Material for pearl culture, nucleus insertion method and material composition for pearl culture

Abstract

The present invention provides a material for culturing pearls, a nucleus insertion method, and a material composition for culturing pearls, the material for culturing pearls comprising at least one selected from the group consisting of pearl nucleuses and mantles, and the at least one selected from the group consisting of pearl nucleuses and mantles containing a protein having an endotoxin content of 10 EU / g or less.

Description

[0001] This application is a divisional application of the international application number PCT / JP2019 / 039666, international application date October 8, 2019, Chinese application number 201980067304.0, entitled "Materials for Pearl Culture, Method for Nucleation, and Composition of Materials for Pearl Culture". Technical Field

[0002] This invention relates to a material for pearl culture, a method for inserting a pearl nucleus, and a composition of materials for pearl culture. Background Technology

[0003] Cultured pearls are produced by inserting a tissue sheet of the mantle (hereinafter also called "mantle sheet") and a pearl nucleus (hereinafter also called "nucleus") into the body of a pearl-producing oyster. This causes a pearl sac to form on the surface of the pearl nucleus, which secretes mother-of-pearl, and a nacreous layer to form on the surface of the pearl nucleus.

[0004] In particular, nucleus insertion involves inserting a nucleus, obtained by cutting other types of shells into a spherical shape, into the mother oyster's body. Therefore, the live defense response caused by the foreign object becomes a heavy burden for the mother oyster. It can be said that the quality of nucleus insertion is an important process that affects the mortality rate of the mother oyster and the quality of the pearl (whether there are spots or scratches).

[0005] To improve the compatibility between the living mother-of-pearl and the nucleus, which is a foreign object, and thus improve the survival rate of the oyster and the ratio of high-quality pearls, various living materials have been disclosed, including a nucleus coated with peptides, fibronectin, chitosan, polylactic acid, etc.

[0006] For example, a pearl culture nucleus is disclosed, characterized in that it has a coating on the surface of the nucleus with a thickness of 0.005 μm or more, which is selected from one or more of the group consisting of collagen, gelatin and their chemical derivatives (see, for example, Patent Document 1).

[0007] Furthermore, a pearl farming nucleus is disclosed, characterized in that it is formed by coating the surface of the farming nucleus with a cell scaffold organic substance (e.g., fibronectin, fetal globulin) that is non-toxic to pearl oysters and has a weaker repulsiveness than calcium crystals (e.g., see Patent Document 2).

[0008] Furthermore, a mantle cell activator for pearl farming, used for processing mantle slices from pearl oysters, is disclosed, characterized in that it contains an artificial polypeptide having an Arg-Gly-Asp sequence in the molecule and having cell adhesion activity and cell activation activity (for example, see Patent Document 5).

[0009] Furthermore, a pearl nucleus is disclosed, characterized by having a positively charged surface (for example, see Patent Document 4).

[0010] Various techniques have been proposed to obtain high-quality pearls with few blemishes and scratches on the surface of the pearls obtained.

[0011] For example, as a method to ensure high-quality mantle sheets and further improve the quality of cultured pearls, a pearl cultivation method is disclosed in which a mantle sheet obtained from the mantle of a shell and a pearl nucleus are transferred to a mother oyster to cultivate pearls. The pearl cultivation method is characterized in that the mantle sheet is obtained by isolating epithelial cells from the epithelial tissue of the mantle and artificially culturing the isolated epithelial cells in vitro (for example, see Patent Document 3).

[0012] As a pearl culture nucleus that is harmless to the mother oyster, has low rejection rate, and can significantly improve the yield of finished products, a pearl culture nucleus is disclosed. It is a nucleus that is inserted into the mother oyster for pearl culture. The nucleus is characterized in that it is formed by finely crushing and sintering the waste shells of the pearl culture mother oyster (for example, see Patent Document 6).

[0013] A method for manufacturing cultured pearls is disclosed, which involves cutting the mother oyster for pearl cultivation, inserting a pearl nucleus and culturing it, and then taking out the resulting cultured pearl. The method for manufacturing cultured pearls is characterized in that, after the mother oyster is cut and before the pearl nucleus is inserted, a nucleus with a lower density than the pearl nucleus is inserted, and the low-density nucleus is taken out after a specified period of cultivation (for example, see Patent Document 7).

[0014] Previous technical documents

[0015] Patent documents

[0016] Patent Document 1: Japanese Patent Application Publication No. 2-203724

[0017] Patent Document 2: Japanese Patent Application Publication No. 1-148135

[0018] Patent Document 3: Japanese Patent Application Publication No. 2006-304628

[0019] Patent Document 4: International Publication No. 2015 / 033972

[0020] Patent Document 5: Japanese Patent Application Publication No. 5-236848

[0021] Patent Document 6: Japanese Patent Application Publication No. 2006-296274

[0022] Patent Document 7: Japanese Patent Application Publication No. 11-056161 Summary of the Invention

[0023] The technical problem to be solved by the invention

[0024] However, the techniques described in Patent Documents 1 to 7 do not necessarily yield sufficiently high yields of high-quality pearls, and new cultivation techniques are still needed to improve the yield of high-quality pearls.

[0025] In view of the above, one embodiment of the present invention aims to solve the problem of providing a pearl culture material, a nucleation method, or a pearl culture material composition that produces pearls with a thick nacre layer and improves the yield of high-quality pearls.

[0026] means for solving technical problems

[0027] The specific methods used to solve the above problems include the following approaches.

[0028] <1> A material for pearl culture, comprising at least one selected from the group consisting of a nacreous nucleus and a mantle.

[0029] Select at least one protein from the group consisting of the above-mentioned pearl nucleus and mantle that contains an endotoxin content of less than 10 EU / g.

[0030] <2> According to the materials for pearl culture described in <1> above, wherein,

[0031] The coating amount of the aforementioned protein in the surface area of ​​the pearl nucleus or mantle is 20 pg / mm². 2 above.

[0032] <3> According to the materials for pearl culture described in <1> or <2> above, wherein,

[0033] The endotoxin levels mentioned above are below 2.5 EU / g.

[0034] <4> The pearl culture material according to any one of <1> to <3> above, wherein,

[0035] The protein described above contains at least a portion of the amino acid sequence of collagen.

[0036] <5> According to the materials for pearl culture described in <4> above, among which,

[0037] The amino acid sequence of the above collagen is the amino acid sequence of the α1 chain of type I collagen.

[0038] <6> The pearl culture material according to any one of <1> to <5> above, wherein,

[0039] At least one of the groups consisting of the above-mentioned pearl nucleus and mantle contains a protein, said protein comprising a repeating sequence of a GXY triplet separable by more than one amino acid and more than one RGD motif, and having a polydispersity of less than 20.

[0040] <7> The pearl culture material according to any one of <1> to <6> above, wherein,

[0041] The weight-average molecular weight of the above proteins obtained by gel permeation chromatography is 30 kDa to 200 kDa.

