Method for evaluating the quality of aged foods and the antibodies used therefor

Abstract

The present invention aims to establish objective inspection standards and provide a new evaluation method for quality control of aged meat. [Solution] A method for evaluating the quality of aged meat is provided, comprising the steps of measuring peptide A in an aqueous extract of aged meat and evaluating the quality of aged meat based on the measured value of peptide A obtained in the measurement step.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the quality of aged foods and an antibody used therefor.

Background Art

[0002] In recent years, aged foods such as aged meat have attracted attention because their umami is elicited by leaving the food to rest. However, their definitions and production methods are ambiguous, and national standards for quality control such as "the most delicious time to eat," "taste period," and "expiration date" have not been established. Currently, the degree of aging, the time to eat, and the expiration date are subjectively evaluated based on the experience and sense of each manufacturer. Aged meat improves in flavor, tenderness, and juiciness through aging. The deliciousness of aged meat is known to be caused by multiple factors, such as the amount of free amino acids such as glutamic acid, aspartic acid, and inosinic acid contained in the aged meat, the amount of fatty acids such as oleic acid and linoleic acid, and volatile organic compounds. So far, analyses of changes in components such as free amino acids and fatty acids, as well as moisture, pH, etc. (Non-Patent Document 1), and changes in the microbiota and aroma on the surface of meat blocks (Non-Patent Document 2) during the aging period have been reported. However, these analyses use special analytical devices such as NMR and LC-MS and are not suitable for quality control applications at the site where aged meat is provided. Also, no attempt has been made to construct objective inspection criteria for a certain "quality control" based on scientific data.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present invention aims to establish objective inspection standards and provide a simple and rapid evaluation method for quality control of aged foods such as aged meat. [Means for solving the problem]

[0005] The inventors have previously aged commercially available beef inner thigh blocks (approximately 11 kg) using a beef aging machine (DRY AGER DX500 (Landig + Lava GmbH & Co. KG)) at 1.5°C and 82% humidity, and conducted sensory evaluations using a single-blind scoring method on samples taken at various time points (0, 10, 20, 30, 40, 50, 60, 90 days) (N=6). As a result, the scores increased from day 0 up to 30 days of aging, but were almost the same as the standard value from day 40 onwards. Furthermore, they found that these results correlated with the recommended aging period for this beef aging machine (25-28 days: Operating Instructions DRY AGER DX500, p.5) and the aging period used by many beef aging manufacturers (14-35 days: Dry-aging of beef executive summary (U.S. Meat Export Federation)).

[0006] It has been reported that a peptide A (peptide with delicious taste) that exhibits a "delicious taste" exists in beef juices (Non-patent Literature 3: Yamasaki et al., Agric. Biol. Chem. (1978)). In order to solve the above problem, the present inventors focused on peptide A and attempted to measure peptide A during the maturation process of aged meat.

[0007] Therefore, after diligent research, the inventors succeeded in producing an "antibody" (anti-peptide A antibody) against the complex of peptide A and carrier protein (Keyhole Limpet Hemocyanin (KLH)), and found that this antibody is applicable to enzyme-linked immunosorbent assay (ELISA), biolayer interferometry (BLI), and immunoscopy. Furthermore, when attempting to detect peptide A using the ELISA method on water extracts of aged meat samples, they found that the period with the highest peptide content coincided with the period considered "most delicious" or "at its peak" in sensory evaluation. The present invention is completed based on the above findings and includes the following embodiments:

[0008] One aspect of the present invention is, [1] A method for evaluating the quality of aged meat, The step of measuring peptide A in the aqueous extract of the aged meat, The present invention relates to an evaluation method that includes a step of evaluating the quality of the aged meat based on the measured value of peptide A obtained in the measurement step. In one embodiment, the evaluation method of the present invention is as follows: [2] The evaluation method described in [1] above, The measurement step is characterized by being a step of quantification using an antibody against peptide A. Furthermore, in one embodiment of the present invention, [3] The evaluation method described in [2] above, The antibody is characterized by being a polyclonal antibody. Furthermore, in one embodiment of the present invention, [4] The evaluation method described in [2] above, The antibody is characterized by being a complex with nanoparticles.

[0009] Furthermore, in one embodiment of the present invention, [5] The evaluation method described in [1] above, The measurement step is characterized by measuring peptide A using the ELISA method. Also, in one embodiment, the evaluation method of the present invention is 〔6〕The evaluation method according to 〔4〕 above, characterized in that the concentration of the water extract of the aged meat is within the range of 1 to 15 μg / mL. Also, in one embodiment, the evaluation method of the present invention is 〔7〕The evaluation method according to 〔1〕 above, characterized in that the aged meat is beef. Also, in one embodiment, the evaluation method of the present invention is 〔8〕The evaluation method according to 〔1〕 above, characterized in that the evaluation step is a step of determining that the quality is high when the measured value of peptide A obtained by the measurement step is higher than the measured value of peptide A contained in the water extract of the aged meat that is not at the right time to eat. Also, in one embodiment, the evaluation method of the present invention is 〔9〕The evaluation method according to 〔1〕 above, characterized in that the quality of the aged meat is deliciousness.

