JP7628288B2 - Method and system for testing the species, variety and/or quality of animal material - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies [Publication 1] Issue date: November 6, 2020 Publication: Electrophoresis, Vol. 64, Supplement, Abstracts of the 71st General Meeting of the Japanese Society for Electrophoresis [Publication 2] Date held: November 12-13, 2020 Meeting name: 71st General Meeting of the Japanese Society for Electrophoresis Meeting location: Online [Publication 3] Issue date: March 17, 2021 Publication: Abstracts of the 2021 Academic Lecture on the Functional Utilization of Silkworm Yarn and Insects (91st Annual Meeting of the Japanese Society of Sericulture) [Publication 4] Date held: March 19-20, 2021 (Participants' slides can be viewed from March 17-26) Meeting name: 2021 Academic Lecture on the Functional Utilization of Silkworm Yarn and Insects (91st Annual Meeting of the Japanese Society of Sericulture) Meeting location: Online

The present invention relates to a method and system for testing animal materials for species, variety, and quality.

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. Furthermore, 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 between old silk thread and 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 of the silk thread of ancient silk textiles.

Identifying the variety of silkworm cocoons is possible to some extent from the appearance such as shape and color while still in the cocoon 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 genomic analysis of the pupa, but when there is no pupa, especially when the silk has become refined silk thread, identification is virtually impossible.

Non-Patent Documents 1 and 2 disclose a method of treating silk thread with trypsin, then analyzing it with LC/MS/MS to perform proteome analysis of the protein components contained in the silk thread and identify the species from which the silk thread originated. 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, subjected to SDS-PAGE, and treated with trypsin. The peptide fragments obtained were analyzed by MALDI-TOF-MS, and the appearance of peaks not present in the wild type was 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 variety of origin, and therefore need to be identified. For example, Non-Patent Document 4 discloses a method of identifying cashmere and other animal hair fibers using MALDI-TOF-MS after trypsin treatment. In addition, Non-Patent Document 5 discloses a method of identifying and quantifying feathers and animal hair using MALDI-TOF-MS.

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 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), Physical properties of silkworm silk introduced with a non-natural amino acid (4-chlorophenylalanine), J. Silk Sci. Tech. Jpn. 23, 27-35 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 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

The methods shown in Non-Patent Documents 1 and 2 are capable of identifying the species of silkworm from which silk thread is derived, but do not identify the silkworm variety. The method shown in Non-Patent Document 3 is capable of distinguishing between wild-type and specific genetically modified organisms, but does not identify the natural species or variety of origin. And neither method is capable of identifying the year of silk production. Furthermore, all of these methods have the problem that trypsin treatment takes several hours to several days, and 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.

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

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 thereof, heating the sample and analyzing the protein and/or peptide components in the treated sample. 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 a plurality of animal materials having 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 identifying the species, variety and/or quality of animal material, comprising:
- an analysis unit that performs analysis of 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 according to (9), further comprising: 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.
(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 according to (9) to (12), wherein the analysis unit is provided with a database, and the database is a database in which the analysis results obtained for a control group consisting of a plurality of animal materials having known species, varieties and/or qualities are registered in advance.

The method of the present invention makes it possible to easily identify the species of origin, variety, and quality of an animal material in a short time. In addition, by using the system of the present invention, it makes it possible to easily identify the species of origin, variety, and quality of an animal material in a short time.

