JP2015172019A - Immunostimulator for seafood - Google Patents
Immunostimulator for seafood Download PDFInfo
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- JP2015172019A JP2015172019A JP2014048805A JP2014048805A JP2015172019A JP 2015172019 A JP2015172019 A JP 2015172019A JP 2014048805 A JP2014048805 A JP 2014048805A JP 2014048805 A JP2014048805 A JP 2014048805A JP 2015172019 A JP2015172019 A JP 2015172019A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
- Y02A40/818—Alternative feeds for fish, e.g. in aquacultures
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract
Description
本発明は、魚介類用の免疫賦活剤に関する。 The present invention relates to an immunostimulant for fish and shellfish.
世界的に魚介類の養殖技術が発展する一方で、それらの疾病、特に感染症による被害が増加している。そのため感染症予防を目的として魚介類の養殖の際に抗生物質や抗菌物質が使用されているが、抗生物質や抗菌物質の乱用による魚介類における薬剤の残留、耐性菌の出現等の問題が発生している。そこでワクチン接種による疾病予防法の開発も行われているが、全ての病原体に対して有効なワクチンは作製できず、またその効果も一般的に３〜４ヶ月間程度と短い。また、ワクチン接種には注射法、浸漬法及び経口法があるが、それぞれ一長一短があり、注射法はワクチン接種の操作が容易でなく、経口法ではワクチン接種の効果が低い等の様々な課題が残されている（非特許文献１）。 While seafood aquaculture technology is developing worldwide, the damage caused by these diseases, especially infectious diseases, is increasing. For this reason, antibiotics and antibacterial substances are used in the cultivation of seafood for the purpose of preventing infectious diseases, but problems such as drug residues in fish and shellfish and the emergence of resistant bacteria due to abuse of antibiotics and antibacterial substances have occurred. doing. Accordingly, disease prevention methods by vaccination have been developed, but effective vaccines against all pathogens cannot be prepared, and the effect is generally short, about 3 to 4 months. In addition, there are injection methods, immersion methods, and oral methods for vaccination, but each has advantages and disadvantages.Injection methods are not easy to operate vaccination, and oral methods have various problems such as low vaccination effect. It remains (Non-Patent Document 1).
エドワジエラ症は、エドワジエラ・タルダ菌（Ｅｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａ）を原因菌とする細菌性疾病であり、多くの魚類、特にウナギ、マダイ、ヒラメ等の重要養殖魚種において大きな被害を及ぼしているが、未だワクチンによる効果的な予防法は開発されていない。 Edwardsiellosis is a bacterial disease caused by Edwardsiella tarda, and has caused great damage in many fish, particularly important cultured fish species such as eel, red sea bream, and flounder, but is still a vaccine. No effective prevention method has been developed.
免疫を賦活する素材を配合した魚類用飼料を給餌することにより、魚類の免疫を高め疾病を予防する方法の開発が行われている。例えば、酵母（特許文献１及び特許文献２）、乳酸菌（特許文献３）、カンゾウ（特許文献４）、多糖類（特許文献５及び特許文献６）等を配合した飼料を用いる方法が報告されている。 Development of a method for improving fish immunity and preventing diseases by feeding fish feeds that contain immunity-stimulating materials has been carried out. For example, a method using a feed containing yeast (Patent Literature 1 and Patent Literature 2), lactic acid bacteria (Patent Literature 3), licorice (Patent Literature 4), polysaccharides (Patent Literature 5 and Patent Literature 6), etc. has been reported. Yes.
しかしこれらの従来の方法で用いている免疫賦活素材の製造は高コストになりがちであり、魚介類用飼料の価格上昇につながる。またこれらの免疫賦活素材は大量製造が難しく、魚介類用飼料に広く使用することは困難である。 However, the production of immunostimulatory materials used in these conventional methods tends to be costly, leading to an increase in the price of feed for seafood. In addition, these immunostimulating materials are difficult to mass-produce and are difficult to widely use in fish and shellfish feed.
本発明は、養殖魚介類の免疫能を効果的に増強する方法を提供することを課題とする。 An object of the present invention is to provide a method for effectively enhancing the immunity of cultured seafood.
本発明者らは、上記事情の課題を解決するために鋭意検討を行った結果、きのこ廃菌床を主成分とする培養基に乳酸菌を播種して培養して得られる乳酸菌発酵物が魚介類の免疫能を賦活し増強することができ、感染症予防に有効であることを見出し、本発明を完成するに至った。 As a result of intensive investigations to solve the above-mentioned problems, the present inventors have found that a fermented lactic acid bacterium obtained by seeding and culturing a lactic acid bacterium in a culture medium mainly composed of a mushroom waste fungus bed is a seafood. It has been found that the immune ability can be activated and enhanced, and is effective in preventing infectious diseases, and the present invention has been completed.
すなわち、本発明は以下を包含する。
[1]きのこ廃菌床を含む培養基で乳酸菌を培養して得られる乳酸菌発酵物を含む、魚介類用の免疫賦活剤。
[2]乳酸菌が、ラクトバチルス属、ペディオコッカス属及びエンテロコッカス属に属する乳酸菌の群から選択される１種以上の乳酸菌である、上記[1]に記載の免疫賦活剤。
[3]乳酸菌が、ラクトバチルス・ファーメンタム キリシマ１Ｒ（受託番号ＮＩＴＥ Ｐ−７８４）、ラクトバチルス・ファーメンタム キリシマ２Ｒ（受託番号ＮＩＴＥ Ｐ−７８５）、ペディオコッカス・ペントサセウス キリシマ１Ｃ（受託番号ＮＩＴＥ Ｐ−７８７）、ペディオコッカス・ペントサセウス キリシマ２Ｃ（受託番号ＮＩＴＥ Ｐ−７８８）、ラクトバチルス・プランタラム ＭＨ（受託番号ＮＩＴＥ Ｐ−１５４８）、ラクトバチルス・プランタラム ＭＥ（受託番号ＮＩＴＥ Ｐ−１５４９）及びエンテロコッカス・フェカリス（ＮＢＲＣ ３９８９）からなる群から選択される少なくとも１つである、上記[1]又は[2]に記載の免疫賦活剤。
[4]きのこ廃菌床が、エノキタケ廃菌床、ヒラタケ廃菌床、又はブナシメジ廃菌床である、上記[1]〜[3]に記載の免疫賦活剤。
[5]培養基が乾燥おから、味噌、及びフスマからなる群より選択される少なくとも１つをさらに含む、上記[1]〜[4]に記載の免疫賦活剤。
[6]感染症予防のための、上記[1]〜[5]に記載の免疫賦活剤。
[7]エドワジエラ属菌又はホワイトスポット病ウイルスによる感染症予防のための、上記[6]に記載の免疫賦活剤。
[8]魚介類が、魚類又は甲殻類である、上記[1]〜[7]に記載の免疫賦活剤。
[9]上記[1]〜[8]に記載の免疫賦活剤を含有する魚介類用飼料。
[10]上記[9]に記載の飼料を魚介類に給餌することによる、魚介類の免疫賦活方法。
That is, the present invention includes the following.
[1] An immunostimulant for fish and shellfish containing a lactic acid bacteria fermentation product obtained by culturing lactic acid bacteria in a culture medium containing a mushroom waste bed.
[2] The immunostimulator according to [1] above, wherein the lactic acid bacterium is one or more lactic acid bacteria selected from the group of lactic acid bacteria belonging to the genus Lactobacillus, Pediococcus and Enterococcus.
[3] Lactic acid bacteria are Lactobacillus fermentum Kirishima 1R (Accession No. NITE P-784), Lactobacillus fermentum Kirishima 2R (Accession No. NITE P-785), Pediococcus pentosaseus Kirishima 1C (Accession No. NITE P) -787), Pediococcus pentosaceus Kirishima 2C (Accession number NITE P-788), Lactobacillus plantarum MH (Accession number NITE P-1548), Lactobacillus plantarum ME (Accession number NITE P-1549) and The immunostimulant according to [1] or [2] above, which is at least one selected from the group consisting of Enterococcus faecalis (NBRC 3989).
[4] The immunostimulant according to [1] to [3] above, wherein the mushroom waste fungus bed is an enokitake waste fungus bed, a oyster mushroom waste fungus bed, or a bunashimeji waste fungus bed.
[5] The immunostimulant according to [1] to [4] above, wherein the culture medium further contains at least one selected from the group consisting of dried okara, miso, and bran.
[6] The immunostimulant described in [1] to [5] above for preventing infectious diseases.
[7] The immunostimulant described in [6] above for preventing infections caused by Edwardsiella spp. Or white spot disease virus.
[8] The immunostimulant described in [1] to [7] above, wherein the seafood is fish or crustacea.
[9] A fish and seafood feed comprising the immunostimulant described in [1] to [8] above.
[10] A method for immunostimulating seafood by feeding the seafood with the feed according to [9] above.
