JP2004271535A - Method for measuring degree of maturing of meat, and maturing method of pork - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring the degree of maturing of meat and a maturing method of pork. <P>SOLUTION: This method for measuring the degree of maturing of meat comprises measuring the degree of maturing by use of a specified substance (e.g., muscle-derived protein, peptide, etc.) as an index. According to this, since the degree of maturing is measured by use of an objective reference, the maturing state can be precisely grasped without individual differences. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a method for measuring the degree of ripening of meat and a method for aging pork. More specifically, the present invention relates to a method for measuring the degree of ripening for obtaining meat having the best palatability and a method for aging pork using the same.

Meat is produced after slaughter through a process called “aging”. During the ripening period, it is believed that the muscles that have become rigid after mortality become softer and have an improved flavor due to the action of enzymes in muscle or the action of increasing calcium concentration in muscle plasma.
Muscles that have stiffened after death can become stiff with time, but 80% of stiffness breaks in storage at 1 ° C. in 10 days for cattle, 5 days for pigs, and 0.5 days for chickens. It is generally said that Japanese beef is aged at 0 ° C. for 2 weeks or more, and for pigs and chickens, the time for distribution and sale at 4 ° C. or less is given to the aging period.

As described above, skeletal muscle, which is the main component of meat, is converted to meat by aging. Aging is an important step, and if the aging is insufficient, meat having good texture and flavor cannot be obtained.
As a method of aging meat, in addition to the conventionally used low-temperature aging method, a high-temperature aging method, a high-temperature aging method immediately before hardening, a high-temperature aging method immediately after hardening, an electrical stimulation method, and the like are known. As an index for measuring the degree of ripening of beef, it has been reported that the shearing titer is reduced and the fragmentation rate of myofibrils is increased in connection with softening due to aging. In addition, decrease in phosphoglyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is a glycolytic enzyme, focusing on an increase in the 30 kDa component, which is a degradation product of troponin T, and changes in muscle plasma proteins during the ripening period Has been reported (Non-Patent Document 1).
However, no effective ripening index has been found for pork, and ripening is carried out based on conventional rules of thumb. As mentioned above, the aging period of pork is considered to be about 5 days, but this is empirical and has not been proven. For example, imported pork shipped chilled by sea is available on the market and may be rated softer and more delicious than short-term domestic pork.

Thus, at present, no clear standard has been obtained for an appropriate aging period for pork. There is also a problem that the indicator substance for measuring the ripening state is not specified, and an objective method for measuring the ripening degree based on the indicator substance is required. Furthermore, not only pork but also various ripened meats are currently circulating in the market, and many kinds of meat are imported, and there are many imported meats whose ripening days and conditions are unknown. Therefore, a method that can measure the degree of ripening of these meats is eagerly desired.
The present inventors have studied a method of aging pork and a substance that can serve as an indicator of the degree of maturation of pork in order to solve these problems, and as a result, have prepared a pork excellent in texture and flavor when aged at a low temperature for a long time. It became clear that it was possible, and it was possible to find an indicator of the maturity of meat. The present invention has been made based on such knowledge, and an object of the present invention is to provide a method for aging pork and a method for measuring the degree of aging of meat, from which pork having good palatability is obtained.

The present invention made in order to solve the above problems,
(a) a method for measuring the degree of maturation of pork, comprising measuring the proportion of small pieces composed of 1 to 4 sarcomere in myofibrils;
(b) a method for measuring the degree of maturation of pork, which comprises subjecting a pork sample to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and measuring a 30 kDa fraction;
(c) a method of measuring the degree of maturation of pork, comprising measuring the amount of phosphoglyceraldehyde triphosphate dehydrogenase (GAPDH) in a pork sample;
(d) a method for measuring the degree of ripening of meat, which comprises measuring peptide fragments in the meat;
(e) the method for measuring the maturity of meat according to (d) above, wherein the peptide fragment to be measured is a peptide fragment consisting of an amino acid sequence represented by the following formula (1), (2) or (3);
Ala-Pro-Pro-Pro-Pro-Ala-Glu-Val-His-Glu-Val-His-Glu-Glu-Val-His- (1)
Val-Pro-Thr-Pro-Asn-Val-Ser-Val-Val-Asp-Leu-Thr- (2)
Ala-Pro-Pro-Pro-Pro-Ala-Glu-Val-His-Glu-Val-Pro-Glu-Glu-Val- (3)
(f) a method for aging pork, which comprises aging pork until any of the following conditions (1) to (4) is met;
(1) In the method described in the above (a), the ratio (%) of the number of myofibrils composed of 1 to 4 sarcomere to the total number of myofibrils is 70 or more;
(2) In the method described in the above (b), the ratio of the 30 kDa fraction to the total protein is 80 or more, or the ratio of the 30 kDa fraction to actin is 0.03 or more;
(3) The method according to (c) above, wherein the ratio of GAPDH to total protein is 1000 or less, or the ratio of GAPDH to myoglobin is 4 or less; or
(4) In the method described in (e) above, a peptide fragment represented by the formula (1) or (2) is detected;
It is.

