a seven-year-old lizard.
(sometimes abbreviated as !3T Gas) and those produced by microorganisms (sometimes abbreviated as !3T Gas). M in song title
TG a s e4. t, for example, Vj fHi
It can be prepared by the method described in item s-14c+64. The latter BTGase is a novel enzyme, and is an invention in which some of the present inventors were involved (¥i
It is related to l1fl162-16!1067)
Its enzymatic properties, production method, etc. will be described in a separate section.

In order to make use of its crosslinking function and enable molding at low temperatures, DQasc generally requires 100U/9 protein (to 1), although there are some differences depending on the protein-containing material. It can be said that 2 to 501 J/9 protein is preferably used in the field where protein is added. Specifically, for example, for soybeans, wheat, livestock meat, and fish meat, 1 to 5 ou/g protein is preferably used.
0u/g protein, 1 to +00 for egg white and loin
tJ/g protein preferably fJ! i~sou/y protein. If too little is used, it will not be possible to differentiate the conventional protein tissue produced without using TGaSe, and if it is too large, the cross-linking reaction will proceed too much, resulting in protein aggregation, and the tissue will become t! ? I can't. If TGase requires other substances to express its S-active effect (MTGaseiyoCa"
dependence), it is natural that such substances also exist.

There is no particular limitation on the method of adding TGaSe, for example,
Gase may be added as a powder into the barrel separately with water and protein-based H, or TGase may be dissolved in water and then added to the protein material. It may be mixed before processing and loaded into a barrel in a dough form.

Of course, additives such as seasonings (meat extracts, spices, flavors), starches, polysaccharides, oils and fats, emulsifiers, etc.
It is possible to add it within a range that does not inhibit the purpose of the present invention, such as inhibiting the action of TGase or inhibiting conversion to 114fE.

Next, extruder processing will be explained.

In extruder processing, shear/υ shearing force is applied,
It only needs to be possible to extrude the protein from a small nozzle after melting it at high temperature and pressure, and it is best to use an extruder.

When using an extruder, any type of extruder can be used, such as a single-sleeve type or a twin-screw type, and there is no particular restriction on the screw configuration as long as it can apply shear force to the screw.

Regarding the temperature, the outlet product temperature is preferably 80 to 250°C, preferably 100 to 180°C, and the extruder inlet (raw material input part) is preferably 60°C or less from the viewpoint of the action of the wood wP element. The residence time in Bareshi is YQ a S
e When it comes into contact with the Lj protein, a crosslinking reaction occurs in a relatively short period of time, so 15 seconds is sufficient to obtain a homogeneous and stable product, but this is related to the suspension of use of the enzyme system, so it usually takes 10 seconds. Above, preferably 20-60 seconds. There is no particular limit to the screw rotation speed, but it is preferably 100
~400 rpm.

When deviating from the above Ex1-Ruda processing conditions (above, no protein structure is obtained, it looks raw (outlet temperature 8
(below 0℃) or loin 1~The odor is too strong (outlet temperature 250℃)
℃ or higher). In particular, if the temperature exceeds 2.10°C (exit product), the product will lack juiciness. Post-treatment of the extruder process may also be carried out by well-known methods, and in this way a protein structure of excellent quality is obtained in conjunction with the final product.

(Novel transglutaminase BT that can be used in the present invention)
Transglutaminase (1) Transglutaminase and its origin Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of γ-carbogycyamide of the glutamine residue in the pebboudo chain. When the ε-amino group of the lysine residue in the protein acts as an acyl acceptor, this TQase generates ε-(γ-Glu)-Ly
s crosslinks are formed. In addition, water functions as an acyl acceptor because it is an enzyme that promotes the reaction in which glutamine residues are deamidated to become glutamic acid residues.

These properties of TGase allow it to be used to gel protein-containing solutions or slurries.

Until now, TGaSe was derived from guinea pig liver (MT
Animal-derived products such as Gase) are known, but animal-derived products are cheap and difficult to obtain for humans, and when gelling proteins, the enzyme concentration and substrate concentration must be adjusted. It is necessary to increase both Ca2+ (
Since it is persistent, its uses are limited.

Novel transglutaminase (B
TGase) is produced by microorganisms, such as Streptoverticillium, but there are currently no reports on TGase derived from microorganisms.

The microorganism-derived BTGasc that can be used in the present invention is available at low cost and is easy to purify, making it highly practical. In addition, by using BTGase, the b enzyme (
B T G a se ) It has the advantage of being able to produce high-quality gelatinized products at very low temperatures and substrate concentrations.

