JP7080034B2 - How to determine the animal species of collagen-derived material - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Publication date: July 1, 2017 Publications of the Japan Society for the Conservation and Restoration of Cultural Properties 39th Annual Meeting of the Kanazawa Research Presentation (Editor's name: Japan Society for the Conservation and Restoration of Cultural Properties 39th) Announced on page 64 of the convention executive committee)

The present invention relates to a method for determining an animal species of a collagen-derived material.

Glue is mainly composed of collagen extracted from animal skins, bones, tendons, etc., and is an adhesive for lacquerware, Buddhist statues, Buddhist implements, wooden buildings, violins and other musical instruments, and a solvent for ink, ink and other pigments in Oriental paintings. , Used as a binder for paints and painting bases. Used since the days of ancient murals and primitive paintings, identification of glue animal species can elucidate the materials and techniques of the work and help determine appropriate display, preservation and restoration methods. Furthermore, the identification of the animal species of glue can distinguish between the original part and the supplementary part, and in some cases can contribute to the examination of the authenticity problem.

Many approaches by mass spectrometry have been reported as a method for determining the animal species from which glue used in paintings and historical materials is derived. This is a method in which collagen in a sample is cleaved with a protease, the generated peptide fragment is detected by a mass spectrometer, and the determination is made based on the difference between species in the amino acid sequence of collagen. For example, the binder applied on the medieval work “Saint John the Evangelist” (Collection of the Prague National Museum) was analyzed by the peptide mass fingerprinting method using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer. , The use of rabbit glue has been clarified (Non-Patent Document 1). Analysis of the altar of the Catholic Parish Church at the end of the 17th century with a high-resolution Fourier transform ion cyclotron resonance mass spectrometer revealed seven peptides specific to fish collagen in the thin protein layer between the silver foil and the glaze. (Non-Patent Document 2). In addition, J. When the glue used in the triptych of Romano Egyptian (180-200 BC), which is a collection of the Paul Getty Museum, was analyzed by a high-performance liquid chromatograph mass spectrometer, bovine-derived collagen was identified. (Non-Patent Document 3).

Further, there is also a method of efficiently identifying the animal species derived from glue by detecting the animal species-specific peptide derived from collagen with a Fourier transform ion cyclotron resonance mass analyzer (Non-Patent Document 4). When 15 kinds of peptides for cattle, 3 kinds for rabbits, and 3 kinds for butterfly sharks were used and applied to rabbit glue called Doer's adhesive of the Toton-Fres family 100 years ago, 2 kinds Rabbit collagen peptide has been identified. He also said that he was able to identify 13 species-specific bovine collagen peptides using only 50 μg of gold leaf samples from the Church of St. Maximum in the 18th century.

Further, there is a method in which an animal fibrous protein group extracted from a sample is cleaved with a protease to form a peptide fragment, and then the amino acid sequence of this fragment is analyzed to distinguish the animal species contained in the sample (Patent Document 1). ). It is a method of discriminating the animal species of leather, but it is a method of discriminating the animal species of a sample by preferentially using a peptide fragment specifically detected in one specific animal species, and cows according to the priority. Animal species are identified in the order of deer → horse → pig → sheep → goat. On the other hand, there is also a method of specifying an animal species by using 6 kinds of peptides selected in advance (Patent Document 2). Collagen contained in leather is digested with trypsin, and the resulting marker peptide is analyzed to determine the animal species of the leather. By combining multiple peptides, it can be easily identified as one of the animal species. ..

International Publication No. 2016/157250 Japanese Unexamined Patent Publication No. 2016-224007

S. Kuckova et al., "Analysis of organic coloring and binding components in color layer of art works", Anal Bioanal Chem (2005), vol. 382, p275-282 S. Dallongeville et al., "Proteomics applied to the authentication of fish glue: Application to a 17th century artwork sample", Analyst, (2013), vol. 138, p5355-5364 J. Mazurek et al., "characterization of binding media in Egyptian Romano portraits using enzyme-linked immunosorbant assay and mass spectrometry", e-PS, (2014), vol. 11, p76-83 S. Dallongeville et al., "Identification of animal glue species in artworks using proteomics: Application to a 18th century gilt sample", Anal. Chem. (2011), vol. 83, p9431-9437 NC Schellmann, "Animal glues: a review of their key properties relevant to conservation", Studies in Conservation, (2007), vol. 52, p55-66

Glue, which forms part of a cultural property, is a verifier in itself. By collating the data obtained from the glue with the literature materials, it helps to decipher the historical information and is likely to fill in the blanks in the literature materials. In paintings, etc., the glue used for the ground coating layer and the coloring layer may be different, and if the animal species of glue used for each layer can be identified, painting research will be assisted and cultural properties will be protected and restored. It can also be used as information for proper use. Therefore, it is desired to develop a method capable of discriminating animal species using a small amount of sample.

On the other hand, glue is classified into bone glue, skin glue and the like according to the raw material, and further classified into bovine glue, pig glue, rabbit glue, deer glue, fish glue and the like according to the animal species. As the name suggests, rabbit glue and the like should be prepared from rabbit skin, but it is stated that some commercially available products are mixed with glue derived from other animal species (non-patent literature). 5). Non-Patent Document 5 warns that raw material information, pretreatments, additives, etc. by the manufacturer are not always accurate. When a plurality of animal species are mixed, glue cannot be identified from the appearance, and it is desired to identify the animal species in the mixture. However, since the methods described in Patent Document 1 and Patent Document 2 target one kind of leather, they are not suitable for identifying glue when two or more different animal species are mixed. Therefore, it is desired to develop a method capable of discriminating each animal species even when two species of animals coexist.

Further, the difference between the raw material information attached to the product and the product composition is not limited to glue. It is hoped that animal species such as gelatin made from collagen can also be identified.

In view of the above situation, it is an object of the present invention to provide a method for determining an animal species used for a collagen-derived material.