1 is a flow diagram showing an outline of the method of the present invention. The method of the present invention comprises 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)). 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 constructed using a database that accumulates the analysis results of known samples 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. 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 includes a robot arm, a reagent dispensing mechanism, etc., and is capable of automatically pre-treating the sample and transferring the treated specimen to the analysis section 3. Mass spectra of boiled cocoon samples A to C that were pretreated at room temperature are shown. Mass spectra of boiled cocoon samples A to C that were irradiated with microwaves during pretreatment are shown. 1 shows the mass spectra of fibers obtained from alpaca wool, (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. 1 shows the mass spectra of fibers obtained from angora wool, (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. 1 shows the mass spectra of fibers obtained from cashmere yarn, (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. 1 shows the mass spectra of fibers obtained from sheep's wool, (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. 1 shows the mass spectra of fibers obtained from quail feathers, (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. 1 shows the mass spectra of fibers obtained from chicken (Plymouth Rock) feathers, (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. 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. The mass spectra of a cowhide sample treated at room temperature, heated for 15 minutes, and heated for 30 minutes are shown in Fig. 1. (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. 1 shows the mass spectra of a cowhide sample treated with microwaves for 5 minutes and 10 minutes, where (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. The 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. The mass spectra of specimens derived from various antler samples are shown. (A) shows the mass spectrum of a heat-treated specimen of a Yezo deer antler, (B) shows the mass spectrum of a water buffalo antler, and (C) shows the mass spectrum of a cow antler. 1 shows the mass spectra of samples derived from spider silk from the spider Nephila orbiculatus. (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. 1 shows the mass spectra of samples derived from spider silk from the orb-weaver spider, where (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.

Methods for identifying the species, variety and/or quality of animal material
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).

The main components of animal materials, particularly animal-derived fibers and leather, are proteins. 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 make them suitable for mass spectrometry, electrophoresis, and the like, using 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 some of the proteins contained in the samples are broken down into smaller molecules, that is, the proteins are fragmented.

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. This has led to the discovery that by acidifying and/or oxidizing a sample under heating conditions under specified conditions and analyzing the resulting protein fragments (proteins and/or peptides), it is possible to identify the species of origin, variety, and quality. Hereinafter, the proteins and/or peptides to be analyzed are collectively referred to simply as "proteins."

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

Figure 1 shows a flow diagram of each step in the method of the present invention. 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 these steps.

2. Explanation of Each Step 2-1 Sample Preparation The "sample" to be tested in the present invention is an "animal material." Here, "animal" 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 mainly used as a material for clothing and accessories, but is not limited to these uses.

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 those spun by silkworms, and includes cocoons and silk threads obtained from cocoons or parts thereof. In this specification, "cocoon" refers in particular to a pupal chamber made of fibers to protect the pupa of silkworms, and is often formed by adhesion, entanglement, etc., using adhesive components (sericin proteins, etc.) of the 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, "silk thread" refers to fibers spun by silk threads that have been subjected to various processing, 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 they are twisted into yarn, twisted yarn, or fibers that have been untwisted again.

In this specification, "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 other families that can spin large amounts of silk threads are preferred as silkworms in this specification. Suitable examples include species belonging to the genera Bombyx, Samia, Antheraea, Saturnia, Attacus, and Rhodinia, specifically, 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.

A "silk gland" is a tubular organ that is a modified salivary gland that has the function of producing, accumulating, and secreting liquid silk. In silkworms, silk glands exist 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: the 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 that has migrated from the posterior silk gland.

Silk is usually refined by the following method, although it varies depending on the type of cocoon used and its purpose, etc. Cocoons produced by silkworms (raw cocoons) are dried with hot air at 60-120°C for about 6 hours and stored in a dried state (heat-treated cocoons). The heat-treated cocoons are boiled in water at 60-98°C for about 20 minutes to make the fibers easier to loosen (boiled cocoons), then cooled in water, the thread ends are hooked with a brush, etc., and silk threads are reeled out using a silk-reeling machine. The silk threads obtained in this way are 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 threads are produced by scouring 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.

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. There is no particular limitation on the species of origin of the animal hair, but any of the following animals that are commonly distributed as materials for clothing and decorative items, such as sheep, cashmere goat, Angora goat, rabbit, camel, alpaca, vicuna, guanaco, llama, musk ox (kiviak), possum, mink, chinchilla, etc., can be used. The animal hair used as a clothing material is generally used after being shorn from the 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.

The majority of the protein in feathers is keratin. There are no particular restrictions on the species from which the feathers come, but feathers from geese and ducks, which are commonly used as materials for clothing and bedding, can be used.

