Method for measuring heme pigment in meat

Abstract

To provide a method for measuring heme pigments in meat more simply than before without using any special equipment. [Solution] Heme pigment is extracted from meat to obtain an extract, the oxidized 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 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 are measured. The myoglobin concentration and hemoglobin concentration in the meat are calculated from the difference between the measured first absorbance and second absorbance, thereby measuring the heme pigment in the meat.

Description

[Technical field]

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

[0002] In the distribution of livestock meat, the occurrence of muscle bleeding (blood spots) and residual blood in carcasses are factors that deteriorate the appearance and odor of the meat, thereby lowering its quality, and it is necessary to reduce them. It is known that blood spots and residual blood are caused by the slaughter method and bleeding conditions (Non-Patent Document 1), but their evaluation has mostly been carried out visually.

[0003] Non-Patent Document 1 reports a method for measuring the amount of blood remaining in the muscle by separating hemoglobin from an extract of the porcine diaphragm leg muscle 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 the extract by bubbling carbon monoxide through it and then measuring the absorbance. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hirohide Ohshika et al., Food Hygiene Research, Vol. 41, No.5, pp. 79-86 (1991) [Non-Patent Document 2] Masahiro Waga et al., Journal of the Japanese Society of Swine Science, Vol. 53, No. 1, pp. 10-15 (2016) Summary of the Invention [Problem to be solved by the invention]

[0005] The method described in Non-Patent Document 1 has problems in that separation by gel filtration takes time and requires complicated operations. In addition, the method described in Non-Patent Document 2 uses carbon monoxide, so it requires a local exhaust ventilation system and is not suitable for daily testing.

[0006] An object of the present invention is to provide a method for measuring heme pigments in meat more simply than conventional methods without using special equipment. [Means for solving the problem]

[0007] The present invention provides the following: (1) A method for measuring heme pigments in meat, comprising the following steps (i) to (v): (i) extracting heme pigment from meat to obtain an extract; (ii) dispensing the extract into 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; (iii) oxidizing the extract; (iv) measuring a first absorbance, which is the absorbance of the first extract, and a second absorbance, which is the absorbance of the second extract, at predetermined wavelengths; (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 (1), wherein the predetermined wavelength in step (iv) is not 520 nm or 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 calculated in step (v) is the myoglobin concentration in the meat. (4) The method according to (1) or (2), wherein the specified wavelength in step (iv) is a wavelength at which the absorbance of metmyoglobin is not affected by pH, and what is calculated in step (v) is the hemoglobin concentration in the meat. (5) The method according to any one of (1) to (4), wherein the step (iii) comprises 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 the step (iii) comprises allowing the extract to stand under aerobic conditions at 20 to 40° C. for 12 to 24 hours. (8) The method according to any one of (1) to (7), comprising filtering each extract after the step (iii) and before measuring the absorbance. (9) A kit for measuring heme pigments 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 an instruction manual describing the measurement wavelength and a formula for calculating the myoglobin or hemoglobin concentration. Effect of the Invention

[0008] According to the present invention, it is possible to measure heme pigments in meat in a simple manner without using any special equipment. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a flow chart showing the steps of the method of the present invention for measuring heme pigments in meat. [Diagram 2] The absorption spectra of horse methemoglobin and metmyoglobin at various pH levels. (A) shows the absorption spectrum of methemoglobin, and (B) shows the absorption spectrum of metmyoglobin. [Diagram 3] A plot of pH vs. 600 nm / 522 nm absorbance for horse metmyoglobin, which confirmed that the pKa value of horse metmyoglobin is 9.02. [Figure 4] 1 is a graph showing measured values ​​of heme pigment concentrations in beef in Example 3, Comparative Example 1, and Comparative Example 2. [Diagram 5] 1 is a graph showing measured values ​​of heme pigment concentration in pork in Example 4, Comparative Example 1, and Comparative Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Structure 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, sheep meat, goat meat, etc. In this specification, poultry meat refers to chicken meat such as chicken, quail, turkey, duck, and goose. In this specification, fish meat refers to red fish meat such as tuna, bonito, and yellowtail. The method of the present invention is applicable to any of 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 livestock, sticking them to let them out of their blood, skinning them, removing their internal organs, and splitting their backs. Blood spots and residual blood can occur due to genetic factors or when the procedures leading up to the bleeding are not appropriate. Residual blood in meat is undesirable from the standpoint of quality, such as taste and shelf life. Blood spots indicate the rupture of capillaries, and residual blood refers to blood remaining in blood vessels.

