WO2021167062A1

WO2021167062A1 - Method for meat modification, composition for modification, and food product containing modified meat

Info

Publication number: WO2021167062A1
Application number: PCT/JP2021/006333
Authority: WO
Inventors: 律彰山田
Original assignee: 味の素株式会社
Priority date: 2020-02-21
Filing date: 2021-02-19
Publication date: 2021-08-26
Also published as: JPWO2021167062A1

Abstract

The present invention relates to: a neutral protease derived from Bacillus amyloliquefaciens, the neutral protease having properties (a) through (e); a method for meat modification, the method including bringing thermolysin into contact with the meat; and a composition for modification that contains the neutral protease and thermolysin.　Property (a): being an enzyme that hydrolyzes a substrate having a peptide bond to free peptides. Property (b): having an optimum pH of 7. Property (c): being stable at a pH between 5 and 7. Property (d): having an optimum temperature of 50°C-55°C. Property (e): being a metalloprotease and inhibited by ethylenediaminetetraacetate (EDTA). 　The present invention is capable of rapidly and easily tenderizing meat and imparting an easy-to-break-up property to the meat.

Description

Meat reforming method and composition for reforming and foods containing modified meat

The present invention relates to a method for modifying meat that can easily impart looseness to meat in a short time and easily, a composition for modifying meat, and a food containing meat to which ease of loosening has been imparted. ..

There are various needs in the fields of meat processing and cooking. For example, the feeling of meat grain and juiciness in hamburger steak, the feeling of pre-preparation in sausage, the softness and juiciness in fried chicken and steak, the ease of loosening of meat and shortening of cooking time in steaked dishes can be mentioned.
In particular, in cooking using chunky meat such as stewed dishes, it is difficult to modify the meat in a short time, so injection processing that injects lipids, proteins, food additives, etc. into lean meat and the original form of meat can be used. Tenderize treatment that cuts fibers short while keeping, complicated pretreatment such as tumbling treatment that makes it easier for fibers to loosen at the same time as physically infiltrating the seasoning liquid, long-term cooking such as vacuum low temperature cooking, pressure cooking In many cases, complicated cooking such as is performed. Therefore, there is a demand for a technique that can easily give the chunky meat easy to loosen in a short time without performing such processing or cooking. In particular, for meat used in stewed dishes, the texture peculiar to stewed dishes that loosens in the mouth is preferred, but in order to realize the texture without complicated operations, it is necessary to stew for a long time. The need for shortening the cooking time is very high.

There are many reports on techniques for softening meat, and among them, methods using proteases include a method of allowing protease and transglutaminase to act on chicken and animal meat (Patent Document 1), and a modification containing protease and sucrose fatty acid ester. A pawnbroker (Patent Document 2), a method of contacting meat with a heat-resistant protease (Patent Document 3), a neutral protease derived from Bacillus amyloliquefaciens and having specific properties, and trisodium citrate or A method for producing a processed ground meat food in which arginine and arginine act on the ground meat (Patent Document 4) and the like are disclosed.
In the techniques described in Patent Document 1 and Patent Document 2, the type of protease is not limited, and there is room for consideration in the superiority of the meat softening effect. Further, in the technique described in Patent Document 3, although a heat-resistant protease is used and a certain superior effect is shown, there are still problems in terms of work complexity and length of processing time. It had been. Further, Patent Document 4 describes that a processed meat product having excellent juiciness and texture can be produced by a neutral protease derived from Bacillus amyloliquefaciens, but meat other than minced meat, For example, its effect on chunky meat has not been investigated.

Further, Patent Document 5 describes a method for modifying hard, streaky, low-quality meat by combining treatment with one or more types of protease and ^{high-pressure treatment under a pressure of 100 kg / cm 2} to 4000 kg / cm ^2. Is disclosed. Patent Document 6 describes a method of softening meat and fish and shellfish with an enzyme-treated solution controlled to a specific viscosity, and Patent Document 7 describes a method of softening meat and seafood with an enzyme-treated solution containing protease, starch and curdran. , A method of softening meat and seafood is disclosed.
Although thermolysin is mentioned as a protease in these patent documents, no detailed study using thermolysin has been made, and no mention is made of the ease of loosening of meat in stewed dishes.

As a method of softening meat in a short time in stewed dishes, a method of adding glutinous rice (Patent Document 8), a method of adding one or more of potassium chloride, trisodium citrate, sorbitol, and trehalose (Patent Document 8). Although 9) are disclosed, there remains a problem in modifying the lumpy meat in a short time with the techniques described in these.

