JPH02100654A - Novel ground krill meat and production thereof - Google Patents

Abstract

PURPOSE:To obtain the subject ground meat excellent in the ability to form boiled fish paste without affecting color, smell and taste of a product by adding a specific amount of a transglutaminase derived from a microorganism to a krill sinew. CONSTITUTION:A transglutaminase derived from a microorganism (preferably produced by a bacterium of the genus Streptoverticillium) in an amount of 0.1-700u/g protein, preferably 1-140u/g protein is added to a krill sinew preferably in steps after a leaching step to afford the objective ground meat.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel krill surimi obtained by allowing microorganism-derived transglutaminase to act on krill muscle, and a method for producing the same.

(Prior Art) Krill surimi is usually produced as follows. In other words, it becomes a raw material! After removing the head, internal organs, and shell from the krill, collecting the muscle parts and dehydrating them by exposing them to water,
Additives are mixed to make surimi. If desired, it is frozen to make frozen surimi.

The quality evaluation of 1q unfrozen or frozen surimi is usually
This is done by actually making a prototype of kamaboko from the fish paste, measuring its physical properties such as elasticity and denting using a machine, and adding sensory tests such as color, odor, and taste using Tokoro 9J.

Krill surimi is produced by the method described above, but its quality is currently very low compared to Juri surimi made from walleye pollock. There are many possible reasons for this, but one reason is that actomyosin (or myosin), the main component of krill, is hundreds of times more unstable and easily denatured than that of walleye. That is, this means that it is very difficult to control 1fflli in the surimi manufacturing process.

Another reason is that protease easily leaks into the muscle from the hepatopancreas of krill, and proteinase that has infiltrated into the muscle is difficult to remove.

Therefore, there is a need for a method for producing surimi that takes into consideration the instability of krill muscle proteins and the contamination of proteases, but there is currently no effective method. Therefore, it was necessary to compensate for these drawbacks with some kind of additive.

(Problems to be Solved by the Invention) Based on the above-mentioned circumstances, the present inventor has investigated various additives to improve the quality of the fourth fish fillet, especially the kamaboko-forming ability. We have not been able to find anything that can be added to foods without affecting the color, odor, or taste of the product, and that can be effective in small amounts.

(Means for Solving the Problems) Under the circumstances described above, the present inventor further continued his intensive research and discovered that transglutaminase (hereinafter referred to as
It is sometimes abbreviated as TGase. ) was found to improve the quality of krill surimi. That is, by adding TGasc during the surimi production process, surimi products'i! i (mainly related to elasticity, water retention, suppleness, and dents)) has been found to be significantly improved.

In addition, ordinary surimi contains phosphate as a quality improver (mainly used to give kamaboko fish cakes dents and elasticity).

) is often added, but we have learned that if TGasef is added as a substitute without adding phosphate, b, it is possible to produce lili-mi of almost the same quality as normal surimi.

Fumi also learned that TGase prevents freezing denaturation of surimi.

Based on this knowledge, the present invention was completed.

The addition of TGase is: - In the boat fishing eggplant production process,
Although it can be added at any step after the meat gathering process, it is practical to add it when mixing additives or bleaching with water, and by doing so, you can obtain great effects with simple operations. .

The fish meat paste obtained in this way can be handled in the same way as normal fish meat paste, and is kept frozen and distributed.

The method for producing unfrozen or frozen fish meat of the present invention is similar to that of conventional surimi, except that transglutaminase is not applied to the krill muscle in the steps after the meat collection step. Since this method conforms to the No. 11 manufacturing method, no particular explanation is required except for the steps mentioned above.

There are various types of transglutaminase depending on its origin, for example, transglutaminase isolated from guinea pig liver (hereinafter referred to as
(sometimes abbreviated as MTGase). The former MTGase is, for example, disclosed in Japanese Unexamined Patent Application Publication No. 58-1
vA can be produced by the method described in No. 49G4. The latter BTGase is the patent application No. 165067 of 1988.
The novel enzyme related to this issue is described in a separate section regarding its enzyme properties, selection method, etc. The transglutaminase used in the present invention is BTGase due to its enzymatic characteristics and availability at low cost in large quantities.

