JPH02250832A - Immunological enhancing agent for fishes and feed for fish-farming - Google Patents

Abstract

PURPOSE:To obtain immunological enhancing agent toughening liver function of fishes and enhancing immunological competence containing glycyrrhizin as active ingredient. CONSTITUTION:The aimed immunological enhancing agent for fishes contains glycyrrhizin expressed by the formula as triterpene-based glucosides contained in root of Glycyrrhiza glabra L. as active ingredient. Administration is performed by mixing in feed and dose of the agent is >=5mg, preferably >=10mg per 1kg body weight of the object fishes. Said immunological enhancing agent is utilized to fish-farming in taking effect of excellent breeding result without affected by poor feed having high POV and hardly attacked with disease even in farming of high density.

Description

[Detailed description of the invention] [Purpose of the invention]

[Field of Industrial Application B] The present invention relates to a fish feed containing an agent and principles for strengthening the liver function of fish and increasing their immunity. [Prior art] (1) Background Today, carp, rainbow trout, eel, sweetfish, mullet, yellowtail, black sea bream, prawn, red sea bream, squid, octopus, abalone, and oysters are found in the inland waters of rivers, lakes, seas, and bays. ,
Cultivation of scallops and other species is actively carried out, and the area is 30,000 km long.
In Japan, which has a long coastline extending over 1,000 yen, aquaculture and cultivated fishing are becoming increasingly important due to restrictions on deep-sea fishing. (2) Problems with the conventional technology However, in these intensive fisheries where large numbers of fish and shellfish are kept in a small aquatic area, the living environment becomes worse than the natural state due to rotting bait and excrement. Therefore, it is common practice to add drugs such as antibiotics to the feed, but the administration of drugs promotes the emergence of resistant bacteria. Not only that, but the drugs accumulate in the bodies of fish and shellfish, and end up being ingested by humans, who are the ultimate predators, which is unfavorable from a public health standpoint (in fact, the amount of antibiotics remaining is strictly regulated). Therefore, if possible, it is desirable to make the feeding of drugs unnecessary by increasing the immunity of fish and shellfish themselves, thereby making them less susceptible to diseases. In response to the above-mentioned problems, the present invention provides means for solving the above-mentioned problems by developing a non-antibiotic drug for increasing the biological defense function of fish and a feed containing the drug. The purpose is to provide.

[Structure of the invention]