本発明に係る乳酸菌発酵物及びそれを含む免疫賦活剤は、魚介類の免疫能を効果的に賦活し増強することができる。 The lactic acid bacteria fermented product and the immunostimulant containing the same according to the present invention can effectively activate and enhance the immunity of fish and shellfish.
以下、本発明を詳細に説明する。
本発明において用いる免疫賦活素材である乳酸菌発酵物は、きのこ廃菌床を主成分とする培養基（培地とも呼ばれる）に乳酸菌を播種し、培養することにより製造することができる。ここできのこ廃菌床は、任意のきのこ（通常は、食用きのこ）の菌を菌床培養することにより製造することができる。きのことしては、例えば、エノキタケ、ヒラタケ、ブナシメジ、ハナビラタケ、エリンギ、シイタケ、ナメコ、ヤマブシタケ、マイタケ、ヌメリスギタケ、ハタケシメジ、ホンシメジ、アラゲキクラゲ、ウスヒラタケ等が挙げられるが、これらに限定されない。きのこを菌床栽培（菌床培養）し、菌糸蔓延及び培養熟成を経て形成された子実体を採取した後の菌床がきのこ廃菌床である。きのこの菌床栽培に用いる培養基は、おがこ、木材チップ、稲わら、コーンコブミール（コーンコブ）等の培地基材、フスマ、おから（乾燥おからなど）、味噌、糠（米糠など）、粉ビート、小麦胚芽、乾燥焼酎粕等の栄養材、水、場合により石灰（消石灰など）、貝化石、カキ殻等のｐＨ調整剤などの添加剤を混合することにより調製することができる。培養基の原料のより好ましい例としてはコーンコブ、米糠、フスマ、おから、石灰、味噌、及び粉ビートが挙げられるが、これらに限定されない。一般的には、栄養材の配合量は培地基材の５〜２５重量％、好ましくは１０〜２０重量％である。加える水の量は、培地基材の乾燥程度により調節するが、通常は６０％〜７０％程度である。きのこの菌床栽培は、用いるきのこの種類に合わせて、常法により適宜行うことができるが、一般的には、例えば、培養容器に充填した培養基を殺菌・冷却した後、きのこの種菌を培養基に接種して培養し、菌糸を菌床に蔓延させ、熟成させ、菌かきして種菌を取り除き、原基形成を促し、子実体を形成させることにより実施することができる。次いで培養基（菌床）から子実体を取り除くことにより得られた廃菌床を、乳酸菌培養のための培養基に用いる。培養基に用いるきのこ廃菌床は、ｐＨ６以上の新鮮なものであることが好ましい。
Hereinafter, the present invention will be described in detail.
A fermented lactic acid bacterium, which is an immunostimulatory material used in the present invention, can be produced by seeding and cultivating lactic acid bacteria in a culture medium (also called a culture medium) mainly composed of mushroom waste bacteria bed. The waste mushroom bed here can be produced by culturing any mushroom (usually edible mushroom) fungus. Examples of mushrooms include, but are not limited to, enokitake, oyster mushrooms, beech shimeji, hanabiratake, eringi, shiitake mushrooms, nameko, yamabushitake, maitake, numerisushitake, mushroom shimeji, hon-shimeji mushroom, jellyfish jellyfish, and oyster mushrooms. The fungus bed after mushroom bed cultivation (fungus bed culture) and collecting fruit bodies formed through hyphal spread and culture ripening is a mushroom waste fungus bed. The culture medium used for the fungus bed cultivation includes mushrooms, wood chips, rice straw, corn cob meal (corn cob) and other medium substrates, bran, okara (dried okara etc.), miso, koji (rice koji etc.), It can be prepared by mixing additives such as flour beet, wheat germ, dried shochu and other nutrients, water, and in some cases, lime (such as slaked lime), shell fossils, oyster shells and the like. More preferred examples of the raw material for the culture medium include, but are not limited to, corn cob, rice bran, bran, okara, lime, miso, and flour beet. Generally, the amount of nutritional material is 5 to 25% by weight, preferably 10 to 20% by weight, based on the medium base material. The amount of water to be added is adjusted depending on the degree of drying of the medium substrate, but is usually about 60% to 70%. Mushroom bed cultivation can be carried out as appropriate according to the type of mushroom to be used, but generally, for example, after sterilizing and cooling the culture medium filled in the culture vessel, the mushroom inoculum is cultured with the culture medium. Inoculating and cultivating, hyphae are spread on the mycelium, matured, scraped to remove the inoculum, promote primordial formation, and form fruit bodies. Next, the waste bacterial bed obtained by removing fruit bodies from the culture medium (bacteria bed) is used as a culture medium for lactic acid bacteria culture. It is preferable that the waste mushroom bed used for the culture medium is fresh having a pH of 6 or more.
発酵飼料製造に用いるきのこ廃菌床は、通常は３０〜１００重量％、好ましくは８０〜９０重量％で乳酸菌培養用の培養基に配合すればよい。乳酸菌培養用の培養基は、きのこ廃菌床に加えて、乳酸菌の増殖を促進させるための任意の栄養材、例えば、フスマ、乾燥おから、味噌、糠（米糠など）、小麦胚芽、乾燥焼酎粕等を１種又は２種以上含むことが好ましい。栄養材の配合量はきのこ廃菌床の量に対して５〜２５重量％、好ましくは１０〜２０重量％である。水分量は培養状態、培養後の操作性からすると６０重量％以下が望ましく、乾燥おから、フスマなどを用い、通常はきのこ廃菌床の量に対して５０重量％程度に調整する。きのこ廃菌床、栄養材等の原料を好ましくは均一に混合して培養基を調製する。 What is necessary is just to mix | blend the mushroom waste fungi bed used for fermented feed normally with the culture medium for lactic acid bacteria culture | cultivation at 30-100 weight%, Preferably 80-90 weight%. The culture medium for lactic acid bacteria culture can be any nutrient for promoting the growth of lactic acid bacteria in addition to the waste mushroom bed, such as bran, dried okara, miso, koji (rice koji, etc.), wheat germ, dried shochu It is preferable that 1 type or 2 types or more are included. The blending amount of the nutrient material is 5 to 25% by weight, preferably 10 to 20% by weight, based on the amount of the mushroom waste fungus bed. The amount of water is preferably 60% by weight or less in view of the culture state and operability after the culture, and is usually adjusted to about 50% by weight with respect to the amount of waste mushroom beds using dry okara or bran. The culture medium is prepared by preferably uniformly mixing raw materials such as mushroom waste fungus bed and nutrients.