There is no difficulty in adding transglutaminase to krill muscle in the additive mixing process during the production process of krill surimi, and it is possible to add transglutaminase to krill muscle together with conventional additives.
It is preferable to add sC. Generally, sugars are added to surimi to maintain its quality, but 13TQas (
The effect of No. 3 is not reduced by the addition of sugars. Also, among the conventional additives, phosphate may be used as usual, or it may be partially or completely replaced by TGaSe, if desired.

The amount of BTGase added to the raw material krill muscle is 0.1
~70001 g protein, preferably 1-140 u/g protein. If the amount added is small, the amount of BTGase bound to the raw material krill muscle will be small and the effect will be small. Also,
It has been found that if the amount is too high, the effect of BTGase appears very quickly, making processing operations such as stirring and molding difficult, and the quality of the obtained processed product deteriorates.

In this addition v1 of BTGase, when the M speech property of BTGase is 2u/mlJ, 0.00% per 100 parts by weight of raw material krill muscle. oo1 to 5,000 tu parts, preferably 0.0
It corresponds to 1 to 1 parts of the enzyme, but the addition and ) fi should be adjusted depending on the degree of purification of the enzyme and the strength of the activity.

Unlike MTGase, BTGase is not dependent on a specific substance to express its activity, so it is often easier to use than MTGase.

To make transglutaminase act on krill muscles during the water exposure process, do the following. In other words, [3T G a
0.001 to 51 ffi parts of s e, preferably 0.001 to 51 ffi parts
Add 1 to 11 parts by weight, stir, dehydrate, and mix with additives to make surimi. There are no particular restrictions on the amount of water used, but it is desirable to use the same amount of water as the raw meat to 5 times as much.

Incidentally, most of the proteins contained in krill muscle surimi are actomyosin, but it also contains a small amount of water-soluble protein and matrix protein, forming a complex protein mixture system. are doing. According to conventional knowledge, the elasticity and water retention of paste products made from surimi are
It is known that it is strongly influenced by the properties of actomyosin.

Therefore, in the method for producing surimi of the present invention, BTG
Ase is thought to mainly act on actomyosin. That is, BTGaSe acts on free myosin, actin, and actomyosin bound to both,
It is presumed that the protein is cross-linked and polymerized. However, for proteins other than actomyosin, BTGas
Since it is quite conceivable that e is a Takeda effect, it is presumed that the quality of surimi is improved as a result of all of these effects being integrated.

(Novel transglutaminase BTGase) (1) Transglutaminase and its origin Transglutaminase ('rGase) is an enzyme that catalyzes the acyl transfer reaction of the remaining γ-carbo-1-diamide group of glutamine in the bebudid chain. be. When the ε-amino group of a lysine residue in a protein acts as an acyl acceptor, TGase generates ε-(γ-Glu)t-y within and between molecules.
s Kurihashi bond is formed. Also, when water functions as an acyl acceptor, it is an enzyme that progresses the reaction in which glutamine residue is deamidated to become glutamic acid residue.

Due to these properties of TGase, TGasC can be used to gel a protein 8 solution or slurry.

TGaSe has been known to be derived from animals such as guinea pig old origin (MTGase), but it is difficult to obtain animal-derived products cheaply and in large quantities. Enzyme 11 when gelling proteins
It is necessary to increase both 11113 and substrate concentration,
Furthermore, since it is Ca2'-dependent, its uses are limited.

The novel transglutaminase (B T
G a se ) can be produced by microorganisms, such as bacteria of the genus Streptoverticillium, but
There are currently no reports on TGase derived from microorganisms.

The microorganism-derived BTGase used in the present invention is available at low cost and is easily purified, so it is highly practical.

In addition, by using BTGase, the enzyme (BTGa
, 5c) It has the advantage that a gelled product of excellent quality can be produced at 9 degrees and at a very low substrate concentration.

