Organic composition having high collagen content and method for producing same

Abstract

The technical problem of the present invention is to provide: an organic composition having a high collagen content; a biological material using the organic composition with high collagen content as a raw material, a biological material using collagen as a main component, or other products; a method for producing the organic composition having a high collagen content; a method for identifying a tooth or bone picked from a mammal as a biological material, a biological material having collagen as a main component, or a raw material of other articles; and methods of using PYD and PYD and PpP as indicators for evaluating the potential risk of suffering from a disease in a mammal and / or whether a mammal is potentially healthy. The solution is that PYD is greater than 100, PYD is greater than 100, and PpP is greater than 740.

Description

Technical Field

[0001] The present invention relates to an organic composition with a high collagen content obtained from teeth or bones extracted from mammals; a biomaterial, a biomaterial with collagen as a main component, or other products using the organic composition with a high collagen content as a raw material; a method for producing the organic composition with a high collagen content; a method for identifying teeth or bones extracted from mammals as a raw material for biomaterials, biomaterials with collagen as a main component, or other products; and a method for using the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of mammals, or the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of mammals and the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of mammals and the quantitative value of the pentosidine content (ng / mg) as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy. Background Art

[0002] Various types of collagen have been developed so far (in fact, "organic composition with high collagen content" is more accurate, but it is usually referred to as "collagen"). Most of them are atelocollagen produced by extracting from soft tissues in living organisms using enzyme treatment. The telopeptides at the N-terminus and C-terminus of the amino acid sequence of collagen are cut off, and the poorly soluble collagen fibers are removed through the purification process. In addition, collagen extracted from hard tissues such as bones or teeth such as demineralized freeze-dried bone allografts (DFDBA) or demineralized dentin matrix (DDM) has also been developed. Collagen is produced by demineralization and is mainly used as a biomaterial.

[0003] Regarding atelocollagen, the quality of collagen is evaluated on the grounds that the antigenic telopeptide is said to be cut by enzyme treatment, and regarding non-denatured collagen from soft tissue (collagen with a triple helix structure by retaining the telopeptide), the quality of collagen is evaluated by how high-purity collagen can be produced by setting the conditions of the purification treatment such as pH. However, when producing collagen from hard tissue, it is necessary to extract collagen by removing minerals through demineralization treatment, i.e., acid treatment, and the yield of collagen is limited, and extraction is labor-intensive. Therefore, almost all the collagen products developed are atelocollagen from soft tissue, non-denatured collagen, or products using the same.

[0004] Most of the collagen contained in biological tissues is type I collagen. Type I collagen has physiological crosslinks and non-physiological crosslinks. Pyridinoline crosslinks are known as physiological crosslink molecules, and crosslink molecules formed by advanced glycation end products (AGEs) represented by pentosidine are known as non-physiological crosslink molecules (Non-patent Document 1). Pyridinoline crosslinks are physiological enzymatic crosslinks formed by the action of lysyl oxidase. They are regular crosslink molecules at specific sites in the amino acid sequence of collagen, and therefore contribute to improving the elasticity or strength of collagen fibers. On the other hand, pentosidine crosslinks are non-physiological / non-enzymatic crosslinks related to blood sugar (aging crosslinks (AGEs crosslinks)). They are crosslink molecules formed between arginine and lysine dispersed in the amino acid sequence of collagen due to the increase in blood sugar due to body temperature. Therefore, they are crosslink molecules randomly formed in adjacent arginine and lysine in the amino acid sequence of collagen, which become the cause of destroying the physiological three-dimensional structure of collagen and reduce the elasticity or strength of collagen fibers. In addition, as described above, the telopeptide of the atelocollagen is cut by enzyme treatment, so it not only basically does not contain pyridinoline cross-linking molecules, but also the insoluble collagen fibers caused by pentosidine cross-linking are removed during the purification process, so it is collagen that basically does not contain pentosidine cross-linking molecules.

[0005] In the past, it is known that by increasing the pyridinoline cross-linking molecules in collagen, the strength or elasticity of the collagen is increased (Non-patent Document 2), and pentosidine cross-linking is known to be aging cross-linking (AGEs cross-linking) (Non-patent Document 3). In addition, there are reports that the amount of pentosidine per unit collagen in human articular cartilage increases linearly with age, and the increase in the ratio of the amount of pentosidine per unit collagen to the amount of pyridinoline (pentosidine content / pyridinoline content) accelerates with age (Non-patent Documents 4 and 5), and there are reports that the ratio of the amount of pentosidine per unit collagen to the amount of pyridinoline (pentosidine content / pyridinoline content) increases significantly in non-calcified lesions of malnutrition calcification of the aorta (Non-patent Document 6).

[0006] Prior art literature Non-patent literature Non-patent document 1: M. Saito, CLINICIAN Vol. 554, 1141-1146, 2006 Non-patent document 2: M. Saito, THE BONE Vol.21, No.1, 53-58, 2007 Non-patent literature 3: Sell, DR et al., VM, J. Biol. Chem. Vol. 284, 21597-21602, 1989 Non-patent document 4: A. Uchiyama et al., J. Biochem. Vol. 110, 714-718, 1991 Non-patent document 5: M. TAKAHASHI et al., Arthritis Rheum. Vol.37, No.5, 724-728, May 1994. Non-patent document 6: H. HOSHINO et al., Atherosclerosis Vol. 112, 39-46, 1995 Summary of the invention

[0007] Problems to be solved by the invention The above-mentioned evaluation of the quality of collagen (organic composition with a high collagen content) is generally based on the purity and source of the collagen in the organic composition with a high collagen content, and is not based on indicators at the molecular level of a certain collagen, that is, indicators at the amino acid sequence level of a certain collagen. Therefore, it is not only difficult to say that it is a correct evaluation of the quality of the collagen itself, but also the quality of the collagen is not evaluated based on the pyridinoline content that improves the quality of collagen by improving the elasticity or strength of collagen fibers, or the pentosidine content that causes the destruction of the physiological three-dimensional structure of collagen and leads to a decrease in the elasticity or strength of collagen fibers, thereby causing a decrease in the quality of collagen, or the pentosidine content and the weight ratio of the pyridinoline content to the pentosidine content.

[0008] Furthermore, in the past, not only has there been no attempt to provide an organic composition with a high collagen content or a biomaterial with collagen as a main component that can be evaluated as containing collagen of intrinsically good quality based on an indicator at the molecular level of a certain collagen, that is, at the amino acid sequence level of a certain collagen, but also no attempt has been made to provide an organic composition with a high collagen content or a biomaterial with collagen as a main component that can be evaluated as containing collagen of intrinsically good quality based on an indicator at the molecular level of a certain collagen, that is, at the amino acid sequence level of a certain collagen, since the residual telopeptide and collagen production are limited and the extraction is laborious, as described above, especially teeth, where collagen extraction is difficult and the production is limited, and bones, where collagen extraction requires a lot of labor due to the large amount of lipids contained.

[0009] However, considering that the telopeptide region of collagen shows high antigenicity, in collagen products, telopeptide-free collagen is sometimes used after the telopeptide is removed by enzyme treatment. However, due to the removal of the telopeptide, the pyridoline cross-linking molecules are lost, resulting in the loss of the softness and toughness unique to collagen. In addition, there is no clear evidence that the antigenicity is reduced by removing the telopeptide, so in the medical application of collagen, the removal of the telopeptide has no advantage in immunogenicity. On the other hand, the proportion of people who are allergic to bovine type I collagen is 2-4%, which is relatively low compared with the incidence of nickel allergy of 10-15% and the incidence of latex allergy of about 6% (Lynn AK et al., J Biomed Mater Res B Appl Biomater. 2004 Nov. 15; 71(2): 343-354). In addition, there are reports that type I collagen does not induce immune response and does not show antigenicity (Courtenay JS et al., Nature, 283, 14, 1980, 666-668; David E et al., J. Exp. Med. 1977, 46; 857-868).

[0010] In addition, in the above-mentioned non-patent documents 1 to 6, various descriptions have been made on pyridinoline cross-linked molecules and pentosidine cross-linked molecules in collagen, and the ratio of pentosidine content to pyridinoline content per unit collagen (pentosidine content / pyridinoline content) has been examined in diseases, but none of these documents is about collagen from teeth, which is not easy to extract. In addition, in hard tissues such as bones and cartilages and soft tissues such as tendons and ligaments that require physiological strength, the amount of pyridinoline cross-linked molecules contained in collagen and the amount of pentosidine cross-linked molecules accompanying them tend to increase (M. TAKAHASHI et al., Anal Biochem Vol. 232, 158-62, 1995), but since these tissues are metabolized tissues, the pyridinoline cross-linked molecules and pentosidine cross-linked molecules contained therein are also metabolized, and therefore, the accumulation amount of pyridinoline cross-linked molecules and pentosidine cross-linked molecules in the host as a mammal throughout life is not reflected. In contrast, teeth, as hard tissues, are also the only tissues among hard tissues that do not participate in the metabolic cycle. Therefore, the pyridinoline cross-linked molecules and pentosidine cross-linked molecules contained in the collagen contained therein are not metabolized, and thus can reflect the accumulation of almost a lifetime amount of pyridinoline cross-linked molecules and pentosidine cross-linked molecules in the host as a mammal. Therefore, in order to accurately evaluate the potential risk of mammalian diseases and whether the mammal is potentially healthy, the collagen (organic composition with high collagen content) from teeth that accumulates a lifetime amount of pyridinoline cross-linked molecules and pentosidine cross-linked molecules in the host as a mammal should be used as an indicator. In addition, the organic composition with high collagen content or the biomaterial with collagen as the main component involved in the present invention is subjected to a strong alkali treatment that is not usually performed on the protein containing collagen under a temperature condition higher than normal temperature, and is manufactured by demineralization treatment, i.e., acid treatment, so that the amount of endotoxin contained is greatly reduced, and substantially no endotoxin is contained or no endotoxin is contained, or the endotoxin contained is substantially inactivated or the endotoxin is inactivated, but in the above-mentioned non-patent documents 1 to 6, not only there is no such record, but also no teaching.

[0011] The object of the present invention is to quantify the pyridinoline content (ng / mg) and the pentosidine content (ng / mg) of collagen using a specified method, calculate the weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the quantitative values ​​PYD of the pyridinoline content (ng / mg) and the quantitative values ​​PEN of the pentosidine content (ng / mg) obtained by those quantitative methods, provide an organic composition with a high collagen content obtained from teeth or bones extracted from mammals, and a biomaterial, a biomaterial or other product with collagen as a main component using the organic composition with a high collagen content as a raw material; and provide the above The method for producing an organic composition with a high collagen content; the method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as the main component, or products, characterized in that it includes: when the above-mentioned PYD or the above-mentioned PYD and the above-mentioned PpP are at a specified value, the process of selecting its teeth or bones as the raw materials for biomaterials, biomaterials with collagen as the main component, or other products; as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, using the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammal, or the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammal ) and a weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal, characterized in that it includes: obtaining a quantitative value PYD of the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal that is an evaluation object for the potential risk of disease and / or whether the mammal is potentially healthy, and a step of calculating a weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the above-obtained PYD and the above-obtained PEN; obtaining A step of obtaining a quantitative value R-PYD of the pyridinoline content (ng / mg) and a quantitative value R-PEN of the pentosidine content (ng / mg) as a reference of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals and unhealthy mammals, and calculating a reference weight ratio value R-PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the R-PYD obtained above and the R-PEN obtained above; and a step of comparing the PYD with the R-PYD, or comparing the PYD with the R-PYD, and the PpP with the R-PpP.