The present inventors can determine the animal species of collagen by examining the trypsin degradation products of collagen of various animals in detail and combining 12 specific peptides contained in the trypsin degradation products. We have found that the use of peptides can identify each animal species when two species of animals coexist, and completed the present invention.

That is, the present invention is a method for discriminating an animal species using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 below as an index for the peptides contained in the trypsin decomposition product of the collagen-derived material.
(1) Using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, the collagen-derived material may be bovine, horse, sheep, pig, rabbit or butterfly shark according to Table 2. (2) Using the four peptides of SEQ ID NOs: 1, 2, 4 and 5 as indicators, the collagen-derived material is sheep, goat, or deer according to Table 3 below. Determine which animal species it is, or
(3) Using the seven peptides of SEQ ID NO: 1, 2, 4, 5, 9, 11 and 12 as indicators, the collagen-derived material is any of bovine, sheep, goat, deer or rabbit according to Table 4 below. Determine if it is an animal species, or (4) use any of the 10 peptides of SEQ ID NO: 1, 2, 4, 5, 6, 8, 9, 11 and 12, and SEQ ID NO: 3 or 7 as indicators. , An animal species of a collagen-derived material, characterized in that it is determined whether the collagen-derived material is an animal species of cattle, horses, sheep, goats, pigs, deer, rabbits or butterfly sharks according to Table 5 below. Provides a method for determining.

Further, the present invention is a method for discriminating an animal species of a peptide contained in a trypsin decomposition product of a collagen-derived material using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 above as an index.
(1) Using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, the collagen-derived material comprises bovine, horse, sheep, pig, rabbit and butterfly shark according to Table 7. Determine which combination of the two species is selected from the group, or (2) use the 12 peptides of SEQ ID NOs: 1 to 12 as indicators, and according to Table 8 below, the animal species of the collagen-derived material is bovine. Determine which combination of the two selected from the group consisting of horses, sheep, pigs, rabbits and sharks, or (3) SEQ ID NOs: 1, 2, 4, 5, 6, 8, 9, 11 and 12 , And 10 kinds of peptides of SEQ ID NO: 3 or 7, and according to Table 9 below, the animal species of the collagen-derived material consists of cattle, horses, sheep, goats, pigs, deer, rabbits and butterfly sharks. Determine which combination of the two species is selected from the group, or (4) Using the 12 peptides of SEQ ID NOs: 1 to 12 as indicators, the animal species of the collagen-derived material is bovine according to Table 10 below. , A method of determining an animal species of a collagen-derived material, characterized in that it determines which combination of two species selected from the group consisting of horses, sheep, goats, pigs, deer, rabbits and butterfly sharks. It is a thing.

Further, the present invention is a method for discriminating an animal species according to Table 6 above, using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 above as an index for peptides contained in a trypsin decomposition product of a collagen-derived material.
(1) Three kinds of collagen-derived materials selected from bovine, horse, sheep, pig, rabbit and butterfly shark using the seven kinds of peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators. Each animal species is determined when the above animal species are included, or (2) 7 kinds of peptides of SEQ ID NO: 3, SEQ ID NO: 5-7, SEQ ID NO: 9-11, and SEQ ID NO: 1-2, SEQ ID NO: 4 , The collagen-derived material includes three or more animal species selected from cattle, horses, sheep, goats, pigs, deer, rabbits and butterfly sharks, using the five peptides of SEQ ID NO: 8 and SEQ ID NO: 12 as indicators. It provides a method for determining an animal species of a collagen-derived material, which comprises determining each animal species in some cases.

Further, in the present invention, when the determined animal species is any combination of two species selected from the group consisting of cows, horses, sheep, pigs, rabbits and butterfly sharks, SEQ ID NO: 3 or SEQ ID NO: 7 is used as butterfly sharks. SEQ ID NO: 5 is a sheep index, SEQ ID NO: 6 is a horse index, SEQ ID NO: 9 is a rabbit index, SEQ ID NO: 10 is a pig index, or SEQ ID NO: 11 is a bovine index. The present invention provides a method for determining an animal species of the collagen-derived material, which comprises specifying the content ratio of bovine, horse, sheep, pig, rabbit, or butterfly shark contained in the collagen-derived material.

Further, the present invention provides a method for determining the animal species of the collagen-derived material, which comprises measuring the presence or absence of the peptide by LC / MS / MS.

The present invention also provides a method for determining an animal species of the collagen-derived material, wherein the collagen-derived material is any one selected from leather, glue, collagen, and gelatin.

The present invention also provides a method for determining the animal species of the collagen-derived material, which is a glue extracted from the cultural property.

According to the method for determining the animal species of collagen-derived material of the present invention, by measuring the presence or absence of 12 specific peptides contained in the trypsin decomposition product of collagen, the animal species of the collagen are cattle, horses, and sheep. , Goat, pig, deer, rabbit, or butterfly shark, and if it is a mixture of two species, which two species it is.

It is a figure which shows the result of LC / MS / MS analysis of the bovine collagen trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the horse collagen trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the porcine collagen trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the sheep collagen trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the rabbit collagen trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the deer glue trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the sturgeon glue trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the goat leather trypsin decomposition product in Example 1. FIG. It is a figure which shows the result of LC / MS / MS analysis of the sample C in Example 2. FIG.

The first aspect of the present invention is a method for discriminating the animal species of a collagen-derived material using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 as an index for the peptide contained in the tryptic decomposition product of the collagen-derived material. Using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, which animal species the collagen-derived material is, bovine, horse, sheep, pig, rabbit, or butterfly shark, is determined. It can be determined. In addition, it is possible to determine whether the collagen-derived material is a sheep, goat, or deer animal species using the four peptides of SEQ ID NOs: 1, 2, 4, and 5 as indicators. In addition, using the seven peptides of SEQ ID NOs: 1, 2, 4, 5, 9, 11 and 12 as indicators, which animal species the collagen-derived material is bovine, sheep, goat, deer or rabbit is determined. It can be determined. Further, using 10 kinds of peptides of SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 11 and 12, the collagen-derived materials are bovine, horse, sheep, goat, pig, deer, and the like. It is possible to determine whether the animal species is a rabbit or a goat. Further, using any 4 or more of all 12 peptides as an index, it is determined whether the collagen-derived material is derived from an animal species such as cattle, horse, sheep, goat, pig, deer, rabbit or butterfly. be able to. Hereinafter, the present invention will be described in detail.

