JP2022174882A - Method and system for testing origin, breed and/or quality of animal material - Google Patents

Abstract

To provide a method and a test system capable of easily identifying the origin, breed and quality of animal materials in a short time.SOLUTION: There is provided a method of identifying origin, breed and/or quality of animal material, including: a) a pretreatment step of contacting a sample made of animal material with a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent, followed by heating; b) a step of analyzing protein and/or peptide components contained in the sample after pretreatment; and c) qualitatively and/or quantitatively analyzing the species, breed and/or quality of the animal material based on the results of step b).SELECTED DRAWING: None

Description

Application for application of Article 30, Paragraph 2 of the Patent Law [Publication 1] Publication date: November 6, 2020 Publication: Electrophoresis Vol. ] Date: November 12 and 13, 2020 Meeting name: The 71st Annual General Meeting of the Japanese Electrophoresis Society Meeting place: Online 3rd year academic lecture on the use of silk and insect functions (The 91st Annual Meeting of the Japanese Society of Sericulture) Lecture Abstracts [Publication 4] Date: March 19th and 20th, 2021 (Slides available for viewing by participants: March 17th to 26th) Meeting name: 2021 Academic lecture on the use of silk and insect functions (The 91st Annual Meeting of the Japanese Society of Sericulture) Meeting place: Online

The present invention relates to methods and systems for testing the origin, breed and quality of animal material.

Animal materials, especially silk fibers, are generally expensive. It was virtually impossible to identify For example, the price of cocoons varies greatly depending on the place of production, the variety, etc. In particular, domestically produced "Koishimaru" is said to be about 10 times more expensive than foreign cocoons at present. Even if the place of origin of the silk thread and the place of origin of the silk thread indicated on the product are different, it is difficult to distinguish the place of origin from the appearance or the like. In addition, even if silkworm silk thread is mixed with valuable wild silk thread (Yamayu, Erisan, Shinjusan, Mugasan, Sakusan, Tensan, Tasarsan, Cricula, etc.), the presence or absence of contamination and the mixing ratio can be reliably determined from the state of the product. I had no means. Also, there was no way to clearly distinguish between old silk threads and silk threads produced within the same year. Furthermore, in the restoration of ancient silk fabrics, there is a desire to use the same kind of silk thread if possible, but it has been difficult to specify the origin of the silk thread of ancient silk fabrics.

The type of silkworm cocoon can be identified to some extent from the appearance of the cocoon, such as shape and color, but it is difficult to confirm. In particular, it is very difficult for anyone other than a skilled craftsman to distinguish from such external appearance. If pupae remain in the cocoon, it is assumed that they can be identified by genome analysis of the pupae, but in the absence of pupae, especially in the state of refined silk thread, identification was virtually impossible.

Non-Patent Documents 1 and 2 disclose a method for identifying the origin species of the silk thread by performing proteome analysis of the protein components contained in the silk thread by analyzing it with LC/MS/MS after trypsinizing the silk thread. ing. In addition, in Non-Patent Document 3, various physical property tests are conducted on domesticated silkworm-derived silk yarn into which non-natural amino acids have been introduced. The silk thread was dissolved in a lithium bromide solution, and the supernatant was subjected to urea treatment, SDS-PAGE, and trypsin treatment. Peptide fragments obtained were analyzed by MALDI-TOF-MS to detect peaks not found in the wild type. It is disclosed that the appearance has been confirmed.

On the other hand, other animal materials, specifically animal fibers (e.g., animal hair or feathers), leather, horns, teeth, etc., are also very rare and expensive, depending on the species and breed of origin. , whose identification is required. For example, Non-Patent Document 4 discloses a method of identifying cashmere and other animal hair fibers using MALDI-TOF-MS after trypsinization. Non-Patent Document 5 discloses a method for identifying and quantifying feathers and animal hairs using MALDI-TOF-MS.

Gu et al. (2019) Identification of silks from Bombyx mori, eri silkworm and chestnut silkworm using western blot and proteomics analyzes. Anal. Sci. 35, 175-180 Gu et al. (2019) Species identification of Bombyx mori and Antheraea pernyi silk via immunology and proteomics. Scientific reports 9:9 Teramoto et al. (2015), Property analysis of domestic silkworm silk containing unnatural amino acid (4-chlorophenylalanine), J. Silk Sci. Tech. Jpn. 23, 27-35 Ohashi et al. (2014), Identification of cashmere and animal hair in textile products by MALDI-TOF mass spectrometry, SENI GAKKAISHI, Vol. 70, No. 5, 105-108 Hollemeyer et al. (2002) Identification and Quantification of Feathers, Down, and Hair of Avian and Mammalian Origin Using Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry, Anal. Chem. Vol. 74, No. 23, pp.5960-5968

Although the methods shown in Non-Patent Documents 1 and 2 make it possible to identify the species of silkworm from which the silk thread is derived, identification of silkworm varieties has not been achieved. Also, the method shown in Non-Patent Document 3 is capable of distinguishing between wild-type and specific genetically modified organisms, but does not identify naturally derived species and cultivars. And neither method can identify the production year of silk. Furthermore, these methods all require several hours to several days for trypsin treatment, and the reagents and techniques used are costly and time-consuming. In addition, the methods for identifying animal hair and feathers described in Non-Patent Documents 4 and 5 also require trypsin treatment, and likewise the reagents and techniques used are costly and time consuming.

An object of the present invention is to provide a method and a test system that can easily identify the species, breed and quality of animal materials in a short period of time.

