JP5719497B2 - Feed additive, feed, production method thereof, drowning preventive agent, and breeding method - Google Patents
Feed additive, feed, production method thereof, drowning preventive agent, and breeding method Download PDFInfo
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- JP5719497B2 JP5719497B2 JP2008085057A JP2008085057A JP5719497B2 JP 5719497 B2 JP5719497 B2 JP 5719497B2 JP 2008085057 A JP2008085057 A JP 2008085057A JP 2008085057 A JP2008085057 A JP 2008085057A JP 5719497 B2 JP5719497 B2 JP 5719497B2
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Classifications
-
- 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
Landscapes
- Feed For Specific Animals (AREA)
- Fodder In General (AREA)
Description
本発明は、畜産又は水産飼料添加剤及び該飼料添加剤を添加した飼料に係わり、特に免疫賦活、感染症予防に著効を示す飼料添加剤、プレミックスと、この飼料添加剤を適宜の割合で添加した飼料の製造方法及び添加剤、プレミックス及び飼料に関する。プレミックスは、Prepared Mix（調製粉）の略で、小麦粉等の粉類（澱粉を含む）に糖類、油脂、脱脂粉乳、卵粉、膨張剤、食塩、香料などを必要に応じて適宜に配合したものである。 The present invention relates to a livestock or aquatic feed additive and a feed to which the feed additive is added, in particular, a feed additive and a premix that are highly effective for immune activation and infection prevention, and an appropriate ratio of the feed additive. It is related with the manufacturing method of the feed added by (1), an additive, a premix, and feed. Premix is an abbreviation for Prepared Mix (prepared powder), and contains sugar, fat, skim milk powder, egg powder, swelling agent, salt, fragrance, etc., as appropriate, in flours (including starch). It is a thing.
近年、畜産や水産産業が発展するに伴って、細菌病並びにウィルス病が多発し、甚大な経済的被害をもたらしている。豚のサルモネラ症、大腸菌症、オーエスキー病などや、鶏のインフルエンザ、ニューカッスル病、サルモネラ症、マレック病など、甲殻類や魚類の疾病については、クルマエビの急性ウィルス血症、ビブリオ病、ブリの類結節症、腸球菌症、アユの冷水病、シュードモナス病、マダイ、カンパチ、ブリなどのイリドウィルス感染症などの発生が多く、経済的被害が大きい。 In recent years, with the development of the livestock and fisheries industries, bacterial diseases and viral diseases frequently occur, resulting in tremendous economic damage. For crustacean and fish diseases such as porcine salmonellosis, colibacillosis, Aujeszky's disease, chicken flu, Newcastle disease, Salmonellosis, Marek's disease, etc. There are many outbreaks of nodule, enterococcal disease, cold water disease of sweetfish, pseudomonas disease, iridovirus infection such as red sea bream, amberjack, yellowtail, etc., and the economic damage is great.
これらの病気のうち細菌性疾病については、治療薬として抗生物質や合成抗菌剤が用いられているが、抗菌性物質に対する耐性菌が出現し、充分な治療効果が得られていない。また、使用した薬剤の畜産動物、甲殻類、魚類への残留による公衆衛生上の問題が生じていることから、化学療法に依存しない予防対策が強く望まれている。一方、ペット、畜産動物、甲殻類及び魚類のウィルス病については、治療薬が開発されておらず、病気が依然として多発している状況にある。 Among these diseases, antibiotics and synthetic antibacterial agents are used as therapeutic agents for bacterial diseases, but resistant bacteria to the antibacterial substances have appeared and sufficient therapeutic effects have not been obtained. In addition, since public health problems are caused by residues of used drugs in livestock animals, crustaceans, and fish, preventive measures that do not depend on chemotherapy are strongly desired. On the other hand, for viral diseases of pets, livestock animals, crustaceans and fish, therapeutic drugs have not been developed, and diseases are still frequently occurring.
