JP5906369B2 - Immunostimulators and feed for fish diseases - Google Patents
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Description
本発明は、ミモザ抽出物を含有する魚病防除剤及び飼料に関するものである。 The present invention relates to a fish disease control agent and feed containing a mimosa extract.
養殖業における細菌感染による病害防除対策として、甘草末を配合した養魚用飼料（特許文献１参照）及び甘草末を含有する養魚用飼料添加物組成、それを用いた養殖魚類の生長促進方法及び飼料（特許文献２参照）、魚病原因菌に対する抗菌剤（特許文献３参照）、魚病防除剤（特許文献４参照）が提案されている。しかしながら、さらに安全で効果が高い魚病防除剤が望まれていた。 As a measure for controlling diseases caused by bacterial infection in the aquaculture industry, a feed for fish farming containing licorice powder (see Patent Document 1) and a feed additive composition for fish farming containing licorice powder, a method for promoting the growth of cultured fish using the same, and feed (Refer patent document 2), The antibacterial agent (refer patent document 3) with respect to a fish disease cause microbe, and the fish disease control agent (refer patent document 4) are proposed. However, a safer and more effective fish disease control agent has been desired.
本発明は、上記事情に鑑みなされたものであり、養殖業における細菌感染による病害防除対策ができ、安全で優れた免疫賦活効果を有する、魚病に対する免疫賦活剤及び飼料を提供することを目的とする。 The present invention has been made in view of the above circumstances, and an object thereof is to provide an immunostimulatory agent and a feed for fish diseases that can be used to control diseases caused by bacterial infection in the aquaculture industry and have a safe and excellent immunostimulatory effect. And
本発明者らは、上記目的を達成するため鋭意検討した結果、ミモザ抽出物、特にミモザタンニンが、優れた魚病の原因菌に対する発育阻止効果、免疫賦活効果を有することを知見し、本発明をなすに至ったものである。 As a result of intensive studies to achieve the above object, the present inventors have found that mimosa extract, particularly mimosa tannin, has an excellent growth inhibitory effect and immunostimulatory effect on the causative bacteria of fish disease, and the present invention. That led to
従って、本発明は下記魚病に対する免疫賦活剤及び飼料を提供する。
［１］．ミモザ抽出物を有効成分とする魚病に対する免疫賦活剤。
［２］．ミモザ抽出物が、タンニンを５０〜９０質量％（固形分）含むものである［１］記載の魚病に対する免疫賦活剤。
［３］．ミモザ抽出物が、ミモザの樹皮の熱水抽出物である［１］又は［２］記載の魚病に対する免疫賦活剤。
［４］．エドワジエラ症の魚病に対するものである［１］、［２］又は［３］記載の魚病に対する免疫賦活剤。
［５］．マダイの魚病に対するものである［１］〜［４］のいずれかに記載の魚病に対する免疫賦活剤。
［６］．［１］〜［５］のいずれかに記載の魚病に対する免疫賦活剤を含有する魚用飼料。
［７］．ミモザ抽出物を含有する、魚病に対する免疫賦活用飼料。
Therefore, this invention provides the immunostimulant and feed with respect to the following fish disease.
[1]. An immunostimulant for fish diseases comprising mimosa extract as an active ingredient.
[2]. The immunostimulant for fish diseases according to [1], wherein the mimosa extract contains 50 to 90% by mass (solid content) of tannin.
[3]. The immunostimulant for fish diseases according to [1] or [2], wherein the mimosa extract is a hot water extract of mimosa bark.
[4]. The immunostimulant for fish disease according to [1], [2] or [3], which is against fish disease of edwardiaerosis.
[5]. The immunostimulator against fish disease according to any one of [1] to [4], which is against fish diseases of red sea bream.
[6]. [1] A fish feed containing the immunostimulant for fish disease according to any one of [5].
[7]. Immunization feed for fish diseases containing mimosa extract.
本発明によれば、魚病に対して、安全で優れた免疫賦活効果を有する免疫賦活剤及び飼料を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the immunostimulant and feed which have a safe and outstanding immunostimulatory effect with respect to a fish disease can be provided.