■Production of BTGase The microorganism that produces BTGase is, for example, Streptoverticillium griseocalneum (3treptocalneum).
verticillium griseocarneu
m) IF012776, Streptoverticillium
3treptoverticillium cinn
amoncu+++ sub sp.

cinnamoneum) I F 012852, Strebtoverticillium limobaraens (Strep
tovert+c+IliumIIobaraense
) I F 013819 etc.

The culture method and M production method for culturing these microorganisms and obtaining transglutaminase are as follows.

Although both liquid culture and solid culture are possible as the culture form, it is advantageous industrially to perform deep aeration agitation culture. In addition, the culture sources to be used include carbon sources, nitrogen sources, inorganic salts, and other trace nutrient sources that are generally used for microbial culture, as well as any nutrient sources that can be used by microorganisms belonging to the genus Streptobercillium. Can be used. Carbon sources for the medium include glucose, ci:I &! In addition to l, lastagen, glycerin, dastrin, starch, etc., fatty acids,
Fats and oils, various types of fa, etc. can be used alone or in combination.

As the nitrogen source, either an inorganic nitrogen source or an if nitrogen source can be used, and examples of the inorganic nitrogen source include ammonium cum nitrate, ammonium sulfate, urea, sodium nitrate, and ammonium chloride. In addition, soybeans are organic nitrogen sources,
Examples include flour, bran, and defatted lees of rice, corn, and wheat, as well as cornstarch liquor, peptone, meat extract, casein, amino acids, and yeast extract. Inorganic salts and fine Ffi nutrients include salts such as phosphoric acid, magnesium, potassium, iron, calcium, and zinc, as well as vitamins, nonionic surfactants, antifoaming agents, and other substances that can inhibit bacterial growth and BTGase.
Any substance that promotes the production of can be used as necessary.

Cultivation is done under aerobic conditions. Temperature increases bacteria growth and BTGas
It is sufficient if the vi range produced by e is sufficient, preferably 25 to 3
The temperature is 5°C. The culture time varies depending on the conditions, but [3T
It is sufficient to culture until the time when Gasc is produced the most, which is usually about 2 to 4 days.

8 T Gas is dissolved in the culture solution in liquid culture, and is collected from the culture filtrate after removing the solid content from the culture solution after the completion of culture 1i.

To purify BTGase from culture filtrate, M f
Any method used for IaV can be used.

For example, ethanol, acetone, isobutylene-
Salting out with ammonium sulfate, common salt, etc.
Methods such as dialysis, ultrafiltration, ion exchange chromatography, adsorption chromatography, gel filtration, adsorbent, and isoelectric point fractionation can be used. In addition, if the degree of purification of BTGase can be improved by appropriately combining these methods, the methods can be combined as appropriate. Enzymes obtained by these methods can be subjected to ultrafiltration concentration, reverse osmosis concentration, vacuum drying, freeze drying, and spraying, with or without the addition of various salts, sugars, proteins, lipids, surfactants, etc. as stabilizers. Liquid or solid BTGase can be obtained by the drying method.

The activity of 13TQase was measured by reacting benzyloxycarbonyl-glutaminylglycine with hydroxylamine as a substrate in the absence of Ca2+, and forming an iron complex with the generated hydroxamic acid in the presence of trichloro[]acetic acid52! +n1ll absorption is measured, and the activity of hydrotetrasamic acid is determined from a calibration curve.

BTGase activity was measured by the method described in Section F unless otherwise specified.

<Activity measurement method> Reagent A 0.2M Tris-HCl buffer (Fll-16
, 0) 0.1M Hydroxylamine 0.01 M Reduced Glutathione 0.03 M Benzyl 1-dicarbonyl L-Glutaminylglycine Reagent 8 3N-Hydrochloric acid 12%-Trichloroacetic acid 5% FeCj! 6H20 (0.IN-11C1
Reagent B is a 1:1:1 mixture of the above solutions.