一方ペット（愛玩動物あるいはコンパニオンアニマルを含む）は高齢化社会の進展や生活様式が核家族化することにともない、その飼育数は世界的に増加の一途をたどっている。そしてこれら動物は野外で飼育されるよりは、家族の一員として飼育されることが多くなっている。この結果、動物の育成環境や食環境が人間のそれと極めて近いものとなる状況が現出している。このためペット等の疾病構造には大きな変化が起こっており、アトピー性皮膚炎をはじめとするアレルギーや自己免疫疾患、糖尿病をはじめとする生活習慣病、骨そしょう症をはじめとする高齢者特有の疾患、免疫低下や不全に基づく感染症、この他がんや白血病などの悪性新生物など、現在人で大きな問題になっている疾患群のすべてがペット等で発生するリスクがあるといって過言ではない。これらペット等における各種疾患群に対しては、主として人に対する医薬品等が代替品として投与される場合が多いが、人とペットでは動物種が異なることから、人用医薬品の有効性は限定的であるとともに、使用にも限界がある。これらの疾患に対して、ペット等の免疫機能を活性化することにより、予防や治療効果を期待することが考えられる。 On the other hand, the number of pets (including pets and companion animals) has been increasing all over the world as the aging society progresses and the lifestyle has become a nuclear family. And these animals are often bred as part of the family rather than being raised outdoors. As a result, a situation has emerged in which the environment for raising animals and the food environment are very close to those of humans. For this reason, there has been a major change in the disease structure of pets, etc., which is peculiar to elderly people such as allergies such as atopic dermatitis, autoimmune diseases, lifestyle-related diseases such as diabetes, and osteoporosis. It is said that all of the disease groups that are currently a major problem in humans, such as cancer diseases, infectious diseases based on decreased immunity and insufficiency, and malignant neoplasms such as cancer and leukemia, are at risk of occurring in pets, etc. It's not too much to say. For these various disease groups in pets, etc., drugs for humans are often administered as substitutes, but the effectiveness of human drugs is limited because humans and pets have different animal species. In addition, there are limits to use. It is conceivable to expect a preventive or therapeutic effect against these diseases by activating immune functions such as pets.
ペット、畜産動物、甲殻類及び魚類の免疫機能の増強と感染症の予防を目的として、ビィフィドバクテリウム・サーモフィラム由来のペプチドグリカン（特許文献１）、バチラス属などグラム陽性菌の細胞壁成分（特許文献２）、スエヒロタケ由来のβ−１，３−グルカン（特許文献３）などの多糖類を利用することは、既に知られている。しかしながら、十分に所望の免疫増強効果を奏し得、飼料又はサプリメントとして実用できる免疫増強物質の開発は未だなされていない現状にある。また、本文献には小麦発酵抽出物の添加方法についての具体的な記述は無い。 Bifidobacterium thermophilum-derived peptidoglycan (patent document 1), cell wall components of gram-positive bacteria such as genus Bacillus for the purpose of enhancing the immune function of pets, livestock animals, crustaceans and fish and preventing infections (patent document) 2) Utilization of polysaccharides such as β-1,3-glucan derived from Suehirotake (Patent Document 3) is already known. However, development of an immunopotentiating substance that can sufficiently exhibit a desired immune enhancing effect and can be used as a feed or a supplement has not yet been made. Moreover, there is no specific description about the addition method of wheat fermented extract in this literature.
既に述べた様に、ペット、畜産動物、甲殻類及び魚類には種々の感染症が多発し、その結果斃死に至るものもあり、甚大な経済的被害をもたらしている。この背景には、これらの畜産又は水産動物が限られた環境で過密に飼育されることによる免疫機能の低下が挙げられる。本発明で使用される食経験が長く、安全性が高いパントエア菌由来のリポ多糖をバイオ技術で大量生産した小麦発酵抽出物には、免疫賦活作用があり、また、ニワトリやブリ、コイ、マウスに経口投与することで感染防除効果を得ていることが特許文献４にある。さらに、βエンドルフィンの誘導作用があり、鎮痛効果があることから（非特許文献１）、ストレスに対する抵抗性効果が期待される。小麦発酵抽出物の有効成分の一つに低分子リポ多糖を含むが、低分子リポ多糖ではポリミキシンBで阻害されるマクロファージ活性化が小麦発酵抽出物では阻害されないことや、低分子リポ多糖ではヨウ素でんぷん反応が陰性であるが、小麦発酵抽出物では陽性であることなど、物質的に異なることが明らかであり、また、生物学的応答性が異なることも知られている。小麦発酵抽出物を飼料に添加し、畜産動物、水産動物に飼料として与えると感染防除効果が得られる事が示されている。そこで、小麦発酵抽出物の感染防除効果と作用機序が異なる既存の免疫賦活物質を混合した飼料を製造すれば、これまでにない高い感染防除効果を示す飼料、サプリメント等を作ることが可能であると考えられた。しかしながら、これまで、小麦発酵抽出物とは機能が異なる免疫賦活物質と混合し、配合した飼料の研究は行われておらず、これらを配合方法の研究もなお十分にはなされていない現状にある。 