JP7722352B2 - Method for modifying meat, composition for modifying meat, and food containing modified meat - Google Patents

Description

The present invention relates to a method for modifying meat that can quickly and easily impart easy-to-shred properties to meat, a composition for modifying meat, and a food product containing meat that has been imparted easy-to-shred properties.

In the fields of meat processing and cooking, there are various needs, such as a juicy and granular texture in hamburger steaks, a firm texture in sausages, a tender and juicy texture in fried chicken and steaks, and easy shredding of meat and reduced cooking time in stews.
In particular, in cooking using chunks of meat, such as stewing, it is difficult to modify the meat in a short period of time, and complicated pretreatments such as injection processing, in which lipids, proteins, food additives, etc. are injected into lean meat; tenderizing, in which the meat fibers are cut short while maintaining its original shape; tumbling, in which seasoning liquid is physically penetrated into the meat while making the fibers easier to separate; prolonged cooking such as vacuum low-temperature cooking; and complicated cooking methods such as pressure cooking are often performed. Therefore, there is a need for a technology that can easily impart ease of separation to chunks of meat in a short period of time without performing such treatments or cooking. In particular, for meat used in stewing, the texture unique to stewing, which falls apart in the mouth, is preferred, but achieving such a texture without complicated operations requires long stewing, and there is a great need to shorten the cooking time.

Many techniques for tenderizing meat have been reported, and among them, methods using proteases include a method in which protease and transglutaminase are allowed to act on poultry and animal meat (Patent Document 1), a modifier containing protease and a sucrose fatty acid ester (Patent Document 2), a method in which meat is brought into contact with a heat-labile protease (Patent Document 3), and a method for producing a processed minced meat food in which a neutral protease derived from Bacillus amyloliquefaciens and having specific properties is allowed to act on minced meat together with trisodium citrate or arginine (Patent Document 4).
The techniques described in Patent Documents 1 and 2 do not limit the type of protease, leaving room for further investigation into the superiority of the meat tenderizing effect. Furthermore, the technique described in Patent Document 3 uses a heat-labile protease and demonstrates certain advantageous effects, but has issues with the complexity of the process and the length of the processing time. Furthermore, Patent Document 4 describes that a neutral protease derived from Bacillus amyloliquefaciens can be used to produce a minced meat food with excellent juiciness and texture, but does not examine its effect on meat other than minced meat, such as chunks of meat.

Furthermore, Patent Document 5 discloses a method for modifying tough, stringy, low-quality meat by combining treatment with one or more types of proteases with high-pressure treatment under a pressure of 100 kg/cm ² to 4000 kg/cm ^2. Patent Document 6 describes a method for tenderizing meat and seafood using an enzyme treatment solution controlled to a specific viscosity, and Patent Document 7 discloses a method for tenderizing meat and seafood using an enzyme treatment solution containing protease, starch, and curdlan.
In these patent documents, thermolysin is mentioned as a protease, but no detailed study has been conducted on the use of thermolysin, and no mention is made of the ease with which meat can be shredded in stews.

Methods disclosed for quickly tenderizing meat in stews include adding glutinous rice (Patent Document 8) and adding one or more of potassium chloride, trisodium citrate, sorbitol, and trehalose (Patent Document 9). However, these techniques leave issues unresolved when it comes to modifying chunks of meat in a short period of time.

Therefore, there is still a need for a modification method that can easily and quickly soften even chunks of meat and make the meat easier to shred.

Patent No. 3235278 Japanese Patent Application Publication No. 7-313108 Special Publication No. 2003-508084 Patent No. 6398198 Japanese Patent Application Publication No. 5-7476 JP 2014-30410 A Patent No. 4850943 Japanese Patent Application Publication No. 8-131122 Patent No. 4809297

The object of the present invention is therefore to provide a method for modifying meat that can quickly and easily tenderize meat and make it easier to shred, and to provide a composition for modifying meat that can quickly and easily tenderize meat and make it easier to shred.

