JP2014183810A - Method for making useful component to be taken into body of aquatic organism, and aquatic organism obtained thereby - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for making a useful component to be taken into a body of an aquatic organism efficiently without giving any external injury on the aquatic organism, the method being capable to reduce the occurrence of death of the aquatic organism after the uptake.SOLUTION: A method for making a useful component to be taken into a body of an aquatic organism includes: a process for immersing the aquatic organism in a protease solution; and a process for immersing the aquatic organisms in a liquid containing the useful component after that. This invention allows the useful component to be taken into the majority of aquatic organisms effectively and inexpensively.

Description

  The present invention relates to a method for incorporating a useful component into an aquatic organism, and an aquatic organism obtained by using the method, and more particularly, a method for efficiently incorporating a target useful component into the body of an aquatic organism. And aquatic organisms obtained by the method.

  If the target substance (useful component) can be efficiently taken into the body of aquatic organisms such as cultured fish from the body surface, various effects can be expected for the aquatic organisms. For example, if antigens can be taken into the body of aquatic organisms, immunity against infectious diseases will increase, and if various medicinal ingredients can be taken into the body of diseased aquatic organisms, recovery will be accelerated.

  Several methods are conventionally known for incorporating useful components into the body of aquatic organisms. For example, a method in which useful ingredients are mixed with food and ingested, a method in which they are mixed with breeding water and taken in from the body surface and / or sputum, and a method in which each individual is forcibly injected by injection or the like can be mentioned.

  However, all of these methods are still insufficient from the viewpoint of substantial efficiency.

  For example, it is considered that the method of ingesting by mixing with food is seemingly efficient for treating a large amount of aquatic organisms and can keep costs low. However, since the intake of useful components of aquatic organisms is affected by the intake of food, the intake of useful components tends to vary due to differences in activity among individuals. In addition, not all of the ingested useful ingredients are reliably absorbed by the body.

  Regarding the method of mixing with the breeding water and taking it from the body surface and / or the cage, it seems that a large amount of aquatic organisms can be treated at a glance and the cost can be kept low. However, it is difficult to efficiently infiltrate useful components into the body by the protective layer on the body surface of aquatic organisms.

  The most reliable method for taking in the body is a method of forcibly injecting it by injection or the like. However, it requires an injection operation for each aquatic organism, and when the target aquatic organism is small, the work becomes particularly complicated, and it is expensive to handle a large number of fish. There is concern about the points that cannot be obtained. Furthermore, since the injection itself is traumatic to the body, it may be drowned after the injection.

  As a method of efficiently incorporating useful components into living aquatic organisms, for example, by immersing aquatic organisms in breeding water in which vaccine components are dissolved and irradiating ultrasonic waves in the environment, the body surface of the aquatic organisms and A method is also known in which a vaccine component is taken directly into the body from sputum (Patent Document 1). However, in this method, the ultrasonic wave distribution may be biased when a large amount of fish is processed.

  Thus, development of a technique that enables more efficient incorporation of useful components into aquatic organisms is desired.

Japanese Patent No. 4910188

  An object of the present invention is to solve the above-mentioned problems, and the object of the present invention is to allow useful components to be efficiently incorporated without causing trauma to aquatic organisms, and to drowning after incorporation. It is an object of the present invention to provide a method for incorporating a useful component into an aquatic organism, which can also reduce the possibility of the above, and an aquatic organism obtained using the method.

  The present inventors temporarily remove the protective tissues present on the body surface and the surface of the cocoon by causing protease to act on the body surface and the surface of the cocoon of living aquatic organisms. The present invention was completed by finding that it can be efficiently incorporated from the surface of the ridge.

The present invention is a method for incorporating a useful component into the body of an aquatic organism,
(A) a step of immersing the aquatic organism in an aqueous protease solution; and (B) a step of immersing the aquatic organism in a liquid containing a useful component after the step (A);
A method comprising

  In one embodiment, the useful ingredient is a water-soluble ingredient.

