JPH09206075A - Low-temperature protease, microorganism producing the same protease, production of the same and softening of meat by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new low-temperature protease capable of keeping high activity even under low temperature conditions and finishing meat so as to be soft and juicy in acting on low-temperature meat and useful as a blending component for detergent compositions. SOLUTION: This low-temperature protease is obtained from culture of Bacillus sp. KSM-506 (FERM P-15326), etc., and has the following enzymatic properties; (1) optimal pH about 7 and 5-9 acting pH range; (2) about 20-30 deg.C optimal temperature and reacting at 0-45 deg.C and stable until 25 deg.C under treating condition of pH7.0 for 20min; (3) 35,000±1,000 estimated molecular weight; (4) 6.0-6.5 isoelectric point; (5) strongly inhibited by EDTA, etc., of chelating agent and completely not influenced by PMSF (phenylmethanesulfonyl fluoride).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel low temperature protease, a microorganism producing the same, a method for producing the low temperature protease, and a meat softening method using the same.

[0002]

BACKGROUND OF THE INVENTION Proteases are a general term for a group of enzymes that catalyze the hydrolysis of peptide bonds, and are widely distributed in microorganisms, animals and plants. Its applications include laundry detergent, automatic dishwashing detergent, contact lens cleaner,
Bath agents, cosmetics for exfoliating, food modifiers (baking, meat softening, seafood processing), beer fining agents, leather tanning agents,
There are gelatin removers for photographic films, digestive aids, and anti-inflammatory agents, and they have been widely used in various fields.

[0003] Among them, the most industrially produced in large quantities and having a large market size are proteases for detergents, such as Alcalase, Savinase (manufactured by Novo Nordisk), Maxacar (manufactured by Gist Brocades), API-21.
(Showa Denko KK), Blap (Henkel KK), Protease K (KAP; Kao KK) and the like are known.

On the other hand, as a conventional method for modifying meat, such as softening the meat or improving the preservability of the meat,
A method using an enzyme is known (Japanese Patent Laid-Open No. 63-21 / 1988).
9351 gazette, JP-A-4-278063, JP-A-5-7476, and JP-A-5-252911). However, most of the above-mentioned detergent proteases have an optimum temperature on the high temperature side, and thus it is hard to say that the enzyme characteristics are sufficiently exerted when tap water is used as it is to wash clothes and the like in a low temperature range. . Also, in enzymes used for meat modification, etc., the optimum temperature of commonly used plant-derived enzymes is 50 to 70 ° C., and from the viewpoint of food hygiene, the temperature is kept in a low temperature range (20 ° C. or lower) until just before cooking It cannot act sufficiently on the meat to be placed, and the satisfactory modification has not been obtained.

[0005]

Therefore, an object of the present invention is to provide a protease which retains high activity even under low temperature conditions, a microorganism producing the same, a method for producing the same, and a method for softening meat using the same. It is a thing.

[0006]

In such an actual situation, the present inventors sought in nature the protease that sufficiently acts in the low temperature region, and as a result of searching, found a microorganism secreting a protease having an activity also in the low temperature region, Further, they have found that the obtained protease has an excellent activity in a low temperature region, and that when it is applied to meat at a low temperature, it is soft and juicy, and the present invention has been completed.

That is, the present invention provides a low temperature protease having the following enzymological properties, a microorganism producing the same, a method for producing the protease and a meat tenderization method using the same.

1) Working temperature and optimum temperature It works at 0 to 45 ° C, and the optimum temperature is about 20 to 30 ° C. 2) Temperature stability It is stable up to 25 ° C under the treatment conditions of pH 7.0 and 20 minutes. 3) Working pH and optimum pH The working pH range is 5-9, and the optimum pH is around 7. 4) Molecular weight Sodium dodecyl sulfate (SDS) -Polyacrylamide gel electrophoresis estimated molecular weight is 35000 ± 10.
00. 5) Effect of metal ions It is slightly inhibited by Cu ²⁺ and Hg ²⁺ ions. 6) Inhibitors ethylenediaminetetraacetic acid (EDTA), ethyleneglycolbis (2-aminoethylether) tetraacetic acid (EGT
It is strongly inhibited by A) and is not inhibited by phenylmethanesulfonyl fluoride, monoiodoacetic acid, diisopropylfluorophosphate.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The low temperature alkaline protease of the present invention can be produced, for example, by culturing a protease-producing bacterium belonging to the genus Bacillus and collecting it from the culture.

