JPH0870818A - Flesh improving agent - Google Patents

Abstract

PURPOSE: To obtain a flesh modifying agent useful for softening edible meat rich in muscle and tendon and bettering a taste, containing a protease acting only on hard protein such as muscle and tendon. CONSTITUTION: This flesh improving agent contains masikinolysin, a protease obtained by culturing Streptomyces griseoloalbus SN-22 strain (FERM P-13,837). The protease has the following properties. (1) Hydrolyzing peptide bonds by CO2 H group of L-Val and L-Ala and cleaving bonds between A1a10-Ala11, Val29-Cys30, Ala32-Cys33, Ala42-Thr43, Ala90-Ser91 and Val109-Ala110 when a reduced alkylated lysozyme is used as a substrate. (2) Optimum pH9.0 and action pH6-11. (3) Optimum temperature is 45 deg.C and an action temperature range is 30-60 deg.C. Hard protein in meat texture can be specifically hydrolyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a meat quality improving agent for softening hard proteins such as lines and tendons in meat tissue to improve the taste of meat. More specifically, it relates to a meat quality improving agent containing a protease that specifically hydrolyzes a hard protein in meat tissue.

[0002]

2. Description of the Related Art As a method of tenderizing meat, it has been known to act plant-derived proteases such as papain, bromelain, and melon-derived proteases (New Food Industry, vol. 36,
No. 2, p53-56 (1994)). Recently, a method of softening meat by combining at least one kind of enzyme selected from proteases such as papain, peptidase, amylase and collagenase with elastase produced by an alkaline Bacillus bacterium has been proposed (Japanese Patent Laid-Open No. Hei 10 (1999) -135242). 5-276899).

Meat is composed of muscle cells composed of myofibrillar proteins and connective tissues which are hard proteins such as streaks and tendons. Myocytes are softened by the action of endopeptidases during aging (Chemistry and Biology, vol.32, No.4, p.
229-237 (1994)), the hard proteins elastin and collagen are not affected by these enzymes and do not soften. When papain, bromelain, etc. are applied to meat, the meat is certainly softened, but the effect is not specific and also acts on muscle cells, and the meat is broken or dissolved. Therefore, the appearance of an enzyme that does not act on muscle cells and specifically softens streaks and tendons is expected. Elastase (Bi produced by alkaline Bacillus bacterium)
ochim. Biophys. Acta, vol. 88
3, p439 to 447 (1986)) has a strong elastin decomposing ability and is considered to be one solution, but this enzyme has a relatively wide substrate specificity and decomposes not only elastin but also meat protein, resulting in deterioration of meat quality. Was a problem.

[0004]

DISCLOSURE OF THE INVENTION According to the present invention, by using an enzyme having a very high substrate specificity and acting only on a hard protein, the meat containing many streaks and tendons is softened and the muscle cell protein is decomposed. A meat quality improver capable of improving the quality of lean meat.

[0005]

Means for Solving the Problems The inventors of the present invention have been earnestly studying a method of softening low-grade meat containing various hard proteins and having many streaks and tendons using various proteases. The present inventors have completed the present invention by finding that a protease produced by a certain actinomycete specifically decomposes hard proteins and hardly decomposes other myofibrillar proteins.

That is, the present invention is a meat quality improving agent containing a protease having the following properties, and more specifically, it contains the protease produced by Actinomyces SN-22 strain, Masikinolysin. It is a meat quality improving agent. 1) Action: Hydrolyze the peptide bond by the carboxyl group of L-valine and L-alanine. 2) Substrate specificity: Ala10-Ala11, Val29-Cy when reductive alkylated lysozyme is used as a substrate.
s30, Ala32-Cys33, Ala42-Thr
43, Ala90-Ser91, Val109-Ala
Cut between 110. 3) Working pH range and optimum pH: When measured at 37 ° C. using succinyl-alanyl-alanyl-alanyl-paranitroanilide as a substrate, the working pH range is 6 to 11, and the optimum pH is 9.0. is there. 4) Working temperature range and optimum temperature: When succinyl-alanyl-alanyl-alanyl-paranitroanilide is used as a substrate and measured in a glycine / sodium hydroxide buffer solution of pH 10.0, the working temperature range is 30 ° C to 60 ° C. C. and the optimum temperature is 45.degree.

5) pH stability: When the residual relative activity after holding at 37 ° C. for 40 minutes was measured, the stable pH range was p.
H8-11. 6) Temperature stability: When the residual enzyme activity after holding at pH 9 for 1 hour was measured, it was stable up to 10 to 40 ° C.
It deactivates at 0 ° C. 7) Inhibitor: Inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, but not microbial alkaline protease inhibitor, elastinal, parachloromercuric benzoic acid, ethylenediaminetetraacetic acid. 8) Isoelectric point: The isoelectric point (pI) by the isoelectric focusing method is 6.4. 9) Molecular weight: The molecular weight measured by SDS-polyacrylamide gel electrophoresis is 26,000 daltons.