  In a further embodiment, the useful ingredient is at least one selected from the group consisting of drugs, nutrition enhancers, pigments, umami ingredients, freshness-preserving agents, antiseptics, antioxidants, deodorants, and fragrances. It is an ingredient.

  In one embodiment, the aquatic organism is a fish.

  In one embodiment, the method of the present invention further includes the step of (C) rearing in a salt water having a sodium chloride concentration of 0.5 wt% to 2 wt% after the step (B).

The present invention is also a method for incorporating useful components into the body of an aquatic organism,
(A ′) a step of immersing the aquatic organism in a liquid containing a protease and a useful component;
A method comprising

  In one embodiment, the useful ingredient is a water-soluble ingredient.

  In a further embodiment, the useful ingredient is at least one selected from the group consisting of drugs, nutrition enhancers, pigments, umami ingredients, freshness-preserving agents, antiseptics, antioxidants, deodorants, and fragrances. It is an ingredient.

  In one embodiment, the aquatic organism is a fish.

  In one embodiment, the method of the present invention further includes (C ′) after the step (A ′), the step of rearing in salt water having a sodium chloride concentration of 0.5 wt% to 2 wt%. .

  The present invention is also an aquatic organism obtained by the above method.

  ADVANTAGE OF THE INVENTION According to this invention, desired useful components, such as a vaccine component, can be efficiently taken in with respect to aquatic organisms, without distinction of freshwater fish and saltwater fish. Furthermore, since this aquatic organism is not damaged at the time of this uptake | capture, possibility that an aquatic organism will be drowned by the method of this invention can also be reduced significantly. The method of this invention can take in a useful component with respect to a large amount of aquatic organisms at once, without requiring operation for each individual of aquatic organisms. Furthermore, according to the method of the present invention, it is possible to provide a more homogeneous environment for individual aquatic organisms in taking up useful components. Thereby, the aquatic organism reinforced with useful components can be produced in large quantities.

It is the graph which showed the time-dependent change of the cumulative mortality rate (%) in each test section when testing the effect of the cold water disease vaccine in sweetfish performed in Example 1 and Comparative Examples 1 and 2. It is the graph which showed the time-dependent change of the cumulative mortality (%) in each test section when the effect with respect to the cold water disease infection in ayu performed in Reference Examples 1 and 2 and Reference Comparative Example 1 was tested. It is the graph which showed the time-dependent change of the cumulative mortality rate (%) in each test section when the effect of the streptococcal vaccine in the Japanese flounder performed in Example 2 and Comparative Examples 3 and 4. It is the graph which showed the time-dependent change of the cumulative mortality rate (%) in each test section when the effect with respect to the streptococcal infection in the flounder performed in Reference Examples 3 and 4 and Reference Comparative Example 2 was tested. It is the graph which showed the average value of the ELISA light absorbency (492nm) for the antibody titer measurement in each test section when testing the effect of the streptococcal vaccine in the ayu performed in Example 3 and Comparative Examples 5 and 6 .

  First, the 1st method for taking in a useful component in the body of the aquatic organism of this invention is demonstrated.

  In the method of the present invention, as a step (A), an aquatic organism is immersed in an aqueous protease solution.

  The target aquatic organism in the present invention refers to all living organisms that live in water. Aquatic organisms may be inhabited in either freshwater or seawater, such as freshwater fish or saltwater fish, such as those that grow in natural waters such as the ocean, rivers, lakes, ponds, and culture troughs, aquaculture facilities Any of those that grow in artificial water such as an aquarium are included. Examples of aquatic organisms include, but are not limited to, teleosts such as ayu, carp, goldfish, flounder, red sea bream, tuna, amberjack, yellowtail, puffer fish, salmon and trout; cartilage fish such as rays and sharks; shrimp and crabs Crustaceans; molluscs such as squid, octopus and shellfish; and echinoderms such as sea cucumber and sea urchin.