[0012] The amount of heme pigment in meat is known as an index of the amount of residual blood contained in meat. Known heme pigments contained in meat are myoglobin (Mb), hemoglobin (Hb) and cytochrome c. Myoglobin exists in the form of oxymyoglobin, deoxymyoglobin, metmyoglobin (OH type) or metmyoglobin (H2O type). Hemoglobin exists in the form of oxyhemoglobin, deoxyhemoglobin, methemoglobin (OH type) or methemoglobin (H2O type). Cytochrome c exists in the form of oxidized cytochrome c or reduced cytochrome c. That is, there are 10 types of heme pigments in meat with different properties. Measuring each of these pigments requires very complicated work. In addition, even if the concentration of each is calculated from absorbance, etc., the calculation formula becomes very complicated, making it difficult to perform accurate calculations.

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

[0014] By oxidation, all Mb contained in meat can be converted to metMb, and all Hb to metHb. It is known that metMb and metHb become OH type (alkaline type) or HO type (acidic type) depending on pH, and their absorbance changes. The present invention is characterized by measuring the total Mb amount and total Hb amount contained in meat by utilizing the absorbance difference due to pH. The amount of cytochrome c in the heme pigment of meat is usually less than 1%, which 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, in the absorbance measurement, 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 can take two forms, OH and HO, depending on the pH, and each has a different absorption spectrum. In this change in absorption with a change in pH, there is a point where no change occurs, i.e., an isosbestic point. The wavelength at which this isosbestic point occurs is different between metMb and metHb. Therefore, if a change occurs with a change in pH at the isosbestic point of metHb, it can be said that the change is due to metMb. Figure 2 shows the absorption spectra of bovine metHb (A) and equine metMb (B) at each pH. As is clear from the absorption 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 absorption 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 occurs in absorbance at 480 nm, 520 nm, or 617 nm at different pHs, it can be said that the change is due to metMb. On the other hand, if a change occurs at 490 nm, 530 nm, or 626 nm at different pH levels, the change 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 A first embodiment of the present invention is a method for measuring heme pigments in meat. The method for measuring heme pigments in meat of this embodiment is characterized by comprising the following steps (i) to (v): (i) extracting heme pigment from meat to obtain an extract; (ii) dispensing the extract into 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; (iii) oxidizing the extract; (iv) measuring a first absorbance, which is the absorbance of the first extract, and a second absorbance, which is the absorbance of the second extract, at predetermined wavelengths; (v) 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 the present embodiment, it is possible to measure heme pigments in meat without requiring special equipment or complicated operations.

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

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

[0020] In the extraction step, the meat may be in a block form, but is preferably cut into small pieces, for example, in a minced form. The cut meat is preferably homogenized in a solvent. The soaked and homogenized meat can be removed from the solvent by centrifugation or filtration through filter paper, gauze, etc. The obtained solvent can be used as an extract in the subsequent measurement.

[0021] 2-2 Process (ii) pH adjustment process The method of the present embodiment includes, as step (ii), a step of dividing 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 or after step (iii) described below.

[0022] The method for adjusting the pH of the first extract and the second extract is not particularly limited. Dilute hydrochloric acid, organic acid solutions (acetic acid, citric acid, etc.), etc. can be used to lower the pH, while sodium hydroxide solution, sodium carbonate solution, potassium carbonate solution, sodium bicarbonate solution, potassium bicarbonate solution, etc. can be used to raise the pH. Note that since the extract before pH adjustment is usually weakly acidic, it is not necessary to lower the pH in many cases. In this case, it is preferable to add the same amount of water as the aqueous solution added to the second extract to raise the pH to the first extract, and make the heme pigment concentration uniform.