Therefore, for example, there is still a demand for a reforming method that can easily soften lumpy meat in a short time and can impart easiness to loosen the meat.

Japanese Patent No. 3235278 Japanese Unexamined Patent Publication No. 7-313108 Special Table 2003-508804 Japanese Patent No. 6398198 Japanese Unexamined Patent Publication No. 5-7476 Japanese Unexamined Patent Publication No. 2014-30410 Japanese Patent No. 4850943 Japanese Unexamined Patent Publication No. 8-131122 Japanese Patent No. 4809297

Therefore, an object of the present invention is to provide a method for modifying meat that can soften meat in a short time and easily to impart easy loosening to the meat, and to provide meat in a short time and easily. It is an object of the present invention to provide a composition for modifying meat, which can be softened to impart looseness to the meat.

As a result of diligent research to solve the above problems, the present inventor uses a neutral protease derived from Bacillus amyloliquefaciens, which has specific characteristics, and a thermolysin. As a result, it has been found that the meat can be softened and easily loosened in a short time and easily, and the present invention has been completed. That is, the present invention is as follows.

[1] A neutral protease derived from Bacillus amyloliquefaciens, which comprises contacting a neutral protease having the following characteristics (a) to (e) and a thermolysin with the meat. Modification method;
(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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).
[2] The amount of neutral protease derived from Bacillus amyloliquefaciens to be brought into contact with meat is 10 U or more and 5,000,000 U or less per 100 g of meat, and the amount of thermolysin to be brought into contact with meat is 100 g of meat. The method according to [1], wherein the amount is 10 U or more and 5,000,000 U or less per unit.
[3] A method for producing a stewed dish, which comprises using the meat modified by the method according to [1] or [2].
[4] A composition for modifying meat, which is a neutral protease derived from Bacillus amyloliquefaciens and contains a neutral protease having the following characteristics (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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).
[5] The content ratio of the neutral protease derived from Bacillus amyloliquefaciens to thermolysin (neutral protease derived from Bacillus amyloliquefaciens: thermolysin) has an enzyme activity ratio of 1: 0. The composition according to [4], which is 000002-1: 500,000.
[6] The composition according to [4] or [5], which is used for modifying meat used in stewed dishes.
[7] A food product containing a neutral protease derived from Bacillus amyloliquefaciens, which has the following characteristics (a) to (e), and meat on which thermolysin acts.
(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 = 5-7,
(D) Optimal temperature = 50 ° C to 55 ° C,
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).
[8] Per 100 g of meat contains a neutral protease derived from Bacillus amyloliquefaciens of 10 U or more and 5,000,000 U or less and a meat on which thermolysin of 10 U or more and 5,000,000 U or less acts. The food product according to [7].
[9] The food according to [7] or [8], which is served as a stewed dish.

According to the present invention, the meat can be softened and the meat can be easily loosened in a short time and easily.
Further, according to the present invention, it is possible to provide a composition for modifying meat that can soften meat and impart easy loosening to the meat in a short time and easily.
Further, according to the present invention, it is possible to provide a food containing meat that has been softened and imparted with ease of loosening.

The present invention provides a method for modifying meat (hereinafter, also referred to as "method for modifying the present invention" in the present specification).
As used herein, the term "meat reforming" refers to softening the meat and imparting ease of loosening to the meat.
The modification method of the present invention comprises contacting meat with a neutral protease 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 the proteases produced by Bacillus amyloliquefaciens are described below (a) to (a). Those having the characteristics of e) are used.
(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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).

As the neutral protease, for example, Bacillus amyloliquefaciens is cultured, and the culture solution is collected, concentrated, and dried. Ion exchange column chromatography, gel filtration column chromatography, or the like is used from the culture solution. It is possible to use a product produced by purification by purification or a product produced by a gene recombination method, but a commercially available enzyme preparation can also be used.
Examples of commercially available enzyme preparations include "Protin SD-NY10" and "Protin NY100" commercially available from Amano Enzyme Co., Ltd.
The neutral protease can be used in various forms such as powder, granular, granular, liquid, and paste.
In the present invention, the above-mentioned neutral protease derived from Bacillus amyloliquefaciens can be used alone or in combination of two or more.

