Method for measuring heme pigments in meat

Abstract

Provided is a method for measuring heme pigments in meat more easily than in the past without using special equipment.　Heme pigments are extracted from meat to obtain an extract, and the post-oxidation extract is dispensed to obtain a first extract and a second extract. The pH of the first extract is adjusted to 5.0-7.5 and the pH of the second extract is adjusted to 8.0-10.0, the extracts are oxidized, and measurements are taken of a first absorbance, which is the absorbance of the first extract at a predetermined wavelength, and a second absorbance, which is the absorbance of the second extract. The myoglobin concentration and the hemoglobin concentration in the meat are calculated from the difference between the measurements taken for the first absorbance and second absorbance, whereby the heme pigments in the meat is measured.

Description

Method for measuring heme pigment in meat

[0001] The present invention relates to a method for measuring heme pigment in meat.

[0002] In the distribution of livestock meat, the occurrence of muscle bleeding (blood spots) and residual blood in carcasses is a factor that reduces the appearance and odor of the meat, thereby lowering its quality, and it is desirable to reduce it further. Although it is known that blood spots and residual blood occur depending on the slaughtering method and bleeding conditions (Non-Patent Literature 1), their evaluation has mostly been done by visual inspection.

[0003] Non-patent document 1 reports a method for measuring the amount of residual blood in muscle by separating hemoglobin from a pig diaphragm leg muscle extract using gel filtration and measuring its absorbance at 578 nm. Non-patent document 2 reports a method for measuring myoglobin, hemoglobin, and cytochrome c in pork by treating a pork extract with carbon monoxide aeration and then measuring its absorbance.

[0004] Hirohide Oshika et al., Food Hygiene Research, Vol. 41, No. 5, pp. 79-86 (1991) Masahiro Waga et al., Journal of the Japanese Society of Swine Surgery, Vol. 53, No. 1, pp. 10-15 (2016)

[0005] The method described in Non-Patent Document 1 had the problem that the separation process by gel filtration was time-consuming and required complicated procedures. Furthermore, the method described in Non-Patent Document 2 used carbon monoxide, requiring a local exhaust ventilation system, and was therefore unsuitable for routine testing.

[0006] The object of the present invention is to provide a method for measuring heme pigment in meat that is simpler than conventional methods and does not require the use of special equipment.

[0007] The present invention provides the following: (1) A method for measuring heme pigment in meat, comprising the following steps (i) to (v): (i) a step of extracting heme pigment from meat to obtain an extract; (ii) a step of dispensing the extract to obtain a first extract and a second extract, adjusting the pH of the first extract to 5.0 to 7.5 and the pH of the second extract to 8.0 to 10.0; (iii) a step of oxidizing the extract; (iv) a step of measuring a first absorbance, which is the absorbance of the first extract at a predetermined wavelength, and a second absorbance, which is the absorbance of the second extract; (v) a step of calculating the myoglobin concentration or hemoglobin concentration in the meat from the difference between the first absorbance and the second absorbance. (2) The method according to (1), wherein the predetermined wavelength in step (iv) is not 520 nm and 550 nm. (3) The method according to (1) or (2), wherein the predetermined wavelength in step (iv) is a wavelength at which the absorbance of methemoglobin is not affected by pH, and the concentration of myoglobin in meat is calculated in step (v). (4) The method according to (1) or (2), wherein the predetermined wavelength in step (iv) is a wavelength at which the absorbance of metmyoglobin is not affected by pH, and the concentration of hemoglobin in meat is calculated in step (v). (5) The method according to any one of (1) to (4), wherein step (iii) includes adding an oxidizing agent to the extract. (6) The method according to (5), wherein the oxidizing agent is at least one selected from sodium nitrite and potassium ferricyanide. (7) The method according to any one of (1) to (6), wherein step (iii) includes letting the extract stand for 12 to 24 hours under aerobic conditions at 20 to 40°C. (8) The method according to any one of (1) to (7), further comprising filtering each extract after step (iii) before absorbance measurement. (9) A kit for measuring heme pigment in meat, comprising at least one pH adjuster and at least one oxidizing agent. (10) The kit according to (9), further comprising filter paper, a filtration filter, and / or instructions describing the measurement wavelength and a formula for calculating the concentration of myoglobin or hemoglobin. This specification incorporates the disclosures of Japanese Patent Application No. 2024-162594, which forms the basis of the priority of this application.

[0008] According to the present invention, it is possible to measure the heme pigment in meat using a simple method without using special equipment.

[0009] This is a flowchart showing the steps of the method for measuring heme pigment in meat according to the present invention. These are the absorbance spectra of horse methemoglobin and metmyoglobin at different pH levels. (A) shows the absorbance spectrum of methemoglobin, and (B) shows the absorbance spectrum of metmyoglobin. This is a plot of horse metmyoglobin at pH and absorbance at 600 nm / 522 nm. From this plot, it was confirmed that the pKa value of horse metmyoglobin is 9.02. This is a graph showing the measured heme pigment concentration in beef in Example 3, Comparative Example 1, and Comparative Example 2. This is a graph showing the measured heme pigment concentration in pork in Example 4, Comparative Example 1, and Comparative Example 2.

