Extracellular fluid replacement agent and method

Abstract

The objective of the present invention is to provide a liquid preparation that can be easily used at the site of pearl nucleation without damaging the health of the mantle, and to provide a method for producing mantle pieces and pearls using the liquid preparation. SOLUTION: The extracellular fluid replenisher has a pH of 7.2 to 7.5 and an osmotic pressure ratio to physiological saline of 2.0 to 4.0.

Description

[Technical Field]

[0001] The present invention relates to an extracellular fluid replenisher, a method for producing mantle flap, and a method for producing pearls. [Background technology]

[0002] Pearl production has long been an important industry in Japan, and technologies to make pearl production more efficient have been developed. The mantle membrane fragments used for transplantation into pearl oysters, known as "cells" or "pieces," play a key role in forming the pearl sac that secretes nacre within the oyster. Therefore, maintaining the health of the transplanted mantle membrane fragments is crucial for improving the quality of the pearls produced.

[0003] One known technology related to mantle flap is a blood cell activation preparation for pearl-producing bivalves that contains a mitogen (see Patent Document 1). Patent Document 1 describes that a significant increase in blood cells was observed at the site of transplantation of pearl oysters into which mantle flap immersed in the preparation was implanted, and the examples in the same document disclose that mantle flap pieces were immersed in a liquid prepared by diluting the preparation with Ringer's solution, physiological saline, phosphate-buffered physiological saline, and artificial seawater. Another known mantle cell activator contains a polypeptide with an amino acid sequence consisting of Arg-Gly-Asp (see Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 2639416 [Patent Document 2] Patent No. 3145454 Summary of the Invention [Problem to be solved by the invention]

[0005] During pearl nucleation, it is necessary to keep the mantle membrane pieces moist from the time they are excised until they are implanted into the mother-of-pearl oyster. Patent Document 1 discloses a method using physiological saline or Ringer's solution (extracellular fluid replacement solution), but the compositions of these liquids were not developed with consideration given to the body fluid composition of mollusks. Therefore, the inventors' investigations revealed that contacting the mantle membrane pieces with these liquids would impair the health of the mantle membrane pieces.

[0006] Furthermore, raw seawater, filtered seawater, or artificial seawater may be used to keep the mantle flap moist, but raw seawater may contain harmful plankton or pathogens, and filtered seawater and artificial seawater require expensive equipment and are time-consuming to produce.

[0007] The objective of the present invention is to provide a liquid preparation that does not impair the health of the mantle and can be easily used at the pearl nucleation site, and a method for producing mantle pieces and pearls using said liquid preparation. [Means for solving the problem]

[0008] That is, the present invention provides an extracellular fluid replenisher having a pH of 7.2 to 7.5 and an osmotic pressure ratio relative to physiological saline of 2.0 to 4.0. The extracellular fluid replenisher may contain sodium ions, potassium ions, magnesium ions, calcium ions, and chloride ions.

[0009] Another extracellular fluid replenisher of the present invention further contains glucose, and may further contain chondroitin sulfate and / or taurine.

[0010] Another extracellular fluid replacement agent of the present invention contains 2 to 3 wt% sodium chloride, 0.05 to 0.15 wt% potassium chloride, 0.3 to 0.4 wt% magnesium sulfate, 0.2 to 0.3 wt% magnesium chloride, 0.05 to 0.2 wt% calcium chloride, 0.01 to 0.05 wt% sodium bicarbonate, and 0.002 to 0.006 wt% sodium dihydrogen phosphate.

[0011] Another extracellular fluid supplement of the present invention contains a dye that makes it possible to distinguish between the mantle that has been in contact with the extracellular fluid supplement and the mantle that has not been in contact with the extracellular fluid supplement.

[0012] Another extracellular fluid replenisher of the present invention contains an antibiotic.

