JP4774484B2 - Fish disease control agent, method for producing the same, and feed - Google Patents

Description

  The present invention relates to a fish disease control agent for edwardieriasis, which contains a specific licorice-derived component as an active ingredient, a method for producing the same, and a feed containing this fish disease control agent.

  Licorice is known as a herbal medicine and is currently used mainly as a raw material for food sweeteners, pharmaceuticals and quasi drugs. In particular, glycyrrhizin, which is a water-soluble component, has excellent pharmacological effects such as immunostimulatory action, anti-inflammatory action, and anti-allergic action, and thus is widely used in foods, pharmaceuticals, cosmetics, and the like. What is said to be a general licorice extract is obtained by hot water or hydrous alcohol extraction, and is often about 5 to 25% as a glycyrrhizin content and about 1 to 5% as a flavonoid content (Non-Patent Document 3, 4).

On the other hand, materials having an immunostimulatory action have been proposed as measures for controlling diseases caused by bacterial infection in the aquaculture industry (see Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2). Furthermore, a feed for fish farming containing licorice powder (see Patent Document 3), a feed additive composition for fish farming containing licorice powder, a method for promoting the growth of cultured fish using the same, and a feed (see Patent Document 4) are proposed. ing.
However, in the aquaculture industry, there has been no proposal of a control effect particularly against edwardiadiasis, which is one of the fish diseases, and an excellent control agent has been desired. In addition, the following is mentioned as a prior art document relevant to this invention.

JP-A-5-262658 JP-A-2-250832 JP-A-2-283247 Japanese Patent Laid-Open No. 11-169099 Kazuhiro Watanabe, "Science", August 1989, p42-44 Tomoko Katayama, Kazumasa Ikoma, Yoshitake Yokota, Masuyo Nakai “MINOPHAGEN MEDICAL REVIEW”, 33 (3), 1988, p54-57 14th revised Japanese Pharmacopoeia commentary, Yodogawa Shoten, published on June 27, 2001 Official Food Addendum 7th Edition, Yodogawa Shoten, published June 9, 1999

  The present invention has been made in view of the above circumstances, can be a disease control measure due to bacterial infection in the aquaculture industry, fish disease control agent having an excellent control effect against edwardiellosis fish disease, and its production method, An object of the present invention is to provide a feed containing the fish disease control agent.

  As a result of earnest research on the disease control action in fish, the present inventor extracted licorice with water under neutral to alkaline conditions, and extracted the precipitate obtained by acid treatment with this extract to obtain ethanol. The obtained ethanol extract was subjected to activated carbon decolorization treatment, and then the crystallization mother liquor after crystallization of glycyrrhizin was subjected to antibacterial action against the causative fungi of fish disease, in particular, Edwardsiella tarda which is a pathogenic bacterium of Edwardsiella disease. ), A remarkable antibacterial action has been found, and the present invention has been made.

  Conventionally, the method used for producing a derivative of glycyrrhizin usually used an alkaline extraction and crystallized crystal, and the mother liquor from which the crystal was removed was usually discarded. In contrast, the mother liquor that has been discarded so far has an antibacterial action against the causative bacteria of fish disease, in particular, a remarkable antibacterial action against Edwardsiella tarda, which is a pathogenic bacterium of Edwardsiella disease. It is a new finding by the present inventors that it is useful as a fish disease control agent.

Accordingly, the present invention provides the following inventions.
[1]. The crystallization mother liquor after crystallization of glycyrrhizin from the activated carbon decolorization solution of the ethanol extract of the precipitate produced by acid treatment of the neutral or alkaline water extract of licorice , the flavonoid content, The control agent with respect to the fish disease of an Edwardsiellosis characterized by consisting of a crystallization mother liquid which is 10-25 mass% per solid content .
[2]. [1] A feed containing the control agent according to [1].
[3]. Licorice is extracted with water under neutral or alkaline conditions, and the precipitate obtained by acid treatment of this extract is extracted with ethanol. The resulting ethanol extract is subjected to activated carbon decolorization, and then glycyrrhizin is crystallized. The method for producing a control agent for fish diseases according to [1], wherein the crystallization mother liquor after collection is collected.

  ADVANTAGE OF THE INVENTION According to this invention, the fish disease control agent which has the outstanding control effect with respect to the fish disease of an Edwardsiella disease, its manufacturing method, and the feed containing a fish disease control agent can be provided.