According to the method for measuring the maturity of the present invention, the maturity is measured based on an objective criterion, so that it is possible to grasp the maturation state accurately and without an individual difference.
Further, the method for aging pork of the present invention is a method of aging using the above-described method, and according to the method for aging pork of the present invention, unlike the conventional empirical method, good taste, aroma and texture are obtained. This has the effect that pork having the formula (1) can be reliably obtained.

As the pork used in the present invention, a carcass prepared by dismantling a pig after slaughtering or a mass of meat collected therefrom is used.
Such pork is pre-cooled (for example, overnight at 0 to 2 ° C.) if necessary, and is bacteriostatic (for example, washed with clean water, hypochlorite solution, etc.) on the meat surface, and then impermeable to gas. Vacuum-packed pork is prepared by storing in a container made of a packaging material, vacuum degassing according to a conventional method, and sealing by means such as heat sealing.
As the gas-impermeable packaging material, any material can be used as long as it is a material that does not substantially transmit gas, but a plastic film (including a sheet) having a high gas-impermeable property is preferred. Is used. Examples of such a plastic material include a single-layer film such as a polypropylene film, a high-density polyethylene film, and a polyester film; a plastic laminate film such as a laminate film composed of unstretched polypropylene and stretched polypropylene; an intermediate layer being an aluminum layer, and an outer layer being A laminated film made of a plastic film is exemplified.
Alternatively, pork may be stored and sealed in a substantially gas impermeable container made of a hard material.

The vacuum-packed pork is stored at an appropriate temperature, preferably 4 ° C., until ripened. At this time, if the pork is vacuum-packaged, it is possible to prevent the rot of the pork and to prevent the water from being scattered, and the degree of vacuum is only required to achieve this purpose.
In the present invention, the present invention is not limited to the above examples, and can be appropriately changed. For example, when packing pork, a method of simultaneously containing an oxygen scavenger and / or an inert gas (for example, nitrogen Gas, argon gas, carbon dioxide gas, etc.) may be used to improve the storage stability of pork.

  The present inventors have found a substance that can serve as an indicator of the degree of maturation of pork when studying a method of aging pork. (A) Proportion of small pieces composed of 1 to 4 sarcomere in myofibrils; (b) 30 kDa fraction when pork sample was subjected to SDS-PAGE analysis; and (c) GAPDH content Can be used as an indicator of the degree of maturation of pork, and peptide fragments that are degradation products of muscle proteins, in particular, peptide fragments having the amino acid sequence represented by the above formula (1), (2) or (3) It became clear that it could be used as an indicator of maturity. The measurement method of the present invention is a method of measuring the maturity by measuring these substances.

The first measuring method of the present invention comprises measuring the proportion of small pieces composed of 1 to 4 sarcomere in myofibrils.
Skeletal muscle, which is the main body of meat, is composed of muscle fibers, and the muscle fibers are composed of a bundle of a plurality of myofibrils. Myofibrils are formed by alternately arranging a large number of A filaments, which are aggregates of myosin molecules, and a large number of I filaments, which are aggregates of actin molecules. The I filament is held by a disk-shaped Z line. One unit between the Z line and the Z line is called sarcomere. As the number of days of storage increases, the Z-ray portion becomes weaker, and thus, by light homogenization, the myofibrils are divided into fragments cut at the Z-ray portion and fragmented. The present inventors measured the proportion of small pieces composed of 1 to 4 sarcomere in myofibrils of a sample prepared by performing a light homogenization operation. When this ratio is compared with the result of the sensory test, the ratio is correlated with the degree of maturation of pork, and in particular, the fragmentation rate of myofibrils reflects the hardness of pork and can be an index of maturity. I found that. This measurement method is to measure the degree of ripening using this index.