As already mentioned, various infectious diseases occur frequently in pets, livestock animals, crustaceans and fish, resulting in drowning, resulting in tremendous economic damage. This background includes a decrease in immune function due to overstocking of these livestock or aquatic animals in a limited environment. The fermented wheat extract produced by mass production of lipopolysaccharide derived from Pantoea bacteria with a long dietary experience and high safety used in the present invention has an immunostimulatory effect, and also has chicken, yellowtail, carp, mouse Patent Document 4 discloses that an infection control effect is obtained by oral administration. Furthermore, since it has an inducing action of β-endorphin and has an analgesic effect (Non-patent Document 1), a resistance effect against stress is expected. One of the active ingredients of fermented wheat extract contains low-molecular-weight lipopolysaccharide. Macrophage activation, which is inhibited by polymyxin B with low-molecular-weight lipopolysaccharide, is not inhibited by fermented wheat extract. It is clear that the starch reaction is negative, but that it is positive in the wheat fermented extract, it is clear that it is materially different and the biological responsiveness is also different. It has been shown that an infection control effect can be obtained when a fermented wheat extract is added to feed and given to livestock and aquatic animals as feed. Therefore, by producing a feed that is mixed with existing immunostimulatory substances that have a different mechanism of action from the infection control effect of the fermented wheat extract, it is possible to produce feeds and supplements that have an unprecedented high infection control effect. It was thought that there was. However, until now, there has been no research on feeds mixed with immunostimulatory substances that have different functions from the fermented wheat extract, and the method of blending them has not been fully studied. .
小麦発酵抽出物は体重ｋｇあたり、０．００５−０．０２ｍｇと従来知られている免疫賦活物質よりも著しく少ない投与で、効果が得られる特徴がある。一方、経口投与で免疫賦活作用を示すには動物の体重ｋｇあたり、0.1-10mg程度を投与する。 The fermented wheat extract is characterized in that the effect is obtained by administration of 0.005-0.02 mg per kg body weight, which is significantly less than conventionally known immunostimulatory substances. On the other hand, in order to show an immunostimulatory effect by oral administration, about 0.1-10 mg is administered per kg body weight of the animal.
最も多く使用され、市販されている、固形飼料を製造する場合には加熱成形が行われる。例えば、混合飼料の製造は、エクストルーダー機（粉砕、混合、混練、加熱、加圧、殺菌、冷却、脱水、押出し、成形が可能な機械）内にて蒸気を吹きかけ、高温高圧下で混練した後に押出して成形させるため、高温になる。小麦発酵抽出物の有効成分の一つであるリポ多糖は120度の高温で分解は無いとされていた。ところが、小麦発酵抽出物の免疫賦活作用は飼料と混合し、これを加熱するとリポ多糖含量が低下する事を我々は見出した。そのため、飼料に小麦発酵抽出物を添加ための検討が必要であるが、小麦発酵抽出物の畜産動物、甲殻類及び魚類の飼料製造法、プレミックス製造法などについてはまったく、検討されていなかった。
本発明の目的はペット、畜産動物、甲殻類及び魚類が本来的に備えている免疫機能を、ごく微量で的確に活性化して感染症を予防し、薬物残留などの公衆衛生上の問題のない、安全な甲殻類及び魚類を育成するための飼料添加剤、この飼料添加剤を添加した飼料の製造方法及びプレミックスあるいは飼料を提供し、ペット等に対するアレルギー疾患、自己免疫疾患、生活習慣病、高齢に伴う機能異常、悪性新生物等に対する予防・治療法に有益な小麦発酵抽出物を配合した添加剤、プレミックス、サプリメント及び各種飼料を提供することにある。 The purpose of the present invention is to activate the immune functions inherently possessed by pets, livestock animals, crustaceans and fish accurately and in minute amounts to prevent infectious diseases, and there are no public health problems such as drug residues. , Providing feed additives for nurturing safe crustaceans and fish, feed production methods and premixes or feeds to which this feed additive is added, allergic diseases for pets, autoimmune diseases, lifestyle-related diseases, An object of the present invention is to provide additives, premixes, supplements, and various feeds that contain a fermented wheat extract useful for prevention and treatment of functional abnormalities associated with aging, malignant neoplasms, and the like.