The mycological properties of the actinomycete SN-22 strain which produces the protease, mashiquinolysin, contained in the meat quality improving agent of the present invention are described below. 1. Morphological characteristics Aerial hyphae are produced from basal hyphae grown on agar medium, which grow well and form spore chains at the tips of linear hyphae. The spore surface is smooth and the size is 0.5-0.7μ in diameter.
m, and the length is 1.0 to 1.5 μm.

2. Growth state on various media (1) Tryptone / yeast extract agar medium (ISP-1)
Growth is moderate, and a small amount of white aerial hyphae are settled. Basal hyphae are white with no formation of melanoid or soluble pigments. (2) Growth on malt extract / yeast extract agar (ISP-2) is vigorous, and white aerial hyphae may be settled, and reddish spores may be settled on the tip. Basal hyphae are light brown in color with no formation of melanoid or soluble pigments. (3) Growth on oatmeal agar (ISP-3) is similar to ISP-2, but produces a purple soluble pigment. (4) Growth on starch / salt agar medium (ISP-4) is I
Similar to SP-2 but slightly yellow soluble pigment is produced. (5) Glycerol-asparagine agar medium (ISP-
Growth in 5) is similar to that of ISP-2, but the spores are white and the basal hyphae are colorless and produce a brown soluble pigment. No melanin pigment is produced in medium containing tyrosine

3. Growth temperature range The growth temperature range is 15 to 35 ° C and the optimum growth temperature is 26.
~ 30 ° C. 4. Utilization of various carbon sources As a result of examination using a medium of Predham Gottlieb, D-xylose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose,
Utilizes D-mannitol and not raffinose.

5. Cell wall component Cell wall component was examined using a decomposed product of whole cells, LL
-Contains diaminopimelic acid with galactose, mannose, ribose, rhamnose and a small amount of glucose. From these results, Le Chevalier (International Journal of Systematic Bacteriology (Int. J. Syst. Bact.) Vo)
l. 20, p435-443 (1970)) type I
Belonged to the cell wall.

[0012] Int. J. Sy
s. Bact. , Vol. 18, p69 to 189, p2
79-392 (1968), ibid. 19, p391
~ 512 (1969), ibid. 22, p265-3
94 (1972) and searched for this strain as Streptomyces.
Griseoalbus) SN-22.
This strain has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, as strain No. 13837 (FERM P-13837).

The protease, mashiquinolysin, contained in the meat quality improving agent of the present invention is produced by inoculating the above strain into a nutrient medium and aerobically culturing. The method for culturing the above-mentioned strain is basically similar to the method for culturing general microorganisms,
Usually, it is suitable to carry out under aerobic conditions such as shaking culture by liquid culture and aeration stirring culture.

The medium used for culture may be any medium containing a nutrient source that can be utilized by microorganisms belonging to the genus Streptomyces, and various synthetic media, semi-synthetic media, natural media, etc. may be used. You can As a medium composition, glucose as a carbon source, sucrose, fructose, glycerin,
Dextrin, starch, molasses and the like can be used alone or in combination. Pharmamedia as a nitrogen source,
Organic nitrogen sources such as peptone, meat extract, soybean flour, casein, amino acids, yeast extract, malt extract, urea and the like, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can be used alone or in combination. In addition, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, and cobalt chloride, heavy metal salts, vitamins such as vitamin B and biotin can be added as required. . When foaming is remarkable during the culture, various known antifoaming agents can be appropriately added to the medium.

The culture conditions may be appropriately selected within the range in which the above-mentioned strain can grow well and produce maxiquinolysin. For example, the pH of the medium is preferably about 5 to 9, usually around neutral. The culture temperature is preferably maintained at a temperature at which the microorganism grows well, usually 26 to 30 ° C., and the culture time may be about 3 to 5 days.

Since Mashquinolysin is an extracellular enzyme, the bacterial cells are removed by filtration or centrifugation after the culture is completed, and the supernatant is used as a crude enzyme solution. The crude enzyme solution can be used as it is as a meat quality improving agent, or a salting-out precipitate with ammonium sulfate or a precipitate with ethanol, acetone or the like can be obtained and used as a dry powder. Further, it can be used after further enhancing the activity purity by a known enzyme purification means such as ion exchange chromatography or gel filtration chromatography.

For example, it can be purified as follows. First, bacterial cells are removed from the culture solution by filtration or centrifugation to obtain a supernatant. Ammonium sulfate is added to this to form a precipitate. The precipitate is dissolved in water, dialyzed, and then purified by DEAE cellulose column chromatography, then butyl Toyopearl 650S column chromatography, and Sephadex G-75 gel filtration to a single band by disc gel electrophoresis and gel filtration chromatography. be able to.