[0023] The pH of the first extract may 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 may be 8.0 to 10.0, preferably 8.5 to 10.0, and more preferably 8.5 to 9.5. Here, the difference between the pH of the first extract and the pH of the second extract is preferably 1.0 or more, particularly 1.5 or more, and more preferably 2.0 or more.

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

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

[0026] The method using an oxidizing agent can cause the heme pigment to become methemoglobin in a short time. Examples of the oxidizing agent that can be used 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 in the case of sodium nitrite, for example, 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 leaving the extract under aerobic conditions at 20 to 40°C for 12 to 24 hours takes time, but allows easy disposal of the reagent after use, and can be used in a process in facilities that do not have disposal means. In this specification, aerobic conditions refer to conditions in which the oxygen concentration is at least 1%. The temperature at which the extract is left standing is preferably 30 to 40°C, particularly 35 to 38°C.

[0028] Whether the oxidation reaction has progressed sufficiently can be confirmed, for example, by visually observing the color change of the extract (from red to brown), or, more preferably, by observing the absorption spectrum with a spectrophotometer.

[0029] 2-4 Filtration process The extract after carrying out the steps (ii) and (iii) may be filtered. For example, by carrying out filter filtration, it is possible to avoid the influence of the precipitate generated in the pH adjustment and oxidation reaction on the subsequent absorbance measurement. The material of the filter used is not particularly limited, but can be, for example, a membrane filter such as cellulose acetate, cellulose mixed ester, polyethersulfone, hydrophilic PTFE, etc. The pore size of the filter is not particularly limited, but can be, for example, 0.45 μm, 0.22 μm, etc. Filter paper may also be used. For example, quantitative filter paper with a particle retention diameter of φ3 μm can be suitably used. The filtration step is preferably carried out when precipitation occurs in the pH adjustment step and oxidation step. The filtration step may also be carried out appropriately not only after the steps (ii) and (iii) but also at the timing when precipitation occurs.

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

[0031] The predetermined wavelength here is preferably a wavelength at which the absorbance of met-hemoglobin is not affected by pH when the amount of Mb is calculated in the subsequent step (v), whereas it is preferably a wavelength at which the absorbance of met-myoglobin is not affected by pH when the amount of Hb is calculated in the subsequent step (v).

[0032] The "wavelength at which the absorbance of methemoglobin is not affected by pH" (hereinafter also referred to as the "metHb isosbestic point") and the "wavelength at which the absorbance of metmyoglobin is not affected by pH" (hereinafter also referred to as the "metMb isosbestic point") vary depending on the animal species, and can be determined in advance by testing using a metHb or metMb standard. Figure 2 shows the absorption spectra of horse metHb (Figure 2A) and metMb (Figure 2B) at each pH. From the results shown in Figure 2A, the metHb isosbestic points are 480 nm, 525 nm, and 617 nm, and therefore, when calculating the amount of Mb, the predetermined wavelength can be 480 nm, 525 nm, or 617 nm. From the results shown in Figure 2B, the metMb isosbestic points are 490 nm, 530 nm, and 626 nm, and therefore, when calculating the amount of Hb, the predetermined wavelength can be 490 nm, 530 nm, or 626 nm.

[0033] It is preferable not to set the predetermined wavelength at 520 nm or 550 nm, because heme pigments include cytochrome c, which has maximum absorption wavelengths at 520 nm and 550 nm, and measuring absorbance at these wavelengths would result in an increase in absorbance due to cytochrome c in the sample, which may result in an inaccurate measurement of the amount of Mb or Hb.