以下、本発明について詳細に説明する。
本発明の魚病に対する免疫賦活剤の有効成分であるミモザ抽出物は、マメ科アカシア（Ａｃａｓｉａ）属に属する植物である。使用部位は、樹皮、根、根茎、葉、茎のいずれの部位でも原料として使用することができるが、樹皮を使用することが好ましい。抽出方法は特に限定されず、溶媒抽出、熱水抽出、水蒸気蒸留、超臨界抽出等の公知の抽出方法を採用することができる。例えば、溶媒抽出の場合、ミモザ樹皮を生のまま、又は乾燥した後に適当な大きさに切断・加工し、抽出溶媒に浸漬、撹拌することによって得ることができる。抽出は必要に応じて加温してもよく、時間は適宜選定され、抽出ｐＨは、極端な酸性又はアルカリ性でなければ特に制限はない。上記抽出方法に用いる溶媒としては、水；メタノール、エタノール、プロパノール、ブタノール等のアルコール類；プロピレングリコール、ブチレングリコール等の多価アルコール類等が挙げられ、これらを１種単独で又は２種以上を適宜組み合わせて用いることができる。抽出後ろ過を行い、適宜公知の精製を行ってもよい。さらに、抽出液は、適宜、濃縮、分離精製、噴霧乾燥等を行うことができる。
Hereinafter, the present invention will be described in detail.
The mimosa extract which is an active ingredient of the immunostimulant for fish diseases of the present invention is a plant belonging to the genus Acasia. The part to be used can be any part of bark, root, rhizome, leaf, stem, but it is preferable to use bark. The extraction method is not particularly limited, and a known extraction method such as solvent extraction, hot water extraction, steam distillation, or supercritical extraction can be employed. For example, in the case of solvent extraction, mimosa bark can be obtained raw or after being dried, cut and processed into an appropriate size, immersed in an extraction solvent, and stirred. The extraction may be heated as necessary, the time is appropriately selected, and the extraction pH is not particularly limited unless it is extremely acidic or alkaline. Examples of the solvent used in the extraction method include water; alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as propylene glycol and butylene glycol, and the like. These may be used alone or in combination of two or more. They can be used in appropriate combinations. After extraction, filtration may be performed to appropriately perform known purification. Furthermore, the extract can be appropriately concentrated, separated and purified, spray dried, and the like.
特に、ミモザ抽出物（固形分）にタンニン量が５０〜９０質量％（固形分）、好ましくは５５〜９０質量％、より好ましくは６０〜８０質量％含まれるものが好ましい。このようなミモザ抽出物としては、ミモザの樹皮の熱水抽出物が挙げられる。また、上記のようなタンニン量のミモザ抽出物を得る方法としては、例えば、ミモザの樹皮を粉砕し、原料の３〜２０倍量、好適には５〜１５倍量の６０〜９０℃、より好ましくは熱水抽出により得ることができる。その後、減圧濃縮等の濃縮、分離精製、噴霧乾燥等を行うことができる。 In particular, it is preferable that the mimosa extract (solid content) contains a tannin amount of 50 to 90 mass% (solid content), preferably 55 to 90 mass%, more preferably 60 to 80 mass%. Examples of such mimosa extract include hot water extract of mimosa bark. Moreover, as a method of obtaining the mimosa extract of the amount of tannins as described above, for example, the bark of mimosa is crushed, and 3 to 20 times the amount of the raw material, preferably 5 to 15 times the amount of 60 to 90 ° C. Preferably it can be obtained by hot water extraction. Thereafter, concentration such as concentration under reduced pressure, separation and purification, spray drying and the like can be performed.
なお、本発明のタンニン含量の測定は、以下の測定方法による。
カテキン１００ｍｇを９９容量％エタノール１００ｍＬに溶解した溶液０．１ｍＬに、蒸留水８．０ｍＬ及びフォーリン・デニス試薬０．５ｍＬを加え、室温で３分間静置した後、１０質量％炭酸ナトリウム溶液１．０ｍＬを加え、室温で３０分間静置し、７６０ｎｍにおける吸光度Ａｏを求める。次にミモザ抽出物１００ｍｇを５０容量％エタノール１００ｍＬに溶解した溶液０．１ｍＬを、同様に処理し吸光度Ａを求める。カテキンの含量を１００質量％として、その相対値（Ａ／Ａｏ×１００）をもってミモザ抽出物のタンニン含量とする。
In addition, the measurement of the tannin content of this invention is based on the following measuring methods.