The enzymological properties of mashiquinolysin precipitated from the crude enzyme solution and purified by various chromatographies are as follows. 1) Action: Hydrolyze the peptide bond by the carboxyl group of L-valine and L-alanine. Succinyl-
When alanyl-alanyl-alanyl-paranitroanilide is used as a substrate, the CONH bond of alanyl-paranitroanilide is hydrolyzed. 2) Substrate specificity: Ala10-Ala11, Val29-C when reductive alkylated lysozyme is used as a substrate.
ys30, Ala32-Cys33, Ala42-Th
r43, Ala90-Ser91, Val109-Al
Cut between a110. When oxidized insulin B chain is used as a substrate, it strongly cleaves between Val12 and Glu13 of B chain and weakly between Gly23 and Phe24.

3) Working pH range and optimum pH: succinyl-alanyl-alanyl-alanyl-paranitroanilide as a substrate, citrate buffer (pH 3.5 to 5),
Phosphate buffer (pH 5-7), Tris / HCl buffer (p
H7-9), glycine / sodium hydroxide buffer (pH
9 to 11), the relative activity when reacted for 20 minutes at 37 ° C. in a sodium hydrogen carbonate / sodium hydroxide buffer solution (pH 10 to 12) is as shown in FIG. 1, and the working pH range is 6 to 11. The optimum pH is 9.0. 4) Working temperature range and optimum temperature: When succinyl-alanyl-alanyl-alanyl-paranitroanilide was used as a substrate and measured in a glycine / sodium hydroxide buffer solution of pH 10.0, the working temperature range was as shown in FIG. Range is 3
The temperature is 0 ° C to 60 ° C, and the optimum temperature is 45 ° C.

5) pH stability: succinyl-alanyl-
Using alanyl-alanyl-paranitroanilide as a substrate,
Citrate buffer (pH 3-5), phosphate buffer (pH 5
~ 7), Tris / hydrochloric acid buffer (pH 7 to 9), glycine / sodium hydroxide buffer (pH 9 to 11), sodium bicarbonate / sodium hydroxide buffer (pH 10 to 1)
When the residual relative activity after being kept in 2) at 37 ° C. for 40 minutes was measured, the stable pH range was pH 8 as shown in FIG.
~ 11. 6) Temperature stability: When succinyl-alanyl-alanyl-alanyl-paranitroanilide was used as a substrate and the residual enzyme activity after holding at pH 9 for 1 hour was measured, it was stable up to 10-40 ° C as shown in Fig. 4. Yes, it is deactivated at 60 ° C.

7) Inhibitors: microbial alkaline protease inhibitors, which are inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride,
Elastinal, parachloromercuric benzoic acid, ethylenediaminetetraacetic acid, chymostatin, N-tosyl-L-lysylchloromethylketone, N-tosyl-L-phenylalanylchloromethylketone, ovomucoid, antipain,
Not inhibited by talopeptin. The concentration of the added inhibitor and the residual activity are shown in Table 1.

[0022]

[Table 1]

8) Isoelectric point: The isoelectric point (pI) by the isoelectric focusing method is 6.4. 9) Molecular weight: 1 band in disk gel electrophoresis and gel filtration with Sephadex G-75 column, and molecular weight measured by SDS-polyacrylamide gel electrophoresis is 26,000 daltons. Therefore, this enzyme is a monomer having a molecular weight of 26,000 daltons.

The content of the enzyme in the meat quality improving agent of the present invention is not particularly limited, but is preferably an amount capable of acting on meat and significantly softening the meat quality. The content of the enzyme varies greatly depending on the degree of purification of the enzyme, but is usually 0.000 in 1 g of the meat improving composition.
001 to 0.5 g, preferably 0.0001 to 0.1
About g is recommended. Further, as a meat quality improving agent, serum albumin, glycerol, calcium chloride, cyclodextrin, dextrin, lactose, sodium citrate, sodium bicarbonate, etc. can be used alone or in combination as an enzyme activity stabilizer or a bulking agent. The meat quality improving agent of the present invention may be in the form of powder, but may also be in the form of a liquid dissolved in a buffer or water.

The method of using the meat quality improving agent of the present invention for meat is not particularly limited, as long as the enzyme effectively acts on the meat from immediately after slaughter until cooking. That is, any method such as immersing the meat in a liquid meat quality improving agent, injecting the liquid meat quality improving agent into the meat, or sprinkling the meat with a powder or liquid meat quality improving agent may be used. Since the meat quality improving agent of the present invention is essentially an enzyme, it needs to be held for a certain period of time so that the enzymatic reaction can proceed sufficiently. Usually, after treating meat with the product of the present invention, it may be stored in a refrigerator for about one night. The higher the temperature, the faster the enzymatic reaction proceeds. Therefore, after processing meat with the product of the present invention,
After keeping at -40 ° C for 30 to 60 minutes, it may be stored in a refrigerator for several hours.

The amount of the meat quality improving agent used in the present invention varies depending on the type of meat to be treated, the amount of connective tissue contained in the meat, the desired degree of softening of the meat, its treatment temperature, treatment time, treatment method and the like. Therefore, although it is not possible to show a universal usage amount, the enzyme activity amount of 0.1 g per 1.0 g of meat is used.
The amount may be about 01 to 10 units.

