Method and system for testing the species, variety and / or quality of animal material

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

[Technical field]

[0001] The present invention relates to a method and system for testing animal material for species origin, breed and quality. [Background technology]

[0002] Animal materials, especially silk fiber, are generally expensive, but it has been virtually impossible to identify which variety of silkworm the silk used as material came from and what kind of refining method was used to make the fabric after it was made. For example, the price of cocoons varies greatly depending on the place of origin and variety, and domestic "Koishimaru" cocoons are currently said to be about 10 times more expensive than foreign cocoons. However, even if the place of origin of the silk thread actually produced differs from the place of origin of the silk thread indicated on the product, it was difficult to distinguish them from the appearance. Also, even if silkworm silk thread was mixed with valuable wild silk thread (Yamamayu, Eri San, Shinju San, Muga San, Saku San, Ten San, Tasar San, Cricula, etc.), there was no way to reliably determine the presence or absence of contamination or the mixture ratio from the state of the product. There was also no way to clearly distinguish old silk thread from silk thread produced within the year. Furthermore, when restoring ancient silk textiles, there is a desire to use silk thread of the same variety if possible, but it has been difficult to identify the origin species of the silk thread of ancient silk textiles.

[0003] Identifying the variety of silkworm cocoons is possible to some extent from the appearance such as shape and color when the cocoon is still in the state, but it is difficult to be definitive. In particular, such identification from appearance is extremely difficult for anyone other than a skilled craftsman. If the pupa remains inside the cocoon, it is expected that identification can be done by genome analysis of the pupa, but when there is no pupa, especially when it has become refined silk thread, identification is virtually impossible.

[0004] Non-Patent Documents 1 and 2 disclose a method for identifying the species of origin of silk thread by treating the silk thread with trypsin, and then analyzing it with LC / MS / MS to perform proteome analysis of the protein components contained in the silk thread. Non-Patent Document 3 discloses various physical property tests on silk thread derived from domesticated silkworms into which unnatural amino acids have been introduced. It discloses that silk thread was dissolved in a lithium bromide solution, and the supernatant was treated with urea, SDS-PAGE, and treated with trypsin to obtain peptide fragments, which were analyzed by MALDI-TOF-MS to confirm the appearance of peaks not present in the wild type.

[0005] 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 variety of origin, and therefore need to be identified. For example, Non-Patent Document 4 discloses a method for identifying cashmere and other animal hair fibers using MALDI-TOF-MS after trypsin treatment. Also, Non-Patent Document 5 discloses a method for identifying and quantifying feathers and animal hair using MALDI-TOF-MS. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Gu et al. (2019) Identification of silks from Bombyx mori, eri silkworm and chestnut silkworm using western blot and proteomics analyses. Anal. Sci. 35, 175-180 [Non-Patent Document 2] Gu et al. (2019) Species identification of Bombyx mori and Antheraea pernyi silk via immunology and proteomics. Scientific reports 9:9 [Non-Patent Document 3] Teramoto et al. (2015), Physical properties of silkworm silk introduced with a non-natural amino acid (4-chlorophenylalanine), J. Silk Sci. Tech. Jpn. 23, 27-35 [Non-Patent Document 4] Ohhashi et al. (2014), Identification of cashmere and animal hairs in textile products using MALDI-TOF mass spectrometry, SENI GAKKAISHI, Vol. 70, No. 5, 105-108 [Non-Patent Document 5] 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 Summary of the Invention [Problem to be solved by the invention]

[0007] The methods described in Non-Patent Documents 1 and 2 are capable of identifying the species of silkworm from which silk thread is derived, but are not capable of identifying the silkworm variety. The method described in Non-Patent Document 3 is capable of identifying wild-type silkworms from specific genetically modified silkworms, but is not capable of identifying the natural species or variety of origin. None of the methods are capable of identifying the year of silk production. Furthermore, all of these methods require several hours to several days to perform trypsin treatment, and there are problems in that the reagents and procedures used are costly and time-consuming. The methods for identifying animal hair and feathers described in Non-Patent Documents 4 and 5 also require trypsin treatment, and similarly require costly and time-consuming reagents and procedures.

[0008] An object of the present invention is to provide a method and test system that can easily identify the species, variety and quality of animal materials in a short period of time. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors have conducted extensive research and found that it is possible to identify the species, variety and quality of a sample by contacting a sample containing animal materials with an acidifying agent, an oxidizing agent or a mixture of these, heating the sample and analyzing the protein and / or peptide components in the sample after the treatment. The present invention is based on this novel finding and specifically provides the following inventions. (1) A method for identifying the species, variety, and / or quality of animal material, comprising the steps of: a) a pretreatment step of contacting a sample made of an 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 the sample; b) analyzing the protein and / or peptide components contained in the pretreated sample; and c) qualitatively and / or quantitatively analyzing the species, variety and / or quality of the animal material based on the results of step b). (2) The method according to (1), wherein the pretreatment agent contains at least one selected from the group consisting of 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, halogens, chlorine dioxide, and manganese dioxide. (3) The method according to (1) or (2), wherein in step b), the analysis is mass spectrometry using an ionization means selected from the group consisting of APCI, CI, EI, ESI, FAB, FD, FI, LILBID, LSIMS, MALDI, PB, PD, SIMS, TSP, or a combination thereof. (4) The method according to (3), wherein the ionization means is an ESI method or a MALDI method. (5) The method of (4), wherein in step b), the analysis comprises MALDI-TOF-MS. (6) The method according to any one of (1) to (5), wherein in step b), the analysis comprises separation by LC, MPLC, HPLC, or capillary electrophoresis. (7) Any of the methods (1) to (6), wherein step c) utilizes analytical results obtained for a control group consisting of multiple animal materials of known species, varieties and / or qualities. (8) The method according to (7), wherein step c) utilizes a database in which analytical results obtained on animal materials of known species, variety and / or quality are registered. (9) A system for identification of the species, variety, and / or quality of animal materials, comprising: - an analysis unit that analyzes protein components contained in a sample containing an animal material that has been heat-treated using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; and -An analysis unit that qualitatively and / or quantitatively analyzes the species, variety and / or quality of the animal material based on the analysis results output from the analysis unit. (10)-The system of (9), further comprising: a processing unit having a heating means, for heat-treating a sample containing animal material using a pretreatment agent comprising at least one selected from the group consisting of an acidifying agent and an oxidizing agent. (11) The system of (9) or (10), wherein the analysis unit includes a mass spectrometer. (12) The system according to (11), wherein the mass spectrometer is a MALDI-TOF-MS mass spectrometer. (13) Any of the systems of (9) to (12), wherein the analysis unit is equipped with a database, and the database is a database in which analytical results obtained for a control group consisting of multiple animal materials having known species, varieties and / or qualities are registered in advance. Effect of the Invention