As a result of intensive research to solve the above-mentioned problems, the inventors discovered that by using a neutral protease derived from Bacillus amyloliquefaciens with specific properties and thermolysin, it is possible to quickly and easily tenderize meat and make it easier to shred, leading to the completion of the present invention. Specifically, the present invention is as follows:

[1] A method for modifying meat, comprising contacting meat with a neutral protease derived from Bacillus amyloliquefaciens, the neutral protease having the following properties (a) to (e), and thermolysin:
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA).
[2] The method according to [1], wherein the amount of neutral protease derived from Bacillus amyloliquefaciens that is contacted with the meat is 10 U or more and 5,000,000 U or less per 100 g of meat, and the amount of thermolysin that is contacted with the meat is 10 U or more and 5,000,000 U or less per 100 g of meat.
[3] A method for producing a stew, comprising using meat modified by the method according to [1] or [2].
[4] A composition for modifying meat, comprising a neutral protease derived from Bacillus amyloliquefaciens, the neutral protease having the following properties (a) to (e), and thermolysin:
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA).
[5] The composition according to [4], wherein the content ratio of the neutral protease derived from Bacillus amyloliquefaciens to thermolysin (neutral protease derived from Bacillus amyloliquefaciens:thermolysin) is 1:0.000002 to 1:500,000 in terms of enzyme activity ratio.
[6] The composition described in [4] or [5], which is used to modify meat for use in stews.
[7] A food product containing a neutral protease derived from Bacillus amyloliquefaciens and having the following properties (a) to (e), and meat treated with thermolysin:
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) Optimum temperature = 50 ° C to 55 ° C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA).
[8] The food according to [7], which contains meat on which 10 U or more and 5,000,000 U or less of neutral protease derived from Bacillus amyloliquefaciens and 10 U or more and 5,000,000 U or less of thermolysin have been reacted per 100 g of meat.
[9] The food product according to [7] or [8], which is served as a stew.

According to the present invention, meat can be softened in a short time and simply, and the meat can be made easy to shred.
Furthermore, the present invention can provide a meat modifying composition that can easily and quickly tenderize meat and make it easier to shred.
Furthermore, the present invention can provide foods containing meat that has been softened and made easy to shred.

The present invention provides a method for modifying meat (hereinafter also referred to as "the modification method of the present invention" in this specification).
In this specification, "meat modification" refers to softening meat and making it easier to shred.
The method for modifying meat of the present invention comprises contacting meat with a neutral protease derived from Bacillus amyloliquefaciens and thermolysin.

The neutral protease derived from Bacillus amyloliquefaciens used in the modification method of the present invention is a protease produced by Bacillus amyloliquefaciens and has the following properties (a) to (e):
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA).

The neutral protease may be produced, for example, by culturing Bacillus amyloliquefaciens, recovering the culture solution, concentrating and drying it, purifying the culture solution using ion exchange column chromatography, gel filtration column chromatography or the like, or by a genetic recombinant method. Alternatively, a commercially available enzyme preparation may be used.
Examples of commercially available enzyme preparations include "Protin SD-NY10" and "Protin NY100" available from Amano Enzyme Co., Ltd.
The neutral protease can be used in various forms such as powder, granules, liquid, paste, etc.
In the present invention, the neutral proteases derived from Bacillus amyloliquefaciens can be used singly or in combination of two or more.

The thermolysin used in the modification method of the present invention is a heat-resistant protease, also known as thermolysin or thermolysin, and is a neutral metalloprotease produced by Bacillus thermoproteolyticus. It is an enzyme that selectively hydrolyzes peptide bonds containing hydrophobic amino acids in proteins, and its CAS registration number is 9073-78-3.
In the present invention, as long as it has the above-mentioned properties, an enzyme from another source such as Geobacillus stearothermophilus may be used, and an enzyme modified by amino acid substitution or the like or a recombinant enzyme may also be used. In addition, the enzyme may be produced by any method such as chemical synthesis, fermentation, extraction from bacterial cells, purification, or the like, and a commercially available enzyme preparation may also be used.
Commercially available enzyme preparations include "Thermolysin" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. and "Thermolysin" manufactured by Promega Corporation.
The thermolysin can be used in various forms such as powder, particles, granules, liquid, paste, etc.
In the present invention, the above thermolysins can be used singly or in combination of two or more.

In the modification method of the present invention, "meat" refers to meat that is used for food, that is, meat derived from livestock, poultry, and other wild animals and that is used for food.
Examples of livestock include pigs, cows, sheep, goats, horses, camels, rabbits, and alpacas; examples of poultry include chickens, pigeons, ducks, geese, and quails; and examples of wild animals include deer, boars, and bears.
Furthermore, in the present invention, meat from various parts of the above animals is used as the meat, which can be cut into the desired size and shape as needed, and examples include chunks of meat, sliced meat (thickly sliced meat, thinly sliced meat), minced meat, etc. Hamburgers and sausages made from processed minced meat are not included in the "meat" of the present invention, but formed meat produced by binding small pieces of meat such as cut meat and scrap meat with a binder and shaping them is included in the "meat" of the present invention.
The modification method of the present invention is suitable for modifying chunks of meat that are difficult to soften in a short time using conventional softening methods, as well as tough, low-quality meat, and is particularly suitable for modifying chunks of meat used in stews.