In the present invention, the "collagen-derived material" broadly includes a material containing collagen as a main component, and is a raw raw skin of animal skin, a tanned skin obtained by tanning the raw skin, and a leather product using the tanned skin; an animal. Glue, which is an extract of boiling water from bones, skins, tendons, etc .; collagen, which is the main protein constituting animal bones, skins, tendons, etc .; contains gelatin, which is a heat denatured / decomposed product of collagen.

In the present invention, in order to determine the animal species, a peptide contained in a tryptic decomposition product of a collagen-derived material is used as a sample. Collagen has a triple helix structure of three polypeptide chains as a basic unit, and has a so-called "collagen-like sequence" primary structure represented by-(Gly-amino acid X-amino acid Y) n-. After the peptide chain having the primary structure is translated in vivo, proline and lysine are converted to hydroxyproline and hydroxylysine, respectively, to form the triple helix structure. Collagen types from type I to type XXVIII are known, and the main component is a collagen type that differs depending on the site constituting the living body, but all of them have the above-mentioned primary structure and triple helix structure in common. Collagen contained in leather, glue and the like is known to contain type I as a main component, and by decomposing this into trypsin, it can be used as a measurement sample for the method for determining an animal species of the present invention. The amino acid sequence of collagen is similar between animals, and is therefore often used as a biomaterial with low antigenicity. However, when the tryptic decomposition products of collagen obtained from various animals were examined in detail, when the 12 kinds of peptides constituting the tryptic decomposition products were combined, bovine, horse, sheep, goat, pig, deer, rabbit or butterfly shark It was found that eight kinds of animal species can be determined.

In the present invention, 12 kinds of peptides shown in Table 1 above were selected for use in determining 8 kinds of animal species. The origin of the peptides shown in Table 1 is shown in Table 11. The amino acid sequence shown in SEQ ID NO: 1 is the 12 amino acids at positions 316 to 327 when the triple helix starting amino acid on the N-terminal side of the type I collagen α1 chain of bovine, horse, pig, deer and rabbit is 1. Equivalent to. Similarly, the amino acid sequence shown in SEQ ID NO: 2 corresponds to the 12 amino acids at positions 316 to 327 when the triple helix starting amino acid on the N-terminal side of the type I collagen α1 chain of sheep and goat is the first amino acid. .. This indicates that the amino acid sequence at the same position in the collagen chain is slightly different depending on the animal species, and is a species-specific peptide depending on the animal species. In the present invention, the peptides produced by tryptic digestion of type I collagen of various animals are examined in detail, and Table 1 is a peptide capable of identifying eight kinds of animals such as bovine, horse, sheep, goat, pig, deer, rabbit and butterfly. Twelve kinds of peptides shown in the above were selected.

As shown in Table 11, SEQ ID NO: 3 and SEQ ID NO: 7 are sequences specific to butterfly sharks, SEQ ID NO: 5 is specific to sheep, SEQ ID NO: 6 is specific to horses, and SEQ ID NO: 9 is specific to rabbits. , SEQ ID NO: 10 is pig-specific, and SEQ ID NO: 11 is bovine-specific. Therefore, if any one of the seven species of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 is used as an index, as shown in Table 2 below, the collagen-derived material is bovine depending on the presence or absence thereof. , Horses, pigs, sheep, rabbits, or sharks. In addition, both SEQ ID NO: 3 and SEQ ID NO: 7 are sequences specific to sturgeon. In the present invention, either SEQ ID NO: 3 or SEQ ID NO: 7 may be used, or both SEQ ID NO: 3 and SEQ ID NO: 7 may be used. According to Table 2, when the peptide represented by SEQ ID NO: 3 is detected, it is determined to be "sturgeon", and when the peptide represented by SEQ ID NO: 5 is detected, it is determined to be "sheep". In addition, all 7 types of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 may be used. For example, when the peptide represented by SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 10 is not detected and the peptide represented by SEQ ID NO: 11 is detected, it is determined to be "cattle". As the number of peptides used increases, the possibility of erroneous determination can be reduced, which is preferable.

On the other hand, when the collagen-derived material is leather or the like, it is difficult to judge from the appearance because the structure is similar to that of a sheep and a goat, and it is not easy to make an accurate judgment even by observation with an electron microscope. As shown in Table 11, the peptide shown in SEQ ID NO: 5 is specific to sheep, but Table 11 does not contain a sequence specific to goats or deer. However, four kinds of peptides of SEQ ID NO: 1, 2, 4 and 5 can be used, and whether it is a sheep, a goat or a deer can be determined from the presence or absence thereof according to Table 3 below. For example, when the tryptic decomposition product of the collagen-derived material contains the peptides of SEQ ID NO: 1 and SEQ ID NO: 4 and does not contain the peptides of SEQ ID NO: 2 and SEQ ID NO: 5, the animal species of the collagen-derived material is referred to as "deer". Is determined. According to Table 3, the above four peptides have two differences between sheep and goat, four differences between sheep and deer, and two between goat and deer. There are differences in location. That is, there are two or more differences between any animal species. Therefore, when the animal species is "deer", it is possible to avoid erroneous determination as a sheep or a goat.

Furthermore, when the collagen-derived material is glue, cattle and rabbits are often used as raw materials. The presence or absence of the seven species of SEQ ID NOs: 1, 2, 4, 5, 9, 11 and 12 is evaluated as an index, and if the judgment is made according to Table 4 below, the collagen-derived material has a difference of 2 or more among any animal species. It is possible to determine whether the animal species of the animal is cattle, sheep, goats, deer or rabbits.