調製した培養基を殺菌（例えば蒸気滅菌等の高温殺菌、又は常温殺菌）し、４０℃以下に冷却後、培養基に乳酸菌を播種し、培養する。乳酸菌としては、任意の乳酸菌を用いることができるが、食用可能な乳酸菌が好ましい。好適な乳酸菌としては、例えば、ラクトバチルス属、ペディオコッカス属、エンテロコッカス属及びロイコノストックス属に属する乳酸菌が挙げられる。そのような乳酸菌は、例えば、ラクトバチルス・ファーメンタム、ラクトバチルス・プランタラム、ペディオコッカス・ペントサセウス、ペディオコッカス・アシディラクティシ、エンテロコッカス・フェカリス、ロイコノストックス・ラクティス等が挙げられるが、これらに限定されない。好適に使用できる乳酸菌の具体例としては、ラクトバチルス・ファーメンタム キリシマ１Ｒ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｆｅｒｍｅｎｔｕｍ ｋｉｒｉｓｈｉｍａ １Ｒ）（受託番号ＮＩＴＥ Ｐ−７８４）、ラクトバチルス・ファーメンタム キリシマ２Ｒ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｆｅｒｍｅｎｔｕｍ ｋｉｒｉｓｈｉｍａ ２Ｒ）（受託番号ＮＩＴＥ Ｐ−７８５）、ペディオコッカス・ペントサセウス キリシマ１Ｃ（Ｐｅｄｉｏｃｏｃｃｕｓ ｐｅｎｔｏｓａｃｅｕｓ ｋｉｒｉｓｈｉｍａ １Ｃ）（受託番号ＮＩＴＥ Ｐ-７８７）、ペディオコッカス・ペントサセウス キリシマ２Ｃ（Ｐｅｄｉｏｃｏｃｃｕｓ ｐｅｎｔｏｓａｃｅｕｓ ｋｉｒｉｓｈｉｍａ ２Ｃ）（受託番号ＮＩＴＥ Ｐ−７８８）、ラクトバチルス・プランタラム ＭＨ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ ＭＨ）（受託番号ＮＩＴＥ Ｐ−１５４８）、ラクトバチルス・プランタラム ＭＥ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ ＭＥ）（受託番号ＮＩＴＥ Ｐ−１５４９）、エンテロコッカス・フェカリス（Ｅｎｔｅｒｏｃｏｃｃｕｓ ｆａｅｃａｌｉｓ）寄託株（ＮＢＲＣ ３９８９）等が挙げられる。なお、ラクトバチルス・ファーメンタム キリシマ１Ｒ（受託番号ＮＩＴＥ Ｐ−７８４；寄託日：２００９年７月２３日）、ラクトバチルス・ファーメンタム キリシマ２Ｒ（受託番号ＮＩＴＥ Ｐ−７８５；寄託日：２００９年７月２３日）、ペディオコッカス・ペントサセウス キリシマ１Ｃ（受託番号ＮＩＴＥ Ｐ−７８７；寄託日：２００９年７月２３日）、ペディオコッカス・ペントサセウス キリシマ２Ｃ（受託番号ＮＩＴＥ Ｐ−７８８；寄託日：２００９年７月２３日）、ラクトバチルス・プランタラム ＭＨ（受託番号ＮＩＴＥ Ｐ−１５４８；寄託日：２０１３年２月２８日）、ラクトバチルス・プランタラム ＭＥ（受託番号ＮＩＴＥ Ｐ−１５４９；寄託日：２０１３年２月２８日）は、独立行政法人製品評価技術基盤機構の特許微生物寄託センター（ＮＰＭＤ）に寄託されている。エンテロコッカス・フェカリス（Ｅｎｔｅｒｏｃｏｃｃｕｓ ｆａｅｃａｌｉｓ）（ＮＢＲＣ ３９８９）は、独立行政法人製品評価技術基盤機構 バイオテクノロジーセンター（Ｂｉｏｌｏｇｉｃａｌ Ｒｅｓｏｕｒｃｅ Ｃｅｎｔｅｒ；ＮＢＲＣ）に寄託され、そのカタログ（ＮＢＲＣ Ｃａｔａｌｏｇｕｅ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｒｅｓｏｕｒｃｅｓ）に掲載されており、そのカタログ番号（ＮＢＲＣ番号）に基づいて分譲を受けることができる。 The prepared culture medium is sterilized (for example, high-temperature sterilization such as steam sterilization, or normal temperature sterilization), cooled to 40 ° C. or lower, and then seeded with lactic acid bacteria and cultured. Although any lactic acid bacteria can be used as the lactic acid bacteria, edible lactic acid bacteria are preferable. Suitable lactic acid bacteria include, for example, lactic acid bacteria belonging to the genus Lactobacillus, Pediococcus, Enterococcus and Leuconostoccus. Examples of such lactic acid bacteria include Lactobacillus fermentum, Lactobacillus plantarum, Pediococcus pentosaceus, Pediococcus acidilactice, Enterococcus faecalis, Leuconostoccus lactis, etc. It is not limited to these. Specific examples of lactic acid bacteria that can be suitably used include Lactobacillus fermentum Kirishima 1R (Accession Number NITE P-784), Lactobacillus fermentum Kiritima 2R (Lactobacillus FermentumRTE P-785), Pediococcus pentosaceus kirisima 1C (Accession number NITE P-787), Pediococcus pentosaceus Kirisima 2C (Pediococcus pentocecus 8 Bacillus pula Lactobacillus plantarum MH (Accession number NITE P-1548), Lactobacillus plantarum ME (Accession number NITE P-1549), Enterococcus faecalis (Entococcus faecalis) Is mentioned. In addition, Lactobacillus fermentum Kirishima 1R (Accession number NITE P-784; Deposit date: July 23, 2009), Lactobacillus fermentum Kirishima 2R (Accession number NITE P-785; Deposit date: July 2009) 23rd), Pediococcus pentosaceus Kirisima 1C (Accession number NITE P-787; Deposit date: July 23, 2009), Pediococcus pentosaceus Kirisima 2C (Accession number NITE P-788; Deposit date: 2009) July 23), Lactobacillus plantarum MH (Accession number NITE P-1548; Deposit date: February 28, 2013), Lactobacillus plantarum ME (Accession number NITE P-1549; Deposit date: 2013) (February 28) It is of the deposited with the Patent Microorganisms Depositary (NPMD). Enterococcus faecalis (NBRC 3989) has been deposited with the Biotechnology Center (NBRC) of the National Institute of Product Evaluation Technology and published in its catalog (NBRC Catalog of Biologics). Sales can be received based on the catalog number (NBRC number).
培養基には、１種の乳酸菌を播種してもよいし、２種以上の乳酸菌を播種してもよい。
乳酸菌の播種（接種）量は、以下に限定するものではないが、１．０ ｘ１０５ ｃｆｕ／ｇ以上（培養基の総重量比）、例えば１．０〜９．０ｘ１０６ｃｆｕ／ｇの量が好ましい。
The culture medium may be seeded with one kind of lactic acid bacteria or two or more kinds of lactic acid bacteria.
The seeding (inoculation) amount of lactic acid bacteria is not limited to the following, but is 1.0 × 10 5 cfu / g or more (total weight ratio of culture medium), for example, 1.0 to 9.0 × 10 6 cfu / g. preferable.
培養基での培養は、用いる乳酸菌に適した培養条件で行えばよい。好適な例では、乳酸菌を培養基に加えて均一に攪拌混合した後、２５〜４０℃（例えば３０〜３５℃）で１５〜９６時間（通常は１５〜４８時間、好ましくは１８〜３０時間）培養すればよい。培養は、１０８ｃｆｕ／ｇ以上、例えば１．０〜９．０ｘ１０９ｃｆｕ／ｇの乳酸菌発酵物（死菌体も含む）を得るまで継続することが好ましい。以上のような培養により得られる発酵物を、本発明では乳酸菌発酵物と称する。 The culture in the culture medium may be performed under culture conditions suitable for the lactic acid bacteria to be used. In a preferred example, lactic acid bacteria are added to the culture medium and stirred and mixed uniformly, and then cultured at 25 to 40 ° C. (eg, 30 to 35 ° C.) for 15 to 96 hours (usually 15 to 48 hours, preferably 18 to 30 hours). do it. The culture is preferably continued until a lactic acid bacteria fermentation product (including dead cells) of 10 8 cfu / g or more, for example, 1.0 to 9.0 × 10 9 cfu / g is obtained. In the present invention, the fermented product obtained by the above culture is referred to as a lactic acid bacteria fermented product.
このようにして得られた乳酸菌発酵物（主に生菌を含む）は、そのままでも免疫賦活素材として飼料等に使用できるが、乾燥（例えば約８０℃程度での乾燥）及び粉末化等の処理を行ってもよい。このような処理を施すことにより、安定性が増し、長期保存も可能になる。そのような処理により乳酸菌が死菌体となった乳酸菌発酵物も本発明において免疫賦活素材として好適に使用できる。 The lactic acid bacteria fermented product thus obtained (mainly including live bacteria) can be used as it is as an immunostimulatory material in feeds, etc., but it can be dried (for example, dried at about 80 ° C.) and powdered. May be performed. By performing such treatment, stability is increased and long-term storage is possible. A fermented lactic acid bacterium in which lactic acid bacteria have become dead cells by such treatment can also be suitably used as an immunostimulatory material in the present invention.
本発明に係る乳酸菌発酵物は、魚介類の免疫能を賦活し増強する作用を有する。本発明において魚介類とは、魚類、甲殻類、貝類、及び頭足類を指す。魚介類の具体例としては、コイ、アユ、マス、フナ、ウナギ、サケ等の淡水魚；ブリ、タイ（マダイなど）、カンパチ、フグ、マグロ、アジ、ヒラメ、カレイ、カワハギ、ホッケ、サバ、サンマ、ムツ、イワシ、アナゴ、タラ等の海水魚；クルマエビ、ウシエビ、イセエビ、サクラエビ、ボタンエビ、タラバガニ、ケガニ、ズワイガニ、ロブスター、シャコ等の甲殻類；カキ、ホタテ、アサリ、シジミ、ハマグリ、サザエ、アカガイ、アワビ等の貝類；タコ、イカ等の頭足類が挙げられる。本発明において対象となる魚介類としては、魚類又は甲殻類であることがより好ましく、例えばマダイ、ヒラメ、又はクルマエビが特に好ましい。 The fermented lactic acid bacterium according to the present invention has an effect of activating and enhancing the immunity of fish and shellfish. In the present invention, seafood refers to fish, crustaceans, shellfish, and cephalopods. Specific examples of seafood include freshwater fish such as carp, ayu, trout, crucian carp, eel and salmon; , Sea cucumbers, sardines, sea lions, cod, etc .; crustaceans such as tiger prawns, lobsters, lobsters, cherry shrimp, button shrimp, king crab, crayfish, snow crab, lobster, mantis; oysters, scallops, clams, shijimi, clams, sazae, Shellfish such as red scallop and abalone; cephalopods such as octopus and squid. In the present invention, the target seafood is more preferably fish or crustaceans, and for example, red sea bream, Japanese flounder, or prawn is particularly preferable.