The thermolysin used in the modification method of the present invention is a thermolysin, which is also called thermolysin, thermolysin, etc., and is a neutral metalloproteinase produced by Bacillus thermoproteolitics. It is an enzyme that selectively hydrolyzes peptide bonds containing hydrophobic amino acids in proteins, and has a CAS Registry Number of 9073-78-3.
In the present invention, other basic enzymes such as Geobacillus stearothermophilus may be used as long as they have the above-mentioned characteristics, and a modifying enzyme or a recombinant enzyme by amino acid substitution or the like may be used. can. Further, it may be produced by any method such as a chemical synthesis method, a fermentation method, an extraction from bacterial cells, a method by purification, or the like, and a commercially available enzyme preparation can also be used.
Examples of commercially available enzyme preparations include "thermolysin" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. and "thermolysin" manufactured by Promega Co., Ltd.
The thermolysin can be used in various forms such as powder, granular, granular, liquid, and paste.
In the present invention, the thermolysin can be used alone or in combination of two or more.

In the reforming method of the present invention, "meat" refers to meat to be used for food, that is, meat derived from livestock, poultry, and other wild animals, which is used for food.
Livestock include pigs, cows, sheep, goats, horses, camels, rabbits, alpaca, etc. Poultry includes chickens, pigeons, ducks, ducks, sardines, etc., and wild animals include deer, boars, etc. , Bears, etc.
Further, in the present invention, as the meat, meat of various parts derived from the above animals is used, and it can be appropriately cut into a desired size and shape and used, and can be used as chunky block meat or sliced meat (thick sliced meat). , Thinly sliced meat), chopped meat and the like. Processed minced meat such as hamburger steak and sausage is not included in the "meat" of the present invention. , Included in the "meat" of the present invention.
The reforming method of the present invention is suitable for reforming chunky meat, which was difficult to soften in a short time by the conventional softening treatment method, and hard, low-quality meat, and is used for stewed dishes. It is particularly suitable for modifying meat.

In the modification method of the present invention, the neutral protease derived from Bacillus amyloliquefaciens and the means or mode of contact between the thermolysin and the meat are such that the neutral protease and the thermolysin act on the meat for a certain period of time. There is no particular limitation as long as it can be done in the state of doing. As a means for bringing the enzyme into contact with the meat, the enzyme is added to the meat, such as by sprinkling the enzyme in the form of powder or granules, or the enzyme in the form of a liquid or paste is applied to the meat, or a solution or dispersion of the enzyme. Alternatively, the suspension may be injected into the meat, or the meat may be immersed in the enzyme solution or dispersion or suspension. From the viewpoint of modifying the meat easily and in a short time, a method of sprinkling a powdered or granular enzyme and contacting the meat, or a method of immersing the meat in a solution, dispersion or suspension of the enzyme and contacting the meat is preferable.
The neutral protease derived from Bacillus amyloliquefaciens and thermolysin may be contacted in a state of being prepared as a composition for modifying meat described later.
Further, the contact between the enzyme and the meat can be appropriately performed at the stage of processing and cooking the meat, and the raw meat may be brought into contact with the enzyme and then cooked, or the heated meat may be cooked. The meat may be cooked again after being brought into contact with the enzyme, but from the viewpoint of convenience of treatment, it is preferably brought into contact with raw meat.

The order in which the neutral protease derived from Bacillus amyloliquefaciens and the thermolysin are brought into contact with the meat is not particularly limited, and the neutral protease derived from Bacillus amyloliquefaciens and the thermolysin derived from Bacillus amyloliquefaciens are not particularly limited. They may be brought into contact first or both may be brought into contact at the same time, but from the viewpoint of simplicity of the reforming treatment, it is preferable to bring them into contact at the same time.

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

In the modification method of the present invention, the amount of thermolysin to be brought into contact with meat is preferably 10 U or more and 5,000,000 U or less, and more preferably 50 U or more and 3,000,000 U or less with respect to 100 g of meat. It is 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 the above-mentioned neutral protease.
In addition, the enzymatic activities of various proteases used in the test examples described later are all defined in the same manner as the above-mentioned neutral proteases.

In the modification method of the present invention, the ratio of the neutral protease amount to the thermolysin amount when contacting the meat with a neutral protease derived from Bacillus amyloliquefaciens and thermolysin (the neutral protease amount: The amount of thermolysin) is preferably 1: 0.000002 to 1: 500,000, more preferably 1: 0.00002 to 1: 50,000, and 1: 0 in terms of the activity ratio of these enzymes. It is more preferably .0001 to 1: 10,000.