[0010] Also, in another aspect, the present invention 〔10〕A polyclonal antibody that can be used in the evaluation method of 〔1〕 above, relates to a polyclonal antibody that specifically binds to the peptide A. Here, in one embodiment, the polyclonal antibody of the present invention 〔11〕The polyclonal antibody according to 〔10〕 above, characterized in that it is obtained by a method for producing a polyclonal antibody using a complex of an antigen peptide shown in SEQ ID NO: 1 and a carrier protein as an immunogen. Also, in one embodiment, the polyclonal antibody of the present invention 〔12〕The polyclonal antibody according to 〔10〕 above, characterized in that the binding affinity (KD value) is 3.00 × 10 -8 M or less.

[0011] In another aspect, the present invention 〔13〕A kit for evaluating the quality of aged meat, comprising the polyclonal antibody described in 〔10〕 above. Here, the kit for evaluating the quality of aged meat of the present invention, in one embodiment 〔14〕The kit according to 〔13〕 above, characterized in that the polyclonal antibody is a complex with nanoparticles.

Advantages of the Invention

[0012] According to the method for evaluating the quality of aged meat according to the present invention, without the need for special analytical equipment or sample preparation such as component analysis of amino acids and fatty acids by NMR, LC-MS, etc., and microbiota analysis, evaluation can be performed simply and at low cost using the water extract of the sample. According to the present invention, it is possible to provide objective and consistent quality control inspection criteria in the manufacturing process of aged meat, which have not been established in the prior art.

Brief Description of the Drawings

[0013] [Figure 1] Figure 1 is a graph showing the results of the sensory evaluation conducted in Example 3 below. The vertical axis indicates deliciousness (points), and the horizontal axis indicates the number of aging days (days). [Figure 2] Figure 2 is a graph showing the measurement results of the binding ability and dissociation ability of the anti-peptide A antibody obtained in Example 4 below using the biolayer interferometry method. The anti-peptide A antibody was tested at concentrations of 10.4 nM, 20.8 nM, 46.2 nM, 83.3 nM, and 166.7 nM. [Figure 3] Figure 3 shows a schematic diagram of the detection mechanism of the ELISA method in which peptide A was immobilized on an immunoplate as an antigen in Example 6 below, and a graph showing the results. The anti-peptide A antibody was reacted with peptide A immobilized on the immunoplate as the primary antibody, and detection was performed with an HRP-labeled secondary antibody against the primary antibody. The vertical axis of the graph indicates absorbance (A450 - A630), and the horizontal axis indicates the concentration (ng / mL) of peptide A immobilized on the immunoplate. [Figure 4] Figure 4 shows a schematic diagram of the detection mechanism of the ELISA method performed in Example 6 below, in which a water extract of aged meat was immobilized on an immunoplate, and a graph showing the results. An anti-peptide A antibody was reacted with the water extract of aged meat immobilized on the immunoplate as the primary antibody, and detection was performed using an HRP-labeled secondary antibody against this primary antibody. The vertical axis of the graph shows the absorbance (A450-A630), and the horizontal axis shows the concentration (μg / mL) of the water extract of aged meat immobilized on the immunoplate at each aging day. [Modes for carrying out the invention]

[0014] 1. Methods for evaluating the quality of aged meat One aspect of the present invention relates to a method for evaluating the quality of aged meat. The evaluation method includes the steps of measuring peptide A in an aqueous extract of aged meat and evaluating the quality of the aged meat based on the measured value of peptide A obtained in the measurement step.

[0015] 1-1.Definition In this specification, "maturation" refers to the process of de-rigor mortis, in which the rigidity of the muscles after slaughter subsides and they become soft again. This process results in softer meat, improved water retention, and enhanced flavor. While the muscles are soft immediately after slaughter, they harden and lose water retention once rigor mortis sets in. As mentioned above, the process by which rigid muscles subside and become soft again is called de-rigor mortis. It is preferable that the meat be served in a state where it has returned to the softness it had immediately after slaughter. In this specification, "aged meat" includes aged meat that has matured naturally during the distribution process (for example, commercially available aged meat), as well as meat that has been intentionally stored for a certain period under certain conditions to enhance the flavor of the meat or to make it tender (for example, commercially available or purchased meat). During the aging process, proteolytic enzymes produced in the muscle or by beneficial microorganisms involved in aging act on the collagen fibers surrounding the myofibrils, causing them to break down. Consequently, the bundles of myofibrils also loosen, resulting in a softer texture. Furthermore, during the aging process, proteolytic enzymes break down muscle proteins into amino acids and peptides, increasing the sources of umami and sweetness. In addition, glycogen is converted into glucose by enzymes.