In the modification method of the present invention, the means or manner of contacting meat with the neutral protease derived from Bacillus amyloliquefaciens and thermolysin is not particularly limited, as long as it can be carried out under conditions that allow the neutral protease and thermolysin to act on the meat for a certain period of time. Examples of means for contacting meat with the enzymes include adding the enzyme to the meat, such as sprinkling a powdered or granular enzyme, applying a liquid or paste of the enzyme to the meat, injecting a solution, dispersion, or suspension of the enzyme into the meat, or immersing the meat in a solution, dispersion, or suspension of the enzyme. From the perspective of modifying meat simply and in a short period of time, the method of contacting meat by sprinkling a powdered or granular enzyme, or the method of contacting meat by immersing the meat in a solution, dispersion, or suspension of the enzyme, is preferred.
The neutral protease derived from Bacillus amyloliquefaciens and thermolysin may be contacted in a state prepared as a meat-modifying composition described below.
Furthermore, the enzyme can be brought into contact with meat as appropriate during the meat processing and cooking stages. Raw meat may be brought into contact with the enzyme and then cooked, or heated meat may be brought into contact with the enzyme and then cooked again, but from the standpoint of ease of processing, it is preferable to bring the enzyme into contact with raw meat.

There are no particular restrictions on the order in which the meat is contacted with the neutral protease derived from Bacillus amyloliquefaciens and thermolysin; either the neutral protease derived from Bacillus amyloliquefaciens or thermolysin may be contacted first, or both may be contacted simultaneously. However, from the perspective of ease of modification treatment, it is preferable to contact both simultaneously.

In the modification method of the present invention, the amount of neutral protease derived from Bacillus amyloliquefaciens to be contacted with meat is preferably 10 units (U) or more and 5,000,000 U or less, more preferably 50 U or more and 3,000,000 U or less, and even more preferably 100 U or more and 1,000,000 U or less, per 100 g of meat.
Regarding the enzymatic activity of the neutral protease, 1 U was defined as the amount of enzyme that causes an increase in the amount of Folin's test solution color substance equivalent to 1 μg of tyrosine per minute using casein as a substrate.

In the modification method of the present invention, the amount of thermolysin to be contacted with meat is preferably 10 U or more and 5,000,000 U or less per 100 g of meat, more preferably 50 U or more and 3,000,000 U or less, and even more preferably 100 U or more and 1,000,000 U or less.
The enzyme activity of thermolysin was defined in the same way as that of the neutral protease described above.
The enzymatic activity of the various proteases used in the test examples described below was also defined in the same manner as for the neutral protease.

In the modification method of the present invention, when meat is contacted with a neutral protease derived from Bacillus amyloliquefaciens and thermolysin, the ratio of the amount of the neutral protease to the amount of thermolysin (amount of the neutral protease:amount of thermolysin) is preferably 1:0.000002 to 1:500,000, more preferably 1:0.00002 to 1:50,000, and even more preferably 1:0.0001 to 1:10,000, in terms of the activity ratio of these enzymes.

In the modification method of the present invention, the time for which the neutral protease derived from Bacillus amyloliquefaciens and thermolysin are brought into contact with meat is not particularly limited as long as it is a time that allows the neutral protease and thermolysin to act sufficiently on the substrate substance, i.e., the protein contained in the meat, but specifically, it is preferably 1 minute to 24 hours (for example, 1 minute to 12 hours, 1 minute to 8 hours, 1 minute to 4 hours, 1 minute to 2 hours, 1 minute to 1 hour, etc.).
In particular, the present invention is characterized by the fact that by using a neutral protease and thermolysin in combination, meat can be tenderized in a short time and easily, making the meat easier to shred, and by setting the reaction time to a short period of time, such as 1 minute to 1 hour, the utility value of the present invention is further improved.
Furthermore, in the modification method of the present invention, the temperature at which the neutral protease derived from Bacillus amyloliquefaciens and thermolysin are brought into contact with meat is not particularly limited as long as it is within a range in which the neutral protease and thermolysin maintain their activity, but specifically, a temperature of 0°C to 80°C (e.g., 0°C to 75°C, 0°C to 70°C, 0°C to 65°C, 0°C to 60°C, 0°C to 55°C, 0°C to 50°C, 0°C to 45°C, etc.) is preferred.
That is, by contacting the enzymes under normal conditions for processing and cooking meat, they can act sufficiently.

The modification method of the present invention can easily soften meat in a short time and make the meat more easily shreddable.
Therefore, the modification method of the present invention is effective in stews using meat, particularly in stews using chunks of meat.