本発明における小麦発酵抽出物とバチルス菌体を添加する対象は、動物飼育用で動物体内に栄養や有用な薬品類を投与する目的で作られた全てのタイプをとることができ、代表的な例としては、家畜用飼料、家畜用プレミックス剤、家畜用ビタミン剤、家畜用医薬品、養魚用飼料、養魚用プレミックス剤、養魚用ビタミン剤、養魚用医薬品などがある。 The subject to which the fermented wheat extract and Bacillus cells in the present invention are added can take all types for animal breeding and for the purpose of administering nutrients and useful chemicals into the animal body. Examples include feed for livestock, premix for livestock, vitamin for livestock, pharmaceutical for livestock, feed for fish farming, premix for fish farming, vitamin for fish farming, pharmaceutical for fish farming and the like.
本発明は小麦粉をグラム陰性菌のパントエア菌で発酵することによって得た小麦発酵物を熱水抽出した素材である、小麦醗酵抽出物と、市販の免疫賦活物質であるバチルス菌体を混合することを特徴とする免疫賦活、感染症予防効果を有する畜産又は水産用飼料添加剤、プレミック、及びこれを添加したことを特徴とするペット、畜産又は水産用飼料、及びその製造方法に係る。 The present invention is a material obtained by hot water extraction of a fermented wheat product obtained by fermenting wheat flour with a Gram-negative bacterium Pantoea, and mixing a wheat fermentation extract and a commercially available immunostimulatory bacillus cell. The present invention relates to a feed additive for livestock or aquaculture that has an immunostimulation and infection prevention effect, a premic, and a pet, a feed for livestock or aquaculture that includes this, and a method for producing the same.
また本発明は、上記小麦発酵抽出物及びバチルス菌体のペット、畜産動物、甲殻類及び魚類に許容される担体を含有する畜産又は水産用飼料添加剤及びその製造法に係る。 The present invention, the wheat fermented extract and Bacillus bacteria pet, livestock animals, according to livestock or aquatic feed additive comprises a carrier that is acceptable for crustaceans and fishes and their preparation.
また本発明は、上記小麦発酵抽出物及びバチルス菌体を有効成分として含有することを特徴とするペット、畜産又は水産用の斃死予防剤に係る。 Moreover, this invention relates to the mortality prevention agent for pets, livestock, or fisheries characterized by containing the said wheat fermented extract and a Bacillus microbial cell as an active ingredient.
また本発明は、上記小麦発酵抽出物及びバチルス菌体に薬学的に許容される担体を含有するペット、畜産又は水産用の斃死予防剤に係る。 The present invention also relates to a mortality preventive agent for pets, livestock or fisheries containing a pharmaceutically acceptable carrier for the wheat fermented extract and Bacillus cells.
また本発明は上記飼料をペット、畜産動物又は水産動物に与えることを特徴とするペット、畜産動物又は水産動物の飼育方法に係る。
The present invention according to the feed pets, pet, characterized in that applied to farm animals or aquatic animals, the breeding method of livestock or aquatic animals.
本発明者らは、小麦発酵抽出物の感染防除作用を増強する種々の市販免疫賦活物質を選るために、小麦発酵抽出物と市販免疫賦活物質を配合し、飼料への添加剤、プレミックス、添加飼料、サプリメントの製造方法を検討し、これを用いて作製した飼料を、ペット、鶏、豚、甲殻類及び魚類に投与し、鶏、豚、甲殻類、魚類が本来的に備えている免疫機能が活性化され、ウィルスや細菌による感染症が防御され、斃死が予防され、しかも安全性が非常に高いことを指標として鋭意研究した結果、バチルス菌体との配合飼料が免疫賦活効果に優れ感染防除作用などを有するという新しい知見を得、この配合飼料の製法にかかる発明を完成した。 In order to select various commercially available immunostimulatory substances that enhance the infection control action of the fermented wheat extract, the present inventors blended the fermented wheat wheat wheat extract and the commercially available immunostimulatory substance, added to the feed, premix Investigate the method of manufacturing supplementary feeds and supplements, and feed the prepared feeds to pets, chickens, pigs, crustaceans and fish, and the chickens, pigs, crustaceans and fishes are inherently provided As a result of diligent research using as an index that immune functions are activated, infectious diseases caused by viruses and bacteria are protected, drowning is prevented, and safety is extremely high. Obtaining new knowledge that it has excellent infection control action, etc., and completed the invention relating to the method for producing this blended feed.