[0010] 1. Composition and Definitions In this specification, “meat” refers to livestock meat, poultry meat, whale meat, and fish meat. In this specification, livestock meat refers to beef, pork, horse meat, lamb, and goat meat, etc. In this specification, poultry meat refers to the meat of chickens such as chickens, quail, turkeys, ducks, and geese. In this specification, fish meat refers to the meat of red-fleshed fish such as tuna, bonito, and yellowtail. The method of the present invention is applicable to livestock meat, poultry meat, and fish meat, but is particularly applicable to livestock meat. Hereinafter, unless otherwise specified, meat refers to livestock meat.

[0011] Meat production at slaughterhouses involves stunning (blunting) livestock, followed by bleeding by sticking, skinning, visceration, and splitting. During this process, blood spots and residual blood can occur due to genetic factors or inadequate procedures leading up to bleeding. In meat, residual blood is undesirable from a quality standpoint, affecting taste and shelf life. Blood spots refer to ruptured capillaries, while residual blood refers to blood remaining within blood vessels.

[0012] The amount of heme pigment in meat is known as an indicator of the amount of residual blood contained in meat. Heme pigments found in meat include myoglobin (Mb), hemoglobin (Hb), and cytochrome c. Myoglobin can be oxymyoglobin, deoxymyoglobin, metmyoglobin (OH type), or metmyoglobin (H２ Hemoglobin exists in the form of oxyhemoglobin, deoxyhemoglobin, methemoglobin (OH type), or methemoglobin (H type). ２ It exists in the form of type O. Cytochrome c exists in the form of oxidized cytochrome c or reduced cytochrome c. In other words, there are 10 different forms of heme pigments with different properties in meat. Measuring each of these pigments requires an extremely complicated procedure. Furthermore, even if we calculate the concentration of each from absorbance, the calculation formula becomes extremely complex, making accurate calculation difficult.

[0013] The inventors have discovered a method for measuring these three heme pigments by reducing a meat extract and passing carbon monoxide through it, thereby converting all Mb to carboxymyoglobin, all Hb to carboxyhemoglobin, and all cytochrome c to reduced cytochrome c (Non-Patent Literature 2). However, this method has the drawback of requiring special equipment such as a local exhaust ventilation system because it uses carbon monoxide.

[0014] Through oxidation, all Mb in meat can be converted to metMb, and all Hb can be converted to metHb. MetMb and metHb can be converted to either the OH form (alkaline form) or H form depending on the pH. ２ It is known that when the absorbance changes to type O (acidic type), a change occurs in its absorbance. This invention is characterized by using these absorbance differences due to pH to measure the total amount of Mb and total amount of Hb contained in meat. The amount of cytochrome c in the heme pigment of meat is usually less than 1%, and is very small compared to the amount of Mb and Hb, so it was excluded from the measurement target as it does not affect the evaluation of residual blood in meat. In addition, it is possible to avoid interference from cytochrome c by avoiding the measurement of absorbance at 520 nm and 550 nm, which are the absorbance peaks of cytochrome c (described later).

[0015] metMb and metHb are converted to OH form and H form depending on the pH. ２MetHb can exist in two forms, each with a different absorbance spectrum. In the absorbance change associated with pH changes, there is a point where the absorbance does not change—the isossificant point. The wavelength at which this isossificant point occurs differs between metMb and metHb. Therefore, if a change occurs at the isossificant point of metHb in response to a change in pH, it can be said that the change originates from metMb. Figure 2 shows the absorbance spectra of bovine metHb (A) and horse metMb (B) at various pH levels. As is clear from the absorbance spectrum in Figure 2A, no change in absorbance occurs with pH at wavelengths of 489 nm, 521 nm, and 617 nm. As is clear from the absorbance spectrum in Figure 2B, no change in absorbance occurs with pH at wavelengths of 495 nm, 522 nm, and 626 nm. Therefore, if a change in absorbance occurs at 480 nm, 520 nm, or 617 nm at different pH levels, it can be said that the change originates from metMb. On the other hand, if changes occur at 490 nm, 530 nm, or 626 nm at different pH levels, these changes can be attributed to metHb. As described above, metMb and metHb can be quantified without interfering with each other by appropriately selecting the measurement wavelength.

[0016] 2. Method for Measuring Heme Pigment in Meat The first embodiment of the present invention is a method for measuring heme pigment in meat. The method for measuring heme pigment in meat according to this embodiment is characterized by comprising the following steps (i) to (v): (i) a step of extracting heme pigment from meat to obtain an extract; (ii) a step of dispensing the extract to obtain a first extract and a second extract, adjusting the pH of the first extract to 5.0 to 7.5 and the pH of the second extract to 8.0 to 10.0; (iii) a step of oxidizing the extract; (iv) a step of measuring the first absorbance, which is the absorbance of the first extract at a predetermined wavelength, and the second absorbance, which is the absorbance of the second extract; (v) a step of calculating the Mb concentration or Hb concentration in the meat from the difference between the first absorbance and the second absorbance.

[0017] According to the method of this embodiment, it is possible to measure the heme pigment in meat without requiring special equipment or complicated operations.