Add 0.5d of reagent A to 0.057 of the enzyme solution, mix and react at 37°C for 10 minutes, then add reagent B to stop the reaction and F.
After the e-complex formation, the absorbance at 525rv is measured. As a control, the absorbance of a similarly reacted product is measured using an enzyme solution that has been heat-inactivated in advance, and the difference in absorbance from the enzyme solution is determined. Separately, create a calibration curve using monoglutamic acid γ-monohydroxamic acid as a substitute for the enzyme solution, and determine the hydroxamic acid stones produced from the difference in absorbance.
Enzyme activity that produced 1 μmol of hydrotetasamic acid per minute was defined as 1 unit.

(■ Enzyme properties of BTGase Purified BTGase obtained as above, namely transglutaminase of Streptoveticillium mobans IF013819 (named BTG-1), Streptoveticillium griseoluneum IF Q 12
776 transglutaminase (named BTG-2)
The enzymatic chemical properties of the transglutaminase (named BTG-3) of Streptoverticillium cinnamoneum subsp. cinnamoneum IF Q 12852 are as follows.

a) Optimum emesis: 37°C when using benzyloxycarbonyl-lutaminylglycine and human [ ], Yacilamine as substrates.
, in a 10-minute reaction, the optimum pH of BTG-1 is between 6 and 7, and the optimum pH of 137G-2 is near 6 and 7.
The optimum pH of -3 is around 6-7.

b)￥ Suitable temperature: When using benzyloxycarbonyl-lutaminylglycine and hydroxylamine as 1g, p.16,
In a 10-minute reaction, the optimal temperature for BTG-1 was around 55°C, the optimal temperature for TG-2 was around 45°C, and the optimal temperature for BTG-2 was around 45°C.
The optimum temperature for TG-3 is around 45°C.

C) pt-1 stability: After treatment at 37°C for 10 minutes, BTG-1 was 11N 5-9
BTG-, 2 is stable in pt-1 5-9, BTG-311 1) H 6-9 1' stable-C Al.

d) Temperature stability: Ill-4 7 te 10 min 1 m 'IIL l'l te 1. i, B TG-1 Ha40'Ct'' has 88% activity remaining, 74% activity remains at 50℃, BTG-24
At 10°C, 86% activity remained; at 50°C, 4556% activity remained.
BTG-34J: 80% activity remains at 40°C, and 53% activity remains at 50°C.

C) Substrate specificity: Using each BTGase, the reaction between various synthesis IIs and hydroxylamine was investigated. Which BTGas
e also does not react when the synthetic substrate is benzyloxycarbonyl asparaginylglycine, benzyloxycarbonylglutamine, or glycylglutaminylglycine. However, the reactivity is the highest when the synthetic substance B is penzyloxypolonylglutaminylglycine. The concentration of various synthetic substrates at this time was 5g1M. 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 Δsp is an abbreviation for asparaginyl group.

Table-1 Table-2 f) Effect of metal ions: The effects were investigated by adding various metal ions to the activity measurement system at a concentration of 111M (the binding is shown in Table-2).
. The activity is affected by both BTGasebCu2"zn"c.

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

Both BTGases include parachloromercury-benzoic acid (abbreviated as PCMB), N-ethylmaleimide (NE
(abbreviated as M), the activity of which is inhibited by heptanoiodoacetic acid.

Table 3 In Table 3, PMSF is an abbreviation for noenylmethylsulfonyl fluoride.

h) Isoelectric point: As determined by ampholine isoelectric focusing, BT
The isoelectric point f) of C,-1 is around 9, the isoelectric point pi of BTG-2 is around 9,7, and the isoelectric point 1) of BTG3 is
[ is around 98.

) Molecular weight: As determined by SDS disk electrophoresis, BTG-
The molecular weight of BTG-1 is approximately 38,000, the molecular weight of BTG20 is approximately 41,000, and the molecular weight of BTG-3 is approximately 41,000.
,000.

j) Comparison with MTGase: Next, the properties of BTGaSe and guinea pig liver-derived transglutaminase (MTGasc) will be compared. Furthermore, M
TGase was prepared by the method described in JP-A-58-149645.