■137'Ga5ef7) Production The microorganisms that produce B T Gase are, for example,
Streptoverticillium griseocalneum (S
treotovertic+Illium gris
eocarnetim) I F 01277
6.S1~Lev] Verdesillium Cinnamonium・
Lieb sp. cinnamoneum (3trcptoverticillium cinn)
amoncum sub sp.

c, innamoneum) I F 012B! ]2
, Strike 1 Knob 1 ~ Verticillium Seven Balaens (3t
rcptoverticill+ummobaraen
sc) I F 013819, etc.

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

Regarding culture formats, both liquid culture and solid culture are classified as "IT".
However, from an industrial perspective, is deep aeration stirring culture possible? -J Uno is advantageous. In addition, as a culture source to be used (in addition to carbon sources, nitrogen sources, inorganic salts, and other trace nutrient sources commonly used for microorganisms, nutrient sources that can be used by microorganisms belonging to the Streptococcus genus All carbohydrates can be used.Glucose, sucrose,
In addition to lastagene, glycerin, phosphorus, starch, etc., fatty acids, fats and oils, 11-organic acids, etc. can be used alone or in combination. Any inorganic nitrogen source can be used as the nitrogen source, and examples of non-nitrogen sources include ammonium nitrate, ammonium sulfate, urea, sodium nitrate, and ammonium chloride. In addition, organic nitrogen sources include powders such as rice, corn, and wheat, bran, defatted lees, cornstarch liquor, peptone, meat extract, kaviin, amino acids, and soybean extract. It will be done. Examples of inorganic salts and microorganisms include phosphoric acid,
In addition to salts such as magnesium, potassium, iron, calcicum, and zinc, salts such as vitamins, nonionic surfactants, and antifoaming agents that promote bacterial growth and BTGase production may be used as necessary. I can do it.

The culture is carried out under aerobic conditions, and the culture temperature is set to B'T when the bacteria grow.
The temperature may be within a range in which gas is produced, preferably 25 to 35°C. Although the culture time varies depending on the conditions, it is sufficient to cultivate the culture for 13 days until the time when BTGase is produced the most, which is usually about 2 to 4 days.

In liquid culture, B rGase is dissolved in the culture medium and J
Three, after the completion of culture, the solid content is removed from the culture solution and collected from the culture filtrate.

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

For example, treatment with organic solvents such as ethanol, acetate, isopropylfurcol, etc., salting out with ammonium sulfate, salt, etc., dialysis, ultrafiltration, ion-exchange chromatography, adsorption chromatography, gel filtration, and adsorbents. , isoelectric point fractionation, and other methods can be used. In addition, if the degree of purification of BFQase is increased by combining these methods = ] S, these methods can be combined as appropriate. Enzymes obtained by these methods can be subjected to ultrafiltration concentration, reverse osmosis shoulder line, vacuum drying, freeze drying, and spraying, with or without the addition of various salts, sugars, proteins, lipids, and surfactants as stabilizers. Liquid or solid 13-ga S depending on the drying method
You can get O.

To measure the activity of 13-engine Ga5e, benzyloxycarbonyl-glutaminylglycine is reacted with human [1-xylamine as a substrate in the absence of Ca2+, and the generated hydroxynamic acid is converted into an iron chain form with F in the presence of trichloroacetic acid. is formed and the absorption at 525 nm is measured, and the activity of the hydroxymic acid is determined from the calibration curve.

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

<Activity measurement method> To the reagent 0.2M t-Lis-HCl buffer (DI-16
, 0) 0, 1M Hydroxylamine 001M Monomorphic Geltade A 0.03M Penzyloxycarbonyl L-Glutaminylglycine Reagent 8 3N-Hydrochloric acid 12%-Trichloroacetic acid 5% Fecj! 6H2 0
(Dissolved in 0.INHCl) - A 1:1:1 mixture of the above solutions was used as reagent B.

Add reagent △0.5d to enzyme solution 005d and mix 37
Incubate the reaction at °C for 10 minutes, then add reagent B to stop the reaction and fC.
After the formation of 11 6n forms, the absorbance at 525 nm is measured. A similar reaction was carried out using an enzyme solution that had been heat-inactivated in advance as a first light, and the absorbance was measured, and the difference in absorbance from the enzyme solution was determined. Separately, a calibration curve was created using L-glutamic acid γ-monohydroxamic acid instead of the enzyme solution, and the field of the generated hydrochylamic acid was determined from the difference in absorbance. J-Sam A Riwo 1
- The fermented mulberry activity obtained was set at 1 medium.