[0018] The method of this embodiment will be described below, step by step. A flowchart of the method of this embodiment is shown in Figure 1. The method of this embodiment includes at least the following steps: (i) extraction step, (ii) pH adjustment step, (iii) oxidation step, (iv) absorbance measurement step, and (v) heme pigment amount calculation step. Steps (ii) and (iii) may be performed in the order of (ii) - (iii), or in the order of (iii) - (ii). It is particularly preferable to perform them in the order of (ii) - (iii). When performing them in the order of (iii) - (ii), it is preferable to include a step to measure pH before step (iv). In either case, it is preferable to perform a filtration step before step (iv).

[0019] 2-1 Step (i) Extraction Step The method of this embodiment includes, as step (i), a step of extracting heme pigment from meat to obtain an extract. Specifically, it includes a step of contacting the meat with a solvent to transfer the heme pigment to the solvent. The solvent used here is not particularly limited as long as it is a solvent that dissolves heme pigment, but can be selected from, for example, water and its solutions. When using an aqueous solution, buffers such as phosphate buffer and MES buffer can be suitably used.

[0020] In the extraction process, the meat may be in block form, but it is preferable that it be finely chopped, for example, minced. The finely chopped meat is further preferably homogenized in the solvent. The immersed and homogenized meat can be removed from the solvent by centrifugation or filtration using filter paper, gauze, etc. The resulting solvent can be used as an extract for subsequent measurements.

[0021] 2-2 Step (ii) pH Adjustment Step The method of this embodiment includes, as step (ii), a step of dispensing the extract obtained in step (i) into at least two portions to obtain a first extract and a second extract, and adjusting the pH of the first extract to 5.0 to 7.5 and the pH of the second extract to 8.0 to 10.0. Step (ii) may be performed before step (iii), which will be described later, or after step (iii).

[0022] The method for adjusting the pH of the first and second extracts is not particularly limited. To lower the pH, dilute hydrochloric acid, organic acid solutions (acetic acid, citric acid, etc.) can be used, while to raise the pH, sodium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, etc. can be used. Since the extract before pH adjustment is usually weakly acidic, it is often not necessary to lower the pH. In this case, it is preferable to add the same amount of water to the first extract as the aqueous solution added to the second extract to raise the pH, thereby equalizing the concentration of the heme dye.

[0023] The pH of the first extract can be 5.0 to 7.5, preferably 5.5 to 7.0, and more preferably 5.8 to 6.8. The pH of the second extract can be 8.0 to 10.0, preferably 8.5 to 10.0, and more preferably 8.5 to 9.5. Here, it is preferable that the pH of the first extract and the pH of the second extract are separated by 1.0 or more, particularly 1.5 or more, and even more preferably 2.0 or more.

[0024] 2-3 Step (iii) Oxidation Step The method of this embodiment includes a step (iii) in which the extract is oxidized. The "extract" referred to here may be each extract after pH adjustment in step (ii), or it may be the extract obtained in step (i). If the extract obtained in step (i) is subjected to the oxidation step, then the pH adjustment step in step (ii) is carried out.

[0025] The oxidation method is not particularly limited as long as the conditions are such that Mb and Hb are sufficiently metmyolated. For example, methods using an oxidizing agent or methods of standing the mixture at aerobic conditions of 20 to 40°C for 12 to 24 hours may be used.

[0026] Methods using oxidizing agents can induce metmyolysis of heme dyes in a short time. Examples of oxidizing agents include sodium nitrite and potassium ferricyanide. The concentration of the oxidizing agent and the reaction time vary depending on the type of oxidizing agent used, but for example, in the case of sodium nitrite, the concentration can be about 0.005 to 0.1% by weight, and the reaction time can be about 30 to 60 minutes.

[0027] The method of allowing the extract to stand for 12 to 24 hours under aerobic conditions at 20 to 40°C, although time-consuming, is suitable for use in facilities without waste disposal facilities because it facilitates the disposal of the used reagent. In this specification, aerobic conditions refer to conditions in which an oxygen concentration of at least 1% is present. The standing temperature of the extract is preferably 30 to 40°C, particularly 35 to 38°C.

[0028] Whether the oxidation reaction has proceeded sufficiently can be confirmed, for example, by visually observing the change in the color of the extract (from red to brown). More preferably, it can also be determined from the absorbance spectrum obtained with a spectrophotometer.

[0029] 2-4 Filtration Step The extract obtained after steps (ii) and (iii) may be filtered. For example, by performing filter filtration, the influence of precipitates formed during pH adjustment and oxidation reactions on subsequent absorbance measurements can be avoided. The material of the filter used is not particularly limited, but can be a membrane filter made of, for example, cellulose acetate, cellulose mixed ester, polyethersulfone, hydrophilic PTFE, etc. The pore size of the filter is not particularly limited, but can be 0.45 μm, 0.22 μm, etc. Filter paper may also be used. For example, quantitative filter paper with a particle holding diameter of φ3 μm can be suitably used. The filtration step is preferably performed when precipitates are formed in the pH adjustment step and the oxidation step. Furthermore, the filtration step may be performed not only after steps (ii) and (iii), but also as appropriate at the timing when precipitates are formed.