[0012] Technical solutions to solve problems The inventors of the present invention have conducted intensive and repeated studies to solve the above-mentioned technical problems, and as a result, have found that, in an organic composition with a high collagen content obtained by subjecting a crushed tooth or bone extracted from a mammal to a strong alkali treatment at a temperature higher than room temperature and lower than the boiling point of water, and subjecting the crushed product after the strong alkali treatment to a demineralization treatment under negative pressure conditions, and in a biomaterial obtained by the above-mentioned, a biomaterial or other product having collagen as a main component, when a quantitative value PYD of a pyridinoline content (ng / mg) per unit collagen and a value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of a weight ratio calculated from a pyridinoline content (ng / mg) per unit collagen and a pentosidine content (ng / mg) per unit collagen are predetermined values, the contained collagen has the property of imparting excellent The invention relates to a method for evaluating the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy by comparing the collagen contained in the teeth of the mammal as an evaluation object for the potential risk of disease and / or whether the mammal is potentially healthy, with the collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without disease, mammals with disease, healthy mammals and unhealthy mammals, and the following inventions are completed.

[0013] (1) An organic composition with a high collagen content, which is an organic composition with a high collagen content obtained from teeth or bones extracted from mammals, comprising the following (i), or (i) and (ii): (i) a quantitative value PYD of the pyridinoline content (ng / mg) of collagen contained in the organic composition having a high collagen content, obtained by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, is PYD>100; (ii) The value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) of the quantitative value PYD of the collagen contained in the organic composition with a high collagen content, obtained by quantification using heptafluorobutyric acid and formic acid as ion pair reagents and a fluorescence detector, is a weight ratio of PpP>740.

[0014] (2) The organic composition with a high collagen content as described in (1), which does not substantially contain endotoxin; or does not contain endotoxin; or the endotoxin contained is substantially inactivated; or the endotoxin is inactivated.

[0015] (3) The organic composition with a high collagen content according to (1), wherein the mammal is one or more mammals selected from the group consisting of cattle, pigs, horses, sheep, deer, dogs, cats and humans.

[0016] (4) A biomaterial or a biomaterial or other product containing collagen as a main component, using the organic composition having a high collagen content as described in any one of (1) to (3) as a raw material.

[0017] (5) A method for producing an organic composition having a high collagen content according to any one of (1) to (3), comprising: A process of crushing teeth or bones extracted from mammals to obtain crushed matter; The step of subjecting the pulverized product obtained above to a strong alkali treatment; and The process of demineralizing the pulverized material after the strong alkali treatment under negative pressure.

[0018] (6) The production method according to (5), wherein the step of subjecting the obtained pulverized product to a strong alkali treatment is a step of subjecting the obtained pulverized product to a strong alkali treatment at a temperature higher than room temperature and lower than the boiling point of water.

[0019] (7) A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as the main component, or other products, comprising: A process for quantifying the pyridinoline content (ng / mg) of collagen contained in teeth or bones extracted from mammals; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the tooth or bone extracted from the mammal; A step of calculating a weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the value PYD of the pyridinoline content (ng / mg) obtained by the quantitative determination and the value PEN of the pentosidine content (ng / mg) obtained by the quantitative determination; and A process for selecting the teeth or bones extracted from mammals as biomaterials, biomaterials with collagen as the main component, or raw materials for other products when the value PYD of the pyridinoline content (ng / mg) obtained by the above quantitative determination or the value PYD of the pyridinoline content (ng / mg) obtained by the above quantitative determination and the value PpP of the weight ratio obtained by the above calculation (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are the specified values.

[0020] (8) A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as the main component, or other products, comprising: A process of crushing teeth or bones extracted from mammals to obtain crushed matter; The step of subjecting the above-obtained pulverized product to a strong alkali treatment; The step of demineralizing the pulverized product after the strong alkali treatment under negative pressure to obtain an organic composition with a high collagen content; A step of quantifying the pyridinoline content (ng / mg) of collagen contained in the organic composition having a high collagen content obtained above; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the organic composition having a high collagen content obtained above; A step of calculating a weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the value PYD of the pyridinoline content (ng / mg) obtained by the quantitative determination and the value PEN of the pentosidine content (ng / mg) obtained by the quantitative determination; and A process for selecting the teeth or bones extracted from mammals as biomaterials, biomaterials with collagen as the main component, or raw materials for other products when the value PYD of the pyridinoline content (ng / mg) obtained by the above quantitative determination, or the value PYD of the pyridinoline content (ng / mg) obtained by the above quantitative determination and the value PpP of the weight ratio obtained by the above calculation (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are the specified values.

[0021] (9) A method as described in (7) or (8), wherein the step of quantifying the pyridinoline content (ng / mg) of collagen contained in the organic composition with a high collagen content and the step of quantifying the pentosidine content (ng / mg) of collagen contained in the organic composition with a high collagen content are carried out by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, and the prescribed values ​​are PYD>100, or PYD>100 and PpP>740, respectively.

[0022] (10) A method for using a quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or a quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and a weight ratio calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, comprising: A step of quantifying the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal as an evaluation target for the potential risk of disease and / or the potential health of the mammal, and obtaining a quantitative value PYD; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the tooth of the mammal to be evaluated to obtain a quantitative value PEN; A step of calculating a weight ratio value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) from the PYD obtained above and the PEN obtained above; A step of quantifying the pyridinoline content (ng / mg) of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PYD; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PEN; A step of calculating a reference value R-PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) of a weight ratio from the R-PYD obtained above and the R-PEN obtained above; and A step of comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD, and the PpP and the R-PpP.

[0023] (11) The method as described in (10), wherein the step of quantifying the pyridinoline content (ng / mg) to obtain a reference quantitative value R-PYD and the step of quantifying the pentosidine content (ng / mg) to obtain a reference quantitative value R-PEN are steps of quantifying the pyridinoline content (ng / mg) to obtain a reference quantitative value R-PYD and quantifying the pentosidine content (ng / mg) to obtain a reference quantitative value R-PEN by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, and the step of comparing the PYD with the R-PYD, or comparing the PYD with the R-PYD, and comparing the PpP with the R-PpP is a step of confirming whether the PYD is PYD>100, or confirming whether the PYD is PYD>100 and the PpP is PpP>740.

[0024] Effects of the Invention According to the present invention, it is possible to provide an organic composition and biomaterial with a high quality collagen content, a biomaterial or other product containing collagen as a main component, which is obtained by subjecting a crushed tooth or bone extracted from a mammal to a strong alkali treatment (preferably, the strong alkali treatment is performed under predetermined conditions such as a temperature condition higher than room temperature) and then subjecting the crushed product after the strong alkali treatment to a demineralization treatment under negative pressure conditions, and which contains collagen having a three-dimensional structure that imparts excellent elasticity and strength. In addition, it is possible to provide a raw material for a biomaterial, a biomaterial or other product containing collagen as a main component, which can be identified. The teeth or bones extracted from a mammal, and further the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammal, or the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammal and the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammal, can be used as an indicator for evaluating the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The present invention is a distribution graph showing the quantitative value PYD of the pyridoline content (ng / mg) of collagen contained in an organic composition having a high collagen content in the dentin of teeth from 18 humans, 36 cattle (including 19 cattle Specified Risk Materials (SRM) and 17 Tokachi Wakagyu (Japanese registered trademark, early-fattening (14-month-old) male Holstein cattle)) and 8 domestic pigs (6-month-old) (a total of 62 cases), obtained by quantification using high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents.

[0026] Figure 2 The distribution graph shows the quantitative values ​​of PYD of pyridinoline content (ng / mg) obtained by quantification using heptafluorobutyric acid and formic acid as ion pair reagents using high performance liquid chromatography (HPLC-Flu) with a fluorescence detector, in which collagen (n=62) contained in an organic composition with a high collagen content in dentin from 18 humans, 36 cattle (19 of which were cattle SRM and 17 were Tokachi Wakani (Japanese registered trademark)), and 8 domestic pigs, Teruplug (n=3, de-telopeptide collagen from bovine dermis, telopeptide-free treatment), High-Grade gelatin (HG gelatin) (n=3, pig skin acid treatment, telopeptide-preserving treatment, low endotoxin collagen), gums of Tokachi Wakani (Japanese registered trademark) (n=3), and muscles of Tokachi Wakani (Japanese registered trademark) (n=3). In the graph, the dotted line indicates the threshold value of PYD.

[0027] Figure 3 This is a distribution graph showing the quantitative value PEN of the pentosidine content (ng / mg) contained in an organic composition with a high collagen content in the dentin of teeth from 18 humans, 36 cattle (including 19 cattle SRM and 17 Tokachi Wakagyu (Japanese registered trademark)), and 8 domestic pigs (a total of 62 cases), obtained by quantification using high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents.

[0028] Figure 4This is a distribution graph showing the values ​​of the weight ratio PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative value PYD of the pyridoline content (ng / mg) and the quantitative value PEN of the pentosidine content (ng / mg) of collagen contained in the organic composition with a high collagen content in the dentin of 18 humans, 36 cattle (including 19 cattle SRM and 17 Tokachi Wakagyu (Japanese registered trademark)) and 8 domestic pigs (a total of 62 cases), obtained by quantification using heptafluorobutyric acid and formic acid as ion pair reagents using high performance liquid chromatography (HPLC-Flu) with a fluorescence detector.

[0029] Figure 5 This is a distribution graph showing the endotoxin values ​​(endotoxin levels; EU / mg) obtained by endotoxin testing (LAL method) for collagen contained in an organic composition with a high collagen content derived from dentin of 6 samples of Tokachi Wakagyu (Japanese registered trademark) teeth, 3 samples of pig skin-derived gelatin, and 3 samples of high-grade gelatin. DETAILED DESCRIPTION

[0030] Hereinafter, the present invention relates to an organic composition with a high collagen content obtained from teeth or bones extracted from mammals, a biomaterial, a biomaterial or other product having collagen as a main component using the above-mentioned organic composition with a high collagen content as a raw material, a method for producing the above-mentioned organic composition with a high collagen content, a method for identifying teeth or bones extracted from mammals as a raw material for biomaterials, biomaterials or other products having collagen as a main component, and a method for using the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the above-mentioned mammals, or the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammals and the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative value of the pyridinoline content (ng / mg) of collagen contained in the teeth of the mammals as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy. In addition, in this specification, the numerical range expressed using "to" or "to" means a range including the numerical values ​​described before and after "to" or "to" as the lower limit and the upper limit.

[0031] The organic composition with high collagen content according to the present invention is an organic composition with high collagen content obtained from teeth or bones extracted from mammals, and has the following (i), or (i) and (ii): (i) a quantitative value PYD of the pyridinoline content (ng / mg) of collagen contained in the organic composition having a high collagen content, obtained by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, is PYD>100; (ii) The value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) of the collagen contained in the organic composition with a high collagen content, obtained by quantification using heptafluorobutyric acid and formic acid as ion pair reagents and a fluorescence detector, is a weight ratio of the quantitative value PYD of the collagen contained in the organic composition with a high collagen content and the quantitative value PEN of the pentosidine content (ng / mg). The value is PpP>740.