To 0.1 mL of a solution obtained by dissolving 100 mg of catechin in 100 mL of 99% ethanol by volume, 8.0 mL of distilled water and 0.5 mL of foreign dennis reagent were added and allowed to stand at room temperature for 3 minutes. Add 0 mL, allow to stand at room temperature for 30 minutes, and determine the absorbance Ao at 760 nm. Next, 0.1 mL of a solution obtained by dissolving 100 mg of mimosa extract in 100 mL of 50% by volume ethanol is treated in the same manner to determine absorbance A. The catechin content is defined as 100% by mass, and the relative value (A / Ao × 100) is defined as the tannin content of the mimosa extract.
以下、本発明について詳細に説明する。
本発明の魚病に対する免疫賦活剤は、ミモザ抽出物を有効成分とする魚病に対する免疫賦活剤である。魚病に対する免疫賦活剤の対象となる魚病としては、エドワジエラ症：エドワジエラ タルダ（Ｅｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａ）、ビブリオ病：ビブリオ ダムセラ（Ｖｉｂｒｉｏ ｄａｍｓｅｌａ）、ビブリオ ハーベイ（Ｖｉｂｒｉｏ ｈａｒｖｅｙｉ）、ビブリオ オーダリー（Ｖｉｂｒｉｏ ｏｒｄａｌｌｉ）、ビブリオ ペスシダ（Ｖｉｂｒｉｏ ｐｅｎａｅｉｃｉｄａ）、ビブリオ スプレンディダス（Ｖｉｂｒｉｏ ｓｐｌｅｎｄｉｄｕｓ）、ビブリオ バルニフィカス（Ｖｉｂｒｉｏ ｖｕｌｎｉｆｉｃｕｓ）、ビブリオ アンギラルム（Ｖｉｂｒｉｏ ａｎｇｕｉｌｌａｒｕｍ）、細菌性腸管白濁症：ビブリオ イチオエンテリー（Ｖｉｂｒｉｏ ｉｃｈｔｈｙｏｅｎｔｅｒｉ）、潰瘍病：ビブリオ パラヘモリティカス（Ｖｉｂｒｉｏ ｐａｒａｈａｅｍｏｌｙｔｉｃｕｓ）、桿菌性壊死症：ビブリオ トゥビアッシ（Ｖｉｂｒｉｏ ｔｕｂｉａｓｈｉｉ）、カラムナリス病：フラボバクテリウム カラムナレ（Ｆｌａｖｏｂａｃｔｅｒｉｕｍ ｃｏｌｕｍｎａｒｅ）、冷水病：フレキシバクター サイクロフィラム（Ｆｌｅｘｉｂａｃｔｅｒ ｐｓｙｃｈｒｏｐｈｉｌｕｍ）、滑走細菌症：フレキシバクター マリティマス（Ｆｌｅｘｉｂａｃｔｅｒ ｍａｒｉｔｉｍｕｓ）、テナチバキュラム・マリティマム（Ｔｅｎａｃｉｂａｃｕｌｕｍ ｍａｒｉｔｉｍｕｍ）、ノカルジア症：ノカルディア アステロイデス（Ｎｏｃａｒｄｉａ ａｓｔｅｒｏｉｄｅｓ）、ノカルジア・セリオラエ（Ｎｏｃａｒｄｉａ ｓｅｒｉｏｌａｅ）、類結節症：フォトバクテリウム ダムセラ（Ｐｈｏｔｏｂａｃｔｅｒｉｕｍ ｄａｍｓｅｌａ）、乳酸桿菌症：カーノバクテリウム ピシコラ（Ｃａｒｎｏｂａｃｔｅｒｉｕｍ ｐｉｓｃｉｃｏｌａ）、ブドウ球菌症：スタフィロコッカス エピデルミデス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ｅｐｉｄｅｒｍｉｄｅｉｓ）、菌血症：ラクトコッカス ガービア（Ｌａｃｔｏｃｏｃｃｕｓ ｇａｒｖｉｅａｅ）、せっそう病：エロモナス サルモニシダ（Ａｅｒｏｍｏｎａｓ ｓａｌｍｏｎｉｃｉｄａ）、連鎖球菌症：エンテロコッカス種（Ｅｎｔｅｒｏｃｏｃｃｕｓ ｓｐ．）、ストレプトコッカス イニエ（Ｓｔｒｅｐｔｏｃｏｃｕｓ ｉｎｉａｅ）、ストレプトコッカス ディスガラクティエ（Ｓｔｒｅｐｔｏｃｏｃｕｓ ｄｙｓｇａｌａｃｔｉａｅ）、ストレプトコッカス・パラユベリス（Ｓｔｒｅｐｔｏｃｏｃｕｓ ｐａｒａｕｂｅｒｉｓ）等が挙げられる。中でも、エドワジエラ症に対して有効である。
Hereinafter, the present invention will be described in detail.