Therefore, the present invention also provides a method for producing a stew, which comprises using meat modified by the above-mentioned modification method of the present invention.
In this specification, the term "stewed dish" refers to a dish in which meat, particularly a chunk of meat, is cooked by immersing it in a liquid, and examples thereof include stew, curry, pot-au-feu, braised pork, hot pot dishes, miso soup, and other soups.
The method for producing the stew of the present invention can further include the usual steps employed in producing stew, such as processing steps such as finely chopping other ingredients and parboiling, a step of mixing the meat modified by the modification method of the present invention with other ingredients, a step of cooking the meat and other ingredients by heat (frying, grilling, boiling, steaming, etc.), a seasoning step, etc.
The method for producing the stew of the present invention may also include a step of cooling the produced stew and storing it by refrigeration or freezing, a sterilization step such as high-pressure heat sterilization, and a step of filling and packaging the stew into containers such as cans, bottles, and retort pouches.
Each of the above steps is carried out in a general manner under conditions normally used in the production of food products.

The present invention also provides a composition for modifying meat (hereinafter also referred to as "the composition of the present invention" in this specification).
In this specification, the term "meat modifying composition" refers to a composition used to tenderize meat and make it easier to shred.
The composition of the present invention contains a neutral protease derived from Bacillus amyloliquefaciens and thermolysin.
The neutral protease and thermolysin contained in the composition of the present invention are as described above in the modification method of the present invention, and the content of each of them in the composition of the present invention is set so that the above-mentioned amounts of neutral protease and thermolysin can be brought into contact with 100 g of meat in the modification method of the present invention.
Furthermore, the content ratio of the neutral protease to thermolysin in the composition of the present invention (neutral protease:thermolysin), in terms of the ratio of their enzyme activities (U), is preferably 1:0.000002 to 1:500,000, more preferably 1:0.00002 to 1:50,000, and even more preferably 1:0.0001 to 1:10,000.

The composition of the present invention can be prepared by mixing a neutral protease derived from Bacillus amyloliquefaciens with thermolysin, and adding common food additives as necessary, to form the composition in various forms, including solid forms such as powder, granules, granules, and tablets; semi-solid forms such as gel, paste, and cream; and liquid forms such as solution, suspension, and dispersion.
Alternatively, the composition may be provided in a solid form such as powder, granules, or particulates, and may be prepared as a ready-to-use composition that is dissolved, dispersed, or suspended in a solvent such as water at the time of use.
Examples of the food additives include solvents such as water and polyhydric alcohols; bases such as polyethylene glycol; excipients such as dextrin and lactose; binders such as gelatin, pregelatinized starch, partially pregelatinized starch, cellulose and its derivatives (crystalline cellulose, hydroxypropyl cellulose, etc.); disintegrants such as crospovidone, povidone, and crystalline cellulose; lubricants such as talc and magnesium stearate; glycerin fatty acid esters, sucrose fatty acid esters, and polyglycerin fatty acid esters. Examples of suitable additives include emulsifiers such as ester, saponin, lecithin, and sodium caseinate; seasonings such as salt, amino acids and their salts, nucleic acids and their salts, yeast extract, and meat extract; proteins such as vegetable protein, gluten, egg white, gelatin, and casein; protein hydrolysates; partial protein hydrolysates; pH adjusters such as organic acid salts (fumarates, etc.); chelating agents such as polymerized phosphates; reducing agents such as glutathione and cysteine; thickening polysaccharides such as alginic acid and xanthan gum; kansui (water liquor); colorants; acidulants; and flavorings.

The modifying composition of the present invention can be produced by adding the above-mentioned food additives to a neutral protease derived from Bacillus amyloliquefaciens and thermolysin as necessary, in accordance with formulation methods well known in the field of formulations, such as the methods described in each article of the formulations in the Seventeenth Edition of the Japanese Pharmacopoeia, General Provisions for Preparations [3].

The composition of the present invention is used to modify meat by contacting it with meat using the means or modes described above in the method of modifying meat of the present invention.
The composition of the present invention is used so that the contact amounts of the neutral protease derived from Bacillus amyloliquefaciens and thermolysin per 100 g of meat are the amounts described above in the modification method of the present invention.

The composition of the present invention can easily tenderize meat in a short time, making the meat easier to shred.
Therefore, the composition of the present invention is suitable for use in stews using meat, particularly chunks of meat.