[0013] The extracellular fluid supplement of the present invention may be an extracellular fluid supplement for shellfish, an extracellular fluid supplement for bivalve mollusks, an extracellular fluid supplement for pearl oysters, or an extracellular fluid supplement for pearl oysters.

[0014] The present invention also provides a method for producing mantle membrane pieces, which comprises the steps of collecting mantle membrane from shellfish and contacting the collected mantle membrane with a liquid, wherein the liquid has a pH of 7.2 to 7.5 and an osmotic pressure ratio of the liquid to physiological saline of 2.0 to 4.0.

[0015] The present invention also provides a method for producing pearls, comprising the steps of harvesting the mantle from an oyster, bringing the harvested mantle into contact with a liquid, and transplanting the liquid-contacted mantle into an oyster other than the oyster, wherein the pH of the liquid is 7.2 to 7.5 and the osmotic pressure ratio of the liquid to physiological saline is 2.0 to 4.0.

[0016] In these methods, the step of contacting the collected mantle with a liquid may be a step of contacting the mantle with the liquid, thereby placing the mantle in an in vitro environment with an osmotic pressure of more than 520 mOsm, and the step of contacting the collected mantle with a liquid may be a step of not placing the mantle in an in vitro environment with an osmotic pressure of less than 520 mOsm. [Effects of the Invention]

[0017] The extracellular fluid replenisher of the present invention has the same pH and osmotic pressure as the body fluids of shellfish, and therefore has the effect of maintaining the health of the mantle membrane fragments with which it comes into contact. The cells contained in the mantle membrane fragments produced by the present invention are less likely to die during or immediately after transplantation, and high-quality pearls can be formed within the bodies of the transplanted mother-of-pearl oysters. [Brief explanation of the drawings]

[0018] [Figure 1] 1 shows microscopic images of pearl oyster hemocytes (A) contacted with the Example for 30 minutes and pearl oyster hemocytes (B) contacted with the Comparative Example for 30 minutes. [Figure 2] 1 shows the results of measuring the amount of active oxygen in pearl oyster blood mixed with the Example or Comparative Example. The double asterisk in the figure indicates that a statistically significant difference was obtained between the Example and Comparative Example (t-test p<0.01). [Figure 3] 1 shows microscopic images of a pearl oyster mantle piece (A) that has been in contact with the Example for 2 hours, and a pearl oyster mantle piece (B) that has been in contact with the Comparative Example for 2 hours. [Figure 4] 1 is a micrograph of pearl oyster blood cells contacted with a diluted example. [Figure 5] 1 shows a micrograph of pearl oyster blood cells that were contacted with a diluted example and then with an undiluted example. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in detail below based on the embodiments for carrying out the invention, but the present invention is not limited to these embodiments.

[0020] The extracellular fluid supplement of the present invention is an extracellular fluid supplement for mollusks. The present invention particularly provides an extracellular fluid supplement for shellfish, particularly bivalve mollusks, and more particularly for pearl oysters.

[0021] In one embodiment, the extracellular fluid replenisher is used to maintain the mantle ex vivo for use in pearl nuclei. Specifically, to prevent the mantle harvested from pearl oysters from drying out, the extracellular fluid replenisher is brought into contact with the mantle ex vivo, thereby replenishing the mantle with extracellular fluid and maintaining its moist state. The contact between the extracellular fluid replenisher and the mantle can be achieved by dripping the extracellular fluid replenisher onto the mantle, immersing the mantle in the extracellular fluid replenisher, applying the extracellular fluid replenisher to the mantle, or the like.

[0022] In one embodiment, the extracellular fluid replenisher has a pH of 7.2 to 7.5 and an osmotic pressure ratio relative to physiological saline of 2.0 to 4.0. Since physiological saline has an osmotic pressure of 285 mOsm / L, the osmotic pressure of an extracellular fluid replenisher having an osmotic pressure ratio relative to physiological saline of 2.0 to 4.0 is 570 to 1140 mOsm / L.