[0010] According to the method of the present invention, it is possible to easily identify the species, variety, and quality of an animal material in a short time. Furthermore, by using the system of the present invention, it is possible to easily identify the species, variety, and quality of an animal material in a short time. [Brief description of the drawings]

[0011] [Figure 1] 1 is a flow diagram showing an outline of the method of the present invention. The method of the present invention includes at least a sample preparation step (step a)), a sample pretreatment step (step b)), and an analysis step (step c)) of the obtained results. [Diagram 2] This is a flow chart showing an example of an embodiment in which AI is used in the method of the present invention. In the illustrated example, an AI is used that is constructed using a database that accumulates the analysis results of known samples as training data. The analysis results of the target unknown sample, as well as the pretreatment and analysis conditions of 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. [Diagram 3] Schematic diagram of an example of the system of the present invention. (A) shows an outline of the first embodiment of the system of the present invention. In the first embodiment of the system of the present invention, the test system 1 comprises an analysis unit 3 and an analysis unit 4. In addition, the test system 1 comprises an input unit 5 for inputting sample information, analysis conditions, etc., an output unit 6 for outputting analysis results, and a control unit 7 for controlling each device in the test system 1. After a 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 embodiment of the system of the present invention, the test system 1 comprises a processing unit 2, an analysis unit 3, and an analysis unit 4. In addition, the test system 1 comprises an input unit 5 for inputting sample information, analysis conditions, etc., an output unit 6 for outputting analysis results, and a control unit 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 unit 2 and pretreatment is performed. The processing section is equipped with a robot arm, a reagent dispensing mechanism, etc., and is capable of automatically performing pre-processing of the sample and transferring the processed specimen to the analysis section 3. [Figure 4] Mass spectra of boiled cocoon samples A to C that were pretreated at room temperature are shown. [Diagram 5] Mass spectra of boiled cocoon samples A to C, which were irradiated with microwaves during pretreatment, are shown. [Figure 6] Mass spectra of fibers obtained from alpaca wool are shown. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 7] Mass spectra of fibers obtained from angora wool are shown. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 8] Mass spectra of fibers obtained from cashmere yarn are shown. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 9] Mass spectra of fibers obtained from sheep's wool are shown. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 10] Mass spectra of fibers obtained from quail feathers are shown. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 11] Mass spectra of fibers obtained from chicken (Plymouth Rock) feathers are shown. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 12] This shows the change in mass spectrum of fibers obtained from chicken (Blabada) feathers depending on the heat treatment time. (A) shows the mass spectrum of a sample pretreated for 1 hour, (B) shows the mass spectrum of a sample pretreated for 3 hours, (C) shows the mass spectrum of a sample pretreated for 5 hours, and (D) shows the mass spectrum of a sample pretreated for 24 hours. [Figure 13] The mass spectra of cowhide 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 sample treated at room temperature, (B) shows the mass spectrum of the sample treated for 15 minutes, and (C) shows the mass spectrum of the sample treated for 30 minutes. [Figure 14] The mass spectra of cowhide samples treated with microwaves for 5 minutes and 10 minutes are shown. (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. [Figure 15] Mass spectra of buffalo leather and cow leather samples heat-treated for 30 minutes are shown. (A) shows the mass spectrum of buffalo leather, and (B) shows the mass spectrum of cow leather. [Figure 16] Mass spectra of samples from various antler samples are shown. (A) shows the mass spectrum of a heat-treated sample from a Hokkaido deer antler, (B) from a water buffalo antler, and (C) from a cow antler. [Figure 17] Mass spectra of samples derived from spider silk from the Nephila orbicularia spider. (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. [Figure 18] Mass spectra of samples derived from spider silk from the orb-weaver spider (A) shows the mass spectrum of a heat-treated sample, (B) of a microwave-treated sample, and (C) of a room temperature-treated sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <Methods for identifying the species, variety and / or quality of animal ingredients> 1. Overview The method of the present invention is a method for identifying the species, variety and / or quality of animal material, comprising the steps of: The method includes: a) a pretreatment step of contacting a sample 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 the sample; b) a step of analyzing protein and / or peptide components contained in the pretreated sample; and c) a step of qualitatively and / or quantitatively analyzing the species, variety and / or quality of the animal material based on the results of step b).

[0013] Proteins are the main components of animal materials, particularly animal-derived fibers and leather. Fibroin, the main component of silk fibers, and keratin, the main component of animal hair and leather, have robust molecular structures, and it has been difficult to prepare them in a state suitable for mass spectrometry, electrophoresis, etc., by conventional treatments such as SDS treatment. However, it has been found that by contacting these samples with a solution containing an acidifier and / or an oxidizer and subjecting them to a heat treatment, at least a portion of the proteins contained in the samples is broken down into smaller molecules, that is, the proteins are fragmented.

[0014] It was found that the fragmentation pattern of the protein differs depending on the quality of the sample, such as the species of origin, variety, and age. As a result, it was found that the species of origin, variety, and quality can be identified by acidifying and / or oxidizing a sample under heating conditions under specified conditions and analyzing the resulting protein fragments (proteins and / or peptides). Hereinafter, the proteins and / or peptides to be analyzed are collectively referred to simply as "proteins."

[0015] In the present invention, "analysis" refers to obtaining output data obtained directly from a sample, so-called raw data, and "analysis" refers to obtaining output information calculated based on the output data.

[0016] A flow diagram of each step in the method of the present invention is shown in Figure 1. The method of the present invention includes at least a sample preparation step, a sample pretreatment step (step a)), an analysis step (step b)), and an analysis step of the obtained results (step c). Each of these steps is described in detail below. Note that the method of the present invention may include other steps in addition to each of these steps.

[0017] 2. Explanation of each process 2-1 Sample preparation The "sample" to be tested in the present invention is an "animal material." The term "animal" here specifically refers to metazoans in general, including sponges, arthropods, and chordates. "Animal material" specifically includes animal-derived fibers (e.g., silk, spider silk, animal hair, feathers, etc.), leather, horns, and teeth. It is a material that is primarily used as a material for clothing and accessories, but is not limited to these uses.