In order to solve the above-mentioned problems, the present inventors have conducted extensive research, and brought a sample containing animal materials into contact with an acidifying agent, an oxidizing agent, or a mixture thereof, followed by heating. It was found that by analyzing the protein and/or peptide components, it was possible to identify the origin species, variety and quality of the sample. The present invention is based on this new finding, and specifically provides the following inventions.
(1) A method for identifying the species, breed and/or quality of animal material,
a) a pretreatment step of contacting a sample made of animal material with a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent, followed by heating;
b) a step of analyzing protein and/or peptide components contained in the sample after pretreatment; and/or quantitatively analyzing;
(2) the pretreatment agent is trifluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, difluoroacetic acid, monofluoroacetic acid, propionic acid, butyric acid, hypochlorite, hydrogen peroxide, hydrogen fluoride, bromoacetic acid; The method of (1), comprising at least one selected from the group consisting of ozone, halogen, chlorine dioxide, or manganese dioxide.
(3) In step b), the analysis is performed by APCI method, CI method, EI method, ESI method, FAB method, FD method, FI method, LILBID method, LSIMS method, MALDI method, PB method, PD method, SIMS method The method of (1) or (2), which is mass spectrometry using an ionization means selected from the group consisting of , TSP method, or a combination thereof.
(4) The method of (3), wherein the ionization means is the ESI method or the MALDI method.
(5) The method of (4), wherein in step b), said analysis comprises MALDI-TOF-MS.
(6) The method of any one of (1) to (5), wherein in step b), the analysis comprises separation by LC, MPLC, HPLC, or capillary electrophoresis.
(7) Any of (1) to (6), wherein step c) utilizes analytical results obtained for a control group consisting of a plurality of animal materials of known origin, breed and/or quality. Method.
(8) The method of (7), wherein step c) utilizes a database in which analysis results obtained for animal materials of known origin, breed and/or quality are registered.
(9) A system for identification of species, breed and/or quality of animal material comprising:
- an analysis unit that analyzes protein components contained in a sample containing animal material heat-treated using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; - An analysis unit that qualitatively and/or quantitatively analyzes the species, breed and/or quality of the animal material based on the analysis results output from the analysis unit.
(10) - a processing unit having a heating means and heat-treating a sample containing animal material using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; The system of (9) further comprising.
(11) The system of (9) or (10), wherein the analysis unit comprises a mass spectrometer.
(12) The system according to (11), wherein the mass spectrometer is a MALDI-TOF-MS mass spectrometer.
(13) The analysis unit has a database, and the database is a database in which analysis results obtained for a control group consisting of a plurality of animal materials having known origin species, breeds and/or qualities are registered in advance. , (9) to (12).

INDUSTRIAL APPLICABILITY According to the method of the present invention, it is possible to easily identify the origin species, variety and quality of animal materials in a short time. In addition, by using the system of the present invention, it becomes possible to easily identify the origin species, breed and quality of animal materials in a short period of time.

1 is a flow diagram outlining the method of the present invention; FIG. The method of the present invention comprises at least sample preparation, a sample pretreatment step (step a)), an analysis step (step b)) and an analysis step of the results obtained (step c)). FIG. 2 is a flow diagram showing an example of how AI is used in the method of the present invention; In the illustrated example, an AI constructed using a database storing analysis results of known samples as training data is used. The analysis result of the target unknown sample, pretreatment and analysis conditions for the analysis are input to the AI, and information such as the origin species, variety, and year of manufacture of the sample determined by the AI is output. 1 is a schematic diagram of an example of a system of the invention; FIG. (A) shows a schematic of a first embodiment of the system of the present invention; In the first aspect of the system of the present invention, the test system 1 comprises an analysis section 3 and an analysis section 4 . It also has an input section 5 for inputting sample information, analysis conditions, etc., an output section 6 for outputting analysis results, and a control section 7 for controlling each device in the test system 1 . After the pretreated sample S1 is set at a predetermined position in the test system 1 from the outside, the test system 1 analyzes the protein components in the sample and analyzes the analysis results. (B) shows a second embodiment of the system of the present invention. In the second aspect of the system of the present invention, the test system 1 comprises a processing section 2, an analysis section 3 and an analysis section 4. FIG. It also has an input section 5 for inputting sample information, analysis conditions, etc., an output section 6 for outputting analysis results, and a control section 7 for controlling each device in the test system 1 . After an untreated sample S2 is set at a predetermined position in the test system 1 from the outside, the sample S2 is taken into the processing section 2 and subjected to pretreatment. The processing unit includes a robot arm, a reagent dispensing mechanism, etc., and can automatically preprocess the sample and deliver the processed sample to the analysis unit 3 . Mass spectra of boiled cocoon samples A to C pretreated at room temperature are shown. Mass spectra of boiled cocoon samples A to C that were subjected to microwave irradiation during pretreatment are shown. 1 shows a mass spectrum of fibers obtained from alpaca yarn. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. 1 shows a mass spectrum of fibers obtained from angora yarn. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. 1 shows a mass spectrum of fibers obtained from cashmere yarn. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. Figure 2 shows the mass spectrum of fibers obtained from sheep wool. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. 1 shows a mass spectrum of fibers obtained from quail feathers. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. Figure 2 shows the mass spectrum of fibers obtained from chicken (plymouth rock) feathers. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. Fig. 2 shows changes in the mass spectrum of fibers obtained from chicken (bravader) feathers with heat treatment time. (A) is a sample with a pretreatment time of 1 hour, (B) is a sample with a pretreatment time of 3 hours, (C) is a sample with a pretreatment time of 5 hours, and (D) is a sample with a pretreatment time of 24 hours. A mass spectrum of a specimen is shown. Mass spectra of bovine leather samples treated at room temperature, heat treated for 15 minutes, and heat treated for 30 minutes are shown. (A) shows the mass spectrum of the specimen treated at room temperature, (B) the specimen heat-treated for 15 minutes, and (C) the specimen heat-treated for 30 minutes. Mass spectra of 5 minute microwaved and 10 minute microwaved samples of bovine hide are shown. (A) shows the mass spectrum of the sample treated with microwaves for 5 minutes, and (B) the mass spectrum of the sample treated with microwaves for 10 minutes. Mass spectra of 30 minute heat treated samples of buffalo and cow hides are shown. (A) shows the mass spectrum of water buffalo leather, and (B) shows the mass spectrum of cow leather. Mass spectra of specimens derived from various horn samples are shown. (A) shows mass spectra of Yezo deer, (B) water buffalo, and (C) heat-treated bovine horn specimens. 1 shows a mass spectrum of a spider silk-derived specimen of Nephila spp. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. Fig. 2 shows a mass spectrum of a sample derived from spider silk of Argiope spider. (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen.

<Method for Identifying Derived Species, Varieties and/or Quality of Animal Materials>
1. Overview The method of the present invention is a method for identifying the origin species, breed and/or quality of animal material,
a) a pretreatment step of contacting a sample made of animal material with a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent and heating; and c) qualitatively and/or quantitatively analyzing the origin, breed and/or quality of the animal material based on the results of step b); A method comprising:

The main component of animal materials, especially animal-derived fibers and leathers, is protein. Fibroin, which is the main component of silk fibers, and keratin, which is the main component of animal hair and leather, have robust molecular structures, and conventional treatments such as SDS treatment are suitable for mass spectrometry, electrophoresis, and the like. It was difficult to state. However, by contacting these samples with a solution containing an acidifying agent and/or an oxidizing agent and subjecting them to heat treatment, at least part of the proteins contained in the samples are reduced to small molecules, that is, the proteins are fragmented. was found to be

It was found that the protein fragmentation patterns differed depending on the quality of the sample, such as origin, variety, and age. Under predetermined conditions, the sample is acidified and/or oxidized under heating conditions, and the resulting protein fragments (proteins and/or peptides) are analyzed to identify the origin species, variety and quality. It was found possible. Hereinafter, proteins and/or peptides to be analyzed are collectively simply referred to as "proteins".