Hereinafter, the present invention will be described in detail.
The licorice used as a raw material for the fish disease control agent of the present invention is a plant belonging to the genus Glycyrihiza, for example, G. glabra, G. uralensis, G. inflata (G. inflata). ) And the like can be used, but it is preferable to use glycyrrhizal inflators (G. inflata). Moreover, although any site | part of a root, a rhizome, a leaf, and a stem can be used as a raw material, it is preferable to use a root and / or a rhizome as a raw material. In addition, these may be raw or dried, but use dry roots and dry rhizomes as raw materials for industrially produced glycyrrhizin extraction raw materials. Can do. In addition, licorice is often called with the name of the production area, and examples thereof include Tohoku licorice, Northwest licorice, Xinjiang licorice, Mongolian licorice, Russian licorice, and Afghanistan licorice.

  As extraction conditions for obtaining the fish disease control agent of the present invention, water extraction is carried out under neutrality and alkalinity with respect to the above licorice. Usually, the pH of the water extract is preferably 7 to 11, particularly preferably 9 to 10. The extraction temperature may be cold water, hot water or hot water, but is preferably 5 to 100 ° C, particularly preferably 50 to 100 ° C. For adjusting the pH, ammonia, sodium hydroxide, potassium hydroxide, or the like can be used. Although extraction time changes with extraction temperature, it is 1 to 72 hours normally, and 2 to 24 hours are especially preferable.

  Next, the precipitate obtained by acid-treating the extract is subjected to ethanol extraction. The obtained ethanol extract is subjected to activated carbon decolorization treatment, and then a crystal mother liquor after crystallization of glycyrrhizin is obtained.

  In the acid treatment, precipitation is performed with an acidic solution such as sulfuric acid, hydrochloric acid, and nitric acid at an acid pH of 2.0 to 4.0 to precipitate glycyrrhizin and the like. Separate the precipitate and filtrate by filtration. 2-10 mass parts ethanol is added with respect to 100 mass parts of obtained precipitates, and an ethanol extract is obtained. The extract is stirred and filtered. 2-10 mass% activated carbon is added with respect to the obtained filtrate, and reflux extraction is performed. Thereafter, filtration is performed using a filter aid such as celite, diatomaceous earth, pearlite, etc., and alkaline water such as ammonia water, sodium hydroxide, potassium hydroxide is added to adjust the pH to 4.0 to 7.0, preferably pH 5.0 to Adjust to 5.5. Further, glycyrrhizin is crystallized by allowing to stand at 3 to 10 ° C., preferably 5 ° C. for 1 to 5 days, preferably 2 days, and then filtered to remove the crystallized glycyrrhizin.

  In this case, the amount of glycyrrhizin in the extract obtained by drying the ethanol extract is 10 to 30% by mass per solid content. In the above crystallization step, 20 to 50% by mass, particularly 30 to 40% by mass, of this glycyrrhizin is crystallized and removed so that the crystallization mother liquor after the crystallization step is dried. The glycyrrhizin content in the extract is preferably 5 to 20% by mass, particularly 8 to 15% by mass, based on the solid content.

  The fish disease control agent of the present invention comprises the above-mentioned crystallization mother liquor, but the crystallization mother liquor can be used as it is, and can also be used after being concentrated by a conventional method. Moreover, it can also be used as a powder or solid substance of a tan licorice extract by drying the extract by an appropriate method.

  The glycyrrhizin content of the fish disease control agent of the present invention is preferably 5 to 20% by mass, particularly 8 to 15% by mass per solid content, and the content of flavonoids is 5 to 25% by mass, particularly 10 to 10% by mass. 20 mass% is preferable. The flavonoid content of the fish disease control agent of the present invention is about 2 to 20 times as high as that of licorice powder used for aquatic feeds and conventional licorice extracts.

  The present invention is a fish disease control agent for edwardiarrhosis comprising a licorice-derived component obtained by a specific production method as an active ingredient. As a causative bacterium of the above-mentioned Edwardsiella disease, Edwardsiella tarda (Edwardsiella tarda) can be mentioned. Since the licorice-derived component obtained by the specific production method of the present invention has an excellent antibacterial action against these bacteria, it is optimal as an antibacterial agent against these causative bacteria.

  The above fish diseases are effective for the above-mentioned diseases occurring in a wide variety of fish and shellfish such as Japanese flounder, Thai, yellowtail, mullet, flounder, eel, eel, carp and tilapia.

  Since the fish disease control agent of the present invention uses an extract obtained from licorice, there is no problem of occurrence of drug-resistant bacteria and side effects due to antibiotics, and it can be administered with confidence. Furthermore, since there is no concern about the human body and the environment due to the residue of the drug, it is particularly suitable as a fish disease control agent for cultured fish.

  The fish disease control agent of the present invention can be administered from juvenile fish to adult fish, and can be administered orally or parenterally. In the case of oral administration, it is possible to administer the fish disease control agent as it is or mixed with any fish feed ingredient and administer as fish feed. Moreover, the dosage is normally 5 to 100 mg per kg body weight of fish, and the administration period is not particularly limited, but the effect can be obtained in 1 to 7 days.