[0032] In the present invention, "mammal" refers to an animal that basically reproduces sexually, most of the existing species are viviparous, and feeds their young with milk. As long as it is such an animal, there is no particular limitation. As such an animal, for example, humans; apes such as orangutans, gorillas, chimpanzees, bonobos, and monkeys belonging to the Gibbon family; monkeys other than monkeys belonging to the Gibbon family, and large mammals such as cows, horses, pigs, wild boars, sheep, goats, antelopes, bears, sea lions, seals, walruses, northern sea lions, fur seals, and whales; small mammals such as dogs, cats, rabbits, mice, squirrels, weasels, raccoons, and mongooses. Preferred mammals include cows, pigs, horses, sheep, deer, dogs, cats, and humans, and cows, pigs, and horses can be listed as more preferred mammals, and cows and pigs can be listed as the most preferred mammals.

[0033] "Collagen" is one of the proteins that mainly constitutes various organs, blood vessels, nerves, skin, ligaments, tendons, bones, cartilages, dentin of teeth, etc. of vertebrates, and forms various tissue frameworks in organisms. It is the main component of the extracellular matrix of multicellular animals. In the present invention, "collagen" mainly refers to type I collagen. In this specification, organic compositions with high collagen content are sometimes referred to as "collagen". In addition, the "collagen" of the present invention is collagen in teeth or bones. Teeth and bones are tissues that bear strong loads such as body weight or bite force, so the collagen in teeth or bones is required to have high strength enough to support the tissue.

[0034] Hard tissue collagen can be determined to be derived from hard tissue by detecting, for example, BMP-2, BMP-4, BMP-7, and osteocalcin, and can be determined to be derived from teeth by detecting dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP).

[0035] Generally, an "organic composition with a high collagen content" refers to an organic composition in which the proportion of collagen contained in the organic composition as a whole is high. De-telopeptide collagen or demineralized freeze-dried bone allograft (DFDBA) and demineralized dentin matrix (DDM) are equivalent to the so-called "organic composition with a high collagen content". In the present invention, an "organic composition with a high collagen content" refers to an organic composition in which the proportion of collagen in the organic composition as a whole is at least 60%, preferably 70% or more, more preferably 80% or more, further preferably 85% or more, further preferably 90% or more, and most preferably 95% or more. In addition, in the present invention, organic compositions other than collagen contained in the organic composition with a high collagen content include the above-mentioned DSP, DGP, DPP, etc.

[0036] In addition, generally, "biomaterial" refers to a material used in vivo, or a material used in contact with biological components such as proteins and cells, and "biomaterial with collagen as the main component" refers to a material with collagen as the main component in the whole biomaterial. Demineralized freeze-dried bone allograft (DFDBA) or demineralized dentin matrix (DDM) is equivalent to the so-called "biomaterial with collagen as the main component". In the present invention, "biomaterial with collagen as the main component" refers to a biomaterial with collagen as the main component in the biomaterial as a whole, wherein the proportion of collagen in the biomaterial is at least 40%, preferably 45% or more, more preferably 50% or more, further preferably 55% or more, further preferably 60% or more, and most preferably 65% ​​or more. In addition, as organic compositions other than collagen contained in the biomaterial, the above-mentioned DSP, DGP, DPP, etc. can be listed.

[0037] In the present invention, the pyridinoline-containing collagen and the pentosidine-containing collagen contained in the organic composition having a high collagen content are pyridinoline constituting a pyridinoline cross-linking molecule which is a physiological cross-linking molecule in collagen, and pentosidine constituting a pentosidine cross-linking molecule which is a non-physiological cross-linking molecule in collagen, respectively. In the present invention, the amount of pyridinoline-containing collagen (ng / mg) and the amount of pentosidine-containing collagen (ng / mg) contained in the organic composition having a high collagen content are quantified, and the quantitative value PYD of the pyridinoline content (ng / mg) is divided by the quantitative value PEN of the pentosidine content (ng / mg) to calculate the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)).

[0038] When PYD, or PYD and PpP, of the organic composition with a high collagen content according to the present invention are within the above-specified values, it can be said that the collagen contained in the composition has excellent elasticity and strength, etc., and is of high quality. The reasons for this are described in detail below.

[0039] It is not an exaggeration to say that pyridinoline crosslinking molecules, which are physiological / enzymatic crosslinking molecules, are programmed mainly for the purpose of improving the basic strength of hard tissues. As mentioned above, they are crosslinking molecules formed by two lysine residues in the telopeptide respectively present at the N-terminus and C-terminus in the amino acid sequence of collagen and one lysine residue in the amino acid sequence other than the two ends of collagen through the enzymatic catalysis of lysyl oxidase. The pyridinoline crosslinking molecules formed by the two lysine residues in the telopeptide respectively present at the N-terminus and C-terminus in the amino acid sequence of collagen are crosslinking molecules between collagen fibers, and therefore, it can be said that they are largely involved in maintaining the three-dimensional structure of tropocollagen (the entire collagen sequence) as a triple helical structure.

[0040] Therefore, if the pyridinoline cross-linking molecules in the collagen contained in the biological tissue increase, the texture of the collagen contained in the biological tissue becomes flexible and the viscosity increases, the strength and calcification of the collagen increase, and it can become an element to increase the activity of the cells constituting the biological tissue, that is, an element to easily maintain the homeostasis of the individual by increasing the activity of tissue stem cells related to tissue repair. Therefore, compared with soft tissue collagen represented by skin, the pyridinoline content in collagen from teeth or bones is higher, and the collagen from teeth or bones is flexible, high in strength and high in elasticity.

[0041] The larger the value of the quantitative value PYD of the pyridinoline content (ng / mg) of the collagen contained in the teeth or bones extracted from mammals which is the raw material of the organic composition with a high collagen content and which is derived from such pyridinoline cross-linked molecules, the higher the quality of the collagen contained in the teeth or bones extracted from mammals, and the organic composition with a high collagen content obtained from such teeth or bones extracted from mammals can be said to be an excellent organic composition.

[0042] like Figure 1 As shown, the quantitative value PYD of the pyridinoline content (ng / mg) of collagen contained in the organic composition with high collagen content in the dentin of teeth as an example of hard tissue (a total of 62 cases) from 18 cases of humans as mammals, 36 cases of cattle as mammals and livestock (among which, 19 cases were cattle SRM (Hayaki Factory, Hokkaido Livestock Corporation Co., Ltd.), 17 cases were Tokachi Wakagyu (Japanese registered trademark, early fattening (14 months old) male Holstein cattle; Tokachi Shimizu-cho Agricultural Cooperative)), and 8 cases were all included in the range of PYD involved in the present invention. Here, "SRM" means Specified Risk Materials. In Japan, tonsils and distal ileum (a part of the small intestine) of all ages, head (excluding tongue and cheek meat) over 30 months old, spine and spinal cord are defined as Specified Risk Materials (Food Safety Committee, Cabinet Office).

[0043] On the other hand, Figure 2 As shown, the quantitative values ​​of pyridinoline content (ng / mg) of soft tissue Teruplug (n=3, de-telopeptide collagen from bovine dermis, telopeptide-free treatment; Olympus Terumo Biomaterials Co., Ltd.), High-Grade Gelatin (n=3, pig skin acid treatment, telopeptide-preserved treatment, low-endotoxin collagen; Nippi Co., Ltd.), gums of Tokachi Wakagyu (Japanese registered trademark) (n=3; Tokachi Shimizu Town Agricultural Cooperative Association), and muscles of Tokachi Wakagyu (Japanese registered trademark) (n=3; Tokachi Shimizu Town Agricultural Cooperative Association) were all below 50.

[0044] Based on the above, the PYD value involved in the present invention can be set to PYD>100 by taking into account the lower limit of the PYD of collagen contained in an organic composition with a high collagen content in the dentin of teeth as hard tissue from 18 humans as mammals, 36 cattle as mammals and 8 pigs as livestock, which are domestic animals.

[0045] That is, in the present invention, the quantitative value PYD of the pyridinoline content (ng / mg) of the collagen contained in the organic composition with a high collagen content, that is, the collagen contained in the teeth or bones extracted from a mammal which serves as the raw material of the organic composition with a high collagen content, is PYD>100, which means that it can be inferred that the collagen contained in the organic composition with a high collagen content is not collagen derived from biological tissues other than teeth or bones in mammals.

[0046] On the other hand, the crosslinks formed by advanced glycation end products (AGEs), represented by pentosidine crosslinking molecules, which are non-physiological crosslinking molecules, are pathological crosslinks caused by blood sugar (aging crosslinks (AGEs crosslinks)). As mentioned above, they are crosslinking molecules formed in arginine and lysine dispersed in the amino acid sequence of collagen due to the increase in blood sugar due to body temperature. When arginine and lysine are close to each other in the amino acid sequence of collagen, pentosidine crosslinking molecules are formed non-specifically (randomly), so it is difficult to form a state where collagen fibers are entangled, and the sequence regularity of collagen fibers is lost, which becomes a cause of destroying the three-dimensional structure of original collagen. Therefore, the increase of pentosidine crosslinking molecules in the amino acid sequence of collagen will lead to the weakening of the flexibility, strength and elasticity of collagen fibers. The formation of this pathological crosslinking molecule is caused by blood sugar, so it is said that its accumulation amount increases depending on the host's eating habits (carbohydrate intake habits). In addition, the sugars that are the raw materials of pentosidine crosslinking molecules are supplied through blood vessels, so collagen, which is the supporting basis of tissues throughout the body, can be crosslinked uniformly regardless of the location of the tissue. That is, it can be said that excessive intake of sugar can lead to a decrease in the quality of collagen, which is the support base of the body, regardless of hard tissue or soft tissue.

[0047] In addition, if the number of pentosidine cross-linked molecules in collagen increases, the collagen contained in the biological tissue becomes brittle and chalky, and the strength and calcification of the collagen contained in the biological tissue decrease. The late products of the Maillard reaction are called advanced glycation end products (AGEs), but pentosidine cross-linked molecules are AGEs. In the collagen of mammals whose blood sugar levels are maintained at a continuously high state due to a high-frequency carbohydrate intake diet, more pentosidine cross-linked molecules are contained than in the collagen of mammals of the same age (months) with a balanced diet. Therefore, it can be said that aging cross-linking is taking place in the collagen of mammals with continuously high blood sugar levels. The generated pentosidine cross-linked molecules destroy the regular sequence order of collagen fibers, impair the flexibility of collagen, make it hard and brittle, and inhibit the metabolism of collagen. As a result, the quality of collagen constituting the whole body deteriorates, causing a decrease in the flexibility of tissues. Collagen is the main component of all hard tissues such as bones and teeth, and is also the main component of all soft tissues including nerves and blood vessels. Therefore, the reduction in the quality of collagen will cause the quality of hard and soft tissues in the body to deteriorate. For example, once the quality of bones is reduced due to glycation, the bones become brittle and osteoporosis occurs. Also, once the quality of blood vessels is reduced due to glycation of blood vessel walls, the arteries become hardened and so-called arteriosclerosis occurs, which causes tissue disorders such as increased blood pressure. The same is true for the three major complications of diabetes, namely "(1) diabetic retinopathy (which, if worsened, leads to blindness)", "(2) diabetic nephropathy (which, if worsened, leads to artificial dialysis)", and "(3) diabetic neuropathy (which, if worsened, leads to necrosis of fingers and toes due to blood circulation disorders, for example)". The causes are said to be "(1) retinal blood circulation disorders", "(2) capillary disorders in the glomeruli of the kidneys, which are blood filtering organs", and "(3) capillary nerve disorders and blood flow disorders in the extremities due to arteriosclerosis", respectively. It can be said that the main cause of these is the quality deterioration caused by the glycation of collagen.