本発明に係る乳酸菌発酵物は、魚介類の免疫能を賦活し増強する作用を有することから、魚介類用の免疫賦活剤として用いることができる。本発明において免疫賦活とは、生体の免疫系を活性化し、免疫機能を高めることを意味する。より具体的には、本発明に係る乳酸菌発酵物による免疫能の増強は、本発明に係る乳酸菌発酵物を魚介類に投与した場合、オプソニン化酵母菌に対する白血球の貪食率（総白血球数に対する貪食白血球数の割合）を、乳酸菌発酵物を投与しない対照と比較して例えば１０％以上、好ましくは２０％以上増加させることにより示され得る。本発明に係る乳酸菌発酵物による免疫能の増強はまた、本発明に係る乳酸菌発酵物を魚介類に投与した場合、血清補体活性（補体第２経路活性）を、乳酸菌発酵物を投与しない対照と比較して例えば２０％以上、好ましくは４０％以上増加させる（ＡＣＨ５０値で例えば２０％以上、好ましくは４０％以上の低下に対応）ことにより示され得る。本発明に係る乳酸菌発酵物による免疫能の増強はまた、本発明に係る乳酸菌発酵物を魚介類に投与した場合、ウサギ赤血球に対する血清又は体表粘液の凝集活性として測定されるレクチン活性（粗レクチン活性）を、乳酸菌発酵物を投与しない対照と比較して例えば１０％以上、好ましくは２０％以上増加させる（ウサギ赤血球を凝集させる最低濃度で例えば１０％以上、好ましくは２０％以上の低下に対応）ことにより示され得る。さらに本発明に係る乳酸菌発酵物による免疫能の増強は、本発明に係る乳酸菌発酵物を魚介類に投与した場合、へい死を引き起こす濃度の病原体（エドワジエラ・タルダ菌、ホワイトスポット病ウイルスなど）の存在下、乳酸菌発酵物を投与しない対照で生残率０％となる時点で例えば２０％以上、好ましくは３０％以上の生残率となることにより示され得る。これらの免疫能増強の指標は、具体的には実施例に記載の方法に従って測定することができる。 Since the fermented lactic acid bacterium according to the present invention has an action of activating and enhancing the immunity of fish and shellfish, it can be used as an immunostimulator for fish and shellfish. In the present invention, immunostimulation means activating the living body's immune system and enhancing the immune function. More specifically, the enhancement of immunity by the lactic acid bacteria fermented product according to the present invention is such that when the lactic acid bacterium fermented product according to the present invention is administered to fish and shellfish, the phagocytosis rate of leukocytes against opsonized yeast (phagocytosis relative to the total leukocyte count) The percentage of white blood cell count) can be shown, for example, by increasing by more than 10%, preferably by more than 20% compared to the control without administration of lactic acid bacteria fermentation. The enhancement of immunity by the lactic acid bacteria fermented product according to the present invention is such that when the lactic acid bacterial fermented product according to the present invention is administered to fish and shellfish, serum complement activity (complement alternative pathway activity) is not administered. For example, it can be indicated by an increase of 20% or more, preferably 40% or more compared to the control (corresponding to a decrease of ACH50 value of 20% or more, preferably 40% or more). The enhancement of immunity by the lactic acid bacteria fermented product according to the present invention is also achieved when the lactic acid bacterial fermented product according to the present invention is administered to fish and shellfish, and the lectin activity (crude lectin) measured as agglutination activity of serum or body mucus on rabbit erythrocytes. Activity) is increased by, for example, 10% or more, preferably 20% or more compared to a control to which no fermented lactic acid bacteria are administered (corresponding to a decrease of, for example, 10% or more, preferably 20% or more at the lowest concentration at which rabbit erythrocytes aggregate) ). Furthermore, the enhancement of immunity by the lactic acid bacteria fermented product according to the present invention is the presence of pathogens (Edwardiaella tarda, white spot disease virus, etc.) at a concentration that causes death when the lactic acid bacterial fermented product according to the present invention is administered to seafood. Below, it can be shown that the survival rate is, for example, 20% or more, preferably 30% or more when the survival rate becomes 0% in the control not administered with the fermented lactic acid bacteria. Specifically, these immunity enhancement indices can be measured according to the methods described in the Examples.
本発明では、本発明に係る乳酸菌発酵物を魚介類に投与することにより、魚介類の免疫能を賦活し増強することができる。典型的には、本発明に係る乳酸菌発酵物を魚介類用飼料に添加し、それを魚介類に給餌することにより、魚介類の免疫能を賦活し増強することができる。本発明に係る乳酸菌発酵物の成分が魚介類の免疫系に直接的又は間接的に作用し、魚介類の免疫能を賦活するものと考えられる。このような本発明の方法によれば、従来より安価に、かつ多くの養殖魚介類の免疫能を一度に賦活・増強することができる。本発明はこのような魚介類の免疫賦活方法も提供する。 In the present invention, the immunity of fish and shellfish can be activated and enhanced by administering the fermented lactic acid bacteria according to the present invention to fish and shellfish. Typically, by adding the fermented lactic acid bacterium according to the present invention to a feed for seafood and feeding it to the seafood, the immunity of the seafood can be activated and enhanced. It is considered that the components of the fermented lactic acid bacterium according to the present invention act directly or indirectly on the immune system of fish and shellfish and activate the immune ability of fish and shellfish. According to such a method of the present invention, it is possible to activate and enhance the immunity of many cultured fish and shellfish at a lower cost than before. The present invention also provides such a method for immunizing fish and shellfish.
本発明に係る魚介類の免疫賦活方法では、本発明に係る乳酸菌発酵物を任意の量で含む魚介類用飼料を給餌に用いることができるが、典型的には、飼料総重量に対して乾燥重量で１重量％〜３０重量％、好ましくは４〜２０重量％の本発明に係る乳酸菌発酵物を含む魚介類用飼料が用いられる。本発明に係る乳酸菌発酵物をそのような量で飼料原料と混合し、魚介類用飼料を製造することができる。 In the immunostimulation method for fish and shellfish according to the present invention, feed for fish and shellfish containing the lactic acid bacteria fermented product according to the present invention in any amount can be used for feeding, but typically dried relative to the total weight of the feed. A feed for seafood containing 1 to 30% by weight, preferably 4 to 20% by weight, of the lactic acid bacteria fermentation product according to the present invention is used. The fermented lactic acid bacteria according to the present invention can be mixed with the feed raw material in such an amount to produce a feed for seafood.
本発明に係る乳酸菌発酵物と混合する飼料原料としては、魚介類用飼料の原料として用いられる通常の飼料基材を用いることができ、特に限定されず、対象となる魚介類の種類や成長段階等に応じて適宜選択することもできる。一例では、魚粉（主成分）、大豆粕、トウモロコシ粉、小麦、及びでん粉を用いた配合飼料を飼料原料として本発明に係る乳酸菌発酵物と混合することができる。別の例では、飼料原料として、魚粉（主成分）、タラ肝油、ダイズレシチン、ビタミン混合物、ミネラル混合物、活性グルテン、及びセルロースを、本発明に係る乳酸菌発酵物と混合してもよい。 As a feed raw material to be mixed with the lactic acid bacteria fermented product according to the present invention, a normal feed base material used as a raw material for fish and shellfish feed can be used, not particularly limited, and the type and growth stage of the target fish and shellfish It can also be appropriately selected according to the like. In one example, a mixed feed using fish meal (main component), soybean meal, corn meal, wheat, and starch can be mixed with the lactic acid bacteria fermentation product according to the present invention as a feed material. In another example, fish meal (main component), cod liver oil, soybean lecithin, vitamin mixture, mineral mixture, active gluten, and cellulose may be mixed with the fermented lactic acid bacteria according to the present invention as feed materials.
魚介類用飼料の製造方法は、特に限定されず、適宜選択することができる。例えば、飼料基材に対し、本発明に係る乳酸菌発酵物を添加した後に、常法に従い混合し、成形してもよい。例えば、エクストルーダを使用し、本発明に係る乳酸菌発酵物を１００℃前後で乾燥し、粉末化して一定量を飼料原料に混合し、それをペレット化することにより、本発明に係る乳酸菌発酵物を含有する魚介類用飼料を製造してもよい。あるいは、飼料基材を混合し、成形して得た配合飼料に対し、本発明に係る乳酸菌発酵物をコーティングする等の方法により、魚介類用飼料を製造することができる。 The method for producing the feed for seafood is not particularly limited, and can be selected as appropriate. For example, after adding the fermented lactic acid bacterium according to the present invention to a feed base material, it may be mixed and molded according to a conventional method. For example, by using an extruder, the lactic acid bacteria fermented product according to the present invention is dried at around 100 ° C., pulverized, mixed with a certain amount of feed material, and pelletized to obtain the lactic acid bacterial fermented product according to the present invention. You may manufacture the feed for fishery products to contain. Or the feed for fishery products can be manufactured by methods, such as coating the lactic-acid-bacteria fermented material which concerns on this invention with respect to the compound feed obtained by mixing and shape | molding a feed base material.