To describe the measurement method more specifically, first, an appropriate amount of a pork sample during the aging period is collected to obtain ground meat. Next, an appropriate buffer (for example, a phosphate buffer) is added to the minced meat, homogenized, centrifuged, and the supernatant is removed. After appropriately washing the precipitate with a buffer, the precipitate is suspended in the buffer to obtain a myofibril suspension. An appropriate amount of this suspension is dropped on a slide glass and observed or photographed under a phase contrast microscope. Then, the total number of myofibrils and the number of myofibrils composed of 1 to 4 sarcomere were measured, and the ratio of the number of myofibrils composed of 1 to 4 sarcomere to the total number of myofibrils (% This value, denoted by MFI, for convenience, is calculated.
When the MFI thus obtained was compared with the results of sensory tests, etc., it was found that ripening was completed when the MFI was 70 or more, preferably 80 or more, and that the pork had good taste, aroma and texture. That is, according to the method for measuring the degree of ripening, MFI is measured by the above-described method, and when the value is 70 or more, preferably 80 or more, it can be determined that ripening is completed.

The second method for measuring the maturity in the present invention comprises subjecting a pork sample to SDS-PAGE analysis and measuring a 30 kDa fraction.
When pork is stored and matured, myofibrillar proteins are converted to protein subunits or lower molecular substances by the action of proteolytic enzymes. However, the present inventors have analyzed pork samples by SDS-PAGE analysis. When ripening progressed, a 30 kDa fraction considered to be derived from troponin T appeared, and it was found that this could be an index of the ripening degree. This measurement method is to measure the degree of ripening using this index.

  To describe the measurement method more specifically, first, an appropriate amount of a pork sample during the aging period is collected to obtain ground meat. Next, a buffer solution having an appropriate ionic strength (for example, a phosphate buffer solution) is added to the minced meat, homogenized, centrifuged, and the precipitate is heat-treated in the presence of SDS, subjected to SDS-PAGE, and subjected to 30 kDa extraction. This is done by measuring minutes. SDS-PAGE may be performed according to a conventional method, and staining after electrophoresis may be performed using a conventional staining agent such as Coomassie brilliant blue.

For the measurement of the stained 30 kDa fraction, the concentration of each band was measured using a densitometer, and the ratio of the concentration of the 30 kDa fraction to the total protein mass (for convenience, referred to as a 30 kDa component index) or the ratio to actin (for convenience, relative to actin) Ratio).
When the thus obtained 30 kDa component index and the ratio of actin are compared with the results of a sensory test, the 30 kDa component index becomes 80 or more, preferably 100 or more, and the actin ratio becomes 0.03 or more, preferably 0.05 or more. The aging was completed, and it was found that the pork had good taste, aroma and texture. That is, according to the method for measuring the degree of maturation, the 30 kDa component index and / or the ratio of actin is measured by the above method, and if the value is equal to or more than the above value, it can be determined that the maturation is completed. it can.

The third method for measuring the maturity in the present invention comprises measuring the GAPDH content in a pork sample.
As pork is stored and matured, some sarcoplasmic proteins are converted to low molecular substances by the action of proteolytic enzymes. The present inventors have found that when a pork sample is subjected to SDS-PAGE analysis, as the ripening proceeds, the GAPDH fraction decreases, and this can be an indicator of the ripening degree. This measuring method measures the degree of ripening using this index.
To explain the measurement method more specifically, first, an appropriate amount of a pork sample during the aging period is collected to obtain ground meat. Then, a buffer solution (for example, a phosphate buffer solution) having an appropriate ionic strength is added to the minced meat, homogenized, centrifuged, and the supernatant is separated. The supernatant is separated, and in the presence of a reducing agent such as 2-mercaptoethanol and SDS. And subjected to SDS-PAGE, and measuring the GAPDH fraction (36 kDa fraction). SDS-PAGE may be performed according to a conventional method, and staining after electrophoresis may be performed using a conventional staining agent such as Coomassie brilliant blue.

For the measurement of the stained GAPDH fraction, the concentration of each band was measured with a densitometer, and the ratio of the concentration of the GAPDH fraction to the total protein mass (for convenience, referred to as the GAPDH index) or the ratio to the myoglobin (for convenience, the ratio of myoglobin to myoglobin) It is easy to measure).
When the GAPDH index and the ratio of myoglobin thus obtained are compared with the results of the sensory test described above, ripening is completed when the GAPDH index becomes 1000 or less, preferably 500 or less, and the myoglobin ratio becomes 4 or less, preferably 2 or less. It turned out that the pork had good taste, aroma and texture. That is, according to the method for measuring the degree of ripening, the GAPDH index and / or myoglobin ratio is measured by the method described above, and if the value is equal to or less than the above value, it can be determined that ripening is completed. .
In the above example, the GAPDH content is measured by SDS-PAGE analysis, but may be measured by other methods. For example, an antibody obtained by using GAPDH or the peptide represented by the formula (2) as an antigen is used. May be used to measure the GAPDH content by a conventional immunoassay.