[0030] 2-5 Step (iv) Absorbance Measurement Step The method of this embodiment includes, as step (iv), a step of measuring the first absorbance, which is the absorbance of the first extract at a predetermined wavelength, and the second absorbance, which is the absorbance of the second extract.

[0031] The predetermined wavelength referred to here is preferably a wavelength at which the absorbance of methemoglobin is not affected by pH when calculating the amount of Mb in the subsequent step (v). On the other hand, when calculating the amount of Hb in the subsequent step (v), it is preferable to use a wavelength at which the absorbance of metmyoglobin is not affected by pH.

[0032] The wavelengths at which the absorbance of methemoglobin is unaffected by pH (hereinafter also referred to as the "metHb isossipation point") and the wavelengths at which the absorbance of metmyoglobin is unaffected by pH (hereinafter also referred to as the "metMb isossipation point") differ depending on the animal species. Therefore, these can be determined in advance by testing with metHb or metMb standards. Figure 2 shows the absorbance spectra of horse metHb (Figure 2A) and metMb (Figure 2B) at various pH values. From the results shown in Figure 2A, the metHb isossipation points are 480 nm, 525 nm, and 617 nm. Therefore, when calculating the amount of Mb, the specified wavelengths can be 480 nm, 525 nm, or 617 nm. As shown in Figure 2B, the metMb isosbestic points are 490 nm, 530 nm, and 626 nm. Therefore, when calculating the amount of Hb, the predetermined wavelengths can be 490 nm, 530 nm, or 626 nm.

[0033] It is preferable not to set the specified wavelength to 520 nm or 550 nm. Heme dyes contain cytochrome c, and its maximum absorption wavelengths are 520 nm and 550 nm. Measuring absorbance at these wavelengths may result in an increase in absorbance originating from cytochrome c in the sample, potentially making it impossible to accurately measure the amount of Mb or Hb.

[0034] Absorbance can be measured, for example, by placing the extract in an absorbance measurement cell with a path length of 10 mm and using a spectrophotometer. Alternatively, multiple extracts may be dispensed into microplates (24-well, 96-well, etc.) and measured using a microplate reader. The spectrophotometer or microplate reader used here is not particularly limited and any that is normally used to measure the absorbance of reagents can be used.

[0035] 2-6 Step (v) Heme pigment amount calculation step The method of this embodiment includes, as step (v), a step of calculating the Mb concentration or Hb concentration in meat from the difference between the first absorbance and the second absorbance.

[0036] As mentioned above, metMb and metHb are converted into OH form and H form depending on the pH. ２It can take two O-type forms, each with a different absorption spectrum. In the absorption change accompanying this pH change, there is a point where the change does not occur, an isosbestic point. Since the change amount of absorbance at this point (wavelength) is concentration-dependent, by previously determining the change amount per concentration, the concentration of heme pigment can be calculated from the change amount (the following formula (I)).

[0037] The change amount per concentration is based on the difference in absorbance of metMb or metHb of unit concentration estimated at any two pH values. In order to estimate the absorbance of metMb and metHb at any pH, it is necessary to determine the ratio of each OH-bonded type or H ２ O-bonded type. The ratio of the OH or H ２ O-bonded type of metMb or metHb is very similar to the ionization behavior of an acid and can be determined from the concentration acid dissociation constant (pKa) and pH of metMb and metHb. There is the following relationship between pH and pKa (the following formula (II)). (Here, [HA] is the concentration of non-dissociated acid HA, that is, the concentration of H ２ O type in metMb (metHb)) From the above formula (II), the following formula (III) is derived. Further taking the log, the following formula (IV) is derived. [[ID=​​​​​​​​​​​When absorbance increases with increasing pH (and thus with decreasing [HA]), (this applies to Mb at a wavelength of 617 nm), the value Abs(pH) that the absorbance Abs at a specific wavelength takes at a specific pH can be expressed by the following formula (IX). Abs(MIN) is the minimum absorbance per unit concentration shown when the HA concentration is lowest, and Abs(MAX) is the maximum absorbance per unit concentration shown when the HA concentration is highest.

[0039] When absorbance decreases with increasing pH (and therefore with decreasing [HA]), (this applies to Hb at a wavelength of 626 nm), the value Abs(pH) that the absorbance Abs at a specific wavelength takes at a specific pH can be expressed by the following formula (X).

[0040] Substituting equation (VIII) into equations (IX) and (X), we can obtain equations (XI) and (XII) below, respectively.

[0041] Substituting equations (XI) and (XII) into the conceptual formula of equation (I), respectively, the Mb concentration [Mb] and Hb concentration [Hb] can be expressed by the following equations (XIII) and (XIV). (Here, pHa refers to any pH (acidic side), pHb refers to any pH (alkaline side), AbsH(a) refers to the absorbance at the metHb isossipation point at pHa (Mb first absorbance), AbsH(b) refers to the absorbance at the metHb isossipation point at pHb (Mb second absorbance), AbsM(a) refers to the absorbance at the metMb isossipation point at pHa (Hb first absorbance), and AbsM(b) refers to the absorbance at the metMb isossipation point at pHb (Hb second absorbance).)