[0018] The animal material used in the present invention includes silk derived from silkworms. The term "silk" as used herein includes all fibers derived from silkworms, particularly including threads spun by silkworms, and includes cocoons and silk threads obtained from cocoons or parts thereof. In this specification, the term "cocoon" particularly refers to a pupal chamber made of fibers to protect the pupa of silkworms, and is often formed by adhesion, entanglement, etc., of adhesive components (sericin proteins, etc.) of fiber components (fibroin proteins) contained in silk. The cocoon as a sample may be a raw cocoon or a heat-treated cocoon, a part of the cocoon cut out as a test piece, or a fiber taken out from the cocoon. In this specification, the term "silk thread" refers to fibers spun by silk threads that have been subjected to various processes, and includes raw silk, degummed silk, and dyed versions of these (including those dyed after being woven). The silk thread sample may be fibers before being made into twisted yarn, twisted yarn, or fibers that have been untwisted again.

[0019] In the present specification, the term "silkworm" refers to a general term for insects that have silk glands and can spin silk threads. Specifically, it refers to species of Lepidoptera, Hymenoptera, Nerionoptera, Trichoptera, Lepidoptera, Diptera, and Termitidae that can spin silk threads mainly in the larval stage for nesting, cocooning, or movement among Lepidoptera. Among Lepidoptera, Bombycidae, Saturniidae, Brahmaeidae, Eupterotidae, Lasiocampidae, Psychidae, Archtiidae, Noctuidae, and the like that can spin large amounts of silk threads are preferred as silkworms in the present specification. Suitable examples include species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically, silkworms, mulberry silkworms (Bombyx mandarina), Samia cynthia (including Samia cynthia ricini and hybrids of Samia cynthia and Samia ricini), Antheraea yamamai, Antheraea pernyi, Saturnia japonica, and Actias gnoma.

[0020] A "silk gland" is a modified tubular organ of a salivary gland that has the function of producing, accumulating, and secreting liquid silk. In silkworms, silk glands are present in pairs on the left and right sides of the silkworm, mainly along the digestive tract of the larva, and each silk gland is composed of three regions: anterior, middle, and posterior silk gland. As mentioned above, the posterior silk gland produces and secretes fibroin, a fiber component of silk. The middle silk gland also produces and secretes sericin, a coating component, and accumulates in its lumen together with fibroin transferred from the posterior silk gland.

[0021] Silk degumming methods vary depending on the type of cocoon used as material, the purpose of use, etc., but it is usually degummed by the following method. Cocoons (raw cocoons) produced by silkworms, etc. are dried with hot air at 60 to 120°C for about 6 hours and stored in a dried state (heat-treated cocoons). The heat-treated cocoons are boiled in water at 60 to 98°C for about 20 minutes to make the fibers easier to loosen (boiled cocoons), cooled with water, the thread ends are hooked with a brush, etc., and reeled out as silk threads using a silk-reeling machine. 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, the main component of silk fibers, and has a relatively hard texture. Degummed thread is produced by degumming the raw silk or the cloth woven from it with sodium carbonate solution or Marcel soap. The former degummed yarn refined with sodium carbonate solution (hereinafter referred to as "sodium carbonate degummed silk") contains sericin and has a hard texture, while the latter degummed yarn refined with Marseille soap (hereinafter referred to as "Marseille soap degummed silk") has the sericin removed and has a soft texture. The method of the present invention can be used to test any of the above raw cocoons, heat-treated cocoons, raw silk, sodium carbonate degummed silk, and Marseille soap degummed silk. Table 1 shows the main components typically contained in raw cocoons, heat-treated cocoons, sodium carbonate degummed silk, and Marseille soap degummed silk.

[0022] [Table 1]

[0023] The animal material used in the present invention includes animal fibers other than silk. Examples of animal fibers other than silk include animal hair, feathers, spider silk, etc. Keratin accounts for the majority of the protein contained in animal hair. The origin of the animal hair is not particularly limited, but any of the animal hairs derived from sheep, cashmere goats, Angora goats, rabbits, camels, alpacas, vicunas, guanacos, llamas, musk oxen (kiviak), possums, minks, chinchillas, etc., which are generally distributed as materials for clothing and decorative items, can be used. The animal hair used as a clothing material is generally used after being cut from an animal and processed into thread through a washing process, a combing process, a dyeing process, a spinning process, etc. The animal hair used as a sample may be at any stage of each of the above processing steps, for example, animal hair before washing, wool after spinning, or a part of the fiber of a woven or knitted product.

[0024] The majority of the protein in feathers is keratin. There is no particular limit to the species from which the feathers are derived, but feathers from geese and ducks, which are commonly used as materials for clothing and bedding, can be used.

[0025] The animal material used in the present invention includes leather. The majority of the protein contained in leather is collagen. The origin of leather is not particularly limited, but any of leathers derived from cattle, sheep, pigs, goats, horses, deer, ostriches, kangaroos, snakes, lizards, crocodiles, etc., which are generally distributed as materials for clothing and ornaments, can be used. The leather used in the present invention may be the skin itself (generally called "skin") that covers the surface of an animal's body, or it may be tanned (generally called "leather"). The "tanning" here refers to a processing process in which animal fats and oils or plant components are applied to the skin or smoked to remove defects such as rotting or hardening of the skin. The leather sample may have undergone any processing process.

[0026] The animal materials used in the present invention include horns and teeth. The main component of horns and teeth is calcium, but they also contain small amounts of protein, mainly consisting of collagen. The origin of horns is not particularly limited, but any of horns derived from deer, buffalo, cow, etc., which are mainly used as materials for clothing and ornaments, can be used. The origin of teeth is not particularly limited, but any of teeth derived from elephants (ivory), mammoths, hippopotamuses, whales, etc., which are mainly used as materials for ornaments, can be used.

[0027] 2-2 a) Pretreatment process In the method of the present invention, "pretreatment" refers to heat treatment of a sample in contact with a "pretreatment agent" to make the sample analyzable in the identification of the sample animal material. The "pretreatment agent" is a solution containing an acidifying agent and / or an oxidizing agent. The solvent constituting the pretreatment agent can dissolve fragmented peptides and the like and has a boiling point of 70°C or higher, particularly 85°C or higher. More specifically, water can be used. The pretreatment agent may be used in one liquid, or two or more liquids may be used simultaneously or successively.