The fourth method for measuring the degree of ripening in the present invention is a method for measuring the degree of ripening of meat, comprising measuring peptide fragments in meat, and particularly preferably the above formula (1), (2) or (3). ) Is measured.
As described above, muscles are decomposed by the action of enzymes during the ripening period and are converted into peptides and amino acids.However, it is understood that peptide fragments generated as ripening progresses can be an indicator of meat ripening degree. I found it. This measurement method focuses on an arbitrary peptide fragment among the generated various peptide fragments, and measures the maturity using this peptide fragment as an index. Particularly preferably, the above formulas (1) and (2) are used. Alternatively, the measurement is performed using a peptide having the amino acid sequence represented by (3) as an index.

  An example of the present measurement method is to measure a peptide having the sequence represented by the formula (1) (hereinafter referred to as P1 peptide) or a peptide having the amino acid sequence represented by the formula (2) (hereinafter referred to as P2 peptide). This will be described more specifically. First, an appropriate amount of pork sample during the ripening period is collected and ground. About 0.1% of a 2-mercaptoethanol solution is added to the minced meat, homogenized with a homogenizer, and then a protein remover (for example, trichloroacetic acid) is added and stirred. After leaving the mixture in a cool place for about one night, it is centrifuged to separate the supernatant. The supernatant (acid-soluble extract from which protein has been removed) can be subjected to conventional measurement means such as HPLC and immunoassay to measure P1 peptide or P2 peptide.

In the above example, the measurement operation by HPLC may be performed according to a conventional method.
When an immunoassay is used, an antibody (monoclonal antibody or polyclonal antibody) against the P1 peptide and / or P2 peptide is prepared, and a conventional immunoassay (for example, Enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, etc.). Examples of the measurement method include a sandwich method using a solid phase carrier, a competition method, and a two-antibody method. Antibodies used in these immunoassays can be prepared by a conventional method.For example, monoclonal antibodies can be prepared, if necessary, by using a protein such as albumin or a polyamine matrix (Proc. Natl.Acad.Sci. USA, 85, 5409-5413, 1988) after immunizing a rat with a P1 peptide or a P2 peptide to which the spleen cells of the rat were fused with rat myeloma cells, and an antibody recognizing the above peptide from the obtained hybridoma. Can be obtained by screening cells that produce E. coli and culturing the antibody-producing cells. The polyclonal antibody can be immunized by administering the above-mentioned P1 peptide or P2 peptide or a complex thereof to an experimental animal (eg, rabbit, goat, sheep, rat, mouse, etc.) together with Freund's ajuvant. Then, it can be obtained from the serum according to a conventional method.

  As will be shown in Examples described later, P1 peptide is useful as an easy-to-detect indicator because it has a large amount of production and significantly increases with aging. On the other hand, the P2 peptide is a peptide showing a tendency to increase up to 20 days after storage and decrease after 30 days, and as described above, the storage period is 10 to 30 days, preferably about 15 to 25 days, when it is most delicious. Therefore, P2 can be used as an index for determining a delicious time.

In addition, since the method of measuring the maturity of the present invention involves measuring a peptide fragment which is a decomposition product of muscle protein, it is not limited to pork, but may be used for other meats (eg, beef, chicken, mutton, horse, goat, It can also be used to measure the degree of ripening of rabbit meat. That is, it is inferred that the P1 peptide is a degraded peptide derived from troponin T and the P2 peptide is a degraded peptide derived from GAPDH. Troponin T and GAPDH are proteins contained not only in pork but also in other meats. For example, in beef, as in pork, P2 peptide is produced as ripening proceeds, and P1 peptide is produced in P1 peptide. A peptide represented by the formula (3) is produced as a corresponding peptide.
As described above, the method for measuring the degree of maturity, which comprises measuring the peptide fragments which are the decomposition products of muscle proteins, can be widely used for measuring the degree of maturity in general meat.