[0016] In this specification, the intentional aging methods and aging periods used to produce "aged meat" are not limited as long as they enhance the flavor and tenderize the meat. Aging methods are not limited to the following, but may include, for example, dry aging of carcasses, hanging aging, wet aging, dry aging, salt aging, and resting aging. The maturation period in intentional maturation methods is appropriately set by those skilled in the art depending on the animal species, age at slaughter, storage method, storage temperature, storage humidity, etc. When the aged meat is beef, in one embodiment, the maturation period (appropriate maturation period) in intentional maturation methods is preferably 10 to 100 days, more preferably 20 to 50 days, and even more preferably 30 to 40 days. For aging, commercially available equipment (for example, a meat aging machine such as the DRY AGER DX500 (Landig + Lava GmbH & Co. KG)) may be used. In one embodiment, aged meat can be intentionally further aged by dry aging under conditions of 0-4°C and 60-90% humidity.

[0017] In this specification, “aged meat” can refer to beef, pork, lamb, chicken, fish, or game meat (such as deer, wild boar, rabbit, pheasant, duck, or mountain pheasant). Examples of beef include Japanese Black, Japanese Brown, Japanese Polled, and Japanese Shorthorn; beef breeds such as Angus, Hereford, Mary Grey, and Brahman; and crossbreeds. Examples of pork include Landrace, Large Yorkshire, Middle Yorkshire, Duroc, Berkshire, Hampshire, and their crossbreeds. Examples of lamb include Suffolk, Romney, Texel, or their crossbreeds. Examples of chicken include White Cornish, White Plymouth Rock, Rhode Island Red, New Hampshire, or their crossbreeds. In a preferred embodiment, aged meat is beef.

[0018] The term "aged meat" does not refer to any specific cut of meat, but can include any cut commonly sold for consumption. Examples of such cuts include, but are not limited to, shoulder, shoulder loin, rib loin, sirloin, tenderloin, belly, thigh, rump, shank, neck, tail, breast, tenderloin, wingtip, mid-wing, and drumstick.

[0019] In this specification, "quality of aged meat" refers to the deliciousness of aged meat. The deliciousness of aged meat increases with the aging period. On the other hand, the deliciousness decreases if the aging period exceeds a certain period. The present invention provides a method for evaluating the quality of aged meat that can determine the period in which the quality (e.g., taste) of the aged meat is at its best. Here, the period in which the quality (e.g., taste) of the aged meat is at its best, as evaluated by the method of the present invention, coincides with the period in which consumers (=sensory evaluators) perceive the aged meat as "most delicious." Therefore, the present invention's method for evaluating the quality of aged meat enables producers and sellers of aged meat to determine the period in which the quality (e.g., taste) of the aged meat is at its best and provide it to consumers. In one embodiment, "quality of aged meat" refers to the deliciousness of the aged meat. In one embodiment, the evaluation of "deliciousness" can be carried out by a comprehensive sensory evaluation that includes aspects such as softness, sweetness, umami, sourness, aroma, and greasiness.

[0020] In this specification, "peptide A" refers to a peptide isolated from beef as a peptide exhibiting a delicious taste (Non-Patent Document 3), and can be identified as a peptide consisting of the amino acid sequence shown in Sequence ID No. 1. In this specification, "peptide A" may refer to the naturally occurring peptide identified as described above, as well as a mutant or modified peptide having amino acid mutations or other modifications, or a part (fragment) thereof.

[0021] In this specification, a variant may include proteins encoded by orthologous or paralogous genes of the gene encoding peptide A, which consists of the amino acid sequence shown in Sequence ID No. 1. A variant means, for example, a peptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence constituting peptide A, and which shows a positive correlation with quality during the maturation process of aged meat. Here, "a few" means three or two amino acid residues. A modified peptide means a peptide in which a chemical structure other than that of peptide A is added to the amino acid sequence of peptide A by chemical modification of a part of the amino acid sequence of the peptide, or a peptide in which a part of the chemical structure in peptide A is removed, and which shows a positive correlation with quality during the maturation process of aged meat.

[0022] In one embodiment, peptide A can be identified as the following polypeptides (a) to (c): (a) Polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) Polypeptides consisting of amino acid sequences in which one or more amino acids are substituted, added, or deleted from the amino acid sequence shown in Sequence ID No. 1. (c) A polypeptide having 90% or more identity (more preferably 95%, 96%, 97%, 98%, or 99% or more) with respect to the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the polypeptides identified by (b) or (c) above are polypeptides that show a positive correlation with quality during the maturation process of aged meat.

[0023] In this specification, "identity" refers to the percentage (%) of identical amino acids in the amino acid sequence of the polypeptide being compared relative to the total number of amino acid residues, after the two amino acid sequences have been aligned and gaps introduced as necessary to maximize the degree of agreement between the two amino acid sequences. The identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA and its default parameters.

[0024] 1-2. Process for measuring peptide A in aqueous extract of aged meat The present invention provides a method for evaluating the quality of aged meat, which includes the step of measuring peptide A in an aqueous extract of aged meat.

[0025] A water extract of aged meat can be obtained by taking a sample from the aged meat whose quality is to be evaluated, crushing the sample in an aqueous solution, and removing impurities. It is preferable to use pure water or ultrapure water as the aqueous solution, with ultrapure water being more preferable.