In the present invention, "analysis" means obtaining output data obtained directly from a sample, so-called raw data, and "analysis" means obtaining output information calculated based on the output data. shall be

FIG. 1 shows a flow diagram of each step in the method of the present invention. The method of the present invention comprises at least sample preparation, a sample pretreatment step (step a)), an analysis step (step b)) and an analysis step of the results obtained (step c)). Each of these steps is described in detail below. In addition to each of these steps, the method of the present invention may include other steps.

2. Description of each step 2-1 Preparation of sample The "sample" to be tested in the present invention is "animal material". The term "animal" as used herein specifically refers to metazoans in general, including sponges, arthropods and chordates. "Animal materials" specifically include animal-derived fibers (eg, silk, spider silk, animal hair, feathers, etc.), leather, horns, and teeth. Although it is mainly used as a material for clothes and accessories, it is not limited to these uses.

Animal materials used in the present invention include silk derived from silkworms. The term "silk" as used herein includes all materials including fibers derived from silkworms, particularly silk thread spun by silkworms, including cocoons and silk threads obtained from cocoons or parts thereof. As used herein, the term “cocoon” particularly refers to a pupal chamber composed of fibers to protect the pupa of the silkworm, and is often an adhesive component (sericin) of the fiber component (fibroin protein) contained in silk. It is formed by adhesion, entanglement, etc. by proteins, etc.). The cocoon as a sample may be a raw cocoon or a heat-treated cocoon, a part cut from the cocoon as a test piece, or a fiber extracted from the cocoon. As used herein, the term “silk thread” refers to various types of processed fibers spun from silk thread, including raw silk, kneaded silk, and dyed products of these (dyed after being made into a woven fabric). including). The silk thread as a sample may be a fiber before being twisted, a twisted thread, or a fiber obtained by untwisting the twisted thread.

As used herein, the term “silicworm” is a general term for insects that have silk glands and are capable of spinning silk threads. Specifically, among the order Lepidoptera, Hymenoptera, Reticulum order, Caddisformer order, Moth order, Diptera order, Termite order, etc., species that can spun silk for nesting, cocooning or movement mainly in the larval stage. Point. In Lepidoptera, the family Bombycidae, the family Saturniidae, the family Brahmaeidae, the family Eupterotidae, the family Lasiocampidae, the family Psychidae, and the tiger moth The Archtiidae, Noctuidae, etc. are preferred as silkworms herein. Species belonging to the genus Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically, silkworm, Bombyx mandarina, Samia cynthia; Preferable examples include hybrids), Antheraea yamamai, Antheraea pernyi, Saturnia japonica, and Actias gnoma.

A "silic gland" is a modified tubular organ of the salivary gland that functions to produce, store and secrete liquid silk. Silkworms have a pair of left and right silk glands along the digestive tract of mainly larvae of the silkworm, and each silk gland is composed of three regions: anterior, middle, and posterior silk glands. As mentioned above, the posterior silk gland produces and secretes fibroin, the fibrous component of silk. In addition, the middle silk gland produces and secretes sericin, which is a covering component, and accumulates in its lumen together with fibroin migrated from the rear silk gland.

The method of refining silk varies depending on the type of cocoon used as a material, the purpose of use, etc., but the following methods are usually used for refining silk. Cocoons (raw cocoons) produced by silkworms or the like are dried with hot air at 60 to 120° C. for about 6 hours and stored in the dried state (heat-treated cocoons). The heat-treated cocoons are boiled in hot water at 60 to 98°C for about 20 minutes to make the fibers easier to loosen (boiled cocoons), then cooled in water, the yarn ends are hooked using a brush or the like, and silk threads are produced using a reeling machine. is brought out. The silk thread obtained here is called raw silk or raw silk. Raw silk usually contains sericin, P25, secondary metabolites, etc. in addition to fibroin, which is the main component of silk fibers, and has a relatively hard texture. A kneaded yarn is produced by scouring the raw silk or the cloth woven from it with a sodium carbonate solution or Marcel soap. The former refined thread refined with sodium carbonate solution (hereinafter referred to as "sodium carbonate refined silk") contains sericin and has a texture that remains firm, while the latter refined thread refined with Marcel soap (hereinafter referred to as "Marcel soap scouring silk") has sericin removed and has a soft texture. The method of the present invention can be used for testing any of the raw cocoons, heat-treated cocoons, raw silk, sodium carbonate scouring silk, and Marcel soap scouring silk. Table 1 shows the major components commonly contained in fresh cocoons, heat-treated cocoons, sodium carbonate refined silk, and Marcel's soap refined silk.

Animal materials used in the present invention include animal fibers other than silk. Examples of animal fibers other than silk include animal hair, feathers, and spider silk. Keratin accounts for most of the protein contained in animal hair. The origin of animal hair is not particularly limited, but sheep, cashmere goats, angora goats, rabbits, camels, alpaca, vicuña, guanaco, llama, and musk ox (kiviak), which are generally distributed mainly as materials for clothing and accessories. , possums, minks, chinchillas, etc. can be used. Animal hair used as a clothing material is generally cut from an animal and processed into thread through washing, combing, dyeing, spinning, and the like before use. The animal hair as a sample may be at any stage of each of the above processing steps, for example, it may be animal hair before washing, it may be woolen yarn after spinning, or a fabric It may be part of the fibers of a product or knitted product.

Keratin accounts for most of the protein in feathers. The origin of feathers is not particularly limited, but feathers derived from geese and ducks, which are generally distributed mainly as materials for clothing and bedding, can be used.

Animal materials used in the present invention include leather. Collagen accounts for most of the protein contained in leather. The origin of leather is not particularly limited, but it is mainly derived from cows, sheep, pigs, goats, horses, deer, ostriches, kangaroos, snakes, lizards, crocodiles, etc., which are generally distributed as materials for clothing and accessories. You can use either one. The leather used in the present invention may be the skin itself (commonly referred to as "skin") that wraps the surface of the animal's body, or may be tanned (generally referred to as "leather"). ) may be The term "tanning" as used herein refers to a processing process in which animal fats and vegetable ingredients are applied to the skin, or smoked, in order to eliminate defects such as rotting and hardening of the skin. Leather as a sample may have undergone any processing step.

Animal materials used in the present invention include horns and teeth. The main component of horns and teeth is calcium, but it also contains a small amount of protein, which is mainly composed of collagen. The origin of the horn is not particularly limited, but any one derived from deer, water buffalo, cow, etc., which is mainly used as a material for clothing and accessories, can be used. The species of tooth origin is not particularly limited, but any one derived from elephant (ivory), mammoth, hippopotamus, whale, or the like, which is mainly used as a material for ornaments, can be used.