In addition, SEQ ID NOs: 1, 2, 4, 5, 6, 8, 9, 11 and 12 are used to determine whether the animal species is cattle, horses, sheep, goats, pigs, deer, rabbits or butterfly sharks. , And 10 species of any one of SEQ ID NO: 3 or 7 are used as an index. Among the peptides of SEQ ID NOs: 1 to 12 shown in Table 1, SEQ ID NO: 10 is a peptide specific to pigs. According to Table 5 below, it is possible to identify an animal species including "pig" with a difference of 2 or more among each animal species without using a pig species-specific peptide as an index. In the present invention, "SEQ ID NO: 3 or 7" may mean either SEQ ID NO: 3 or SEQ ID NO: 7, and both SEQ ID NO: 3 and SEQ ID NO: 7 may be used. Table 5 shows both SEQ ID NO: 3 and SEQ ID NO: 7.

According to the present invention, according to Table 6, using any 4 or more of the 12 peptides of SEQ ID NOs: 1 to 12 as an index, the collagen-derived material is bovine, horse, sheep, goat, pig, deer, rabbit or butterfly shark. It is possible to determine which of the eight animal species is. By using the four peptides of SEQ ID NOs: 1, 2, 4 and 5 as an index as described above, it is possible to identify which animal species is sheep, goat or deer. Similarly, if the five species of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 12 are used as indicators, it is any of cattle, horses, pigs, sheep, deer, rabbits, and sturgeon. Can be specified. When the collagen-derived material is leather or the like and it is difficult to distinguish between "cow" and "horse" from the appearance, the animal species can be determined by using these five peptides as indicators. Further, if the five species of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 12 are used as indicators, which of cow, horse, pig, sheep, goat, deer, and sturgeon is used? Can be identified. When it is difficult to distinguish between "sheep" and "goat" from the appearance of the collagen-derived material, the animal species can be determined by using these five peptides as indicators. The above is an example. Peptides that can specify the animal species to be determined in this way can be selected from Table 6, and the animal species can be determined according to Table 6. In the present invention, the peptide used for identifying the animal species is selected from the 12 types of peptides shown in Table 6, and may be 5 or more, 6 or more, or 7 or more as long as it is 4 or more, and any of them may be used. It is optional to combine the peptides. If all 12 kinds of peptides are used, the animal species can be specified with the difference in the presence or absence of 2 to 6 kinds of peptides among the above 8 kinds of animal species.

In the present invention, since the peptide is used as an index in the determination of the animal species, the treatment can be performed in a short time as compared with the case of using DNA, and the stability is higher than that of DNA, so that the determination accuracy can be maintained high. Since collagen has Gly-amino acid X-amino acid Y as a basic unit and the sequences are similar between animal species, species specificity does not occur when a peptide having a small number of amino acids is selected. However, according to the present invention, by using the above 12 kinds of peptides having 12 to 24 amino acids after trypsin digestion and further combining them, it is possible to determine the animal species with high accuracy.

The second aspect of the present invention is the case where two or more animal species are mixed with the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 above as an index for the peptides contained in the trypsin decomposition product of the collagen-derived material. It is a method of discriminating the animal species of. In the case of leather, a single animal species is targeted, but collagen and glue extracted from animals, gelatin, which is a denatured / decomposed product of collagen, etc., may be mixed. However, according to the present invention, the tryptic decomposition product of the extracted collagen was used as a sample to determine whether two animal species, bovine, horse, sheep, goat, pig, deer, rabbit or butterfly, were mixed. Can be identified. The peptides used for the determination index are the peptides of SEQ ID NOs: 1 to 12 shown in Table 1. As shown in Table 11 above, there are species-specific peptides and peptides that are commonly contained in other animals, but by combining these, each animal can be identified even when two animal species are mixed. can do.

For example, using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, the animal species of the collagen-derived material consists of a group consisting of cattle, horses, sheep, pigs, rabbits, and butterfly sharks. It is possible to determine which of the two selected combinations. As shown in Table 11 above, the seven peptides of SEQ ID NOs: 3, 5 to 7, and 9 to 11 are animal species-specific peptides. Therefore, if the seven peptides of SEQ ID NOs: 3, 5 to 7, and 9 to 11 are used as indicators, any of the six animals having this specific peptide, that is, bovine, horse, pig, sheep, rabbit, and sturgeon. The animal species can be identified for the mixture of the two species. Table 7 shows the relationship between the combination of animal species and the sequence number of the peptide as an index. In addition, both SEQ ID NO: 3 and SEQ ID NO: 7 are peptides specific to sturgeon. Either one of SEQ ID NO: 3 and SEQ ID NO: 7 may be used, or both SEQ ID NO: 3 and SEQ ID NO: 7 may be used. According to the present invention, for example, when the peptides of SEQ ID NO: 3 and SEQ ID NO: 6 are detected in the tryptic decomposition product of the collagen-derived material, it is determined that this sample is a mixture of "horse and butterfly shark". Can be done . If the tryptic degradation product of the collagen -derived material contains the peptides of SEQ ID NO: 9 and SEQ ID NO: 10 and does not contain the peptides of SEQ ID NOs: 3, 5-7, and 11, the animal species of the collagen-derived material is ". It is judged as "pig and rabbit".

Further, in addition to the peptides shown in Table 7 above, other peptides may be included in the index, and two animal species can be specified. For example, all 12 peptides shown in Table 11 can be used as indicators to identify animal species for any two mixtures of six species: bovine, horse, pig, sheep, rabbit, and butterfly shark. Table 8 shows the relationship between the combination of animal species and the sequence number of the peptide as an index. Even when all seven peptides are used as indicators according to Table 7, for example, "pig and rabbit" and "pig and sheep" have only two differences in the presence or absence of the peptide. On the other hand, when all 12 kinds of peptides shown in Table 8 were used and the animal species were determined according to Table 8, there were 4 places regarding the presence or absence of peptides between "pig and rabbit" and "pig and sheep". It is possible to identify the animal species with the difference between the two.