魚介類用飼料は任意の形状であってよく、対象となる魚介類の種類や成長段階等に応じて適宜選択することができる。魚介類用飼料の形状としては、例えば固形状（モイストペレットを含む）、粉末状（練り餌を含む）、及びペースト状等が挙げられる。 The feed for fish and shellfish may have an arbitrary shape, and can be appropriately selected according to the type and growth stage of the target fish and shellfish. Examples of the shape of the feed for seafood include solid forms (including moist pellets), powder forms (including paste bait), and paste forms.
本発明は、上記のような、本発明に係る乳酸菌発酵物を含有する魚介類用飼料も提供する。本発明はまた、本発明に係る乳酸菌発酵物を含有する、免疫賦活用の魚介類用飼料の製造方法も提供する。 The present invention also provides a feed for seafood containing the fermented lactic acid bacteria according to the present invention as described above. The present invention also provides a method for producing an immunostimulating feed for seafood containing the fermented lactic acid bacteria according to the present invention.
本発明に係る乳酸菌発酵物を、飼料に配合して与える等により魚介類に投与することにより、魚介類の免疫能の賦活・増強効果を得ることができる。本発明に係る乳酸菌発酵物による魚介類の免疫能の賦活・増強効果は、具体的には、例えば、血清補体活性の増加、白血球の貪食率の増加、又はレクチン活性の増加によって確認することができる。 By administering the fermented lactic acid bacteria according to the present invention to fish and shellfish by mixing them with feed and the like, it is possible to obtain an effect of stimulating and enhancing the immunity of fish and shellfish. Specifically, the effect of stimulating and enhancing the immunity of fish and shellfish by the fermented lactic acid bacteria according to the present invention should be confirmed by, for example, increasing serum complement activity, increasing leukocyte phagocytosis, or increasing lectin activity. Can do.
さらに本発明に係る乳酸菌発酵物は、このような免疫能の賦活及び増強により、魚介類の疾病を予防する効果も有する。本発明に係る乳酸菌発酵物は、特に、感染症、すなわち病原体感染による疾病を効果的に予防する効果を有する。感染症は特に限定されないが、細菌又はウイルスによる感染が好ましい。好適な具体例としては、以下に限定されるものではないが、例えば、エドワジエラ属菌、典型的にはエドワジエラ・タルダ菌の感染に起因する疾病（エドワジエラ症など）や、ホワイトスポット病ウイルスの感染に起因するホワイトスポット病等が挙げられる。これらエドワジエラ症やホワイトスポット病等は、効果的なワクチン開発が困難とされていた疾病であり、これらの感染症の予防に対して高い有効性を示す本発明に係る乳酸菌発酵物は、非常に有用である。 Further, the fermented lactic acid bacterium according to the present invention has an effect of preventing diseases of fish and shellfish by such activation and enhancement of immunity. The fermented lactic acid bacterium according to the present invention has an effect of effectively preventing infectious diseases, that is, diseases caused by pathogen infection. Infectious diseases are not particularly limited, but infection by bacteria or viruses is preferred. Preferable specific examples include, but are not limited to, for example, diseases caused by infection with Edwardsiella spp., Typically Edwardsiella tarda, and infection with white spot disease virus. White spot disease and the like caused by These Edwardsiellosis and white spot disease are diseases for which it was considered difficult to develop effective vaccines, and the fermented lactic acid bacteria according to the present invention showing high effectiveness for the prevention of these infections are very Useful.
本発明に係る乳酸菌発酵物は、高い感染症予防効果を示すことから、魚介類用の、感染症予防剤又は感染症予防のための免疫賦活剤としても用いることができる。このため本発明に係る乳酸菌発酵物の投与対象としては、感染症のリスクが高い魚介類も好適である。例えば、エドワジエラ症が知られる魚介類（例えば、ウナギ）なども好適な対象となる。またホワイトスポット病は甲殻類全般で発生が知られるウイルス病であるため、甲殻類は広く好適な投与対象となる。 Since the fermented lactic acid bacterium according to the present invention exhibits a high infectious disease preventing effect, it can also be used as an infectious disease preventive agent or an immunostimulator for infectious disease prevention for fish and shellfish. For this reason, fish and shellfish with a high risk of infectious disease are also suitable as the administration target of the lactic acid bacteria fermentation product according to the present invention. For example, seafood (for example, eel) with known Edwardsiella disease is also a suitable target. White spot disease is a viral disease that is known to occur in all crustaceans, and therefore crustaceans are widely suitable for administration.
以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
（実施例１）
＜乳酸菌発酵物の製造＞
（１）乳酸菌発酵物１の製造
エノキタケ廃菌床８５部、乾燥おから１０部及び味噌５部を発酵タンク内で均一に混合して培養基を調製し、撹拌しながら蒸気を用いて３０分間培養基を殺菌した。培養基を室温まで冷却した後、乳酸菌ラクトバチルス・ファーメンタム キリシマ１Ｒ（受託番号ＮＩＴＥ Ｐ−７８４）及びペディオコッカス・ペントサセウス キリシマ１Ｃ（受託番号ＮＩＴＥ Ｐ−７８７）を合わせて１０６ｃｆｕ／ｇのオーダーになるように添加した。培養基を３０〜３５℃に維持しながら約２０時間培養し、５．６ｘ１０９ｃｆｕ／ｇの乳酸菌発酵物（生菌）を得た。得られた乳酸菌発酵物（生菌）を、乾燥機を用い約８０℃で乾燥し、乳酸菌発酵物１を得た。
Example 1
<Production of fermented lactic acid bacteria>
(1) Production of fermented lactic acid bacteria 1 85 parts enokitake mushroom bed, 10 parts dried okara and 5 parts miso are mixed evenly in a fermentation tank to prepare a culture medium, and agitation for 30 minutes using steam while stirring. Sterilized. After cooling the culture medium to room temperature, the lactic acid bacteria Lactobacillus fermentum Kirishima 1R (Accession No. NITE P-784) and Pediococcus pentosaceus Kirishima 1C (Accession No. NITE P-787) are combined and ordered in the order of 10 6 cfu / g. It added so that it might become. The culture was maintained for about 20 hours while maintaining the culture medium at 30 to 35 ° C. to obtain a 5.6 × 10 9 cfu / g lactic acid bacteria fermentation product (viable bacteria). The obtained fermented lactic acid bacteria (viable bacteria) was dried at about 80 ° C. using a dryer to obtain fermented lactic acid bacteria 1.
（２）乳酸菌発酵物２の製造
ヒラタケ廃菌床９０部、及び乾燥おから１０部を発酵タンク内で均一に混合して培養基を調製し、撹拌しながら蒸気を用いて３０分間培養基を滅菌した。培養基を室温まで冷却した後、乳酸菌エンテロコッカス・フェカリス（ＮＢＲＣ ３９８９）を１０６ｃｆｕ／ｇのオーダーになるように添加した。培養基を３０〜３５℃に維持しながら約２０時間培養し、６．７ｘ１０９ｃｆｕ／ｇの乳酸菌発酵物（生菌）を得た。得られた乳酸菌発酵物（生菌）を、乾燥機を用い約８０℃で乾燥し、乳酸菌発酵物２を得た。
(2) Manufacture of lactic acid bacteria fermented product 2 90 parts of oyster mushroom waste fungus bed and 10 parts of dried okara were mixed uniformly in the fermentation tank to prepare a culture medium, and the culture medium was sterilized with steam for 30 minutes while stirring. . After the culture medium was cooled to room temperature, lactic acid bacteria Enterococcus faecalis (NBRC 3989) was added to the order of 10 6 cfu / g. While maintaining the culture medium at 30 to 35 ° C., it was cultured for about 20 hours to obtain 6.7 × 10 9 cfu / g lactic acid bacteria fermentation product (viable bacteria). The obtained fermented lactic acid bacteria (live bacteria) was dried at about 80 ° C. using a dryer to obtain fermented lactic acid bacteria 2.
（３）乳酸菌発酵物３の製造
ブナシメジ廃菌床９０部、及びフスマ１０部を発酵タンク内で均一に混合して培養基を調製し、撹拌しながら蒸気を用いて３０分間培養基を滅菌した。培養基を室温まで冷却した後、乳酸菌ペディオコッカス・ペントサセウス キリシマ２Ｃ（受託番号ＮＩＴＥ Ｐ−７８８）を１０６ｃｆｕ／ｇのオーダーになるように添加した。培養基を３０〜３５℃に維持しながら約２０時間培養し、５．１ｘ１０９ｃｆｕ／ｇの乳酸菌発酵物（生菌）を得た。得られた乳酸菌発酵物（生菌）を、乾燥機を用い約８０℃で乾燥し、乳酸菌発酵物３を得た。
(3) Production of Fermented Product 3 of Lactic Acid Bacteria 90 parts of Buna shimeji mushroom bed and 10 parts of bran were uniformly mixed in a fermentation tank to prepare a culture medium, and the culture medium was sterilized with steam for 30 minutes while stirring. After the culture medium was cooled to room temperature, the lactic acid bacterium Pediococcus pentosaceus Kirishima 2C (accession number NITE P-788) was added to the order of 10 6 cfu / g. The culture medium was maintained for about 20 hours while maintaining the culture medium at 30 to 35 ° C. to obtain a fermented product of lactic acid bacteria (viable bacteria) of 5.1 × 10 9 cfu / g. The obtained fermented lactic acid bacteria (viable bacteria) was dried at about 80 ° C. using a dryer to obtain fermented lactic acid bacteria 3.