Furthermore, the present invention provides a food product containing meat that has been softened and made easy to shred (hereinafter also referred to as "the food product of the present invention" in this specification).
The food of the present invention contains meat on which a neutral protease derived from Bacillus amyloliquefaciens and thermolysin have been reacted.
"Meat treated with a neutral protease derived from Bacillus amyloliquefaciens and thermolysin" refers to meat that has been modified by contact with a neutral protease derived from Bacillus amyloliquefaciens and thermolysin.
The neutral protease derived from Bacillus amyloliquefaciens and thermolysin used in preparing the meat contained in the food product of the present invention are as described above in the modification method of the present invention.
Furthermore, when preparing the meat contained in the food product of the present invention, the amount of the neutral protease and thermolysin to be contacted with the meat (amount to be contacted per 100 g of meat), the method of contacting the neutral protease and thermolysin, the contact time, and the temperature during contact are also as described above in the modification method of the present invention.

The foods of the present invention can be prepared by cooking methods commonly used in the food industry using meat treated with a neutral protease derived from Bacillus amyloliquefaciens and thermolysin. For example, the foods can be prepared by adding other ingredients, preservatives, seasonings, and other food additives to the meat as needed, and then frying, grilling, boiling, steaming, and other common cooking methods. These foods can be provided as processed foods that can be further cooked as ingredients, such as boiled or steamed meat, or as processed foods used as side dishes, such as stew, curry, pot-au-feu, braised pork, roast beef, roast pork, roast chicken, grilled meat, roast pork on skewers, steak, cutlet, and the like.
Furthermore, the storage form of the food of the present invention is not particularly limited, and it can be stored in various forms such as room temperature, refrigerated, or frozen, and may also be prepared as a retort pouch food.

The food product of the present invention contains meat that has been softened and made easy to shred, and has a good texture.
Therefore, the food product of the present invention can be particularly suitably served as a stew dish using chunks of meat.

The present invention will be explained in more detail below with reference to the following test examples and examples. The present invention is not limited in any way by these examples.

[Test Example 1]
The effects of various proteases on tenderizing and flaking meat were examined as follows.

(i) Domestic young chicken breast meat was cut into 20 g pieces and placed in a vinyl bag, and the protease for each test group shown in Table 1 was added and mixed by shaking. In Table 1, "-" indicates that no protease was added.
The neutral protease derived from Bacillus amyloliquefaciens used had the following properties (a) to (e):
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA).
The amount of each protease added to 20 g of chicken breast meat was 10,000 U (50,000 U per 100 g of chicken breast meat). The mixture was allowed to stand at 20° C. for 15 minutes to allow each protease to come into contact with the chicken breast meat.
(ii) Next, the chicken breast meat contacted with each protease was cooked in a pot as follows to prepare cream stew.
<Method for preparing cream stew>
After lightly frying the surface of the chicken breast, water was added and boiled, followed by simmering for 15 minutes. After that, a commercially available roux (House Foods Corporation) was dissolved and simmered for 5 minutes. Milk was then added and simmered for another 5 minutes. The total simmering time was 25 minutes. The ratio of water, milk, and roux was the recommended ratio for commercially available roux (water:milk:roux = 700:100:90 (weight ratio)), and a model system using only chicken breast without vegetables was used.
(iii) Chicken breast meat was removed from the prepared chicken breast cream stew and subjected to sensory evaluation.
The sensory evaluation was carried out by four panelists on softness and ease of loosening using a scoring system ranging from -5 to 5 points, with the non-additive group (test group 1) being scored as 0. The scores were rounded to the first decimal place, and the evaluation results were shown as a numerical value to the first decimal place obtained by rounding the average of the four panelists.
In this sensory evaluation, "tenderness" refers to the chewing force required to chew the entire piece of meat, and "ease of coming apart" refers to the sensation of the meat falling apart like white fish when chewed. Ease of coming apart is a texture that is particularly preferred in stews, and generally improves with prolonged simmering. Ease of coming apart is a texture that is felt when the myofibrils of the meat break down into bundles to a certain extent. If the meat is hard overall, the myofibrils do not break down, making it difficult to sense ease of coming apart. Conversely, in the case of meat with a soft texture such as liver or foie gras, where the entire meat is crushed evenly, ease of coming apart is not very noticeable.
The softness and ease of disintegration were scored in comparison with the control group and defined as follows:
<Softness rating>
-5 points: Extremely hard -4 points: Very hard -3 points: Hard -2 points: Slightly hard -1 point: Slightly hard 0 point: No difference from the non-additive group 1 point: Slightly soft 2 points: Slightly soft 3 points: Soft 4 points: Very soft 5 points: Extremely soft <Score for ease of disintegration>
-5 points: Extremely difficult to disintegrate -4 points: Very difficult to disintegrate -3 points: Difficult to disintegrate -2 points: Somewhat difficult to disintegrate -1 point: Slightly difficult to disintegrate 0 point: No difference from the untreated group 1 point: Slightly easy to disintegrate 2 points: Somewhat easy to disintegrate 3 points: Easy to disintegrate 4 points: Very easy to disintegrate 5 points: Extremely easy to disintegrate (iv) The evaluation results are shown in Table 1.