[0042] <8> The pearl culture material according to any one of <1> to <7> above, wherein,

[0043] The proteins mentioned above include those derived from recombinant yeast.

[0044] <9> A pearl culture material composition comprising any one of the pearl culture materials described in <1> to <8> above.

[0045] <10> A method for inserting a pearl nucleus, wherein the pearl nucleus is inserted into the pearl pouch of a mother oyster from which pearls have been removed, using the pearl culture material described in any one of <1> to <8> above.

[0046] <11> A method for making pearls, which uses mother-of-pearl that has been nucleated by the method described in <10> above to make pearls.

[0047] Invention Effects

[0048] According to one embodiment of the present invention, a pearl culture material, a nucleation method, or a pearl culture material composition can be provided that yields pearls with a thick nacre layer and improves the yield of high-quality pearls. Detailed Implementation

[0049] The present invention will now be described in detail. The description of the constituent elements described below is based on a representative embodiment of the present invention, but the present invention is not limited to this embodiment.

[0050] In this invention, the numerical range represented by "~" refers to the range encompassed by the values ​​recorded before and after "~" as the minimum and maximum values, respectively. Within the numerical ranges recorded in stages in this invention, the upper or lower limit value recorded within a certain numerical range can be replaced with the upper or lower limit value of other numerical ranges recorded in stages. Furthermore, within the numerical ranges recorded in this invention, the upper or lower limit value recorded within a certain numerical range can be replaced with the values ​​shown in the embodiments.

[0051] In this invention, a combination of two or more preferred methods is a more preferred method.

[0052] In this invention, the amount of each component in the composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances equivalent to each component are present in the composition.

[0053] In this invention, "peptide" refers to a collective term for compounds formed by the bonding of two or more amino acids through peptide bonds.

[0054] In this invention, "polypeptide" refers to a general term for compounds formed by peptide bonding of 10 or more amino acids. When there are 10 or more amino acids, "peptide" and "polypeptide" can be used with the same meaning.

[0055] In this invention, "protein" refers to a polypeptide with a molecular weight of 5000 or more. When the molecular weight is 5000 or more, "protein" and "polypeptide" can be used with the same meaning.

[0056] In this invention, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) of proteins are expressed in Da (Daltons).

[0057] Furthermore, unless otherwise specified, the weight-average molecular weight (Mw) and number-average molecular weight (Mn) in this invention are calculated using a gel permeation chromatography (sometimes called "GPC" or "gel permeation chromatography") analyzer (HLC-8220GPC, manufactured by TOSOH CORPORATION) with a Shodex Asahipak GS-620 7G-p column (7.5 mm inner diameter, 50 cm length, manufactured by SHOWA DENKO KK) as a solvent, using a differential refractometer, and using pullulan as a standard substance.

[0058] In this invention, "adhesiveness" refers to the "adhesiveness" or "adhesiveness" used in relation to the operability of inserting pearl culture materials into the nucleus.

[0059] In this invention, "nacre" refers to the thickness of the nacre layer, which can be a single layer or multiple layers. The thickness of the nacre layer can be determined based on the thickness of the pearl's cross-section. The thickness of the nacre layer can be confirmed by subtracting the mass of the pearl nucleus before culture from the mass of the harvested pearl; the larger this value, the thicker the nacre layer. Furthermore, the thicker the nacre layer, the superior the quality of the pearl's nacre layer.

[0060] In this invention, "whiteness" can be determined by experts engaged in pearl farming with the naked eye, but it can also be determined using measuring equipment.

[0061] In this invention, "high-quality pearl" means a pearl that at least meets the above-mentioned "nacre thickness".

[0062] In this invention, a "spot" refers to a region that is blue, gray, or brown compared to the overall color of the pearl's outer surface. In this invention, a "scratch" refers to a protrusion or dent formed on the surface of the pearl.

[0063] (Materials for pearl farming)

[0064] The pearl culture material involved in this invention comprises at least one selected from the group consisting of pearl nucleus and mantle, and the at least one selected from the group consisting of pearl nucleus and mantle contains a protein with an endotoxin content of less than 10 EU / g (hereinafter also referred to as "low endotoxin protein").

[0065] Using pearl culture materials containing low-endotoxin proteins and / or including the mantle membrane can increase the yield of high-quality pearls with thicker nacre layers. Furthermore, it can also increase the yield of high-quality pearls with high whiteness.

[0066] Pearl cultivation is carried out as follows: Typically, a tissue sheet of the mantle (mantle sheet) and a pearl nucleus are inserted into the breeding nest of a pearl-producing oyster. The tissue sheet covers the area around the nucleus to form a "pearl pouch". Secretions are secreted from the epithelial cells of the mantle that form the pearl pouch, thereby forming a nacreous layer on the surface of the pearl nucleus to make a pearl.

[0067] In particular, nucleus insertion, a surgical procedure involving cutting a spherical nucleus from another type of shell (which is considered a foreign object) and inserting it into the shell, is a huge burden on the mother oyster. Since the quality of this insertion technique directly affects the mortality rate of the oyster after insertion, as well as the quality of the pearl (whether it has blemishes or scratches), nucleus insertion can be considered a crucial process.

[0068] Spots and scratches on pearls are caused by organic matter between the pearl nucleus and the nacreous layer. The causes of these spots and scratches are said to include, for example, pigmented tissue fragments such as blood cells and reproductive cells within the pearl pouch, as well as organic matter secreted by inflamed epithelial cells. (Published by Kouseisha-kouseikaku Corporation, edited by Masahiko Awaji, Akira Furuma, and Daisuke Matsubara, "Frontiers of Pearl Research and Prospects for High-Quality Pearl Production," p. 33)

[0069] To improve the survival rate of oysters and the proportion of high-quality pearls, techniques have been proposed such as coating the pearl nucleus with substances (e.g., peptides, fibronectin, chitosan, polylactic acid) that are expected to improve biocompatibility between the living oyster and the pearl nucleus (as a foreign object) (e.g., see Patent Document 2), and adding antibiotics to the coating to kill bacteria considered to be one of the causes of inflammatory reactions (e.g., see Patent Document 1). Although the quality of pearls has been improved through these techniques, their quality is insufficient and they have not been widely used.

[0070] To further improve the biocompatibility of pearl nuclei with living mollusks, the inventors focused on endotoxins released from bacteria that remain even after sterilization and can trigger foreign body reactions when introduced into living organisms. Endotoxins are lipopolysaccharides that make up the cell walls of Gram-negative bacteria. They are known to cause various in vivo reactions, such as fever, even in very small amounts entering the bloodstream of mammals; however, there are no clear reports regarding their effects on mollusks such as mollusks.

[0071] The inventors used a nucleus and / or mantle coated with proteins that minimize endotoxins for nucleation. As a result, they found that the pearl nacre was thicker and the yield of higher-value, higher-quality pearls was increased. Furthermore, they found that the number of blemishes and scratches in the pearls was reduced, and in addition, the yield of higher-quality pearls with greater whiteness was easily increased.