The animal material used in the present invention includes leather. The majority of the protein contained in leather is collagen. There is no particular limitation on the species of origin of leather, but any of the leathers derived from cattle, sheep, pigs, goats, horses, deer, ostriches, kangaroos, snakes, lizards, crocodiles, etc., which are commonly used as materials for clothing and decorative items, can be used. The leather used in the present invention may be the skin that covers the surface of an animal's body (generally called "skin"), or it may be leather that has been tanned (generally called "leather"). "Tanning" here refers to a processing process in which animal fats and oils or plant ingredients are applied to the leather or smoked to remove defects that cause the leather to rot or harden. The leather sample may have undergone any processing process.

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. There are no particular limitations on the species of origin of the horns, but any of the horns derived from deer, buffalo, cow, etc., which are mainly used as materials for clothing and ornaments, can be used. There are no particular limitations on the species of origin of the teeth, but any of the teeth derived from elephants (ivory), mammoths, hippopotamuses, whales, etc., which are mainly used as materials for ornaments, can be used.

2-2 a) Pretreatment Step In the method of the present invention, "pretreatment" refers to heat treatment of a sample in contact with a "pretreatment agent" in order to make the sample analyzable in the identification of the animal material sample. The "pretreatment agent" is a solution containing an acidifying agent and/or an oxidizing agent. As the solvent constituting the pretreatment agent, one that can dissolve fragmented peptides and the like and has 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 in one liquid, or two or more liquids may be used simultaneously or successively.

In this specification, the term "acidifying agent" refers to an agent for placing a sample under an acidic condition, i.e., 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. The type of the agent is not particularly limited as long as it 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 of these, can be used. Also, 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 10.0 N (normal), particularly 0.1 to 2.0 N, and furthermore 0.2 to 1.0 N.

In this specification, the term "oxidizing agent" refers to any agent capable of oxidizing a sample, particularly proteins, and is not particularly limited in type. Preferably, hydrogen peroxide, ozone, hypochlorite, halogen alone, chlorine dioxide, manganese dioxide, etc., or a combination of these can be used. When using hydrogen peroxide water as the oxidizing agent, it is preferable to use it 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.

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, but for example, means such as immersion, coating, spraying, solid-phase-gas-phase contact with a vaporized substance, etc. can be used. When heat-treating the sample by heating the pretreatment agent, it is preferable to seal the pretreatment agent in which the sample has been immersed in a container in order to prevent the sample from drying out due to evaporation of the solvent.

The method of the present invention requires that the pretreatment agent be heated while in contact with the sample. There are no particular limitations on the heating temperature, but when the sample is analyzed in the liquid phase, it is preferable to raise the temperature to 60 to 100°C, particularly 70 to 98°C, and even more preferably 85 to 95°C. Examples of methods for such heating include sealing the pretreatment agent in which the sample is immersed in it in a container and heating it with a Peltier element, hot water bath, oil bath, etc., or irradiating the sample 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.

When heating via a pretreatment agent, the heating time can be 30 minutes to 24 hours, particularly 1 hour to 3 hours. When heating via microwave irradiation, the heating time can be 1 minute to 15 minutes, particularly 3 minutes to 7 minutes.

It was confirmed that for silk, proteins such as fibroin can be fragmented by contacting the sample with a pretreatment agent at room temperature, enabling analysis by mass spectrometry. Furthermore, it was found that applying heat during pretreatment of silk samples causes the proteins to be further 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 analysis by mass spectrometry, etc. possible.

The sample after pretreatment may be used with fibers or fiber clumps remaining, or the supernatant obtained by subjecting the pretreatment agent after reaction to centrifugation, filtration, etc. may be used. In addition, when mass spectrometry is performed in the subsequent analysis step, it is preferable to use about 0.01 to 100 mg of sample, especially 0.05 to 5.0 mg, since this is a system suitable for microanalysis.