[0048] Furthermore, if the amount of advanced glycation end products (AGEs) represented by pentosidine cross-linked molecules increases, AGEs are easy to bind to RAGE, which is a receptor for AGEs in cells constituting tissues throughout the body, and as a result, an in vivo environment is established in which its signal is easily activated. As a result, NF-κB signals related to inflammation are promoted, and for over-glycosylated tissues, AGEs that cause chronic inflammation, represented by pentosidine cross-linked molecules, function as chronic inflammation triggers. This mechanism is believed to be the cause of the deterioration of chronic inflammatory diseases represented by inflammatory bowel disease (IBD) and collagen diseases. As mentioned above, the glycation of collagen is a factor that gradually erodes the body from multiple directions, and it has unknowingly reduced the quality of life (QOL), becoming a major factor that hinders the healthy longevity of organisms. In addition, it has been reported that the pyridinoline cross-linked molecules and pentosidine cross-linked molecules in collagen contained in human biological tissues increase with age (Shimizu, "Non-enzymatic glycation modification of dentin collagen protein associated with age", Osaka University Knowledge Archive. 2015; Walters C. et al., Calcif. Tissue. Int. Vol. 35, 401-405, 1983).

[0049] The smaller the quantitative value PEN of the collagen contained in the organic composition with a high collagen content derived from such pentosidine cross-linked molecules, that is, the collagen contained in the teeth or bones extracted from a mammal which serves as the raw material of the organic composition with a high collagen content, the higher the quality of the collagen contained in the teeth or bones extracted from a mammal, and the organic composition with a high collagen content obtained from the teeth or bones extracted from a mammal can be said to be an excellent organic composition.

[0050] On the other hand, in the case of humans, as they grow older, they tend to live an omnivorous lifestyle, so compared with healthy livestock animals whose feed is managed to a certain quality throughout their lives, the accumulation of pentosidine cross-linked molecules is higher, and it is known that the quality of collagen tends to deteriorate with this. Figure 3 This tendency is also seen in sugars, so the standard indicator for pentosidine content (ng / mg) should not be based on humans, but on healthy livestock animals that live with standard sugar intake habits.

[0051] Therefore, if Figure 3As shown, the quantitative value PEN of the pentosidine content (ng / mg) of collagen contained in an organic composition with a high collagen content in the dentin of teeth, which is an example of hard tissue, from 44 livestock animals (including 19 cattle SRM (Hakai Factory, Hokkaido Livestock Corporation), 17 Tokachi Wakagyu (Japanese registered trademark; Tokachi Shimizu-cho Agricultural Cooperative), and 8 livestock pigs (Hakai Factory, Hokkaido Livestock Corporation)) can be set to 0<PEN<0.4 in consideration of the upper limit value of the 44 livestock animals.

[0052] In the present invention, "livestock animals" refer to mammals whose feed is managed to a certain quality, and cattle, pigs, horses, sheep, goats and deer can be listed as preferred livestock animals.

[0053] In addition, in the present invention, the weight ratio PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of the quantitative value PYD of the pyridinoline content (ng / mg) of the collagen contained in the organic composition with a high collagen content derived from pyridinoline cross-linking molecules, i.e., the tooth or bone extracted from a mammal which is the raw material of the organic composition with a high collagen content, and the quantitative value PEN of the pentosidine content (ng / mg) of the collagen contained in the organic composition with a high collagen content derived from pentosidine cross-linking molecules, i.e., the tooth or bone extracted from a mammal which is the raw material of the organic composition with a high collagen content, is a specified value. Considering the properties of the pyridinoline cross-linking and pentosidine cross-linking, it can be inferred that the collagen contained in the organic composition with a high collagen content or the biomaterial with collagen as the main component is flexible, high-strength, high-elasticity, and is a high-quality collagen that maintains the sequence regularity and three-dimensional structure of the collagen fibers. However, in this case, the premise is that PYD is a predetermined value, that is, the collagen contained in the organic composition with a high collagen content or the biomaterial containing collagen as a main component is not collagen derived from biological tissues other than teeth and bones.

[0054] In the present invention, the pyridinoline content (ng / mg) and the pentosidine content (ng / mg) of the collagen contained in the organic composition with a high collagen content, that is, the collagen contained in the teeth or bones extracted from mammals which are the raw materials of the organic composition with a high collagen content, can be quantified by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents. Furthermore, with respect to the organic composition with a high collagen content according to the present invention, the value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of the quantitative value PYD of the pyridinoline content (ng / mg) and the quantitative value PEN of the pentosidine content (ng / mg) of the collagen contained in the organic composition with a high collagen content obtained by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, is considered that livestock animals are used as indicators for PEN, according to Figure 4 , considering the lower limit value of 44 livestock animals, it can be set to PpP>740.

[0055] The sample for quantitative determination of the pyridoline content (ng / mg) and the pentosidine content (ng / mg) can be prepared by a conventional preparation method known in the art, and can be roughly carried out as follows, for example.

[0056] (1) Dry collagen powder obtained by the method for producing an organic composition having a high collagen content according to the present invention is weighed in a test tube and swollen with a small amount of 6N hydrochloric acid under degassing.

[0057] (2) Add 6N hydrochloric acid to the dried collagen and deaerate, then seal the container.

[0058] (3) Hydrolysis was carried out by heating at 110°C for one day and one night.

[0059] (4) Cool the sample to room temperature.

[0060] (5) The excess hydrochloric acid is removed by drying under reduced pressure while heating, thereby obtaining a collagen hydrolyzate.

[0061] (6) The obtained collagen hydrolyzate was solubilized with a 10% methanol aqueous solution.

[0062] (7) Centrifuge at 12,000 rpm for 5 minutes at room temperature and use the supernatant as the sample.

[0063] (8) Add a known amount of standard substance to the sample, calculate the extraction rate and standard curve, and appropriately calibrate the collagen content.

[0064] The quantification of the pyridoline content (ng / mg) and the pentosidine content (ng / mg) by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents can also be performed using a conventional quantitative method known in the art, and can be performed roughly as follows, for example.

[0065] (1) Ultrapure water containing 0.05% heptafluorobutyric acid (mobile phase A) and a 1:1 mixture of methanol and ethanol containing 0.05% formic acid (mobile phase B) were used as mobile phases, and detection was performed by gradient setting.

[0066] (2) Specific liquid delivery conditions were as follows: a detection column temperature of 40° C., a flow rate of 0.5 mL / min, a ratio of 90% mobile phase A to 10% mobile phase B, and liquid delivery for 0.5 min.

[0067] (3) Then, the concentration of mobile phase B was increased to 22% over 1 minute, and after applying a concentration gradient, the liquid was sent while maintaining this state for 7.5 minutes.

[0068] (4) Next, the mobile phase B concentration was increased to 80% over 1 minute, and then the liquid was sent for a further 1 minute.

[0069] (5) Finally, after sending the solution at a ratio of 10% mobile phase A to 90% mobile phase B, equilibrate with 90% mobile phase A to 10% mobile phase B for 4 minutes.

[0070] (6) The above (1) to (5) were performed for a total of 15 minutes for one sample. The separation column used was Cadenza CD-C18, 150 mm in length and 3 mm in inner diameter, and a fluorescence detector was used for detection. The first half of the sample was monitored at an excitation wavelength of 295 nm and a fluorescence wavelength of 395 nm for 8 minutes, and the second half of the sample was monitored at an excitation wavelength of 325 nm and a fluorescence wavelength of 385 nm for 7 minutes. Through these monitoring, pyridinoline was detected at a retention time of about 6.5 minutes, and pentosidine was detected at a retention time of about 9.1 minutes.

[0071] Next, the organic composition with a high collagen content involved in the present invention is subjected to a strong alkali treatment, which is not normally applied to proteins containing collagen, at a temperature higher than room temperature, and is manufactured through a demineralization treatment, i.e., an acid treatment. Therefore, as shown in Figure 6, the amount of endotoxin contained is sufficiently reduced or the activity of the endotoxin contained is sufficiently reduced, and the composition does not substantially contain endotoxin or does not contain endotoxin, or the endotoxin contained is substantially inactivated or the endotoxin is inactivated.

[0072] The amount or activity that is "reduced" is typically a "statistically significant" amount or activity, including all integers between these in the absence of the composition (in the absence of the agent or compound), or the amount generated by a control composition, or the activity stimulated by a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reduction.

[0073] "Substantially free of endotoxins" generally refers to containing at most trace amounts of endotoxins (e.g., amounts that have no clinically harmful physiological effects on the subject), preferably amounts that cannot be detected. Endotoxins are toxins associated with specific bacteria, typically Gram-negative bacteria, but endotoxins are also found in Gram-positive bacteria such as Listeria monocytogenes. The most important endotoxins are lipopolysaccharides (LPS) or lipolysaccharides (LOS) found in the outer membrane of various Gram-negative bacteria, which show core pathogenic characteristics in the ability of these bacteria to cause disease. A small amount of endotoxins in the human body can also produce fever, hypotension, inflammation, and activation of blood coagulation among other harmful physiological effects. Therefore, in most cases, it is desirable to remove most or all traces of endotoxins from the organic composition with a high collagen content involved in the present invention and the biomaterial with collagen as the main component involved in the present invention, because even a small amount of endotoxins can cause harmful effects in the human body.

[0074] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus Amebocyte Lysate assay (LAL method) using blood from horseshoe crabs is a very sensitive test for detecting the presence of endotoxins. In this test, very low levels of lipopolysaccharide (LPS) cause detectable coagulation of horseshoe crab lysate caused by a powerful enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially free of endotoxin, the endotoxin level may be about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055 0.032、0.033、0.034、0.035、0.036、0.037、0.038、0.039、0.04、0.041、0.042、0.043、0.044、0.045、0.046、0.047、0.048、0.049、0.05、0.051、0.052、0.053、0.054、0.055、0.056、0.057、0.058、0.059、0.06、0.061、0.062、0.063、0.064、0.065、0.066、0 .067, 0.068, 0.069, 0.07, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.08, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.09, 0.091, 0.092, 0.093, 0.094, 0.095, 0.096, 0.097, 0.098, 0.099, 0.11, 0.12. 12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0 .84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6. 6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10 EU / mg or less of protein.

[0075] On the other hand, "substantially inactivated" means that the toxicity of the endotoxin is reduced when measured by any standard test, which means that the endotoxin can be judged to be at least 95% inactivated, preferably 98% inactivated, and more preferably 100% inactivated.

