JP2013066399A - Method for immunostimulation of fish and shellfish and method for raising fish and shellfish - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for immunostimulation of fish and shellfish, which simply and inexpensively activates immunological effect of fish and shellfish.SOLUTION: The method for immunostimulation of fish and shellfish includes administering a yeast extract having ≥20 mass% content of 5'-nucleotides and ≥10 mass% content of a free amino acid to fish or feeding a feed mixed with the yeast extract to fish and shellfish. The method for raising fish and shellfish is provided.

Description

  The present invention relates to a method for immunizing fish and shellfish and a method for raising fish and shellfish.

In recent years, many types of seafood have been artificially raised (cultured) for food and ornamental purposes. In artificial breeding, breeding is usually carried out in a more dense state than when seafood inhabit natural rivers and lakes. In such a case, improvement in production efficiency is achieved, but on the other hand, if an infection caused by bacteria or viruses occurs, the rate of infectious disease is high in fish and shellfish raised in the same area. There was a problem.
In recent years, drugs such as antibiotics and vaccines for fish and shellfish have been used for the purpose of preventing infection with bacteria and viruses. However, there are problems with many viruses for which vaccines have not yet been developed, problems with the generation of bacteria and viruses that acquire drug resistance, the problem of rising breeding costs due to drug prices, and the use of drugs in edible seafood As a result, there were safety issues, and further solutions were required.

Therefore, recently, a method has been proposed in which a naturally occurring substance having an immunostimulatory action is administered to fish and shellfish. Β-glucan is widely known as a substance having an immunostimulatory action.
For example, in Cited Document 1, a yeast extract composition containing 5′-nucleotides, free amino acids, β-glucan and mannan has a feeding promoting action in piglets, calves and chicks, and in mice and guinea pigs. It is described as having an immunopotentiating effect.

JP 7-184595 A

  As described above, there is a problem that there are many viruses for which no vaccine has been developed in the breeding of seafood, but countermeasures are urgently needed because there are few effective vaccines especially in crustaceans. For example, a typical virus that infects crustaceans is White Spot Syndrome Virus (WSSV). WSSV is a highly infectious and lethal virus in crustaceans such as shrimp and is transmitted not only by vertical infection but also by horizontal infection. It was.

However, the immunostimulatory yeast extract composition described in Cited Document 1 is whether or not the fish and shellfish acquire antiviral ability and antiviral ability against the viruses as described above when administered to fish and shellfish. That is, it is not clear whether fish and shellfish have immunostimulatory ability.
This invention is made | formed in view of the said situation, Comprising: It aims at providing the immune activation method of fish and shellfish which can activate the immune action of fish and shellfish simply and cheaply.

  As a result of diligent research to solve the above problems, the present inventors administer a yeast extract containing 5′-nucleotides and a free amino acid in a specific ratio, or feed a feed containing the yeast extract. Thus, it was found that the immune action of seafood can be activated easily and inexpensively, and the present invention has been completed.

That is, the present invention provides a method for immunizing fish and shellfish and a method for raising fish and shellfish having the following characteristics.
(1) administering a yeast extract having a 5′-nucleotide content of 20% by mass or more and a free amino acid content of 10% by mass or feeding a feed containing the yeast extract. A method for immunostimulating fish and shellfish characterized by the above.
(2) The method for immune activation of fish and shellfish according to (1), wherein the 5′-nucleotides contain inosinic acid and guanylic acid.
(3) The method for immune activation of seafood according to (1) or (2), wherein the contents of β-glucan and mannan are each less than 1% by mass.
(4) The method for immunizing fish and shellfish according to any one of (1) to (3), wherein the fish and shellfish are carp or shrimp.
(5) The method for immunizing fish and shellfish according to any one of (1) to (4), wherein the yeast extract is a yeast extract obtained from one or more selected from the group consisting of torula yeast, beer yeast and baker's yeast. .
(6) administering a yeast extract having a 5'-nucleotide content of 20% by mass or more and a free amino acid content of 10% by mass or feeding a feed containing the yeast extract. A method for raising seafood, characterized by

  According to the seafood immunity activation method and seafood breeding method of the present invention, a yeast extract containing 5′-nucleotides and a free amino acid in a specific ratio is administered or a feed containing the yeast extract is fed. By this, it becomes possible to activate the immune action of seafood simply and inexpensively. Therefore, by using the immunostimulation method of the present invention, it is possible to make fish and shellfish resistant to infectious diseases caused by bacteria, viruses, etc., so that it is possible to achieve an improvement in production efficiency in industrial production of fish and shellfish.