[0072] Although the reasons for achieving the above effects are not yet clear, the following is a hypothesis.

[0073] It is speculated that if the nucleus and / or mantle coated with proteins that have reduced endotoxins are used for nucleation, the compatibility between the mother-of-pearl and the pearl nucleus can be improved, thereby (1) the pearl pouch is formed earlier, the nacre layer is formed earlier, the roll becomes thicker, and the pearl quality is improved. (2) It is speculated that in the pearl pouch formed by the suppression of immune response, the secretion efficiency of secretions such as calcium carbonate secreted from the tissue sheets of the mantle for the formation of the nacre layer is improved, thus the roll becomes thicker and the pearl quality is improved.

[0074] In addition, it is speculated that by increasing the compatibility between the mother-of-pearl and the pearl nucleus, pearl pouches can form earlier, reducing the probability of reproductive cells being drawn into the nacreous layer, thus making it easier to reduce blemishes and scratches. Furthermore, it is speculated that in pearl pouches formed with suppressed immune responses, organic substances such as melanin are less likely to be produced, and blemishes are also less likely to appear.

[0075] The following describes the various structures that constitute the pearl culture materials involved in this invention.

[0076] <<Protein>>

[0077] The pearl culture material involved in this invention includes a pearl nucleus and a mantle, and at least one of the pearl nucleus and mantle contains a protein with an endotoxin content of less than 10 EU / g (low endotoxin protein).

[0078] When pearl culture is carried out using at least one of the groups consisting of a nucleus and mantle containing low endotoxin proteins, the yield of high-quality pearls with thick nacre can be increased.

[0079] From the viewpoint of further increasing the yield of high-quality pearls with thicker nacre layers, the endotoxin content is preferably 8 EU / g or less, more preferably 6 EU / g or less, even more preferably 2.5 EU / g or less, and especially preferably substantially free of endotoxins.

[0080] The pearl nucleus and mantle in the pearl culture materials involved in this invention may also contain proteins with an endotoxin content exceeding 10 EU / g.

[0081] In this instruction manual, “EU / g” represents the quantitative unit of endotoxin, which is the value determined according to the biological activity of standard endotoxins specified in the United States Pharmacopeia, and indicates that 1 EU / ml ≈ 0.1 ng / ml.

[0082] In this invention, the amount of endotoxin can be determined by using a dissolving reagent (Limulus amebocyte lysate reagent) (product name: "PYROGENT"). TM The kinetic turbidimetric method (manufactured by LONZA) was used to determine the quantification.

[0083] From the viewpoint of further improving the yield of high-quality pearls, the pearl culture material involved in this invention preferably contains a pearl nucleus containing a protein with an endotoxin content of less than 10 EU / g.

[0084] There are no particular limitations on the method for setting the endotoxin content of proteins contained in the nucleus and mantle of pearl to below 10 EU / g. For example, known methods such as adsorption and ultrafiltration can be used to reduce and remove endotoxins contained in proteins such as natural gelatin used as raw materials.

[0085] Furthermore, the low-endotoxin protein can be a protein derived from a recombinant eukaryote. From the viewpoint that proteins can be obtained in an endotoxin-free environment, proteins derived from recombinant eukaryotes are preferred over low-endotoxin proteins purified from natural proteins such as gelatin, which may contain endotoxins.

[0086] Examples of hosts for recombinant eukaryotes include yeast, silkworms, Chinese hamster ovary (CHO) cells, and insect cells. However, yeast is preferred from the perspective of relatively easy access to high-yield strains and the construction of large-scale production systems.

[0087] In the pearl nucleus and mantle, at least a portion of the surface of the pearl nucleus and mantle is coated with low endotoxin proteins.

[0088] From the viewpoint of further improving the yield of high-quality pearls, the coating ratio (coating rate) of low-endotoxin proteins relative to the total surface area of ​​the pearl nucleus or mantle is preferably 20% or more, more preferably 30% or more, further preferably 40% or more, particularly preferably 50% or more, most preferably 60% or more, and very preferably 65% ​​or more. The entire surface of the pearl nucleus or mantle can be coated with low-endotoxin proteins.

[0089] Regarding the pearl nucleus and mantle membrane contained in the pearl culture material involved in this invention, from the viewpoint of further improving the yield of high-quality pearls, the protein coating amount relative to the total surface area of ​​the pearl nucleus or mantle membrane is preferably 20 pg / mm². 2 The above, more preferably 50 pg / mm 2 ~10000pg / mm 2 Further preferably 50 pg / mm 2 ~500pg / mm 2 .

[0090] Regarding the amount of low endotoxin protein coating on the surface area of ​​the pearl nucleus or mantle, after dissolving and hydrolyzing the low endotoxin protein on the surface of the pearl nucleus or mantle, the amount of amino acids contained in the protein obtained by hydrolysis was quantified using liquid chromatography-mass spectrometry (LC / MS), and determined based on the difference between the amino acids obtained by hydrolyzing the uncoated pearl nucleus or mantle.

[0091] More specifically, it can be determined using the following method.

[0092] (1) Elution of surface proteins

[0093] For example, prepare 10 pearl nuclei or mantles, immerse the pearl nuclei or mantles in about 10 mL of water at 20°C and let them stand for 18 hours to prepare a sample solution of aquaculture material.

[0094] (2) Hydrolysis of surface proteins

[0095] The aquaculture material sample solution was concentrated using a centrifugal evaporator, and 1 mL of 6 mol / L (N) hydrochloric acid was added to redissolve it. The solution was then allowed to stand at 110℃ for 22 hours.

[0096] (3) Redissolution of proteins

[0097] After passing the above 6 mol / L (N) hydrochloric acid through nitrogen gas and causing it to evaporate, 0.2 mL of 0.02 mol / L (N) dilute hydrochloric acid is added.

[0098] (4) Quantification of amino acids by liquid chromatography-mass spectrometry (LC / MS)

[0099] Using a liquid chromatography-mass spectrometry (LC / MS) apparatus (Waters Corporation, UPLC / MS (SQD)), the amino acids contained in the sample were quantified under the following conditions, and the difference between the amount of amino acids in the blank nucleus or mantle (a pearl nucleus or mantle free of low endotoxin proteins) was determined.

[0100] Column: Imtakt Corporation, Intrade amino acid 75

[0101] MS Electrospray (ESI) (Positive Mode)

[0102] The types of amino acids quantified can be appropriately selected according to the type of protein. When using Cellnest (manufactured by Fujifilm Corporation) as a low endotoxin protein, it is preferable to quantify proline (Pro).

[0103] As a method for containing low endotoxin proteins in the pearl nucleus and mantle involved in the present invention (hereinafter also referred to as the "coating method"), for example, a solution of the pearl culture material composition described later (hereinafter also referred to as the "coating solution") is prepared and coated onto the pearl nucleus or mantle by a known coating method.