Immunostimulant agent for fish diseases of the present invention is an immunostimulant agent for fish diseases containing, as an active ingredient, mimosa extract. Fish diseases that are the target of immunostimulants against fish disease include: Edwardsiella disease: Edwardsiella tarda, Vibrio disease: Vibrio damsela, Vibrio harveyi, i Vibrio penaeicida, Vibrio splendidus, Vibrio vulnificus, idiopathic intestine Cass (Vibrio p) rahaemoliticus), gonococcal necrosis: Vibrio tubashishii, columnaris disease: Flavobacterium columnar (Flavobacterium columnar), cold water disease: flexibacteria ), Tenachibacrum maritimum, Nocardiosis: Nocardia asteroides, Nocardia seriolae, Nodule: Photobacterium damacera (Photobacto) Rium damsela), Lactobacilli: Carnobacterium picicola, Staphylococci: Staphylococcus epidermidides, Bacteremia: Lactococcus gaemocidal salmonicida), streptococci: Enterococcus sp., Streptococcus inier, Streptococcus dysgalactiae, Streptococcus sp., Streptococcus sp. ruberis) and the like. Above all, it is effective against Edwardsiellosis.
上記の魚病は、マダイ、ヒラメ、タイ類、ブリ類、ボラ、カレイ、アイナメ、ウナギ、コイ、ティラピア、ギンザケ、ニジマス、トラフグ、マグロ、メバル、アジ、サバ、カワハギ、アナゴ、チョウザメ等の広範囲な種類の魚介類に発生する上記記載の病害に有効であるが、マダイの魚病に好適であり、特にマダイのエドワジエラ症に対して有効である。なお、マダイのエドワジエラ症と、ヒラメのエドワジエラ症とは別の株である。 The above fish diseases are widespread such as red sea bream, Japanese flounder, Thai, yellowtail, mullet, flatfish, eel, eel, carp, tilapia, coho salmon, rainbow trout, tiger puffer, tuna, sea bream, horse mackerel, mackerel, riverfish, anago, sturgeon, etc. It is effective against the above-mentioned diseases that occur in various types of fish and shellfish, but is suitable for fish diseases of red sea bream, and particularly effective against red sea bream edible dystonia. The red sea bream edovadierosis and the flounder edovadierosis are different strains.
本発明の魚病に対する免疫賦活剤は、ミモザから得られる抽出物を用いるため、抗生物質による薬剤耐性菌の出現や副作用の問題がなく、安心して投与することができる。さらに、薬剤の残留による人体や環境への影響の心配がないため、特に、養殖魚に対する魚病の防除剤として好適である。 Since the immunostimulant for fish diseases of the present invention uses an extract obtained from mimosa, there is no problem of appearance of drug-resistant bacteria and side effects due to antibiotics, and administration can be performed with confidence. Furthermore, since there is no concern about the influence on 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.
本発明の魚病に対する免疫賦活剤は、稚魚から成魚に投与することができ、投与方法は経口投与が可能である。経口投与する場合は、魚病に対する免疫賦活剤をそのまま又は任意の魚用飼料成分と混合し、魚用飼料として投与することが可能である。また、その投与量は、通常、魚体重１ｋｇあたり１０〜１０００ｍｇが好ましく、５０〜２００ｍｇがより好ましい。投与期間は特に限定されないが、７〜２８日間で効果を得ることができる。 The immunostimulant for fish diseases of the present invention can be administered from juvenile fish to adult fish, and the administration method can be administered orally. In the case of oral administration, it is possible to administer an immunostimulant against fish disease as it is or mixed with any fish feed ingredient and administered as a fish feed. The dose is usually preferably 10 to 1000 mg, more preferably 50 to 200 mg per kg of fish body weight. The administration period is not particularly limited, but an effect can be obtained in 7 to 28 days.
本発明のミモザ抽出物は、後述する実施例における結果からも明らかであるように、優れた免疫賦活効果を有するため、ミモザ抽出物を有効成分とする免疫賦活剤を提供することができる。 Since the mimosa extract of the present invention has an excellent immunostimulatory effect, as will be apparent from the results in Examples described later, an immunostimulant containing the mimosa extract as an active ingredient can be provided.