[0023] In another embodiment, the extracellular fluid replenisher has an osmotic pressure ratio relative to physiological saline of 2.8 to 3.8. The osmotic pressure of the extracellular fluid replenisher in this embodiment is 798 to 1083 mOsm / liter.

[0024] In another embodiment, the extracellular fluid replenisher has an osmotic pressure of 600 to 1100 mOsm / L, and the osmotic pressure ratio of the extracellular fluid replenisher to physiological saline is about 2.1 to about 3.9.

[0025] In another embodiment, the extracellular fluid replenisher has an osmolality of 800 to 1050 mOsm / L, and the osmolality ratio of the extracellular fluid replenisher to physiological saline is about 2.8 to about 3.7.

[0026] In one embodiment, the extracellular fluid replenisher contains at least sodium ions, potassium ions, magnesium ions, calcium ions, and chloride ions. These ions may be contained in the extracellular fluid replenisher to achieve the above-mentioned osmotic pressure. In one example, the extracellular fluid replenisher may contain, per liter, 350 to 550 millimoles of sodium ions, 5 to 20 millimoles of potassium ions, 40 to 70 millimoles of magnesium ions, 5 to 20 millimoles of calcium ions, and 400 to 650 millimoles of chloride ions.

[0027] In another embodiment, the extracellular fluid replenisher contains at least sodium ions, potassium ions, magnesium ions, calcium ions, chloride ions, and ionized phosphate. These ions may be contained in the extracellular fluid replenisher to achieve the above-mentioned osmolality. In one example, the extracellular fluid replenisher may contain, per liter, 350 to 550 millimoles of sodium ions, 5 to 20 millimoles of potassium ions, 40 to 70 millimoles of magnesium ions, 5 to 20 millimoles of calcium ions, 400 to 650 millimoles of chloride ions, and 2 to 5 millimoles of ionized phosphate.

[0028] Here, ionized phosphate includes dihydrogen phosphate ion, hydrogen phosphate ion, and phosphate ion, which are ionic species generated by dissociation of phosphoric acid. Since the pH of the extracellular fluid supplement in one embodiment is 7.2 to 7.5, the phosphate contained in the extracellular fluid supplement in this form is considered to be dihydrogen phosphate ion and hydrogen phosphate ion, but it may also contain phosphate ion.

[0029] In another embodiment, the extracellular fluid replenisher may contain sulfate ions in addition to the above ions. In one example, the extracellular fluid replenisher may contain 25 to 35 millimoles of sulfate ions per liter.

[0030] In one embodiment, the extracellular fluid replenisher is prepared by dissolving salts such as sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, calcium sulfate, sodium bicarbonate, and sodium dihydrogen phosphate in water to contain the above-mentioned ions.

[0031] In manufacturing the extracellular fluid replenisher, salts are dissolved in water in amounts sufficient to achieve the pH and osmotic pressure within the above-mentioned ranges. For example, 20g to 30g of sodium chloride, 0.5g to 1.5g of potassium chloride, 3g to 4g of magnesium sulfate, 2g to 3g of magnesium chloride, 0.5g to 1.5g of calcium chloride, 0.1g to 0.5g of sodium bicarbonate, and 0.02g to 0.06g of sodium dihydrogen phosphate are dissolved in 1 liter of water.

[0032] That is, in the above example, the extracellular fluid replacement agent is produced by dissolving 2 to 3 wt% sodium chloride, 0.05 to 0.15 wt% potassium chloride, 0.3 to 0.4 wt% magnesium sulfate, 0.2 to 0.3 wt% magnesium chloride, 0.05 to 0.2 wt% calcium chloride, 0.01 to 0.05 wt% sodium bicarbonate, and 0.002 to 0.006 wt% sodium dihydrogen phosphate in water.

[0033] The water used in the production of the extracellular fluid replenisher is not limited as long as it does not impair the health of the mantle when it is in contact with the extracellular fluid replenisher, and specific examples include, but are not limited to, distilled water, sterilized water, purified water, deionized water, reverse osmosis water, and ultrapure water.