It is a graph which shows the survival rate of the prawn which infected Vibrio nigripulchritudo in Experimental example 1. FIG. It is a graph which shows the survival rate of the prawn which infected with White Spot Syndrome Virus in Experimental example 1. FIG. It is a graph which shows the result of the expression analysis in the head kidney tissue of the carp type I interferon gene in Experimental example 2. It is a graph which shows the result of the expression analysis in the head kidney tissue of the carp interferon-gamma1 gene in Experimental example 2. It is a graph which shows the result of the expression analysis in the head kidney tissue of the carp interferon gamma2 gene in Experimental example 2.

In the present invention, the seafood is not particularly limited, and may be any of fish, crustaceans, shellfish, cephalopods and the like. Specifically, freshwater fish such as carp, ayu, trout, crucian and salmon; saltwater fish such as yellowtail, thailand, amberjack, puffer fish, tuna and horse mackerel; Shellfish such as scallops, clams, swordfish, clams, and turban shells; cephalopods such as octopus and squid. Among them, the seafood in the present invention is preferably fish or crustaceans, and more preferably carp or shrimp.
In the present invention, immunostimulation means activating the immune system inherent in the living body and enhancing the immune function.

  The method for immunostimulating fish and shellfish according to the present invention comprises administering a yeast extract having a 5'-nucleotide content of 20% by mass or more and a free amino acid content of 10% by mass or more. Feeding the blended feed. The reason why such an effect can be obtained is not clear, but it contains 5′-nucleotides and free amino acids in a relatively large content compared to the conventional yeast extract, so that these directly contribute to the immune system of seafood. Or it acts indirectly and it is thought that the immunity of fish and shellfish is activated.

  In the present invention, the yeast extract is an extract of various components of yeast, and includes amino acids, peptides, nucleic acids, minerals, and the like. Moreover, the content ratio of various components can be adjusted according to the kind of yeast, culture conditions, and extraction conditions.

The yeast used as a raw material for the yeast extract used in the present invention is not particularly limited, and edible yeasts usually used in the field of food production can be used. An edible yeast-derived yeast extract is preferable, unlike the case where a drug is administered, because there is no need to worry about accumulation of the administered product in seafood.
Specific examples of such yeast include torula yeast used in the production of food and feed, bakery yeast used in bread production, and beer yeast used in beer production.
Among them, baker's yeast or torula yeast is preferable because of its good growth ability, Saccharomyces spp. Or Candida spp. Is more preferable, and Saccharomyces cerevisiae (Saccharomyces cerevisiae). Or Candida utilis is particularly preferred.

  As a raw material of the yeast extract, one type of yeast may be used, or a plurality of types of yeast may be used. For example, after Saccharomyces cerevisiae and Candida utilis are cultured and collected separately, the obtained cells are mixed to form a mixed cell and extracted from the mixed cell The yeast extract obtained by mixing the yeast extract obtained by extracting each cultured yeast separately may be sufficient. In addition, culture | cultivation of yeast can be performed by a conventional method.

  The method for extracting the yeast extract is not particularly limited, and any of the methods usually used when extracting the extract from biological materials such as yeast may be used. Examples of the extraction method include an autolysis method and an enzymatic decomposition method. Here, the self-digestion method is a method of solubilizing and extracting yeast by the action of an enzyme inherent in yeast, and a yeast extract having a high free amino acid content can be obtained. On the other hand, the enzymatic decomposition method is a method of inactivating an enzyme or the like possessed by yeast by heat treatment or the like and then adding a degrading enzyme to solubilize and extract the yeast. Since an enzyme reaction can be easily controlled by adding an appropriate enzyme from the outside, the content of free amino acids and nucleic acids can be adjusted.

  The enzyme used in the enzymatic decomposition method is not particularly limited as long as it is an enzyme that is usually used for decomposing biological components, and any enzyme can be used. Examples of the enzyme include an enzyme capable of degrading the cell wall of yeast, a proteolytic enzyme, a nucleolytic enzyme, and the like, and various components can be efficiently extracted from the yeast by appropriately using them together.