[0076] In addition, the biomaterial, biomaterial with collagen as the main component, or other products using the organic composition with a high collagen content according to the present invention as a raw material are not particularly limited as long as they are biomaterials, biomaterials with collagen as the main component, or other products using the organic composition with a high collagen content according to the present invention as a raw material.

[0077] Furthermore, in order to directly define the organic composition with a high collagen content according to the present invention by its structure and / or characteristics, it is necessary to attempt to modulate or produce a large amount of the organic composition with a high collagen content according to the present invention and to determine it by structure or characteristics using various methods, but such experiments require multiple experiments even for a capable technician in the field, and are time-consuming and accompanied by huge economic expenditures (refer to the Patent and Utility Model Examination Manual, Part II, Chapter 2, "2205 Judgment on 'impossible / impractical situations' in the examination of claims of product inventions that describe the method for manufacturing the product", from the last line of page 16 to lines 1 to 4 of page 17). That is, in order to directly identify the organic composition with a high collagen content involved in the present invention through its structure or characteristics, a person skilled in the art needs to spend uneconomical time and money at the time of application, and requiring such specific work is too strict for applicants seeking an early filing date under the first-to-file principle in the context of rapid technological development and fierce competition for patent acquisition on an international scale (page 10 of Supreme Court No. 1204, 1998, or pages 10-11 of Supreme Court No. 2658, 1998, etc.).

[0078] Next, the method for manufacturing an organic composition with a high collagen content according to the present invention is described. In addition, in the method for manufacturing an organic composition with a high collagen content according to the present invention, the same or equivalent structure as the organic composition with a high collagen content obtained from teeth or bones extracted from mammals according to the present invention, and the biomaterials, biomaterials with collagen as the main component, and other products using the organic composition with a high collagen content as raw materials are omitted for re-description.

[0079] The method for producing an organic composition having a high collagen content according to the present invention comprises the following steps (i) to (iii): (i) a step of crushing a tooth or bone extracted from a mammal to obtain a crushed product (a crushed product preparation step); (ii) a step of subjecting the pulverized product obtained in the pulverized product preparation step (i) to a strong alkali treatment (strong alkali treatment step); (iii) A step of demineralizing the pulverized product obtained in the strong alkali treatment step (ii) under negative pressure (negative pressure demineralization step).

[0080] The pulverized product preparation step (i) in the method for producing an organic composition with a high collagen content according to the present invention is a step of obtaining a pulverized product by using a conventional pulverizing means known in the art such as a grinder from teeth or bones extracted from mammals. In the present invention, the particle size of the pulverized product is preferably 0.05 to 3 mm, more preferably 0.1 to 2 mm.

[0081] The strong alkali treatment step (ii) in the method for producing an organic composition with a high collagen content according to the present invention is a step of stirring the pulverized product obtained in the pulverized product preparation step (i) in a strong alkaline aqueous solution. The strong alkaline aqueous solution can be any conventional strong alkaline aqueous solution known in the art as appropriate, and examples of such a strong alkaline aqueous solution include sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, calcium hydroxide aqueous solution, sodium carbonate aqueous solution, and the like.

[0082] In addition, the strong alkali treatment step (ii) may be a step of subjecting the pulverized product obtained in the pulverized product preparation step (i) to a strong alkali treatment under a temperature condition higher than room temperature and lower than the boiling point of water. Specifically, the strong alkali treatment step (ii) in the present invention is preferably a step of subjecting the pulverized product to a strong alkali treatment under a condition of 50 to 90° C., more preferably a step of subjecting the pulverized product to a strong alkali treatment under a condition of 60 to 80° C., further preferably a step of subjecting the pulverized product to a strong alkali treatment under a condition of 65 to 75° C., and most preferably a step of subjecting the pulverized product to a strong alkali treatment under a condition of 70° C.

[0083] The negative pressure demineralization step (iii) in the method for producing an organic composition with a high collagen content according to the present invention is a step of demineralizing the pulverized product after strong alkali treatment obtained in the strong alkali treatment step (ii) under negative pressure conditions, and specifically includes a step of performing the demineralization while reducing pressure. By performing the demineralization while reducing pressure, the gas (mainly CO) generated during the treatment can be removed. 2 ) and the air mixed therein are removed, so that the demineralization liquid quickly penetrates into the crushed material, and as a result, the time required for demineralization can be shortened. This chemically significant effect is that the demineralization can be completed quickly without increasing the temperature, thereby indirectly protecting substances that are relatively weak to acid. The demineralization step (iii) under negative pressure in the present invention includes a step of performing demineralization while reducing pressure. For example, a step of performing demineralization at normal pressure and a step of performing demineralization while reducing pressure can be combined. In addition, the demineralization treatment can be carried out through one to several steps including the commonly used demineralization treatment steps known in the art. For example, as the demineralization liquid used, phosphoric acid, hydrochloric acid, nitric acid, sulfuric acid, formic acid, ethylenediaminetetraacetic Acid (EDTA), ethanol and hydrochloric acid, ethanol and EDTA aqueous solution, etc. can be used alone or in combination.

[0084] In addition, the method for producing an organic composition with a high collagen content according to the present invention may include not only the above-mentioned pulverized product preparation step (i), the strong alkali treatment step (ii) and the negative pressure demineralization step (iii), but also other steps within the scope that does not impair the characteristics of the present invention. As such steps, for example, further pulverization steps or drying steps, reduced pressure drying steps, washing steps, heating steps, cooling steps, neutralization steps, mixing steps, dissolution steps, etc. can be listed.

[0085] Next, a first embodiment of a method for identifying teeth or bones extracted from mammals as raw materials for the biomaterials, biomaterials with collagen as the main component, or other products involved in the present invention is described. In addition, in the method of the first embodiment of the present invention, the same or equivalent structures as the organic composition with a high collagen content obtained from teeth or bones extracted from mammals involved in the present invention, the biomaterial, the biomaterial with collagen as the main component, and other products using the above-mentioned organic composition with a high collagen content as raw materials, and the method for manufacturing the organic composition with a high collagen content involved in the present invention can be marked with the same symbols, etc., and repeated description is omitted.

[0086] The method of the first embodiment of the present invention comprises the following steps (iv) to (vii): (iv) a step of quantifying the pyridinoline content (ng / mg) of collagen contained in teeth or bones extracted from mammals (pyridinoline content quantification step); (v) a step of quantifying the pentosidine content (ng / mg) of collagen contained in the tooth or bone extracted from the mammal (pentosidine content quantification step); (vi) a step of calculating a weight ratio value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) from the value PYD of the pyridoline content (ng / mg) quantified in the pyridoline content quantification step (iv) and the value PEN of the pentosidine content (ng / mg) quantified in the pentosidine content quantification step (v) (weight ratio calculation step); (vii) A step of selecting the teeth or bones extracted from mammals as biomaterials, biomaterials with collagen as a main component, or raw materials for other products when the value PYD of the pyridinoline content (ng / mg) quantified in the pyridinoline content quantification step (iv) or the value PYD of the pyridinoline content (ng / mg) quantified in the pyridinoline content quantification step (iv) and the value PpP of the weight ratio calculated in the weight ratio calculation step (vi) (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are predetermined values ​​(tooth / bone selection step).

[0087] In the pyridinoline content quantification step (iv) and the pentosidine content quantification step (v) in the method of the first embodiment of the present invention, there is no particular limitation on the method for quantifying the pyridinoline content (ng / mg) and the pentosidine content (ng / mg) of collagen contained in teeth or bones extracted from mammals, as long as they can be quantified. As such a quantitative method, for example, high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents can be used.

[0088] In the weight ratio calculation step (vi) of the method of the first embodiment of the present invention, the quantitative value PYD of the pyridinoline content (ng / mg) of collagen contained in the teeth or bones extracted from a mammal obtained in the pyridinoline content quantification step (iv) is divided by the quantitative value PEN of the pentosidine content (ng / mg) of collagen contained in the teeth or bones extracted from a mammal obtained in the pentosidine content quantification step (v), and the weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of the quantitative value of the pyridinoline content (ng / mg) to the quantitative value of the pentosidine content (ng / mg) is calculated.

[0089] In the tooth / bone selection step (vii) of the method of the first embodiment of the present invention, when the value PYD of the pyridinoline content (ng / mg) quantified in the pyridinoline content quantification step (iv), or the value PYD of the pyridinoline content (ng / mg) quantified in the pyridinoline content quantification step (iv) and the value PpP of the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated in the weight ratio calculation step (vi) are specified values, the above-mentioned tooth or bone extracted from a mammal is selected as a biomaterial, a biomaterial with collagen as the main component, or a raw material for other products.

[0090] The "prescribed value" in the tooth / bone selection step (vii) is defined by a quantitative method for the pyridinoline content (ng / mg) and the pentosidine content (ng / mg) of collagen contained in teeth or bones extracted from mammals. For example, when the pyridinoline content quantification step (iv) and the pentosidine content quantification step (v) are quantitative steps performed by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, the prescribed values ​​are PYD>100, or PYD>100 and PpP>740, respectively.

[0091] In addition, the method of the first embodiment of the present invention, like the method for producing an organic composition with a high collagen content according to the present invention, may have not only the above-mentioned pyridinoline content quantification step (iv), pentosidine content quantification step (v), weight ratio calculation step (vi) and tooth / bone selection step (vii), but may also have other steps within the scope that does not impair the characteristics of the present invention.

[0092] Next, a second embodiment of a method for identifying teeth or bones extracted from mammals as raw materials for the biomaterials of the present invention, biomaterials with collagen as the main component, or other products will be described. The method of the second embodiment of the present invention comprises the following steps (i) to (vii):

[0093] (i) a step of crushing a tooth or bone extracted from a mammal to obtain a crushed product (a crushed product preparation step); (ii) a step of subjecting the pulverized product obtained in the pulverized product preparation step (i) to a strong alkali treatment (strong alkali treatment step); (iii) a step of demineralizing the pulverized product obtained in the strong alkali treatment step (ii) under negative pressure to obtain an organic composition having a high collagen content (negative pressure demineralization step); (iv) a step of quantifying the pyridinoline content (ng / mg) of collagen contained in the organic composition having a high collagen content obtained in the demineralization step (iii) under negative pressure (pyridinoline content quantification step); (v) a step of quantifying the pentosidine content (ng / mg) of collagen contained in the organic composition having a high collagen content obtained in the demineralization step (iii) under negative pressure (pentosidine content quantification step); (vi) a step of calculating a weight ratio value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) from the value PYD of the pyridoline content (ng / mg) quantified in the pyridoline content quantification step (iv) and the value PEN of the pentosidine content (ng / mg) quantified in the pentosidine content quantification step (v) (weight ratio calculation step); (vii) A step of selecting the teeth or bones extracted from mammals as biomaterials, biomaterials with collagen as a main component, or raw materials for other products when the value PYD of the pyridinoline content (ng / mg) quantified in the pyridinoline content quantification step (iv) or the value PYD of the pyridinoline content (ng / mg) quantified in the pyridinoline content quantification step (iv) and the value PpP of the weight ratio calculated in the weight ratio calculation step (vi) (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are predetermined values ​​(tooth / bone selection step).