  The protease used in the present invention may be any enzyme that degrades protein, for example, any of endopeptidase and exopeptidase, for example, any of aminopeptidase and carboxypeptidase. Good. Furthermore, from the viewpoint of practically cost, availability, and safety, for example, a protease used for food processing may be used. Examples of proteases include papain, bromelain, actinase, and trypsin. For example, papain preparations are commercially available under the trade names such as Sumiteam P from Shin Nippon Chemical Industry Co., Ltd. and Papain w-40 from Amano Enzyme Inc.

  In the present invention, a protease aqueous solution is prepared by dissolving a predetermined amount of protease in breeding water (for example, seawater, river water, tap water, and ion exchange water).

  In addition, since the action of protease varies depending on the type, in order to temporarily and efficiently remove the protective tissue existing on the surface of the aquatic organism and / or the surface of the cocoon, an aqueous protease solution can be obtained by combining a plurality of proteases. May be prepared.

  The content (concentration) of the protease in the aqueous protease solution is not necessarily limited because it varies depending on conditions such as the type of aquatic organisms, the growth stage, the number of individuals, or the density. For example, when a protease such as the papain preparation is used. The concentration to be prepared is preferably 0.01 mg / ml to 10 mg / ml, more preferably 0.1 mg / ml to 5 mg / ml. When the concentration of the protease is less than 0.01 mg / ml, the protease does not sufficiently function in the body surface or the protective layer of the coral for aquatic organisms, and the incorporation of useful components described later cannot be sufficiently achieved. There is. On the other hand, when the concentration of the protease exceeds 10 mg / ml, the influence on the protective layer such as the body surface of the aquatic organism is increased, and the aquatic organism may be drowned. Alternatively, for example, the concentration prepared when using a protease such as the papain preparation is preferably 4 units (U) / ml to 4000 units (U) / ml, more preferably 40 units (U) / ml to 2000 units (U) / ml.

  The protease aqueous solution may contain other additives (for example, glucose) necessary for the growth of aquatic organisms, if necessary. Furthermore, in order to not reduce the activity of aquatic organisms during immersion, the protease aqueous solution is supplied with sufficient oxygen in advance and / or during immersion using means well known in the art such as an air pump. Also good. Further, a water temperature ordinarily required for the growth of the aquatic organism may be set in advance using a heater or the like.

  Such immersion of aquatic organisms in an aqueous protease solution may be performed, for example, for each individual in a predetermined volume of water tank, or may be performed for a plurality of individuals at once. In consideration of work efficiency, it is preferable to immerse a plurality of individuals at once.

  The immersion time of the aquatic organism is not necessarily limited because it varies depending on the type of aquatic organism, the growth stage, the number and density of the aquatic organism, the preparation concentration of the aqueous protease solution, and the like, but preferably 1 minute to 30 minutes, more preferably 5 minutes. ~ 20 minutes.

  By dipping in an aqueous protease solution, the protein in the protective layer present on the surface of the aquatic organism and / or the surface of the cocoon is degraded. As a result, an environment in which useful components described later can be more easily taken up is formed in aquatic organisms.

  After soaking, the aquatic organisms are removed from the aqueous protease solution. The extracted aquatic organism is directly subjected to the next step without being washed.

  After immersion in the protease aqueous solution, the aquatic organism is immersed in a liquid containing useful components.

  The useful components used in the present invention are usually taken into the body of aquatic organisms by feeding, injection or any other method for the purpose of promoting the growth of aquatic organisms, preventing or treating diseases, etc., and improving the value of products. Or a material containing the substance. Examples of such useful ingredients include drugs (including vaccine ingredients, hormone ingredients), nutrition enhancers, pigments, umami ingredients, freshness-preserving agents, anti-corruption agents, antioxidants, deodorants, and fragrances, As well as combinations thereof. The useful component is preferably water-soluble.

  In the present invention, a useful component is a liquid containing a useful component (hereinafter referred to as “useful component”) by dissolving or suspending a predetermined amount in breeding water (for example, seawater, river water, tap water, and ion-exchanged water). Liquid)) is prepared. Such a useful component liquid is either an aqueous solution or a suspension depending on the kind of useful component contained.