[0104] There are no particular limitations on the coating method. For example, methods such as immersing the pearl nucleus or mantle in a coating solution, spraying the coating solution onto the surface of the pearl nucleus or mantle, or coating the surface of the pearl nucleus or mantle with a brush containing the coating solution can be used.

[0105] There is no particular limitation on the concentration of low endotoxin protein contained in the above coating solution. For example, it is preferably 0.0001% to 1% by mass relative to the total mass of the coating solution, and more preferably 0.0005% to 0.5% by mass.

[0106] The low-endotoxin protein contained in the pearl culture materials involved in this invention can be a freeze-dried form. When the low-endotoxin protein is a freeze-dried form, it is preferably a freeze-dried form that has been freeze-dried together with the activator described later.

[0107] The low endotoxin protein contained in the pearl culture substrate involved in this invention preferably contains at least a portion of the amino acid sequence of collagen, and more preferably contains the amino acid sequence of the collagen domain (i.e., the domain that forms a triple helix, hereinafter also simply referred to as the "collagen domain") of collagen.

[0108] The collagen domain also contains repeating sequences of the GXY triplet (described later) that can be separated by more than one amino acid and more than one RGD motif.

[0109] The proportion (total proportion) of the region derived from the amino acid sequence of collagen (e.g., the amino acid sequence of sequence number 1) relative to the full length of the amino acid sequence of the low endotoxin protein is preferably 50% or more, more preferably 60% or more, further preferably 70% or more, especially preferably 80% or more, most preferably 90% or more, and very preferably 95% or more.

[0110] There are no particular restrictions on the source of collagen; it can originate from any biological species. It can also be collagen derived from two or more biological species. Examples of collagen sources include fish (tilapia), cattle, pigs, and humans.

[0111] As mentioned above, collagen can be natural collagen contained in cowhide, pigskin, or fish skin, but it is preferably insoluble in solvents such as water. Therefore, in the extraction of collagen, treatments such as the use of proteolytic enzymes and heat-based gelation are required, resulting in a wide molecular weight distribution of the obtained collagen. Furthermore, commercially available gelatin typically has a molecular weight distribution of tens of thousands to millions, so even when using conventional methods to extract natural collagen, the likelihood of a polydispersity of less than 20 is low.

[0112] In one embodiment of the present invention, low endotoxin protein can be obtained by using fractionation methods such as size exclusion chromatography to separate and purify only protein molecules within a specific molecular weight range from collagen extracted from natural collagen sources derived from cowhide, pigskin, or fish skin.

[0113] In another embodiment of the present invention, the low-endotoxin protein can be obtained by production from a recombinant cell containing a protein having a gene incorporating a repetitive sequence of the GXY triplet described later and containing one or more RGD motifs. In this case, cells of bacteria such as *Escherichia coli*, cells of yeast such as *S. cerevisiae*, or cells of insects such as silkworms can be used as the host cell for incorporating the low-endotoxin protein; however, from the viewpoint of reducing endotoxin levels, yeast cells are preferred, and the low-endotoxin protein is more preferably a protein derived from the recombinant yeast gene.

[0114] Expression vectors can be selected from a range of known vectors based on the host and the size of the introduced protein. In the case of production by recombinant cells, proteins with high molecular weight uniformity can be obtained.

[0115] There are no particular restrictions on the type of collagen; it can be any type of collagen. For example, examples of collagen include type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, or type XXVIII collagen, etc.

[0116] The amino acid sequence of the aforementioned collagen can be any subtype of amino acid sequence from the various polypeptides that constitute the collagen. For example, in the case of type I collagen, it can be the amino acid sequence of the type I collagen α1 chain or the type I collagen α2 chain; in the case of type V collagen, it can be the amino acid sequence of the type V collagen α1 chain, the type V collagen α2 chain, or the type V collagen α3 chain.

[0117] If it is natural collagen, then from the point of view of availability, and if it is recombinant collagen, then from the point of view of manufacturing performance, the amino acid sequence of the collagen is preferably the amino acid sequence of type I collagen, and more preferably the amino acid sequence of type I collagen α1 chain.

[0118] The low endotoxin protein is preferably a polypeptide containing the amino acid sequence of sequence number 1, and more preferably a polypeptide composed of the amino acid sequence of sequence number 1.

[0119] The low endotoxin protein contained in the pearl nucleus or mantle can be a single polypeptide or a combination of two or more polypeptides.

[0120] Furthermore, the amino acid sequence of the human type I collagen α1 chain shown in Serial No. 2 contains only two RGD sequences (positions 745-747 and 1093-1095) within its full length of 1464 amino acids. When a protein prepared based on the amino acid sequence of the human type I collagen α1 chain is used as the protein involved in this invention, a more preferred protein with cell adhesion-promoting ability can be obtained by changing the number of RGD sequences to the preferred range described above.

[0121] Furthermore, it is believed that by removing the telopeptides present at both ends of the molecule and which facilitate association between collagen molecules, the association between molecules is inhibited, and the operability during nucleation is further improved.

[0122] Based on the above viewpoint, the low-endotoxin protein contained in the pearl farming material involved in this invention can include multiple (preferably 4 to 20, more preferably 6 to 16) regions, for example, of 20 to 60 amino acid lengths in the amino acid sequence of sequence number 2, containing Arg at position 745 to Asp at position 747. In this case, the multiple regions can be identical or different (i.e., although the regions surrounding RGD at positions 745 to 747 may be repeated, their boundary points may be different), and even if they are directly connected, one or more other amino acid residues can be sandwiched between them.

[0123] Preferably, each region is independently a region of 34 to 50 amino acids in length, containing Gly from position 722 to position 755. Furthermore, from the viewpoints of expression efficiency in recombinant cells, solubility in water, and inhibition of deliquescence, the total amino acid length is preferably 200 to 800 amino acids, more preferably 300 to 600 amino acids.

[0124] More specifically, the low-endotoxin protein contained in the pearl culture material involved in this invention can be the protein shown in Serial No. 1. This protein is 571 amino acids in length, including 12 regions containing approximately several dozen amino acids, namely Arg at position 745 to Asp at position 747 in Serial No. 2.

[0125] The protein constituting the low endotoxin protein preferably contains a repeating sequence of a GXY triplet that can be separated by more than one amino acid and more than one RGD motif, with a polydispersity of less than 20.

[0126] -GXY Triple-

[0127] In this invention, "GXY triplet" represents a unit of amino acid sequence containing three amino acids from the N-terminal side to the C-terminal side: "G (glycine)", "X (any amino acid other than G)" and "Y (any amino acid other than G)".

[0128] Here, "X" and "Y" independently represent any amino acid other than G. "X" and / or "Y" are preferably "P (proline)" and / or "4-hydroxyproline".