The protease-producing bacterium of the present invention belonging to the genus Bacillus is not particularly limited as long as it belongs to the genus Bacillus and produces the above-mentioned protease of the present invention. For example, the KSM-506 strain showing the following mycological properties is mentioned. To be

(A) Morphological properties (a) Cell size: 0.8 × 3-5 μm bacilli (b) Polymorphism: None (c) Motility: + (d) Spores: 0. 6-0.8 × 0.8-1.0 μm oval, no swelling of sporangia (e) Gram stainability: Positive (f) Anti-acidity: None (g) Growth morphology on broth agar plate: growth Is good, rim is leafy (h) Growth on broth agar slope: growth is good, rim is leafy (i) Broth liquid culture: good growth (j) Gelatin stab culture: good growth, liquefaction Yes (k) Litmus milk: grows and liquefies

(B) Physiological properties (a) Reduction of nitrate: positive (b) Denitrification reaction: negative (c) MR test: negative (d) VP test: negative (e) Indole formation: negative (f) Sulfide Hydrogen generation: Negative (g) Starch hydrolysis: Positive (h) Use of citric acid: Negative (i) Use of inorganic nitrogen source: Use of nitrate is not allowed, Use of ammonium salt is not allowed (j) Generation: Growth but no pigment production (k) Urease: Negative (l) Oxidase: ± (m) Catalase: Positive (n) Growth temperature range: 10-50 ° C (o) Growth pH range: 6.0 9.0 (p) Oxygen attitude: Aerobic (q) OF test (Hugh Leifson method): Oxidized form (r) Tolerance to sodium chloride: Can not grow at a salt concentration of 10% (s) Lysine hydrolysis: Negative (T) Ornithine water Solution: negative (u) Arginine hydrolysis: positive (v) Liquefaction of gelatin: Positive (w) Esculin hydrolysis: positive (x) DNAase: negative (y) generated and the gas generating acid from sugars:

[0013]

[Table 1]

As is clear from the above results, it is appropriate that the present strain belong to Bacillus firmus, but it is different in some respects and different from other known strains. Therefore, this strain was used as described above for Bacillus sp . KSM-5.
Named 06, F at the Institute of Biotechnology, Institute of Biotechnology, AIST
Deposited as ERM P-15326.

To obtain a low temperature protease using this strain, the cells may be inoculated into a medium and cultured according to a conventional method.

As the medium to be used, any medium can be used as long as it can be used as a medium for ordinary microorganisms and the cells can grow. It is preferable to include an appropriate amount of nitrogen source.

The carbon source and the nitrogen source are not particularly limited, but examples of the carbon source include assimilable carbon sources such as arabinose, xylose, glucose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, and the like. Examples thereof include mannitol, inositol, glycerin, soluble starch, inexpensive molasses, invert sugar and the like, and assimilable organic acids such as acetic acid. As the nitrogen source, corn gluten meal, soybean powder, corn steep liquor, casamino acid, yeast extract,
Pharmamedia, sardine meal, meat extract, peptone, high pro, adipower, corn meal, soybean meal, coffee meal, cottonseed oil meal, cultivator, amiflex and adipron, zest, azix and the like can be mentioned. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , M
Inorganic salts such as n ²⁺ , Zn ²⁺ , Co ²⁺ , Na ⁺ , and K ⁺ , and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Collection and purification of the target low-temperature protease from the culture can be carried out according to general enzyme collection and purification means. That is, cells are separated from the culture by centrifugation, filtration, or the like, and the culture filtrate is subjected to a conventional separation means, for example, a salting-out method, an isoelectric focusing method,
Protein precipitation by solvent precipitation method (methanol, ethanol, isopropyl alcohol, acetone, etc.),
It can also be obtained by concentrating by ultrafiltration.

The KSM-506-derived low-temperature protease thus obtained has the following enzymatic properties.

(Enzymatic properties) (1) Effect of inhibitor This enzyme solution was added to Tris buffer (pH 7.0) containing various inhibitors so that the concentration was 1 mM, and the mixture was allowed to stand at 25 ° C for 20 minutes. The residual activity was measured. The residual activity of the treated group was determined by setting the enzyme activity treated in the same manner without adding an inhibitor as 100%. Table 2 shows the results.