[Means for Solving the Problems] (1) Concept In the process of conducting intensive studies to solve the above problems, the present inventor focused on the liver function and immune-enhancing effects of glycyrrhizin on mammals, and applied it to fish. When we observed the pharmacological effects of the present invention, not only were effects similar to those observed in mammals observed in lower fish species, but also significant differences were observed in an immunity test against streptococci. is based on this knowledge. (21 Overview The gist of the present invention is to utilize glycyrrhizin as an immune enhancer for fish, and to include glycyrrhizin in feed for fish farming as a simple means for that purpose. Below, various elements constituting the invention will be described. (3) Glycyrrhizin Glycyrrhizin (formula below) is a legume that grows wild from southern Europe to central Asia.
a 1labra L, , G, 11abra L, v
ar, 11anduljfera Re1. et He
rd. G, echinata L,, G, uralens
It is a triterpene-based glycoside contained in the roots of fs fi#sch et DC, etc.), and its aglycone is a glucuronic acid dimer. G4+ Effect of glycyrrhizin on fish (a) Measurement items (a) Liver function and immune enhancement function: Three weeks after the start of glycyrrhizin administration, 5 fish were taken from each group and weight gain rate, hematocrit value (red blood cell volume percentage), serum Protein content, serum albumin content, GOT (glutamate-oxaloacetate transaminase) value, GPT (glutamate-virupate transaminase) value, complement value, lysozyme activity, adrenal macrophage phagocytic activity, and TNP-BSA
The agglutinated antibody titer was measured. (T?) Infection protection ability: Four weeks after the start of glycyrrhizin administration, the cells were challenged with live Streptococcus S-2433 strain and changes in infection protection ability were observed. (b) Experimental method (a) Test fish: Young yellowtail fish with an average weight of 15.8 g purchased from a fish farmer in Usa-cho, Tosa City, Kochi Prefecture were used. (υ) Rearing method: Test fish were housed in an FRP aquarium with an 8L sail and reared while being aerated. (8) Test glycyrrhizin: High purity product with purity of 98% or more. 2) Glycyrrhizin administration method: Glycyrrhizin was added to minced squid in an amount of 12% of the fish body weight at doses of 0 (control), 2.5, 5.0, 25.0 and 50.
0II1g as a spreading agent ((Stash) manufactured by Dainippon Pharmaceutical ■)
The drug was also administered once on -day for 35 days (5 weeks). (e) Immunization method: Before administration of glycyrrhizin, TNP-modified bovine serum albumin (TNP-BSA) was intraperitoneally inoculated into the test fish. Control plots were similarly inoculated with saline. (f) Collection of blood and plasma: 3 from the second antigen vaccination
Every week, 10 fish from each group were taken, and blood was collected by cardiac puncture using a heparin-treated syringe. This blood was stored overnight at 4°C and then centrifuged to collect plasma. (g) Measurement of plasma components: Hematocrit values were measured by a conventional method. Serum protein level was measured by the Biulet method. Measurement of GOT and GPT values in serum was performed at <G
The test was carried out using OT UV Test Wako) and <GPT UV Test Wako) (both manufactured by Wako Pure Chemical Industries, Ltd.). (H) Measurement of serum complement value: Using rabbit red blood cells, Sa
It was measured by the method of Fati et al. (1982). (S) Measurement of hypophagic activity of adrenal cells: Immediately remove the adrenal glands from the blood sampled fish, and remove
After cutting into small pieces using scissors in Is (Flow Laboratory) medium, filtering through a platinum mesh to 1×10
A leukocyte suspension with a concentration of 7 cells/- was prepared. The prize edible particles were latex sensitized with each antigen (0.81μi, D
ifco) and FXC were used. The sensitized latex was prepared by the method of Takagi and Hideshi (19g2).
Mix 0.1 − of phagocytic particles adjusted to 10′ cells/−,
The reaction was carried out at 25°C for 3 hours. After the reaction, 100 μg of the mixture was taken out and centrifuged at an angular velocity of 400×g for 5 minutes, and the resulting precipitate was suspended in 10-fetal bovine serum to prepare a smear. After drying, this smear was fixed with 10% formalin and ethanol, peroxidase staining and Giemsa staining, and microscopic examination.
The number of particles taken up by each phagocyte was calculated. (J) Measurement of agglutination antibody titer: Measurement was carried out by a microtiter method using sheep red blood cells (SRBC) sensitized with THP-BS^ as an antigen. Antigen sensitization was performed using glutaraldehyde-treated 5R8C according to a conventional method. (l) Experimental infection test: Four weeks after administration of glycyrrhizin, the test fish were tested for infection with streptococci. First, 5tr was cultured on a BH1 plate at 30°C for 24 hours.
Eptococcus sp., strain 2433, was suspended in sterile phosphate buffer at a concentration of 5.2 x 10' CFU/-, and 0.1- of this suspension was added to 10 cells in each section.
Inoculate the tail of the test fish intraperitoneally. This experimental infection was continued for 10 days and changes in survival rate were examined. In addition, bacteria were isolated from each kidney of each dead fish, and using anti-5 treptococcus serum, it was confirmed that the fish had died due to streptococcus disease. (c) Experimental results (a) Body weight change: The weight change in the glycyrrhizin administration group and the non-administration group are shown in the attached Figure 1. As shown in the figure, it can be seen that as the amount of glycyrrhizin administered increases, the difference between the curves at the start of the experiment and at the end of the experiment tends to increase, indicating that administration of glycyrrhizin is effective in increasing the weight of young fish. Although the cause of this is unknown, it may be considered to be one of the following. ■ Glycyrrhizin improved feed quality. ■ Improves constitution due to synergistic effect with immune enhancing effect (increase in complement value). ■ Detoxifying effect of glycyrrhizin. ■ Effect of glycyrrhizin on improving liver function. (b) Changes in blood properties: Changes in hematocrit value and serum protein level due to administration of glycyrrhizin are shown in FIG. As shown in the figure, the hematocrit value and the serum protein level both increased with the administration of glycyrrhizin, indicating that the administration improved the properties of the blood. Incidentally, if you feed feed with a high content of oxidized fats and oils, such as minced sardines,
Although it is known that the hematocrit value decreases, administration of glycyrrhizin improves or eliminates this drawback. (Improvement of liver function: Glycyrrhizin administration increases serum G
The effect on the values of OT and GPT is shown in FIG. As shown in the figure, GOT and GPT values decreased by administration of glycyrrhizin and in proportion to the dose.
The fact that glycyrrhizin improves liver function can be seen. -i. Feed for cultivating yellowtail is of poor quality because it contains a large amount of oxidized oil and bacteria, and tends to reduce the liver function of the fish, which often leads to streptococcal infections. This shows the possibility of overcoming this problem through functional enhancement.The food used in this experiment was mainly squid minced sand eel, as mentioned above, but the sardine minced meat that is actually used will probably make a bigger difference. (d) Immune-enhancing effect: Figure 4 shows the effects of glycyrrhizin administration on complement value, lysozyme activity, phagocytosis rate, and agglutination antibody titer, which are indicators related to immunity. In particular, the increase in macrophage phagocytosis rate in the glycyrrhizin-dosing group was remarkable.From these results, glycyrrhizin is effective against fish pathogens, similar to what has already been shown against -11V (AIDS virus, etc.). It is also speculated that it has the effect of strengthening immunity. Therefore, it is expected that the resistance of the treated fish to pathogenic bacteria and viruses will increase, and that it will have the effect of keeping the fish in good health. (e) Infection-preventive effect against streptococcal disease: Figure 5 shows that Streptococcus S-
2433 shares of L Om. From this graph showing the relationship between glycyrrhizin dosage and survival rate when
Although glycyrrhizin does not exhibit a dramatic effect on antibiotics, it is clear that at doses of 50 mg/g or higher, it has a significant effect on increasing the survival rate. Incidentally, in actual fish farms, even if a disease occurs, L
Dm. Since this level is not considered to be basolateral, even this mild effect is sufficient for practical purposes. (51 Dosage and Feed Composition The fish liver function agent and fortification and fish culture feed according to the present invention are administered to target fish in an amount of 5 mg or more, preferably 1011 g or more per IKg of body weight. If desired, sodium salt or potassium salt is used. It may be applied in the form of easily soluble salts such as , or sparingly soluble salts such as calcium salts or aluminum salts.Since the target is fish, it is usually administered by mixing it into the feed. As feed, fish meal, pupa meal, minced raw fish meat, grain flour, wheat gluten, etc. are used depending on the type of fish, but in any case, known binders such as pregelatinized starch, wheat glutin, and casein are used. It is necessary to use sodium, sodium alginate, sodium polyacrylate, sodium carboxymethyl cellulose, various vegetable gums, soybean whey, etc. to give it a viscosity that does not easily disintegrate in water. [Action] Inventor's research It was discovered that glycyrrhizin also has effects on fish such as improving liver function, detoxification, and strengthening immunity.Therefore, if it is used in fish farming,
It is not adversely affected by poor quality feed with a high POV, and exhibits good rearing results, being less susceptible to diseases even in high-density aquaculture. Moreover, glycyrrhizin is a natural product and unlike antibiotics, it does not disrupt the natural microbial balance or accumulate in fish bodies, so it can be suitably used as a safe feed additive. [Examples] Hereinafter, embodiments of the invention will be explained using examples, but the examples are merely for explanation and do not mean any restriction or limitation on the idea of the invention. 1 kg of raw sardines, 50 g of pregelatinized starch, 2 glycyrrhizin
0 mg and vitamin E (antioxidant > 10 mg) were mixed with minced meat, the sown material was extruded into a radon shape, and was lightly dried with hot air to obtain about 1 kg of raw feed for young yellowtail fish. [Effects of the Invention] As explained above and demonstrated. As described above, the present invention provides a means for solving the above problems by developing a non-antibiotic drug for enhancing the biological defense function of fish and a feed containing the drug. (Margins below)