2-2 a) Pretreatment step In the method of the present invention, "pretreatment" refers to contacting a sample with a "pretreatment agent" in order to make the sample analyzable in the identification of the animal material. It refers to heat treatment in the state. A "pretreatment agent" is a solution containing an acidifying agent and/or an oxidizing agent. As a solvent constituting the pretreatment agent, a solvent capable of dissolving fragmented peptides and the like and having a boiling point of 70° C. or higher, particularly 85° C. or higher can be used. More specifically, water can be used. The pretreatment agent may be used as one liquid, or two or more liquids may be used simultaneously or consecutively.

As used herein, the term "acidifying agent" refers to an agent for subjecting a sample to pH less than 7.0, particularly pH 5.0 or less, and pH 1.0 to 3.0, that is, under acidic conditions. The type is not particularly limited as long as it can be converted. Suitably, trifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, propionic acid, butyric acid, hydrogen fluoride, bromoacetic acid, etc., or combinations thereof can be used. A plurality of these acids can also be used consecutively. When used as a pretreatment agent, the acidifying agent is preferably used at a concentration of 0.01 to 10.0N (regulation), particularly 0.1 to 2.0N, more preferably 0.2 to 1.0N.

As used herein, the term “oxidizing agent” is not particularly limited as long as it is an agent capable of oxidizing a sample, particularly protein. Preferably, hydrogen peroxide, ozone, hypochlorite, elemental halogens, chlorine dioxide, manganese dioxide, etc., or combinations thereof can be used. When hydrogen peroxide water is used as the oxidizing agent, it is preferably used at a concentration of 0.01 to 10.0M, particularly 0.1 to 2.0M, more preferably 0.2 to 1.0M.

The means for bringing the pretreatment agent into contact with the sample is not particularly limited as long as the pretreatment agent is evenly in contact with the sample. Means such as phase contact can be used. When the sample is heat-treated by heating the pretreatment agent, it is preferable to seal the pretreatment agent in which the sample is immersed in a container in order to prevent drying of the sample due to evaporation of the solvent.

The method of the present invention requires heating the pretreatment agent while it is in contact with the sample. The heating temperature is not particularly limited, but when the sample is analyzed as a liquid phase, the temperature is preferably raised to 60 to 100°C, particularly 70 to 98°C, and further to 85 to 95°C. As a method for performing such heating, for example, a method of sealing a pretreatment agent in which a sample is immersed in a container and heating it with a Peltier element, a hot water bath, an oil bath, or the like, or a method of irradiating a sample with microwaves is used. be able to. Alternatively, if the sample is analyzed as a gas phase, the temperature of the sample can be, for example, 100-120°C.

The heating time can be 30 minutes to 24 hours, particularly 1 hour to 3 hours, when heating via a pretreatment agent. In the case of microwave irradiation, the heating time can be 1 to 15 minutes, particularly 3 to 7 minutes.

For silk, it was confirmed that proteins such as fibroin can be fragmented by contacting the sample with a pretreatment agent at room temperature, and analysis by mass spectrometry is possible. Furthermore, it was found that by applying heat during the pretreatment of the silk sample, the protein was further fragmented and showed more diverse mass spectral patterns. In addition, some animal materials other than silk were not sufficiently fragmented by pretreatment at room temperature, but by adding heat during pretreatment, protein fragmentation progressed. , mass spectroscopy, etc. can be performed.

The sample after pretreatment may be used in a state in which fibers or fiber clumps remain, or the supernatant obtained by centrifuging or filtering the pretreatment agent after reaction may be used. When mass spectrometry is performed in the subsequent analysis step, the system is suitable for microanalysis, so the sample used is preferably about 0.01 to 100 mg, particularly 0.05 to 5.0 mg.

Pretreatment conditions such as components of the pretreatment agent, reaction time and temperature can be appropriately selected depending on the type of sample and the purpose of identification. It was found that the signal obtained in the analysis step described below differs depending on the pretreatment conditions. By referring to known results, etc., it is possible to select conditions that facilitate the desired test. For example, if the purpose of the test is to identify cultivars, pretreatment conditions can be selected that increase the difference in signals obtained between cultivars. In addition, the same sample can be pretreated under a plurality of different conditions, and analyzed and compared.

The sample may optionally be treated with a reducing agent before or after treatment with an acidifying agent and/or an oxidizing agent. Examples of reducing agents include dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl(phosphine) hydrochloride (TCEP), tributylphosphine, cysteine hydrochloride, hydroxysulfite, and the like, which are commonly used in protein analysis. Any agent can be used.

It is preferable not to use an enzymatic reaction for the pretreatment step in the method of the present invention. Traditionally, silk analysis has involved fragmentation of proteins with proteases such as trypsin. Although the use of enzymes has the effect of fragmenting proteins, there are problems in that the reagents used are more expensive than oxidizing/reducing agents and the reaction takes a long time.

INDUSTRIAL APPLICABILITY According to the method of the present invention, it is possible to fragment proteins contained in animal materials in a short time and at low cost by using a pretreatment agent and heating without enzymatic treatment.

2-3 b) Analysis Process The sample processed in the pretreatment process is subjected to the analysis process. In the analysis step, proteins fragmented in the pretreatment step are analyzed. Protein analysis means include known means such as electrophoresis, amino acid sequencing, liquid chromatography (LC), mass spectrometry, amino acid composition analysis, X-ray diffraction, nuclear magnetic resonance spectrum (NMR), and Fourier transform infrared absorption spectrum (FTIR). can be used, but mass spectrometry, which can easily analyze the properties of macromolecular substances, can be preferably used. Mass spectrometry is a method of ionizing a minute amount of a compound in a high vacuum chamber, separating and detecting the generated ions by mass.

Means for ionizing compounds include, for example, atmospheric pressure chemical ionization (APCI), chemical ionization (CI), electron ionization (EI), electrospray ionization. , ESI), fast atom bombardment (FAB), field desorption ionization (FD), field ionization (FI), laser-induced liquid secondary ion desorption (FI). spectrometry, LILBID), liquid secondary ion mass spectrometry (LSIMS), matrix assisted laser desorption ionization (MALDI), particle beam (PB), plasma desorption plasma desorption (PD), secondary ion mass spectrometry (SIMS) or thermospray (TSP) or a combination of these ionization methods. In particular, the MALDI method or the ESI method, which are widely used for analysis of macromolecules, especially proteins, can be preferably used. It was found that the mass spectrometry spectrum of silk protein after pretreatment (hereinafter referred to as "mass spectrum") produced different patterns depending on the ionization means. It is also possible to select conditions that facilitate the achievement of test objectives. For example, if the purpose of the test is to identify the cultivar, then an ionization means can be selected that increases the cultivar-to-cultivar difference in the mass spectrum pattern obtained. Also, the same pretreated sample may be analyzed in parallel using a plurality of ionization means to obtain respective mass spectra.