  The content (concentration) of useful components in the useful component solution is not necessarily limited because it varies depending on conditions such as the type of aquatic organisms, the growth stage, the number of individuals, or the density, and can be arbitrarily determined by those skilled in the art depending on the types of useful components. Concentrations can be prepared.

  The useful component liquid may contain other useful components and / or other additives necessary for the growth of aquatic organisms (for example, glucose) as necessary. Further, the useful component liquid is supplied with sufficient oxygen in advance and / or during immersion so as not to reduce the activity of aquatic organisms during immersion, using means well known in the art such as an air pump. May be. Further, a water temperature ordinarily required for the growth of the aquatic organism may be set in advance using a heater or the like.

  The immersion of aquatic organisms in such useful component liquids may also be performed for each individual in a predetermined volume of water tank, or a plurality of individuals may be performed in a lump. In consideration of work efficiency, it is preferable to immerse a plurality of individuals at once.

  The immersion time of the aquatic organism is not necessarily limited because it varies depending on the type of aquatic organism, the growth stage, the number and density of the aquatic organism, the preparation concentration of the useful component liquid, etc., but preferably 1 minute to 60 minutes, more preferably 5 to 30 minutes.

  After such immersion, aquatic organisms are removed from the useful component liquid.

  In the present invention, after being immersed in the useful component liquid, aquatic organisms are kept isotonic with body fluids of the organisms, or salt water having a sodium chloride concentration of 0.5 to 2% by weight (for example, , Seawater diluted with river water, tap water and / or ion-exchanged water, or saline prepared artificially to reach the concentration range, and isotonic or nearly isotonic with body fluids of aquatic organisms You may breed with breeding water. This is because the protective layer such as the surface of aquatic organisms removed or weakened by immersion in the protease aqueous solution can be recovered earlier.

  Such breeding is temporarily performed to recover the protective layer. The breeding period is not necessarily limited because it varies depending on the type of aquatic organism, the growth stage, the number of individuals, the density, and the like, but is, for example, about 1 to 3 days.

  In this way, useful components can be taken into the body of aquatic organisms.

  Next, the 2nd method for taking in a useful component in the body of the aquatic organism of this invention is demonstrated.

  In the second method of the present invention, an aquatic organism is immersed in a liquid containing a protease and a useful component (hereinafter referred to as “protease-useful component liquid”). Such a protease-useful component solution is either an aqueous solution or a suspension depending on the kind of useful component contained.

  Unlike the first method, there is only one kind of liquid to be immersed, and the protease and useful components coexist in the liquid. The kind and preparation concentration of the protease that can be used in the second method of the present invention, the kind and preparation concentration of useful components, and other materials that can be added are the same as in the first method.

  The immersion time of the aquatic organism is not necessarily limited because it varies depending on the type of aquatic organism, the growth stage, the number and density of the aquatic organism, the preparation concentration of the useful component aqueous solution, etc., but preferably 1 minute to 60 minutes, more preferably 5 minutes. ~ 30 minutes.

  Thereby, protein decomposition in the body surface of the aquatic organism and / or the protective layer of the cocoon, and incorporation of useful components from the body surface and / or the cocoon can be achieved by a single dipping operation.

  After immersion for a predetermined time, the aquatic organism is removed from the protease-useful component solution.

  In the present invention, after immersion in the protease-useful component solution, aquatic organisms are mixed with salt water (for example, river water, tap water and / or ions) having a sodium chloride concentration of 0.5 wt% to 2 wt%. Seawater diluted with exchange water, or saline prepared artificially so as to be in the concentration range, may be reared in the body fluid of aquatic organisms and isotonic or nearly isotonic). Such breeding techniques and breeding means are appropriately selected by those skilled in the art as those in the first method.

  In this way, useful components can be taken into the body of aquatic organisms.