Further, the method for measuring the degree of maturation has a feature that the operation is simple. That is, in the above-described method for measuring the 30 kDa fraction, extraction of the 30 kDa fraction requires a solvent having an appropriate ionic strength. However, peptide fragments such as P1 peptide and P2 peptide are water-soluble peptides, and are extracted with water. Therefore, the sample preparation operation is simple. If the sample is a high ionic strength solution (high salt concentration solution), a desalting step may be required to avoid interference with the measurement due to salt. There is the advantage that no salt operation is required.
Furthermore, since peptide fragments such as P1 peptide and P2 peptide are also contained in the broth (drip) leached during storage of meat, the drip can be used as a measurement sample. The measurement sample can be prepared without destroying the meat.
Recently, meat is often packaged in trays and retailed, and drip absorbent paper made of a water-absorbing polymer is generally used between the tray and the meat. If a device for measuring or detecting P1 peptide or the like is incorporated in the drip-absorbing paper as an indicator, the indicator indicates the aging state of the meat, so that the meat can be sold while promoting the aging state of the meat.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the examples. The samples and aging conditions used in the following tests are as follows.

Samples and Aging Conditions Four LWD (Landrace × Large Yorkshire × Duroc) pigs were used as test pigs. After slaughter, the test pigs were cooled overnight at 0 to 2 ° C. as carcasses, and a total of eight loin parts were collected from each individual. Each loin was bacteriostatically treated with sodium hypochlorite (available chlorine: 10 ppm), divided into four parts (shoulder, middle shoulder, middle thigh, thigh side), and vacuum-packed using a cryo-back film. The sample thus prepared was sacrificed at 4 ° C for the purpose of ripening, stored for 2, 5, 10, 15, 20, 25, and 30 days, and then stored frozen at -20 ° C until analysis.

Example 1
The aged pork obtained by storage was subjected to a sensory test, and changes in the taste of the pork after long-term aging were examined. Further, changes in amino acid composition and hardness during the aging period were examined.
(1) Sensory test The sensory test sample was stored frozen at -20 ° C after aging until the test. The frozen sensory test sample was cut to a thickness of 12 mm with a band saw, spontaneously thawed, and the loin core was taken out. In the sensory test, a roasted core from the same part of the same carcass (eg, the left and right shoulders of carcass A) was used to eliminate differences between carcasses and between parts. The roasted core was heated on a hot plate at 180 ° C. for one minute, one minute, the back one minute, and repeated one more time for a total of four minutes. The inspection was performed four times by trained panelists (9 to 11 persons) using a two-point comparison method based on four items of taste intensity, scent intensity, softness, and palatability (overall evaluation). However, the intensity of the taste was measured using a nasal plug to distinguish the flavor from the flavor.

  Table 1 shows the results. As shown in Table 1, the pork during the other storage periods had significantly stronger taste and aroma than the second day. On the 2nd to 30th days of storage, no significant difference was observed in the intensity of the taste on the 10, 20, and 30th days, but the taste and aroma became significantly stronger and became significantly softer with aging. Tended to be. Under the influence, the palatability tended to be significantly improved by aging. No significant difference was observed up to the 30th day of storage, but after the 30th day of storage, sour odor was generated in pork, indicating a limit of ripening due to problems with storage stability and the like. From this, it became clear that in the early ripening period, hard pork with weak taste and fragrance became stronger and softer due to ripening and the palatability improved. From the results of the sensory tests, it became clear that the taste of pork was improved by long-term aging, and when stored at 4 ° C., aging for about 15 to 25 days was optimal.

(2) Changes in Amino Acid Composition of Meat During Aging For amino acid analysis, a part of the left side of the loin frozen and stored at -20 ° C after aging was used. The sample was naturally thawed, and after removing fat and connective tissue, it was minced with a 3 mm-diameter plate and stored frozen at -20 ° C until analysis. 22 ml of 0.1% mercaptoethanol was added to 5 g of the cryopreserved minced meat for analysis, and homogenized for 1 minute. 3 ml of 50% trichloroacetic acid was added to this suspension, mixed, left standing in a refrigerator overnight, filtered by centrifugation. 1N-NaOH was added to 1 ml of the supernatant to adjust the pH to 2.3, and the volume was adjusted to 5 ml to obtain a sample for free amino acid analysis. Further, 1 ml of concentrated hydrochloric acid was added to 1 ml of the supernatant, and the mixture was heated at 110 ° C. for 24 hours using a block heater (hydrochloric acid hydrolysis). After heating, the solution was dehydrochlorinated and the volume was increased to 10 ml, which was used as a hydrolyzate amino acid analysis sample. Beckman (SYSTEM 6300E) amino acid analyzer was used for sample analysis. The amino acid composition of the peptide was calculated by subtracting the free amino acid amount from the hydrolyzate amino acid amount.