[0028] In this specification, the term "acidifying agent" refers to an agent for placing a sample under an acidic condition, that is, a pH of less than 7.0, particularly a pH of 5.0 or less, and furthermore a pH of 1.0 to 3.0, and the type of the agent is not particularly limited as long as the agent can acidify the sample. Preferably, trifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid, formic acid, acetic acid, nitric acid, hydrochloric acid, propionic acid, butyric acid, hydrogen fluoride, bromoacetic acid, etc., or a combination thereof can be used. In addition, a plurality of these acids can be used consecutively. When used as a pretreatment agent, the acidifying agent is preferably used at a concentration of 0.01 to 10.0N (normal), particularly 0.1 to 2.0N, and furthermore 0.2 to 1.0N.

[0029] In the present specification, the "oxidizing agent" is not particularly limited as long as it is an agent capable of oxidizing a sample, particularly a protein. Preferably, hydrogen peroxide, ozone, hypochlorite, a halogen element, chlorine dioxide, manganese dioxide, or a combination thereof can be used. When hydrogen peroxide is used as the oxidizing agent, it is preferably used at a concentration of 0.01 to 10.0 M, particularly 0.1 to 2.0 M, and further preferably 0.2 to 1.0 M.

[0030] The means for contacting the sample with the pretreatment agent is not particularly limited as long as the pretreatment agent is in contact with the sample evenly, and can be, for example, immersion, coating, spraying, solid-phase-gas-phase contact with a vaporized substance, etc. When the sample is heat-treated via 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 the sample from drying due to evaporation of the solvent.

[0031] The method of the present invention requires heating the pretreatment agent in contact with the sample. The heating temperature is not particularly limited, but when the sample is analyzed in the liquid phase, the temperature is preferably increased to 60 to 100°C, particularly 70 to 98°C, and further 85 to 95°C. Examples of the method for performing such heating include a method in which the pretreatment agent in which the sample is immersed is sealed in a container and heated with a Peltier element, a hot water bath, an oil bath, or the like, or a method in which the sample is irradiated with microwaves. Alternatively, when the sample is analyzed in the gas phase, the temperature of the sample can be, for example, 100 to 120°C.

[0032] The heating time may be 30 minutes to 24 hours, particularly 1 hour to 3 hours, when heating is performed via a pretreatment agent, or 1 minute to 15 minutes, particularly 3 minutes to 7 minutes, when heating is performed by microwave irradiation.

[0033] For silk, it was confirmed that proteins such as fibroin can be fragmented by contacting the sample with a pretreatment agent at room temperature, making it possible to analyze the samples by mass spectrometry. Furthermore, it was found that applying heat during pretreatment of silk samples causes the proteins to be more fragmented, resulting in more diverse mass spectrum patterns. It was also found that proteins in some animal materials other than silk were not sufficiently fragmented by pretreatment at room temperature, but that applying heat during pretreatment promoted protein fragmentation, making it possible to analyze the samples by mass spectrometry, etc.

[0034] The pretreated sample may be used in a state in which fibers or fiber aggregates remain, or the supernatant obtained by subjecting the pretreated agent after the reaction to centrifugation, filtration, etc. When mass spectrometry is performed in the subsequent analysis step, the amount of the sample used is preferably about 0.01 to 100 mg, particularly 0.05 to 5.0 mg, since this is a system suitable for trace analysis.

[0035] Pretreatment conditions such as the 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 has been found that the signals obtained in the analysis process described below differ depending on the pretreatment conditions. By referring to known results, etc., conditions that make the target test easier can be selected. For example, if the purpose of the test is to identify the variety, pretreatment conditions that increase the difference in the signals obtained between varieties can be selected. In addition, the same sample can be pretreated under multiple different conditions and analyzed and compared.

[0036] Before or after the treatment of the sample with the acidifying agent and / or the oxidizing agent, the sample may be treated with a reducing agent as necessary. As the reducing agent, any reducing agent usually used in protein analysis, such as dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl(phosphine) hydrochloride (TCEP), tributylphosphine, cysteine ​​hydrochloride, hydroxysulfite, etc., can be used.

[0037] It is preferable not to use an enzyme reaction in the pretreatment step in the method of the present invention. Conventionally, in silk analysis, protein fragmentation has been performed using proteases such as trypsin. Although the use of enzymes is effective in fragmenting proteins, there are problems in that the reagents used are expensive compared to oxidizing / reducing agents, and the reaction takes a long time.

[0038] 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 heat, without using enzyme treatment.

[0039] 2-3 b) Analysis process The sample treated in the pretreatment step is subjected to the analysis step. In the analysis step, the proteins fragmented in the pretreatment step are analyzed. Any known means such as electrophoresis, amino acid sequencing, liquid chromatography (LC), mass spectrometry, amino acid composition analysis, X-ray diffraction, nuclear magnetic resonance spectroscopy (NMR), and Fourier transform infrared spectroscopy (FTIR) can be used as a means of analyzing proteins, but mass spectrometry, which can easily analyze the characteristics of polymeric substances, is particularly suitable. Mass spectrometry is a method in which a trace amount of a compound is ionized in a high vacuum chamber, and the generated ions are separated and detected by mass.

[0040] Examples of methods for ionizing compounds include 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 beam ion desorption (LILBID), liquid secondary ion mass spectrometry (LSIMS), matrix assisted laser desorption ionization (MALDI), particle beam (PB), plasma desorption (PD), and secondary ion mass spectrometry (SIM). Examples of ionization methods include SIMS (simulation spectrometry, SIMS) or thermospray (TSP), or a combination of these ionization methods. In particular, the MALDI method or ESI method, which are widely used for the analysis of polymers, especially proteins, can be preferably used. Since it has been found that the mass spectrometry spectrum (hereinafter referred to as "mass spectrum") of silk protein after pretreatment produces different patterns depending on the ionization means, it is also possible to select conditions that are easier to achieve the test purpose by referring to the analysis results of known samples. For example, if the purpose of the test is to identify the variety, an ionization means that will result in a large difference between the varieties in the mass spectrum pattern obtained can be selected. In addition, the same pretreated sample may be analyzed in parallel using multiple ionization means to obtain the mass spectrum of each.