  It is more preferable to use endo-type protease and exo-type protease in combination as the proteolytic enzyme. This is because the free amino acid content in the yeast extract can be increased. Further, it is more preferable to use 5'-phosphodiesterase as the nucleolytic enzyme. This is because, among the nucleic acid components, the content of 5'-ribonucleotides, which are main taste components, in the yeast extract can be increased.

The content of 5′-nucleotides in the yeast extract used in the present invention is not particularly limited as long as it is 20% by mass or more, but is preferably 20 to 60% by mass. Although the mechanism of action is not clear, immunostimulation becomes better when the content of 5′-nucleotides in the yeast extract is relatively large, such as 20% by mass or more. Although an upper limit is not specifically limited, It is preferable that it is 60 mass% or less.
Specifically, 5′-ribonucleotides are preferable as 5′-nucleotides, and examples of the 5′-ribonucleotides include 5′-inosinic acid, 5′-guanylic acid, 5′-adenylic acid, 5 Examples include '-uridylic acid, 5'-cytidylic acid, and metal salts thereof.
The yeast extract used in the present invention is the total amount of inosinic acid and guanylic acid, that is, the total amount of 5′-inosinic acid, 5′-guanylic acid, and their metal salts, etc. contained in the yeast extract. It is more preferable that the yeast extract has a large amount. In particular, the combined value of inosinic acid content and guanylic acid content in the yeast extract (hereinafter referred to as IG nucleic acid content) is more preferably 1% by mass or more, and further preferably 5% by mass or more. It is particularly preferably 20% by mass or more. Although an upper limit is not specifically limited, It is preferable that it is 40 mass% or less.

  The free amino acid content of the yeast extract used in the present invention is not particularly limited as long as it is 5% by mass or more, but is preferably 10% by mass or more. Although an upper limit is not specifically limited, It is preferable that it is 40 mass% or less.

The yeast extract used in the present invention may contain proteins, peptides, minerals, organic acids, vitamins and the like as components other than the 5′-nucleotides and free amino acids described above, but β-glucan and mannan. It is preferable not to contain the component derived from the cell wall of yeast, such as.
A yeast extract containing no β-glucan and mannan can be produced by a conventional method. For example, a yeast extract that does not contain β-glucan and mannan is obtained by fractionating a yeast extract that has been enzymatically digested and extracted by an autolysis method, an enzymatic degradation method, etc., and removing an insoluble fraction derived from the cell wall. Can do.

  The form of the yeast extract is not particularly limited, and various forms of yeast extract can be used in consideration of the administration form described later, the type of the target seafood, and the like. For example, any of powder, paste, and liquid may be used.

In the present invention, the method of administering the yeast extract to the seafood is not particularly limited. For example, the method of directly injecting the yeast extract itself into the body of the seafood using a sonde, a syringe, etc. Examples include a method in which a yeast extract is dissolved or dispersed in water and spontaneously ingested by fish and shellfish.
In addition, in the method of feeding fishery products with a feed containing a yeast extract, the feed is not particularly limited as long as it contains a yeast extract as described above, and even a feed consisting only of a yeast extract may be used. It is also possible to feed a feed containing a yeast extract in a general seafood feed. As the seafood feed, a feed that is usually used for raising seafood can be used. When yeast extract is used by being contained in fish and shellfish feed, the yeast extract may be contained at the production stage of fish and shellfish feed, and contained or supported by sprinkling or coating the produced fish and shellfish feed. You may let them.

  Among them, the amount of yeast extract in fish and shellfish can be easily adjusted, and the amount of yeast extract to be used can be reduced. It is preferable to use a method of direct injection into the body, and a method of feeding a feed containing a yeast extract is particularly preferable because administration can be performed easily.