FIG. 1 shows changes in the total amount of free amino acids and the total amount of peptides. As shown in FIG. 1, the total amount of free amino acids and the total amount of peptides increased with aging. In addition, the content of individual amino acids increased with aging. It has been reported that the dipeptides Car and Ans have a taste buffering action. Although the concentrations of these substances did not increase by aging, Car was present in a considerable amount in the meat, and it was presumed that Car contributed to improving the taste.
As described above, free amino acids and peptides increased due to aging, and it was presumed that these components were greatly involved in improving the taste due to aging.

(3) Changes in meat hardness (Tenderness) The sample was sliced with a band saw with a frozen thickness of about 15 mm and packaged in a vacuum using a cryo-back film. After the natural thawing, the sample was heated in a constant temperature water bath at 80 ° C. for 20 minutes, immediately cooled with running water, the sample was taken out, the surface of the sample was wiped off, wrapped in Saran wrap, and refrigerated in a refrigerator overnight. The next day, it was cut into a size of 10 × 10 × 50 mm in parallel with the muscle fibers, and the hardness was measured with a tensipressor (TTP-50B, manufactured by Taketomo). One carcass was measured 30-40 times.

  The change in meat hardness during storage at 4 ° C. is shown in FIG. The hardness of the meat decreased with the storage period, indicating that the meat was softened by aging. In particular, the hardness of meat decreased remarkably in the early stage of storage, but the change was small after 10 days of storage, particularly 15 days after storage. This tendency was consistent with the change in tenderness of meat in the sensory test in Table 1. On the other hand, after the 35th day, the meat was excessively aged, so that it was considered that the meat tended to be further softened and lacked in taste. On the 10th day of storage, more preferably for 15 to 30 days of storage, the softness was just good, and it was considered that aging was appropriate.

Example 2
Observation of myofibril morphology and fragmentation rate Myofibrils were prepared by the following method.
The longissimus thoracic muscle from which fat and connective tissue have been removed is minced with a plate having a diameter of 3 mm, 1 g of the obtained minced meat is collected, 100 mM KCl, 20 mM KH2PO4, 5 mM EDTA-2Na, 1 mM MgCl2, 1 mM NaN3 buffer (pH 7.0). Was added and homogenized for 1 minute under ice-cooling using a Polytron (PT10-35, manufactured by KINEMATICA) (scale 5). After the suspension was centrifuged, the supernatant was discarded, and the precipitate was washed twice with the above buffer solution and filtered with gauze. To the washed precipitate, 10 ml of the same buffer was added to suspend. To 1 ml of the myofibril suspension thus obtained, 3 ml of buffer was added and mixed. One drop of the diluted suspension was dropped on a slide glass, placed on a cover glass, observed at 1,000 times with a phase contrast microscope using an oil immersion lens, and photographed.
According to the method of Takahashi et al. (J. Food Sci., 32, 409-413, 1967), the percentage of the number of myofibrils consisting of 1 to 4 sarcomere (MFI) in the total number of myofibrils in the photograph taken. I asked.

The morphological changes of myofibrils during storage of pork are shown in FIG. The myofibrils were divided into pieces cut at the Z-ray part as the storage days progressed, and many myofibrils having 4 or less sarcomere were found. From day 10 to day 20, it was observed that the percentage of fragmented myofibrils increased significantly.
Next, changes in the fragmentation rate (MFI) of muscle fibers during the storage period are shown in FIG. It was observed that the fragmentation rate of myofibrils increased with aging, and particularly increased greatly from day 10 to day 20. Since the measurement of the fragmentation rate of myofibrils is considered to reflect the hardness of pork, it is effective as an aging index. Considering the results of the sensory test and the change in hardness, the MFI is about It was found that ripening was almost completed at 70 or more, and that the optimal ripening time was 80 or more.

Example 3
Measurement of the 30 kDa fraction by SDS-PAGE As the myofibrillar protein, the myofibril suspension obtained in Example 2 was used. The myofibril suspension was dissolved in 0.5 M Tris-HCl buffer (pH 6.8) containing 2.5% SDS and 5% 2-mercaptoethanol, and heat-treated in boiling water for 3 minutes. The electrophoresis was performed at room temperature and 20 mA for 100 minutes using a separation gel concentration of 12% and a concentrated gel concentration of 5%. Quantitative analysis from the SDS-PAGE image was performed with a densitometer (Shimadzu Chromatoscanner CS-9000). That is, after staining the SDS-PAGE gel, the gel was placed on a stage of a densitometer, and the concentration of each band was measured by transmitted light at 570 nm. In addition, in order to remove the influence of the non-protein component of the myofibrillar protein and to correct the variation, the amount of each protein was measured by the Lowry method. That is, each fraction was diluted with distilled water, dissolved in a solution containing copper sulfate-sodium citrate-sodium carbonate-sodium hydroxide, a phenol reagent was added, and the absorbance was measured at 750 nm. Bovine albumin was used as a standard.