[0034] Furthermore, in one embodiment, the extracellular fluid supplement may contain glucose, chondroitin sulfate, and / or taurine in addition to the above-mentioned ions. By including these components, the mantle may be maintained in a better health state ex vivo. In this embodiment, the extracellular fluid supplement may contain 0.1 g to 1.0 g of glucose, 0.01 g to 0.1 g of sodium chondroitin sulfate, and / or 0.01 g to 0.1 g of taurine per liter.

[0035] Furthermore, in another embodiment, the extracellular fluid supplement may contain an antibiotic. By containing an antibiotic, the risk of proliferation of unwanted bacteria in the extracellular fluid supplement or on the mantle surface with which the extracellular fluid supplement comes into contact can be reduced. Any antibiotic may be used, and multiple antibiotics may be combined.

[0036] Furthermore, in another embodiment, the extracellular fluid supplement may contain a dye. The dye is preferably one that does not impair the health of the mantle that has been contacted with the extracellular fluid supplement and that allows for differentiation between mantles that have been contacted with the extracellular fluid supplement and mantles that have not been contacted with the extracellular fluid supplement. This allows for easy determination of whether or not an excised mantle has been contacted with the extracellular fluid supplement.

[0037] The pigments contained in the extracellular fluid supplement of this embodiment are not limited, but specific examples include Monascus pigment, lac pigment, cochineal pigment, beetroot red, annatto pigment, paprika pigment, anthocyanin pigments, carotenoid pigments, xanthene pigments, phycobilin pigments, chlorophyll pigments, tar-based synthetic pigments, methylbromine (mercurochrome solution), etc. Examples of tar-based pigments include Food Red No. 2 (amaranth), Food Red No. 3 (erythrosine), Food Red No. 40 (Allura Red AC), Food Red No. 102 (New Coccin), Food Red No. 104 (phloxine), Food Red No. 105 (Rose Bengal), Food Red No. 106 (Acid Red), Food Yellow No. 4 (tartrazine), and Food Yellow No. 5 (Sunset Yellow FCF). In one embodiment, the extracellular fluid supplement may contain 0.01 to 0.1 wt.% of the pigment.

[0038] The extracellular fluid supplement of the present invention has the same pH and osmotic pressure as the body fluid of shellfish, thereby maintaining the health of the mantle flap. Therefore, the content of glucose, chondroitin sulfate, taurine, antibiotics, or pigments contained in one embodiment of the extracellular fluid supplement is not limited as long as the pH and osmotic pressure of the supplement are within the above-mentioned ranges.

[0039] The present invention also provides a method for producing mantle pieces. One embodiment of the method of the present invention includes the steps of harvesting mantle pieces from shellfish and contacting the harvested mantle pieces with a liquid, wherein the liquid has a pH of 7.2 to 7.5 and an osmotic pressure ratio of the liquid to physiological saline of 3.0 to 3.3.

[0040] The shellfish used in the method of the present invention may be pearl oysters. Pearl oysters refer to shellfish, particularly bivalves, used in pearl production, and specifically include Pinctada fucata, White-lipped oyster, Black-lipped oyster, Mabe, Ikecho oyster, Tridacna punctata, Tridacna gigas, and Giant clam. In the method of producing mantle pieces of the present invention, pearl oysters produced for cutting out mantle pieces for transplantation are preferably used, and these shellfish are also called "cell oysters" or "piece oysters."

[0041] The method of the present invention includes a step of harvesting the mantle from the shellfish. The shellfish is opened, the mantle is cut with scissors or the like, and cut into mantle pieces approximately 2 to 5 mm wide to obtain the mantle pieces. The mantle to be contacted with the liquid may include not only the mantle immediately after collection, but also mantle pieces obtained by cutting. That is, in the step of contacting the collected mantle with the liquid, the mantle may be contacted with cut mantle pieces, or the mantle may be contacted with the liquid immediately after collection and then cut into mantle pieces.