Pretreatment conditions such as the components of the pretreatment agent, reaction time, and temperature can be selected appropriately 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 it easier to perform the desired test can be selected. For example, if the purpose of the test is to identify the variety, pretreatment conditions that will 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 then analyzed and compared.

Before or after the treatment of the sample with the acidifying agent and/or oxidizing agent, the sample may be treated with a reducing agent as necessary. The reducing agent may be any of the reducing agents normally used in protein analysis, such as dithiothreitol, 2-mercaptoethanol, tris(2-carboxyethyl(phosphine) hydrochloride (TCEP), tributylphosphine, cysteine hydrochloride, and hydroxysulfite.

In the pretreatment step of the method of the present invention, it is preferable not to use an enzymatic reaction. Conventionally, in the analysis of silk, 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.

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 Step The sample treated in the pretreatment step is subjected to the analysis step. In the analysis step, the protein fragmented in the pretreatment step is analyzed. Any known means such as electrophoresis, amino acid sequence, 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.

Methods 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 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 (SIMMS). Examples of ionization methods include SIMS (simulation ionization mass spectrometry) or TSP (thermospray), or a combination of these ionization methods. In particular, the MALDI or ESI methods, 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 method that produces 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.

Any known method can be used to separate 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.

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 conductive double-sided carbon tape and dried before analysis. The conductive double-sided carbon tape that can be used is usually one that is available for use with scanning electron microscopes (SEM) (for example, manufactured by Nissin EM Co., Ltd.).

Separation may be performed prior to or in place of mass spectrometry using liquid chromatography (LC), middle pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), capillary electrophoresis, etc.

2-4 c) Analysis step: This is 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 b). Preferably, the analysis step is carried out using the results obtained by carrying out an analysis step under the same conditions for a control group consisting of a plurality of animal materials having known species, variety and/or quality.

It has been found that the data obtained in the analysis process for samples 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, variety, degumming conditions, production year, etc. are known, and accumulating the obtained spectral patterns as fingerprints for each control. Alternatively, the database may be constructed by acquiring mass spectra for a large number of controls under specified pretreatment and analysis conditions. However, when using this database, the pretreatment and analysis processes for unknown samples may be limited to the specified pretreatment and analysis conditions. The mass spectrum data accumulated in the database may be image data of the mass spectrum or a collection of numerical data on 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.

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 unknown sample of interest with the constructed database. If necessary, the pretreatment and analysis conditions used to obtain the analysis results may also be compared. Furthermore, the analysis result of the sample to be compared does not have to be one from one unknown sample; 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.

Alternatively, in the method of the present invention, the analysis step can be performed by comparing the mass spectrum of a standard analyzed at approximately the same time (e.g., on the same day) under the same conditions as the target sample. 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.

The matching may be performed manually, but is preferably performed using a computer. For example, the matching is performed 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 that accumulates the analysis results of known samples 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.

2-5 Other Steps 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, signals derived from dyes or impurities may appear in the analysis results, particularly in the mass spectrum, making it difficult to distinguish them 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 of these, 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.

The analysis and characterization steps 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. For example, naturally occurring impurity components may be separately sampled in the washing step and the results of the analysis may be analyzed together. In addition, the analysis results are not limited to those of the sample using the pretreatment agent, and may also be analyzed together with the results of the analysis of the same sample treated separately using a reducing agent or a basifying agent.

3. Examples of Application of the Test Method Below, examples of identification of animal materials to which the method of the present invention can be applied are listed, but the method of the present invention is not limited to the following examples and can be applied to a variety of identifications.

3-1 Identification of Silk 3-1-1 Identification of Differences in the Manufacturing and Degumming Methods of Silk Yarn 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, to obtain a sodium carbonate-degummed silk and a Marseille soap-degummed silk sample. Therefore, in identifying silk yarn, the analysis results of the target silk yarn sample can be collated with the analysis results of raw silk, sodium carbonate-degummed silk, and Marseille soap-degummed silk obtained in advance from the same type of cocoon under the same conditions, thereby making it possible to objectively identify the degumming methods of each thread, which have been distinguished sensorily 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.