本発明は、上記魚病に対する免疫賦活剤を含有する魚用飼料や、ミモザ抽出物を含有する魚病に対する免疫賦活用飼料を提供する。ミモザ抽出物、対象となる魚病や、投与方法等は同じである。 The invention, and fish feed containing immunostimulant to said fish diseases, provides an immunostimulatory feed for fish diseases containing mimosa extract. Mimosa extract, target fish disease, administration method, etc. are the same.
ミモザ抽出物以外の魚用飼料成分としては、魚粉、小麦粉、でん粉、魚油等が挙げられ、１種単独で又は２種以上を適宜組み合わせて用いることができる。飼料中のミモザ抽出物（魚病に対する免疫賦活）の含有量は０．０３〜１０質量％が好ましく、より好ましくは０．１〜２．０質量％である。
Examples of fish feed components other than mimosa extract include fish meal, wheat flour, starch, fish oil and the like, and these can be used alone or in combination of two or more. The content of the mimosa extract ( immune activation against fish disease) in the feed is preferably 0.03 to 10% by mass, more preferably 0.1 to 2.0% by mass.
以下、実施例及び比較例を示し、本発明を具体的に説明するが、本発明は下記の実施例に制限されるものではない。 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.
[実施例１]
ミモザ（樹皮）１０００ｇを粉砕し、ミモザ（樹皮）の１０倍量で熱水抽出した。固体と液体とを分離して抽出液を得た。得られた抽出液を濃縮し、濃縮液（固形４５質量％）を得て、これを噴霧乾燥し、ミモザ抽出物１５０ｇ（タンニン含量６５質量％）を得た。
得られたミモザ抽出物について、下記評価を行った。
[Example 1]
1000 g of mimosa (bark) was crushed and extracted with hot water in an amount 10 times that of mimosa (bark). A solid and a liquid were separated to obtain an extract. The obtained extract was concentrated to obtain a concentrated liquid (solid 45% by mass), which was spray-dried to obtain 150 g of mimosa extract (tannin content 65% by mass).
The following evaluation was performed about the obtained mimosa extract.
<魚病原因菌に対する発育阻止効果>
ミモザ抽出物含有寒天培地（ミモザ抽出物濃度：０．００２〜１．０００％（ｗ／ｖ）、２倍希釈系列）に種々の菌液を滴下し（５μＬ／滴）、２〜４日間、２５℃で培養した。目視でコロニーの発育が確認できなかったミモザ抽出物最小濃度を、最小発育阻止濃度（ＭＩＣ）とした。寒天培地には、ミモザ抽出物を溶解した培地２４ｍＬを直径９ｃｍのシャーレに注いだものを使用した。また、発育陽性培地として、ミモザ抽出物不含寒天培地も同様に作成し、細菌の発育と滴下菌量を確認した。結果を表１に示す。下記９種類(１５株)の魚病原因菌（細菌）に対し、発育阻止効果が認められた。
<Growth inhibitory effect against fish disease-causing bacteria>
Various bacterial solutions were dropped into agar medium containing mimosa extract (concentration of mimosa extract: 0.002 to 1.000% (w / v), 2-fold dilution series) (5 μL / drop) for 2 to 4 days. Cultured at 25 ° C. The minimum concentration of mimosa extract for which colony growth could not be confirmed visually was defined as the minimum growth inhibitory concentration (MIC). As the agar medium, 24 mL of a medium in which the mimosa extract was dissolved was poured into a petri dish having a diameter of 9 cm. In addition, a mimosa extract-free agar medium was similarly prepared as a growth positive medium, and the growth of bacteria and the amount of dripped bacteria were confirmed. The results are shown in Table 1. The following nine types (15 strains) of fish disease-causing bacteria (bacteria) were found to have a growth inhibitory effect.
<マダイの免疫に対する効果>
エドワジエラ症の原因細菌であるＥｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａは宿主食細胞内で抵抗性を示すため、本症の予防には、食細胞のポテンシャルキリング活性（ＰＫ）の向上が有効と考えられている。ミモザ抽出物がＰＫに及ぼす影響を確認した。
１００Ｌ水槽４槽に平均体重６６．７ｇのマダイを５尾ずつ収容した。水温２５℃に馴致した後、各水槽のマダイに、実施例１で得られたミモザ抽出物を表２の割合で添加したモイストペレット（ＭＰ）を給餌率４質量％／日で４週間毎日投与した。最終投与日の翌日、下記方法により血液のポテンシャルキリング活性を測定した。結果を表２に併記する。
<Effects of red sea bream on immunity>
Since Edwardsiella tarda, the causative bacterium of Edwardsiella disease, exhibits resistance in host phagocytic cells, it is considered that improvement of phagocyte potential killing activity (PK) is effective in preventing this disease. The effect of mimosa extract on PK was confirmed.