Further, in the present invention, 10 kinds of SEQ ID NOs: 1, 2, 4 to 6,8, 9, 11 and 12 and SEQ ID NO: 3 or 7 are used as indexes, and the animal species of the collagen-derived material are described according to Table 9 below. , A cow, a horse, a sheep, a goat, a pig, a deer, a rabbit, and a combination of two species selected from the group of sharks. As described above, in the present invention, "SEQ ID NO: 3 or 7" may be either SEQ ID NO: 3 or SEQ ID NO: 7, and both SEQ ID NO: 3 and SEQ ID NO: 7 may be used. means. Table 9 includes both SEQ ID NO: 3 and SEQ ID NO: 7. According to Table 9, the animal species can be identified with a difference of 1 to 6 between the combinations of each animal. SEQ ID NO: 10 is a peptide specific to "pig", but it has a characteristic that a mixture of two kinds can be specified by a combination of a plurality of peptides without using the peptide.

Further, in the present invention, using any three or more of the 12 peptides of SEQ ID NOs: 1 to 12 shown in Table 1 as an index, the animal species of the collagen-derived material are bovine, horse, sheep, according to Table 10 below. It is possible to determine which combination of the two species is selected from the group consisting of goats, pigs, deer, rabbits and sharks. Since the three species of SEQ ID NOs: 5 to 7 are specific to sheep, horse and butterfly, respectively, the sample containing the peptides of SEQ ID NO: 5 and SEQ ID NO: 6 can be identified as "sheep and horse", and the animal species can be identified. The animal species can be identified as "sheep and butterfly shark" from the sample containing the peptide of SEQ ID NO: 7 and 5. In the present invention, at least 3 kinds, preferably 7 kinds, more preferably 10 kinds, and particularly preferably all 12 kinds are used. As the number of peptides as an index increases, the possibility of erroneous determination can be reduced. The combinations of all 12 peptides and each animal species are shown in Table 10 below. When the combination of animal species is determined using 10 peptides as an index according to Table 9, there is only one difference between "cattle and pigs" and "cattle and deer", but it contains SEQ ID NO: 10. According to Table 10, when all 12 kinds of peptides are used as an index, the animal species can be identified with two differences, so that the possibility of erroneous determination can be reduced.

The third aspect of the present invention is to use the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 as an index for the peptides contained in the tryptic decomposition products when the collagen-derived material contains three or more animal species. It is a method of discriminating an animal species according to Table 6. Three or more animals using the species-specific peptides of cattle, horses, sheep, pigs, rabbits and sharks identified by the seven species of SEQ ID NOs: 3, SEQ ID NOs: 5-7 and SEQ ID NOs: 9-11 as indicators. Each animal species can be identified even when species are mixed.

The method for evaluating the presence or absence of the peptide is not particularly limited. For example, the peptide can be separated from the tryptic decomposition product of collagen by liquid chromatography or the like, and the presence or absence thereof can be evaluated while confirming by a peptide sequence or the like. Further, it may be LC / MS, LC / MS / MS, or the like. The present invention is preferably analyzed by LC / MS / MS. According to LC / MS / MS, the tryptic decomposition product can be separated by the affinity with the column in the LC part, ionized in the MS part, and then further separated and detected by mass, so that a series of operations can be performed. It can be done quickly. In particular, if a triple quadrupole mass spectrometer is used, precursor ions are selected in the first quadrupole mass analyzer, and the ions are destroyed in the second quadrupole chamber to destroy the ions in the third quadrupole. Since it is possible to set a multiple reaction monitoring method (MRM mode) for detecting a specific ion from the product ions obtained by the destruction, it is possible to make a highly specific determination. Further, according to the MRM mode, since a plurality of channels can be set in one measurement, it is possible to analyze multiple components in one analysis.

In the present invention, it is analyzed whether or not the tryptic decomposition product contains 12 kinds of peptides of SEQ ID NOs: 1 to 12 specified in Table 1 above, and the result is matched with any of Tables 2 to 10 above, and the animal It determines the species. In LC / MS / MS analysis, the precursor ion of the peptides of SEQ ID NOs: 1 to 12 can be identified, the generated product ion (measurement ion) can be selected, and the MRM channel can be set to detect the peptide. can. Further, the certainty can be further improved by identifying another product ion (confirmed ion) and separately setting and measuring the corresponding MRM channel. By detecting the measurement ion and the confirmation ion, the presence or absence of the measurement ion can be reliably evaluated.

In the present invention, only 12 kinds of peptides are used for determining 8 kinds of animal species. By presetting the detection conditions for precursor ions, measurement ions, and confirmation ions corresponding to each peptide and simultaneously analyzing the above 12 species in one analysis, it is possible to easily determine the animal species.

Further, when the trypsin decomposition product of collagen is analyzed by LC / MS / MS to determine the animal species, an electronic device for determination is further arranged in the LC / MS / MS analyzer, and a storage unit of the electronic device is provided. The contents of Tables 2 to 10 are stored in advance, the results of SEQ ID NOs: 1 to 12 of the LC / MS / MS analyzer are input to the calculation unit, and the animal species in Tables 2 to 10 are associated with the input results. It is possible to determine the animal species. The determination result may be printed or displayed on a display or the like.

In the present invention, based on the LC / MS / MS analysis results, the two mixed animal species are any combination of the two selected from the group consisting of cattle, horses, sheep, pigs, rabbits and sturgeon. If it is determined to be, the compounding ratio of the animal species can be specified.

When the determined animal species includes any of cattle, horses, sheep, pigs, rabbits, and sturgeon, species-specific peptides have been detected in the samples. A calibration curve or the like showing the relationship with the concentration is prepared in advance from the peak area, peak height, peak intensity, etc., and the concentration of each animal species contained in the sample is determined according to this calibration curve, and then the blending ratio is determined. The same applies when three or more species-specific peptides are contained. In the present invention, SEQ ID NO: 3 or SEQ ID NO: 7 is used as an index of butterfly, SEQ ID NO: 5 is used as an index of sheep, SEQ ID NO: 6 is used as an index of horse, SEQ ID NO: 9 is used as an index of rabbit, and SEQ ID NO: 10 is used as an index of pig. It can be used as an index, or SEQ ID NO: 11 can be used as a bovine index.