[0041] Any known method can be used to separate the ionized molecules based on their mass, but time of flight mass spectrometry (TOF-MS), which is particularly suitable for separating polymers, is preferably used. MALDI-TOF-MS is preferably used for the analysis of protein components in the present invention.

[0042] When MALDI-TOF-MS is used as the analytical method, the pretreated sample may be subjected to analysis together with the pretreatment agent or the supernatant may be placed in a cuvette or the like. Alternatively, the fibers remaining in the pretreatment agent may be attached to a conductive double-sided carbon tape and dried before analysis. The conductive double-sided carbon tape that can be used is usually one available for use with scanning electron microscopes (SEM) (for example, manufactured by Nissin EM Co., Ltd.).

[0043] Prior to or instead of mass spectrometry, separation may be performed by liquid chromatography (LC), middle pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), capillary electrophoresis, or the like.

[0044] 2-4 c) Analysis process b) A step of qualitatively and / or quantitatively analyzing the species, variety and / or quality of the sample based on the results (raw data) obtained in the analysis step. Preferably, the analysis step is carried out using the results obtained by performing an analysis step under the same conditions on a control group consisting of a plurality of animal materials having known species, variety and / or quality.

[0045] It has been found that the data obtained in the analysis process for a sample containing pretreated silk, particularly the mass spectrum pattern, varies depending on at least the parameters derived from the sample and the parameters derived from the pretreatment and analysis conditions shown in Table 2. Therefore, for example, a database useful for analyzing unknown samples can be constructed by acquiring a large number of mass spectra under various pretreatment and analysis conditions for a control group consisting of a large number of silk threads or cocoons whose origin species, variety, degumming conditions, production year, etc. are known, and accumulating the obtained spectral patterns as fingerprints of each control. Alternatively, the database may be constructed by acquiring mass spectra for a large number of controls under predetermined pretreatment and analysis conditions. However, when using this database, the pretreatment and analysis processes of the unknown sample may be limited to the predetermined pretreatment and analysis conditions. The mass spectrum data accumulated in the database may be image data of the mass spectrum or a set of numerical data of the m / z and signal intensity of each detected peak. Note that the parameters for constructing the database are not limited to those listed in Table 2.

[0046] [Table 2]

[0047] In the method of the present invention, the analysis step can be performed by comparing the analysis results, particularly the mass spectrum, 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 compared. In addition, the analysis result of the sample to be compared does not have to be one from one unknown sample, and for example, multiple analysis results, such as multiple mass spectra, may be obtained by changing the pretreatment and analysis conditions, and these may be compared in a composite manner with the database.

[0048] Alternatively, in the method of the present invention, the analysis step can be performed by comparing the mass spectrum of the target sample with that of a standard analyzed at approximately the same time (e.g., on the same day) under the same conditions. The standard used can be appropriately selected according to the purpose of identification. For example, when identifying the year of manufacture of the animal material of the sample, a single or multiple animal materials of the same type manufactured in different years can be used as standards, and the mass spectrum of the sample can be compared with the mass spectrum of each standard.

[0049] The matching may be performed manually, but is preferably performed using a computer. The matching is performed, for example, by inputting data such as the analysis results of the target sample, extracting data in the database that is closest to the input data, and outputting the extracted data and its background information (such as the origin species, variety, and year of manufacture of the sample). The matching may also be performed using artificial intelligence (AI). When using AI, a trained model can be constructed through machine learning using a known neural network using the database as training data, and desired information such as the origin species, variety, and year of manufacture of the sample can be output from data input from an unknown sample using this. FIG. 2 shows a flow diagram of an example of an embodiment in which AI is used in the method of the present invention. In the example shown in FIG. 2, an AI constructed using a database in which the analysis results of known samples are accumulated as training data is used. The analysis results of the target unknown sample, as well as the pretreatment and analysis conditions of 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 embodiment in which AI is used is not limited to the example shown in FIG. 2.

[0050] 2-5 Other processes Depending on the type of target sample and the desired information, other steps may be added in addition to the above steps. For example, when testing a dyed product or a product containing naturally occurring impurities, the analysis results, particularly the mass spectrum, may show signals derived from the dye or impurities, which may be difficult to distinguish from signals derived from the target fragmented protein. When using such a sample, it is preferable to add a decolorization step and / or a washing step before the pretreatment step. Specific means for the decolorization step and the washing step include, for example, a means of immersing the sample in an organic solvent such as acetonitrile, acetone, ethanol, or methanol or an aqueous solution thereof, a means of immersing in an alkaline solution such as aqueous ammonia or a sodium hydroxide solution, a means of washing with a surfactant such as SDS, CTAB, or Marcel soap, a means of washing with a bleaching agent such as thiourea dioxide, and a means of immersing in a reducing agent solution such as hydroxysulfite. The above means may be used alone or in combination. In addition, as long as there is no influence of side reactions, for example, they may be mixed appropriately and used, for example, by mixing an organic solvent with a surfactant.

[0051] The analysis step and analytical step in the method of the present invention do not necessarily have to be performed on only the protein and / or peptide components of the sample, and for example, naturally occurring impurity components may be separately sampled in the washing step and the results of analyzing the sample may be analyzed together. Furthermore, the analysis results are not limited to those of the sample using the pretreatment agent, and for example, the analysis results of the same sample treated separately using a reducing agent or a basifying agent may be analyzed together.

[0052] 3. Examples of application of test methods Examples of identification of animal materials to which the method of the present invention can be applied are given below, but the method of the present invention is not limited to the following examples and can be applied to a variety of identifications.

[0053] 3-1 Identification of silk 3-1-1 Identification of differences in the manufacturing and refining methods of silk thread and cocoons In the method of the present invention, different patterns can be obtained, particularly in mass spectra, for a section obtained from one raw cocoon, heat-treated silk obtained by heat-treating the section, and further refined with sodium carbonate and Marseille soap, respectively, for a sodium carbonate-refined silk sample, and a Marseille soap-refined silk sample. Therefore, in identifying silk threads, the analysis results of the target silk thread sample can be collated with the analysis results of raw silk, sodium carbonate-refined silk, and Marseille soap-refined silk obtained in advance from the same type of cocoon under the same conditions, thereby making it possible to objectively identify the refining methods of each thread, which have been sensuously distinguished by touch, texture, etc., until now. In addition, in identifying cocoons, the analysis results of the target cocoon sample can be collated with the analysis results of raw cocoons and heat-treated cocoons obtained in advance from the same type of cocoon under the same conditions, thereby making it possible to objectively identify the cocoons.