In the modification method of the present invention, the neutral protease derived from Bacillus amyloliquefaciens and the time for contacting the thermolysin with the meat are sufficient for the neutral protease and the thermolysin to be a substrate substance, that is, a protein contained in the meat. The time is not particularly limited as long as it can act on, but specifically, 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 2 hours). Minutes to 1 hour, etc.) is preferable.
In particular, the present invention is characterized in that by using a neutral protease and thermolysin in combination, the meat can be softened and easily loosened in a short time and easily, and the meat can be easily loosened for about 1 minute to 1 hour. The utility value of the present invention is further improved by setting the action time to be short.
Further, in the modification method of the present invention, the neutral protease derived from Bacillus amyloliquefaciens and the temperature at which the thermolysin is brought into contact with the meat are also within the range in which the neutral protease and the thermolysin maintain their activities. If there is, it is not particularly limited, but specifically, it is 0 ° C to 80 ° C (for example, 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 preferable.
That is, they can be sufficiently allowed to act by contacting the enzymes under normal meat processing / cooking conditions.

According to the modification method of the present invention, the meat can be softened and the meat can be easily loosened in a short time and easily.
Therefore, the modification method of the present invention is effective in a stewed dish using meat, particularly a stewed dish using chunky meat.

Therefore, the present invention also provides a method for producing a stewed dish, which comprises using the meat modified by the modification method of the present invention.
In the present specification, the “simmered dish” refers to a dish in which meat, particularly chunky meat, is cooked in a state of being immersed in a liquid. , Soup, etc.
The method for producing a stewed dish of the present invention is modified by a normal process adopted in the production of a stewed dish, for example, a processing step such as shredding other ingredients, boiling underneath, or the above-mentioned modification method of the present invention. Further, a step of mixing the meat and other ingredients, a step of cooking the meat and other ingredients by heating (frying, baking, boiling, steaming, etc.), a seasoning step, and the like can be further included.
Further, the method for producing a stewed dish of the present invention includes a step of cooling the manufactured stewed dish and storing it by refrigerating, freezing, etc., a sterilization process such as high-pressure heat sterilization, and a container such as a can, a bottle, and a retort pouch. The process of filling and packaging can also be included.
Each of the above steps is carried out by a general method under the conditions normally carried out in the production of food.

The present invention also provides a composition for modifying meat (hereinafter, also referred to as “composition of the present invention” in the present specification).
As used herein, the term "meat reforming composition" refers to a composition used to soften meat and impart ease of loosening to the meat.
The composition of the present invention contains a neutral protease derived from Bacillus amyloliquefaciens, and a 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 each content thereof in the composition of the present invention is the modification method of the present invention. In 100 g of meat, the above-mentioned amounts of the neutral protease and thermolysin are set as an amount that can be brought into contact with each other.
The content ratio of the neutral protease to the thermolysin in the composition of the present invention (the neutral protease: thermolysin) is preferably 1: 0.000002 to 1: 500 in terms of the enzyme activity (U) ratio. It is 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 is a mixture of a neutral protease derived from Bacillus amyloliquefaciens and thermolysin, and if necessary, general food additives are added to make a powdery, granular, granular composition. It can be in various forms such as solids such as granules and tablets; semi-solids such as gels, pastes and creams; liquids such as solutions, suspensions and dispersed liquids.
Further, it can be provided in a solid form such as powder, granule, or granule, and can be used as a time-prepared type composition which is dissolved, dispersed or suspended in a solvent such as water at the time of use.
Examples of the food additive include water, solvents such as polyhydric alcohol; bases such as polyethylene glycol; excipients such as dextrin and lactose; gelatin, pregelatinized starch, partially pregelatinized starch, cellulose and derivatives thereof (crystalline cellulose). , Hydroxypropyl cellulose, etc.); Disintegrants such as crospovidone, povidone, crystalline cellulose, etc .; Starch, magnesium stearate, etc. Emulsifiers such as lecithin, sodium caseinate; seasonings such as salt, amino acids and salts thereof, nucleic acids and salts thereof, yeast extract, livestock meat extract; proteins such as plant protein, gluten, egg white, gelatin, casein; protein hydrolyzate; protein Partial decomposition products; pH adjusters such as organic acid salts (fumarate etc.); chelating agents such as polymerized phosphates; reducing agents such as glutathione and cysteine; thickening polysaccharides such as alginic acid and xanthan gum; casein; pigments; Acidulants; fragrances and the like can be mentioned.

The modifying composition of the present invention is a formulation well known in the field of formulation by adding the above-mentioned food additives to the neutral protease and thermolysin derived from Bacillus amyloliquefaciens, if necessary. It can be manufactured according to the means for making it, for example, the method described in each article of the 17th revised Japanese Pharmacopoeia General Regulations for Pharmaceuticals [3] Pharmaceuticals.

The composition of the present invention is used for modifying meat by contacting it with meat by the means or embodiment described above in the modification method of the present invention.
In the composition of the present invention, the contact amount of the neutral protease derived from Bacillus amyloliquefaciens and the thermolysin is the same amount as described above in the modification method of the present invention with respect to 100 g of meat, respectively. , Used.