  The aquatic organisms extracted through the first or second method of the present invention are not particularly washed with water, for example, in the original breeding environment (for example, the ocean, rivers, lakes, ponds, culture troughs, aquaculture) Returned to the facility, aquarium, etc.) Or when using the said aquatic organism as a fishery processed food, you may move to the next process for fishery processing.

  In addition, although the example at the time of using the living aquatic organism was demonstrated in said 1st method and 2nd method, this invention is not necessarily limited above. That is, the incorporation of useful components can be applied to aquatic organisms that are already dead after landing for the purpose of processing fisheries, for example.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

(Example 1: Incorporation of cold water disease vaccine into sweetfish)
As a vaccine-producing strain, Flavobacterium syphilophilum PH-0424 strain isolated from a cold water ayu of a farm in Hiroshima Prefecture in 2004 was used as a CGY medium (0.25 wt% cashitones, 0.15 wt% gelatin, and 0.05% by weight yeast extract) and inactivated by adding formalin to 0.3 (v / v)% after shaking culture (110 rpm) at 15 ° C. until late in logarithmic growth By doing so, a cold water disease vaccine was obtained.

  Thirty artificially-produced sweetfish with an average weight of 2.0 g were used as one of the experimental plots. An aqueous solution prepared by dissolving 1 g of papain preparation (Sumiteam P, manufactured by Shinnippon Kagaku Kogyo Co., Ltd.) in 5 L of fresh water was placed in a water tank, and the ayu of the section was immersed in the water tank at 14.0 ° C. for 15 minutes. Thereafter, all sweetfish were taken out from the water tank, and these sweetfish were immersed in 5 L of the cold water disease vaccine suspension diluted to 1/10 volume ratio with fresh water at 14.0 ° C. for 15 minutes.

  All sweetfish were removed from the vaccine suspension, transferred to a normal breeding aquarium and raised for 15 days.

After 15 days from the vaccine treatment, all sweetfish in this experimental group were immersed in a cold water fungus culture solution (PH-1037 strain culture solution; 10 ^6.8 CFU / mL) for 30 minutes. In addition, the PH-1037 strain was isolated from a cold water ayu that occurred in a farm in Hiroshima in 2010. psychromium.

  Thereafter, the sweetfish was returned to the normal breeding environment, and the breeding of the sweetfish was continued for 14 days, and the number of dead sweetfish during that period was counted. FIG. 1 shows the number of dead individuals calculated as a percentage of the cumulative number of deaths during the breeding period.

(Comparative Example 1: Incorporation of cold water disease vaccine into sweetfish)
Ayu was bred in the same manner as in Example 1 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved, but only immersed in a vaccine suspension. Thereafter, in the same manner as in Example 1, sweetfish was dipped in a culture solution of cold water disease bacteria, and then raised in a state where the sweetfish was returned to a normal breeding environment, and the number of dead sweetfish during that period was counted. FIG. 1 shows the number of dead individuals calculated as a percentage of the cumulative number of deaths during the breeding period.

(Comparative example 2: control group (non-immune group))
As a control group, sweetfish was bred in the same manner as in Example 1 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved and was not immersed in a vaccine suspension. Thereafter, in the same manner as in Example 1, sweetfish was dipped in a culture solution of cold water disease bacteria, and then raised in a state where the sweetfish was returned to a normal breeding environment, and the number of dead sweetfish during that period was counted. FIG. 1 shows the number of dead individuals calculated as a percentage of the cumulative number of deaths during the breeding period.

  As shown in FIG. 1, the cumulative mortality after 14 days was 63.3% in the control group of Comparative Example 2 and 73.3% in the experimental group of Comparative Example 1 treated with the vaccine alone. It became 44.8% in the experimental section of Example 1, and it can be seen that the effect of the vaccine was enhanced against the sweetfish in the corresponding experimental section by treating with the protease aqueous solution before the vaccine treatment.