A tryptic decomposition product of collagen for determining an animal species can be prepared by a conventional method. For example, collagen or gelatin may be extracted from leather, glue or other collagen-derived materials and decomposed with trypsin. The reason for limiting to tryptic decomposition products is that trypsin is easily available and has excellent reproducibility of collagen decomposition. Although collagenase may be used for the decomposition of collagen, collagenase is not used in the present invention. This is because collagenase decomposes collagen to Gly-amino acid X-amino acid Y and loses the animal species specificity of the produced peptide.

When the collagen-derived material is leather, it is desirable to heat a part of leather such as raw leather or tannin tanned leather in water to extract collagen contained in the leather prior to trypsin treatment. For example, in order to improve the extraction efficiency, a piece of leather is cut into 1 mm square pieces, which are put into 1 to 20 times as much water and heated. The heat treatment has a temperature of 50 to 100 ° C, preferably 60 to 90 ° C, and particularly preferably 70 to 90 ° C. The heating time depends on the size of the leather piece, but is 5 minutes to 2 hours, preferably 10 minutes to 1 hour, and particularly preferably 20 minutes to 50 minutes. As a result, collagen contained in leather changes like gelatin. If the leather is a processed product such as chrome tanning, it is necessary to remove the influence of chrome tanning. After cutting the leather pieces into small pieces, add 1 to 20 times the amount of 0.25% calcium hydroxide solution to the leather, shake for 10 minutes to 24 hours, wash with water, and then heat-treat the water-washed test pieces in the same manner as above. And trypsin treatment.

When the collagen-derived material is glue, it may be decomposed by adding trypsin after being heated and dissolved in water. The same applies when the collagen-derived material is collagen or gelatin. In addition, the collagen-derived material may be glue extracted from a cultural property, or may contain pigments or other inorganic substances. The insoluble matter may be removed by centrifugation, filtration, or the like after being dissolved by heating in water.

In the trypsin treatment, trypsin is allowed to act on a solution in which collagen is changed to gelatin at a temperature of 15 to 60 ° C. The reaction time is 1 to 24 hours, preferably 2 to 16 hours, and particularly preferably 4 to 16 hours. At this time, a buffer solution or the like may be added to the reaction solution. Examples of the buffer solution include Tris-hydrochloric acid buffer solution and sodium phosphate buffer solution. Further, reagents that can be added include stabilizers such as calcium chloride and denaturants such as sodium dodecyl sulfate and urea. It can be centrifuged to remove insoluble solids and the like to prepare a test solution for determining an animal species.

Next, the present invention will be specifically described with reference to examples, but these examples do not limit the present invention in any way.

The LC / MS / MS conditions of the samples prepared in each example were as follows.
(1) Equipment LC / MS (1200 series manufactured by Agilent Technologies) and triple quadrupole mass spectrometer (3200QTRAP manufactured by AB SIX Co., Ltd.) were used.
(2) HPLC conditions Analytical column: Ascentis Express C18 HPLC column Particle diameter 5 μm, length 15 cm, diameter 2.1 mm (manufactured by Spellco)
Mobile phase: solution A; 0.1% formic acid, solution B; 100% acetonitrile,
Gradient conditions: 0-2 minutes: Solution A 98%, 2.1-6 minutes: Solution A 98-40%, 6.1-8 minutes: Solution A 40-10%, Solution 8.1-10 minutes: Solution A 98%
Flow velocity: 500 μl / min
Column temperature: 40 ° C
(3) Mass spectrometry conditions Ionization: ESI, positive analysis mode: Multiple Reaction Monitoring (MRM) mode Ion spray voltage: 4.0 kV
Ion source temperature: 700 ° C
(4) MRM Channels The 12 types of peptides shown in SEQ ID NOs: 1 to 12 and their detection channels are shown in Table 12 below.

(Example 1)
(I) Analysis of bovine skin-extracted collagen was performed. 50 μg of bovine skin-extracted collagen was heated in 100 mM Tris-hydrochloric acid (pH 7.6) at a temperature of 60 ° C. for 30 minutes in 1 mM calcium chloride, and then a trypsin digestion reaction was carried out with 2.5 μg of trypsin for 16 hours (total liquid volume). 100 μL). Then, formic acid was added to the reaction solution to a concentration of 1.0% to stop the reaction, and the mixture was filtered through a 0.45 μm filter to prepare a sample for LC / MS / MS analysis.

(Ii) A collagen sample prepared from bovine skin using pepsin was digested with trypsin according to the above (i) to obtain a sample for LC / MS / MS analysis, and LC / MS / MS analysis was performed. The MRM chart is shown in FIG. Peaks of the peptides of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 11 shown in Table 6 above were detected, but peaks of other peptides were not detected. In FIGS. 1 to 8, P means the peak of the SEQ ID NO:.

(Iii) A collagen sample was prepared from the skin of horses and pigs using pepsin instead of the collagen extracted from bovine skin, and trypsin was digested in the same manner as in (i) above to prepare a sample for LC / MS / MS analysis. LC / MS / MS analysis was performed. The MRM charts are shown in FIGS. 2 and 3. In addition, LC / MS / MS analysis was performed on the skin-extracted collagen of sheep and rabbit (manufactured by Sigma-Aldrich) in the same manner as that of bovine skin-extracted collagen. The MRM charts are shown in FIGS. 4 and 5.

In addition, deer glue (provided by Amanosan Cultural Heritage Research Institute), sturgeon glue (Seki Mizuki et al., "Research on manufacturing method of isinglass using sturgeon float bag (swimbladder)", Contract research report 2012. Contractor: Fujikin Co., Ltd. Was dissolved in distilled water at a temperature of 60 ° C. for 4 hours, and then LC / MS / MS analysis was performed in the same manner as for bovine skin-extracted gelatin. The MRM charts are shown in FIGS. 6 and 7.