[0094] In addition, in the method of the second embodiment of the present invention, the above-mentioned organic composition with a high collagen content obtained from teeth or bones extracted from mammals, the biomaterial, the biomaterial with collagen as the main component and other products using the above-mentioned organic composition with a high collagen content as a raw material, the method for manufacturing the organic composition with a high collagen content involved in the present invention, and the method for identifying teeth or bones extracted from mammals as the raw material for biomaterials, biomaterials with collagen as the main component or other products, are the same or equivalent to the first embodiment. The same symbols are marked, and repeated description is omitted.

[0095] The crushed material preparation step (i), the strong alkali treatment step (ii) and the negative pressure demineralization step (iii) as the components of the method of the second embodiment of the present invention correspond to the crushed material preparation step (i), the strong alkali treatment step (ii) and the negative pressure demineralization step (iii) as the components of the method for producing an organic composition with a high collagen content according to the present invention. In addition, the pyridinoline content quantification step (iv), the pentosidine content quantification step (v), the weight ratio calculation step (vi) and the tooth / bone selection step (vii) as the components of the method of the second embodiment of the present invention correspond to the pyridinoline content quantification step (iv), the pentosidine content quantification step (v), the weight ratio calculation step (vi) and the tooth / bone selection step (vii) as the components of the first embodiment of the method for identifying teeth or bones extracted from mammals as biomaterials, biomaterials containing collagen as a main component or raw materials for other products according to the present invention.

[0096] In addition, the method of the second embodiment of the present invention can also be similar to the method of producing an organic composition with a high collagen content according to the present invention, and the method of identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as the main component, or other products according to the first embodiment of the present invention, and can have not only the above-mentioned crushed material preparation step (i), strong alkali treatment step (ii), demineralization step under negative pressure (iii), pyridoline content quantification step (iv), pentosidine content quantification step (v), weight ratio calculation step (vi) and tooth / bone selection step (vii), but can also have other steps within the scope that does not impair the characteristics of the present invention.

[0097] Next, the present invention relates to a method for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, comprising the following steps (viii) to (xiv): (viii) a step of quantifying the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal to be evaluated for potential risk of disease and / or potential health of the mammal, to obtain a quantitative value PYD (evaluation target pyridinoline content quantification step); (ix) a step of quantifying the pentosidine content (ng / mg) of collagen contained in the tooth of the mammal as the evaluation object to obtain a quantitative value PEN (evaluation object pentosidine content quantification step); (x) a step of calculating a weight ratio value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) from PYD obtained in the evaluation object pyridoline content quantitative step (viii) and PEN obtained in the evaluation object pentosidine content quantitative step (ix) (evaluation object weight ratio calculation step); (xi) a step of quantifying the pyridinoline content (ng / mg) of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PYD (reference pyridinoline content quantification step); (xii) a step of quantifying the pentosidine content (ng / mg) of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PEN (reference pentosidine content quantification step); (xiii) a step of calculating a reference value R-PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) as a weight ratio from R-PYD obtained in the reference pyridoline content quantification step (xi) and R-PEN obtained in the reference pentosidine content quantification step (xii) (reference weight ratio calculation step); (xiv) A step of comparing the PYD obtained in the evaluation object pyridinoline content quantification step (viii) with the R-PYD obtained in the reference pyridinoline content quantification step (xi), or comparing the PYD obtained in the evaluation object pyridinoline content quantification step (viii) with the R-PYD obtained in the reference pyridinoline content quantification step (xi), and the PpP calculated in the evaluation object weight ratio calculation step (x) with the R-PpP calculated in the reference weight ratio calculation step (xiii) (PYD / weight ratio comparison step).

[0098] In addition, in a method for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a mammal suffering from a disease involved in the present invention and / or whether the mammal is potentially healthy, the above-mentioned organic composition with a high collagen content obtained from teeth or bones extracted from a mammal involved in the present invention, a biomaterial, a biomaterial with collagen as a main component, and other products using the above-mentioned organic composition with a high collagen content as a raw material, a method for manufacturing an organic composition with a high collagen content involved in the present invention, and a method for identifying teeth or bones extracted from a mammal as a raw material for a biomaterial, a biomaterial with collagen as a main component, or other products, the first embodiment and the second embodiment have the same or equivalent structures, are marked with the same symbols, and repeated description is omitted.

[0099] In the present invention, "potential risk of disease" refers not only to the state of having a disease, but also to the state where the host's defense response to the disease is at its maximum and the patient has not yet become ill, and is at risk. It refers to the state where it is almost impossible to confirm that the patient has a disease based on subjective and objective observation results or existing routine examination observation results.

[0100] In addition, in the present invention, "potentially healthy" refers to a state that has not yet reached an unhealthy state, and it is almost impossible to confirm that it is an unhealthy state in subjective and objective observation results, and in existing normal examination and observation results. In the present invention, "potentially unhealthy" refers to a state that has not yet reached a healthy state, and it is almost impossible to confirm that it is a healthy state in subjective and objective observation results, and in existing normal examination and observation results.

[0101] The pyridinoline content quantification step (viii) of the evaluation object in the method of the present invention, which uses the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy, is a step equivalent to the pyridinoline content quantification step (iv) of the first embodiment and the second embodiment of the method of the present invention for identifying teeth or bones extracted from a mammal as raw materials for biomaterials, biomaterials with collagen as a main component, or other products.

[0102] In addition, the pentosidine content quantification step (ix) as an evaluation object in the method of the present invention, which uses the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy, is a step equivalent to the pentosidine content quantification step (v) of the first embodiment and the second embodiment of the method of the present invention for identifying teeth or bones extracted from a mammal as a raw material for a biomaterial, a biomaterial with collagen as a main component, or other products.

[0103] In addition, the evaluation object weight ratio calculation step (x) in the method of the present invention for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy is a step equivalent to the weight ratio calculation step (vi) of the first embodiment and the second embodiment of the method of the present invention for identifying teeth or bones extracted from a mammal as a raw material for a biomaterial, a biomaterial with collagen as a main component, or other products.

[0104] In addition, the reference pyridinoline content quantification step (xi) in the method of the present invention for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy, corresponds to the method of the present invention for identifying a raw material of a biomaterial, a biomaterial having collagen as a main component, or other product, obtained from a mammal. The present invention relates to a method for quantifying the pyridinoline content of a tooth or bone extracted from a mammal, a pyridinoline content quantification step (iv) of the first embodiment and a pyridinoline content quantification step (viii) of the evaluation object in a method for using the quantitative value of the pyridinoline content of collagen contained in the tooth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the tooth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the tooth of a mammal and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a disease in a mammal and / or whether the mammal is potentially healthy.

[0105] In addition, the reference pentosidine content quantification step (xii) in the method of the present invention for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, corresponds to the method of the present invention for identifying the raw materials of a biomaterial, a biomaterial having collagen as a main component, or other products, The first embodiment and the second embodiment of the method for extracting teeth or bones from mammals, the quantification step (v) of the pentosidine content, and the evaluation object quantification step (ix) in the method of the present invention using the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals, or the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy.

[0106] In addition, the "collagen contained in the teeth of one or more mammals selected from the group consisting of mammals not suffering from the disease, mammals suffering from the disease, healthy mammals and unhealthy mammals" in the reference pentosidine content quantification step (xii) may be "collagen contained in the teeth of one or more mammals selected from the group consisting of mammals not suffering from the disease, mammals suffering from the disease, healthy mammals and unhealthy mammals" which is different from the reference pyridinoline content quantification step (xi), or may be the same "collagen contained in the teeth of one or more mammals selected from the group consisting of mammals not suffering from the disease, mammals suffering from the disease, healthy mammals and unhealthy mammals".

[0107] Furthermore, in any process, “mammals not suffering from the disease” include “mammals not suffering from the disease themselves”, “mammals suffering from the disease” include “mammals suffering from the disease themselves”, “healthy mammals” include “healthy mammals themselves”, and “unhealthy mammals” include “unhealthy mammals themselves”.

[0108] In addition, the reference weight ratio calculation step (xiii) in the method of the present invention using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value of the pentosidine content (pyridinoline content / pentosidine content) as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy corresponds to the method of the present invention for identifying the raw material of a biomaterial, a biomaterial having collagen as a main component, or other products. , the weight ratio calculation step (vi) of the first embodiment and the second embodiment of the method for extracting teeth or bones from mammals, and the evaluation object weight ratio calculation step (x) in the method of the present invention using the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals, or the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the weight ratio calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the quantitative value of the pentosidine content (pyridinoline content / pentosidine content) as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy.

[0109] In addition, the PYD / weight ratio comparison step (xiv) in the method of the present invention for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy is a step of comparing the weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated in the evaluation object weight ratio calculation step (x) with the reference value R-PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of the weight ratio calculated in the reference weight ratio calculation step (xiii).

[0110] The method of the present invention involves using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of the mammal and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy. The pyridinoline content quantitative step (viii), the pentosidine content quantitative step (ix), the reference pyridinoline content quantitative step (xi), and the reference pentosidine content quantitative step (xii) can be performed on extracted teeth of a mammal that is a potential risk of suffering from a disease, a mammal that is not suffering from a disease, a mammal that is suffering from a disease, a healthy mammal, and an unhealthy mammal, for example, and can also be performed in a cutting, contact, or non-contact manner.

[0111] In addition, the method of the present invention for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, can also have other steps in addition to the evaluation target pyridinoline content quantitative step (viii), evaluation target pentosidine content quantitative step (ix), evaluation target weight ratio calculation step (x), reference pyridinoline content quantitative step (xi), reference pentosidine content quantitative step (xii), reference weight ratio calculation step (xiii) and PYD / weight ratio comparison step (xiv) as described above, in the same manner as the first and second embodiments of the method of the present invention for producing an organic composition having a high collagen content and the method of the present invention for identifying a tooth or bone extracted from a mammal as a raw material for a biomaterial, a biomaterial having collagen as a main component, or other products.

[0112] The method of using the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals or the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the quantitative value of the pentosidine content of collagen contained in the teeth of mammals as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, which is related to the present invention, is limited to "teeth" because both pyridinoline crosslinks and pentosidine crosslinks in type I collagen are crosslinking reactions accelerated by factors such as body heat generated by body temperature and sugars supplied through blood vessels, and are crosslinking reactions that occur uniformly throughout the body rather than locally, and among them, the collagen contained in teeth (particularly dentin) is considered to be the most accurate reflection of the accumulation of pyridinoline crosslink molecules and pentosidine crosslink molecules. Teeth are the only tissue in the body that does not participate in the metabolic cycle, and once formed, they are not absorbed and decomposed, so pyridinoline crosslink molecules and pentosidine crosslink molecules are accumulated in the collagen contained in teeth (particularly dentin).

[0113] In addition, the collagen in teeth (especially dentin) is insoluble compared to bones. When digested with pepsin under the conditions of 0.01N hydrochloric acid, 4°C, and 72 hours of treatment time, about 35% of the collagen in adult bovine bones can be dissolved, but only 5.6% of the collagen in adult bovine tooth dentin can be dissolved. In addition, under the conditions of pH 2, the insoluble collagen in skin and Achilles tendon swells to 4 to 8 times the volume, the insoluble collagen in adult bovine bones swells to 1.2 times the volume, and the insoluble collagen in adult bovine dentin does not swell at all (Nagai Yu and Fujimoto Daisaburo, eds., Collagen Experimental Methods, Kodansha Scientific, p. 21-22). Moreover, the content rate of pyridinoline cross-linked molecules in tooth dentin collagen is the highest in the body. Therefore, it is reasonable to evaluate the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy using the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the value PpP (pyridinoline content / pentosidine content) of the weight ratio calculated from the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value PEN of the pentosidine content as an indicator.