As a means for separating ionized molecules by mass, any known method can be used, but time of flight mass spectrometry (TOF-MS), which is particularly suitable for separating macromolecules, is preferably used. can. Analysis of protein components in the present invention preferably includes MALDI-TOF-MS.

When MALDI-TOF-MS is used as an analysis means, the sample after pretreatment may be subjected to analysis together with the pretreatment agent, or the supernatant may be placed in a cuvette or the like. On the other hand, the fibers remaining in the pretreatment agent can be adhered to a conductive carbon double-sided tape, dried, and analyzed. As the conductive carbon double-sided tape, those generally available for scanning electron microscopes (SEM) can be used (for example, manufactured by Nissin EM Co., Ltd.).

Prior to or instead of mass spectrometry, liquid chromatography (LC), middle pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), Separation by capillary electrophoresis or the like may be performed.

2-4 c) Analysis step b) A step of qualitatively and/or quantitatively analyzing the origin species, variety and/or quality of the sample based on the results (raw data) obtained in the analysis step. Preferably, the analysis step is performed using the results obtained through the analysis step under the same conditions for a control group consisting of a plurality of animal materials having known origin species, breeds and/or qualities.

It was found that the data obtained in the analysis process, especially the mass spectrum pattern, of the sample containing pretreated silk varies at least according to the parameters derived from the sample and the parameters derived from the pretreatment and analysis conditions shown in Table 2. did. Therefore, for example, for a control group consisting of a large number of silk threads or cocoons with known origin species, breeds, scouring conditions, manufacturing years, etc., a large number of mass spectra were obtained under various pretreatment and analysis conditions, and the obtained spectrum By accumulating the pattern of each control as a fingerprint, it is possible to construct a database useful for analysis of unknown samples. Alternatively, the database may be constructed by acquiring mass spectra under defined pretreatment and analytical conditions for a number of controls. However, when using this database, the pretreatment and analysis steps for unknown samples can be limited to the predetermined pretreatment and analysis conditions. The mass spectrum data stored in the database may be mass spectrum image data or a set of numerical data of m/z and signal intensity of each detected peak. The parameters for database construction are not limited to those listed in Table 2.

In the method of the present invention, the analysis step can be performed by collating analysis results, particularly mass spectra, obtained from the target unknown sample with the constructed database. If necessary, the pretreatment and analysis conditions used to obtain the analysis results may be checked. In addition, the analysis result of the sample to be collated does not need to be one from one unknown sample, for example, by changing the pretreatment and analysis conditions, multiple analysis results, for example, multiple mass spectra are obtained, These may be cross-checked against the database.

Alternatively, in the method of the present invention, the analysis step can be performed by comparing the mass spectrum of a sample analyzed under the same conditions as the target sample and at approximately the same time (for example, on the same day). The preparation to be used can be appropriately selected according to the purpose of identification. For example, when identifying the production year of animal material in a sample, use the same kind of animal material of the same production year as a standard, and compare the mass spectrum of the sample with the mass spectrum of each standard. be able to.

Verification may be done manually, but is preferably done using a computer. For collation, for example, data such as the analysis results of the target sample is input, the data closest to the input data in the database is extracted, and the extracted data and its background information (sample origin species, breed , year of manufacture, etc.). Matching may also be performed using artificial intelligence (AI). When using AI, it is possible to construct a learned model through machine learning using a known neural network using the database as teacher data, and using this, from data input from an unknown sample, the Desired information such as the origin of the sample, the variety, and the year of manufacture can be output. FIG. 2 shows a flow diagram of an example of how AI is used in the method of the present invention. In the example shown in FIG. 2, an AI constructed using a database of accumulated analysis results of known samples as training data is used. The analysis result of the target unknown sample, pretreatment and analysis conditions for the analysis are input to the AI, and information such as the origin species, variety, and year of manufacture of the sample determined by the AI is output. Note that the mode using AI is not limited to the example shown in FIG.

2-5 Other Steps Depending on the type of target sample and desired information, other steps may be added in addition to the above steps. For example, when testing dyed products or products containing naturally-occurring impurities, the analysis results, especially the mass spectrum, show signals from the dyes and impurities, and the signal from the fragmented protein of interest appears. Distinction can be difficult. When using such a sample, it is preferable to add a destaining step and/or a washing step before the pretreatment step. Specific means for the decolorization step and washing step include, for example, means for immersing the sample in an organic solvent such as acetonitrile, acetone, ethanol, and methanol, or an aqueous solution thereof, and immersion in an alkaline solution such as ammonia water and sodium hydroxide solution. washing with surfactants such as SDS, CTAB and Marcel soap; washing with bleach such as thiourea dioxide; and immersion in a reducing agent solution such as hydroxysulfite. . The above means may be used alone or in combination. If there is no adverse effect such as side reactions, they may be mixed as appropriate, for example, by mixing an organic solvent and a surfactant.

The analysis step and the analysis step in the method of the present invention do not necessarily have to be performed only with the protein and/or peptide components of the sample. The results may be analyzed together. In addition, not only the analysis results of the sample using the pretreatment agent, for example, the analysis results of the same sample separately treated using a reducing agent and a basifying agent may be analyzed together. .

3. Examples of application of the test method Hereinafter, identification of animal materials to which the method of the present invention is applied is listed as an example, but the method of the present invention is not limited to the following examples, and can be applied to various identifications. is.

3-1 Identification of Silk 3-1-1 Identification of Differences in Manufacturing and Refining Methods of Silk Threads and Cocoons Different patterns can be obtained, especially for the mass spectra, for sodium carbonate degummed silk and marcel soap degummed silk samples degummed with sodium and marcel soap, respectively. Therefore, in the identification of silk threads, the analysis results of the desired silk thread sample are collated with the analysis results of raw silk, sodium carbonate refined silk and Marcel soap refined silk obtained in advance from cocoons of the same type under the same conditions. It is possible to objectively identify the scouring method of each yarn, which has been sensuously distinguished by touch, texture, etc. In addition, in the identification of cocoons, the analysis results of the target cocoon sample should be objectively identified by previously comparing the analysis results of the cocoons of the same species under the same conditions obtained from raw cocoons and heat-treated cocoons. can be done.