An SDS-PAGE image of the myofibrillar protein is shown in FIG. Two new bands appeared around the molecular weight of 30 kDa after the 5th day of storage, and these bands tended to become darker as the number of days of storage increased. Conversely, among the bands near the molecular weight of 40 kDa, the band of 37 kDa considered to be troponin-T disappeared after 15 days, and the band of 40 kDa estimated from the molecular weight marker position tended to be thin.
The newly emerging 30 kDa band was measured with a densitometer to see changes during storage. FIG. 6 shows the change in the 30 kDa fraction in terms of the ratio (30 kDa component index) to the total amount of myofibril protein, and FIG. 7 shows the ratio in terms of actin (actin ratio).
As shown in FIG. 6, the 30 kDa component index increased in a quadratic curve until day 20, and did not change thereafter. Considering the results of the sensory test and the change in hardness, it was found that ripening was almost completed when the 30 kDa component index was about 80, and that 100 or more was the optimal ripening time.
Further, as shown in FIG. 7, the ratio of actin to act rapidly increased from the 15th day. Considering the results of the sensory test and the change in hardness, it was found that ripening was almost completed when the ratio of actin to actin was about 0.03, and that the optimum ripening time was 0.05 or more.

Example 4
Measurement of GAPDH Fraction by SDS-PAGE As a specimen for measuring muscle plasma protein, a centrifuged supernatant obtained at the time of myofibril preparation described in Example 2 was used. The sarcoplasmic proteins were dissolved in 0.5 M Tris-HCl buffer (pH 6.8) containing 2.5% SDS and 5% 2-mercaptoethanol, and heat-treated in boiling water for 3 minutes. Then, electrophoresis was performed under the same conditions as described in Example 3, and quantitative analysis was performed from the SDS-PAGE image.
FIG. 8 shows an SDS-PAGE image of the sarcoplasmic protein. A tendency was observed that the band around 36 kDa disappeared after 10 days of storage, particularly after 15 days of storage. The 36 kDa band is considered to be a subunit of GAPDH (144 kDa).

The band of the GAPDH subunit was measured with a densitometer to see changes during storage. FIG. 9 shows the change in the GAPDH subunit in terms of the ratio to the total amount of sarcoplasmic proteins (GAPDH index), and FIG. 10 shows the change in the ratio to myoglobin (ratio to myoglobin). As shown in FIG. 9, the GAPDH index decreased sharply until day 15, and hardly changed thereafter. Considering the results of the sensory test and changes in hardness, it was found that ripening was almost completed when the GAPDH index was about 1000, and that the optimal ripening time was 500 or less.
In addition, as shown in FIG. 10, the ratio of myoglobin rapidly decreased until day 10 and gradually decreased until day 15, but did not change thereafter. In view of the results of the sensory test and the change in hardness, it was found that ripening was almost completed when the ratio of myoglobin to 4 was 4, and that the optimal ripening time was 2 or less.

Example 5
Measurement of P1 peptide and P2 peptide To 5 g of minced meat of the longissimus dorsi, 22 ml of 0.1% mercaptoethanol was added and homogenized for 1 minute. 3 ml of 50% trichloroacetic acid was added to this suspension, mixed, left standing in a refrigerator overnight, filtered by centrifugation. This supernatant (acid-soluble extract obtained by deproteinization) was used for peptide analysis. Separation and fractionation of the peptide were performed by HPLC using an ODS column (Sensepak VP-318, 4.6 × 250 mm). Distilled water containing 0.09% TFA as solution A and 80% acetonitrile containing 0.08% TFA as solution B were used. The peptide was eluted by a gradient method of 50% of solution A and 50% of solution B. The flow rate was 1.0 ml / min, the column temperature was 40 ° C., and 100 μl of the sample was injected during the analysis and 500 μl was injected during the fractionation. The peptide concentrated after continuous fractionation was analyzed for amino acid sequence using a protein sequencer (477A, manufactured by Applied Biosystems), and homology search was performed to examine the derived protein.

  When the acid-soluble extract was eluted by HPLC using an ODS column, amino acids were eluted at first, and then low-molecular peptides were eluted. FIG. 11 shows a chromatogram showing the separation of peptides having different storage periods. As the storage period becomes longer, the peak of the separated peptides tends to increase, and the oligopeptides increase with aging, indicating that these peptides can be used as an indicator of the ripening degree. Further, as described above, it has been found that the taste becomes stronger and the taste becomes better by aging, and this data also suggests that the aging leads to an increase in the amount of the peptide, thereby making the taste better.