]) [3 T Gase U) Fermentation properties Purification B T Gase, beaten Stjib 1 to Vedicillium Separance II: 043
819 1-lance glutaminose (BT G-1
), Streptoverticillium griseoluneum I F 0 1277G transglutamibuse (named BTG-2), Streptoverticillium
Cinna [Neum Sub SP Cinnamonium IF
0 12852 transglutaminapi (BTG-3
The enzymatic chemical properties of (named) are as follows.

a) to) pto': When benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine are used as substrates, 37
℃, 10 minutes reaction, +3 T G-1 optimal p)l is 6
The optimal pH of BTG-2 is around 6-7, and the optimal pH of [3 T ('+-3 is around 6-7
It's near 1.

b) Suitable temperature: When using benzyloxycarbonyl-rutaminylglycine and human [1xylamine as Ill-16, the optimal temperature for BTG-1 is around 55 °C in a 10-minute reaction, The optimal temperature for BTG-2 is around 45°C, and the optimal temperature for BTG-3 is around 45°C.

c) pH stability: After treatment at 37°C for 10 minutes, BTG-1 was stable at pH 5 to 9, BTG2 was stable at pH 5=9, and BTG-1 was stable at pH 5=9.
TG-3 is stable at I)l-1 6-9.

d) Disturbance A degree stability D117F 10 minutes % T-t. i,BTG”IC
, L40'CT" has 88%'gIII remaining and 50%
At 40°C, BT G-2 has 86% activity remaining, 56% activity remains at 50°C, and BT G-3 has 80% activity remaining at 40°C. % activity remains; at 50°C, 53% activity remains.

0) Substrate specificity: Using each of the 13 TGases, reactions between various synthetic substrates and hydroxylamine were investigated. None of BTGaSe reacts when the synthetic substrate is benzyloxycarbonyl aspara-1=nylglycine, penzyloxycarbonylglutamine, or glycylglutaminylglycine. However, the reactivity is the highest when the synthetic substrate is benzyl A-oxyluponylglutaminylglycine. The concentration of various synthetic M at this time was 5 mM. The results are shown in Table-1.

In addition, CBZ in Table 1 is an abbreviation for penzyloxycarbonyl group, Gln is an abbreviation for glutamyl group, and Gly
is an abbreviation for glycyl group, and △sp is aspara A dinyl 4
It is an abbreviation of

Table 1 shows). Both BTGases are c, 2+ln
” activity is inhibited.

Table 2 f) Effects of metal ions: To investigate the effects of each tumor metal ion added to the activity measurement system at 1mM 1 degree, the results are shown in Table 2g) Effects of inhibitors: Each harmful agent Add to sea urchin, 25℃, 30℃ to make 1mM.
After standing for a minute, the activity was measured (results are shown in Table 3).
. The activity of BTGase is also inhibited by parachloromer A (abbreviated as 11(+)0M13), N-ethylmaleimide (abbreviated as NEM), and monoiodoacetic acid.

In Table 3, r-'MSF is an abbreviation for phenylmethylsulfonyl fluoride.

h) Isoelectric point: As determined by ampholine isoelectric focusing, B
The isoelectric point pl of T G -1 is close to 9 i (j, and BTG
The isoelectric point pl of -2 is around 9.7, and 13 T G-
The isoelectric point pt of No. 3 is around 9.8.

) Molecular weight: As determined by SDS disk electrophoresis, BTG-
The molecular weight of BTG-1 is approximately 38,000, the molecular weight of BTG2 is approximately 41,000, and the molecular weight of BTG-3 is approximately 41,000.
．． It is 000.

j) Comparison with MTGase: Next, the properties of 13TGase and guinea pig liver-derived transglutaminase (MTGase) will be compared. In addition, MTGaSe is disclosed in Japanese Patent Application Laid-Open No. 58-149645.
'JI was manufactured by the method described in No.

Table 4 shows a comparison of the chemical properties of each enzyme, and Table 5 shows Ca
The effect on the activity of 2' is shown. As is clear from Tables 4 and 5, the M engineering Qas that has been studied in the past
E and R'r Gas derived from actinomycetes are enzymatically +! In 1″C1, various types of ′ are observed, particularly differences in temperature stability, minute:f amount, isoelectric point, and group f″ specificity. Furthermore, in the presence of ca2-) and in the presence of J1, there is a clear difference in the action of each [3T Gase. Therefore, each B T G a
It is thought that s e has a different property from M'r G a S e.

Table-4 Table-b (4) B T Gas (D Production Example a)
Production of BTG-1 1-Leptoverticillium [Varaens I Fo
13819, medium composition: Volibebone 0.2%, glycine,
200 d
The seed culture was inoculated and cultured at 30°C for 148 hours, and the resulting seed culture was mixed with 20% polypeptone, 2.0% lastagen, 0.2% dipotassium phosphate, 0.1% magnesium sulfate, and 0.2% yeast extract. %, medium 201 (pH 7
), cultured at 30°C for 3 days, filtered, and added to T8 nutrient solution 18.
It's 51i9. The activity of this product is 0.35u/d.