  Examples of fish feed ingredients include fish meal, wheat flour, starch, fish oil, and the like, which can be used singly or in appropriate combination of two or more. The content of the fish disease control agent in the feed is preferably 0.1 to 5% by mass, more preferably 0.2 to 1% by mass.

  EXAMPLES Hereinafter, although an Example and a comparative example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following Example. In the following examples, “%” indicates mass% unless otherwise specified.

[Example 1]
The rhizome of licorice (Glycyrrhiza inflata) was crushed into chips. 1.0 kg of this licorice chip was extracted overnight with 10 L of 3% aqueous ammonia (pH 10, temperature: room temperature), and then separated into solid and liquid. The obtained extract was subjected to an acid precipitation treatment with a 1% sulfuric acid solution to precipitate glycyrrhizin and the like, and the filtrate was separated into a precipitate and an extract filtrate. Among these, 1 L of ethanol was added to the precipitate, stirred at room temperature for 1 hour, and filtered using celite. Activated carbon 60g was added to the obtained filtrate, and reflux extraction was performed at room temperature for 1 hour. Then, filtration using celite was performed. Ammonia water was added to the obtained filtrate to adjust the pH to 5.0 to 5.5. The filtrate was allowed to stand at 5 ° C. for 2 days to crystallize glycyrrhizin, and then centrifuged to separate the crystals and the filtrate. The obtained filtrate was concentrated under reduced pressure, neutralized to about 7.0 with sodium carbonate, further concentrated under reduced pressure, and spray-dried to obtain 150 g of a licorice extract as a yellow-brown extract powder. This licorice extract had a glycyrrhizin content of 11% and a flavonoid content of 15%. About the obtained licorice extract, the forced attack test was performed using the Edwardsiella disease causative microbe, and the cumulative mortality and colonization rate of Japanese flounder were measured, and the fish disease control effect confirmation test was performed. The results are shown in Table 2.

<Evaluation of fish disease control effect>
The licorice extract obtained in Example 1, wheat gluten and water were added to a commercially available EP feed (containing 1% licorice powder) to prepare test feeds shown in Table 1. The feed composition of each section is as shown in Table 1.
34 flounder (average weight 304 g) preliminarily raised in a circular running water tank (diameter 5 m, water depth 0.5 m) using commercially available EP feed (containing 1% licorice powder) as test fish were accommodated in each test area 34 units. The above-mentioned commercially available EP was fed at a daily feeding rate ((feeding amount (kg) / fish body weight (kg)) × 100 / test period (day)) at 0.8% and acclimated to the test environment for one week.
Feeding of the test feed was carried out for 2 weeks after acclimatization, and the daily feeding rate was 0.88% so that the dose of the licorice extract of Example 1 was as shown in Table 1.
The UF806 strain derived from Edwardsiella tarda flounder was attacked with 1.54 × 10 ⁴ CFU / tail in the abdominal cavity of 20 fish in each test section, and a flowing water tank (2 × 1 × 1 m, water temperature 17.4-19. (2 ° C) were housed in 4 groups, and observed without feeding for 3 weeks. For dead fish and all surviving fish at the end of the observation, bacterial isolation from the kidney was attempted using SS agar medium, and the cumulative flounder mortality and Edwardsiella tarda colonization rate of each test area were determined.

From Table 2, the cumulative mortality in Test Groups 2 to 4 (Examples) reared with the licorice extract of Example 1 was compared with Test Group 1 (Comparative Example) reared with only the licorice powder. Was significantly lower.
Moreover, the colonization rate in the test groups 2 to 4 bred with the licorice extract of Example 1 is significantly lower than that in the test group 1 bred with only the licorice powder.
From these results, it was confirmed that the licorice extract of Example 1 has a significantly stronger disease control effect than licorice powder.

[Formulation Example 1]
Fish feed having the composition shown in Table 3 below was prepared.

Claims (3)

The crystallization mother liquor after crystallization of glycyrrhizin from the activated carbon decolorization solution of the ethanol extract of the precipitate produced by acid treatment of the neutral or alkaline water extract of licorice , the flavonoid content, The control agent with respect to the fish disease of an Edwardsiellosis characterized by consisting of a crystallization mother liquid which is 10-25 mass% per solid content .   A feed containing the control agent according to claim 1.   Licorice is extracted with water under neutral or alkaline conditions, and the precipitate obtained by acid treatment of this extract is extracted with ethanol. The resulting ethanol extract is subjected to activated carbon decolorization, and then glycyrrhizin is crystallized. The method for producing a control agent for fish diseases according to claim 1, wherein the crystallization mother liquor after collection is collected.