[0042] One embodiment of the method of the present invention for producing mantle pieces includes the steps of collecting the mantle from shellfish, contacting the collected mantle with a liquid, and cutting the mantle that has been contacted with the liquid to obtain mantle pieces, wherein the pH of the liquid is 7.2 to 7.5 and the osmotic pressure ratio of the liquid to physiological saline is 3.0 to 3.3.

[0043] Another embodiment of the method for producing mantle pieces of the present invention is a method comprising the steps of harvesting the mantle from a shellfish, cutting the harvested mantle to obtain mantle pieces, and contacting the mantle pieces with a liquid, wherein the pH of the liquid is 7.2 to 7.5 and the osmotic pressure ratio of the liquid to physiological saline is 3.0 to 3.3.

[0044] Here, the pH of the liquid to be brought into contact with the mantle (including the mantle piece) is 7.2 to 7.5, and the osmotic pressure ratio of the liquid to physiological saline is 2.0 to 4.3. Since the osmotic pressure of physiological saline is 285 mOsm / liter, the osmotic pressure of a liquid whose osmotic pressure ratio to physiological saline is 2.0 to 4.0 is 570 to 1140 mOsm / liter. The liquid to be brought into contact with the mantle is the above-mentioned extracellular fluid replenisher.

[0045] The step of contacting the harvested mantle with a liquid may be a step of placing the mantle (including the mantle fragment) in an in vitro environment with an osmotic pressure of more than 520 mOsm by contacting the mantle (including the mantle fragment) with a liquid. At the same time, the step of contacting the harvested mantle (including the mantle fragment) with a liquid may also be a step of not placing the mantle (including the mantle fragment) in an in vitro environment with an osmotic pressure of 520 mOsm or less.

[0046] Even if the mantle is brought into contact with a liquid of 570 to 1140 mOsm / L immediately after collection, the liquid is diluted by the shellfish's body fluids, seawater, etc., so the mantle is not necessarily placed in an osmotic environment of 570 to 1140 mOsm / L. As will be shown in the Examples below, it has been revealed that when shellfish tissue is placed in an in vitro environment with an osmotic pressure of 520 mOsm / L or less, its function is significantly impaired, while when placed in an environment with an osmotic pressure higher than that, the tissue function is well maintained.

[0047] In one embodiment, the step of contacting the harvested mantle with a liquid may be a step of contacting the mantle (including the mantle fragment) with a liquid, thereby placing the mantle (including the mantle fragment) in an osmotic environment of 600 to 1000 mOsm / liter ex vivo.

[0048] The present invention also provides a method for producing pearls, which comprises the steps of harvesting the mantle from an oyster, contacting the harvested mantle with a liquid, and transplanting the liquid-contacted mantle (including a mantle fragment) into a separate oyster. The separate oyster may be a mother-of-pearl oyster.

[0049] Furthermore, one embodiment of a method for producing pearls includes the steps of harvesting the mantle from an oyster, contacting the harvested mantle with a liquid, transplanting the liquid-contacted mantle (including a mantle fragment) into a separate oyster, and cultivating the transplanted separate oyster. The separate oyster may be a mother-of-pearl oyster.

[0050] The step of transplanting the mantle (including mantle fragments) that has been brought into contact with the liquid into a separate oyster from the oyster also includes the step of transplanting the mantle (including mantle fragments) that has been brought into contact with the liquid together with a pearl nucleus into a separate oyster from the oyster.