The enzyme activity is 1 μmol / min / ml of paranitroaniline when measured by the following method.
The amount of enzyme produced was defined as 1 unit (u). Enzyme sample solution 100 μl, pH 10 50 mM glycine
After mixing 1 ml of sodium hydroxide buffer and holding at 37 ° C. for 10 minutes, 2 mM succinyl-alanyl-alanyl-alanyl-paranitroanilide (50% methanol solution)
Add 100 μl and react at 37 ° C. for 20 minutes. afterwards,
Add 200 μl of 1M acetate buffer (pH 4.0) 410
The absorbance at nm is measured.

The effects of the present invention will be more specifically described below with reference to examples, but the present invention is not limited to these. In the following description, the indicated concentration is% by weight unless otherwise specified.

[0029]

【Example】

Example 1 1% glucose, defatted soybean powder 0.5%, yeast extract
A medium containing 0.3% and 0.3% malt extract (pH 7.
0) 70 ml was put in a shaker flask with a shoulder (Sakaguchi flask) and steam pressure sterilized at 120 ° C. for 15 minutes, and Streptomyces griseo-Albus SN-22 strain was inoculated at 30 ° C.
The cells were cultivated with shaking for 84 hours. This culture solution was centrifuged to remove the bacterial cells and obtain a supernatant. The enzyme activity of this solution is 2.
It was 5 u / ml, and this was used as a crude enzyme solution for the following purification.

Ammonium sulfate was added to 10 liters of the above crude enzyme solution so as to be 40% saturated, and the precipitate formed after standing at 4 ° C. for 1 hour was collected by centrifugation. The precipitate was dissolved in 1 liter of water, dialyzed against pure water at low temperature for 24 hours, and then subjected to DEAE column chromatography. The adsorbed enzyme protein was eluted with 0 to 1 M saline, and the active fraction was collected and re-dissolved.
Salting-out precipitation was performed with% saturated ammonium sulfate, followed by gel filtration with Sephadex G-75 column chromatography to collect active fractions. This was freeze-dried to obtain 200 mg of enzyme protein. The specific activity was 13 u / mg, the yield from the filtrate was 10.3%, and the purity was increased 91 times or more.

Example 2 100 μl (1 of the purified mashiquinolysin obtained in Example 1
0 u) and 1 ml of 50 mM glycine-sodium hydroxide buffer of pH 10 100 μl of 2 mM succinyl-alanyl-alanyl-alanyl-paranitroanilide solution
In addition, the mixture was reacted at 37 ° C. for 20 minutes, and the reaction solution was mixed with a silica gel thin layer chromatography plate (Art 5
715) was spotted, developed with butanol: acetic acid: water (4: 1: 2), and the enzymatic reaction product was examined by ninhydrin color development to confirm the formation of paranitroaniline.

Example 3 205 μg of the purified mashiquinolysin obtained in Example 1 was placed in a glass tube, 200 μl of 6N hydrochloric acid was added, and the tube was sealed.
Hydrolysis was performed at 100 ° C. for 48 hours to analyze the amino acid composition. The results are shown in Table 2. Asx, Gly, Va
The feature is that a large amount of 1 is included. Asx represents aspartic acid or asparagine, and Glx represents glutamic acid or glutamine.

[0033]

[Table 2]

Example 4 The hydrolysis site for the oxidized insulin B chain was determined using the machinquinolysin obtained in Example 1. 50 mM
250 μl of 1 mg / ml solution of oxidized insulin B chain (manufactured by Sigma) dissolved in Tris / HCl buffer (pH 9)
2.4 μg of enzyme preparation was added to and reacted at 37 ° C.
Sampling is performed every 30, 60, and 240 minutes, and hydrochloric acid is added so that the final concentration is 0.1N (6N hydrochloric acid is added to 5N).
After the reaction was stopped, 9 μl of the reaction was decomposed and fractionated by high performance liquid chromatography (HPLC).

Conditions of HPLC Column: Nucleosil 5C18, 4 × 150 m
m Developing solvent: A = 0.05% trifluoroacetic acid B = 80% acetonitrile + 0.05% trifluoroacetic acid gradient A 100% → 50%, 45 minutes Detection: 210 nm Flow rate: 0.8 ml / min Column temperature: 40 ° C.

The amino acid sequence was determined for the four separated peaks to determine the hydrolysis site. This enzyme was found to strongly cleave between Val12 and Glu13 of oxidized insulin B chain and weakly between Gly23 and Phe24. The state of hydrolysis by various proteases is shown in FIG.

Example 5 The hydrolysis site for reductive alkylated lysozyme (manufactured by Wako Pure Chemical Industries) was examined. 50 mM Tris / HCl buffer (pH
The enzyme preparation obtained in Example 1 was added to the reduced alkylated lysozyme dissolved in 9) to obtain the reduced alkylated lysozyme: enzyme =
Mix at a ratio of 100: 1 (w: w) and react at 30 ° C,
Sampling was performed every 15, 30, 60, and 120 minutes, the reaction was stopped with hydrochloric acid, and then analyzed by HPLC.