[0129] The proportion (total proportion) of the region occupied by the GXY triplet relative to the full length of the amino acid sequence of the protein involved in this invention is preferably 50% or more, more preferably 60% or more, further preferably 70% or more, especially preferably 80% or more, most preferably 90% or more, and very preferably 95% or more.

[0130] Furthermore, the GXY triplet can be repeated along the full-length amino acid sequence of low endotoxin proteins.

[0131] Among low-endotoxin proteins, those in the presence of the GXY triplet exhibit a structure similar to that of collagen proteins with high biocompatibility. Therefore, it is believed that inserting at least one of the following groups—consisting of a pearl nucleus and mantle containing low-endotoxin proteins—into the nucleus improves the yield of high-quality pearls.

[0132] In this invention, the "GXY triplet" can be linked in series without any amino acids between the GXY triplets, or it can contain one or more amino acids between the GXY triplets. Preferably, the "GXY triplet" is linked in series without any amino acids between the GXY triplets.

[0133] -RGD motif-

[0134] In this invention, the "RGD motif" represents a motif consisting of three amino acids—"R (arginine)," "G (glycine)," and "D (aspartic acid)"—present from the N-terminal to the C-terminal side. It is believed that because the "RGD motif" interferes with cell adhesion, for example, in the case of inserting a pearl nucleus containing low-endotoxin proteins, the expansion of mantle cells relative to the surface of the pearl nucleus is promoted, resulting in high-quality pearls with fewer blemishes.

[0135] In addition, the term "motif" used in relation to proteins refers to an amino acid sequence that is characterized functionally or structurally.

[0136] The low endotoxin protein may contain 1 to 100 “RGD motifs”, preferably 5 to 75 “RGD motifs”, more preferably 10 to 50 “RGD motifs”, and even more preferably 10 to 25 “RGD motifs”.

[0137] Furthermore, the "RGD motif" is preferably included in a ratio of 1 to 100 amino acids constituting the low endotoxin protein; more preferably, it is included in a ratio of 1 to 20 to 75 amino acids constituting the low endotoxin protein; even more preferably, it is included in a ratio of 1 to 30 to 60 amino acids constituting the low endotoxin protein; and even more preferably, it is included in a ratio of 1 to 45 to 55 amino acids constituting the low endotoxin protein.

[0138] By including the RGD motif within the aforementioned range in low-endotoxin proteins, cell adhesion-promoting capacity is enhanced, thereby contributing to the yield of high-quality pearls.

[0139] The “GXY triplet” and the “RGD motif” can exist in an overlapping manner. That is, the first G in the GXY triplet can be the second G in the RGD motif.

[0140] The weight-average molecular weight of the low endotoxin protein is preferably 30 kDa to 200 kDa, more preferably 30 kDa to 100 kDa, even more preferably 40 kDa to 75 kDa, and particularly preferably 50 kDa to 60 kDa. When the weight-average molecular weight of the low endotoxin protein is within the above range, an excellent balance is achieved between good water solubility and inhibition of deliquescence due to hygroscopicity.

[0141] From the viewpoint of achieving an excellent balance between good solubility in water and inhibition of deliquescence due to hygroscopicity, the number average molecular weight of low endotoxin protein is preferably 15kDa to 100kDa, more preferably 20kDa to 80kDa, even more preferably 30kDa to 60kDa, and particularly preferably 40kDa to 50kDa.

[0142] -Polydispersion-

[0143] From the viewpoint of further improving the yield of high-quality pearls and providing excellent operability during nucleation, the polydispersity of low endotoxin proteins is preferably less than 20, more preferably less than 10, even more preferably less than 5, and especially preferably less than 2.

[0144] It is believed that if the polydispersity of low-endotoxin proteins is within the aforementioned range, the interaction between the pearl nucleus and mantle tissue at the living tissue level in the mother-of-pearl is made more uniform, thereby further increasing the yield of high-quality pearls. Furthermore, in the aforementioned interactions, bonds formed by intermolecular interactions are preferentially formed compared to bonds between polar hydrophilic groups, thus hindering the absorption of moisture from the air, which is the main cause of stickiness, and improving the operability during nucleation.

[0145] In this invention, polydispersity refers to the value obtained by dividing the weight-average molecular weight (Mw) by the number-average molecular weight (Mn) (Mw / Mn).

[0146] <Pearl Nut>

[0147] The pearl culture material involved in this invention includes a pearl nucleus. The pearl nucleus is usually inserted into the mother oyster along with the mantle, and a secretion mainly composed of calcium carbonate is secreted from the epithelial cells of the mantle, thereby forming a nacreous layer on the surface of the pearl nucleus.

[0148] There are no particular restrictions on the materials used for pearl nuclei, as long as a nacreous layer forms on the surface of the nucleus. Examples include freshwater bivalves such as the *Genus Lamprotula*, *Fusconaia flava*, and *Fusconaia ebenus*. Furthermore, materials such as polypropylene, polycarbonate resins, glass, quartz, and calcium carbonate ceramics can also be used as pearl nuclei.

[0149] The size of the pearl nucleus (maximum diameter) can be appropriately selected according to the size of the target pearl, for example, it can be 4mm to 10mm.

[0150] There are no particular restrictions on the shape of the pearl nucleus; it can be spherical, hemispherical, oval, cocoon-shaped, disc-shaped, star-shaped, teardrop-shaped, or irregular in shape.

[0151] <Mantle>

[0152] The pearl culture material involved in this invention includes the mantle membrane. The mantle membrane is a small piece of the mantle membrane of the oyster (hereinafter, sometimes also referred to as "mantle membrane piece"), which is inserted into the mother oyster together with the pearl nucleus. The epithelial cells of the mantle membrane secrete a secretion with calcium carbonate as the main component, thereby forming a nacreous layer on the surface of the pearl nucleus.

[0153] There are no particular restrictions on the types of shellfish used to make the mantle, including Pinctada martensii, Pinctada maxima, Pinctada salina, Pinctada ventricosa, and snails; freshwater shellfish such as Abalone, Conch, Conch vulture, Conch simonii, and Conch brevis; and freshwater shellfish such as Pond Butterfly Mussel, Triangular Sail Mussel, and Crested Mussel. The mantle can originate from the same species as the mother shell or from a different species.

[0154] In this invention, "shells of different species from mother shells" not only includes shells that are different from mother shells in terms of taxonomic classification, but also shells that are in the same taxonomic classification as mother shells but are different in variety or system.

[0155] When using a mantle derived from a different species of oyster than the mother oyster (hereinafter also referred to as "heterogeneous mantle"), examples of combinations of mother oyster and heterogeneous mantle include: a combination of mother oyster being Pinctada martensii and heterogeneous mantle being Pteranodon davidii; a combination of mother oyster being Pinctada martensii and heterogeneous mantle being the aforementioned snail; and a combination of mother oyster being Pinctada martensii and heterogeneous mantle being the aforementioned freshwater oyster, etc.