[0026] The sample taken from aged meat can be 500 to 5000 mg, more preferably 500 to 1000 mg. From the perspective of providing aged meat as a product, a smaller sample amount is preferable. On the other hand, a sample amount less than 500 mg is undesirable because the measurement of peptide A will not be stable. The method of taking a sample from aged meat is not limited as long as peptide A can be measured, but it is preferable to remove the dried surface portion and use the lean meat portion with low fat content.

[0027] The method for crushing the collected sample in an aqueous solution is not limited as long as peptide A can be measured, and a commercially available homogenizer can be used, for example. When crushing the sample using a bead crusher (e.g., TAITEC μT-12), processing can be done, for example, at 2000-3000 rpm, 20-30°C, and for 15-60 seconds. Known methods can also be used to remove impurities other than peptide A from the aqueous solution after sample crushing. For example, the supernatant can be recovered as an aqueous extract by centrifugation (e.g., 10,000-20,000 rpm, 20-30°C, 3-10 minutes).

[0028] In this specification, "measuring peptide A" means measuring peptide A contained in an aqueous extract of aged meat to obtain a measurement value. The measurement method is not limited to any method that can detect and measure peptide A contained in an aqueous extract of aged meat, and known methods can be used. Examples include, but are not limited to, ELISA, immunostigmatic methods, Western blotting, flow cytometry, immunochromatography, quartz crystal microbalance method, surface plasmon resonance method, mass spectrometry, and high-performance liquid chromatography. In a preferred embodiment, the step of measuring peptide A is to measure the antibody response level of an anti-peptide A antibody to peptide A to obtain a measurement value.

[0029] In one embodiment, the anti-peptide A antibody may be used in combination with known techniques for improving the detection sensitivity of the antigen. For example, nanoparticles capable of supporting multiple anti-peptide A antibodies can be used. Techniques for improving sensitivity by supporting multiple antibodies using nanoparticles are well known, and include techniques using bio-nanoparticles based on hepatitis B virus surface antigen L protein and lipid bilayers, virus-like particles, metal nanoparticles such as gold nanoparticles, resin nanoparticles, magnetic nanoparticles, or combinations thereof. A suitable example using nanoparticles is a complex of an anti-peptide A antibody and a bionanopapule. The bionanopapule is not limited as long as it presents the antibody on the bionanopapule surface to improve the detection sensitivity of the antigen. For example, ZZ-tag presenting bionanopapules (ZZ-BNC) can be used (see Japanese Patent Publication No. 2008-162981; Japanese Patent Publication No. 2009-120533; Masumi Iijima and Shunichi Kuroda, "Collection of DDS Carrier Production Protocols" (2015), CMC Publishing, Chapter 4, 4.3 Bionanopapules; Iijima, M. et al., "Bionanocapsule-based enzyme-antibody conjugates for enzyme-linked immunosorbent assay" Analytical Biochemistry, 396, 257-261, (2010), etc.). ZZ-BNCs that can present the antibody on the bionanopapule surface with the orientation aligned are particularly preferred. Techniques for aligning such antibodies are also publicly known and can be implemented by referring to Iijima, M. et al., “Nanocapsules incorporating IgG Fc-binding domain derived from Staphylococcus aureus protein-A for displaying IgGs on immunosensor chips.” Biomaterials, 32, 1455-1464, (2011). and Iijima, M. et al., “Nano-visualization of oriented-immobilized IgGs on immunosensors by high-speed atomic force microscopy.” Scientific Reports, 2, 790, (2012). Methods for manufacturing bio-nanopapules are publicly known, and commercially available products can also be used. Those skilled in the art can manufacture complexes of anti-peptide A antibodies and bio-nanopapules by referring to publicly known information.Furthermore, a method for detecting peptide A using a complex of an anti-peptide A antibody and a biocapsule can also be carried out in accordance with known methods. This may involve using bio-nanocapsules directly labeled with enzymes or fluorescent dyes, or detecting peptide A using a labeled secondary antibody against an anti-peptide A antibody (primary antibody). In the evaluation method of the present invention, using a complex of anti-peptide A antibody and nanoparticles improves sensitivity, making it easier to determine the optimal time for consumption. This effect is particularly desirable for a simple and rapid quality evaluation method (kit).

[0030] In a preferred embodiment, peptide A is measured by the ELISA method. In this case, the "concentration of the water extract" is preferably in the range of 1 to 15 μg / mL, more preferably in the range of 3.25 to 10 μg / mL, and even more preferably in the range of 3.25 to 7.5 μg / mL. Here, the concentration of the water extract can be expressed as the weight of the freeze-dried water extract relative to the volume of the aqueous solution.

[0031] In this specification, "measured value" refers to a value obtained by a method for measuring peptide A contained in a water extract of aged meat. The measured value may be treated as a quantitative value or as a qualitative value. The measured value may be an absolute value expressed in weight, such as ng (nanograms) or μg (micrograms), representing the amount of peptide A in the sample, or a relative value expressed as absorbance relative to a control value or fluorescence intensity from a labeled molecule.