[0021]

[Table 2]

From Table 2, chelators EDTA and E
It is considered to be a metalloprotease because it is strongly inhibited by GTA and is not affected by serine enzyme inhibitors such as PMSF at all.

(2) Effect of metal ion The present enzyme solution was added to Tris buffer (pH 7.0) containing various metal salts added to 2.5 mM, and the residual activity was allowed to stand at 25 ° C. for 20 minutes. It was measured. The residual activity of the treated group was determined by setting the enzyme activity treated in the same manner without addition of a metal salt as 100%. The results are shown in Table 3.

[0024]

[Table 3]

From Table 3, this enzyme activity is shown to be Cu ²⁺ and Hg ^2+.
A slight inhibitory effect on ions is observed.

(3) Optimal pH The buffer solution for activity measurement was changed to a buffer solution having various pH values of 0.1 M, and the reaction was carried out at 25 ° C. for 15 minutes. As shown in FIG. 1, the result shows that the optimum pH of this enzyme is around 7. In addition, pH 4.0-6.0 is an acetate buffer solution, pH 6.0-8.0
Is phosphate buffer, pH 7.5-9.0 is Tris buffer, pH
Glycine-NaOH buffer was used for 9.0 to 10.0.

(4) Temperature stability This enzyme was added to 100 mM Tris buffer (pH 7.0), heat-treated at each temperature for 20 minutes, and then ice-cooled. Residual activity was determined at 25 ° C using azocasein as a substrate, and the results are shown in Fig. 2.
It was shown to. It can be seen that this enzyme is stable up to 25 ° C. (90% remains).

(5) Optimum temperature The reaction was carried out for 20 minutes using 0.1 M phosphate buffer pH 7.0 at each temperature in the range of 5 to 75 ° C. The temperature showing the highest activity was determined as the optimum temperature. As is clear from FIG. 3, the optimum temperature of this enzyme was 20 to 30 ° C.

(6) Molecular Weight The molecular weight of this enzyme was measured by SDS-polyacrylamide gel electrophoresis. The molecular weight markers include phosphorylase b (molecular weight: 94,000), bovine serum albumin (molecular weight: 67,000), and egg white ovalbumin (molecular weight: 4) of a low molecular weight marker kit (Pharmacia).
3,000), carbonic anhydrase (molecular weight:
30,000), soybean trypsin inhibitor (molecular weight: 20,100), γ-lactalbumin (molecular weight:
14,400) was used. As a result, a single band was obtained and its molecular weight was estimated to be 35,000 ± 1,000.

(7) Isoelectric point The isoelectric point of the present protease was determined by the isoelectric focusing method. The pH gradient of acrylamide gel (6.0%) is based on the amphoteric electrolyte Pharmalyte (pH 3-1).
0; manufactured by Pharmacia) was used. As a result, the isoelectric point of this protease was found to have two major bands at pH 6.0 to 6.5.

In the present invention, the above-mentioned method or Japanese Patent Laid-Open No.
The enzyme solution obtained by the method described in JP-A-3-219351 can be used as it is for meat, but it can also be concentrated and purified by a known method in order to increase the specific activity. It is also possible to freeze-dry the enzyme solution and mix it as an enzyme powder. By freeze-drying, the enzyme activity can be kept stable for a long period of time, and it can be widely used.

The protease's proteolytic activity was determined using azocasein as a substrate. That is, 0.2 ml of an enzyme solution appropriately diluted with 0.8 ml of 0.1 M phosphate buffer (pH 7.0) was added, and the mixture was incubated at 25 ° C., and then 1 ml of a 2% azocasein solution was added to start the reaction.
After reacting for 20 minutes, the reaction was stopped by adding 2 ml of a 5% TCA solution. The reaction solution was filtered with filter paper and the absorbance at 440 nm was measured. One unit of the enzyme amount was an amount that produced 1 μmol of the azo dye in the trichloroacetic acid-soluble portion in 1 minute.