Conditions of HPLC Column: Wakosil 5C4, 2.6 × 150 mm Solvent: A = 0.05% trifluoroacetic acid B = 80% acetonitrile + 0.05% trifluoroacetic acid gradient A 100% → B 100%, 45 minutes Flow rate: 0.3ml / min Detection: 210nm

The 9 peaks thus obtained were fractionated, the amino acid sequence was analyzed, and the hydrolysis sites were determined as shown below. Machiquinolysin is reductively alkylated lysozyme Ala10-Ala11, Val29-Cys3.
0, Ala32-Cys33, Ala42-Thr4
3, Ala90-Ser91 and Val109-Al
The part between a110 was cut. FIG. 6 shows these hydrolysis sites. From the above results, the present enzyme is L-valine and L-valine.
It can be seen that it is a unique protease that cleaves the carboxyl terminus of alanine.

Example 6 Using the commercially available beef streaks and streaks collected from chicken thighs, the effect of the machinquinolysin of the present invention obtained in Example 1 on the streaks of animals was examined. For comparison, porcine pancreatic elastase (Wako Pure Chemical Industries, Ltd.), Clostri
The test was also performed simultaneously on collagen histamine derived from collagen histolyticum (collagenase Amano, manufactured by Amano Pharmaceutical Co., Ltd.), papain derived from papaya (Papain W-40, manufactured by Amano Pharmaceutical Co., Ltd.), and bromelain derived from pineapple (manufactured by Amano Pharmaceutical Co., Ltd.). The enzyme concentration used was a large excess in order to confirm the action of the enzymes.

The cow streaks are 2 mm × 2 cm, the chicken streaks are the obtained string-shaped pieces, which are cut into 2 cm long pieces, and placed in a bean test tube having a capacity of about 1 ml. Mashiquinolysin is 40 mM Tris / HCl buffer (pH 9.0). ), A concentration of 6 mg / ml, and elastase derived from swine pancreas is 40 mM Tris / HCl buffer (pH
8.8) concentration 2.24 mg / ml, collagenase 4
Concentration 2.15 mg / ml in 0 mM McIlvaine's buffer (pH 7.0), papain 40 mM McI
lvaine buffer solution (pH 7.0) with a concentration of 10.2 m
For g and bromelain, 0.5 ml of each enzyme solution having a concentration of 9.8 mg / ml was added to 40 mM McIlvaine's buffer (pH 7.0) and reacted at 30 ° C. for 16 hours. The streaks after the enzymatic reaction were taken out, and the state of the streaks after the treatment was observed under an actual microscope at a magnification of 60 times.

As a result, in both papain and bromelain treatment, both streaks were not affected by the enzyme at all, and were in the same state as before the enzyme treatment. It was clarified that papain and bromelain do not show enzymatic action on streaks. The chicken line was completely dissolved by the collagenase treatment, and the surface of the beef line was dissolved and became muddy. On the other hand, mashiquinolysin and elastase showed almost the same effects on the streaks. That is, although the original shape of the streaks was retained in both streaks by both enzyme treatments, the surface changed to transparent and a clear effect on streaks was confirmed. From the above, it has been clarified that mashiquinolysin is not as potent as collagenase, but exhibits the same action on the connective tissue streak as that of elastase derived from porcine pancreas.

Example 7 The effects of mashiquinolysin and various proteases used in Example 6 on myofibrillar proteins were examined. A commercially available beef thigh meat (5.12 g) was added to the R. Yang et al. (Agric.
Biol. Chem. , Vol. 36, No. 12, p
2087 to 2095 (1972)) to obtain 6.4 g of crude myofibrillar protein (wet weight, protein content by Lowry method 12.6%).

To 10 mg of the obtained myofibrillar protein (122 mg of crude myofibrils) was added 2 ml of a buffer solution (the same buffer solution as in Example 6 was used for each treated enzyme), and the mixture was stirred uniformly and then each enzyme solution was added to 0.2 mg. 2 ml was added and the reaction was carried out at 37 ° C. for 1 hour. The enzyme concentration of each enzyme is 24
2 μg / ml, porcine pancreatic elastase 258 μg / ml,
Clostridium histolyticum-derived collagenase 248 μg / ml, papain 256 μg
/ Ml and Bromelain 278 μg / ml.

The enzyme reaction solution was subjected to SDS-polyacrylamide gel electrophoresis to examine the behavior of myofibrillar proteins by various enzymes. FIG. 7 shows an electropherogram of SDS-polyacrylamide gel electrophoresis. As shown in FIG. 7, papain and bromelain satisfactorily decomposed myosin heavy chain (MHC in the figure) of myofibrillar protein having a molecular weight of about 200,000, and the protein band disappeared. Porcine pancreatic elastase, which appeared not to degrade myofibrillar proteins, also completely degraded myosin heavy chains.