(Reference Example 1: Effect of protease treatment on sweetfish)
Twenty artificially-produced sweetfish with an average body weight of 4.7 g were used as one of the experimental plots. An aqueous solution prepared by dissolving 0.5 g of papain preparation (Sumiteam P, manufactured by Shinnippon Kagaku Kogyo Co., Ltd.) in 10 L of fresh water was placed in a water tank, and the ayu of the section was immersed in the water tank at 15.5 ° C. for 30 minutes.

All sweetfish were taken out from the papain aqueous solution, and all sweetfish in this experimental section were immersed in a cold disease bacterial culture solution (PH-0424 strain culture solution; 10 ^6.9 CFU / mL) for 1 hour.

  Thereafter, the sweetfish was returned to the normal breeding environment, and the breeding of the sweetfish was continued for 14 days, and the number of dead sweetfish during that period was counted. FIG. 2 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

(Reference Example 2: Effect of prosthesis treatment and recovery treatment on sweetfish)
Ayu was soaked with an aqueous protease solution in the same manner as in Reference Example 1. All sweetfish were taken out from the protease aqueous solution and reared in fresh water for 1 day (recovery treatment).

  Thereafter, in the same manner as in Reference Example 1, sweetfish was dipped in a culture solution of cold water disease bacteria, and then raised in a state where the sweetfish was returned to a normal breeding environment, and the number of dead sweetfish during that period was counted. FIG. 2 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

(Reference Comparative Example 1: Control group (non-immune group))
As a control, sweetfish was bred in the same manner as in Reference Example 1 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved. Thereafter, in the same manner as in Reference Example 1, sweetfish was dipped in a culture solution of cold water disease bacteria, and then raised in a state where the sweetfish was returned to a normal breeding environment, and the number of dead sweetfish during that period was counted. FIG. 2 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

  As shown in FIG. 2, the cumulative mortality after 14 days was 45% in the control group of Reference Comparative Example 1, 70% in the experimental group of Reference Example 1 where only protease treatment was performed, and protease treatment and recovery treatment were performed. It was 20% in the experimental section of Reference Example 2. From this, the protease treatment makes it easier for ayu to take up not only the vaccine as in Example 1 but also the bacteria in the aqueous solution. It can be seen that the treatment of Reference Example 1 in which treatment was performed increased the intake of cold water disease bacteria. On the other hand, in the experimental group of Reference Example 2 in which the protease treatment and the recovery treatment were performed, it can be seen that the body surface mucus lost by the protease treatment was recovered after one day.

(Example 2: Uptake of streptococcal vaccine into Japanese flounder)
Twenty artificially produced flounder with an average weight of 28.5 g was used as one of the experimental plots. An aqueous solution prepared by dissolving 5 g of papain preparation (Sumiteam P, manufactured by Shin Nippon Chemical Industry Co., Ltd.) in 1 L of seawater was placed in a water tank, and the flounder of the section was immersed in this water tank at 19.0 ° C. for 15 minutes. Thereafter, all the flounder were removed from the aquarium, and these flounder were placed at 19.0 ° C. in 1 L of a suspension containing a streptococcal vaccine diluted with seawater to a volume ratio of 1/10 (M Buckinye; manufactured by Kyoritsu Pharmaceutical Co., Ltd.). Soaked for 30 minutes.

  All flounder was taken out from the vaccine suspension, transferred to a normal breeding aquarium and raised for 14 days.

After 14 days from the vaccine treatment, all the flounder in this experimental section were cultured in Streptococcus bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) Streptococcus iniae Psi402 strain (Matsuoka et al., Fish Disease Research, 2007, No. 42) , Pp.181-189) were each intraperitoneally injected so as to be 10 ^4.1 CFU / individual.