本発明においては、小麦発酵抽出物を、そのまま、あるいは公知の担体、安定剤等を加えて、更に必要に応じてビタミン、アミノ酸類、ミネラル等の各種養分、抗酸化剤、抗生物質、抗菌剤及びその他の添加剤等を加えて、ペット、畜産又は水産用の飼料添加剤となしてもよく、その形状としては、粉体、顆粒、ペレット、懸濁液等の適宜の状態に調製すればよい。本発明の飼料添加剤を給餌する場合は、ペット、畜産又は水産に対し、単独で給餌しても良いし、飼料に混合して給餌しても良い。給餌時期は、疾患予防のために常時給餌しても、また飼養後半に添加してもよい。 In the present invention, the wheat fermented extract is used as it is, or by adding a known carrier, stabilizer, etc., and if necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants, antibiotics, antibacterial agents And other additives, etc., may be used as a feed additive for pets, livestock or aquaculture, and the shape thereof may be adjusted to an appropriate state such as powder, granules, pellets, suspension, etc. Good. When feeding the feed additive of the present invention, it may be fed alone, mixed with feed, or fed to pets, livestock or fisheries. The feeding time may be constantly fed for disease prevention or added in the latter half of the feeding.
また、本発明の飼料は特に限定されるものではなく、液体、粉末飼料、固型飼料、モイストペレット飼料、ドライペレット飼料、ＥＰ（Extruder Pellet）飼料、生餌など、どのような飼料でもよい。 The feed of the present invention is not particularly limited, and may be any feed such as liquid, powdered feed, solid feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, and live feed.
本発明において小麦発酵抽出物の飼料添加剤又は飼料等への配合量は広い範囲から選択できるが、好ましくは飼料添加剤又は飼料に対して０．０００１〜０．１重量％、特に好ましくは０．００２〜０．００５重量％であるが、これに限定されるものではない。小麦発酵抽出物の給餌量は適宜決定すれば良いが、例えばイヌ、豚、鶏、甲殻類及び魚類の体重１ｋｇあたり１日量として0.１〜１０ｍｇ、好ましくは０．５〜２ｍｇを投与するのがよいが、これに限定されるものではない。 In the present invention, the blending amount of the fermented wheat extract into the feed additive or feed can be selected from a wide range, but is preferably 0.0001 to 0.1% by weight, particularly preferably 0, based on the feed additive or feed. 0.002 to 0.005% by weight, but is not limited thereto. The feed amount of the fermented wheat extract may be determined as appropriate. For example, 0.1 to 10 mg, preferably 0.5 to 2 mg is administered as a daily dose per kg body weight of dogs, pigs, chickens, crustaceans and fish. However, it is not limited to this.
本発明で用いるバチルス菌体としては、特に限定されないが、例えば、Bacillus mesentericus、Bacillus subtilis、Bacillus cereus、Bacillus licheniformis、Bacillus coagulans、Bacillus polyfermenticusが挙げられる。 The Bacillus cells used in the present invention are not particularly limited, and examples thereof include Bacillus mesentericus, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus coagulans, and Bacillus polyfermenticus.
本発明においてバチルス菌体の飼料添加剤又は飼料等への配合量は広い範囲から選択できるが、好ましくは飼料添加剤又は飼料に対して０．００１〜１重量％、特に好ましくは０．２〜０．５重量％であるが、これに限定されるものではない。小麦発酵抽出物の給餌量は適宜決定すれば良いが、例えばイヌ、豚、鶏、甲殻類及び魚類の体重１ｋｇあたり１日量として１〜１００ｍｇ、好ましくは５〜２０ｍｇを投与するのがよいが、これに限定されるものではない。なお、バチルス菌体は生菌が好ましいが死菌であっても良い。 In the present invention, the amount of bacillus cells to the feed additive or feed can be selected from a wide range, but is preferably 0.001 to 1% by weight, particularly preferably 0.2 to 1% with respect to the feed additive or feed. Although it is 0.5 weight%, it is not limited to this. The feed amount of the fermented wheat extract may be appropriately determined. For example, 1 to 100 mg, preferably 5 to 20 mg as a daily dose per kg body weight of dogs, pigs, chickens, crustaceans and fish may be administered. However, the present invention is not limited to this. The Bacillus cells are preferably live bacteria, but may be dead bacteria.