The composition of the present invention can easily soften the meat in a short time and impart the meat to be easily loosened.
Therefore, the composition of the present invention is preferably used in meat, especially stewed dishes using chunky meat.

Furthermore, the present invention provides a food containing meat that has been softened and imparted with ease of loosening (hereinafter, also referred to as "food of the present invention" in the present specification).
The food product of the present invention contains a neutral protease derived from Bacillus amyloliquefaciens, and meat on which thermolysin is acted.
"Neutral protease derived from Bacillus amyloliquefaciens and meat on which thermolysin acts" refers to neutral protease derived from Bacillus amyloliquefaciens and modified contact with thermolysin. Meat.
The neutral protease derived from Bacillus amyloliquefaciens used for preparing the meat contained in the food of the present invention and the thermolysin are as described above in the modification method of the present invention.
Further, in the preparation of the meat contained in the food of the present invention, the amount of the neutral protease and thermolysin to be brought into contact with the meat (contact amount with respect to 100 g of meat), the method of contacting the neutral protease and the thermolysin, the contact time, The temperature at the time of contact is also the same as described above in the reforming method of the present invention.

The food product of the present invention can be prepared by a cooking means commonly used in the field of food products, using a neutral protease derived from Bacillus amyloliquefaciens and meat on which thermolysin is acted, for example, as described above. If necessary, other ingredients and food additives such as preservatives and seasonings are added to the meat, and the meat is prepared by ordinary cooking means such as roasting, baking, boiling, and steaming. Processed foods such as meat that are further used for cooking as ingredients, and processed foods such as stew, curry, potov, boiled, roast beef, roast pork, roast chicken, roasted meat, roasted pork, roasted chicken, steak, cutlet, etc. Can be provided as.
Further, 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 storage, refrigerated storage, and frozen storage, and may be prepared as a retort pouch food.

The food product of the present invention contains meat that has been softened and imparted with ease of loosening, and exhibits a good texture.
Therefore, the food product of the present invention can be particularly preferably provided as a stewed dish using chunky meat.

The present invention will be described in more detail with reference to test examples and examples below. The present invention is not limited to these examples and the like.

[Test Example 1]
For various proteases, the effect of imparting softness and the effect of imparting looseness to meat were examined as follows.

(I) Domestic young chicken breast meat was cut into 20 g pieces, placed in a vinyl bag, and the proteases of each test group shown in Table 1 were added and shaken. In Table 1, "-" indicates that protease is not added.
As the neutral protease derived from Bacillus amyloliquefaciens, those having the following characteristics (a) to (e) were used.
(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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that 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). Each protease was brought into contact with chicken breast meat by allowing it to stand at 20 ° C. for 15 minutes.
(Ii) Next, using chicken breast meat contacted with each protease, it was cooked in a pan as follows to prepare a cream stew.
<How to prepare cream stew>
After lightly frying the surface of chicken breast meat, water was added, and after boiling, it was simmered for 15 minutes, then commercially available roux (manufactured by House Foods Co., Ltd.) was dissolved, simmered for 5 minutes, then milk was added and simmered for another 5 minutes. The total boiling time was 25 minutes. The ratio of water, milk, and roux was the recommended ratio of commercially available roux (water: milk: roux = 700: 100: 90 (weight ratio)), and a model system of chicken breast meat without vegetables was used.
(Iii) Chicken breast meat was taken out from the prepared chicken breast meat cream stew and subjected to sensory evaluation.
The sensory evaluation was performed on a panel of 4 people by a scoring method of -5 to 5 points with the additive-free group (test group 1) as 0 points for softness and ease of loosening. The grades were given to the first decimal place, and the evaluation results were shown by the numerical values up to the first decimal place, rounded off from the average value of the four people.
In this sensory evaluation, "softness" means the chewing force required to chew the whole meat, and "easiness to loosen" means the feeling of loosening like a white fish when chewing the meat. Means. The ease of loosening is a texture that is particularly preferred in stewed dishes, and is generally improved by boiling for a long time. In addition, the ease of unraveling is the texture that is felt when the myofibrils of the meat collapse as a bundle to some extent. Even with a soft texture such as liver or foie gras, where the whole meat is crushed evenly, the ease of loosening is not felt so much.
The scores for softness and ease of loosening were defined as follows by comparison with the additive-free group.
<Score of softness>
-5 points: Extremely hard -4 points: Very hard -3 points: Hard -2 points: Slightly hard -1 point: Slightly hard 0 points: No difference from the additive-free group 1 point: Slightly soft 2 Point: Slightly soft 3 points: Soft 4 points: Very soft 5 points: Extremely soft <Score of ease of loosening>
-5 points: Very noticeably hard to loosen -4 points: Very hard to loosen -3 points: Hard to loosen -2 points: Slightly hard to loosen -1 point: Slightly hard to loosen 0 points: No difference from additive-free group 1 point : Slightly easy to unravel 2 points: Slightly easy to unravel 3 points: Easy to unravel 4 points: Very easy to unravel 5 points: Very significantly easy to unravel (iv) The evaluation results are shown in Table 1.