＜乳酸菌凍結乾燥物の製造＞
試験区の効果と比較するため乳酸菌発酵物に含まれるものと同様の乳酸菌の凍結乾燥物を作製した。先ず、５重量％ＭＲＳブイヨン培地（関東化学製）に乳酸菌を播種し、３５℃で２０時間培養した。これを遠心分離し、沈殿物を精製水で洗浄し、５重量％トレハロース及び５重量％スキムミルク調整液に加え、懸濁し凍結し、凍結乾燥した。乳酸菌ラクトバチルス・ファーメンタム キリシマ１Ｒについては１．７ｘ１０11ｃｆｕ／ｇ、乳酸菌ペディオコッカス・ペントサセウス キリシマ２Ｃについては３．２ｘ１０11ｃｆｕ／ｇ及び乳酸菌エンテロコッカス・フェカリス（ＮＢＲＣ ３９８９）については３．２ｘ１０11ｃｆｕ／ｇの凍結乾燥物（生菌）を得た。これらは下記の評価試験に供した。
<Production of lyophilized lactic acid bacteria>
In order to compare with the effect of the test group, a lyophilized product of lactic acid bacteria similar to that contained in the fermented lactic acid bacteria was prepared. First, lactic acid bacteria were inoculated in a 5% by weight MRS broth medium (manufactured by Kanto Chemical) and cultured at 35 ° C. for 20 hours. This was centrifuged, the precipitate was washed with purified water, added to 5% by weight trehalose and 5% by weight skimmed milk preparation, suspended, frozen and lyophilized. Lactic acid bacteria Lactobacillus fermentum Kirishima 1R is 1.7 × 10 11 cfu / g, Lactic acid bacteria Pediococcus pentosaceus Kirishima 2C is 3.2 × 10 11 cfu / g and Lactobacillus enterococcus faecalis (NBRC 3899) is 3.2 × 10 11 A freeze-dried product (live bacteria) of cfu / g was obtained. These were subjected to the following evaluation tests.
（実施例２）
＜自然免疫能評価試験＞
試験魚としてマダイ稚魚（平均体重３８．９ｇ）を用い、乳酸菌発酵物１が魚の自然免疫能におよぼす影響を飼育試験で評価した。
マダイ稚魚に乳酸菌発酵物１の添加量の異なる飼料を１５日間又は３０日間与えて飼育した。飼育試験で与えた飼料の組成を表１に、飼育試験条件を表２に示した。
(Example 2)
<Innate immunity evaluation test>
A red sea bream fry (average body weight 38.9 g) was used as a test fish, and the effect of the fermented lactic acid bacteria 1 on the natural immunity of the fish was evaluated in a breeding test.
The red sea bream larvae were fed with different amounts of lactic acid bacteria fermented product 1 for 15 days or 30 days and reared. Table 1 shows the composition of the feed given in the breeding test, and Table 2 shows the breeding test conditions.
１５日間又は３０日間の飼育試験終了後、各区より試験魚を５尾ずつ取り上げ、フェノキシエタノール（３００ｐｐｍ）で麻酔し、血液、体表粘液及び腎臓（頭腎）を採取した。採取した血液は室温で放置し、凝固後に遠心分離（４℃、３０００ｒｐｍ、１５分）し、血清を回収した。このマダイ血清試料について、血清補体活性（補体第２経路活性、ＡＣＨ５０値）を以下のとおり測定した。まず、Ｍｇ・ＥＤＴＡを１０ｍＭ濃度となるよう加えた０．１％ゼラチンベロナール緩衝液（ｐＨ７．５）を用いてウサギ保存血液を遠心洗浄（４℃、３００ｒｐｍ、５分）し、得られたウサギ赤血球をＭｇ・ＥＧＴＡ添加０．１％ゼラチンベロナール緩衝液（ｐＨ７．５）に２×１０８細胞／ｍＬとなるよう懸濁した（赤血球浮遊液）。次に、１２５、１００、８０、６２．５、５０及び２５μＬのマダイ血清試料に対して０、２５、４５、６２．５、７５及び１００μＬのＭｇ・ＥＤＴＡ添加０．１重量％ゼラチンベロナール緩衝液（ｐＨ７．５）をそれぞれ加え、マダイ血清の希釈系列を作製した。これらのマダイ血清希釈液に上記で調製した赤血球浮遊液５０μＬをそれぞれ加え、２０℃で２４時間インキュベート後、ＥＤＴＡを１０ｍＭ濃度となるよう添加した０．１％ゼラチンベロナール緩衝液（ｐＨ７．５）を７００μＬ加え、反応を停止させ、遠心分離（４℃、３００rpm、５分）した液層の吸光度（ＯＤ４１４）を測定した。両対数グラフのＸ軸に試験に用いたマダイ血清の希釈倍率、Ｙ軸にＹ／（１−Ｙ）をプロットし、得られた曲線よりＹ＝０．５（溶血率５０％）となるｘを求め、ＡＣＨ５０（Ｕｎｉｔ/ｍＬ）とした。 After the 15-day or 30-day breeding test, 5 test fish were taken from each group and anesthetized with phenoxyethanol (300 ppm), and blood, body surface mucus and kidney (head kidney) were collected. The collected blood was allowed to stand at room temperature, and after clotting, centrifuged (4 ° C., 3000 rpm, 15 minutes) to collect serum. Serum complement activity (complement alternative pathway activity, ACH50 value) was measured for this red sea bream serum sample as follows. First, rabbit stored blood was centrifuged and washed (4 ° C., 300 rpm, 5 minutes) using 0.1% gelatin veronal buffer (pH 7.5) to which Mg · EDTA was added to a concentration of 10 mM. Rabbit erythrocytes were suspended in a 0.1% gelatin veronal buffer solution (pH 7.5) supplemented with Mg · EGTA to a concentration of 2 × 10 8 cells / mL (erythrocyte suspension). Next, 0.1 wt% gelatin veronal buffer supplemented with 0, 25, 45, 62.5, 75 and 100 μL Mg • EDTA for 125, 100, 80, 62.5, 50 and 25 μL red sea bream serum samples. Each solution (pH 7.5) was added to prepare a dilution series of red sea bream serum. To each of these red sea bream serum dilutions, 50 μL of the erythrocyte suspension prepared above was added, incubated at 20 ° C. for 24 hours, and then 0.1% gelatin veronal buffer (pH 7.5) added with EDTA to a concentration of 10 mM. Was added to stop the reaction, and the absorbance (OD414) of the centrifuged liquid layer (4 ° C., 300 rpm, 5 minutes) was measured. Plotting the dilution ratio of red sea bream serum used in the test on the X-axis of the log-log graph and Y / (1-Y) on the Y-axis. And determined as ACH50 (Unit / mL).
またマダイ血清試料及び体表粘液の粗レクチン活性（レクチン活性）をウサギ赤血球に対する凝集活性として測定した。ウサギ保存血液２０００μＬをプラスチックチューブに取り、ＰＢＳを１ｍＬ加え、２０００回転で２分間遠心後、上澄みを捨てた。この操作を３、４回繰り返し、ウサギ保存血液から赤血球画分を得た。これにＰＢＳを１０ｍＬ加え、ウサギ赤血球浮遊液を作製した。次に９６穴マイクロプレートの各ウェルに、ＰＢＳを５０μＬずつ分注し、そこへ、総タンパク濃度をＢＣＡ法によりあらかじめ算出しておいた、マダイ血清試料及び体表粘液５０μＬをマイクロプレートの一列目に加え、マイクロピペットで数回吸引吐出した。一列目のうち、５０μＬを二列目に移し、マイクロピぺットで数回吸引吐出した。この操作を三列目へ、四列目へと同様に行い、測定対象サンプルの２倍希釈系列を作製した。これらの各ウェルにウサギ赤血球浮遊液を５０μＬずつ分注し、２８℃で９０分間インキュベートした。各ウェルの底部へのウサギ赤血球の凝集沈降の有無を顕微鏡観察し、赤血球凝集沈降を起こすことのできた血清及び体表粘液の最低タンパク濃度（μｇ／ｍＬ）を算出し、活性値とした。 In addition, the crude lectin activity (lectin activity) of red sea bream serum samples and body surface mucus was measured as agglutination activity on rabbit erythrocytes. 2000 μL of rabbit stored blood was taken in a plastic tube, 1 mL of PBS was added, centrifuged at 2000 rpm for 2 minutes, and the supernatant was discarded. This operation was repeated three or four times to obtain a red blood cell fraction from rabbit stored blood. 10 mL of PBS was added to this to prepare a rabbit erythrocyte suspension. Next, 50 μL of PBS was dispensed into each well of the 96-well microplate, and the total protein concentration was previously calculated by the BCA method, and then the red sea bream serum sample and 50 μL of body surface mucus were placed in the first row of the microplate. In addition, the sample was aspirated and discharged several times with a micropipette. Of the first row, 50 μL was transferred to the second row and aspirated and discharged several times with a micropipette. This operation was performed in the same manner for the third row and the fourth row, and a 2-fold dilution series of the sample to be measured was prepared. 50 μL of rabbit erythrocyte suspension was dispensed into each well and incubated at 28 ° C. for 90 minutes. The presence or absence of agglutination and sedimentation of rabbit erythrocytes at the bottom of each well was observed under a microscope, and the minimum protein concentration (μg / mL) of serum and body surface mucus that could cause erythrocyte aggregation and precipitation was calculated and used as the activity value.