The meat softening method of the present invention can be carried out by directly coating the low temperature protease or a mixture of the low temperature protease and the optional ingredients directly on the meat pieces, and by spraying the meat pieces. It is preferable to appropriately adjust the form of use according to the circumstances. The amount of the low temperature protease that acts on the meat piece is not particularly limited and is determined by the amount of meat when used. That is, it is preferable that the low temperature protease act on 0.5 to 5.0 units, particularly 1.0 to 3.0 units, per 1 g of meat.

As other optional components, for example,
Examples include starches, proteins, sugars, seasonings and the like. By using these components, for example, when the low temperature protease is in a liquid state, the softening agent can be made into a powder form by a method of including it in the components and spraying it. Further, examples of the usage form of these include the following forms.

(1) Used by being mixed with a powder base material such as fried chicken powder and powder seasoning. (2) It is used by being mixed with a liquid cooking oil or fat such as soybean oil or corn oil. (3) Used by blending with refined lard, shortening and other plastic oils and fats. (4) Used by blending with a W / O emulsion composition such as margarine. (5) Used by blending with an O / W emulsion composition. (6) Dashi, used by mixing with liquid seasonings such as sauce.

The meat softening method of the present invention uses animal meat such as beef, pig and sheep, chicken meat such as chicken, turkey, duck and goose, or fish meat such as white fish such as horse mackerel, salmon, cod and flounder. It is effective when cooking, but it is particularly effective for meat and poultry. Further, as a meat part, it is effective when a part containing a lot of relatively hard proteins such as kata and peach is used. Further, as the form of meat, as described above, there is a shape as a piece of meat (thick cut meat, thin cut meat,
Minced meat) is particularly effective when used, but in particular, pieces of meat with a certain size (such as steak, meat for grilled meat, meat for fried, fried or teriyaki, meat for salt grilled, mackerel, or munière) The effect is great when using.

In addition, since the meat tenderizing method of the present invention is sufficiently effective when treated at low temperature for a short time, it can be used for room temperature conditions (preferably 0 to 30 ° C.) for meat pieces refrigerated or thawed after frozen storage. The treatment may be performed for a short time (preferably 1 to 30 minutes, more preferably 1 to 15 minutes). Then, the meat piece may be cooked after such treatment. As the heat treatment method, any cooking method such as baking, frying, frying, boiling or steaming may be used, and particularly a remarkable effect is obtained in cooking such as baking or frying.

The meat product containing the meat pieces treated by the meat softening method of the present invention may be already cooked (cooked food) or cooked to be cooked at the time of eating. It may be one (uncooked food). That is, in the cooked food, it is sufficient that it is treated with the low temperature protease in the heating step when producing it, while in the unheated food, the low temperature protease acts during the heating cooking when eating. As described above, the food may be previously treated such as adhering. The product containing the piece of meat treated with the low temperature protease of the present invention retains the tenderness and juiciness of meat even after storage and has a good flavor.

In the present invention, the meat piece means a meat having a relatively large shape (eg, meat cut from raw meat such as livestock meat).
The surface area of a piece of meat is about 1 cm ² or more). Therefore, a piece of meat (for example, hamburger) formed by connecting minced meat and shaping it is not meant.

[0040]

EFFECTS OF THE INVENTION Since the low temperature protease of the present invention has an optimum action temperature in a low temperature region, for example, by simply treating a piece of meat immediately after freezing and thawing at room temperature for a short time, the meat after cooking can be softened It can be finished juicy without impairing the flavor. Further, the present enzyme can be advantageously used at low temperature as a blending component of a detergent composition.

[0041]

EXAMPLES Next, the present invention will be described more specifically by way of examples, which are merely examples and do not limit the present invention.

Example 1 (Production of Low Temperature Protease) 20 ml of sterilized artificial seawater was added to the shellfish White bai that lived in the Southern Ocean and crushed with a homogenizer. The resulting suspension was smeared on various artificial sea horizontal plate media and cultured at 20 ° C. for 72 to 96 hours. The composition of the artificial seawater medium used was as shown below, and skim milk was added to determine the production of protease. After culturing, a colony having a transparent zone formed due to the decomposition of skim milk was selected around the grown colony.

[0043]

[Table 4] Artificial seawater medium (pH 7.6) (ingredient) (blending amount) Bactopeptone (manufactured by Difco) 2.5 g Yeast extract (manufactured by Difco) 1.25 g Meat extract (manufactured by Difco) 1.25 g Glucose 0.5g Agar 20.0g Artificial seawater (Jamarin Laboratory) 1000ml

The Bacillus sp. KSM-5 obtained
06 was brought into contact with the liquid medium shown below and cultured at 5 ° C. for 96 hours to produce the present enzyme.