As shown in Table 1, the addition of thermolysin improved the ease of loosening most (Test Group 4).
On the other hand, the softness was most improved by adding papain, but the softness given was a liver-like uniform softness, and the ease of loosening was not felt so much (Test Group 2). Similarly, a high softness-imparting effect is obtained in the neutral protease-added group (test group 8) derived from Aspergillus oryzae and the acidic protease-added group (test group 5) derived from Aspergillus sp. Although it was observed, there was not much improvement in the ease of loosening.

From the above results, it was clarified that the action on the meat tissue differs depending on the type of protease, and the effect of softening and the effect of imparting looseness are different.
Therefore, it was suggested that the thermolysin, which showed the effect of improving the looseness most, may be used in combination with other proteases to further improve the looseness.

[Test Example 2]
In Test Example 1, thermolysin, which had the highest effect of imparting looseness to meat, was examined for its combined effect with other proteases.

(I) In the same manner as in Test Example 1, domestic young chicken breast meat was cut into 20 g pieces, placed in a vinyl bag, and the proteases of each test group shown in Table 2 were added and shaken. In Table 2, "-" in the enzyme 1 column and "-" in the enzyme 2 column indicate that each enzyme is not added. Test group 2 was a group to which thermolysin was added alone, and test groups 3 to 11 were groups to which thermolysin was added in combination with other proteases.
As the thermolysin and other proteases shown in Table 2, the same ones used in Test Example 1 were used. 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 to stand at 20 ° C. for 15 minutes to contact each protease, the chicken breast meat to which each protease was contacted was cooked in a pan to prepare a cream stew. The cooking method of the cream stew was the same as in Test Example 1.
(Iii) Chicken breast meat was taken out from the prepared chicken breast meat cream stew and subjected to sensory evaluation. The sensory evaluation was performed on a panel of 4 people with a scoring method of -5 to 5 points with the enzyme-free group (test group 1) as 0 points for softness and ease of loosening, and was the same as in Test Example 1. The average value of the scores of 4 people was calculated by the method of.
Regarding the ease of loosening, thermolysin was based on the evaluation results of the test group to which only thermolysin was added (test group 2) and the test group to which a protease other than thermolysin was added alone (test groups 2 to 11 of test example 1). The theoretical value of the evaluation of the test group added in combination with other proteases was calculated as the sum of the evaluation results (average value of the scores) in the test group added individually.
For example, for the test group 3 in Table 2, the theoretical value for the evaluation of looseness is "2" for the test group to which only thermolysin was added (test group 2 in Table 2), and only papain. The evaluation result of the looseness of the added test group (test group 2 in Table 1) is "0.2", and the sum of these is calculated as "2 + 0.2 = 2.2". Therefore, "2.2" is the theoretical value for the evaluation of the ease of loosening for the test group 3 in Table 2. The difference between the theoretical value calculated in this way and the actual sensory evaluation result was obtained. For example, for the test group 3 in Table 2, the sensory evaluation result is "0.1" and the theoretical value is "2.2", so the difference is "0.1-2.2 = -2.1". Is calculated.
If the difference between the theoretical value obtained as described above and the actual sensory evaluation result is larger than zero, an effect better than the theoretical value obtained as an additive effect of the effect when added alone, that is, a synergistic effect is obtained. It means that it was obtained. For the test plots where the synergistic effect was obtained, "○" was added to the synergistic effect column, and for the test plots where the synergistic effect was not obtained, "x" was added to the synergistic effect column.
(Iv) Table 2 shows the evaluation results of Test Example 2.

As shown in Table 2, it was confirmed that the combined use of a neutral protease derived from the genus Bacillus and thermolysin tends to greatly improve the looseness (test groups 7 and 9).
In Test Group 7, the difference between the score of ease of loosening obtained by sensory evaluation and the theoretical value was 1.1 points, and a neutral protease derived from Bacillus amyloliquefaciens was used in combination with thermolysin. It was shown that a synergistic effect of improving the ease of loosening can be obtained.
However, in Test Group 9, the difference between the score of ease of loosening obtained by the sensory evaluation and the theoretical value was -0.7 points, and no synergistic improvement in ease of loosening was observed.
In addition, the combined use of papain and thermolysin greatly improved the softness, but the texture became uniform like a lever, and the ease of loosening decreased on the contrary (Test Group 3).