Table 4 shows a comparison of the chemical properties of each enzyme, and Table 5 shows Ca
The effect on 2+ activity is shown. As is clear from Tables 4 and 5, MTGa, which has been mainly studied in the past,
There are various differences in enzymatic chemical properties between se and actinomycete-derived BTGaSe, especially in temperature stability and molecular fJ.
, rj electric point, and substrate specificity. Also, Ca2
BTGase is clearly different from MTGase in that it acts both in the presence and absence of 4.

Therefore, each enzyme of BTGase is considered to have different properties from MTGase.

Table-4 Table-5 (4) BTGase (7) Iffi Example a) Production of B-rG-1 Streptoverticillium mobaraens IF0138
19, medium composition: polypeptone 0.2%, glycose 0
．． 5% dipotassium phosphate, 0.2% magnesium sulfate, and 0.1% magnesium sulfate. 20%, Lastagen 2.0%, Dipotassium phosphate 0.2%, Magnesium glaate 0.1%, Yeast extract 0.2%, Adekanol (trade name, Asahi Denka product) as an antifoaming agent 0. Medium 2ON (DH7) consisting of 0.05%
In addition, culture at 30°C for 3 days, slightly filtered, and culture solution 18. F+
I got it. The activity of this thing is 0.35u/jIi! There is r.

The culture solution was adjusted to I) H6,5 with hydrochloric acid, and 0.05M
CG5 equilibrated with phosphate buffer (pH 6,5)
0 (trade name, Organo Co., Ltd. product) column.

Transglutaminase was adsorbed by this operation.

Furthermore, after washing away impure proteins with the same buffer solution, a temperature gradient of 0.05 to 0.5M of the same buffer solution was created, and the eluate was fractionated and collected by passing through the solution, and fractions with high specific activity were collected. Collected. It was passed through a column of diluted blue Sephas so that the conductivity was less than 10 m5. With this operation, transglutaminase is adsorbed! ζ0 more 0.05M phosphoric acid! ! After washing away impure proteins with buffer solution (1) H7), 0-1M
We created a salt concentration scent of 100%, passed the solution through it, collected the eluate, and collected those with high specific activity. Concentrate using UF 6000 membrane,
0.05M phosphate buffer (pH 7) containing 0.5M sodium chloride
) and equilibrated with buffer.

The obtained concentrate was passed through a column containing Sephadex G-75 <Pharmacia Fine Chemicals Co., Ltd., which had been equilibrated with the same buffer solution in advance, and the eluate was fractionated by flowing the same buffer solution. As a result, the active fraction was eluted as a peak. The specific activity of this product was 625 times that of the liquid, and the recovery rate was 47%.

b) Production of BTG-2 In the same manner as in the case of BTG-1, Streptoverbucilium griseocalneum I F O1277f3 was added to 3
After culturing at 0°C for 3 days, the culture solution was filtered.

The activity of this product was 0.28 LJ/d.

'M element was purified in the same manner as in the case of BTG-1, and S
D S Fisk electrophoresis-C single enzyme was obtained.

c) Production of BTG-3 In the same manner as in the case of BTG-1, St. Butoverdicillium cinnamoneum subsp.
18.51 of a culture solution was obtained. The enzyme activity of this thing is 0.5
It was u/d.

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

Hereinafter, the present invention will be explained in detail with reference to Examples.

In this actual droplet example, BTG-1 was mainly used, but BTG-2 and BTG-3 were also used.
19 results were obtained that were almost the same as -1.

(Example) In the Examples, the kamaboko-forming ability was measured using a rheometer as follows, and the section is Jyushabu.

The elasticity and dent were measured using a rheometer and dent (manufactured by Fudo II Co., Ltd.) using a 5φ flange 17. The shape of the rimpu is 23mm in diameter and 30mm in height.
When the 1III110 Plunge V- is pushed into the kamaboko, the force required for the kamaboko to rupture is expressed as the elasticity (y), and the distance the plunger moves until the kamaboko breaks is expressed as the concavity (+11 m).