As shown in Table 1, the addition of thermolysin resulted in the greatest improvement in ease of loosening (Test Group 4).
On the other hand, the addition of papain improved tenderness the most, but the softness imparted was a uniform softness similar to that of liver, and the ease of separation was not felt to be significant (Test Group 2). Similarly, the addition of neutral protease derived from Aspergillus oryzae (Test Group 8) and acidic protease derived from Aspergillus sp. (Test Group 5) also showed a high effect of imparting tenderness, but did not significantly improve the ease of separation.

These results demonstrate that different types of proteases have different effects on meat tissue, and that the tenderizing effect and the effect of making meat easier to shred are separate.
Therefore, it was suggested that thermolysin, which showed the greatest effect in improving the ease of disintegration, may be able to further improve the ease of disintegration by using it in combination with other proteases.

[Test Example 2]
In Test Example 1, thermolysin was found to have the greatest effect of making meat easier to shred, and the combined effects of thermolysin and other proteases were examined.

(i) Using the same method as in Test Example 1, domestic young chicken breast meat was cut into 20 g pieces and placed in a vinyl bag, and the protease of each test group shown in Table 2 was added and mixed by shaking. In Table 2, a "-" in the Enzyme 1 column and a "-" in the Enzyme 2 column indicate that the respective enzyme was not added. Test group 2 was a group where thermolysin was added alone, and test groups 3 to 11 were groups where thermolysin was added in combination with other proteases.
The thermolysin and other proteases shown in Table 2 were the same as those used in Test Example 1. The amount of each protease added was 10,000 U per 20 g of chicken breast meat (50,000 U per 100 g of chicken breast meat).
(ii) After allowing the mixture to stand at 20°C for 15 minutes to allow the mixture to come into contact with each protease, the mixture was cooked in a pot using the chicken breast meat that had been contacted with each protease to prepare cream stew. The cooking method for the cream stew was the same as in Test Example 1.
(iii) Chicken breast meat was removed from the prepared chicken breast cream stew and subjected to a sensory evaluation. The sensory evaluation was carried out by four panelists on a scale of −5 to 5 points, with the enzyme-free group (Test Group 1) being given a score of 0, for tenderness and ease of shredding. The average score of the four panelists was calculated in the same manner as in Test Example 1.
Regarding ease of loosening, based on the evaluation results of the test plot to which only thermolysin was added (test plot 2) and the test plots to which a protease other than thermolysin was added alone (test plots 2 to 11 in Test Example 1), the theoretical evaluation value for the test plot to which thermolysin was added in combination with other proteases was calculated as the sum of the evaluation results (average score) for the test plots to which each protease was added alone.
For example, for Test Plot 3 in Table 2, the theoretical value for the ease of disentanglement is calculated as "2 + 0.2 = 2.2," where the test plot to which only thermolysin was added (Test Plot 2 in Table 2) has an evaluation result of "2" and the test plot to which only papain was added (Test Plot 2 in Table 1) has an evaluation result of "0.2." Therefore, "2.2" is the theoretical value for the ease of disentanglement for Test Plot 3 in Table 2. The difference between the theoretical value calculated in this way and the actual sensory evaluation result was determined. For example, for Test Plot 3 in Table 2, the sensory evaluation result was "0.1" and the theoretical value was "2.2," so the difference was calculated as "0.1 - 2.2 = -2.1."
If the difference between the theoretical value obtained as described above and the actual sensory evaluation result is greater than zero, it means that a better effect than the theoretical value obtained as the additive effect of the effects when added alone, i.e., a synergistic effect, was obtained. Test plots in which a synergistic effect was obtained were marked with "○" in the synergistic effect column, and test plots in which a synergistic effect was not obtained were marked with "×" in the synergistic effect column.
(iv) The evaluation results of Test Example 2 are shown in Table 2.

As shown in Table 2, it was confirmed that the use of a neutral protease derived from the genus Bacillus in combination with thermolysin tended to significantly improve the ease of loosening (Test Groups 7 and 9).
In test group 7, the difference between the score for ease of loosening obtained by sensory evaluation and the theoretical value was 1.1 points, indicating that the combined use of neutral protease derived from Bacillus amyloliquefaciens and thermolysin can achieve a synergistic effect of improving ease of loosening.
However, in test group 9, the difference between the score for ease of disintegration obtained by the sensory evaluation and the theoretical value was −0.7 points, and no synergistic improvement in ease of disintegration was observed.
Furthermore, the combined use of papain and thermolysin significantly improved the tenderness of the meat, but it developed a uniform texture similar to that of liver, and the ease of separation decreased (Test Group 3).