The amount of the yeast extract to be taken into the seafood body is not particularly limited as long as it can exert an immunostimulatory action, and is appropriately determined in consideration of the type and concentration of the yeast extract, the type of seafood, etc. can do. For example, in the case of feeding a yeast extract-containing feed, it is preferable to adjust the yeast extract dosage per day to 5 to 2000 mg per 1 kg of seafood weight, and 10 to 1000 mg. Is more preferable, and it is further more preferable that it is 10-500 mg. In addition, in the case of administration by injection, it is preferable to prepare so that the yeast extract dose per time is 5 to 2000 mg, more preferably 10 to 1000 mg per 1 kg of seafood. 10 to 800 mg is more preferable.
In the present invention, since 5′-nucleotides and free amino acids which are considered to be active ingredients are contained in a relatively large amount as described above, the amount of the yeast extract used in fish and shellfish can be made relatively small. Since the cost concerning an extract can be reduced, it is preferable.
Administration of the yeast extract or feeding of the yeast extract-containing feed may be performed all at once, or may be performed in a plurality of times. When performed multiple times a day, it is preferable that the sum of the multiple times of yeast extract intake falls within the range of yeast extract intake per day.

The fish and shellfish breeding method of the present invention administers a yeast extract having a content of 5′-nucleotides of 20% by mass or more and a free amino acid content of 10% by mass or more, or contains the yeast extract The immunity of fish and shellfish can be activated in the same manner as the above-described immune activation method for fish and shellfish.
In the fish and shellfish breeding method of the present invention, 5′-nucleotides, free amino acids, their contents, administration method, feeding method, and intake amount are the same as in the above-described fish and shellfish immunostimulation method.
The method for raising seafood other than the administration is not particularly limited, and the seafood can be raised in seawater or fresh water by a normal method.

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
In this example shown below, “Vertex IG20” (trade name, manufactured by Tablemark) and “CW-I” (trade name, manufactured by Tablemark) were used as yeast extracts. These are all yeast extracts derived from Saccharomyces cerevisiae, which is a baker's yeast. Table 1 shows the amounts of free amino acids, total amino acids, and nucleic acids contained in these yeast extracts. In addition, the amount of nucleic acids indicates the total amount (calculated as a heptahydrate) of 5′-sodium inosinate and sodium 5′-guanylate as a content ratio (% by mass) in the yeast extract.
As is apparent from Table 1, IG20 is a yeast extract having a high free amino acid content and a high IG content. CW-I contains free amino acids and IG, but its concentration is not high, while it is a yeast extract containing β-glucan, which is an insoluble component derived from the cell wall.

[Experimental Example 1]
We examined the survival rate of tiger prawns fed with feed containing yeast extract against bacteria and viruses.
First, prawn (Marsupenaaeus jamonicus) having an average weight of about 10 g was bred in a 60 cm water tank filled with 20 ° C. artificial seawater. Feed the mixed sample containing 0.1% by mass or 0.5% by mass of the yeast extract Vertex IG20 or CW-I per day for a total of 7 days, and then artificially infect bacteria or viruses. And the survival rate after the infection test was examined. The control group (control group) was fed with a mixed sample containing no yeast extract.

Specifically, in the bacterial infection group, Vibrio nigripulcitudo was injected directly into the peritoneal cavity 100 μL (1 × 10 ⁶ cfu / individual) 24 hours after the end of the feeding period. V. Nigripulchritudo was cultured at 27 ° C. for 14 hours in a water bath and adjusted to the above concentration.
In the virus-infected group, first, tissues (Ella, hepatopancreas, heart, lymphoid organ) were extracted from prawns infected with White Spot Syndrome Virus (WSSV), homogenized in PBS, and 1/1000 in PBS. A diluted homogenizing solution was prepared. 50 mL of the homogenized solution was added to a tank containing 4 L of seawater. The prawns 24 hours after the end of the feeding period were immersed in the tank for 2 hours, and returned to the original water tank after 2 hours.
V. FIG. 1 shows the survival test results of the prawns infected with nigripulchritudo, and FIG. 2 shows the survival test results of the prawns infected with WSSV. In the WSSV infection experiment, the vertex IG20 feeding group and the control group were followed for 13 days, and only the CW-I feeding group was followed for 10 days.

From the results of FIG. On the 11th day after the infection with nigripulchritudo, the survival rate of the Vertex IG20 feeding group was 65%, whereas the survival rate of the CW-I feeding group was 40%, and the survival rate of the control group was 13%. It is clear from the results in FIG. 2 that, on the 13th day after the infection with WSSV, the survival rate of the vertex IG20 0.1 mass% or 0.5 mass% feeding group was 71% and 59%, respectively. It was. In addition, the survival rate on the 10th day of the CW-I 0.1% by mass feeding group was 42%, and the survival rate on the 13th day of the control group was 12%, compared with the vertex IG20 feeding group. It is clear that the survival rate is significantly lower.
From the above results, it was confirmed that in the prawns fed with Vertex IG20, the survival rate when infected with viruses and bacteria was improved, and that Vertex IG20 had an immunostimulatory effect.