また、腎臓からはパーコールを用いた密度勾配遠心法によりマクロファージを含む白血球集団を分離し、オプソニン化酵母菌に対する貪食率（総白血球数に対する貪食白血球数の割合）を測定した。貪食率の測定は、酵母菌にあらかじめ用意しておいたマダイ血清試料を加え、２５℃で１時間インキュベートし、オプソニン化酵母液（１０６／ｍＬ）を作製した。次に試験魚の腎臓を取り出し、セルストレイナー（１００メッシュ）で細胞を濾し、比重１.０４及び１．０７ｇ／ｍＬに調整し、プラスチックチューブに重層したパーコールに注入して、遠心分離（４℃、１６００ｒｐｍ、３０分）後、分離された白血球層のみを回収した。これをＲＰＭＩ１６４０培地で数回遠心洗浄して、細胞濃度を１０６細胞／ｍＬに調整した。このようにして調製した白血球懸濁液にオプソニン化酵母液を等量加え、２５℃で２時間インキュベート後、塗抹標本を作製し、ギムザ染色して顕微鏡観察した。視野内の全白血球数に対するオプソニン化酵母貪食白血球数の割合（％）を算出し、貪食率とした。 In addition, leukocyte populations including macrophages were separated from the kidney by density gradient centrifugation using Percoll, and the phagocytosis rate against opsonized yeast (ratio of phagocytic leukocyte count to total leukocyte count) was measured. To measure the phagocytosis rate, a red sea bream serum sample prepared in advance was added to the yeast and incubated at 25 ° C. for 1 hour to prepare an opsonized yeast solution (10 6 / mL). Next, the kidney of the test fish is taken out, the cells are filtered with a cell strainer (100 mesh), adjusted to a specific gravity of 1.04 and 1.07 g / mL, injected into Percoll layered on a plastic tube, and centrifuged (4 ° C., After 1600 rpm for 30 minutes, only the separated leukocyte layer was recovered. This was centrifugally washed several times with RPMI 1640 medium to adjust the cell concentration to 10 6 cells / mL. An equal amount of opsonized yeast solution was added to the leukocyte suspension thus prepared and incubated at 25 ° C. for 2 hours. A smear was prepared, stained with Giemsa, and observed with a microscope. The ratio (%) of the number of opsonized yeast phagocytic leukocytes to the total number of leukocytes in the visual field was calculated and used as the phagocytosis rate.
３０日間の飼育試験後の稚魚の白血球の貪食率と血清補体活性（ＡＣＨ５０値）の測定結果を表３に示す。また１５日間又は３０日間の飼育試験後の稚魚の血清及び体表粘液の粗レクチン活性の測定結果を表４に示す。 Table 3 shows the measurement results of the leukocyte phagocytosis rate and serum complement activity (ACH50 value) of the juvenile fish after the 30-day breeding test. In addition, Table 4 shows the measurement results of the crude lectin activity of fry serum and body surface mucus after a 15-day or 30-day breeding test.
表３に示すとおり、白血球の貪食率は乳酸菌発酵物の添加量の増加に伴い上昇した。同様に血清補体活性も乳酸菌発酵物の添加量の増加と共に増加した。 As shown in Table 3, the phagocytosis rate of leukocytes increased as the amount of lactic acid bacteria fermented product increased. Similarly, serum complement activity increased with increasing amount of fermented lactic acid bacteria.
一方、乳酸菌発酵物の添加量の異なる飼料を１５日間与えたマダイ稚魚の血清及び体表粘液中の粗レクチン活性は対照区及び試験区間で同様のレベルを示したが、３０日間の飼育試験後には血清、体表粘液ともに試験区では対照区と比較して粗レクチン活性が顕著に増加し、また飼料中の乳酸菌発酵物の添加量の１０％以上への増加に伴い粗レクチン活性はさらに増加した。比較区の凍結乾燥乳酸菌は過剰量にもかかわらず、粗レクチン活性の増加については僅かな効果にとどまった。なお表４に示す粗レクチン活性値は、数値が高い程、粗レクチン活性が低いことを示す。これらの結果より、本発明に係る乳酸菌発酵物にはマダイ稚魚の自然免疫能を増強する効果があり、マダイの健全性を高めることができることが示された。 On the other hand, the crude lectin activity in the sera and body surface mucus of red sea bream fry fed with different amounts of fermented lactic acid bacteria for 15 days showed similar levels in the control and test sections, but after 30 days of feeding test In both the serum and the body surface mucus, the crude lectin activity increased significantly in the test group compared to the control group, and the lectin activity increased further as the amount of lactic acid bacteria fermented product in the feed increased to 10% or more. did. The lyophilized lactic acid bacteria in the comparative group had little effect on the increase in the crude lectin activity despite the excessive amount. The crude lectin activity values shown in Table 4 indicate that the higher the numerical value, the lower the crude lectin activity. From these results, it was shown that the fermented lactic acid bacterium according to the present invention has an effect of enhancing the natural immunity of red sea bream, and can improve the soundness of red sea bream.
（実施例３）
＜各種乳酸菌を用いた乳酸菌発酵物の貪食率に対する効果＞
乳酸菌としてペディオコッカス・ペントサセウス キリシマ２Ｃ（受託番号ＮＩＴＥ Ｐ−７８８）、ラクトバチルス・ファーメンタム キリシマ２Ｒ（受託番号ＮＩＴＥ Ｐ−７８５）、ラクトバチルス・プランタラム ＭＨ（受託番号ＮＩＴＥ Ｐ−１５４８）、ラクトバチルス・プランタラム ＭＥ（受託番号ＮＩＴＥ Ｐ−１５４９）及びエンテロコッカス・フェカリス（ＮＢＲＣ ３９８９）のいずれか１種を使用すること以外は、実施例１の乳酸菌発酵物１と同様にして乳酸菌発酵物を製造した。この乳酸菌発酵物を用いて表１の試験区１の組成（乳酸菌発酵物を５重量％配合）に従って調製した飼料をマダイ稚魚に給餌しながら、実施例２と同様にして、３０日間試験を行い、白血球の貪食率を調べた。
(Example 3)
<Effects on phagocytosis rate of fermented lactic acid bacteria using various lactic acid bacteria>
As lactic acid bacteria, Pediococcus pentosaceus Kirishima 2C (Accession number NITE P-788), Lactobacillus fermentum Kirisima 2R (Accession number NITE P-785), Lactobacillus plantarum MH (Accession number NITE P-1548), Lact A lactic acid bacteria fermented product is produced in the same manner as in the lactic acid bacteria fermented product 1 of Example 1 except that any one of Bacillus plantarum ME (Accession No. NITE P-1549) and Enterococcus faecalis (NBRC 3989) is used. did. Using this fermented lactic acid bacterium, the feed prepared according to the composition of Test Zone 1 in Table 1 (containing 5% by weight of lactic acid bacterium fermented product) was fed to red sea bream, and the test was conducted for 30 days in the same manner as in Example 2. The leukocyte phagocytosis rate was examined.
その結果、貪食率は、表１の対照区と同じ飼料を用いた対照区の２８．５％に対して、試験区ではペディオコッカス・ペントサセウス キリシマ２Ｃで４２．１％、ラクトバチルス・ファーメンタム キリシマ２Ｒで３８．７％、ラクトバチルス・プランタラム ＭＨで４４．１％、ラクトバチルス・プランタラム ＭＥで４１．３％、エンテロコッカス・フェカリス（ＮＢＲＣ ３９８９）で４３．６％を示し、上記乳酸菌発酵物を含む飼料を用いた試験区では貪食率が有意に増加した。すなわち様々な乳酸菌種を用いて製造した本発明に係る乳酸菌発酵物が、魚の自然免疫能の増強に有効であることが示された。 As a result, the phagocytosis rate was 28.5% in the control group using the same feed as the control group in Table 1, whereas 42.1% in the Pediococcus pentosaceus Kirishima 2C in the test group and Lactobacillus fermentum Kirishima 2R showed 38.7%, Lactobacillus plantarum MH 44.1%, Lactobacillus plantarum ME 41.3%, Enterococcus faecalis (NBRC 3989) 43.6% The phagocytosis rate increased significantly in the test plots using feed containing food. That is, it was shown that the fermented lactic acid bacteria according to the present invention produced using various lactic acid bacteria species is effective for enhancing the natural immunity of fish.