3-1-2 Identification of Derived Species According to the method of the present invention, different patterns can be obtained, particularly for mass spectra, depending on the derived species of silk. Therefore, the origin species of the sample can be identified by collating the analysis results of the target sample with a database that stores the results of the analysis of silk of each origin species under the same conditions as fingerprints in advance. In addition, when the sample is silk thread, even when fibers obtained from a plurality of species are mixed, the types and mixture ratio can be determined.

3-1-3 Identification of cultivar In the method of the present invention, different patterns can be obtained, particularly for mass spectra, depending on the silk cultivar. Therefore, the cultivar of the sample can be identified by collating the analysis results of the target sample with a database that stores in advance the results of the analysis of each cultivar of silk under the same conditions as fingerprints. In addition, even when fibers obtained from a plurality of varieties of silk thread are mixed, the varieties and mixture ratio can be determined. As a result, for example, it is possible to objectively judge the authenticity of silk products that claim to be of an expensive breed such as Koishimaru.

3-1-4 Identification of Quality According to the method of the present invention, different patterns are obtained, particularly for mass spectra, depending on the quality of the silk, especially the elapsed time since production. More specifically, silk threads or cocoons that have been produced for a long time have peak shifts in which each peak of the mass spectrum shifts toward the high molecular weight side compared to new cocoons. Silk fibers are prone to oxidation over time. This peak shift is presumed to be derived from protein oxidation. Therefore, the year of manufacture of a sample can be identified by comparing the results of analysis of the same type of silk thread or cocoon of each year of manufacture under the same conditions as a database with the results of analysis of the target sample. can be done. Since fiber oxidation affects qualities such as texture and strength, identification of the year of manufacture of silk is useful for estimating its quality.

3-2 Identification of Animal Hair According to the method of the present invention, different patterns can be obtained, particularly for mass spectra, depending on the species and breed of animal hair. Therefore, it is possible to identify the origin species and breed of the sample by collating the analysis results of the target sample with a database that holds the analysis results of the animal hair of each origin species under the same conditions as fingerprints in advance. . In addition, even when fibers obtained from a plurality of species are mixed, the types and mixture ratio can be determined. As a result, for example, it is possible to objectively determine the authenticity of animal hair products that claim to be of expensive breeds such as cashmere.

The method of the invention also makes it possible to determine the quality of the animal hair, especially the age since it was produced. Specifically, the year of manufacture of the sample is identified by comparing the analysis results of the same type of animal hair in each year of manufacture as a database under the same conditions in advance with the analysis results of the target sample. can do.

3-3 Identification of Leather According to the method of the present invention, different patterns, especially for mass spectra, can be obtained depending on the species and variety of leather origin. Therefore, the origin species of the sample can be identified by collating the analysis result of the target sample with a database that holds the analysis result of the leather of each origin species under the same conditions as a fingerprint in advance. As a result, for example, it is possible to objectively determine the authenticity of leather products that claim to be of an expensive variety such as crocodile leather.

The method of the invention also makes it possible to determine the quality of the leather, especially the age of the leather. Specifically, the year of manufacture of the sample is identified by collating the analysis results of the same type of leather in each year of manufacture as a database in advance with the analysis results of the target sample under the same conditions. be able to.

3-4 Identification of Horns or Teeth According to the method of the present invention, different patterns, especially for mass spectra, can be obtained depending on the species and varieties of horns or teeth. Therefore, the species of origin of the sample can be identified by collating the analysis results of the target sample with a database in which the results of the analysis of the horns or teeth of each origin species under the same conditions are held as fingerprints in advance. As a result, for example, it is possible to objectively determine the authenticity of rare breeds such as ivory.

<System for identification of species, breed and/or quality of animal materials>
The system of the present invention is a system for testing animal material comprising: a.
- an analysis unit that analyzes protein components contained in a sample containing animal material heat-treated using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; - An analysis unit that qualitatively and/or quantitatively analyzes the species, breed and/or quality of the animal material based on the analysis results output from the analysis unit.

Unless otherwise specified, the system of the present invention is a system for carrying out the method of the present invention, and is described in the section <Method for Identifying Derived Species, Varieties and/or Quality of Animal Material> above. It has features and common features.

The system of the present invention, in addition to the analysis unit and the analysis unit,
- A processing unit that has a heating means and heat-treats a sample containing animal material using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; good.

A schematic of the system of the present invention is illustrated in FIG. (A) shows a first embodiment of the system of the present invention. In the first aspect of the system of the present invention, the test system 1 comprises an analysis section 3 and an analysis section 4 . It also has an input section 5 for inputting sample information, analysis conditions, etc., an output section 6 for outputting analysis results, and a control section 7 for controlling each device in the test system 1 . Preferably, the control unit 7 is a computer and the input unit 5 and the output unit 6 are user interfaces. After the pretreated sample S1 that has been heat-treated in contact with the "pretreatment agent" from the outside is set at a predetermined position in the test system 1, the test system 1 analyzes the protein components in the sample and , analyze the analysis results.

FIG. 3B shows a second embodiment of the system of the invention. In the second aspect of the system of the present invention, the test system 1 comprises a processing section 2, an analysis section 3 and an analysis section 4. FIG. It also has an input section 5 for inputting sample information, analysis conditions, etc., an output section 6 for outputting analysis results, and a control section 7 for controlling each device in the test system 1 . Preferably, the control unit 7 is a computer and the input unit 5 and the output unit 6 are user interfaces. After an untreated sample S2 is set at a predetermined position in the test system 1 from the outside, the sample S2 is taken into the processing section 2 and subjected to pretreatment. The processing unit 2 includes heating means for heat-treating the sample. The processing unit 2 may also include a robot arm, a reagent dispensing mechanism, etc. In this case, it is possible to automatically preprocess the sample and deliver the processed sample to the analysis unit 3. .

The following configuration is a configuration common to the first mode (FIG. 3(A)) and the second mode (FIG. 3(B)). The analysis unit 3 includes a device for analyzing protein components of the pretreated sample S1. Any device may be used as long as the protein component of the pretreated sample can be analyzed, and known means such as an electrophoresis tank, an amino acid sequencer, a liquid chromatography (LC) device, and a mass spectrometer are used. However, a mass spectrometer that can easily analyze the properties of macromolecular substances can be preferably used. In particular, a MALDI-TOF-MS mass spectrometer, which is widely used for analysis of macromolecules, especially proteins, can be preferably used. The analysis unit may include multiple types of analysis devices.