[0045]

[Table 5] Liquid culture medium (pH 7.6) (ingredient) (blending amount) Bactopeptone (manufactured by Difco) 5 g Yeast extract (manufactured by Difco) 2.5 g Glucose 10 g Artificial seawater (manufactured by Yasu Pharmaceutical) 1000 ml

After completion of the culture, the obtained culture solution was centrifuged (8,000 rpm, 5 minutes) to remove the cells. After salting out by adding saturated ammonium sulfate to the main culture supernatant, gel filtration chromatography using GCL-300m as a carrier, anion exchanger chromatography using QAE-Toyopearl, PHENYL-C.
As a result of purification to a single band by SDS electrophoresis by hydrophobic chromatography using ellulofine and anion exchanger chromatography using Mono Q, the specific activity increased by 643 times, and the activity recovery rate was 5%. %Met.

The low temperature protease had the above-mentioned enzymatic properties.

Example 2 (Meat Softening Test of Low Temperature Protease) Using the above enzyme powder, imported beef from Australia, peach meat (100 g)
The softening experiment was performed. Place about 20 g of meat sliced in a meat slicer to a thickness of 5 mm in a refrigerator (4 ° C.), sprinkle 12.5 units of enzyme powder on it and sprinkle with 10 g of meat, cover with wrap, and keep in the refrigerator for a certain period of time. I left it. Then, on a hot plate at 200 ° C for 1 minute on one side,
After returning, it was baked for 45 seconds, cut into a strip having a width of 3 cm, and the cutting stress (SFV) was measured with a meat shear. As a control, papain powder (manufactured by Amano) was used. The results are shown in FIG.

As shown in FIG. 4, the enzyme showed proper softening after 5 minutes and kept in good condition for 1 hour thereafter. On the other hand, when using papain to obtain a softening effect at a low temperature in a short time (within at least 5 minutes), 10
It was necessary to treat with more than 10 units of enzyme per g of meat, and even 5 minutes of papain 2 units / 10g meat caused bitterness on the surface before softening, and became unusable due to flavor. .

[Brief description of drawings]

FIG. 1 is a diagram showing the relationship between pH and relative activity of a low temperature protease.

FIG. 2 is a graph showing the temperature and stability of cold protease.

FIG. 3 is a diagram showing an optimum temperature of a low temperature protease.

FIG. 4 is a diagram showing the relationship between the treatment time of low temperature protease and the cutting force.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Hirofumi Takigawa Inventor Hirofumi Takigawa 20 Kasuga-gun, Kashima-gun, Ibaraki 20 Kaiga Co., Ltd. (72) Inventor Keiichi Watanabe 2-11-542 Wakakusu, Saga, Saga Prefecture

Claims (5)

[Claims] 1. A low temperature protease having the following enzymatic properties. 1) Working temperature and optimum temperature It works at 0 to 45 ° C, and the optimum temperature is about 20 to 30 ° C. 2) Temperature stability It is stable up to 25 ° C under the treatment conditions of pH 7.0 and 20 minutes. 3) Working pH and optimum pH The working pH range is 5-9, and the optimum pH is around 7. 4) Molecular weight Sodium dodecyl sulfate (SDS) -Polyacrylamide gel electrophoresis estimated molecular weight is 35000 ± 10.
00. 5) Effect of metal ions It is slightly inhibited by Cu ²⁺ and Hg ²⁺ ions. 6) Inhibitors ethylenediaminetetraacetic acid, ethylene glycol bis (2
Strongly inhibited by -aminoethyl ether) tetraacetic acid and not inhibited by phenylmethanesulfonyl fluoride, monoiodoacetic acid, diisopropylfluorophosphoric acid. 2. A microorganism which belongs to the genus Bacillus and produces the low temperature protease according to claim 1. 3. Bacillus sp. K
The microorganism according to claim 2, which is named SM-506 and is deposited as FERM P-15326. 4. The method for producing a protease according to claim 1, wherein the microorganism according to claim 2 is cultured, and the low temperature protease is collected from the culture. 5. A meat tenderization method using the low temperature protease according to claim 1.