[Experiment 2]
Changes in the expression level of immune-related genes in carp inoculated with yeast extract were examined.
First, carp (Cyprinus carpio L.) having an average weight of about 100 g was bred in a water tank filled with 20 ° C. artificial seawater, and pre-breeding was carried out for 1 day before the start of the test. The yeast extract Vertex IG20 was adjusted with PBS (phosphate buffered saline, pH 7.0) so that the inoculation amount was 0.1 mg yeast extract 50 mg / kg body weight, and 0.1 cc was administered daily using an oral sonde. One dose was orally administered for a total of 3 days. Similarly, 0.1 cc PBS was orally administered to the control group (control group).
These carp were collected 1 day, 3 days and 5 days after the end of the administration period, and the expression level of immune-related genes was examined as follows.

Primers were prepared for the type I IFN, IFN-γ1, and IFN-γ2 genes from the known carp (Cyprinus carpio L.) cDNA sequence, and the method of Molecular Kitology (Molecular Immunology) used for PCR was used. 46 (2009) 2548-2556). First, RNA was extracted in accordance with a conventional method using carp head kidney tissue (about 20 μg), and RT-PCR reaction was performed using this RNA using a thermal cycler C1000 (Bio-rad, USA). CDNA in the head and kidney tissue was synthesized. Furthermore, PCR was performed with a thermal cycler C1000 (Bio-rad, USA) using β-actin and type I IFN, IFN-γ1, and IFN-γ2 genes, respectively. After completion of PCR, electrophoresis was performed on a 1.5% agarose gel, and then the band was stored as a TIFF image file by digital photography. The saved file was quantified using Science Lab 99 Image Gauge software (manufactured by Fuji Film), and the amplification rate of the PCR product was determined from the density of the band. Based on this, the expression rate of each gene relative to the β-actin gene was determined, and a graph was prepared. FIG. 3 shows the results of an expression analysis of a type I IFN gene, FIG. 4 shows an interferon γ1 gene, and FIG. 5 shows an interferon γ2 gene. The controls in FIGS. 3 to 5 are results one day after the end of oral administration of PBS.
Type I IFN is known to be mainly produced by virus-infected cells and is a cytokine considered to be involved in antiviral action. On the other hand, interferon γ1 / 2 is known to have an action of mainly activating macrophages, and is a cytokine thought to play an important role in the cellular immune response.

  As a result of the above expression analysis, type I IFN gene in carp head kidney tissue administered with vertex IG20 showed a significant increase in expression at the 1% level on the first and third days after the administration period (FIG. 3). . Interferon γ1 and γ2 both showed a significant increase in the expression level at the 1% level on the third day after the administration period (FIGS. 4 and 5). From the above results, it was confirmed that in the carp administered with vertex IG20, the expression level of the immune-related gene was changed, and vertex IG20 had an immunostimulatory effect.

  The fish and shellfish immunostimulation method of the present invention can be suitably used in the fishery industry.

Claims (6)

  It is characterized by administering a yeast extract having a 5′-nucleotide content of 20% by mass or more and a free amino acid content of 10% by mass or feeding a feed containing the yeast extract. A method for immunizing fish and shellfish.   The method for immunizing fish and shellfish according to claim 1, wherein the 5'-nucleotides contain inosinic acid and guanylic acid.   The method for immunizing fish and shellfish according to claim 1 or 2, wherein the contents of β-glucan and mannan are each less than 1% by mass.   The method for immunizing fish and shellfish according to any one of claims 1 to 3, wherein the fish and shellfish are carp or shrimp.   The method for immunizing fish and shellfish according to any one of claims 1 to 4, wherein the yeast extract is a yeast extract obtained from one or more selected from the group consisting of torula yeast, beer yeast, and baker's yeast.   It is characterized by administering a yeast extract having a 5′-nucleotide content of 20% by mass or more and a free amino acid content of 10% by mass or feeding a feed containing the yeast extract. How to raise seafood.

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