[0051] The mantle membrane fragments produced by the above-mentioned method are transplanted into pearl oysters, and the transplanted pearl oysters are cultured to produce pearls. It is preferable that the mantle membrane be kept in contact with the liquid for as long as possible from the time it is harvested from the pearl oyster until it is transplanted into the pearl oyster as a mantle membrane fragment. The production method of the present invention allows for the production of many high-quality pearls. [Example]

[0052] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0053] The following example was prepared by dissolving raw materials in distilled water to obtain the following composition: 26.22 g of sodium chloride, 1.08 g of potassium chloride, 3.18 g of magnesium sulfate, 2.2 g of magnesium chloride, 1.12 g of calcium chloride, 0.3 g of sodium bicarbonate, 0.044 g of sodium dihydrogen phosphate, 0.3 g of glucose, 0.05 g of chondroitin sulfate, 0.05 g of taurine, 0.08 g of penicillin, 0.08 g of streptomycin, 0.10 g of sulfanilamide, and 0.50 g of Scarlet 3R Water-Soluble (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) per liter of distilled water.

[0054] An exemplary extracellular fluid replenisher contains, per liter, 456 millimoles of sodium ion, 530 millimoles of chloride ion, 50 millimoles of magnesium ion, 10 millimoles of calcium ion, 4 millimoles of ionized phosphate (dihydrogen phosphate, hydrogen phosphate, and / or phosphate), and 26 millimoles of sulfate ion.

[0055] The pH of the example was measured to be 7.2 to 7.5. The osmotic pressure of the example was measured to be 1,000 mOsm / liter, and the osmotic pressure ratio to physiological saline was 3.51.

[0056] As a comparative example, Japanese Pharmacopoeia Ringer's solution (manufactured by Fuso Pharmaceutical Industries, Ltd.) was used. The pH of the comparative example was measured to be 6.17. The osmotic pressure of the comparative example was measured to be 290 mOsm / L, and the osmotic pressure ratio to physiological saline was 1.02.

[0057] Blood was collected from the adductor muscles of pearl oysters produced at the Fisheries Research Center of the Ehime Prefectural Agriculture, Forestry and Fisheries Research Institute. The collected blood was mixed with four times the volume of the Example or Comparative Example and allowed to stand for 30 minutes. The appearance of the blood cells was then observed using a phase-contrast microscope. A 100x magnified microscopic image is shown in Figure 1. Agranular blood cells in pearl oysters are known to secrete matrix components, such as fibronectin-like proteins, extracellularly, which serve as a scaffold for epithelial cells to form pearl sacs. Blood cells exposed to the Example (A) were observed to actively secrete extracellular matrix, while blood cells exposed to the Comparative Example (B) showed almost no matrix secretion.

[0058] The amount of active oxygen in the blood mixed with the Example or Comparative Example was measured using the iron-xylenol orange method. The measurement results of the amount of active oxygen are shown in Figure 2. The amount of active oxygen produced in the blood mixed with the Example was significantly lower than that produced in the blood mixed with the Comparative Example, indicating that the production of active oxygen was suppressed by the Example.

[0059] Additionally, mantle membrane fragments for pearl grafting were collected from pearl oysters produced for pearl cultivation at the Fisheries Research Center of the Ehime Prefectural Agriculture, Forestry and Fisheries Research Institute. The mantle membrane fragments were immersed in either the Example or Comparative Example for two hours, fixed in Davidson's fixative, embedded in paraffin, and tissue sections were prepared. These were stained with hematoxylin and eosin and observed under a microscope. The microscopic images are shown in Figure 3. Numerous gaps were observed in the mantle membrane fragment (B) exposed to the Comparative Example, but no gaps like those seen in (B) were observed in the mantle membrane fragment (A) exposed to the Example.

[0060] These mantle membrane pieces were used to produce pearls. The mantle membrane pieces, which had been soaked in either the Example or Comparative Example for two hours, were then transplanted into 160 mother-of-pearl oysters, along with commercially available pearl nuclei. The pearls were recovered six months later and sorted into grades (Grade 1, Grade 2, and trash) based on their quality. The results are shown in Table 1. When the Example was used, about twice as many Grade 1 pearls were produced as compared to the Comparative Example. This demonstrates that high-quality pearls can be produced using the Example.