[0054] 3-1-2 Identification of the species of origin In the method of the present invention, different patterns, particularly mass spectra, can be obtained depending on the species of silk origin. Therefore, the species of origin of the sample can be identified by comparing the analysis results of the target sample with a database that holds the analysis results of each species of silk under the same conditions as fingerprints. In addition, when the sample is silk thread, even if fibers obtained from multiple species are mixed, the types and mixture ratios can be determined.

[0055] 3-1-3 Variety Identification In the method of the present invention, different patterns, particularly mass spectra, can be obtained depending on the silk variety. Therefore, the variety of the sample can be identified by comparing the analysis results of the target sample with a database that holds the analysis results of each variety of silk under the same conditions as fingerprints. Furthermore, even if fibers obtained from multiple varieties are mixed in a silk thread, the varieties and the mixture ratio can be determined. This makes it possible to objectively determine the authenticity of silk products that claim to be expensive varieties such as Koishimaru.

[0056] 3-1-4 Quality Identification In the method of the present invention, different patterns are obtained, particularly in the mass spectrum, depending on the quality of the silk, particularly the time since production. More specifically, a peak shift is observed in the mass spectrum of silk threads or cocoons that have been produced for a long time, in which each peak shifts to the high molecular weight side, compared to new cocoons. Silk fibers are prone to oxidation over time. This peak shift is presumed to be due to oxidation of proteins. Therefore, the production year of a sample can be identified by comparing the analysis results of the target sample with a database that contains analysis results under the same conditions for the same type of silk thread or cocoon produced in each production year. Since oxidation of fibers affects the quality of the silk, such as texture and strength, identifying the production year of silk is useful for estimating its quality.

[0057] 3-2 Identification of animal hair In the method of the present invention, different patterns, particularly mass spectrum, can be obtained depending on the origin and variety of the animal hair. Therefore, the analysis result of the target sample can be compared with a database that holds the analysis results of each animal hair under the same conditions as fingerprints, thereby identifying the origin and variety of the sample. In addition, even if fibers obtained from multiple species are mixed, the types and mixture ratios can be determined. This makes it possible to objectively determine the authenticity of animal hair products that claim to be expensive varieties such as cashmere.

[0058] The method of the present invention can also determine the quality of animal hair, particularly the time since production. Specifically, the production year of a sample can be identified by comparing the analysis results of the target sample with a database of analysis results of the same type of animal hair produced in the same year under the same conditions.

[0059] 3-3 Identification of leather In the method of the present invention, different patterns, particularly mass spectra, can be obtained depending on the species and variety of leather. Therefore, the species of origin of the sample can be identified by comparing the analysis results of the target sample with a database that contains the analysis results of leather of each species under the same conditions as fingerprints. This makes it possible to objectively determine the authenticity of leather products that claim to be of expensive varieties, such as crocodile leather.

[0060] The method of the present invention can also determine the quality of leather, particularly the time since its manufacture. Specifically, a database of analysis results, under the same conditions, for the same type of leather produced in each production year is stored in advance, and the analysis results of the target sample are collated to identify the production year of the sample.

[0061] 3-4 Identification of horns or teeth In the method of the present invention, different patterns, particularly mass spectra, can be obtained depending on the species and variety of the horn or tooth. Therefore, the species of origin of the sample can be identified by comparing the analysis results of the target sample with a database that holds the analysis results of the horns or teeth of each species under the same conditions as fingerprints. This makes it possible to objectively determine the authenticity of rare varieties such as ivory.

[0062] <System for identification of species, variety and / or quality of animal ingredients> The system of the present invention is a system for testing animal material, comprising: - an analysis unit that analyzes protein components contained in a sample containing an animal material that has been heat-treated using a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; and -An analysis unit that qualitatively and / or quantitatively analyzes the species, variety and / or quality of the animal material based on the analysis results output from the analysis unit.

[0063] Unless otherwise specified, the system of the present invention is a system for carrying out the method of the present invention and has features in common with those described in the above section <Method for identifying the species, variety and / or quality of animal material>.

[0064] The system of the present invention includes, in addition to the analysis unit and the analysis unit, - a processing section having a heating means, which 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;

[0065] An outline of the system of the present invention is illustrated in FIG. 3. (A) shows a first embodiment of the system of the present invention. In the first embodiment of the system of the present invention, the test system 1 comprises an analysis unit 3 and an analysis unit 4. It also comprises an input unit 5 for inputting sample information, analysis conditions, etc., an output unit 6 for outputting the analysis results, and a control unit 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 a pretreated sample S1 that has been heat-treated while in contact with a "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 analyzes the analysis results.

[0066] FIG. 3B shows a second embodiment of the system of the present invention. In the second embodiment of the system of the present invention, the test system 1 includes a processing unit 2, an analysis unit 3, and an analysis unit 4. The test system 1 also includes an input unit 5 for inputting sample information and analysis conditions, an output unit 6 for outputting analysis results, and a control unit 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 unit 2 and pretreatment is performed. The processing unit 2 includes a heating means for performing heat treatment of the sample. The processing unit 2 may also include a robot arm, a reagent dispensing mechanism, etc., in which case it is possible to automatically perform pretreatment of the sample and transfer the treated specimen to the analysis unit 3.

[0067] The following configuration is common to the first embodiment (FIG. 3(A)) and the second embodiment (FIG. 3(B)). The analysis unit 3 is equipped with a device for analyzing the protein components of the pretreated sample S1. Any device may be used as long as it is capable of analyzing the protein components of the pretreated sample, and any known means such as an electrophoresis tank, an amino acid sequencer, a liquid chromatography (LC) device, a mass spectrometer, etc. may be used, but a mass spectrometer that can easily analyze the characteristics of polymeric substances is preferably used. In particular, a MALDI-TOF-MS mass spectrometer, which is widely used for analyzing polymers, especially proteins, is preferably used. The analysis unit may be equipped with multiple types of analysis devices.