In addition, 0.5 g of goat leather was cut into test pieces, which were cut into 1 mm square pieces, soaked in 10 mL of 0.25% calcium hydroxide solution, shaken for 2 hours, and then the finely cut pieces were washed with water 7 times. did. Five shredded pieces were collected, 200 μL of water was added, and the mixture was heated at 80 ° C. for 30 minutes. Then, water was removed, 50 μg of trypsin was added in 0.1 M Tris-hydrochloric acid buffer (pH 7.6) and 1 mM calcium chloride, and a digestion reaction was carried out at 37 ° C. for 4 hours. After that, formic acid is added to the reaction solution to 1.0% to stop the reaction, and after centrifugation, insoluble solids and the like are removed, and then filtered through a 0.45 μm filter to decompose goat leather trypsin. Got LC / MS / MS analysis was performed on the obtained goat leather trypsin decomposition product. The MRM chart is shown in FIG.

Table 13 shows the relationship between the peptides detected in FIGS. 1 to 8 and the animal species. When the above 12 kinds of peptides were detected, it was found that they could be identified as any one of bovine, horse, pig, sheep, goat, deer, rabbit, and sturgeon.

(Example 2)
Twenty kinds of commercially available glue / gelatin products A to T were analyzed as samples, and LC / MS / MS analysis was performed in the same manner as in Example 1. Table 14 shows the raw material display of the sample and the detected peptides. Sample A had the peaks of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 11, but did not have other peaks. Therefore, it was determined to be "cow". In addition, Sample C had the peaks of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 11, but did not have other peaks. SEQ ID NO: 9 is a "rabbit" species-specific peptide and SEQ ID NO: 11 is a "cattle" species-specific peptide. From the animal species patterns in Tables 6 and 10, it was determined to be a mixture of "cattle" and "rabbit". In addition, sample D, sample E, sample F, sample G, sample H, sample I, sample J, sample K, sample L, sample N, sample O, sample P, sample Q, sample R, sample S, and sample T are also available. , Species-specific peptides and the animal species patterns in Tables 6 and 10 allowed each animal species to be identified. The analysis results are shown in Table 14.

On the other hand, sample B had peaks of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 10 to SEQ ID NO: 12. SEQ ID NO: 10 is a "pig" species-specific peptide and SEQ ID NO: 11 is a "cattle" species-specific peptide. Among the above peptides, the peptides of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12 are derived from "porcine", and the peptides of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 11 are derived from "bovine". do. "Sheep" and "goat" can be selected from Table 6 as the animal species having the remaining SEQ ID NO: 2. The "sheep" is identified by the peptides of SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 12, and the "goat" is identified by the peptides of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 12. Sample B does not contain "sheep" because the peptide of SEQ ID NO: 5 is absent. From these, it was determined that Sample B contained three species, "pig", "cow", and "goat".

Similarly, sample M had peaks of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 to SEQ ID NO: 12. SEQ ID NO: 10 is a "pig" species-specific peptide, SEQ ID NO: 11 is a "cattle" species-specific peptide, and SEQ ID NO: 6 is a "horse" species-specific peptide. From these results, the animal species was determined to be a mixture of "cattle", "pig" and "horse".

FIG. 9 shows a chart of MRM of sample C. Only the peaks of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 11 are detected in the chart.

As shown in Table 14, the samples in which the animal species labeled as raw materials and the animal species found in the analysis results of the present invention did not match were B, C, F, G, H, I, M, N, and S. .. Among these, only animal species different from the raw material labeled animals were detected in the samples F, H, I, N and S. In each of the samples F, G, H, I, and N, the raw material display animal is a rabbit, but the analysis result shows that it is either "pig" or "cattle". In Non-Patent Document 5, rabbit glue should be originally prepared from rabbit skin, but it is consistent with the description that it may actually be marketed by mixing glue derived from other animal species.

(Example 3)
The amount ratios of the samples C, G, and M in which two or more animal species were found to be mixed in Example 2 were calculated. The measurements of Sample C and Sample G were as follows. Type I collagen concentration A calibration curve showing the relationship between the peak areas and concentrations of SEQ ID NO: 11 and SEQ ID NO: 9 was prepared for bovine collagen and rabbit collagen with known concentrations. Next, using this calibration curve, the bovine type I collagen concentration was obtained from the peak area of SEQ ID NO: 11 of the sample, and the rabbit type I collagen concentration was obtained from the peak area of SEQ ID NO: 9. Calculated. In addition, for the measurement of sample M, a calibration curve showing the relationship between the peak areas and concentrations of SEQ ID NO: 11, SEQ ID NO: 10, and SEQ ID NO: 6 was prepared for bovine collagen, pig collagen, and horse collagen having a known type I collagen concentration. .. Then, using this calibration line, bovine type I collagen concentration from the peak area of SEQ ID NO: 11, pig type I collagen concentration from the peak area of SEQ ID NO: 10, and horse type I collagen from the peak area of SEQ ID NO: 6. After determining each concentration, the blending ratio of the three species of animals was calculated. The results are shown in Table 15.

Among the samples used in Example 3, the samples in which the animal species indicated as raw materials were one species but two or more species were detected were samples C, G, and M. As shown in Table 15, in Sample C and Sample M, the mixing ratio of the animals mixed with the raw material labeling animals was 20% or less, and the possibility of accidental contamination was estimated. On the other hand, in the sample G, the blending ratio of the animal species not described in the raw material label was 50% by mass or more, suggesting intentional contamination.

(Example 4)
A sample of a painting with a tempera on a canvas (Camille Pissarro, title "Harvest", National Museum of Western Art, collection number p.1984-0003, 1882) with a ground coating was used as a sample.