[0032] In this specification, "unit amount" refers to an arbitrarily defined amount of sample. For example, this could be volume (expressed as μL or mL) or weight (expressed as μg, mg, or g). While the unit amount is not specifically defined, it is preferable to keep the unit amount measured by the measurement method constant. For example, when comparing water extracts derived from meat before maturation or in the early stages of maturation with test samples from AIH patients, keeping the unit amount constant allows for more accurate detection. In particular, when measuring the antibody response level of autoantibodies as an absolute value, it is necessary to keep the unit amount constant.

[0033] 1-3. Process for evaluating the quality of aged meat based on the measured value of peptide A obtained in the measurement process. The present invention provides a method for evaluating the quality of aged meat, which includes a step of evaluating the quality of aged meat based on the measured value of peptide A obtained in the measurement step. The inventors have found that there is a positive correlation between the content of peptide A in aged meat and the quality of the aged meat. Therefore, the quality of aged meat is evaluated based on the measured value of peptide A.

[0034] Herein, one embodiment of the method for evaluating the quality of aged meat according to the present invention includes a step of comparing the measured value of peptide A in aged meat whose quality is to be evaluated with the measured value of peptide A in aged meat before aging, during the initial stage of the aging process, during the optimal aging period, or during the period beyond the aging period, thereby enabling the evaluation of quality. The optimal aging period refers to the period when aged meat is at its most delicious or ready to eat. The initial stage of the aging process refers to the aging period from the start of aging until the optimal aging period begins. On the other hand, the period beyond the optimal aging period refers to the aging period that has exceeded the optimal aging period. The initial stage of the aging process, the optimal aging period, and the over-aging period of aged meat may vary depending on the animal species and cut from which it originates. Those skilled in the art can determine these periods for each type of aged meat based on publicly available information. Alternatively, as shown in Example 3 below, the initial stage of the aging process, the optimal aging period, and the over-aging period can be determined by aging the meat using known methods and performing sensory evaluations at each stage of aging.

[0035] When the aged meat is beef, in one embodiment, the initial stage of the aging process can be defined as the period from the start of aging to approximately 20 days in an intentional aging method. Also, when the aged meat is beef, in one embodiment, the extended aging period can be defined as the period from approximately 40 days after the start of aging in an intentional aging method.

[0036] For comparison, the measured values ​​of peptide A in aged meat before aging, during the early stages of aging, during the optimal aging period, or during the over-aging period may be those measured in advance, or the measured values ​​of peptide A in aged meat before aging, during the early stages of aging, during the optimal aging period, or during the over-aging period may be used. Therefore, in one embodiment, the method for evaluating the quality of aged meat according to the present invention further includes a step of measuring the measured value of peptide A in aged meat before aging, during the initial stage of the aging process, during the appropriate period of the aging process, or during the period beyond the aging process. Furthermore, in one embodiment, the method for evaluating the quality of aged meat according to the present invention further includes a step of measuring the antibody response level between peptide A and anti-peptide A antibody in aged meat before aging, during the initial stage of the aging process, during the appropriate period of the aging process, or during the period beyond the aging process.

[0037] In this process, comparing the measured value of peptide A obtained in the measurement step with the measured value of peptide A in aged meat before aging, in the early stages of the aging process, during the optimal aging period, or during the over-aging period, for example, if the measured value of peptide A in the aged meat whose quality is to be evaluated is equivalent to the measured value of peptide A in aged meat during the optimal aging period, the quality can be evaluated as good. Alternatively, if the measured value of peptide A in the aged meat whose quality is to be evaluated is significantly higher than the measured value of peptide A in the aged meat before aging, in the early stages of aging, or in the later stages of aging, the meat can be evaluated as being of good quality. Alternatively, by pre-measuring peptide A in aged meat during the appropriate maturation period, a threshold can be set for determining good quality, and the quality of the aged meat to be evaluated can be assessed based on whether the measured value of peptide A exceeds that threshold.

[0038] 2. Anti-peptide A polyclonal antibody Another aspect of the present invention provides an anti-peptide A polyclonal antibody. The anti-peptide A polyclonal antibody according to the present invention can specifically and sensitively detect peptide A contained in the aqueous extract of aged meat.

[0039] Polyclonal antibodies can be produced according to known methods. When administering an antigen to an animal to produce polyclonal antibodies, it is preferable to use a complex of the antigen peptide shown in SEQ ID NO: 1 and a carrier protein as the immunogen. Specifically, it is preferable to use a complex of peptide A (H-CKGDEESLA-AMIDE (SEQ ID NO: 2); 9 amino acids; molecular weight 947.2), in which a carbon atom is added to the N-terminus of the amino acid sequence of peptide A (H-KGDEESLA-OH (SEQ ID NO: 1); 8 amino acids; Yamasaki et al., Agric. Biol. Chem. (1978)) and the C-terminus is amidated, and a carrier protein (Keyhole Limpet Hemocyanin (KLH)) as the immunogen. In one embodiment, the anti-peptide A polyclonal antibody according to the present invention is obtained by a method for producing a polyclonal antibody that uses a complex of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and a carrier protein KLH as an immunogen.