本発明によれば、安全性が高く、安価に多方面の用途に対応することが可能な小麦発酵抽出物とバチルス菌体を含有した飼料、飼料添加剤、プレミックス、サプリメントを製造することができる。 According to the present invention, it is possible to produce a feed, a feed additive, a premix, and a supplement containing a fermented wheat extract and Bacillus cells that are highly safe and can be used for various purposes at low cost. it can.
小麦発酵抽出物の安定性はこれまで知見がなく、加熱、分散を伴う飼料、飼料添加剤、プレミックス製造に適した混合、成形方法はこれまで検討が必要であることが着想されておらず、これまでの免疫賦活剤混合技術の知見から容易に推察される事実ではない。 There has been no knowledge of the stability of fermented wheat extract so far, and it has not been conceived that studies on mixing and forming methods suitable for feed, feed additives, premix production with heating and dispersion are necessary. This is not a fact that can be easily inferred from the knowledge of conventional immunostimulant mixing techniques.
また、本製造方法によって作成した小麦発酵抽出物とバチルス菌体添加飼料は、感染防除効果を有することを実施例で示した。また、小麦発酵抽出物とバチルス菌体がそれぞれ単独よりも併用することが有効な効果を出すことを実施例で示した。 Moreover, it showed by the Example that the fermented wheat extract and bacillus cell addition feed produced with this manufacturing method have an infection control effect. Moreover, it showed by the Example that a wheat fermented extract and a Bacillus microbial cell show the effect that it uses together rather than each independently.
以下に実施例を挙げて本発明について説明するが、これら実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
小麦発酵抽出物は、飼料との混合を容易にするために、ブドウ糖と混合して造粒・乾燥・整粒し、乳糖と混合したものを供試した。投与時には小麦発酵抽出物が規定量となるように測量し、室温にて適量の水に溶解した。バチルス菌体はBacillus cereusを使用した。溶解後、ただちにＥＰ(エクストルーダペレット)飼料に添加し、均一に混ざるようによく混合して小麦発酵抽出物とバチルス菌体混合飼料とした。なお、ＥＰ飼料との混合は作り置きせず、投与日毎に作製した。 In order to facilitate mixing with the feed, the fermented wheat extract was mixed with glucose, granulated, dried and sized, and then mixed with lactose. At the time of administration, the wheat fermented extract was weighed so as to be a prescribed amount and dissolved in an appropriate amount of water at room temperature. Bacillus cereus was used as the Bacillus cell. Immediately after dissolution, the mixture was added to EP (extruder pellet) feed and mixed well so as to be uniformly mixed to obtain a wheat fermented extract and Bacillus cell body mixed feed. In addition, the mixture with EP feed was not made, and it produced for every administration day.
カワハギに対する小麦発酵抽出物とバチルス菌体混合飼料の疾病予防効果
試験方法：平均体重２６ｇのカワハギを20,000尾ずつ本発明区と対照区に分養し、海面生簀にて約３ヶ月間飼育した。この期間の水温は28.5〜18.3℃であった。発明区には、実施例１で製造した小麦発酵抽出物とバチルス菌体入り混合飼料を、小麦発酵抽出物を1日あたり２０〜４０μｇ/魚体重１ｋｇの量で、バチルス菌体を1日あたり０．０１ｍｇ〜１ｍｇ/魚体重１ｋｇの量で、投与間隔を４日投与４日休みとして投与した。なお、休みの期間は対照区と同じ小麦発酵抽出物とバチルス菌体を添加しない混合飼料を投与した。その後、試験期間中の死亡数を計測して生残率を求めた。
Disease prevention effect of fermented wheat extract and Bacillus fungus mixed feed on eelfish Test method: 20,000 eelfish with an average weight of 26 g were divided into the present invention zone and the control zone, and bred in sea surface ginger for about 3 months. The water temperature during this period was 28.5 to 18.3 ° C. The invention zone includes the fermented wheat extract and the mixed feed containing Bacillus cells produced in Example 1, the fermented wheat extract in an amount of 20-40 μg / kg of fish weight per day, and the Bacillus cells per day. The dose was 0.01 mg to 1 mg / kg body weight of fish and the administration interval was 4 days and 4 days off. During the rest period, the same wheat fermented extract and the mixed feed without the addition of Bacillus cells as in the control group were administered. Thereafter, the number of deaths during the test period was measured to determine the survival rate.