[0042] By inputting pH a, pH b, pre-measured Abs (MAX), and Abs (MIN) into the above formula (XIII), and further inputting the values ​​of AbsH(a) (Mb 1st absorbance) and AbsH(b) (Mb 2nd absorbance) measured in step (iv), the Mb concentration of the extract can be calculated. Similarly, by inputting pH a, pH b, pre-measured Abs (MAX), and Abs (MIN) into the above formula (XIV), and further inputting the values ​​of AbsM(a) (Hb 1st absorbance) and AbsM(b) (Hb 2nd absorbance) measured in step (iv), the Hb concentration of the extract can be calculated. From the Mb concentration or Hb concentration calculated here and the weight of meat used in step (i), the amount of Mb and Hb contained in the meat per unit weight can be calculated.

[0043] 2-7 Other Steps The method of this embodiment may include other steps in addition to steps (i) to (v) and the filtration step. An example of other steps is the step of determining the measurement wavelength in step (iv). In step (iv), the measurement wavelength is required to be the metHb isossippic point or metMb isossippic point. The metHb isossippic points and metMb isossippic points differ depending on the animal species from which the material originates, but if they are known in advance, that wavelength can be used. On the other hand, if the metHb isossippic points and metMb isossippic points of that animal species are unknown, they must be determined in advance by testing. Specifically, this can be determined by obtaining the absorbance spectrum of metHb or metMb under multiple pH conditions and identifying the point at which the absorbance does not change with pH.

[0044] Another example of a different process is the process of pre-determining the values ​​necessary for the calculation in process (v). In process (v), Abs(MAX) and Abs(MIN) values ​​are required to calculate the amount of Mb and Hb in the meat. These values ​​differ depending on the animal species, but if they are known in advance, those values ​​can be used. On the other hand, if the Abs(MAX) and Abs(MIN) values ​​for that animal species are unknown, it is necessary to determine them in advance by testing. Specifically, this can be determined by adjusting a solution containing a unit concentration of Mb or Hb to a pH that is sufficiently high and sufficiently low compared to pKa, and measuring the absorbance of each at a predetermined measurement wavelength.

[0045] According to the method of this embodiment, it is possible to measure the heme pigment in meat by simply adjusting the pH, adding an oxidizing agent if necessary, and measuring the absorbance, without using special equipment or complicated procedures.

[0046] 3. Kit for Measuring Heme Pigment in Meat A second embodiment of the present invention is a kit for measuring heme pigment in meat. The kit of this embodiment is characterized by comprising at least one pH adjusting agent and at least one oxidizing agent. More specifically, the kit of this embodiment is a kit for use in the method described in section "2. Method for Measuring Heme Pigment in Meat". In this embodiment, unless otherwise specified and unless otherwise contradicted, the definitions of terms used are the same as those described in "2. Method for Measuring Heme Pigment in Meat".

[0047] Examples of pH adjusting agents included in the kit of this embodiment include dilute hydrochloric acid, phosphoric acid, citric acid, succinic acid, tartaric acid, acetic acid, lactic acid, etc., which lower the pH, and sodium hydroxide solution, potassium carbonate, sodium bicarbonate, sodium carbonate, dipotassium hydrogen phosphate, potassium dihydrogen, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc., which raise the pH.

[0048] In the kit of this embodiment, any oxidizing agent capable of oxidizing heme dye can be used, but for example, sodium nitrite, potassium ferricyanide, etc., can be preferably used.

[0049] The kit of this embodiment may further include filter paper. The filter paper can be used to remove residue after cutting and homogenizing the meat. Gauze may be used instead of filter paper.

[0050] The kit of this embodiment may include a filtration filter. The filtration filter can be used to remove any precipitate that may form when a pH adjuster and an oxidizing agent are added to the heme pigment extract from meat.

[0051] The kit of this embodiment may include instructions describing the measurement wavelength and the formula for calculating the concentration of myoglobin or hemoglobin. When measuring the absorbance of heme pigment extract from meat, the appropriate measurement wavelength differs depending on the animal species from which the meat originates. Therefore, it is necessary to inform the user of the appropriate measurement wavelength for each type of meat to which it is applied. Furthermore, the formula for calculating the amount of Mb and Hb in the meat from the obtained pH and absorbance data also differs depending on the animal species from which the meat originates. Therefore, it is necessary to inform the user of the appropriate calculation formula for each type of meat to which it is applied. Preferably, the instructions include a detailed explanation of the measurement wavelength and the calculation formula. Alternatively, the instructions may display a function to indicate the appropriate measurement wavelength based on the animal species, and a QR code (registered trademark) for connecting to an application that automatically calculates the amount of Mb and Hb in the meat from the obtained pH and absorbance data.

[0052] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited to these embodiments.

[0053] <Example 1: Measurement of Absorbance Spectra at Various pH Levels of Heme Dyes> The absorbance spectra of standard metHb (derived from bovine) and metMb (derived from horse) were obtained using the following method. Both the reagent Hb (derived from bovine) and Mb (derived from horse) were treated as oxidized forms. These were added to various pH buffers to a concentration of 1 mg / mL, and the solutions filtered through a 0.45 μm filter were used as samples for metHb and metMb at various pH levels. The absorbance spectra of these samples from 450 to 750 nm were measured using a UV-Vis spectrophotometer UV-2700 (Shimadzu Corporation) in a cell with a path length of 10 mm.