Among the various peptides in FIG. 11, P1 and P2 peptides were selected as effective ripening indexes. Changes in the amounts of P1 and P2 peptides during the ripening period are shown in FIGS. 12 and 13, respectively. As shown in FIG. 12, the amount of the P1 peptide produced is large and significantly increases with ripening, so that it is suitable as a ripening index for easy detection. In addition, as shown in FIG. 13, P2 was a peptide that increased until the 20th day of ripening and then decreased slightly. As described above, it has been found that the 15th to 25th days of ripening are the most delicious times, and it has been found that the P2 peptide is suitable as an aging index for knowing the delicious times.
Each peptide was fractionated and purified by the same HPLC as described above, and the amino acid sequence was determined using a peptide sequencer. As a result, it was found that P1 was a peptide of 16 residues having the amino acid sequence represented by the formula (1) (SEQ ID NO: 1) and was a degradation product derived from troponin T. P2 was a 12-residue peptide (SEQ ID NO: 2) having an amino acid sequence represented by the formula (2), and was found to be a GAPDH-derived degradation product. The P2 peptide was homologous to porcine GAPDH.

  Separately, beef was stored at 4 ° C., subjected to HPLC in substantially the same manner as described above, and fractions corresponding to the P1 peptide and the P2 peptide were collected and purified. The sequence was determined. As a result, also in the beef, the fraction corresponding to the P2 peptide had the same amino acid sequence as the P2 peptide. Further, it was revealed that the peptide corresponding to the P1 peptide had the amino acid sequence represented by the formula (3) (SEQ ID NO: 3). It was shown that these can be used as an index of maturity.

It is a figure which shows the change of the total amount of free amino acids and the total amount of peptides during the ripening period. It is a figure which shows the change of the hardness of the meat during the aging period. It is a figure which shows the change of the form of a myofibril during a ripening period. It is a figure showing change of MFI during a ripening period. It is a figure which shows the change of SDS-PAGE of a myofibrillar protein during a ripening period. It is a figure which shows the change of the 30 kDa component index during the aging period. It is a figure which shows the change of the ratio to actin during the ripening period. It is a figure which shows the change of SDS-PAGE of a sarcoplasmic protein during the ripening period. It is a figure which shows the change of the GAPDH index during a ripening period. It is a figure which shows the change of the ratio to myoglobin during a ripening period. It is a figure which shows the change of the HPLC elution pattern of the acid-soluble extract of a pork sample. It is a figure which shows the change of the amount of P1 peptides during a ripening period. It is a figure which shows the change of the amount of P2 peptides during the ripening period.

Claims (6)

  A method for measuring the degree of maturation of pork, comprising measuring the proportion of small pieces composed of 1 to 4 sarcomere in myofibrils.   A method for measuring the degree of maturity of pork, which comprises subjecting a pork sample to SDS-polyacrylamide gel electrophoresis analysis and measuring a 30 kDa fraction.   A method for measuring the degree of maturity of pork, comprising measuring the amount of phosphoglyceraldehyde triphosphate dehydrogenase (GAPDH) in a pork sample.   A method for measuring the degree of ripening of meat, comprising measuring peptide fragments in the meat. The method according to claim 4, wherein the peptide fragment to be measured is a peptide fragment having an amino acid sequence represented by the following formula (1), (2) or (3).
Ala-Pro-Pro-Pro-Pro-Ala-Glu-Val-His-Glu-Val-His-Glu-Glu-Val-His- (1)
Val-Pro-Thr-Pro-Asn-Val-Ser-Val-Val-Asp-Leu-Thr- (2)
Ala-Pro-Pro-Pro-Pro-Ala-Glu-Val-His-Glu-Val-Pro-Glu-Glu-Val- (3) A method for aging pork, comprising aging pork until one of the following conditions (1) to (4) is met.
(1) The method according to claim 1, wherein the ratio (%) of the number of myofibrils composed of 1 to 4 sarcomere to the total number of myofibrils is 70 or more;
(2) The method according to claim 2, wherein the ratio of the 30 kDa fraction to the total amount of the protein is 80 or more, or the ratio of the 30 kDa fraction to the actin is 0.03 or more;
(3) The method according to claim 3, wherein the ratio of GAPDH to total protein is 1000 or less, or the ratio of GAPDH to myoglobin is 4 or less; or
(4) In the method according to claim 5, the peptide fragment represented by the formula (1) or (2) is detected.

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