[0040] In one embodiment, the anti-peptide A polyclonal antibody according to the present invention has a binding affinity (KD value) of 4.00 × 10⁻¹⁰. -8 It is less than or equal to M, preferably 3.00 × 10 -8 M or less, more preferably 2.80 × 10 -8 It is M or less. The binding affinity (KD value) can be measured using biolayer interferometry under the conditions shown in Example 5 below.

[0041] In one embodiment, the anti-peptide A polyclonal antibody of the present invention can be provided as a complex with nanoparticles. The nanoparticles and the complex with the polyclonal antibody using them are as described above.

[0042] 3. Kit for evaluating the quality of aged meat Another aspect of the present invention provides a kit for evaluating the quality of aged meat. The kit for evaluating the quality of aged meat according to the present invention measures peptide A by methods such as ELISA, immunostigmatism, or immunochromatography, and allows for simple and rapid quality evaluation of aged meat on-site. The kit for evaluating the quality of aged meat according to the present invention includes, in addition to the anti-peptide A polyclonal antibody, any components that can be appropriately included depending on the peptide A measurement method, such as a buffer, preservative, diluent, blocking solution, washing solution, labeled secondary antibody, and instructions for use.

[0043] The present invention will be described in detail below using the following examples, but the present invention is not limited to the following examples. [Examples]

[0044] (Example 1. Preparation of aged meat) Commercially available beef inner thigh blocks (Japanese Black cattle, approximately 11 kg) were divided into approximately 1.3 kg portions. These portions were then aged using a DRY AGER DX500 (Landig + Lava GmbH & Co. KG) at 1.5°C and 82% humidity for 0, 10, 20, 30, 40, 50, 60, and 90 days. Samples were taken at various points during each aging process (0, 10, 20, 30, 40, 50, 60, and 90 days). For sample collection, the top 2 cm of each block was trimmed to remove the dry portion, and a 500 mg sample was taken from the lean, inner part of the meat.

[0045] (Example 2. Preparation of aqueous extract) 5 mg of meat samples were collected at various time points during the maturation process (0, 10, 20, 30, 40, 50, 60, and 90 days). 1.5 mL of ultrapure water was added to each sample, and the mixture was crushed using a bead crusher (TAITEC μT-12) at 2500 rpm, 25°C, and 30 seconds. The crushed solution was centrifuged (15000 rpm, 25°C, 5 minutes), the supernatant was collected, freeze-dried, and weighed. The freeze-dried product was then prepared by adding ultrapure water to a concentration of 10 mg / mL to obtain the aqueous extract.

[0046] (Example 3. Sensory evaluation) Approximately 5 g of meat samples were taken at different stages of the aging process (0, 10, 20, 30, 40, 50, 60, and 90 days). These samples were cooked at 200°C for 1 minute on each of the four sides, and sensory evaluation was conducted using a single-blind scoring method. The baseline value (0 days) was set at 0 points, and the "overall evaluation = deliciousness" of the meat at each aging stage was evaluated on a scale of -2 to 2 points (very unappetizing: -2, somewhat unappetizing: -1, average: 0, somewhat delicious: 1, very delicious: 2). The "overall evaluation = deliciousness" was a comprehensive evaluation including aspects such as tenderness, sweetness, umami, acidity, aroma, and fattiness. The panel consisted of six men and women aged 21 to 49. As shown in Figure 1, the scores increased from day 0 up to 30 days of aging, but were almost the same as the baseline value from 40 days onward. Furthermore, these results correlated with the recommended aging period for this meat aging machine (25-28 days: Operating Instructions DRY AGER DX500, p.5), as well as the aging period used by many other common meat aging manufacturers (14-35 days: Dry-aging of beef executive summary (U.S. Meat Export Federation)).

[0047] (Example 4. Preparation of anti-peptide A antibody) A complex was created by adding a carbon atom to the N-terminus of peptide A (H-KGDEESLA-OH (SEQ ID NO: 1); 8 amino acids; Yamasaki et al., Agric. Biol. Chem. (1978)), a peptide A (H-CKGDEESLA-AMIDE (SEQ ID NO: 2); 9 amino acids; molecular weight 947.2), which is the "shelf life" peptide in beef juice, and a carrier protein (Keyhole Limpet Hemocyanin (KLH)). After administering this complex to rabbits four times, total blood was collected on day 49, and anti-peptide A antibodies were purified using peptide affinity column chromatography (Cosmo Bio Contract Peptide Antibody). The chromatography test conditions and the concentration and amount of the obtained antibodies were as follows: Purification type: Peptide column Replacement buffer: PBS_GlycOH Amount of serum used: 10mL Preservative added: Proclin 300 (15 ppm) Antibody concentration: 1.32mg / mL Antibody amount: 1.0mL