As described above, the effect on meat tissue varies depending on the type of protease used in combination with thermolysin. However, it was found that the effect of combining thermolysin with a neutral protease derived from Bacillus amyloliquefaciens in improving the ease of shredding was significantly greater and synergistic than when thermolysin was combined with other proteases. This finding makes the shredding effect particularly useful in stews, where shredding is preferred.

[Examples 1 to 15] Modification of chicken breast meat and preparation of cream stew Using the same method as in Test Example 1, domestic young chicken breast meat was cut into 20g pieces and placed in a vinyl bag. The neutral protease derived from Bacillus amyloliquefaciens and thermolysin were added in the amounts shown in Table 3, shaken, and allowed to stand at 20°C for 15 minutes to bring the protease into contact with the chicken breast meat pieces, thereby modifying the chicken breast meat. In Table 3, the "-" in the Bacillus amyloliquefaciens-derived neutral protease column and the thermolysin column indicates that the respective enzymes were not added.
In each example, the same neutral protease and thermolysin derived from Bacillus amyloliquefaciens as those used in Test Example 1 were used.
Next, the chicken breast meat modified by contact with each of the proteases was cooked in a pot to prepare cream stew. The cooking method for the cream stew was the same as in Test Example 1.

Chicken fillets were removed from the resulting chicken fillet cream stew and used as samples for sensory evaluation. Cream stew was prepared in the same manner as above without adding any of the proteases, and the chicken fillets were removed and used as controls.
The sensory evaluation was carried out by four panelists on softness and ease of disintegration using a scoring system ranging from -5 to 5 points, with the control being scored as 0 points. The evaluation results were shown as the average score calculated in the same manner as in Test Example 1.
Regarding ease of disintegration, samples with an average score of 3 points or more were rated as "○", samples with an average score of 2 points or more but less than 3 points were rated as "△", and samples with an average score of less than 2 points were rated as "×".
The evaluation results are also shown in Table 3.

As shown in Table 3, when chicken breast meat that had been contacted with the neutral protease derived from Bacillus amyloliquefaciens and thermolysin was added and then cooked, the ease of shredding of the cooked chicken breast meat was confirmed to be improved (Examples 1 to 15).
These results suggest that adding 10 U to 5,000,000 U of both Bacillus amyloliquefaciens-derived neutral protease and thermolysin per 100 g of meat can improve the ease of separation. Furthermore, it was found that the ease of separation of chicken breast meat was further improved by adding 100 U or more of both enzymes.
The results of the above examples suggest that by contacting meat with the neutral protease and thermolysin derived from Bacillus amyloliquefaciens in amounts of 10 U to 5,000,000 U per 100 g of meat, it is possible to impart to the meat an easy-to-shred texture that is particularly preferred in stews.

Example 16 Beef Round Curry American beef round was diced into 2 cm cubes and placed in a vinyl bag, to which a neutral protease derived from Bacillus amyloliquefaciens and thermolysin were added, followed by shaking and allowing to stand for 15 minutes at 20° C. The same neutral protease derived from Bacillus amyloliquefaciens and thermolysin as those used in Test Example 1 were used, and the amount of each protease added was 50,000 U per 100 g of beef round.
Next, the beef that had been contacted with each protease in the above-mentioned treatment (i.e., beef round meat that had been acted on by each protease) was cooked in a pot as follows to prepare curry.
Specifically, the beef thigh meat was lightly fried on the surface, then water was added, and after boiling, the mixture was simmered for 15 minutes. After that, a commercially available roux (manufactured by House Foods Corporation) was dissolved and the mixture was simmered for an additional 10 minutes. The total simmering time was 25 minutes. The ratio of water to roux was the ratio recommended for the commercially available roux (water:roux = 850:115 (weight ratio)), and a curry containing only beef thigh meat without any vegetables was prepared as Example 16.
In addition, curry was prepared as Comparative Example 1 using beef round meat that had been treated in the same manner but without contacting it with the protease.