[0156] There are no particular limitations on the type of oyster used to insert the nucleus into the pearl culture material involved in this invention; any oyster can be used that is the same as the oyster used to make the mantle.

[0157] The size of the mantle can be appropriately selected according to the size of the nacre, for example, it can be about 2mm to 4mm square (2mm×2mm to 4mm×4mm).

[0158] (Method for inserting a pearl nucleus)

[0159] The method of inserting a pearl nucleus according to the present invention can be a method of inserting (hereinafter, sometimes referred to as "re-insertion") a pearl nucleus into the pearl bag of a mother oyster from which the pearl has been removed, using the pearl culture material according to the present invention.

[0160] There are no particular restrictions on the methods for forming pearls in mother-of-pearl. They can be formed through general pearl farming methods or in the artificial environment described later.

[0161] The method for inserting a pearl nucleus according to the present invention can also be applied, for example, to the formation of pearls in in vitro systems such as test tubes, reactors, or cell culture media, and the insertion of the nucleus into the pearl pouch of the mother-of-pearl from which the pearl has been removed. In this case, the mother-of-pearl from which the pearl was removed and the mother-of-pearl from which the nucleus was inserted again can be of the same species or different species.

[0162] In the method for inserting a pearl nucleus according to the present invention, there are no particular limitations on the mother oyster, and the oysters used for making the mantle as described above can be cited. Furthermore, there are no particular limitations on the mother oyster used for re-insertion of the nucleus, and in addition to marine oysters such as Pinctada martensii, Pinctada salina, and Pinctada fusiformis, freshwater oysters such as Sailfin bream can also be cited, and any type of oyster can be preferably used.

[0163] Furthermore, the method for making pearls according to the present invention can be a method of making pearls using mother-of-pearl that has undergone nucleation by the re-nucleation method according to the present invention.

[0164] There are no particular restrictions on the methods for making pearls. Pearls can be made using general pearl farming methods, or they can be made in an artificial environment, such as in a test tube or reactor, or in an in vitro system such as a cell culture medium.

[0165] Furthermore, as one embodiment of the pearl farming material involved in the present invention, the pearl farming material involved in the present invention can be used to insert a mantle containing no protein with an endotoxin content of less than 10 EU / g into the mother oyster.

[0166] <Materials Composition for Pearl Culture>

[0167] The pearl culture material composition of this invention comprises the pearl culture materials involved in this invention. By using the pearl culture material composition of this invention for pearl culture, the yield of high-quality pearls can be improved.

[0168] There are no particular restrictions on the shape of the material composition used in pearl farming; it can be liquid, solid, or semi-solid.

[0169] Regarding the pearl farming material composition involved in this invention, without impairing the effects involved in this invention, it may contain excipients, solvents, inorganic salts such as NaCl, buffers; HEPES, PBS, etc., thickeners, pH adjusters, stabilizers, light absorbers, antibiotics; penicillin compounds, cephalosporin compounds, macrolide compounds, tetracycline compounds, neoquinolone compounds, etc., colorants; phenol red, eosin, etc., additives.

[0170] There are no particular limitations on the solvents mentioned above; examples include water, alcohol compounds, soybean oil, olive oil and other animal and vegetable oils, mineral oils and synthetic oils.

[0171] From the viewpoint of excellent compatibility with low endotoxin proteins, water is preferred as a solvent.

[0172] There are no particular limitations on the excipients mentioned above, and examples include sugar compounds such as maltose, inositol, mannitol, lactose, sucrose, and trehalose, amino acid compounds such as phenylalanine, cellulose derivatives such as hydroxypropyl cellulose, and organic acid salts such as magnesium stearate.

[0173] When the material composition for pearl culture is in solid form, it can be obtained by dissolving a low-endotoxin protein in a suitable solvent of appropriate selection and then coating it onto at least one of the pearl nucleus and mantle.

[0174] For example, physiological saline, various buffer solutions, glucose, inositol, mannitol, lactose and other sugar solutions, and ethylene glycol compounds such as ethylene glycol and polyethylene glycol are preferred as the above-mentioned solutions.

[0175] Furthermore, when the low-endotoxin protein contained in the pearl culture material composition of the present invention is a freeze-dried form, it is preferable that the pearl culture material composition also contains a suitable solvent, such as disinfectant, physiological saline, glucose solution, electrolyte solution or amino acid solution.

[0176] As a component included in the pearl farming material composition involved in this invention, it is preferred to have a reduced amount of endotoxin.

[0177] Example

[0178] The present invention will be further described in detail below through embodiments; however, the present invention is not limited to the following embodiments as long as it does not depart from its spirit. Furthermore, unless otherwise specified, "parts" and "%" refer to mass.

[0179] The coating aqueous solutions 1 to 7 were prepared as follows. The endotoxin content of the Cellnest human type I collagen-like recombinant peptide (product name: Cellnest, manufactured by Fujifilm Corporation, lyophilized form) used below was determined using the previously described method for determining endotoxin content (LONZA's "PYROGENT"). TM -5000”; below the detection limit (below 2.5 EU / g) in kinetic turbidimetric assay.

[0180] The Cellnest human type I collagen-like recombinant peptide (hereinafter also referred to as "collagen-like peptide") used below has the following structure: it is formed by cleaving four types of fragments from the human type I collagen α1 chain, which contain RGD sequences with high cell adhesion, and then linking these fragments together, with three more fragments linked together. More specifically, it consists of 571 amino acids with the amino acid sequence described in sequence number 1 and repeating GXY triplets, containing 12 RGD motifs.

[0181] Furthermore, the weight-average molecular weight (Mw), number-average molecular weight (Mn), polydispersity (Mw / Mn), and viscosity of the collagen-like peptides used below were determined according to the following methods.

[0182] An aqueous solution of Cellnest human type I collagen-like recombinant peptide (manufactured by Fujifilm Corporation) (after thawing a frozen standard) was heated at 40°C for 30 minutes to completely dissolve the collagen-like peptide. The solution was then diluted with 100 mM phosphate buffer to bring the protein concentration in the aqueous solution to 0.2% by mass. Finally, the solution was filtered through a 0.45 μm filter to prepare the sample solution.

[0183] The above sample solution was analyzed using a high-speed gel permeation chromatography (GPC) analyzer (product name: HLC-8220GPC, manufactured by TOSOHCORPORATION) under the following conditions. The pullulan-converted weight-average molecular weight (Mw) and number-average molecular weight (Mn) were determined, and the polydispersity (Mw / Mn) was calculated based on the obtained weight-average molecular weight and number-average molecular weight. The results are as follows.