[0068] The analysis unit 4 qualitatively and / or quantitatively analyzes the origin species, variety, 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, a collation unit 42, and a database 43 that collect input information from the input unit 5 and analysis results from the analysis unit 3. The database 43 is a database in which analysis results obtained for a control group consisting of a plurality of animal materials having known origin species, variety, and / or quality are registered in advance, and the collation unit 42 includes software that compares the data collected in the data collection unit 41 with the data in the database 43. As the software, for example, MALDI biotyper (manufactured by Bruker Daltonics) can be used as an existing software. For example, the collation unit compares the data collected from the analysis unit with the database, extracts the closest data in the database, and outputs the extracted data and its background information (such as the origin species, variety, refining state, and production year of the sample). The collation unit 42 may include AI. The AI ​​referred to here can suitably use a trained model that has been trained using machine learning with the database as training data, and by inputting collected data into the AI, it outputs qualitative and / or quantitative information regarding the species, variety and / or quality of the sample.

[0069] Alternatively, the analysis unit 4 may not use the database 43, but may directly use mass spectral data of a standard analyzed under the same conditions as the target sample at approximately the same time (e.g., on the same day), and perform comparison in the comparison unit 42 with the data collected in the data collection unit 41.

[0070] The system of the present invention may be any system that includes an analysis unit and an analysis section and is applicable to carrying out the above-described method of the present invention, and should not be construed as being limited to the above-mentioned first and second aspects. EXAMPLES

[0071] Example 1: Analysis of boiled cocoons 1. Silk degumming Boiled and reeled silk was prepared from cocoons (samples A to C) of three different genetic strains of silkworms using the following procedure. 120 mg of fiber pieces cut from raw cocoons were placed in a 50 mL beaker and dried at 120°C for 10 minutes, then at 60°C for 6 hours to prepare heat-treated silk. The pieces were then placed in water at 98-100°C, heated for 20 minutes, and then dried.

[0072] 2. Sample pretreatment 100 μL of 2.5% formic acid solution was placed in a sealable 1.5 mL polypropylene tube, and 1 mg of each of samples A to C was added to it. The tube was sealed and floated on 100 to 200 mL of water filled in a plastic container, and then placed in a microwave oven set to 750 W and irradiated with microwaves for 5 minutes (microwave-treated sample).

[0073] In addition to the samples that had been pretreated as described above, each sample was pretreated at room temperature as follows. 100 μL of 2.5% formic acid solution was placed in a sealable 1.5 mL polypropylene tube, and 1 mg of each of samples A to C was added to it. The tube was sealed and shaken for 5 minutes to mix (room temperature treated samples).

[0074] 3. Analysis by MALDI-TOF-MS The solution after each treatment was quenched, and 1 μL was dropped onto conductive carbon double-sided tape (manufactured by Nissin EM Co., Ltd.), which was then set on the sample stage of a MALDI-TOF-MS mass spectrometer (Autoflex III or Microflex, Bruker Daltonics) and dried. Mass spectra were obtained from m / z 1,000 to 20,000 in linear positive mode.

[0075] The mass spectra of each sample obtained are shown in Figures 4 and 5. Figure 4 shows the mass spectra of samples A to C that were pretreated at room temperature. Figure 5 shows the mass spectra of samples A to C that were pretreated with microwave irradiation. It was found that for boiled cocoons, even with pretreatment at room temperature, different mass spectrum patterns were obtained for each sample, making it possible to distinguish between them. It was found that by irradiating microwaves, many peaks were observed in the mass spectrum of each sample, making it possible to distinguish in detail. It was also found that by combining the data from room temperature treatment and the data from microwave irradiation, more detailed distinctions were possible.

[0076] Example 2: Analysis of animal hair The following pretreatment was carried out on fiber samples from alpaca, angora, cashmere, and sheep wool. 100 μL of 2.5% formic acid solution was placed in a sealable 1.5 mL polypropylene tube, and 1 mg of each sample was added to it. The tube was sealed, placed in a heat block, and heated at 90°C for 3 hours (heat-treated sample).

[0077] In addition to the above pretreatment, each specimen was subjected to a pretreatment by microwave irradiation and a pretreatment 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 each pretreatment.

[0078] The mass spectra of each sample obtained are shown in Figures 6 to 9. Figure 6 shows the mass spectrum of alpaca, Figure 7 shows the mass spectrum of angora, Figure 8 shows the mass spectrum of cashmere, and Figure 9 shows the mass spectrum of sheep. In all of Figures 6 to 9, (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a room temperature treated sample. In all samples, protein fragmentation was hardly observed in the room temperature treated sample, whereas many fragmentation peaks were observed in the heat-treated sample. In addition, many fragmentation peaks were observed in the microwave-treated samples of alpaca, cashmere, and sheep. Fragmentation peaks were also observed, although in small amounts, in the microwave-treated sample of angora. From these results, it was found that the origin of animal hair can be identified by pretreatment involving heat treatment or microwave treatment.

[0079] Example 3: Feather analysis For 1 mg each of quail and chicken (Plymouth Rock) feather samples, heat-treated samples, microwave-treated samples, and room temperature-treated samples were prepared under the same conditions as in Examples 1 and 2. Separately, chicken (Blabber) feather samples were prepared by changing the pretreatment time to 1 hour, 3 hours, 5 hours, and 24 hours. Mass spectra were obtained for each pretreated sample under the same conditions as in Example 1.

[0080] Figure 10 shows the mass spectra for quail and Figure 11 shows the mass spectra for chicken (Plymouth Rock) under various pretreatment conditions. In both Figures 10 and 11, (A) shows the mass spectrum of a heat-treated sample, (B) shows the mass spectrum of a microwave-treated sample, and (C) shows the mass spectrum of a sample treated at room temperature. In the case of quail, protein fragmentation was observed even at room temperature. On the other hand, in the case of chicken, protein fragmentation was hardly observed in the room temperature-treated sample, but many fragmentation peaks were observed in the heat-treated and microwave-treated samples.

[0081] FIG. 12 shows the change in mass spectrum depending on the heat treatment time of chicken (Blabada). In FIG. 12, (A) shows the mass spectrum of a sample pretreated for 1 hour, (B) shows the mass spectrum of a sample pretreated for 3 hours, (C) shows the mass spectrum of a sample pretreated for 5 hours, and (D) shows the mass spectrum of a sample pretreated for 24 hours. Many fragmentation peaks were observed when the heat treatment time was 3 to 5 hours, but the fragmentation peaks disappeared after 24 hours of treatment. It was found that about 3 hours of heat treatment is optimal for feather samples. In addition, the pattern shown by the chicken (Blabada) sample heat-treated for 3 hours shown in FIG. 12(B) was different from that shown by the chicken (Prismaroc) heat-treated sample shown in FIG. 11(A). This shows that even if the feathers are from the same species, the mass spectrum patterns obtained are different if they are from different breeds, and the method of the present invention can also be used to distinguish the breed of feathers.