Example 17 Braised Pork Belly Domestic pork belly was diced into 3 cm cubes and placed in a vinyl bag, to which a neutral protease derived from Bacillus amyloliquefaciens and thermolysin were added, followed by shaking and allowing to stand for 15 minutes at 20° C. The same neutral protease derived from Bacillus amyloliquefaciens and thermolysin as those used in Test Example 1 were used, and the amount of each protease added was 50,000 U per 100 g of pork belly.
Next, the pork belly meat that had been contacted with each protease in the above-mentioned treatment (i.e., the pork belly meat on which each protease had acted) was cooked in a pot as described below to prepare braised pork belly meat.
Specifically, the pork belly was lightly parboiled, water and a commercially available sauce for braised pork belly (manufactured by Moranbong Co., Ltd.) were added, and the mixture was simmered with the lid on for 40 minutes, and then the lid was removed and the mixture was simmered for an additional 15 minutes to prepare braised pork belly, which was designated Example 17. The total simmering time was 55 minutes. The ratio of water to sauce was the recommended ratio for the commercially available sauce (water:sauce = 400:120 (weight ratio)).
In addition, braised pork cubes were prepared in the same manner as above without contacting with the protease, and this was used as Comparative Example 2.

The beef thigh meat and pork belly meat were taken from each of the beef thigh meat curries of Example 16 and Comparative Example 1, and each of the braised pork belly meats of Example 17 and Comparative Example 2, and subjected to sensory evaluation.
The sensory evaluation was conducted focusing on the ease of shredding of the meat, and was conducted by a panel of four people using the following evaluation criteria. The results of the sensory evaluation were decided by discussion among the four people. For pork belly, the ease of shredding of the meat layer was evaluated, out of the fat layer and the meat layer.
<Evaluation criteria>
◯: Easier to disentangle than the comparative example △: Slightly easier to disentangle than the comparative example ×: Easier to disentangle than the comparative example or harder to disentangle than the comparative example The evaluation results are shown in Table 4.

As shown in Table 4, the beef in the curry of Example 16 and the pork belly in the braised pork in Example 17 were both evaluated as being easier to shred than the beef in the curry of Comparative Example 1 and the pork belly in Comparative Example 2. This confirmed that the beef and pork belly meat in the curry and braised pork that had been treated with the neutral protease and thermolysin derived from Bacillus amyloliquefaciens were clearly made easier to shred.

The results shown in the above examples demonstrate that contacting meat with neutral protease and thermolysin derived from Bacillus amyloliquefaciens can make the meat easier to shred, regardless of the type, part of the meat, or cooking method.
Such an effect of imparting ease of separation is particularly useful in stews, where the ease of separation of meat is preferred as a texture.

As described above in detail, the present invention provides a method for modifying meat that can easily tenderize meat in a short time and make the meat more easily shreddable.
Furthermore, the present invention can provide a composition for modifying meat that can easily tenderize meat in a short time and make the meat more easily shreddable.
Furthermore, the present invention can provide foods containing meat that has been softened and made easy to shred.
The present invention is extremely useful in the food industry, particularly in stews where meat that falls apart easily is preferred as a texture.

This application is based on patent application No. 2020-028861 filed in Japan, the contents of which are incorporated in their entirety into this specification.

Claims (9)

A method for modifying meat, comprising contacting meat with a neutral protease derived from Bacillus amyloliquefaciens, the neutral protease having the following properties (a) to (e), and thermolysin:
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA). The method of claim 1, wherein the amount of neutral protease derived from Bacillus amyloliquefaciens contacted with the meat is 10 U or more and 5,000,000 U or less per 100 g of meat, and the amount of thermolysin contacted with the meat is 10 U or more and 5,000,000 U or less per 100 g of meat. A method for producing a stew, comprising using meat modified by the method of claim 1 or 2. A composition for modifying meat, comprising a neutral protease derived from Bacillus amyloliquefaciens, the neutral protease having the following properties (a) to (e), and thermolysin;
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA). The composition according to claim 4, wherein the ratio of the content of the neutral protease derived from Bacillus amyloliquefaciens to that of thermolysin (neutral protease derived from Bacillus amyloliquefaciens:thermolysin) is 1:0.000002 to 1:500,000 in terms of enzyme activity ratio. The composition described in claim 4 or 5, which is used to modify meat for use in stews. A method for producing a food product, comprising cooking meat that has been modified by contacting it with thermolysin and a neutral protease derived from Bacillus amyloliquefaciens, the neutral protease having the following properties (a) to (e):
(a) an enzyme that hydrolyzes a substrate having a peptide bond to release a peptide;
(b) the optimum pH is 7;
(c) stable at pH 5 to 7;
(d) the optimum temperature is 50°C to 55°C;
(e) It is a metalloprotease and is inhibited by ethylenediaminetetraacetic acid (EDTA). The method according to claim 7, wherein the meat is modified by contacting 10 U to 5,000,000 U of a neutral protease derived from Bacillus amyloliquefaciens and 10 U to 5,000,000 U of thermolysin per 100 g of meat . 9. The method according to claim 7 or 8, wherein the meat is a chunk of meat served as a stew.