The analysis unit 4 qualitatively and/or quantitatively analyzes the origin species, breed and/or quality of the animal material based on the analysis results obtained by the analysis unit 3 . The analysis unit 4 includes a data collection unit 41 that collects input information from the input unit 5 and analysis results from the analysis unit 3, a collation unit 42, and a database 43. FIG. The database 43 is a database in which the analysis results obtained for a control group consisting of a plurality of animal materials with known origin species, breeds and/or qualities are registered in advance. It has software for collating the aggregated data with the data in the database 43 . Existing software such as MALDI biotyper (manufactured by Bruker Daltonics) can be used. The collation unit, for example, collates the data collected from the analysis unit with the database, extracts the closest data in the database, and extracts the extracted data and its background information (sample origin species, breed, scouring state, manufacturing year, etc.). The collation part 42 may be provided with AI. The AI here can suitably use a learned model machine-learned using the database as teacher data, and by inputting the collected data into the AI, the origin species, breed and / or quality of the sample result in qualitative and/or quantitative information about

Alternatively, the analysis unit 4 does not use the database 43, but directly uses the mass spectrum data of the sample analyzed under the same conditions as the target sample (for example, on the same day). The data aggregated by the data collection unit 41 may be collated.

The system of the present invention includes an analysis unit and an analysis unit, and may be any system that is applicable to the implementation of the method of the present invention described above, and should not be construed as being limited to the first and second aspects. .

<Example 1 Analysis of boiled cocoons>
1. Scouring of Silk For three types of silkworm cocoons (samples A to C) of different genetic strains, boiled cocoon reeling threads were prepared by the following procedure. 120 mg of fiber pieces cut from the raw cocoons were placed in a 50 mL beaker and dried by heating at 120° C. for 10 minutes and then at 60° C. for 6 hours to prepare heat-treated silk. Further, it was placed in water of 98-100° C. and heated for 20 minutes, and then dried.

2. Sample Pretreatment 100 μL of 2.5% formic acid aqueous solution was placed in a sealable 1.5 mL polypropylene tube, and 1 mg of each of Samples A to C was added thereto. The tube was sealed and floated on 100 to 200 mL of water filled in a plastic container, then placed in a microwave oven set at 750 W and irradiated with microwaves for 5 minutes (microwave-treated sample).

Separately from the samples subjected to the above pretreatment, each sample was subjected to the following room temperature pretreatment. 100 μL of a 2.5% formic acid aqueous solution was placed in a sealable 1.5 mL polypropylene tube, and 1 mg each of Samples A to C was added thereto. The tube was sealed and mixed by shaking for 5 minutes (room temperature treated samples).

3. Analysis by MALDI-TOF-MS The solution after each treatment was quenched, and 1 μL was dropped onto a conductive carbon double-sided tape (manufactured by Nisshin EM Co., Ltd.) and analyzed with a MALDI-TOF-MS mass spectrometer (Autoflex III or Microflex, It was placed on a Bruker Daltonics sample stage and allowed to dry. Mass spectra were acquired from m/z 1,000 to 20,000 in linear positive mode.

Mass spectra of each sample obtained are shown in FIGS. FIG. 4 shows mass spectra of samples A to C pretreated at room temperature. FIG. 5 shows mass spectra of samples A to C which were subjected to microwave irradiation during pretreatment. As for the boiled cocoons, different mass spectral patterns were obtained for each sample, even if it was pretreated at room temperature. It was found that by performing microwave irradiation, many peaks were observed in the mass spectrum of each sample, and detailed discrimination became possible. It was also found that more detailed discrimination can be made by combining the data of room temperature treatment and the data of microwave irradiation.

<Example 2 Animal hair analysis>
Fiber samples from alpaca, angora, cashmere and sheep yarns were subjected to the following pretreatments. 100 μL of 2.5% formic acid aqueous solution was placed in a sealable 1.5 mL polypropylene tube, and 1 mg of each sample was added thereto. The tube was sealed, placed on a heat block, and heated at 90° C. for 3 hours (heat-treated specimen).

Separately from the above pretreatment, each sample was pretreated by microwave irradiation and pretreated at room temperature in the same manner as in Example 1. Mass spectra were obtained under the same conditions as in Example 1 for the specimens after various pretreatments.

Mass spectra of the obtained specimens are shown in FIGS. 6-9. 6 shows the mass spectrum of alpaca, FIG. 7 shows the mass spectrum of Angora, FIG. 8 shows the mass spectrum of cashmere, and FIG. 9 shows the mass spectrum of sheep. In any of FIGS. 6 to 9, (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. In any sample, almost no protein fragmentation was observed in the room-temperature-treated specimen, whereas many fragmentation peaks were observed in the heat-treated specimen. Many fragmentation peaks were also observed in microwave treated specimens in alpaca, cashmere and sheep. A minor peak of fragmentation was also observed in the Angola microwave treated sample. From these results, it was found that the pretreatment involving heat treatment or microwave treatment enabled identification of the origin species of animal hair.

<Example 3 Feather analysis>
Heat-treated samples, microwave-treated samples, and room-temperature-treated samples were prepared under the same conditions as in Examples 1 and 2 using 1 mg each of quail and chicken (Plymouth Rock) feather samples. Separately from the above, each heat-treated specimen was prepared by changing the pretreatment time to 1 hour, 3 hours, 5 hours and 24 hours for feathers of chicken (Bravader). A mass spectrum was obtained under the same conditions as in Example 1 for each sample after pretreatment.

FIG. 10 shows mass spectra of quail, and FIG. 11 shows mass spectra of chicken (Plymouth Rock) under various pretreatment conditions. In both FIGS. 10 and 11, (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. In quail, protein fragmentation was seen even with room temperature treatment. In chicken, on the other hand, almost no protein fragmentation was observed in the room-temperature-treated specimens, but numerous fragmentation peaks were observed in the heat-treated and microwave-treated specimens.

FIG. 12 shows changes in the mass spectrum of chickens (Bravader) depending on the heat treatment time. In FIG. 12, (A) is a sample with a pretreatment time of 1 hour, (B) is a sample with a pretreatment time of 3 hours, (C) is a sample with a pretreatment time of 5 hours, and (D) is the pretreatment time. indicates the mass spectrum of the sample at 24 hours. Many fragmentation peaks were observed when the heat treatment time was 3 to 5 hours, but the fragmentation peaks disappeared after treatment for 24 hours. For feather samples, heat treatment was found to be optimal for about 3 hours. In addition, compared with the chicken (Prismalok) heat-treated specimen shown in FIG. 11 (A), the pattern shown by the chicken (Braverder) specimen subjected to heat treatment for 3 hours shown in FIG. 12 (B) was different. . From this, it was shown that the obtained mass spectral patterns of feathers derived from different breeds differ even if the feathers are of the same biological species, and that the method of the present invention can also be used to identify the breed of feathers. .