[0054] The compositions of the various pH buffers were as follows: 0.1 M sodium acetate buffer (pH 4.5), 0.1 M MES buffer (pH 5.8), 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M sodium phosphate buffer (pH 7.5), 0.1 M Tris buffer (pH 8.6), 0.1 M sodium carbonate buffer (pH 9.6), and 0.1 M sodium carbonate buffer (pH 10.4).

[0055] Figure 2 shows the absorbance spectra of metHb (A) and metMb (B) at various pH levels. From the absorbance spectra in Figure 2A, it is clear that no change in absorbance occurs with pH at wavelengths of 480 nm, 525 nm, and 620 nm. From the absorbance spectra in Figure 2B, it is clear that no change in absorbance occurs with pH at wavelengths of 490 nm, 530 nm, and 630 nm.

[0056] <Example 2: Determination of the pKa value of metMb> The pKa value of horse metMb was determined by the following method. Horse metMb was prepared (Masahiro Waga, Azabu University Doctoral Dissertation (2017)), and metMb aqueous solutions of the same concentration at pH 4 to 12.0 were prepared, and the absorbance at 600 nm / 522 nm was measured for each (optical path length 10 mm). Two independent measurements were performed for each pH solution. Figure 3 shows the plot of the absorbance at 600 nm / 522 nm for metMb at each pH. From the plot, it was confirmed that the pKa value of horse metMb is 9.02.

[0057] <Example 3: Heme pigment measurement in beef> (1) Preparation of bovine Mb After mincing the beef to 3 mm, 1 part by weight of distilled water was added to 1 part by weight of the meat, and homogenized at 10,000 rpm for 1 minute using an ACE homogenizer. The obtained homogenate was centrifuged at 18,000 × g, 1 °C for 20 minutes to obtain the supernatant. The supernatant was fractionated by ammonium sulfate at a saturation of 60-90%, and the resulting pellet was used as a sample for hydrophobic chromatography. Using FPLC, the fraction that eluted at a 40% ammonium sulfate saturation on a HiTrap Phenyl HP 5 mL column was collected as Mb.

[0058] (2) Bovine Hb Bovine Hb was measured using a commercially available standard (Hemoglobin, FreezeDried (MP Biomedicals, OH, USA)).

[0059] (3) Determination of isossipitive points of bovine metHb and bovine metMb MetHb and metMb were prepared from the bovine Hb and Mb obtained in (1) and (2) above (Waga et al., 2017), and the absorbance spectra at each pH were measured in the same manner as in Example 1. As a result, it was confirmed that the isossipitive points of bovine metHb were 489 nm, 521 nm and 617 nm, and the isossipitive points of bovine metMb were 494 nm, 523 nm and 626 nm.

[0060] (4) Determination of pKa values ​​of bovine metMb and bovine metHb The pKa values ​​of each were determined in the same manner as in Example 2, except that bovine metMb or bovine metHb was used instead of horse metMb. The pKa value of bovine metMb was 9.02, and the pKa value of bovine metHb was 8.22.

[0061] (5) Determination of Abs(MAX) and Abs(MIN) Bovine metMb and bovine metHb solutions at pH 4–12 (1 mg / mL) were prepared. For bovine metMb, the absorbance at 617 nm was measured, and the highest absorbance was defined as Abs(MAX), and the lowest absorbance as Abs(MIN). For bovine metHb, the absorbance at 626 nm was measured, and the highest absorbance was defined as Abs(MIN), and the lowest absorbance as Abs(MAX). The values ​​are shown in Table 1.

[0062]

[0063] (6) Measurement of heme pigment in beef After mincing the beef to 3 mm, 3 parts by weight of distilled water was added to 1 part by weight of meat, and homogenized at 10,000 rpm for 1 minute using an ACE homogenizer. The obtained homogenate was centrifuged at 18,000 × g, 1 °C for 15 minutes to obtain the supernatant. The supernatant was filtered through filter paper (ADAVANTEC No. 1) to remove fat and obtain the filtrate (heme pigment extract). The filtrate was dispensed into two 3 mL bottles, and 3 mL of distilled water was added to one bottle and 3 mL of 1 N sodium hydroxide solution was added to the other bottle and mixed. 10 mg of sodium nitrite was added to both and mixed. The filtrate was passed through a 0.45 μm membrane filter (DISMIC 25CS045AN, Toyo Filter Paper), and the pH and absorbance at 617 nm and 626 nm of each obtained filtrate were measured. Absorbance was measured using a UV-Vis spectrophotometer (UV-2700, Shimadzu Corporation) with the filtrate placed in a quartz cell with a 10 mm path length. The measurements were performed at room temperature using a quartz glass cell with a 10 mm path length.

[0064] The measured pH and absorbance were substituted into the following formulas (XV) and (XVI) to calculate the Mb and Hb concentrations in each filtrate. (In the formula, A617a represents the first absorbance of Mb, A617b represents the second absorbance of Mb, pHa represents the acidic pH, and pHb represents the alkaline pH.) (In the formula, A626a represents the first absorbance of Hb, A626b represents the second absorbance of Hb, pHa represents the acidic pH, and pHb represents the alkaline pH.) The Mb concentration and Hb concentration are shown in Figure 4, along with the results of Comparative Examples 1 and 2 described later.