[0048] (Example 5. Affinity analysis of anti-peptide A antibody) The binding affinity (KD value) of the anti-peptide A antibody to the antigen (peptide A) was analyzed using biolayer interferometry (BLItz; Sartorius, Gottingen, Germany). The streptavidin (SA) sensor chip (Sartorius) was hydrated with ultrapure water at 25°C for at least 10 minutes before measurement. All samples were diluted in PBS-BT buffer (5% bovine serum albumin (BSA) and 0.1% (v / v) Tween-20 phosphate buffer (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 2 mM KH2PO4 [pH 7.4])). Peptide A (50 μg / mL; Cosmo Bio), with biotin added to the N-terminus of peptide A, was bound to an SA sensor tip for 300 seconds, and then reacted with anti-peptide A antibody (0-166.7 nM) for 120 seconds to measure the binding ability of peptide A to the anti-peptide A antibody. The reaction was then carried out in PBS-BT buffer for 120 seconds to measure the dissociation ability. Figure 2 shows graphs of the binding and dissociation ability measurements. (BLitz Pro Software) Global analysis was performed using 1.3.0.5 (Sartorius) to calculate the binding rate constant (ka), dissociation rate constant (kd), and affinity (KD value). The calculation results are shown in Table 1. As shown in Table 1, the binding affinity (KD value) of this antibody to the antigen (peptide A) is 2.38 ± 0.36 × 10⁻¹⁶. -8 It was revealed that he is M. [Table 1]

[0049] (Example 6. Quantification of peptide A using ELISA and evaluation of the optimal eating time for aged meat) The aqueous extracts obtained in Example 2 were prepared at concentrations of 0 μg / mL, 3.25 μg / mL, 7.5 μg / mL, and 15 μg / mL. The antigen (peptide A (100 μL, 0-20 ng / mL) or aqueous extracts of aged meat from each aging process (100 μL, 0-15 μg / mL)) was fixed in each well of a 96-well microtiter plate (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C for 12 hours, washed three times with PBST buffer (PBS containing 0.1% (v / v) Tween-20), incubated in 100 μL of 5% (v / v) skim milk at 25°C for 30 minutes, and blocked. The plate was washed three times with PBST, 100 μL / well of anti-peptide A IgG (0.4 μg / mL) was added and reacted at 25°C for 30 minutes, and then washed three times with PBST. Subsequently, 100 μL / well of horseradish peroxidase-labeled donkey anti-rabbit secondary antibody (2 μg / mL: Novus Biologicals, Centennial, CO, USA) was reacted at 25°C for 30 minutes, and the plates were washed three times with PBST. A 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) substrate kit (Pierce, Rockford, IL, USA) was used for a colorimetric reaction at 25°C for 4 minutes, and the reaction was stopped by adding 100 μL / well of 2N H2SO4. Using a microplate reader MPR-A400 (AS ONE, Osaka, Japan), the absorbance at 450 nm was measured with the absorbance at 630 nm as the background. A well containing 0 μg / mL of antigen was subtracted as a blank background. The measurement was performed in triple replication.

[0050] First, as shown in Figure 3, we plotted the absorbance of peptide A at each concentration, which allowed us to draw a calibration curve and demonstrate that the obtained anti-peptide A IgG can be used to quantify peptide A. Next, the absorbance of the water extract of aged meat at each aging stage was plotted. The results are shown in Figure 4. As shown in Figure 4, it was found that the period with the highest peptide A content for each aging period sample (30 days) coincided with the period considered "most delicious" and "ready to eat" in sensory evaluation (30 days). Furthermore, when comparing the measurement results on day 30 of aging with the measurement results for each aging period (0-90 days), significant differences were observed at all water extract concentrations of 3.25, 7.5, and 15 μg / mL (p<0.05; t-test). It was also found that when the water extract concentration of aged meat was in the range of 1-7 μg / mL, the difference between the period considered "most delicious" and "ready to eat" (30 days) and other periods became clearer.

[0051] (Example 7. Quantification of peptide A by ELISA using ZZ-BNC and improvement of sensitivity in evaluating the optimal eating time of aged meat) The bio-nanocapsules used were those capable of aligning the orientation of antibodies and presenting them on the bio-nanocapsule surface. Specifically, ZZ-L protein-expressing yeast cells (Saccharomyces cerevisiae AH22R- containing the ZZ-BNC expression plasmid (pGLD-ZZ50)) were disrupted, the crude extract was heat-treated, and then ZZ-BNCs were purified by rabbit IgG-immobilized affinity column chromatography and gel filtration column chromatography. Antigens (peptide A (100 μL, 0–20 ng / mL) or aqueous extracts of aged meat from each aging process (100 μL, 0–15 μg / mL)) were immobilized in each well of a 96-well microtiter plate (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C for 12 hours. After washing three times with PBST buffer (PBS containing 0.1% (v / v) Tween-20), the plates were incubated in 100 μL of 5% (v / v) skim milk at 25°C for 30 minutes and then blocked. The plates were washed three times with PBST, 100 μL / well of anti-peptide A IgG (0.4 μg / mL) was added and reacted at 25°C for 30 minutes, followed by three washes with PBST. Subsequently, 100 μL / well of a conjugate pre-mixed with ZZ-BNC (2 μg / mL) and horseradish peroxidase-labeled donkey anti-rabbit secondary antibody (2 μg / mL: Novus Biologicals, Centennial, CO, USA), reacted at 25°C for 30 minutes, was added and reacted at 25°C for 30 minutes, followed by three washes with PBST. A colorimetric reaction was performed at 25°C for 4 minutes using 100 μL / well of a 3,3',5,5'-tetramethylbenzidine (TMB) substrate kit (Pierce, Rockford, IL, USA), and the reaction was stopped by adding 100 μL / well of 2N H2SO4. Using a microplate reader MPR-A400 (AS ONE, Osaka, Japan), the absorbance at 450 nm was measured with the absorbance at 630 nm as the background. A well containing 0 μg / mL of antigen was subtracted as a blank background. The measurement was performed in triple replication.