[0082] Example 4: Analysis of leather As samples, glove strings were used as cowhide leather, and pieces of bag were used as buffalo leather. In order to remove coloring agents and the like adhering to each sample, each sample was washed with 70% ethanol solution for 5 minutes three times and then dried. Heat-treated samples were prepared for 1 mg of cowhide leather sample in the same manner as in Example 2, except that the pretreatment time was 15 minutes and 30 minutes. In addition, microwave-treated samples were prepared for 1 mg of cowhide leather sample in the same manner as in Example 1, except that the pretreatment time was 5 minutes and 10 minutes. Room temperature-treated samples were prepared in the same manner as in Example 1. Heat-treated samples were prepared for 1 mg of buffalo leather sample in the same manner as in Example 2, except that the pretreatment time was 30 minutes. Mass spectra were obtained for each sample after pretreatment under the same conditions as in Example 1.

[0083] Figure 13 shows the mass spectra of cow leather samples treated at room temperature, heat treated for 15 minutes, and heat treated for 30 minutes. In Figure 13, (A) shows the mass spectrum of the sample treated at room temperature, (B) shows the mass spectrum of the sample treated for 15 minutes, and (C) shows the mass spectrum of the sample treated for 30 minutes. No protein fragmentation was observed in the sample treated at room temperature. On the other hand, many fragmentation peaks were observed in the heat treated sample after 30 minutes of treatment. It was found that fragmentation of leather samples progresses with short heat treatments.

[0084] Figure 14 shows the mass spectra of cowhide samples treated with microwaves for 5 minutes and 10 minutes. In Figure 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. It was found that for leather samples, fragments disappeared as the microwave treatment time increased.

[0085] Figure 15 shows the mass spectra of buffalo leather and cow leather samples heat-treated for 30 minutes. In Figure 15, (A) shows the mass spectrum of buffalo leather, and (B) shows the mass spectrum of cow leather. The mass spectrum patterns obtained for cow leather and buffalo leather were significantly different. It was found that it is possible to distinguish the species originating from leather samples by performing pretreatment and analysis under the same conditions.

[0086] Example 5: Analysis of horns A guitar pick was used as a cow's antler, a buffalo button as a water buffalo's antler, and a Yezo deer button as a Yezo deer's antler. Each sample was filed and used in powder form. A heat-treated sample was prepared from 1 mg of each sample in the same manner as in Example 2. Mass spectra were obtained for each pretreated sample under the same conditions as in Example 1.

[0087] Figure 16 shows the mass spectrum of each sample. In Figure 16, (A) shows the mass spectrum of the antlers of a Hokkaido deer, (B) shows the mass spectrum of the antlers of a water buffalo, and (C) shows the mass spectrum of a cow's antler. Multiple fragmentation peaks were observed in each sample. Since the mass spectrum patterns obtained from each sample differed greatly, it was found that it was possible to distinguish the species from which the antler samples originated.

[0088] Example 6: Analysis of spider silk The dragline of the Nephila orbiganii spider and the egg capsule of the Orbigan spider 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. Mass spectra were obtained for the pre-treated samples under the same conditions as in Example 1.

[0089] Figure 17 shows the mass spectrum of the Nephila orb-weaver spider samples, and Figure 18 shows the mass spectrum of the Orb-weaver spider samples. In both Figures 17 and 18, (A) shows the mass spectrum of the heat-treated sample, (B) the microwave-treated sample, and (C) the room temperature treated sample. For Nephila orb-weaver spider samples, fragmentation peaks were observed in the heat-treated sample, but no fragmentation was observed in the microwave-treated and room temperature treated samples. For Orb-weaver spider samples, fragmentation peaks were observed in all samples. No significant difference was observed in the patterns between the room temperature and microwave-treated samples. The large difference in the mass spectrum patterns obtained for Nephila orb-weaver spider and Orb-weaver spider spider demonstrated that it is possible to distinguish the species of origin of spider silk. [Industrial Applicability]

[0090] The method and system of the present invention can be applied to quality testing of textile products, clothing products, or decorative items made using animal materials, and can be used in a variety of industrial fields such as sericulture, livestock farming, textile industry, and apparel industry. [Explanation of symbols]

[0091] 1. Test system 2. Processing section 3…Analysis Department 4…Analysis department 41…Data collection section 42…Collation section 43…Database 5. Input section 6. Output section 7...Control section

Claims

1. 1. A method for identifying the species, variety and / or quality of animal material, comprising the steps of: a) a pretreatment step of heating a sample made of an animal material in contact with a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; b) performing mass spectrometry of protein and / or peptide components contained in the pretreated sample; and c) qualitatively and / or quantitatively analyzing the species, variety and / or quality of the animal material based on the results of step b); The method does not include enzyme treatment.

2. 2. The method of claim 1, wherein the pretreatment agent comprises at least one selected from the group consisting of 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, halogens, chlorine dioxide, or manganese dioxide.

3. The method according to claim 1 or 2, wherein in step b), the analysis comprises MALDI-TOF-MS.

4. 4. The method according to claim 1, wherein step c) utilizes analytical results obtained on a control group of animal materials of known species, variety and / or quality.

5. 5. The method according to claim 4, wherein step c) utilizes a database containing analytical results obtained on animal material of known species, variety and / or quality.

6. 1. A system for identification of the species, variety and / or quality of animal material, comprising: a processing section having a heating means, which heat-treats a sample containing animal material in contact with a pretreatment agent containing at least one selected from the group consisting of an acidifying agent and an oxidizing agent; - an analysis unit including a mass spectrometer for analyzing protein and / or peptide components contained in the sample pretreated by the processing unit; and An analysis unit that qualitatively and / or quantitatively analyzes the species, variety and / or quality of the animal material based on the analysis results output from the analysis unit.

7. The system of claim 6, wherein the mass spectrometer is a MALDI-TOF-MS mass spectrometer.

8. The system according to claim 6 or 7, wherein the analysis unit is provided with a database, the database being a database in which analytical results obtained for a control group consisting of a plurality of animal materials having known species, varieties and / or qualities are registered in advance.

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