[0034] The absorbance can be measured, for example, by placing the extract in a cell for measuring absorbance with a path length of 10 mm and using a spectrophotometer. Alternatively, for example, a plurality of extracts may be dispensed into a microplate (24 wells, 96 wells, etc.) and measured using a microplate reader. The spectrophotometer or microplate reader used here is not particularly limited, and any spectrophotometer or microplate reader that is usually used for measuring the absorbance of a test solution can be used.

[0035] 2-6 Step (v) Heme pigment amount calculation step The method of the present embodiment includes, as step (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.

[0036] As described above, metMb and metHb can take two forms, OH and HO, depending on the pH, and each has a different absorption spectrum. In the change in absorbance that accompanies this change in pH, there exists a point where no change occurs, i.e. an isosbestic point. The amount of change in absorbance at this point (wavelength) is concentration-dependent, so by calculating the amount of change per concentration in advance, the concentration of the heme pigment can be calculated from the amount of change (formula (I) below).

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[0037] The change per concentration is the difference in absorbance of metMb or metHb estimated at unit concentration at any two pH points. To estimate the absorbance of metMb and metHb at any pH, it is necessary to calculate the proportion of OH- or HO-bound metMb or metHb. The proportion of OH- or HO-bound metMb or metHb is very similar to the ionization behavior of acids, and can be calculated from the concentration, acid dissociation constant (pKa) and pH of metMb and metHb. The pH and pKa have the following relationship (equation (II) below).

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[0038] When absorbance increases with increasing pH (with decreasing [HA]) (this applies to Mb at a wavelength of 617 nm), the value Abs(pH) of absorbance at a specific wavelength at a specific pH can be expressed by the following formula (IX): Abs(MIN) is the minimum absorbance per unit concentration when the HA concentration is the lowest, and Abs(MAX) is the maximum absorbance per unit concentration when the HA concentration is the highest.

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[0039] When absorbance decreases with increasing pH (with decreasing [HA]) (this is the case for Hb at a wavelength of 626 nm), the value Abs(pH) of the absorbance at a specific wavelength at a specific pH can be expressed by the following formula (X):

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[0040] Here, when formula (VIII) is substituted into formula (IX) and formula (X), the formulas can be represented by the following formulas (XI) and (XII), respectively.

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[0041] When formulas (XI) and (XII) are respectively substituted into the conceptual formula (I), the Mb concentration [Mb] and the Hb concentration [Hb] can be expressed by the following formulas (XIII) and (XIV).

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[0042] The Mb concentration of the extract can be calculated by inputting pHa, pHb, Abs(MAX) and Abs(MIN) measured in advance into the above formula (XIII), and further inputting the values ​​of AbsH(a) (first Mb absorbance) and AbsH(b) (second Mb absorbance) measured in step (iv) into the above formula (XIV). The Hb concentration of the extract can be calculated by inputting pHa, pHb, Abs(MAX) and Abs(MIN) measured in advance into the above formula (XIV), and further inputting the values ​​of AbsM(a) (first Hb absorbance) and AbsM(b) (second Hb absorbance) measured in step (iv). The amount of Mb and the amount of Hb contained in the meat per unit weight can be calculated from the Mb concentration or Hb concentration calculated here and the weight of the meat used in step (i).

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

[0044] Another example of the other step is a step of determining in advance the values ​​required for the calculation in step (v). In step (v), the values ​​of Abs(MAX) and Abs(MIN) are required to calculate the amount of Mb and Hb in meat. These values ​​vary 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) of the animal species are unknown, those values ​​must be determined in advance by testing. Specifically, the values ​​can be determined by adjusting a solution containing a unit concentration of Mb or Hb to a sufficiently high and sufficiently low pH compared to the pKa, and measuring the absorbance of each at a specified measurement wavelength.

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

[0046] 3. Kit for measuring heme pigments in meat The second embodiment of the present invention is a kit for measuring heme pigments in meat. The kit of this embodiment is characterized by including at least one pH adjuster 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 pigments in meat." In this embodiment, unless otherwise specified, or unless otherwise contradictory, the definitions of the terms used are the same as those described in "2. Method for measuring heme pigments in meat."