[0114] On the other hand, the basis for excluding bones is explained. As an indicator of osteoporosis, the values ​​of type I collagen C-terminal peptide (ICTP) in blood (serum) or NTx (type I collagen cross-linked N-terminal peptide) in urine are widely used, but these are substances that are released into the blood and urine as collagen decomposition products produced by the activity of matrix metalloproteinases (MMPs), and are substances containing pyridinoline cross-linked molecules. It can be seen from this that the pyridinoline cross-linked molecules and pentosidine cross-linked molecules contained in the collagen contained in bones are partially eliminated through metabolism, and when using the removed bones, since bones are organs containing a large amount of lipids derived from yellow bone marrow, degreasing is not easy, and it is very difficult to purify high-purity collagen.

[0115] Furthermore, when the pyridinoline content of collagen contained in the teeth of a mammal is high and the pentosidine content is low, the mammal has collagen that improves the individual's own homeostasis and is not likely to cause chronic inflammation, and can be evaluated as having a low risk of various potential diseases and can be judged as potentially healthy. On the other hand, when the pyridinoline content of collagen contained in the teeth of a mammal is low and the pentosidine content is high, the mammal has collagen that reduces the individual's own homeostasis and is likely to cause chronic inflammation, and can be evaluated as having a high risk of various potential diseases and can be judged as potentially unhealthy. Therefore, as an indicator for evaluating the potential risk of a mammal developing a disease and / or whether the mammal is potentially healthy, the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the value PpP (pyridinoline content / pentosidine content) of the weight ratio calculated from the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value PEN of the pentosidine content can be used. Furthermore, the method of using the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the value PpP (pyridinoline content / pentosidine content) of the weight ratio calculated from the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value PEN of the pentosidine content as an indicator for evaluating the potential risk of the mammal suffering from the disease involved in the present invention, and the method of using the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the value PpP (pyridinoline content / pentosidine content) of the weight ratio calculated from the quantitative value PYD of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value PEN of the pentosidine content as an indicator for evaluating whether the mammal involved in the present invention is potentially healthy may be independent of each other or may be related to each other.

[0116] Hereinafter, the organic composition with high collagen content obtained from teeth or bones extracted from mammals according to the present invention, biomaterials, biomaterials with collagen as the main component, and other products using the organic composition with high collagen content as raw materials, the method for producing the organic composition with high collagen content according to the present invention, the method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as the main component, or other products, and the method for using the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals, or the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the weight ratio (pyridinoline content / pentosidine content) calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of mammals and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of the disease of the mammal according to the present invention and / or whether the mammal is potentially healthy will be described based on examples. In addition, the technical scope of the present invention is not limited to the embodiments shown in these examples.

[0117] Example Example 1 Determination of the amount of pyridinoline and pentosidine in collagen from teeth (mainly dentin) of humans, cattle and pigs The following procedures were used to measure the collagen of teeth (mainly dentin) (n=62), and the Teruplug from soft tissue (n=3, The amounts of pyridinoline and pentosidine in de-telopeptide collagen from bovine dermis, telopeptide-free treatment; Olympus Terumo Biomaterials Co., Ltd.), high-grade gelatin (n=3, pig skin acid treatment, telopeptide-preserving treatment, low-endotoxin collagen; Nippi Co., Ltd.), gums of Tokachi Wakagyu (Japanese registered trademark) (n=3; Tokachi Shimizu Agricultural Cooperative Association), and muscles of Tokachi Wakagyu (Japanese registered trademark) (n=3; Tokachi Shimizu Agricultural Cooperative Association).

[0118] 1. Preparation of collagen powder [1-1] Use a pulverizer to pulverize hard tissue into particles of approximately 1 mm in size.

[0119] [1-2] The attached soft tissue was removed by stirring in a 1% sodium carbonate aqueous solution at 70°C for several hours.

[0120] [1-3] Perform demineralization. Specifically, demineralization is performed for several hours under reduced pressure using a demineralization solution containing 1M phosphoric acid and ethanol, and then replaced with the above demineralization solution diluted several times, and demineralization is performed for several hours under reduced pressure. Next, the above demineralization solution diluted 5 times is used to completely demineralize by shaking for about a day and night at room temperature. Then, washing is performed several times at room temperature using 5% ethanol.

[0121] [1-4] The demineralized hard tissue was transferred to a mortar and ground in 70% ethanol to thereby perform micronization.

[0122] [1-5] Stir in 100% ethanol to wash, degrease and dehydrate.

[0123] [1-6] The residual ethanol in the collagen is completely removed by drying to obtain a dry collagen powder.

[0124] 2. Preparation of samples for analysis [2-1] The dried collagen powder obtained in [1-6] is hydrolyzed. Specifically, the dried collagen powder is weighed in a test tube, and after being fully swollen with a small amount of 6N hydrochloric acid under degassing, an appropriate amount of 6N hydrochloric acid is added to the dried collagen, and the test tube is sealed by welding under degassing. The sealed test tube is heated overnight at 110°C for one day and night to perform hydrolysis, and then cooled to room temperature, the test tube is cut, and the remaining hydrochloric acid is removed by vacuum drying while heating to obtain a collagen hydrolyzate.

[0125] [2-2] The obtained collagen hydrolyzate was dissolved in a 10% methanol aqueous solution, centrifuged at 12,000 rpm for 5 minutes at room temperature, and the supernatant was used as a sample.

[0126] [2-3] A known amount of standard substance is added to the sample, and the extraction rate and standard curve are calculated to appropriately calibrate the collagen content.

[0127] 3. Quantification of pyridinoline and pentosidine contents and calculation of their weight ratio by high performance liquid chromatography with fluorescence detection (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents Ultrapure water (mobile phase A) containing 0.05% heptafluorobutyric acid and a 1:1 mixture of methanol and ethanol (mobile phase B) containing 0.05% formic acid were used as mobile phases, and the liquid was sent with a gradient setting for detection. Specifically, the detection column temperature was 40°C, the flow rate was 0.5 mL / min, and the ratio of mobile phase A 90% and mobile phase B 10% was started, and the liquid was sent for 0.5 minutes. Then, the mobile phase B was increased to 22% in 1 minute, and after applying the concentration gradient, the liquid was sent for 7.5 minutes while maintaining this state. Next, the mobile phase B was increased to 80% in 1 minute, and the liquid was sent for another 1 minute. Finally, after sending the liquid at a ratio of mobile phase A 10% and mobile phase B 90%, the mobile phase B was then reduced to 10%, and the mobile phase A was 90% and mobile phase B was balanced for 4 minutes. Then, for each sample, a total of 15 minutes were used for detection. The separation column used was Cadenza CD-C18, 150 mm in length and 3 mm in inner diameter, and a fluorescence detector was used for detection. The first half of 8 minutes was monitored at an excitation wavelength of 295 nm and a fluorescence wavelength of 395 nm, and the second half of 7 minutes was monitored at an excitation wavelength of 325 nm and a fluorescence wavelength of 385 nm. Through this monitoring, the contained pyridinoline was detected around the retention time of about 6.5 minutes, and the contained pentosidine was detected around the retention time of about 9.1 minutes. From the obtained pyridinoline content and pentosidine content, their weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) was calculated.

[0128] In addition, regarding human teeth, permanent teeth were used as the measurement object, but considering the formation period of permanent tooth dentin, it can be considered that dentin begins to form in the tooth germ in the jawbone at an average age of about 10 years old, and the formation of saccharification cross-links also begins with this (Research on the Eruption Period of Japanese Children's Deciduous Teeth and Permanent Teeth II, Journal of the Japanese Society of Pediatric Dentistry, 57 (3), 363-373, 2019, 363). On the other hand, regarding the teeth of the mandible of bovine SRM, it can be said that the same situation is also true after an average of 18 months of birth. Therefore, for the specified teeth of human and bovine SRM, the following Table 1 is used as a formula for converting age to months.

[0129] [Table 1] The quantitative values ​​PYD of the pyridinoline content (ng / mg) and the quantitative value PEN of the pentosidine content (ng / mg) contained in the organic composition with a high collagen content from the dentin of 18 human teeth, which were quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, and the value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of the weight ratio calculated from the quantitative value PYD of the pyridinoline content (ng / mg) and the quantitative value PEN of the pentosidine content (ng / mg) are shown in the following Table 2.

[0130] [Table 2] The quantitative values ​​of the pyridinoline content (ng / mg) PYD and the pentosidine content (ng / mg) PEN contained in the organic composition with a high collagen content in the dentin of teeth from bovine SRM19, obtained by quantification using heptafluorobutyric acid and formic acid as ion pair reagents using high performance liquid chromatography with a fluorescence detector (HPLC-Flu), and the weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative values ​​of the pyridinoline content (ng / mg) PYD and the quantitative values ​​of the pentosidine content (ng / mg) PEN are shown in the following Table 3.

[0131] [Table 3] The quantitative values ​​PYD of the pyridinoline content (ng / mg) and PEN of the pentosidine content (ng / mg) contained in the organic composition with a high collagen content in the dentin of 17 teeth of Tokachi Wakagyu (a registered trademark in Japan), which were quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, and the value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) of the weight ratio calculated from the quantitative values ​​PYD of the pyridinoline content (ng / mg) and PEN of the pentosidine content (ng / mg) are shown in Table 4 below.

[0132] [Table 4] The quantitative values ​​PYD of the pyridinoline content (ng / mg) and PEN of the pentosidine content (ng / mg) contained in the organic composition with a high collagen content from the dentin of teeth of 8 domestic pigs, which were quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, and the weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative values ​​PYD of the pyridinoline content (ng / mg) and PEN of the pentosidine content (ng / mg) are shown in the following Table 5.

[0133] [Table 5] In addition, the distribution of the quantitative values ​​of the pyridinoline content (ng / mg) of collagen contained in the organic composition with high collagen content in the dentin of teeth of 18 human cases, 36 bovine cases (including 19 bovine SRM cases and 17 Tokachi Wakagyu (Japanese registered trademark) cases) and 8 domestic pigs is shown in the figure. Figure 1 In addition, the distribution of the quantitative values ​​of PYD of the pyridinoline content (ng / mg) obtained by quantification by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents was shown in the table below: collagen contained in an organic composition with a high collagen content in the dentin of teeth from 18 humans, 36 cattle (19 of which were cattle SRM and 17 were Tokachi Wakagyu (Japanese registered trademark)), and 8 domestic pigs (n=62), Teruplug from soft tissue (n=3, from bovine dermis), High-Grade gelatin (n=3), gums of Tokachi Wakagyu (Japanese registered trademark) (n=3), and muscles of Tokachi Wakagyu (Japanese registered trademark) (n=3) was obtained. Figure 2 .

[0134] like Figure 1 As shown, the quantitative values ​​PYD of the pyridinoline content (ng / mg) of collagen contained in an organic composition with a high collagen content from the dentin of teeth as an example of hard tissue (a total of 62 cases) from 18 humans as mammals, 36 cattle as mammals and livestock (including 19 cattle SRM and 17 Tokachi Wakagyu (Japanese registered trademark)) and 8 livestock pigs as mammals were all within the range of PYD involved in the present invention.