As described above, the action on meat tissue differs depending on the type of protease used in combination with thermolysin, but the effect of improving the ease of loosening by using a neutral protease derived from Bacillus amyloliquefaciens in combination with thermolysin is It was found to be clearly larger and synergistic than the combination of thermolysin and other proteases. In particular, in stewed dishes in which the ease of loosening of meat is preferred, the effect of imparting ease of loosening based on the findings is extremely useful.

[Examples 1 to 15] Modification of chicken breast meat and preparation of cream stew By the same method as in Test Example 1, domestic young chicken breast meat is cut into 20 g pieces, placed in a vinyl bag, and Bacillus amilo. Neutral protease and thermolysin derived from Bacillus amyloliquefaciens were added in the amounts shown in Table 3 and shaken, allowed to stand at 20 ° C. for 15 minutes, and the chicken breast meat pieces were brought into contact with the protease. The chicken breast was modified. In Table 3, "-" in the neutral protease column and the thermolysin column derived from Bacillus amyloliquefaciens indicates that each enzyme is not added.
In each example, the same neutral protease and thermolysin derived from Bacillus amyloliquefaciens were used as those used in Test Example 1.
Next, a cream stew was prepared by cooking in a pan using chicken breast meat modified by contacting each of the above proteases. The cooking method of the cream stew was the same as in Test Example 1.

Chicken breast meat was taken out from the obtained cream stew of chicken breast meat and used as a sample for sensory evaluation. In addition, all of the above proteases were treated in the same manner without addition, cooked to prepare a cream stew, and chicken breast meat was taken out and used as a control.
The sensory evaluation was performed on a panel of 4 people by a scoring method of -5 to 5 points with a control of 0 points for softness and ease of loosening, and the score was calculated by the same method as in Test Example 1. The evaluation result is shown by the average value.
Regarding the ease of loosening, "○" was given to samples with an average score of 3 points or more, "△" was given to samples with 2 points or more and less than 3 points, and samples with less than 2 points were given. Was determined to be "x".
The evaluation results are also shown in Table 3.

As shown in Table 3, when cooked using chicken breast meat that has been contacted by adding thermolysin to a neutral protease derived from Bacillus amyloliquefaciens, the ease of loosening of the chicken breast meat after cooking Improvement was confirmed (Examples 1 to 15).
From these results, it was suggested that the ease of loosening can be imparted to 100 g of meat by adding 10 U to 5,000,000 U of both the neutral protease and thermolysin derived from Bacillus amyloliquefaciens. In addition, it was found that the ease of loosening of chicken breast meat was further improved by adding 100 U or more of both enzymes.
From the results of the above examples, the neutral protease and thermolysin derived from Bacillus amyloliquefaciens are brought into contact with the meat in an amount of 10 U to 5,000,000 U per 100 g of the meat, respectively, and then boiled. It was suggested that the meat can be given the ease of loosening, which is a texture that is particularly preferred in cooking.

[Example 16] Beef thigh curry A US beef thigh is die-cut into 2 cm squares, placed in a vinyl bag, and a neutral protease and thermolysin derived from Bacillus amyloliquefaciens are added. It was shaken and allowed to stand at 20 ° C. for 15 minutes for contact. As the neutral protease and thermolysin derived from Bacillus amyloliquefaciens, the same proteases used in Test Example 1 were used, and the amount of each protease added was 50, per 100 g of beef thigh meat. It was set to 000U.
Next, using the beef to which each protease was contacted in the above treatment (that is, beef thigh meat on which each protease acted), the curry was prepared by cooking as follows in a pan.
That is, the surface of the beef thigh meat was lightly fried, then water was added, and after boiling, it was simmered for 15 minutes, then a commercially available roux (manufactured by House Foods Co., Ltd.) was dissolved, and the meat was simmered for another 10 minutes. The total boiling time was 25 minutes. The ratio of water to roux was the recommended ratio of commercially available roux (water: roux = 850: 115 (weight ratio)), and a curry containing only beef thigh meat without vegetables was prepared and used as Example 16.
A curry was prepared using beef thigh meat treated in the same manner without contacting the above protease, and used as Comparative Example 1.