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

[0048] In the kit of this embodiment, any agent capable of oxidizing heme pigment can be used as the oxidizing agent, and for example, sodium nitrite, potassium ferricyanide, etc. can be suitably used.

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

[0050] The kit of the present embodiment may include a filter, which can be used to remove precipitates that may be generated when a pH adjuster and an oxidizing agent are added to a heme pigment extract from meat.

[0051] The kit of this embodiment may include an instruction manual that describes the measurement wavelength and the calculation formula for the concentration of myoglobin or hemoglobin. When measuring the absorbance of a heme pigment extract of meat, the appropriate measurement wavelength differs depending on the animal species from which the meat is derived. Therefore, it is necessary to teach the user the appropriate measurement wavelength for each meat to be applied. In addition, the formula for calculating the amount of Mb and Hb in meat from the obtained pH and absorbance data also differs depending on the animal species from which the meat is derived. Therefore, it is necessary to teach the user the appropriate calculation formula for each meat to be applied. The instruction manual preferably includes a specific explanation of the measurement wavelength and the calculation formula. Alternatively, the instruction manual may display a function for indicating the measurement wavelength appropriate for the animal species, and a QR code (registered trademark) for connecting to an app that automatically calculates the amount of Mb and Hb in meat from the obtained pH and absorbance data. EXAMPLES

[0052] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these.

[0053] <Example 1: Measurement of absorption spectrum of heme pigment at each pH> The absorption spectra of the metHb (bovine origin) and metMb (horse origin) samples were obtained using the following method. Both Hb (bovine origin) and Mb (horse origin) reagents were treated as oxidized forms. These were added to various pH buffer solutions at 1 mg / mL, and the solutions filtered through 0.45 μm filters were used as samples of metHb and metMb at each pH. The absorption spectra of these samples were measured from 450 to 750 nm using a UV-2700 UV-visible spectrophotometer (Shimadzu Corporation) in a cell with a path length of 10 mm.

[0054] The compositions of the various pH buffer solutions were as follows: 0.1M Sodium acetate buffer (pH 4.5) 0.1M MES buffer (pH5.8) 0.1M Sodium phosphate buffer (pH 6.6) 0.1M Sodium phosphate buffer (pH 7.5) 0.1M Tris buffer (pH 8.6) 0.1M Sodium carbonate buffer (pH 9.6) 0.1M Sodium carbonate buffer (pH 10.4)

[0055] Figure 2 shows the absorption spectra of metHb (A) and metMb (B) at each pH. The absorption spectra in Figure 2A show that there is no change in absorbance due to pH at wavelengths of 480 nm, 525 nm, and 620 nm. The absorption spectra in Figure 2B show that there is no change in absorbance due to pH at wavelengths of 490 nm, 530 nm, and 630 nm.

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

[0057] <Example 3: Measurement of beef heme pigment> (1) Preparation of bovine Mb After mincing beef to 3 mm, 1 part 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 resulting homogenate was centrifuged at 18,000 × g and 1 ° C for 20 minutes to obtain the supernatant. The supernatant was fractionated with ammonium sulfate at 60-90% saturation, and the resulting pellet was used as a sample for hydrophobic chromatography. Using FPLC, the fraction eluted at 40% ammonium sulfate saturation was collected as Mb using a HiTrap Phenyl HP 5 mL column.

[0058] (2) Bovine Hb A commercially available bovine Hb standard (Hemoglobin, Freeze Dried (MP Biomedicals, Ohio, USA)) was used.

[0059] (3) Determination of the isobesity 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 absorption spectra at each pH were measured in the same manner as in Example 1. As a result, it was confirmed that the isosbestic points of bovine metHb were 489 nm, 521 nm, and 617 nm, and the isosbestic points of bovine metMb were 494 nm, 523 nm, and 626 nm.