A 1 mg sample was heated in 0.1 M Tris-hydrochloric acid buffer (pH 7.6) at 60 ° C. in 1 mM calcium chloride (total liquid volume 100 μl) for 30 minutes, and then enzymatically treated with 2.5 μg of trypsin. Then, formic acid was added to the reaction solution to a concentration of 1.0% to stop the reaction, and after centrifugation, insoluble solids and the like were removed, and LC / MS / MS analysis was performed. As a result, the peptides of SEQ ID NO: 1, 2, 4, 5, 11 and 12 were detected in the sample. It contained a bovine-specific peptide (SEQ ID NO: 11) and a sheep-specific peptide (SEQ ID NO: 5), and was determined to be bovine and sheep from this detection pattern. When the compounding ratio was calculated from the peak area of SEQ ID NO: 5 and the peak area of SEQ ID NO: 11, it was a mixture of 63.8% by mass of cattle and 36.2% by mass of sheep.

According to a previous study (Miho Takashima, National Museum of Western Art, Research Bulletin No. 16, 35-45, 2012.3.31), the ground coating was obtained by observing the cross section of the ground coating of the painting of Example 4 with an electron microscope and an optical microscope. It has been reported to have a two-layer structure. Since glue derived from two kinds of animal species was detected, and even a small amount of sheep glue was 1/3 or more, there is a possibility that different glue is used for each layer of the ground coating layer, and there is a possibility that different glue is used for each layer of the ground coating layer. It was suggested that the glue on the coating layer may be different.

Claims (7)

A method for discriminating an animal species using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 below as an index for the peptides contained in the tryptic decomposition products of collagen-derived materials.
(1) Using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, the collagen-derived material may be bovine, horse, sheep, pig, rabbit or butterfly shark according to Table 2. (2) Using the four peptides of SEQ ID NOs: 1, 2, 4 and 5 as indicators, the collagen-derived material is sheep, goat, or deer according to Table 3 below. Determine which animal species it is, or
(3) Using the seven peptides of SEQ ID NO: 1, 2, 4, 5, 9, 11 and 12 as indicators, the collagen-derived material is any of bovine, sheep, goat, deer or rabbit according to Table 4 below. Determine if it is an animal species, or (4) use any of the 10 peptides of SEQ ID NO: 1, 2, 4, 5, 6, 8, 9, 11 and 12, and SEQ ID NO: 3 or 7 as indicators. , An animal species of a collagen-derived material, characterized in that it is determined whether the collagen-derived material is an animal species of cattle, horses, sheep, goats, pigs, deer, rabbits or butterfly sharks according to Table 5 below. (Note that when specifying the table, the one enclosed in square brackets is not referred to.) .

A method for discriminating an animal species using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 below as an index for the peptides contained in the tryptic decomposition products of collagen-derived materials.
(1) Using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, the collagen-derived material comprises bovine, horse, sheep, pig, rabbit and butterfly shark according to Table 7. Determine which combination of the two species is selected from the group, or (2) use the 12 peptides of SEQ ID NOs: 1 to 12 as indicators, and according to Table 8 below, the animal species of the collagen-derived material is bovine. Determine which combination of the two selected from the group consisting of horses, sheep, pigs, rabbits and sharks, or (3) SEQ ID NOs: 1, 2, 4, 5, 6, 8, 9, 11 and 12 , And 10 kinds of peptides of SEQ ID NO: 3 or 7, and according to Table 9 below, the animal species of the collagen-derived material consists of cattle, horses, sheep, goats, pigs, deer, rabbits and butterfly sharks. Determine which combination of the two species is selected from the group, or (4) Using the 12 peptides of SEQ ID NOs: 1 to 12 as indicators, the animal species of the collagen-derived material is bovine according to Table 10 below. , A method for determining an animal species of a collagen-derived material, which comprises determining which combination of two species is selected from the group consisting of horses, sheep, goats, pigs, deer, rabbits and butterfly sharks . In the specification of the table, the one enclosed in square brackets is not referred to.) .

With respect to the peptides contained in the trypsin decomposition product of the collagen-derived material, the animal species are discriminated according to Table 6 using the peptides of SEQ ID NOs: 1 to 12 shown in Table 1 below as an index.
(1) Using the seven peptides of SEQ ID NO: 3, SEQ ID NO: 5 to 7, and SEQ ID NO: 9 to 11 as indicators, the collagen-derived material is selected from bovine, horse, sheep, pig, rabbit, and butterfly shark. Each animal species is determined when the above animal species are included, or (2) 7 kinds of peptides of SEQ ID NO: 3, SEQ ID NO: 5-7, SEQ ID NO: 9-11, and SEQ ID NO: 1-2, SEQ ID NO: 4 , The collagen-derived material comprises three or more animal species selected from bovine, horse, sheep, goat, pig, deer, rabbit and butterfly shark, using the five peptides of SEQ ID NO: 8 and SEQ ID NO: 12 as indicators. A method for determining the animal species of a collagen-derived material, which comprises determining each animal species in some cases (note that, in the specification of the table, the one enclosed in square brackets is not referred to) .

When the determined animal species is any combination of two species selected from the group consisting of cows, horses, sheep, pigs, rabbits and butterfly sharks, SEQ ID NO: 3 or SEQ ID NO: 7 is used as an index of butterfly sharks, and the sequences are sequenced. The collagen-derived material has No. 5 as a sheep index, SEQ ID NO: 6 as a horse index, SEQ ID NO: 9 as a rabbit index, SEQ ID NO: 10 as a pig index, or SEQ ID NO: 11 as a bovine index. The method for determining an animal species of a collagen-derived material according to claim 2 or 3, wherein the content ratio of the contained bovine, horse, sheep, pig, rabbit, or butterfly shark is specified. The method for determining an animal species of a collagen-derived material according to any one of claims 1 to 3, wherein the presence or absence of the peptide is measured by LC / MS / MS. The method for determining an animal species of a collagen-derived material according to any one of claims 1 to 3, wherein the collagen-derived material is any one selected from leather, glue, collagen, and gelatin. The method for determining the animal species of the collagen-derived material according to any one of claims 1 to 3, wherein the collagen-derived material is glue extracted from a cultural property.

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