[Brief explanation of drawings]

Figure 1 is a graph showing the weight gain of yellowtail fry due to the administration of glycyrrhizin. Figure 2 is a graph showing the change in hematocrit value and serum protein level due to the administration of glycyrrhizin. Figure 3 is a graph showing the increase in body weight of yellowtail fry due to the administration of glycyrrhizin. GOT
A graph showing the influence on the value of GPT and GPT, FIG.
Graph showing the correlation between administration of glycyrrhizin and changes in complement value, lysozyme activity, hypophagia rate, and agglutinating antibody titer, 5th
The figure shows Streptococcus S-2433 after 4 weeks from the start of administration.
It is a graph showing the relationship between glycyrrhizin dosage and survival rate when the LDia amount of the strain is inoculated. The vertical and horizontal parameters of each graph and other explanations are already written in each figure. Added amount of glycyrrhizin (g fat/1 kg body weight/day) Added amount of glycyrrhizin (mg/I kg body weight/day) Added amount of glycyrrhizin (g fat/I kg body weight/day) Added amount of glycyrrhizin (II g/I kg body weight/day) Added glycyrrhizin Amount (mg/body weight IKg/day)

Claims (1)

[Claims] 1. An immune enhancer for fish containing glycyrrhizin as an active ingredient. 2. Fish feed containing glycyrrhizin as an active ingredient.