[0060] (4) Determination of pKa of bovine metMb and bovine metHb Except for using bovine metMb or bovine metHb instead of horse metMb, the pKa values ​​of each were determined in the same manner as in Example 2. 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 were each prepared at 1 mg / mL with pH 4 to 12. For bovine metMb, the absorbance at 617 nm was measured, and the highest absorbance was Abs(MAX) and the lowest absorbance was Abs(MIN). For bovine metHb, the absorbance at 626 nm was measured, and the highest absorbance was Abs(MIN) and the lowest absorbance was Abs(MAX). The values ​​were as shown in Table 1.

[0062] [Table 1]

[0063] (6) Measurement of heme pigment in beef After mincing 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 resulting homogenate was centrifuged at 18,000×g and 1°C for 15 minutes to obtain a supernatant. The mixture was filtered through filter paper (ADAVANTEC No.1) to remove fat, and a filtrate was obtained (heme pigment extract). The filtrate was dispensed into 2 x 3 mL bottles, and 3 mL of distilled water was added to one bottle, and 3 mL of 1N sodium hydroxide solution was added to the other bottle, and mixed. 10 mg of sodium nitrite was added to both bottles 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 filtrate obtained were measured. The absorbance was measured by placing the filtrate in a quartz cell with a path length of 10 mm and using a UV-visible spectrophotometer (UV-2700, Shimadzu Corporation). The measurement was performed at room temperature using a quartz glass cell with a path length of 10 mm.

[0064] The measured pH and absorbance were substituted into the following formulas (XV) and (XVI), respectively, to calculate the Mb concentration and Hb concentration in each filtrate.

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[0065] <Comparative Example 1: Measurement of beef heme pigment (Mb equivalent)> A heme pigment extract was obtained from beef in the same manner as in Example 3(6). Assuming that all the heme pigment was Mb, the absorbance of the extract at 525 nm was measured. The absorbance of several solutions containing known concentrations of Mb was measured at 525 nm to create a calibration curve, and the Mb concentration in the extract was then calculated. Here, Mb and Hb cannot be measured separately, and the amount can be calculated as the Mb equivalent of the heme pigment. Figure 4 shows the amount of heme pigment (Mb equivalent) together with the results of Example 3 and Comparative Example 2 described later.

[0066] <Comparative Example 2: Measurement of beef heme pigment (carbon monoxide method)> The heme pigment in beef was measured using a method according to the description in 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, 10 mg of sodium hydrosulfite (Na2S2O4) was added, and the mixture was stirred using a vortex mixer. The filtrate after stirring 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 poured into 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 system. After reducing the heme pigment extract or heme pigment solution, approximately 10 mL of CO gas was passed through the bag, which was then sealed and mixed by inversion, and allowed to stand for 5 minutes.

[0068] After the heme pigment solutions diluted to various concentrations were similarly treated with CO, the absorption spectrum was measured at 350 to 750 nm using a spectrophotometer, and the extinction coefficient per concentration was obtained from the absorbance at each wavelength and the concentration (mg / mL) of the measurement solution. The measurement was performed at room temperature using a quartz glass cell with an optical path length of 10 mm. The Mb concentration, amount, Hb concentration, and cytochrome c concentration were calculated from the absorption spectrum of the heme pigment extract and the extinction coefficient at each of the above wavelengths. Figure 4 shows the Mb concentration, amount, Hb concentration, and cytochrome c concentration together with the results of 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 the conventional method (Mb equivalent) or the amount of heme pigment measured by the carbon monoxide method. It was demonstrated that the method of Example 3 can measure heme pigment in beef.

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

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

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

[0073] (4) Determination of pKa of porcine metMb and porcine metHb Except for using porcine metMb or porcine metHb instead of horse metMb, the pKa values ​​of each were determined in the same manner as in Example 2. 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) Porcine metMb and porcine metHb were each prepared at 1 mg / mL solutions with pH 4 to 12. For porcine metMb, the absorbance at 617 nm was measured, and the highest absorbance was Abs(MAX) and the lowest absorbance was Abs(MIN). For porcine metHb, the absorbance at 626 nm was measured, and the highest absorbance was Abs(MIN) and the lowest absorbance was Abs(MAX). The values ​​were as shown in Table 2.