（実施例４）
＜マダイの感染試験＞
乳酸菌発酵物の病原菌感染予防効果を評価するため、養殖魚類の感染症エドワジエラ症を引き起こすエドワジエラ・タルダ菌（Ｅｄｗａｒｄｓｉｅｌｌａ ｔａｒｄａ）を用いて、マダイ稚魚の感染試験を実施した。平均体重４０ｇのマダイ稚魚に、実施例１で製造した乳酸菌発酵物２を用いて調製した乳酸菌発酵物添加量の異なる飼料を６週間（４２日間）給餌しながら飼育した。飼料組成は表１の対照区、試験区１、試験区２及び比較区に従った。飼育条件は、給餌期間を除き表２に従った。６週間の飼育終了後、マダイの腹腔内にリン酸緩衝液で濃度調整した菌濃度３．６ｘ１０５ｃｆｕ／ｍＬのエドワジエラ・タルダ菌溶液０．１ｍＬを打注し、マダイ稚魚のへい死数の推移を経時的に観察した。
Example 4
<Red sea bream infection test>
In order to evaluate the effect of fermented lactic acid bacteria on the infection of pathogenic bacteria, an infection test was carried out on juvenile red sea bream using Edwardsiella tarda, which causes infectious diseases of cultured fish, Edwardsiella disease. A red sea bream with an average body weight of 40 g was bred while feeding for 6 weeks (42 days) with different amounts of fermented lactic acid bacteria prepared using the fermented lactic acid bacteria 2 produced in Example 1. The feed composition was in accordance with the control group, test group 1, test group 2 and comparative group of Table 1. Rearing conditions were in accordance with Table 2 except for the feeding period. After the end of 6 weeks of breeding, 0.1 ml of a solution of 3.6 × 10 5 cfu / mL of Edwardsiella tarda fungus adjusted with phosphate buffer was injected into the abdominal cavity of red sea bream, and the number of larvae of red sea bream larvae Were observed over time.
結果を表５に示す。乳酸菌発酵物２無添加の飼料を与えた対照区では打注２日目にへい死尾数が急増し、６日目までに１２尾全てがへい死した。これに対し、乳酸菌発酵物２を５重量％又は１０重量％添加した飼料（それぞれ試験区１、試験区２）を与えた場合、打注２日目以降のへい死数が大きく低減し、打注２日目から明らかにエドワジエラ菌感染予防効果が認められた（表５）。一方、凍結乾燥乳酸菌エンテロッコス・フェカリスを含む飼料を与えた比較区では、僅かに効果が認められたものの試験区と比べるとはるかに効果が低かった。 The results are shown in Table 5. In the control group to which the lactic acid bacteria fermented product 2 additive-free feed was given, the number of dead tails increased sharply on the 2nd day of injection, and all 12 birds died by the 6th day. On the other hand, when feed containing 5% by weight or 10% by weight of lactic acid bacteria fermented product 2 (test zone 1 and test zone 2 respectively) was given, the number of deaths from the second day after the injection was greatly reduced. From the second day, the effect of preventing E. coli infection was clearly observed (Table 5). On the other hand, the comparative group to which the feed containing freeze-dried lactic acid bacteria Enteroccos faecalis was found to be slightly effective, but the effect was much lower than that of the test group.
この結果より、本発明に係る乳酸菌発酵物はマダイ稚魚の自然免疫能を増強させ、病原菌の感染を予防する効果を有すること、それによりマダイの健全性を高めることができることが示された。 From this result, it was shown that the fermented lactic acid bacterium according to the present invention has the effect of enhancing the natural immunity of red sea bream and preventing infection with pathogenic bacteria, thereby improving the soundness of red sea bream.
（実施例５）
＜他の魚類での感染試験＞
実施例１で製造した乳酸菌発酵物２を５重量％添加した飼料をヒラメに６週間給餌したこと以外は実施例４と同様にして、エドワジエラ・タルダ菌感染試験を行った。その結果、対照区（乳酸菌発酵物無添加飼料）では６日目で生残率（生存率）はゼロとなったが、試験区（乳酸菌発酵物添加飼料）では生残率が５０．０％となり、ヒラメでも本発明に係る乳酸菌発酵物のエドワジエラ・タルダ菌感染に対する予防効果が認められた。
(Example 5)
<Infection tests with other fish>
An Edwardsiella / Talda infection test was carried out in the same manner as in Example 4 except that 5% by weight of the lactic acid bacteria fermented product 2 produced in Example 1 was added to the flounder for 6 weeks. As a result, the survival rate (survival rate) was zero on the 6th day in the control plot (feed without lactic acid bacteria fermented product), but the survival rate was 50.0% in the test plot (feed with fermented lactic acid bacteria). Thus, even in Japanese flounder, the preventive effect of the lactic acid bacteria fermented product according to the present invention against the infection with Edwardsiella tarda was recognized.
さらに、エドワジエラ・タルダ菌の代わりにホワイトスポット病ウイルス（以下、ＷＳＳＶ）を用いて、ＷＳＳＶによる感染試験を実施した。平均体重０．８ｇのクルマエビに、実施例１で製造した乳酸菌発酵物２を１．０重量％、２．５重量％及び５．０重量％添加した飼料を８週間給餌しながら飼育した。比較区では、上述した乳酸菌エンテロコッカス・フェカリス（ＮＢＲＣ ３９８９）の凍結乾燥物を添加した飼料を同様に給餌した。飼料組成を表６に、飼育条件を表７に示した。 Furthermore, an infection test using WSSV was performed using white spot disease virus (hereinafter, WSSV) instead of Edwardsiella tarda. Breast shrimp having an average body weight of 0.8 g were bred for 8 weeks while feeding the lactic acid bacteria fermented product 2 produced in Example 1 with 1.0 wt%, 2.5 wt% and 5.0 wt%. In the comparative group, the feed to which the lyophilized product of the lactic acid bacterium Enterococcus faecalis (NBRC 3989) was added was similarly fed. Table 6 shows the feed composition and Table 7 shows the breeding conditions.
８週間の飼育終了後、クルマエビの第３腹節部に、クルマエビがへい死する濃度のＷＳＳＶ液を０．１ｍＬ打注し、クルマエビのへい死数の推移を経時的に観察した。 After 8 weeks of breeding, 0.1 mL of a WSSV solution at a concentration at which the prawns die was injected into the third abdominal node of the prawns, and changes in the number of dead prawns were observed over time.
その結果、ウイルスに感染してから８日目に、対照区（乳酸菌発酵物無添加飼料）ではクルマエビの生残数はゼロとなったが、試験区１の生残数は２０尾中５尾、試験区２では８尾、試験区３では９尾となり、明らかに乳酸菌発酵物のＷＳＳＶ感染予防効果が認められた。乳酸菌エンテロッコス・フェカリス凍結乾燥物添加飼料を用いた比較区では生残数３尾で若干の効果は認められたものの乳酸菌発酵物添加飼料と比べてはるかに効果は低かった。 As a result, on the 8th day after infection with the virus, the number of surviving prawns was zero in the control plot (feed without fermented lactic acid bacteria), but the surviving number in the test plot 1 was 5 out of 20. The test group 2 had 8 fish and the test group 3 had 9 fish, and the effect of preventing fermentation of lactic acid bacteria by the WSSV infection was clearly observed. In the comparative plot using the lactic acid bacterium Enterococcus faecalis lyophilized product-added feed, although a slight effect was observed with the survival number of 3 fish, the effect was much lower than that of the lactic acid bacterium fermented product-added feed.
本発明に係る乳酸菌発酵物は、安全性が高く、安価でかつ大量製造が可能である。本発明に係る乳酸菌発酵物は、それを給餌することにより魚介類の免疫機能を増強し、病原体の感染を予防し、予防困難な魚介類の疾病を防止するために用いることができる。本発明に係る乳酸菌発酵物は、特に、効果的なワクチン製造が困難とされているエドワジエラ・タルダ菌やホワイトスポット病ウイルスに起因する魚類感染症に対して高い有効性を有することから、感染症予防用に有利に使用できる。 The lactic acid bacteria fermented product according to the present invention has high safety, is inexpensive, and can be mass-produced. The fermented lactic acid bacteria according to the present invention can be used to enhance the immune function of seafood by feeding it, prevent infection of pathogens, and prevent diseases of seafood that are difficult to prevent. The fermented lactic acid bacterium according to the present invention has high effectiveness against fish infections caused by Edwardsiella tarda and white spot disease virus, which are considered difficult to produce effective vaccines. It can be advantageously used for prevention.
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