[0065] <Comparative Example 1: Measurement of Heme Pigment in Beef (Mb Equivalent)> A heme pigment extract was obtained from beef using the same method as in Example 3 (6). Assuming that the heme pigment is entirely Mb, the absorbance at 525 nm in the extract was measured. The absorbance at 525 nm was measured for several solutions containing Mb of known concentration, and a calibration curve was created. After that, the Mb concentration in the extract was calculated. Here, it is not possible to measure Mb and Hb separately, so it can be calculated as the Mb equivalent of the heme pigment. Figure 4 shows the amount of heme pigment (Mb equivalent) along with the results for Example 3 and Comparative Example 2 described later.

[0066] <Comparative Example 2: Measurement of Heme Pigment in Beef (Carbon Monoxide Method)> The heme pigment in beef was measured using a method in accordance with Non-Patent Document 2. A heme pigment extract was obtained from beef using the same method as in Example 3 (6). 3 mL of the heme pigment extract was diluted with 3 mL of water and 10 mg of hydrosulfite sodium (Na) was added. ２ S ２ O ４ The mixture was mixed with ) using a vortex mixer. The filtrate after mixing was filtered through a 0.45 μm membrane filter (DISMIC 25CS045AN, Toyo Roshi), and the absorbance was measured.

[0067] 5 mL of formic acid was added dropwise to 15 mL of concentrated sulfuric acid in an Erlenmeyer flask to generate carbon monoxide (CO) bubbles. This gas was collected in a gas collection bag by water displacement in a local exhaust ventilation system. After reducing the heme dye extract or heme dye solution, approximately 10 mL of CO gas was passed through, the flask was sealed tightly, mixed by inversion, and allowed to stand for 5 minutes.

[0068] Heme dye solutions diluted to various concentrations were similarly treated with CO, and then the absorption spectra from 350 to 750 nm were measured using a spectrophotometer. The absorption coefficient per unit concentration was obtained from the absorbance at each wavelength and the concentration (mg / mL) of the measured solution. The measurements were performed at room temperature using a quartz glass cell with a path length of 10 mm. From the absorption spectra of the heme dye extract and the absorption coefficients at each of the above wavelengths, the Mb concentration, volume, Hb concentration, and cytochrome c concentration were calculated. Figure 4 shows the Mb concentration, volume, Hb concentration, and cytochrome c concentration, along with the results for Example 3 and Comparative Example 1.

[0069] As shown in Figure 4, the amount of heme pigment (Mb + Hb) measured in Example 3 was not significantly different from the amount of heme pigment measured by conventional methods, namely the Mb equivalent of heme pigment and the carbon monoxide method. This demonstrates that the method in Example 3 can measure heme pigment in beef.

[0070] <Example 4: Heme pigment measurement in pork> (1) Preparation of porcine Mb Pork (pork shoulder) was minced to 3 mm, and 1 part by weight of distilled water was added to 1 part by weight of meat. Homogenization was performed using an ACE homogenizer at 10,000 rpm for 1 minute. The obtained homogenate was centrifuged at 18,000 × g, 1 °C for 20 minutes to obtain the supernatant. The supernatant was fractionated by ammonium sulfate at a saturation of 60-90%, and the resulting pellet was used as a sample for hydrophobic chromatography. Using FPLC, the fraction that eluted at a 40% ammonium sulfate saturation on a HiTrap Phenyl HP 5 mL column was collected as Mb.

[0071] (2) Preparation of porcine hemoglobin: Three times the volume of distilled water was added to the porcine blood and stirred. The mixture was then centrifuged at 18,000 × g, 1°C for 20 minutes to obtain the supernatant, which was used as hemoglobin.

[0072] (3) Determination of isossipative points of porcine metHb and porcine metMb The isossipative points of porcine metHb and porcine metMb were determined in the same manner as described in the supplement to Example 3 (3). As a result, it was confirmed that the isossipative points of porcine metHb were 499 nm, 523 nm and 617 nm, and the isossipative points of porcine metMb were 494 nm, 522 nm and 626 nm.

[0073] (4) Determination of pKa values ​​of porcine metMb and porcine metHb The pKa values ​​were determined in the same manner as in Example 2, except that porcine metMb or porcine metHb was used instead of horse metMb. The pKa value of porcine metMb was 8.76, and the pKa value of bovine metHb was 8.31.

[0074] (5) Determination of Abs(MAX) and Abs(MIN) Solutions of 1 mg / mL of porcine metMb and porcine metHb at pH 4 to 12 were prepared. For porcine metMb, the absorbance at 617 nm was measured, and the highest absorbance was defined as Abs(MAX), and the lowest absorbance as Abs(MIN). On the other hand, for porcine metHb, the absorbance at 626 nm was measured, and the highest absorbance was defined as Abs(MIN), and the lowest absorbance as Abs(MAX). The values ​​are shown in Table 2.