Five red sea bream with an average weight of 66.7 g were housed in four 100 L water tanks. After acclimatization to a water temperature of 25 ° C., moist pellets (MP) to which the mimosa extract obtained in Example 1 was added at a ratio shown in Table 2 were administered daily for 4 weeks at a feeding rate of 4 mass% / day. did. The day after the last administration day, blood potential killing activity was measured by the following method. The results are also shown in Table 2.
［ポテンシャルキリング活性］
マイクロチューブにＮＢＴ溶液（コントロール）及びＺｙｍｏｓａｎ加ＮＢＴ溶液を１５μＬずつ分注し、魚より採血した血液１００μＬを毛細管（長さ７５ｍｍ、φ２ｍｍ、ミナトメディカル製）に充填し１０００Ｇ、４℃で５分間遠心分離を行った。遠心分離機より毛細管を取り出し、白血球層と赤血球層の境界面及び白血球層の上方２ｃｍで毛細管を切断し、白血球を含む部分を、５０μＬＲＰＭＩ１６４０溶液を用いて新しいマイクロチューブに移動させた。得られた白血球溶液をピペッティングにて白血球細胞を分散し、ＮＢＴ溶液及びＺｙｍｏｓａｎ加ＮＢＴ溶液を１５μＬずつ加え、２５℃でインキュベート行った。その後、１５質量％ＮａＣｌ溶液を３０μＬ加えた。次に、ＤＭＦ４００μＬを各マイクロチューブに加えてピペットを用いて混和し、１５００Ｇ，４℃、１５分間遠心分離を行った。上清２５０μＬを石英マイクロプレートに移し、５４０ｎｍの吸光値（ＯＤ）を測定し、下記式を用いて評価を行った。
ポテンシャルキリング活性（ＰＫ）
＝Ｚｙｍｏｓａｎ加ＮＢＴ溶液のＯＤ−ＮＢＴ溶液のＯＤ
[Potential Killing Activity]
NBT solution (control) and Zymosan-added NBT solution are dispensed into microtubes in a volume of 15 μL each, and 100 μL of blood collected from the fish is filled into a capillary tube (length 75 mm, φ2 mm, manufactured by Minato Medical) and centrifuged at 1000 G, 4 ° C. for 5 minutes. Separation was performed. The capillary tube was taken out from the centrifuge, the capillary tube was cut at 2 cm above the boundary surface between the leukocyte layer and the erythrocyte layer and the leukocyte layer, and the portion containing leukocytes was moved to a new microtube using a 50 μL RPMI1640 solution. White blood cells were dispersed by pipetting the obtained white blood cell solution, and 15 μL each of NBT solution and Zymosan-added NBT solution were added and incubated at 25 ° C. Thereafter, 30 μL of 15 mass% NaCl solution was added. Next, 400 μL of DMF was added to each microtube, mixed using a pipette, and centrifuged at 1500 G, 4 ° C. for 15 minutes. 250 μL of the supernatant was transferred to a quartz microplate, the absorbance value (OD) at 540 nm was measured, and evaluation was performed using the following formula.
Potential killing activity (PK)
= OD of Zymosan-added NBT solution-OD of NBT solution
マダイのＰＫは、ミモザ抽出物投与の方がミモザ抽出物無投与より有意に高かった。ミモザ抽出物の投与により、マダイの免疫が向上することがわかった。 The red sea bream PK was significantly higher with the mimosa extract than without the mimosa extract. It was found that administration of mimosa extract improves red sea bream immunity.