[0184] Weight-average molecular weight (Mw): 56,494

[0185] Number average molecular weight (Mn): 48,787

[0186] Polydispersity (Mw / Mn): 1.16

[0187] -GPC Measurement Conditions-

[0188] Column: Shodex Asahipak GS-620 7G-p, inner diameter 7.5mm, length 50cm (manufactured by SHOWA DENKO KK)

[0189] Elution buffer: 100mM phosphate buffer (pH=6.9)

[0190] Flow rate: Sample: 1.0 mL / min, Reference value: 0.1 mL / min

[0191] Column temperature (set): 40℃

[0192] Injection volume: 100μL

[0193] Detector: RI / UV (210nm)

[0194] Carbohydrates for molecular weight correction: Pulllan (manufactured by SHOWA DENKO KK, Shodex P-82)

[0195] <<Preparation of Coating Solution 1>>

[0196] A 0.1% (w / w) coated aqueous solution was prepared by adding water for injection (manufactured by HIKARI PHARMACEUTICAL CO., LTD.) to 100 mg of Cellnest human type I collagen-like recombinant peptide (product name: Cellnest, manufactured by Fujifilm Corporation, lyophilized form). Furthermore, the Cellnest human type I collagen-like recombinant peptide has the amino acid sequence of SEQ ID NO. 1.

[0197] <<Preparation of Coating Solution 2>>

[0198] A 0.1% (w / w) coated aqueous solution of the above-mentioned recombinant peptide with an endotoxin content of 5 EU / g was prepared by adding water for injection (manufactured by HIKARI PHARMACEUTICAL CO., LTD.) and endotoxin (manufactured by the Pharmaceutical and Medical Device Regulatory Science Society of Japan, a standard endotoxin listed in the Japanese Pharmacopoeia) to 100 mg of Cellnest human type I collagen-like recombinant peptide.

[0199] <<Preparation of Coating Solution 3>>

[0200] In the preparation of the above-mentioned coating solution 2, the endotoxin (the standard endotoxin manufactured by the Pharmaceutical and Medical Device Regulatory Science Society of Japan and listed in the Japanese Pharmacopoeia) was changed to the amount added as listed in Table 1. Otherwise, a 0.1% by mass coating aqueous solution 3 with an endotoxin content of 10 EU / g of the above-mentioned recombinant peptide was prepared in the same manner as the above-mentioned coating solution 2.

[0201] <<Preparation of Coating Solution 4>>

[0202] In the preparation of the above coating solution 2, the endotoxin (the standard endotoxin manufactured by the Pharmaceutical and Medical Device Regulatory Science Society of Japan and listed in the Japanese Pharmacopoeia) was changed to the amount added as listed in Table 1. Otherwise, a 0.1% by mass coating aqueous solution 4 with an endotoxin content of 30 EU / g of the above recombinant peptide was prepared in the same manner as the above coating solution 2.

[0203] <<Preparation of Coating Solution 5>>

[0204] In the preparation of the above coating solution 2, the endotoxin (the standard endotoxin manufactured by the Pharmaceutical and Medical Device Regulatory Science Society of Japan and recorded in the Japanese Pharmacopoeia) was changed to the amount added as recorded in Table 1. Otherwise, a 0.1% by mass coating aqueous solution 5 with an endotoxin content of 100 EU / g of the above recombinant peptide was prepared in the same manner as the above coating solution 2.

[0205] <<Preparation of Coating Solution 6>>

[0206] In the preparation of the above-mentioned coating solution 2, the endotoxin (the standard endotoxin manufactured by the Pharmaceutical and Medical Device Regulatory Science Society of Japan and listed in the Japanese Pharmacopoeia) was changed to the amount added as listed in Table 1. Otherwise, a 0.1% by mass coating aqueous solution 6 with an endotoxin content of 1,000 EU / g of the above-mentioned recombinant peptide was prepared in the same manner as the above-mentioned coating solution 2.

[0207] <<Preparation of Coating Solution 7>>

[0208] In the preparation of the above-mentioned coating solution 2, the endotoxin (the standard endotoxin manufactured by the Pharmaceutical and Medical Device Regulatory Science Society of Japan and listed in the Japanese Pharmacopoeia) was changed to the amount added as listed in Table 1. Otherwise, a 0.1% by mass coating aqueous solution 7 with an endotoxin content of 10,000 EU / g of the above-mentioned recombinant peptide was prepared in the same manner as the above-mentioned coating solution 2.

[0209] <<The Creation of Coated Core 1>>

[0210] Untreated pearl nuclei (approximately 7 mm) with a diameter of 2.3 points were completely immersed in the prepared coating aqueous solution 1 and stirred at 37°C for 2 hours. Then, after filtration through a 16-mesh metal filter, the nuclei were dried for 24 hours at 25°C and 55% humidity, thus producing the coated nuclei 1.

[0211] The amount of recombinant peptide coated on the surface of the above-mentioned core 1 was determined by the following method, and the result was 339 pg / mm². 2 .

[0212] -Methods for determining the coating amount of recombinant peptides-

[0213] (1) Elution of surface proteins

[0214] Prepare at least 10 pearl nuclei or mantles, immerse the pearl nuclei or mantles in about 10 mL of water and let stand for 18 hours to prepare a culture material sample solution.

[0215] (2) Hydrolysis of surface proteins

[0216] 1 mL of 6 mol / L (N) hydrochloric acid was added to the aquaculture material sample solution and allowed to stand at 110 °C for 22 hours.

[0217] (3) Redissolution of proteins

[0218] After passing 6 mol / L (N) hydrochloric acid through nitrogen gas and allowing it to evaporate, 0.2 mL of 0.02 mol / L (N) dilute hydrochloric acid was added.

[0219] (4) Quantification of amino acids using LC / MS

[0220] Proline was quantified using LC / MS (model: UPLC / MS (SQD), manufactured by Waters Corporation). The difference between the blank nucleus (a pearl nucleus free of low endotoxin proteins) and the peptide amount was determined based on a pre-prepared calibration curve. The amount of peptide per 1 mm was also calculated. 2 The amount of coating.

[0221] <<Production of Coated Cores 2 to 7>>

[0222] In the fabrication of the coating core 1, the coating aqueous solution 1 was changed to the coating aqueous solution 2 to coating aqueous solution 7. Otherwise, the coating core 2 to coating core 7 were fabricated in the same manner as the coating core 1.

[0223] (Examples 1-1 to 1-3 and Comparative Examples 1-1 to 1-5)

[0224] The following culture experiments were conducted using coated nuclei 1 to coated nuclei 7 prepared above. The harvested pearls were evaluated as follows (1) to (3), and the evaluation results are recorded in Table 1.

[0225] [Aquaculture Experiment]

[0226] Approximately 140 Pinctada martensii oysters (natural, 2 years old) were prepared and cultured using the coated nucleus and mantle membrane prepared above. After 8 months of culture, the nucleus was removed from the oysters, and pearls were harvested.

[0227] (1) Pearl yield of the product

[0228] Pearl farming experts visually categorize harvested pearls into first-grade, second-grade, and substandard pearls. First-grade and second-grade pearls are considered high-quality and are acceptable grades for commercial purposes.