On the other hand, Clostridium hist
Collagenase derived from Olyticum did not act on myosin heavy chain protein at all under these conditions. The machinolysin of the present invention seems to act slightly on the myosin heavy chain from the figure, but unlike porcine pancreatic elastase, almost all the bands of myosin heavy chain were left. From the above observations, it was revealed that mashiquinolysin has little effect on myofibrillar proteins and acts on hard proteins, and therefore there is no concern of softening the meat quality and excessively degrading the meat quality.

Example 8 2 kg of beef thigh meat on the market was purchased, and the thickness was 2 cm and the slice was 5
Cut to 0 g and add 3% by volume of meat weight of quinoquinicin enzyme solution or buffer with a syringe (needle: 25G).
Inject at approximately 1/3 of the thickness above and below the meat at mm intervals, 5 ℃
After leaving it in the refrigerator overnight at 95
After steaming at ℃ for 15 minutes, rheometer (CR-200D,
(Manufactured by Sun Kagaku Co., Ltd.), the hardness of the meat was determined as the maximum load under the conditions of a penetration rate of 50 mm / min and a compression distance of 5 mm using a cylindrical plunger of 4 mm in diameter.

The processing conditions of each test section are as follows. 1) No treatment 2) 20 mM Tris / HCl buffer (pH 8) was injected 3) 0.2 u of machininolysin (10 u of enzyme used, 0.77 mg of purified enzyme) was dissolved in 2) buffer per 1 g of meat Injected enzyme solution 4) Injected enzyme solution prepared by dissolving 0.5u of machininolysin (25u of used enzyme, 1.92mg of purified enzyme) in 2g of buffer solution per 5g of meat 5) Per 1g of meat An enzyme solution prepared by dissolving 1.0 u of machiquinolysin (50 u of enzyme used, equivalent to 3.85 mg of purified enzyme) in a buffer solution of 2 was injected. The results of the treated meat of each test group are shown in Table 3 as the maximum shear load.

[0049]

[Table 3]

As shown in Table 3, it was found that the meat quality improving agent of the present invention containing maxiquinolysin exhibited a remarkable softening effect on meat. It was also found that the amount of enzyme used was 0.2 u or more per 1 g of meat, which could significantly soften the meat.

Example 9 Since the present invention relates to the action of the enzyme, it was considered that the softening action of the meat would be increased by raising the temperature of action on the meat to about 30 ° C. Therefore, after pouring the enzyme solution into the meat, the refrigerator was used. It was incubated at 30 ° C. for 30 minutes before being stored overnight at 30 ° C. The test section and test conditions are the same as in Example 8 except pre-incubation at 30 ° C. for 30 minutes. The results of the treated meat of each test section are shown in Table 4 as the maximum shear load.

[0052]

[Table 4]

As shown in Table 4, even if the enzyme solution was injected into the meat as a reaction condition and preincubated at 30 ° C. for 30 minutes, no remarkable effect was observed on the meat softening. The pre-incubation made the meat a little harder in the buffer-treated group, but in Example 8 in the mashiquinolysin-injected group,
In the same manner as above, if the amount of enzyme is 0.2u or more per 1g of meat,
A significant softening effect on the meat was observed.

Comparative Example 1 In order to compare the effect of the present invention with other enzymes, the softening effect of papain (papaya-derived, 400 u / mg, manufactured by Amano Pharmaceutical Co., Ltd.) on meat was examined. The experimental conditions were the same as in Example 8. However, the amount of enzyme used was determined from a preliminary test.
That is, purchase 2 kg of beef thigh meat on the market, and have a thickness of 2 c
m, cut into 50 g pieces, and use a syringe (needle: 25
G) at 4 mm intervals above and below the meat at about 1/3 of the thickness, left overnight in the refrigerator at 5 ° C, and then steamed in 3 liters of ion-exchanged water at 95 ° C for 15 minutes, and then put into a rheometer (CR -2
00D, manufactured by Sun Kagaku Co., Ltd.), using a cylindrical plunger of 4 mm in diameter, an intrusion speed of 50 mm / min,
The hardness of the meat was determined as the maximum load under the condition that the compression distance was 5 mm.

The processing conditions of each test section are as follows. 1) Untreated 2) 20 mM Tris-HCl buffer (pH 8) injected 3) 5 u papain per 1 g meat 2) Enzyme solution dissolved in buffer 4) 4) 10 u papain per 1 g meat 2) Injected enzyme solution dissolved in buffer solution 5) Injected 15 u papain per 1 g meat with enzyme solution dissolved in 2) buffer solution Result of treated meat of each test section as maximum shear load It shows in Table 5.