[0075] [Table 2]

[0076] (6) Measurement of heme pigment in pork After mincing three pork samples (samples A to C) at 3 mm each, 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 resulting homogenate was centrifuged at 18,000×g and 1°C for 15 minutes to obtain the supernatant. The mixture was filtered through filter paper (ADAVANTEC No.1) to remove fat, and a filtrate was obtained (heme pigment extract). The filtrate was dispensed into two 3 mL bottles, one of which was filled with distilled water and the other with 3 mL of 1N sodium hydroxide solution, and mixed. Both were mixed with 10 mg of sodium nitrite. 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 filtrate were measured. The absorbance was measured by placing the filtrate in a quartz cell with a path length of 10 mm and using a spectrophotometer (UV-2700, Shimadzu Corporation). The measurement was performed at room temperature using a quartz glass cell with a path length of 10 mm.

[0077] The measured pH and absorbance were substituted into the following formulas (XVII) and (XVIII), respectively, to calculate the Mb concentration and Hb concentration in each filtrate.

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[0078] <Comparative Example 3: Measurement of heme pigment in pork (carbon monoxide method)> The heme pigment in beef was measured using a method according to the description in 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, 10 mg of sodium hydrosulfite (Na2S2O4) was added, and the mixture was stirred using a vortex mixer. The filtrate after stirring 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 poured into 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 system. After reducing the heme pigment extract or heme pigment solution, approximately 10 mL of CO gas was passed through the bag, which was then sealed and mixed by inversion, and allowed to stand for 5 minutes.

[0080] After the heme pigment solutions diluted to various concentrations were similarly treated with CO, the absorption spectrum was measured at 350 to 750 nm using a spectrophotometer, and the absorption coefficient per concentration was obtained from the absorbance at each wavelength and the concentration (mg / mL) of the measurement solution. The measurement was performed at room temperature using a quartz glass cell with an optical path length of 10 mm. The Mb concentration, amount, and Hb concentration were calculated from the absorption spectrum of the heme pigment extract and the absorption coefficient at each of the above wavelengths. Figure 5 shows the Mb concentration and Hb concentration together with the results of Example 4.

[0081] As shown in Figure 5, the amount of heme pigments (Mb + Hb) measured in Example 4 was almost equivalent to the amount of heme pigments (Mb + Hb) measured by the conventional carbon monoxide method. It was demonstrated that the method of Example 4 can measure heme pigments in pork.

Claims

1. The method comprises the following steps (i) to (v): (i) extracting heme pigment from meat to obtain an extract; (ii) dispensing the extract into 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; (iii) oxidizing the extract; (iv) measuring a first absorbance, which is the absorbance of the first extract, and a second absorbance, which is the absorbance of the second extract, at a predetermined wavelength; (v) calculating the myoglobin concentration or the hemoglobin concentration in the meat from the difference between the first absorbance and the second absorbance; The predetermined wavelength in step (iv) is an isosbestic point of methemoglobin, and step (v) is a step of calculating a myoglobin concentration; The predetermined wavelength in step (iv) is an isosbestic point of metmyoglobin, and step (v) is a step of calculating a hemoglobin concentration, The predetermined wavelength is not the maximum absorption wavelength of cytochrome c. Method for measuring myoglobin or hemoglobin concentration in meat.

2. 2. The method of claim 1, wherein step (iii) comprises adding an oxidizing agent to the extraction liquid.

3. 3. The method of claim 2, wherein the oxidizing agent is at least one selected from sodium nitrite and potassium ferricyanide.

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

5. A kit for use in the method of claim 1, comprising: A kit for determining myoglobin or hemoglobin concentration in meat, comprising at least one pH adjusting agent and at least one oxidizing agent.

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