  Thereafter, the flounder was returned to a normal breeding environment, and flounder breeding was continued for 13 days as it was, and the number of dead flounder individuals during that period was counted. FIG. 3 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

(Comparative Example 3: Uptake of streptococcal vaccine into Japanese flounder)
Flounder was reared in the same manner as in Example 2 except that it was not immersed in the aqueous solution in which the papain preparation was dissolved, but only immersed in the vaccine suspension. Thereafter, intraperitoneal injection of the Psi402 strain was performed in the same manner as in Example 2, and then the flounder was reared in a normal breeding environment, and the number of dead flounder individuals during that period was counted. FIG. 3 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

(Comparative Example 4: Control group (non-immune group))
As a control group, flounder was bred in the same manner as in Example 2 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved and was not immersed in a vaccine suspension. Thereafter, intraperitoneal injection of the Psi402 strain was performed in the same manner as in Example 2, and then the flounder was reared in a normal breeding environment, and the number of dead flounder individuals during that period was counted. FIG. 3 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

  As shown in FIG. 3, the cumulative mortality rate after 13 days was 95.0% in the control group of Comparative Example 4 and 45.0% in the experimental group of Comparative Example 3 treated with the vaccine alone. In the experimental group of Example 2, it was 20.0%, and it was found that the effect of the vaccine was enhanced against the Japanese flounder in the corresponding experimental group by treating with the protease aqueous solution before the vaccine treatment.

(Reference Example 3: Effect of protease treatment on Japanese flounder)
Ten artificially produced flounder with an average weight of 28.5 g was used as one of the experimental plots. An aqueous solution composed of 2 L of seawater in which 2 g of papain preparation (Sumiteam P, manufactured by Shinnippon Chemical Co., Ltd.) is dissolved and diluted with fresh water to a volume ratio of 1/4 is charged into the aquarium. Was immersed at 26.5 ° C. for 15 minutes.

All flounder was taken out from the papain aqueous solution, and all flounder in this experimental group was immersed in a streptococcal culture solution (Psi402 strain culture solution: 10 ^8.1 CFU / mL) for 30 minutes.

  Thereafter, the flounder was returned to the normal breeding environment, and flounder breeding was continued for 2 weeks as it was, and the number of flounder dead during that period was counted. FIG. 4 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

(Reference Example 4: Effect of enzyme treatment and recovery treatment on Japanese flounder)
In the same manner as in Reference Example 3, flounder immersion treatment with a papain aqueous solution was performed. All flounder was taken out from the papain aqueous solution and reared in seawater for 1 day (recovery treatment).

  Thereafter, flounder was immersed in a streptococcal culture solution in the same manner as in Reference Example 3, and then flounder was raised in a normal breeding environment, and the number of dead flounder individuals during that period was counted. FIG. 4 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

(Reference Comparative Example 2: Control group (non-immune group))
As a control, flounder was bred in the same manner as in Reference Example 3 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved. Thereafter, flounder was dipped in a streptococcal culture solution in the same manner as in Reference Example 1, and then flounder was raised in a normal breeding environment, and the number of dead flounder individuals during that period was counted. FIG. 4 shows the number of dead individuals calculated as a ratio of the cumulative number of deaths during the breeding period.

  As shown in FIG. 4, the cumulative mortality rate after 2 weeks was 10% in the control group of Reference Comparative Example 2, 90% in the experimental group of Reference Example 3 where only protease treatment was performed, and protease treatment and recovery treatment. It was 10% in the experimental section of Reference Example 4 performed. From this, by the treatment with protease, flounder can easily take up not only the vaccine as in Example 2 but also bacteria in an aqueous solution. It can be seen that the treatment of Reference Example 3 in which treatment was performed increased the uptake of streptococci. On the other hand, in the experimental section of Reference Example 4 in which the protease treatment and the recovery treatment were performed, it was found that the body surface mucus lost by the protease treatment was recovered after one day.