When water is added to the water-retaining shell, the elasticity and dents of the kamaboko decrease. Add water to each piece and adjust the elasticity to about 800g. It is assumed that the more water W added at this time, the higher the water retention capacity of the surimi.

Example 1 Remove the head, internal organs, and shell from 100 parts of krill immediately after landing, collect the remaining muscle (corresponding to the tail meat of krill, and call it shucked meat), add three times the amount of clean water as the shucked meat, and After stirring for 3 minutes, the mixture was dehydrated. Sugar 4 as an additive to this dehydrated meat
parts, 4 parts of sorbitol, 0.2 parts of phosphate (from a 1:1 mixture of tripolyphosphate and pyrophosphate), t3
0.02 part of TG-i was added, mixed, and frozen to obtain frozen surimi. The control did not contain BTG-1.

(b) After meals for 100 parts of both prototype surimi,
It was heated in a 90°C water bath for 20 minutes and cooled with water. The product thus obtained was a type of kamaboko, and all physical properties of this product were measured.

(b) On the other hand, both of the trial surimi were frozen at -30°C to obtain frozen surimi, stored at -20°C for one month, thawed, and prepared kamaboko in the same manner as in (a);
The physical properties of this product were measured.

Table 1 Table 2 From the results in Table 1, the surimi to which B'rG-1 was added had smooth edges and dents, and was of desirable quality.

Example 2 The shelled meat was obtained by the method of Example 1 immediately after landing and stored at a temperature of 2 to 30°C for 4 hours, and 6 specimens of minced meat were added with BTG-1 according to Example 1. is.

The quality of each surimi is shown in Table 2.

From this result, it can be seen that it is desirable that the storage temperature of Oni 1 Ami is lower, preferably 10° C. or lower.

Example 3 The following additives were added to the dehydrated krill meat obtained in Example 1 to produce a well-mixed surimi, which was stored at 20° C. for one month, then thawed and its quality was measured.

Sorbitol with Rinple Number Sorbitol Table - 3 Addition 8 parts 8 parts, Phosphorus M salt 0.2 parts 8 parts, BTGo, 02 parts Add kamaboko 31' in the same way! ! L., compared their quality. The results are shown in Table 4.

Table 4 Example 4 Frozen surimi of the present invention produced by the method of Example 1 Same <
After storing for one month at -20℃, add 40 parts of water to 100 parts of surimi, add 3 parts of salt to the total weight, and stir well with a shukumasu Iff L, actual flow example. Kamaboko was prepared according to 1 (a) and the quality was evaluated.

Furthermore, from the results of 5 or more items of thawed condensed water in the frozen surimi of the control, it became clear that the surimi of the present invention retained more water than the control.

(Effects of the Invention) The present invention has made it possible to produce completely new krill surimi compared to conventional krill surimi. In other words, water droplets have been found to significantly improve the quality of krill surimi, and will provide one of the effective ways to use krill resources in the future.

Within procedural amendments

Claims (7)

[Claims] (1) A method for producing krill surimi, which comprises adding microorganism-derived transglutaminase at 0.1 to 700 u/g protein to krill muscle. (2) The method for producing krill surimi according to claim 1, characterized in that microorganism-derived transglutaminase is added to the krill muscle in a step after the water exposure step. (3) The method for producing krill surimi according to claim 1 or 2, wherein the temperature of the meat is maintained at 10° C. or lower in the surimi production process. (4) The method for producing krill surimi according to claim 1 or 2, characterized in that the raw material is krill that has been caught within 8 hours. (5) The method for producing krill surimi according to claim 1 or 2, characterized in that no phosphate is used as an additive. (6) The method for producing krill surimi according to claim 1, wherein the transglutaminase is produced by a bacterium of the genus Streptoverticillium. (7) A novel krill surimi produced by the method according to any one of claims 1 to 6.