<Example 4 Analysis of leather>
Glove straps were used as bovine leather, and bag pieces were used as water buffalo leather. All samples were washed with a 70% ethanol solution three times for 5 minutes and dried in order to remove adhering coloring agents and the like. Heat-treated specimens were prepared in the same manner as in Example 2, except that the pretreatment time was changed to 15 minutes and 30 minutes for 1 mg of the bovine leather sample. Further, with respect to 1 mg of the bovine leather sample, a microwave-treated sample was prepared by the same method as in Example 1, except that the pretreatment time was changed to 5 minutes and 10 minutes. Room temperature treated specimens were prepared in the same manner as in Example 1. A heat-treated specimen was prepared in the same manner as in Example 2, except that the pretreatment time was set to 30 minutes for 1 mg of a water buffalo leather sample. A mass spectrum was obtained under the same conditions as in Example 1 for each sample after pretreatment.

FIG. 13 shows the mass spectra of bovine leather treated at room temperature, heated for 15 minutes, and heated for 30 minutes. In FIG. 13, (A) shows the mass spectrum of the specimen treated at room temperature, (B) the specimen heat-treated for 15 minutes, and (C) the specimen heat-treated for 30 minutes. No protein fragmentation was observed in room temperature treated specimens. On the other hand, many fragmentation peaks were observed in the heat-treated sample after 30 minutes of treatment. For leather samples, it was found that fragmentation progresses with short-term heat treatment.

FIG. 14 shows the mass spectra of a 5-minute microwaved sample and a 10-minute microwaved sample of bovine hide. In FIG. 14, (A) shows the mass spectrum of the sample treated with microwaves for 5 minutes, and (B) shows the mass spectrum of the sample treated with microwaves for 10 minutes. For leather samples, it was found that fragments disappeared with longer microwave treatment times.

FIG. 15 shows mass spectra of 30-minute heat-treated samples of buffalo leather and cow leather. In FIG. 15, (A) shows the mass spectrum of water buffalo leather, and (B) shows the mass spectrum of cow leather. The obtained mass spectral patterns of bovine and buffalo leathers were significantly different. By performing pretreatment and analysis under the same conditions, it was found that it was possible to distinguish species derived from leather samples.

<Example 5 Analysis of antlers>
A guitar pick was used as a cow horn, a water buffalo button was used as a buffalo horn, and a Yezo deer button was used as a Yezo deer horn. Each sample was filed and used as a powder. A heat-treated sample was prepared in the same manner as in Example 2 for 1 mg of each sample. A mass spectrum was obtained under the same conditions as in Example 1 for each sample after pretreatment.

FIG. 16 shows the mass spectrum of each sample. In FIG. 16, (A) shows the mass spectrum of Yezo deer, (B) Buffalo, and (C) Bovine horn. Multiple fragmentation peaks were observed in all samples. Since the patterns of the mass spectra obtained from each sample were significantly different, it was found that it was possible to distinguish the origin species of the horn sample.

<Example 6 Analysis of spider silk>
Nephila spider dragline and argiope spider egg sac were used as samples. For each sample, heat-treated samples, microwave-treated samples, and room-temperature-treated samples were prepared under the same conditions as in Examples 1 and 2. A mass spectrum was acquired under the same conditions as in Example 1 for the pretreated sample.

FIG. 17 shows the mass spectrum of Nephila spp., and FIG. 18 shows the mass spectrum of Argiope spp. In both FIGS. 17 and 18, (A) shows the mass spectrum of the heat-treated specimen, (B) the microwave-treated specimen, and (C) the room-temperature treated specimen. In the Nephila spider, fragmentation peaks were observed in heat-treated specimens, but no fragmentation was observed in microwave- and room-temperature treated specimens. Fragmentation peaks were observed in all specimens of Argiope. No significant difference was observed in the pattern between the room-temperature-treated specimen and the microwave-treated specimen. Since the patterns of the mass spectra obtained by Nephila and Argiope are significantly different, it is possible to distinguish the origin species of spider silk.

INDUSTRIAL APPLICABILITY The method and system of the present invention can be applied to quality testing of textile products, clothing products, or ornaments using animal materials, and can be used in various industrial fields such as the sericulture industry, the livestock industry, the textile industry, and the apparel industry. Available.

DESCRIPTION OF SYMBOLS 1... Test system 2... Processing part 3... Analysis part 4... Analysis part 41... Data collection part 42... Collation part 43... Database 5... Input part 6... Output part 7... Control part

Claims (13)

A method for identifying the species, breed and/or quality of animal material, comprising:
a) a pretreatment step of contacting a sample made of animal material with a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent, followed by heating;
b) a step of analyzing protein and/or peptide components contained in the sample after pretreatment; and/or quantitatively analyzing; The pretreatment agent is trifluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, difluoroacetic acid, monofluoroacetic acid, propionic acid, butyric acid, hypochlorite, hydrogen peroxide, hydrogen fluoride, bromoacetic acid, ozone, halogen , chlorine dioxide, or manganese dioxide. In step b), the analysis is performed by APCI method, CI method, EI method, ESI method, FAB method, FD method, FI method, LILBID method, LSIMS method, MALDI method, PB method, PD method, SIMS method, TSP method 3. The method according to claim 1 or 2, wherein the method is mass spectrometry using an ionization means selected from the group consisting of or combinations thereof. 4. The method of claim 3, wherein the ionization means is ESI or MALDI. 5. The method of claim 4, wherein in step b) said analysis comprises MALDI-TOF-MS. A method according to any one of claims 1 to 5, wherein in step b) said analysis comprises separation by LC, MPLC, HPLC or capillary electrophoresis. A method according to any one of claims 1 to 6, wherein step c) utilizes analytical results obtained on a control group consisting of a plurality of animal materials of known origin, breed and/or quality. . 8. The method according to claim 7, wherein step c) utilizes a database containing analytical results obtained for animal material of known origin, breed and/or quality. A system for identification of species, breed and/or quality of animal material comprising:
- an analysis unit that analyzes protein components contained in a sample containing animal material heat-treated using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; - An analysis unit that qualitatively and/or quantitatively analyzes the species, breed and/or quality of the animal material based on the analysis results output from the analysis unit. - a processing unit that has heating means and heat-treats a sample containing animal material using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; 10. The system according to Item 9. 11. The system of claim 9 or 10, wherein the analysis portion comprises a mass spectrometer. 12. The system of claim 11, wherein said mass spectrometer is a MALDI-TOF-MS mass spectrometer. The analysis unit is provided with a database, and the database is a database in which analysis results obtained for a control group composed of a plurality of animal materials having known origin species, breeds and/or qualities are registered in advance. The system according to any one of items 9-12.

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