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 origin of silk. 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 silk of each species of origin 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 the mixture ratios can be determined.

3-1-3 Identification of variety In the method of the present invention, different patterns, particularly mass spectra, can be obtained depending on the variety of silk. 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.

3-1-4 Identification of quality 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.

3-2 Identification of Animal Hair In the method of the present invention, different patterns, particularly mass spectra, can be obtained depending on the origin and variety of the animal hair. Therefore, the origin and 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 animal hair of each origin under the same conditions as fingerprints. 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.

The method of the present invention can also determine the quality of animal hair, particularly the time that has passed since it was produced. Specifically, 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 animal hair produced in each production year.

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

The method of the present invention can also determine the quality of leather, particularly the time that has passed since its manufacture. Specifically, a database of analysis results, conducted under the same conditions for the same type of leather from each manufacturing year, is stored in advance, and the analysis results of the target sample are compared to the database, allowing the manufacturing year of the sample to be identified.

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 horns or teeth. 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 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.

<System for identifying the 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 performs analysis of 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.

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>.

The system of the present invention includes, in addition to the analysis unit and the analysis unit,
A processing section may further comprise a processing means having a heating means for 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 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 and analysis conditions, 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.

(B) of FIG. 3 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 in 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 pass the treated specimen to the analysis unit 3.

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

The analysis unit 4 qualitatively and/or quantitatively analyzes the species of origin, 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 aggregate the input information from the input unit 5 and the analysis results from the analysis unit 3. The database 43 is a database in which the analysis results obtained for a control group consisting of multiple animal materials having known species of origin, 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. The collation unit, for example, 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 species of origin, variety, refining state, and year of manufacture of the sample). The collation unit 42 may be equipped with 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 about the species of origin, variety, and/or quality of the sample.

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

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-mentioned method of the present invention, and should not be interpreted as being limited to the above-mentioned first and second aspects.

Example 1: Analysis of boiled cocoons
1. Silk degumming For cocoons (samples A to C) of three different genetic strains of silkworms, boiled and reeled silk was prepared as follows. 120 mg of fiber pieces cut from raw cocoons were placed in a 50 mL beaker and heated and dried at 120°C for 10 minutes and 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.

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).

Separate from 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 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).

3. Analysis by MALDI-TOF-MS After each treatment, the solution 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.

The mass spectra obtained for each sample 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. For boiled cocoons, even with pretreatment at room temperature, different mass spectrum patterns were obtained for each sample, and it was found that these could be distinguished. By irradiating with microwaves, many peaks were observed in the mass spectrum of each sample, making detailed distinction possible. It was also found that by combining the data from room temperature treatment and the data from microwave irradiation, more detailed distinction was possible.

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 the solution. The tube was sealed, placed in a heat block, and heated at 90° C. for 3 hours (heat-treated sample).

In addition to the above pretreatment, each sample was pretreated by microwave irradiation and room temperature pretreatment in the same manner as in Example 1. Mass spectra were obtained for the samples after each pretreatment under the same conditions as in Example 1.

The mass spectra obtained for each sample 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. Furthermore, many fragmentation peaks were observed in the microwave-treated samples of alpaca, cashmere, and sheep. Fragmentation peaks were also observed, although in trace amounts, in the microwave-treated sample of angora. These results demonstrate that pretreatment involving heat or microwave treatment makes it possible to identify the species of origin of animal hair.

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 to prepare heat-treated samples. Mass spectra were obtained for each pretreated sample under the same conditions as in Example 1.

Figure 10 shows the mass spectrum for quail, and Figure 11 shows the mass spectrum for chicken (Plymouth Rock) under various pretreatment conditions. In both Figures 10 and 11, (A) shows the mass spectrum for a heat-treated sample, (B) for a microwave-treated sample, and (C) for a sample treated at room temperature. In quail, protein fragmentation was observed even at room temperature. On the other hand, in chicken, almost no protein fragmentation was observed in the room temperature-treated sample, but many fragmentation peaks were observed in the heat-treated and microwave-treated samples.