[0135] On the other hand, Figure 2As shown, the quantitative values ​​of pyridinoline content (ng / mg) PYD of soft tissue-derived Teruplug (n=3, derived from bovine dermis), High-Grade gelatin (n=3), gums of Tokachi Wakagyu (Japanese registered trademark) (n=3), and muscles of Tokachi Wakagyu (Japanese registered trademark) (n=3) were all below 50.

[0136] As can be seen from the above, the PYD value involved in the present invention is set to PYD>100, taking into account the lower limit of the PYD of collagen contained in an organic composition with a high collagen content in the dentin of teeth as hard tissue from 18 humans as mammals, 36 cattle as mammals and 8 domestic pigs as domestic animals.

[0137] In addition, the distribution of the quantitative values ​​of pentosidine content (ng / mg) contained in the collagen-rich organic composition of the dentin of teeth from 18 humans, 36 cattle (including 19 cattle SRM and 17 Tokachi Wakagyu (Japanese registered trademark)), and 8 domestic pigs, which was quantified by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, is shown in the figure. Figure 3 .

[0138] like Figure 3 As shown, the quantitative value PEN of the pentosidine content (ng / mg) of collagen contained in an organic composition with a high collagen content in the dentin of teeth as an example of hard tissue from 44 livestock animals (including 19 bovine SRMs, 17 Tokachi Wakagyu (Japanese registered trademark), and 8 livestock pigs) was set to 0<PEN<0.4 in consideration of the upper limit value of the 44 livestock animals.

[0139] In addition, the distribution of the values ​​of the weight ratio PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) calculated from the quantitative value PYD of the pyridinoline content (ng / mg) and the quantitative value PEN of the pentosidine content (ng / mg) obtained by quantification by high performance liquid chromatography with a fluorescence detector (HPLC-Flu) using heptafluorobutyric acid and formic acid as ion pair reagents, of collagen contained in the organic composition with a high collagen content in the dentin of teeth of 62 cases in total, namely, 18 cases of humans, 36 cases of cattle (among which, 19 cases were cattle SRM and 17 cases were Tokachi Wakagyu (Japanese registered trademark)), and 8 cases of domestic pigs, is shown in the figure. Figure 4 .

[0140] like Figure 4As shown, the weight ratio of the quantitative value PYD of the pyridinoline content (ng / mg) to the quantitative value PEN of the pentosidine content (ng / mg) is PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)). Considering that livestock animals are used as indicators for PEN, PpP>740.

[0141] Example 2: Endotoxin test on collagen from bovine teeth (mainly dentin), gelatin from pig skin, and high-grade gelatin Endotoxin tests were performed on 6 samples of collagen from teeth (mainly dentin) of Tokachi Wakagyu (Japanese registered trademark, early-fattened (14-month-old) male Holstein cattle; Tokachi Shimizu Agricultural Cooperative Association), 3 samples of gelatin from porcine skin (Merck), and 3 samples of high-grade gelatin. The endotoxin test was performed by colorimetry using LAL reagent prepared from horseshoe crab amoeba cell lysate from horseshoe crab blood. The results are shown in Figure 5 .

[0142] In addition, samples of collagen from the teeth of six samples of Tokachi Wakagyu (Japanese registered trademark) were prepared by the following method.

[0143] (1) For collagen with residual mineral components, a demineralization solution containing 500 mM EDTA was used. The demineralization solution was changed while shaking at 37° C. for 2 days to completely remove the mineral components. The collagen was then washed with ultrapure water to remove EDTA.

[0144] (2) The obtained collagen is completely dried to obtain dry collagen.

[0145] (3) The dried collagen was completely degraded using proteinase K (Proteinase K, Takara Bio) at 56°C.

[0146] (4) Then, the proteinase K was inactivated by heating at 95°C for 5 minutes.

[0147] (5) Centrifuge at 12,000 rpm at room temperature and use the supernatant as the collagen sample.

[0148] like Figure 5As shown, the endotoxin level of the collagen from the teeth of 6 cases of Tokachi Wakaushi (Japanese registered trademark) was less than 0.125, the endotoxin level of the gelatin from pig skin was about 1.25 in 3 cases, and the endotoxin level of the high-grade gelatin was less than 0.125 in 3 cases. Considering that the endotoxin standard value of famotidine injection is 15EU / mg, the endotoxin standard value of thiamine hydrochloride injection is 6.0EU / mg, the endotoxin standard value of roxatidine acetate hydrochloride injection is 4.0EU / mg, the endotoxin standard value of pyridoxine hydrochloride injection is 3.0EU / mg, and the endotoxin standard value of morphine hydrochloride injection is 1.5EU / mg, it is not to mention that the endotoxin level of the collagen from the teeth of 6 cases of Tokachi Wakaushi (Japanese registered trademark) and the endotoxin level of the high-grade gelatin in 3 cases are extremely excellent, it can also be said that the endotoxin level of the gelatin from pig skin in 3 cases is also excellent.

[0149] That is, it is known that the amount of endotoxin contained in the high-collagen organic composition of the present invention is sufficiently reduced or the activity of the contained endotoxin is sufficiently reduced, or that the endotoxin is substantially contained or is contained, or the contained endotoxin is substantially inactivated or the endotoxin is inactivated.

Claims

1. An organic composition with a high collagen content, which is an organic composition with a high collagen content obtained from teeth or bones extracted from mammals, characterized in that: Having the following (i), or (i) and (ii): (i) a quantitative value PYD of the pyridinoline content (ng / mg) of collagen contained in the organic composition having a high collagen content, obtained by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, is PYD>100; (ii) The value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) of the quantitative value PYD of the collagen contained in the organic composition with a high collagen content, obtained by quantification using heptafluorobutyric acid and formic acid as ion pair reagents and a fluorescence detector, is a weight ratio of PpP>740.

2. The organic composition with high collagen content according to claim 1, characterized in that: Substantially free of endotoxin; or free of endotoxin; or substantially inactivated endotoxin; or inactivated endotoxin.

3. The organic composition with high collagen content according to claim 1, characterized in that: The mammal is one or more mammals selected from the group consisting of cattle, pigs, horses, sheep, deer, dogs, cats and humans.

4. A biomaterial using the organic composition with a high collagen content according to any one of claims 1 to 3 as a raw material, a biomaterial containing collagen as a main component, or other products.

5. A method for producing an organic composition with a high collagen content, which is used to produce the organic composition with a high collagen content according to any one of claims 1 to 3, the method comprising: A process of crushing teeth or bones extracted from mammals to obtain crushed matter; The step of subjecting the obtained pulverized material to a strong alkali treatment; and The step of demineralizing the pulverized material after the strong alkali treatment under negative pressure.

6. The manufacturing method according to claim 5, characterized in that: The step of subjecting the obtained pulverized product to a strong alkali treatment is a step of subjecting the obtained pulverized product to a strong alkali treatment under a temperature condition higher than normal temperature and lower than the boiling point of water.

7. A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as a main component, or other products, characterized in that: include: A process for quantifying the pyridinoline content (ng / mg) of collagen contained in teeth or bones extracted from mammals; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the tooth or bone extracted from the mammal; a step of calculating a weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the quantitatively obtained pyridinoline content (ng / mg) value PYD and the quantitatively obtained pentosidine content (ng / mg) value PEN; and When the quantitatively obtained value PYD of the pyridinoline content (ng / mg), or the quantitatively obtained value PYD of the pyridinoline content (ng / mg) and the calculated value PpP of the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are specified values, the tooth or bone extracted from a mammal is selected as a raw material for a biomaterial, a biomaterial with collagen as the main component, or other products.

8. A method for identifying teeth or bones extracted from mammals as raw materials for biomaterials, biomaterials with collagen as a main component, or other products, characterized in that: include: A process of crushing teeth or bones extracted from mammals to obtain crushed matter; The step of subjecting the obtained pulverized material to a strong alkali treatment; The step of demineralizing the pulverized product after the strong alkali treatment under negative pressure to obtain an organic composition with a high collagen content; A step of quantifying the pyridinoline content (ng / mg) of collagen contained in the organic composition having a high collagen content; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the obtained organic composition having a high collagen content; a step of calculating a weight ratio value PpP (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) from the quantitatively obtained pyridinoline content (ng / mg) value PYD and the quantitatively obtained pentosidine content (ng / mg) value PEN; and A process for selecting the tooth or bone extracted from a mammal as a biomaterial, a biomaterial with collagen as a main component, or a raw material for other products when the quantitatively obtained value PYD of the pyridinoline content (ng / mg), or the quantitatively obtained value PYD of the pyridinoline content (ng / mg) and the calculated value PpP of the weight ratio (pyridinoline content (ng / mg) / pentosidine content (ng / mg)) are specified values.

9. The method according to claim 7 or claim 8, characterized in that: The step of quantifying the pyridinoline content (ng / mg) of collagen contained in the obtained organic composition with a high collagen content and the step of quantifying the pentosidine content (ng / mg) of collagen contained in the obtained organic composition with a high collagen content are performed by high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, and the specified values ​​are PYD>100, or PYD>100 and PpP>740.

10. A method for using the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal, or the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the weight ratio calculated from the quantitative value of the pyridinoline content of collagen contained in the teeth of a mammal and the quantitative value of the pentosidine content as an indicator for evaluating the potential risk of a mammal suffering from a disease and / or whether the mammal is potentially healthy, characterized in that: include: A step of quantifying the pyridinoline content (ng / mg) of collagen contained in the teeth of a mammal as an evaluation target for the potential risk of disease and / or the potential health of the mammal, and obtaining a quantitative value PYD; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the tooth of the mammal as the evaluation object to obtain a quantitative value PEN; A step of calculating a weight ratio value PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) from the obtained PYD and the obtained PEN; A step of quantifying the pyridinoline content (ng / mg) of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PYD; A step of quantifying the pentosidine content (ng / mg) of collagen contained in the teeth of one or more mammals selected from the group consisting of mammals without a disease, mammals with a disease, healthy mammals, and unhealthy mammals to obtain a reference quantitative value R-PEN; A step of calculating a reference value R-PpP (pyridoline content (ng / mg) / pentosidine content (ng / mg)) of a weight ratio from the obtained R-PYD and the obtained R-PEN; and A step of comparing the PYD and the R-PYD, or comparing the PYD and the R-PYD, and the PpP and the R-PpP.

11. The method according to claim 10, characterized in that: The step of quantifying the pyridinoline content (ng / mg) to obtain a reference quantitative value R-PYD and the step of quantifying the pentosidine content (ng / mg) to obtain a reference quantitative value R-PEN are steps of quantifying the pyridinoline content (ng / mg) to obtain a reference quantitative value R-PYD and quantifying the pentosidine content (ng / mg) to obtain a reference quantitative value R-PEN by using high performance liquid chromatography (HPLC-Flu) with a fluorescence detector using heptafluorobutyric acid and formic acid as ion pair reagents, and the step of comparing the PYD with the R-PYD, or comparing the PYD with the R-PYD and comparing the PpP with the R-PpP is a step of confirming whether the PYD is PYD>100, or confirming whether the PYD is PYD>100 and the PpP is PpP>740.

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