[0056]

[Table 5]

As shown in Table 5, papain was used in an amount of enzyme of 10 u or more per 1 g of meat, and the amount of papain was 5 per
Meat was significantly softened at a risk of%. In the case of papain treatment, the degree of softening of meat with the increase in the amount of enzyme was not as remarkable as that of mashiquinolysin. In addition, the enzyme solution injection site was dissolved, and many cracks were formed on the surface of the meat along the trace of the injection needle. This is probably because papain digests myofibrils, and it was found that papain is not suitable for meat processing.

Comparative Example 2 In order to compare the effect of the present invention with other enzymes, the meat softening effect of elastase (porcine pancreas origin, 251 u / mg, manufactured by Wako Pure Chemical Industries, Ltd.) was examined. The experimental conditions were the same as in Example 4. However, the amount of enzyme used was determined from a preliminary test. That is, purchase 2 kg of beef thigh meat on the market
cm, cut into 50 g pieces, and use a syringe (needle:
25 G) at 4 mm intervals at the top and bottom of the meat at about 1/3 of the thickness, left in the refrigerator at 5 ° C overnight, and then steamed in 3 liters of ion-exchanged water at 95 ° C for 15 minutes, and then the rheometer (CR
-200D, manufactured by Sun Kagaku Co., Ltd.), using a cylindrical plunger with a diameter of 4 mm, an intrusion speed of 50 mm / mi
The hardness of the meat was determined as the maximum load under the conditions of n and a compression distance of 5 mm.

The processing conditions of each test section are as follows. 1) Untreated 2) 20 mM Tris / HCl buffer (pH 8) injected 3) 5 u elastase per 1 g of meat 2) Enzyme solution dissolved in buffer 2) 4) 10 u elastase per 1 g of meat Injected with the enzyme solution dissolved in the buffer of 2) 5) Injected 15u of elastase per 1g of meat with the enzyme solution dissolved in the buffer of 2) The result of the treated meat of each test section is the maximum shear load Are shown in Table 6.

[0060]

[Table 6]

As shown in Table 6, elastase is meat 1
By using the enzyme amount of 14 u per g, the meat was significantly softened at a risk rate of 5% with respect to the untreated group. In the case of elastase treatment, the tenderization of meat is not remarkable, and considering that elastase is derived from porcine pancreas, it is pointed out that there is a price problem and it is not practical to use it in the food industry. It was

Example 10 The taste of meat softened with each enzyme was evaluated by sensory test by 15 panelists. The meat used was the untreated group in Example 8, the meat-quinolysin-treated meat was 0.5 u per 1 g of meat of Example 8, the papain-treated meat was the papain 10 u-treated group per 1 g of meat of Comparative Example 1, and the elastase-treated meat. For the meat of Comparative Example 2, the meat of 14 u treatment area was used per 1 g of meat. The evaluation was carried out by a 2-point comparison method in which no treatment was used as a comparison control, and a 5-point method in which 1 point was extremely bad and 5 points was the most delicious. The results are shown in Table 5.

[0063]

[Table 7]

As shown in Table 7, it was only the treatment with mashiquinolysin of the present invention that the taste was significantly improved compared with the untreated group. Elastase has an insufficient meat-softening effect, and because papain treatment also digests myofibrils,
The taste evaluation was not high. From the above, the effect of the present invention is clear.

[0065]

[Brief description of drawings]

Figure 1: Streptomyces griseoarbus SN
It is a figure which shows the relationship between the action pH and activity of the protease and Machiquinolysin which a -22 strain produces.

[Figure 2] Streptomyces griseoarbus SN
It is a figure which shows the stable pH range of the protease and mashiquinolysin which a -22 strain produces.

[Fig. 3] Streptomyces griseoarbus SN
FIG. 3 is a diagram showing the relationship between the action temperature and the activity of the protease, mashiquinolysin, produced by strain -22.

Figure 4: Streptomyces griseoarbus SN
It is a figure which shows the action temperature stability of the protease and mashiquinolysin which a -22 strain produces.

Fig. 5 Streptomyces griseoarbus SN
It is a figure which shows the hydrolysis site of the oxidized insulin B chain by the protease produced by strain -22, mashiquinolysin and several proteases.

[Fig. 6] Streptomyces griseoarbus SN
FIG. 5 is a diagram showing hydrolysis of reductively alkylated lysozyme by a protease produced by strain -22, mashiquinolysin.

[Figure 7] Streptomyces griseoarbus SN
FIG. 3 is a diagram of SDS-polyacrylamide gel electrophoresis showing hydrolysis of myofibrillar protein by protease produced by strain -22, mashiquinolysin and various proteases.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuhiko Okamura 4-9-1 Jonan, Fujisawa-shi, Kanagawa Central Research Institute (72) Inventor Hiroshi Tone 4-9-1 Jonan, Fujisawa-shi, Kanagawa Mercian Co., Ltd. Central Research Laboratory (72) Inventor Sawao Murao 2-17-31 Horikami Midoricho, Sakai City, Osaka Prefecture

Claims (3)