Adjust the culture solution to 6.5 ml of salt,
CG-50 (trade name, Organo Co., Ltd. product) that had been equilibrated with 0.05M phosphate buffer (DH6,5) in advance.
column.

This procedure-C transglutaminase was adsorbed.

Furthermore, after washing away impure proteins with the same buffer solution,
i7 of the same buffer at 05-0.5M! A diluted solution was prepared, and the eluate was fractionated and recovered by passing the solution through it, and fractions with high specific activity were collected. It was diluted so that the conductivity was 10 m5 or more and passed through a blue Sepharose column. Transglutaminase was adsorbed by this operation. Further, impure proteins were washed away with 0.05M phosphate buffer (pH 7), and then a salt concentration range of 0 to 1M was created and the eluate was collected, and both fractions with high specific activity were collected. Concentrate using UF 6000 membrane to 0.
0.05M phosphate buffer containing 5M NaCl (pl-17
) was equilibrated using mild VJJ solution. lko.

1! ′? I got it! The condensed solution was passed through a column containing Sephadex Q-75 (Courtesy of Pharmacia Fine Chemicals! IJ) that had been equilibrated with the same buffer solution, and the same buffer solution was added to the column. The eluate was fractionated at 11 t. As a result, the active fraction was eluted as a single peak. Specific activity of this substance tJ
The recovery rate was 625 times that of the culture medium filtrate, and the recovery rate was 47%.

b) Production of BTG-2 In the same manner as in the case of BTG-1, Streptoverticillium glileocarneum IFO42776 was soaked and filtered with culture N at 30°C for 30 minutes to obtain culture solution 192.

The viscosity of this b was 0.28 LJ/+m.

[3T(,1) The enzyme was purified using the same method as S
IJi-enzyme was obtained by DS disk electrophoresis.

C) Production of BTG-3 In the same manner as in the case of BTG-1, Streptoverticillium cinnamoneum subsp. cinnamoneum I
l”012f152 was cultured at 30°C for 3 days and filtered.
18.5 p of culture solution was obtained. The enzyme activity of this product is 0.
It was 5u/m.

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

The present invention will be further explained below with reference to Examples.

Example 1 Defatted soybean flour (N3160%) 20Kg/to 1 between feeds, water alone or TGasc active 1g of soybean flour per person
Feed each enzyme aqueous solution containing 1.10.50.100u per F! To L8! ? /H into the hopper of a twin-screw extruder (screw diameter: 37 m, manufactured by Uniruna), and subjected to g-temperature and high-pressure treatment.

Note that TGaSe is the BTG named BTG-1.
Ase was used.

”°The conditions for the barrel exit are 200°C.
, screw rotation speed '250 p p m, feed material residence time in the barrel approximately 35 seconds, die size 1 willow φ×
It had 2 holes. .

When treated under these conditions, the temperature of the product was 177°C.

Dry the extruder-treated product at 60°C for 3 hours and
Under different conditions of G a sc activity! i1! Each organized tissue (also

Next, this mulch was crushed and divided into bulk portions, and the ID value for use in hamburger steaks was determined using the particle size classifications under the 6-mesh sieve and on the 12-mesh sieve.

The hamburger steak was made according to a common method used at home, by mixing minced beef and pork, onions, breadcrumbs, and whole eggs in a thermosynthetic mold and then heating. However, the livestock meat portion was 60% of the total, and the rehydrated products of each of the above-mentioned textured products were substituted for this. The replacement rate was set to 1/3 of meat.

The evaluation results for each hamburger steak are shown in Table 1. The evaluation was performed using panel N-10 on a 10-point scale, using only the meat containing no textured material as a control (scoring 5). Evaluation criteria: 10 points: Very good, 7 points: Fairly good, 5 points: Fair (1 to 1 roll),
3 points: Slightly bad, 1 fish: 11 Always bad.

(Effect of the invention) When using an extruder that converts protein-containing protein into a structured protein, it can be processed under relatively mild conditions compared to conventional extruder processing, resulting in a structured material with good flavor and color. (You can.

Furthermore, when the protein structure produced by the method of the present invention is added to livestock meat, ground meat, etc., it is possible to produce foods with excellent taste, texture, etc.

lMinistry of Industry Going to work engineering circle

Claims (1)

[Claims] (1) A method for producing a protein tissue product, which comprises adding transglutaminase to a protein-containing material and subjecting it to extruder treatment.