[0075]

[0076] (6) Measurement of heme pigment in pork Three pork samples (samples A to C) were each minced to 3 mm, and 3 parts by weight of distilled water were added to 1 part by weight of meat. Homogenization was performed using an ACE homogenizer at 10,000 rpm for 1 minute. The resulting homogenate was centrifuged at 18,000 × g, 1 °C for 15 minutes to obtain the supernatant. The supernatant was filtered through filter paper (ADAVANTEC No. 1) to remove fat and obtain the filtrate (heme pigment extract). The filtrate was dispensed into two 3 mL bottles, and 3 mL of distilled water was added to one bottle and 3 mL of 1 N sodium hydroxide solution was added to the other bottle and mixed. 10 mg of sodium nitrite was added to both and mixed. The filtrate was passed through a 0.45 μm membrane filter (DISMIC 25CS045AN, Toyo Filter Paper), and the pH and absorbance at 617 nm and 626 nm of each obtained filtrate were measured. Absorbance was measured using a spectrophotometer (UV-2700, Shimadzu Corporation) by placing the filtrate in a quartz cell with a 10 mm optical path length. The measurement was performed at room temperature using a quartz glass cell with a 10 mm optical path length.

[0077] The measured pH and absorbance were substituted into the following formulas (XVII) and (XVIII) to calculate the Mb and Hb concentrations in each filtrate. (In the formula, A617a represents the first absorbance of Mb, A617b represents the second absorbance of Mb, pHa represents the acidic pH, and pHb represents the alkaline pH.) (In the formula, A626a represents the first absorbance of Hb, A626b represents the second absorbance of Hb, pHa represents the acidic pH, and pHb represents the alkaline pH.) The Mb concentration and Hb concentration are shown in Figure 5, along with the results of Comparative Example 3 described later.

[0078] <Comparative Example 3: Measurement of Heme Pigment in Pork (Carbon Monoxide Method)> The heme pigment in beef was measured using a method in accordance with Non-Patent Document 2. A heme pigment extract was obtained from pork using the same method as in Example 4 (6). 3 mL of the heme pigment extract was diluted with 3 mL of water, and 10 mg of hydrosulfite sodium (Na) was added. ２ S ２ O ４ The mixture was mixed with ) using a vortex mixer. The filtrate after mixing was filtered through a 0.45 μm membrane filter (DISMIC 25CS045AN, Toyo Roshi), and the absorbance was measured.

[0079] 5 mL of formic acid was added dropwise to 15 mL of concentrated sulfuric acid in an Erlenmeyer flask to generate carbon monoxide (CO) bubbles. This gas was collected in a gas collection bag by water displacement in a local exhaust ventilation system. After reducing the heme dye extract or heme dye solution, approximately 10 mL of CO gas was passed through, the flask was sealed tightly, mixed by inversion, and allowed to stand for 5 minutes.

[0080] Heme dye solutions diluted to various concentrations were similarly treated with CO, and then the absorption spectra from 350 to 750 nm were measured using a spectrophotometer. The absorption coefficient per unit concentration was obtained from the absorbance at each wavelength and the concentration (mg / mL) of the measured solution. The measurements were performed at room temperature using a quartz glass cell with a path length of 10 mm. From the absorption spectra of the heme dye extract and the absorption coefficients at each of the above wavelengths, the Mb concentration, volume, and Hb concentration were calculated. Figure 5 shows the Mb concentration and Hb concentration, along with the results from Example 4.

[0081] As shown in Figure 5, the amount of heme pigment (Mb + Hb) measured in Example 4 was found to be approximately equivalent to the amount of heme pigment (Mb + Hb) measured by the conventional carbon monoxide method. This demonstrates that the method of Example 4 can measure heme pigment in pork. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A method for measuring heme pigment in meat, comprising the following steps (i) to (v): (i) extracting heme pigment from meat to obtain an extract; (ii) dispensing the extract to obtain a first extract and a second extract, adjusting the pH of the first extract to 5.0 to 7.5 and the pH of the second extract to 8.0 to 10.0; (iii) oxidizing the extract; (iv) measuring the first absorbance, which is the absorbance of the first extract at a predetermined wavelength, and the second absorbance, which is the absorbance of the second extract; (v) calculating the myoglobin concentration or hemoglobin concentration in the meat from the difference between the first absorbance and the second absorbance.

2. The method according to claim 1, wherein the predetermined wavelength in step (iv) is a wavelength at which the absorbance of methemoglobin is not affected by pH, and the concentration of myoglobin in meat is calculated in step (v).

3. The method according to claim 1, wherein the predetermined wavelength in step (iv) is a wavelength at which the absorbance of metmyoglobin is not affected by pH, and the value calculated in step (v) is the hemoglobin concentration in meat.

4. The method according to claim 1, wherein step (iii) includes adding an oxidizing agent to the extract.

5. The method according to claim 4, wherein the oxidizing agent is at least one selected from sodium nitrite and potassium ferricyanide.

6. The method according to claim 1, wherein step (iii) comprises allowing the extract to stand for 12 to 24 hours under aerobic conditions at 20 to 40°C.

7. A kit for measuring heme pigment in meat, comprising at least one pH adjuster and at least one oxidizing agent.

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