(Example 3: Uptake of streptococcal vaccine into ayu)
25 artificially-produced sweetfish with an average body weight of 12.5 g were used as the experimental section. An aqueous solution prepared by dissolving 0.4 g of papain preparation (Sumiteam P, manufactured by Shinnippon Kagaku Kogyo Co., Ltd.) in 2 L of fresh water was placed in a water tank, and ayu of the section was immersed in the water tank at 20.0 ° C. for 15 minutes. Thereafter, all the sweetfish were taken out from the water tank, and these sweetfish were added to 1L of a suspension containing a streptococcal vaccine (M Bakynier; manufactured by Kyoritsu Pharmaceutical Co., Ltd.) diluted with fresh water to a volume ratio of 1/10 at 20.0 ° C. Soaked for 10 minutes.

  All sweetfish were taken out from the vaccine suspension, transferred to a normal breeding tank and raised for 13 days.

  After 13 days from the vaccine treatment, 5 sweetfish were taken out from this experimental group, blood was collected from the tail blood vessels, and the blood was centrifuged (5000 g, 5 minutes) to collect serum. From the obtained serum, the antibody titer was measured by ELISA using anti-Ayu IgM monoclonal antibody (Funakoshi Co., Ltd.). The obtained results are shown in FIG.

(Comparative Example 5: Uptake of streptococcal vaccine into ayu)
Ayu was bred in the same manner as in Example 3 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved, but only immersed in a vaccine suspension. Thereafter, serum was obtained in the same manner as in Example 3, and the antibody titer was measured by ELISA using an anti-Ayu IgM monoclonal antibody. The obtained results are shown in FIG.

(Comparative Example 6: control group (non-immune group))
As a control group, sweetfish was bred in the same manner as in Example 3 except that it was not immersed in an aqueous solution in which a papain preparation was dissolved and was not immersed in a vaccine suspension. Thereafter, serum was obtained in the same manner as in Example 3, and the antibody titer was measured by ELISA using an anti-Ayu IgM monoclonal antibody. The obtained results are shown in FIG.

  As shown in FIG. 5, the antibody titer was the highest in the case of the sweetfish of Example 3 treated with the protease and the vaccine, indicating that the uptake of the vaccine component was promoted by the protease treatment.

  According to the present invention, useful components can be efficiently taken into the body of aquatic organisms. This makes it possible to efficiently incorporate useful components at a low cost for the majority of aquatic organisms. Furthermore, since the present invention can be applied regardless of the growth stage of aquatic organisms, it can also be applied to, for example, juvenile fish that have conventionally been difficult to take in by injection. Thus, the present invention is useful in, for example, the fishery field.

Claims (12)

A method for incorporating useful components into the body of aquatic organisms,
(A) a step of immersing the aquatic organism in an aqueous protease solution; and (B) a step of immersing the aquatic organism in a liquid containing a useful component after the step (A);
Including the method.   The method according to claim 1, wherein the useful component is a water-soluble component.   The useful component is at least one component selected from the group consisting of a drug, a nutrient enhancer, a pigment, a savory component, a freshness-preserving agent, an antiseptic, an antioxidant, a deodorant, and a fragrance. Item 3. The method according to Item 1 or 2.   The method according to claim 1, wherein the aquatic organism is a fish.   The method according to any one of claims 1 to 4, further comprising the step of (C) rearing in a salt water having a sodium chloride concentration of 0.5 wt% to 2 wt% after the step (B). A method for incorporating useful components into the body of aquatic organisms,
(A ′) a step of immersing the aquatic organism in a liquid containing a protease and a useful component;
Including the method.   The method according to claim 6, wherein the useful component is a water-soluble component.   The useful component is at least one component selected from the group consisting of a drug, a nutrient enhancer, a pigment, a savory component, a freshness-preserving agent, an antiseptic, an antioxidant, a deodorant, and a fragrance. Item 8. The method according to Item 6 or 7.   The method according to claim 6, wherein the aquatic organism is a fish.   The method according to any one of claims 6 to 9, further comprising the step of (C ') rearing in a salt water having a sodium chloride concentration of 0.5 wt% to 2 wt% after the step (A'). Method.   An aquatic organism obtained by the method according to any one of claims 1 to 5.   An aquatic organism obtained by the method according to claim 6.

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