[0229] The ratio of the number of first- and second-grade pearls selected to the total number of pearls harvested is defined as "yield relative to the number of pearls harvested (yield 1)". Furthermore, the ratio of the number of first- and second-grade pearls selected to the number of pearl nuclei inserted is defined as "yield relative to the number of pearl nuclei inserted (yield 2)". The higher the values ​​of yield 1 and yield 2, the better the pearl yield of the product.

[0230] (2) Thickness of the nacreous layer (roll)

[0231] The mass of the pearl nucleus before cultivation is subtracted from the mass of the harvested pearls to calculate the mass of the resulting pearl layers, and the average mass of the pearl layers is also calculated. A higher average mass value indicates a thicker pearl layer and a superior "curl" appearance.

[0232] (3) Whiteness

[0233] Regarding whiteness, only the first-grade pearls selected in (1) above were used for evaluation. The experts in (1) above compared the whiteness of the first-grade pearls and the standard pearls with the naked eye, and evaluated the first-grade pearls according to the following criteria, and calculated the average value of each evaluation value as the whiteness. The higher the whiteness value, the better the whiteness.

[0234] -Evaluation Criteria-

[0235] 1: Pearls have a slightly bluish-creamy appearance and require bleaching and color adjustment before being processed into jewelry.

[0236] 2: Pearls have a creamy appearance. Although they need to be bleached to be processed into jewelry, they do not require color adjustment.

[0237] 3: Pearls have a very light cream color and do not require bleaching or color adjustment in order to be processed into jewelry.

[0238]

[0239] <<Preparation of Coating Coat 1 to Coating Coat 7>>

[0240] In the above-prepared coating aqueous solution 1, the outer film sheet cut into 2mm square (2mm×2mm) was completely immersed at 25°C for 1 minute to produce the coating outer film 1.

[0241] Furthermore, the above-mentioned coating aqueous solution 1 was changed to coating aqueous solution 2 to coating aqueous solution 7. Otherwise, coating membrane 2 to coating membrane 7 were manufactured in the same manner as the coating membrane 1.

[0242] Using the obtained coated mantle 1 to coated mantle 7 and untreated nucleus, the same culture experiment as in Example 1-1 was conducted. In the culture experiment using coated mantle 1 to coated mantle 3, as in Example 1-1, the obtained pearls had a thick nacre layer and excellent roll. Furthermore, the whiteness was also excellent.

[0243] On the other hand, in the culture experiments using coated mantle 4 to coated mantle 7, similar to Comparative Example 1-1, the thickness and whiteness of the nacre layer of the obtained pearls were different.

[0244] Furthermore, the results of the same culture experiment as in Example 1-1, conducted by combining the coated core 1 and the coated mantle 1, showed that, similarly to Example 1-1, the obtained pearls had a thick nacre layer and excellent roll. Furthermore, the whiteness was also excellent.

[0245] <<Preparation of Coating Solution 21>>

[0246] A 1% by weight coated aqueous solution was prepared by adding water for injection (manufactured by HIKARI PHARMACEUTICAL CO., LTD.) to 2,000 mg of Cellnest human type I collagen-like recombinant peptide (product name: Cellnest, manufactured by Fujifilm Corporation, lyophilized form).

[0247] <<Preparation of Coating Solution 22>>

[0248] A 0.025% by weight coated aqueous solution was prepared by adding 100 mg of Cellnest human type I collagen-like recombinant peptide (product name: Cellnest, manufactured by Fujifilm Corporation, lyophilized form) to water for injection (manufactured by HIKARI PHARMACEUTICAL CO., LTD.).

[0249] <<Preparation of Coating Solution 23>>

[0250] A 0.0125% by weight coated aqueous solution was prepared by adding 100 mg of Cellnest human type I collagen-like recombinant peptide (product name: Cellnest, manufactured by Fujifilm Corporation, lyophilized form) to water for injection (manufactured by HIKARI PHARMACEUTICAL CO., LTD.).

[0251] <<Preparation of Coating Solution 24>>

[0252] A 0.0625% by mass aqueous solution of 100 mg of Cellnest human type I collagen-like recombinant peptide (product name: Cellnest, manufactured by Fujifilm Corporation, lyophilized form) was prepared by adding water for injection (manufactured by HIKARI PHARMACEUTICAL CO., LTD.) to 24.

[0253] <<Production of Coated Cores 21 to 24>>

[0254] In the preparation of the above-described coating core 1, coating aqueous solutions 21 to 24 were used instead of coating aqueous solution 1. Otherwise, coating cores 21 to 24 were prepared in the same manner as coating core 1. The amount of recombinant peptides coated on the surface of the obtained coating cores 21 to 24 was determined following the same steps as described above. The coating amount of recombinant peptides in coating cores 21 to 24 is as follows.

[0255] Coating amount of recombinant peptide coated on nucleus 21: 20-130 pg / mm 2

[0256] Coating amount of recombinant peptide coated on nucleus 22: 147 pg / mm 2

[0257] Coating amount of recombinant peptide coated on nucleus 23: 83 pg / mm 2

[0258] Coating amount of recombinant peptide coated on core 24: 29 pg / mm 2

[0259] (Examples 2-1 to 2-4)

[0260] Using the coated cores 21 to 24 prepared above, a culture experiment was conducted in the same manner as in Example 1-1. The harvested pearls were evaluated in the same way as in Example 1. The evaluation results are recorded in Table 2.

[0261]

[0262] As clearly shown in Tables 1 and 2, the pearl culture materials of this invention produce pearls with a thicker nacre layer, i.e., superior "curl," compared to the comparative examples. Furthermore, the pearl culture materials of this invention exhibit superior "whiteness" and a higher yield relative to the number of nuclei inserted and the total number of harvested pearls. Using the pearl culture materials of this invention for pearl culture yields high-quality pearls.

[0263] The publication of Japanese Patent Application No. 2018-193668, filed on October 12, 2018, is incorporated herein by reference in its entirety.

[0264] All documents, patent applications and technical standards described herein are incorporated herein by reference to the same extent as those specifically and separately described therein.

Claims

1. A material for pearl culture, comprising at least one selected from the group consisting of a pearl nucleus and a mantle, at least one selected from the group consisting of the pearl nucleus and the mantle containing a protein having an endotoxin amount of 10 EU / g or less, wherein the protein is a polypeptide comprising gelatin, wherein the coating amount of the protein in the surface area of the pearl nucleus or the mantle is 20 pg / mm 2 above.

2. The material for pearl culture according to claim 1, wherein The amount of endotoxin is 2.5 EU / g or less.

3. A material for pearl culture comprising the material for pearl culture according to claim 1 or claim 2.

4. A method for inserting a pearl nucleus into a pearl bag of a mother-of-pearl shell from which a pearl has been removed, using the material for pearl culture according to claim 1 or claim 2.

5. A method for producing a pearl, using a mother-of-pearl shell into which a nucleus has been inserted by the method according to claim 4.

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