[0061] [Table 1]

[0062] Dilutions were prepared by diluting the Example with distilled water to 90% to 10% by volume. Blood collected from the adductor muscle of a pearl oyster was mixed with four times the volume of the Example, dilution, or distilled water. After leaving the mixture to stand for 30 minutes, the appearance of the blood cells was observed using a phase-contrast microscope. A 100x magnified microscopic image is shown in Figure 4. Substrate secretion was confirmed from blood cells exposed to dilutions ranging from 100 v / v% (Example) to 50% (50 v / v%), but no active secretion of substrate was confirmed from blood cells exposed to dilutions of 40% (40 v / v%) or less.

[0063] Furthermore, blood cells immersed in diluted solutions with dilutions of 40% by volume (40 v / v %) to 10% by volume (10 v / v %) for 30 minutes were mixed with four times the volume of the Example and left to stand for another 30 minutes, and the appearance of the blood cells was observed using a phase-contrast microscope. A 100x magnified microscopic image is shown in Figure 5. No secretion of substrates was observed from any of the blood cells that came into contact with the diluted solutions of 40% by volume (40 v / v %) to 10% by volume (10 v / v %). This suggests that contact with these diluted solutions caused irreparable damage to the blood cells.

[0064] Table 3 shows the results of measuring the osmotic pressure of pearl oyster blood mixed with four-fold dilutions of 90% to 10% by volume. The osmotic pressure of the Example and the 90% to 50% by volume dilutions of blood was 600 to 1000 mOsm / L. On the other hand, the osmotic pressure of the 40% to 10% by volume liquid or the 40% to 10% by volume mixture of blood and distilled water was 520 mOsm / L or less. Therefore, while good substrate secretion was observed from hemocytes placed in an in vitro environment with an osmotic pressure of 600 to 1000 mOsm / L, hemocytes placed in an in vitro environment with an osmotic pressure of 520 mOsm / L or less were damaged to the extent that they lost their substrate secretion ability.

[0065] [Table 2]

Claims

1. collecting the mantle from the shellfish; contacting the harvested mantle with a liquid; The pH of the liquid is 7.2 to 7.5, and the osmotic pressure ratio of the liquid to physiological saline is 2.0 to 4.

0. A method for producing mantle flap.

2. The method of claim 1 further comprises a step of transplanting the mantle piece produced by the method of claim 1 into a shellfish of a different individual. How to produce pearls.

3. The step of contacting the collected mantle with a liquid is a step of placing the mantle in an in vitro osmotic environment exceeding 520 mOsm / liter by contacting the mantle with the liquid.

3. The method according to claim 1 or 2.

4. The method according to claim 3, wherein the step of contacting the harvested mantle with a liquid is a step of not placing the mantle in an in vitro environment with an osmotic pressure of 520 mOsm / liter or less.

5. The method described in claim 1 or 2, wherein the liquid contains sodium ions, potassium ions, magnesium ions, calcium ions, and chloride ions.

6. The method described in claim 5, wherein the liquid further contains glucose.

7. The method described in claim 6, wherein the liquid further contains chondroitin sulfate and / or taurine.

8. A method according to claim 1 or 2, wherein the liquid contains 2 to 3 wt% sodium chloride, 0.05 to 0.15 wt% potassium chloride, 0.3 to 0.4 wt% magnesium sulfate, 0.2 to 0.3 wt% magnesium chloride, 0.05 to 0.2 wt% calcium chloride, 0.01 to 0.05 wt% sodium bicarbonate, and 0.002 to 0.006 wt% sodium dihydrogen phosphate.

9. The liquid contains a dye, and the dye makes it possible to distinguish between the mantle that has come into contact with the liquid and the mantle that has not come into contact with the liquid.

3. The method according to claim 1 or 2.

10. The method described in claim 1 or 2, wherein the liquid contains an antibiotic.

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