[Claims] 1. A meat quality improving agent comprising a protease having the following properties. 1) Action: Hydrolyze the peptide bond by the carboxyl group of L-valine and L-alanine. 2) Substrate specificity: Ala10-Ala11, Val29-Cy when reductive alkylated lysozyme is used as a substrate.
s30, Ala32-Cys33, Ala42-Thr
43, Ala90-Ser91, Val109-Ala
Cut between 110. 3) Working pH range and optimum pH: When measured at 37 ° C. using succinyl-alanyl-alanyl-alanyl-paranitroanilide as a substrate, the working pH range is 6 to 11, and the optimum pH is 9.0. is there. 4) Working temperature range and optimum temperature: When succinyl-alanyl-alanyl-alanyl-paranitroanilide is used as a substrate and measured in a glycine / sodium hydroxide buffer solution of pH 10.0, the working temperature range is 30 ° C to 60 ° C. C. and the optimum temperature is 45.degree. 5) pH stability: The stable pH range is pH 8 to 11 when the residual relative activity after holding at 37 ° C. for 40 minutes is measured. 6) Temperature stability: When the residual enzyme activity after holding at pH 9 for 1 hour was measured, it was stable up to 10 to 40 ° C.
It deactivates at 0 ° C. 7) Inhibitor: Inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, but not microbial alkaline protease inhibitor, elastinal, parachloromercuric benzoic acid, ethylenediaminetetraacetic acid. 8) Isoelectric point: The isoelectric point (pI) by the isoelectric focusing method is 6.4. 9) Molecular weight: The molecular weight measured by SDS-polyacrylamide gel electrophoresis is 26,000 daltons. 2. Streptomyces
ces), which is produced by a microorganism belonging to the genus ces and contains a protease having the following properties. 1) Action: Hydrolyze the peptide bond by the carboxyl group of L-valine and L-alanine. 2) Substrate specificity: Ala10-Ala11, Val29-C when reductive alkylated lysozyme is used as a substrate.
ys30, Ala32-Cys33, Ala42-Th
r43, Ala90-Ser91, Val109-Al
Cut between a110. 3) Working pH range and optimum pH: When measured at 37 ° C. using succinyl-alanyl-alanyl-alanyl-paranitroanilide as a substrate, the working pH range is 6 to 11, and the optimum pH is 9.0. is there. 4) Working temperature range and optimum temperature: When succinyl-alanyl-alanyl-alanyl-paranitroanilide is used as a substrate and measured in a glycine / sodium hydroxide buffer solution of pH 10.0, the working temperature range is 30 ° C to 60 ° C. C. and the optimum temperature is 45.degree. 5) pH stability: The stable pH range is pH 8 to 11 when the residual relative activity after holding at 37 ° C. for 40 minutes is measured. 6) Temperature stability: When the residual enzyme activity after holding at pH 9 for 1 hour was measured, it was stable up to 10 to 40 ° C.
It deactivates at 0 ° C. 7) Inhibitor: Inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, but not by microbial alkaline protease inhibitor, elastinal, parachloromercuric benzoic acid, ethylenediaminetetraacetic acid. 8) Isoelectric point: The isoelectric point (pI) by the isoelectric focusing method is 6.4. 9) Molecular weight: The molecular weight measured by SDS-polyacrylamide gel electrophoresis is 26,000 daltons. 3. Streptomyces griseoloalb
us) SN-22 strain (FERM P-13837) produced, and containing a protease having the following properties, a meat quality improving agent. 1) Action: Hydrolyze the peptide bond by the carboxyl group of L-valine and L-alanine. 2) Substrate specificity: Ala10-Ala11, Val29-C when reductive alkylated lysozyme is used as a substrate.
ys30, Ala32-Cys33, Ala42-Th
r43, Ala90-Ser91, Val109-Al
Cut between a110. 3) Working pH range and optimum pH: When measured at 37 ° C. using succinyl-alanyl-alanyl-alanyl-paranitroanilide as a substrate, the working pH range is 6 to 11, and the optimum pH is 9.0. is there. 4) Working temperature range and optimum temperature: When succinyl-alanyl-alanyl-alanyl-paranitroanilide is used as a substrate and measured in a glycine / sodium hydroxide buffer solution of pH 10.0, the working temperature range is 30 ° C to 60 ° C. C. and the optimum temperature is 45.degree. 5) pH stability: The stable pH range is pH 8 to 11 when the residual relative activity after holding at 37 ° C. for 40 minutes is measured. 6) Temperature stability: When the residual enzyme activity after holding at pH 9 for 1 hour was measured, it was stable up to 10 to 40 ° C.
It deactivates at 0 ° C. 7) Inhibitor: Inhibited by diisopropylphosphofluoridate and phenylmethanesulfonyl fluoride, but not microbial alkaline protease inhibitor, elastinal, parachloromercuric benzoic acid, ethylenediaminetetraacetic acid. 8) Isoelectric point: The isoelectric point (pI) by the isoelectric focusing method is 6.4. 9) Molecular weight: The molecular weight measured by SDS-polyacrylamide gel electrophoresis is 26,000 daltons.