[Example 17] Kakuni pork belly Die-cut domestic pork belly into 3 cm squares, put them in a vinyl bag, and add a neutral protease and thermolysin derived from Bacillus amyloliquefaciens. It was shaken and allowed to stand at 20 ° C. for 15 minutes for contact. As the neutral protease and thermolysin derived from Bacillus amyloliquefaciens, the same proteases used in Test Example 1 were used, and the amount of each protease added was 50, per 100 g of pork belly. It was set to 000U.
Next, using the pork belly to which each protease was brought into contact in the above treatment (that is, the pork belly on which each protease acted), the pork belly was cooked in a pan as follows to prepare a stewed pork belly.
That is, the above pork belly is lightly boiled, water and a commercially available kakuni sauce (manufactured by Moranborg Co., Ltd.) are added, and the pork belly is covered and simmered for 40 minutes. Kakuni was prepared and used as Example 17. The total boiling time was 55 minutes. The ratio of water to dripping was the recommended ratio of commercially available dripping (water: dripping = 400: 120 (weight ratio)).
In addition, the pork kakuni was prepared by cooking in the same manner without contacting the above protease, and used as Comparative Example 2.

The beef thighs and pork ribs were taken out from the curry of each beef thigh of Example 16 and Comparative Example 1 and the pork ribs of Example 17 and Comparative Example 2, respectively, and subjected to sensory evaluation.
The sensory evaluation was carried out focusing on the ease of loosening of the meat, and was evaluated by a panel of four people according to the following evaluation criteria. The sensory evaluation results were decided through discussions among four people. For pork ribs, the ease of loosening in the meat layer was evaluated among the fat layer and the meat layer.
<Evaluation criteria>
〇: Easy to unravel compared to the comparative example Δ: Slightly unraveled compared to the comparative example ×: Evaluated as easy to unravel as the comparative example or harder to unravel than the comparative example is shown in Table 4.

As shown in Table 4, the beef in the curry of Example 16 and the pork ribs in the kakuni of Example 17 are both easier to loosen than the beef in the curry of Comparative Example 1 and the pork ribs in Comparative Example 2. Beef thighs and pork ribs in curry and kakuni prepared using beef thighs and pork ribs on which neutral protease and thermolysin from Bacillus amyloliquefaciens acted, respectively. It was confirmed that the ribs were clearly given the ease of loosening.

From the results shown in the above examples, by contacting the neutral protease and thermolysin derived from Bacillus amyloliquefaciens with the meat, it is easy to loosen the meat regardless of the type and part of the meat and the cooking menu. It has been shown that it can be given.
This effect of imparting looseness is particularly useful in stewed dishes in which the looseness of meat is preferred as a texture.

As described in detail above, according to the present invention, it is possible to provide a method for modifying meat that can easily soften meat in a short time and impart ease of loosening to the meat.
Further, according to the present invention, it is possible to provide a composition for modifying meat that can easily soften meat in a short time and impart ease of loosening to the meat.
Further, according to the present invention, it is possible to provide a food containing meat that has been softened and imparted with ease of loosening.
The present invention is extremely useful in the food field, especially in stewed dishes in which the ease of loosening of meat is preferred as a texture.

This application is based on Japanese Patent Application No. 2020-028861 filed in Japan, the contents of which are all included in the present specification.

Claims

A method for modifying meat, which is a neutral protease derived from Bacillus amyloliquefaciens and which comprises contacting a neutral protease having the following characteristics (a) to (e) and thermolysin with meat. ;
(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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).
The amount of neutral protease derived from Bacillus amyloliquefaciens in contact with meat is 10 U or more and 5,000,000 U or less per 100 g of meat, and the amount of thermolysin in contact with meat is 10 U or more per 100 g of meat. The method according to claim 1, which is 5,000,000 U or less.
A method for producing a stewed dish, which comprises using the meat modified by the method according to claim 1 or 2.
A composition for modifying meat, which is a neutral protease derived from Bacillus amyloliquefaciens and contains a neutral protease having the following characteristics (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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).
The content ratio of the neutral protease derived from Bacillus amyloliquefaciens to thermolysin (neutral protease derived from Bacillus amyloliquefaciens: thermolysin) has an enzyme activity ratio of 1: 0.000002 to 1. : The composition according to claim 4, which is 500,000.
The composition according to claim 4 or 5, which is used for modifying meat used in stewed dishes.
A food product containing a neutral protease derived from Bacillus amyloliquefaciens, which has the following characteristics (a) to (e), and meat on which thermolysin has acted;
(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 = 5-7,
(D) The optimum temperature is 50 ° C to 55 ° C.
(E) A metalloproteinase that is inhibited by ethylenediaminetetraacetic acid (EDTA).
17. Foods listed in.
The food according to claim 7 or 8, which is served as a stewed dish.