<マダイのエドワジエラ症に対する効果−１>
マダイのエドワジエラ症の感染予防に、ミモザ抽出物の投与が有効かどうか確認した。
１００Ｌ水槽６槽に平均体重５５．０ｇのマダイを１０尾ずつ収容した。水温２５℃に馴致した後、各水槽のマダイに、実施例１のミモザ抽出物を表３の割合で添加したモイストペレット（ＭＰ）を、給餌率４質量％／日で４週間毎日投与した。４週間投与した翌日、ミモザ抽出物を投与して１．５時間後にＥ．ｔａｒｄａ（ＭＥＥ０３０９株）の菌液（１０7ＣＦＵ／ｍＬ）でマダイを１．５時間浸漬攻撃した。その後水槽に再収用し、攻撃後２４時間目の血液と腎臓における菌量を測定した。
<Effects of red sea bream on Edwardsiellosis-1>
It was confirmed whether mimosa extract was effective in preventing infection of red sea bream edodezieriosis.
Ten red sea bream with an average weight of 55.0 g were accommodated in six 100 L water tanks. After acclimatization to a water temperature of 25 ° C., moist pellets (MP) to which mimosa extract of Example 1 was added at a ratio shown in Table 3 were administered daily for 4 weeks at a feeding rate of 4 mass% / day. The day after administration for 4 weeks, 1.5 hours after administration of the mimosa extract. The red sea bream was attacked by immersion for 1.5 hours with a bacterial solution (10 7 CFU / mL) of tarda (MEE0309 strain). Thereafter, the cells were collected again in a water tank, and the amount of bacteria in blood and kidney was measured 24 hours after the attack.
ミモザ抽出物０．３３質量％添加を４週間投与（水槽５）と、ミモザ抽出物０．６７質量％添加を２週間投与（水槽３）とにおける体内菌量は、ミモザ抽出物無投与（水槽１）のそれよりも有意に低かった。ミモザ抽出物の投与により、体内菌量が低下することが確認された。 The amount of bacteria in the body after administration of 0.33% by weight of mimosa extract for 4 weeks (water tank 5) and administration for 0.67% by weight of mimosa extract for 2 weeks (water tank 3) It was significantly lower than that of 1). It was confirmed that the amount of bacteria in the body decreased by administration of the mimosa extract.
<マダイのエドワジエラ症に対する効果−２>
マダイのエドワジエラ症の発生予防に、ミモザ抽出物の投与が有効かどうかを確認した。５００Ｌ水槽２槽に平均体重７９．１ｇのマダイを１８尾ずつ収容した。水温２５℃に馴致した後、各水槽のマダイに、実施例１のミモザ抽出物を表４の割合で添加したモイストペレット（ＭＰ）を給餌率４質量％／日で毎日投与した。４週間投与した翌日、ミモザ抽出物を投与して１．５時間後にＥ．ｔａｒｄａ（ＭＥＥ０３０９株）の菌液（１０7ＣＦＵ／ｍＬ）でマダイを１．５時間浸漬攻撃した。その後水槽に再収用し、攻撃後２８日間、マダイの累積死亡率を観察した。攻撃後も観察終了まで表４のミモザ抽出物を投与した。
<Effects of red sea bream on Edwardsiellosis-2>
We confirmed whether administration of mimosa extract was effective in preventing the occurrence of edwardiadiosis in red sea bream. 18 red sea bream with an average weight of 79.1 g were accommodated in two 500 L water tanks. After acclimatization to a water temperature of 25 ° C., moist pellets (MP) to which mimosa extract of Example 1 was added at a rate shown in Table 4 were administered daily at a feeding rate of 4 mass% / day. The day after administration for 4 weeks, 1.5 hours after administration of the mimosa extract. The red sea bream was attacked by immersion for 1.5 hours with a bacterial solution (10 7 CFU / mL) of tarda (MEE0309 strain). After that, it was re-collected in the water tank, and the cumulative mortality of red sea bream was observed for 28 days after the attack. The mimosa extract of Table 4 was administered after the challenge until the end of the observation.
また、ミモザ抽出物投与と、ミモザ抽出物無投与の累積死亡率（％）を図１に示す。ミモザ抽出物投与の方が、ミモザ抽出物無投与に比べ累積死亡率が低かった。以上のことから、本症の発生予防にミモザ抽出物の投与が有効であることが確認された。 In addition, FIG. 1 shows the cumulative mortality rate (%) when the mimosa extract was administered and when the mimosa extract was not administered. The cumulative mortality rate was lower when mimosa extract was administered than when mimosa extract was not administered. From the above, it was confirmed that administration of mimosa extract is effective in preventing the occurrence of this disease.
［配合例１］
下記表５に示す組成の魚用飼料を作製した。
[Formulation Example 1]
Fish feed having the composition shown in Table 5 below was prepared.
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