[0052] As shown in Figure 5, a calibration curve could be drawn by plotting the absorbance for peptide A at each concentration, demonstrating that the resulting complex of anti-peptide A IgG and ZZ-BNC enables the quantification of peptide A. Figure 5 also shows the calibration curve obtained from measurements of anti-peptide A IgG alone, without the formation of a complex with ZZ-BNC. When comparing the measurement results using a complex of anti-peptide A IgG and ZZ-BNC with the measurement results using anti-peptide A IgG alone (without complexing with ZZ-BNC) at each concentration of peptide A, significant differences were observed at all concentrations of 2.5, 5, 10, and 20 ng / mL (p<0.05; t-test).

[0053] Next, the absorbance of the water extract of aged meat at each aging stage was plotted. The results are shown in Figure 6. The left panel of Figure 6 shows the measurement results of anti-peptide A IgG alone, without complex formation with ZZ-BNC (Figure 4). The right panel of Figure 6 shows the measurement results using a complex of anti-peptide A IgG and ZZ-BNC. As shown in Figure 6, the detection sensitivity improved compared to the case without ZZ-BNC, and the difference from the uneaten period became easier to visualize. Also, similar to the case without ZZ-BNC, the period with the highest peptide A content in the samples for each aging period (30 days) coincided with the period considered "most delicious" and "at its best" in sensory evaluation (30 days). Furthermore, when comparing the measurement results on day 30 of aging with the measurement results for each aging period (0-90 days), significant differences were observed at all water extract concentrations of 3.25, 7.5, and 15 μg / mL (p<0.05; t-test). Furthermore, when the concentration of the water extract of aged meat was within the range of 1-7 μg / mL, the difference between the period considered "most delicious" or "best time to eat" (30 days) and other periods was more clearly defined.

[0054] We also investigated whether the presence or absence of ZZ-BNC increased absorbance. Specifically, for each concentration of water extract from samples of each maturation period, we compared the measurement results using a complex of anti-peptide A IgG and ZZ-BNC with the measurement results of anti-peptide A IgG alone without the complex with ZZ-BNC. As a result, at a concentration of 1.25 μg / mL, a significant difference was observed among the samples from 10 to 90 days. At a concentration of 7.5 μg / mL, a significant difference was observed among all samples from 0 to 90 days. At a concentration of 15 μg / mL, a significant difference was observed among the samples from 10 to 90 days (p<0.05; t-test).

Claims

1. A method for evaluating the quality of aged meat, The step of measuring peptide A in the aqueous extract of the aged meat, A step to evaluate the quality of the aged meat based on the measured value of peptide A obtained in the above measurement step. An evaluation method that includes this.

2. The evaluation method according to claim 1, The measurement step is a step of quantification using an antibody against peptide A. Evaluation method.

3. The evaluation method according to claim 2, A method for evaluating whether the aforementioned antibody is a polyclonal antibody.

4. The evaluation method according to claim 2, An evaluation method in which the antibody is a complex with nanoparticles.

5. The evaluation method according to claim 1, An evaluation method wherein the measurement step is the measurement of peptide A by the ELISA method.

6. The evaluation method according to claim 4, An evaluation method wherein the concentration of the aqueous extract of the aged meat is within the range of 1 to 15 μg / mL.

7. The evaluation method according to claim 1, An evaluation method in which the aged meat is beef.

8. The evaluation method according to claim 1, An evaluation method wherein the evaluation step is a step in which the quality is judged to be high if the measured value of peptide A obtained by the measurement step is higher than the measured value of peptide A contained in the water extract of aged meat that is not yet ready to eat.

9. The evaluation method according to claim 1, The evaluation method for determining the quality of the aforementioned aged meat as its deliciousness.

10. A polyclonal antibody that can be used in the evaluation method of claim 1, A polyclonal antibody that specifically binds to the aforementioned peptide A.

11. A polyclonal antibody according to claim 10, A polyclonal antibody obtained by a method for producing polyclonal antibodies using a complex of the antigen peptide shown in SEQ ID NO: 1 and a carrier protein as an immunogen.

12. A polyclonal antibody according to claim 10, The binding affinity (KD value) is 3.00 × 10 -8 Polyclonal antibodies with a M value of 0 or less.

13. A kit for evaluating the quality of aged meat, comprising the polyclonal antibody described in claim 10.

14. The kit according to claim 13, A quality evaluation kit for aged meat, wherein the polyclonal antibody is a complex with nanoparticles.

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