Figure 12 shows the change in mass spectrum depending on the heat treatment time of chicken (Blabada). In Figure 12, (A) shows the mass spectrum of a sample with a pretreatment time of 1 hour, (B) shows the mass spectrum of a sample with a pretreatment time of 3 hours, (C) shows the mass spectrum of a sample with a pretreatment time of 5 hours, and (D) shows the mass spectrum of a sample with a pretreatment time of 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 the optimal heat treatment time for feather samples was about 3 hours. In addition, the pattern shown by the chicken (Blabada) sample that was heat treated for 3 hours shown in Figure 12 (B) was different from that shown in Figure 11 (A) of the heat-treated chicken (Prismaroc). This shows that even if the feathers are from the same species, the mass spectrum patterns obtained will be different if they are from different breeds, and that the method of the present invention can also be used to distinguish the breed of feathers.

Example 4: Analysis of leather
As samples, glove strings were used as cowhide leather, and pieces of a bag were used as buffalo leather. In order to remove any adhering coloring agent, 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. 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.

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 room temperature treated sample. On the other hand, many fragmentation peaks were observed in the heat treated sample after 30 minutes of treatment. It was found that fragmentation progresses with short heat treatment of leather samples.

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 a sample treated with microwaves for 5 minutes, and (B) shows the mass spectrum of a sample treated with microwaves for 10 minutes. It was found that fragments of leather samples disappeared as the microwave treatment time increased.

Figure 15 shows the mass spectra of buffalo leather and cow leather samples that were 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 by performing pretreatment and analysis under the same conditions, it is possible to distinguish the species originating from the leather sample.

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 from each pretreated sample under the same conditions as in Example 1.

Figure 16 shows the mass spectrum of each sample. In Figure 16, (A) shows the mass spectrum of the antlers of a Yezo deer, (B) shows the mass spectrum of the antlers of a water buffalo, and (C) shows the mass spectrum of the antlers of a cow. Multiple fragmentation peaks were observed in each sample. As the mass spectrum patterns obtained for each sample differed significantly, it was found that it was possible to distinguish the species from which the antler samples originated.

Example 6: Analysis of spider silk
The dragline of the spider Nephila orbicularis and the egg capsule of the orb 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.

Figure 17 shows the mass spectrum of the samples from the Nephila orbiganja spider, and Figure 18 shows the mass spectrum of the samples from the Orbiganja orbiganja spider. In both Figures 17 and 18, (A) shows the mass spectrum of the heat-treated sample, (B) shows the mass spectrum of the microwave-treated sample, and (C) shows the mass spectrum of the room temperature treated sample. For the Nephila orbiganja spider, fragmentation peaks were observed in the heat-treated sample, but no fragmentation was observed in the microwave-treated and room temperature treated samples. For the Orbiganja orbiganja spider, 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 the Nephila orbiganja spider and the Orbiganja orbiganja spider demonstrated that it is possible to distinguish the species of origin of spider silk.

The method and system of the present invention can be applied to quality testing of textile products, clothing products, and decorative items made from animal materials, and can be used in a variety of industrial fields, including sericulture, livestock farming, textiles, and apparel.

Reference Signs List 1 Test system 2 Processing section 3 Analysis section 4 Analysis section 41 Data collection section 42 Collation section 43 Database 5 Input section 6 Output section 7 Control section

Claims (8)

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 . 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, and manganese dioxide. The method according to claim 1 or 2 , wherein in step b), the analysis comprises MALDI-TOF-MS. 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. 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. 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 equipped with a mass spectrometer that analyzes protein and/or peptide components contained in the sample pretreated in 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. The system of claim 6 , wherein the mass spectrometer is a MALDI-TOF-MS mass spectrometer. 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.