試験結果：本発明区及び対照区における投与３ヶ月後の生残率及び主な死因を表１に示した。試験期間中の主な死因は、β溶血性連鎖球菌症であり、本発明区の生残率は対照区と比較して有意（ｐ＜0.05，0.01）に高かった。 Test results: Table 1 shows the survival rate and main causes of death 3 months after administration in the present invention group and the control group. The main cause of death during the test period was β-hemolytic streptococci, and the survival rate of the invention group was significantly higher (p <0.05, 0.01) than the control group.
この結果から、カワハギに対する本発明の疾病予防効果を有することが明らかとなった。
マダイに対する小麦発酵抽出物とバチルス菌体混合飼料の疾病予防効果
試験方法：平均体重４２ｇのマダイを16,500尾ずつ本発明区と対照区に分養し、海面生簀にて約３ヶ月間飼育した。発明区には、実施例１で製造した小麦発酵抽出物とバチルス菌体入り混合飼料を、小麦発酵抽出物を1日あたり２０μｇ/魚体重１ｋｇの量で、バチルス菌体を1日あたり０．０１ｍｇ〜１ｍｇ/魚体重１ｋｇの量で、投与間隔を４日投与４日休みとして投与した。なお、休みの期間は対照区と同じ小麦発酵抽出物とバチルス菌体を添加しない混合飼料を投与した。その後、試験期間中の死亡数を計測して生残率を求めた。
Disease prevention effect of fermented wheat extract and Bacillus mixed diet for red sea bream Test method: Red sea bream with an average body weight of 42 g was divided into 16,500 fish in the present invention zone and the control zone and reared in sea surface ginger for about 3 months. In the invention zone, the mixed fermented wheat fermented extract and Bacillus cell mixture produced in Example 1, the wheat fermented extract in an amount of 20 μg / kg of fish body weight per day, and the Bacillus cell mass in the amount of 0. The dose was 01 mg to 1 mg / kg body weight of fish, and the administration interval was 4 days and 4 days off. During the rest period, the same wheat fermented extract and the mixed feed without the addition of Bacillus cells as in the control group were administered. Thereafter, the number of deaths during the test period was measured to determine the survival rate.
試験結果：本発明区及び対照区における投与３ヶ月後の生残率および主死因を表２に示した。試験期間中の主な死因は、イリドウィルス症であり、本発明区の生残率は対照区と比較して有意（ｐ＜0.05，0.01）に高かった。 Test results: Table 2 shows the survival rate and the main cause of death 3 months after administration in the present invention group and the control group. The main cause of death during the test period was iridovirosis, and the survival rate of the invention group was significantly higher (p <0.05, 0.01) than the control group.
この結果から、マダイに対する本発明の疾病予防効果を有することが明らかとなった。
ブリに対する小麦発酵抽出物とバチルス菌体の疾病予防効果
試験方法：ブリを約5,000尾ずつ３群(１〜３区)に分け、海面生簀にて２ヶ月間飼育した。本発明の１区は実施例１で製造した小麦発酵抽出物とバチルス菌体入り混合飼料を、小麦発酵抽出物を1日あたり２０μｇ/魚体重１ｋｇの量で、バチルス菌体を1日あたり０．０１ｍｇ〜１ｍｇ/魚体重１ｋｇの量で、毎日の連続投与区、２区は１区と同量で４日間投与３日間無投与の間欠投与区とした。なお、無投与の期間は対照区と同じ小麦発酵抽出物とバチルス菌体を添加しない混合飼料を投与した。その後、試験期間中の死亡数を計測して生残率を求めた。
Disease prevention effect of fermented wheat extract and Bacillus fungus on yellowtail Test method: About 5,000 fish were divided into 3 groups (1st to 3rd districts) and reared in sea surface ginger for 2 months. The first section of the present invention is the mixed feed containing the fermented wheat extract and Bacillus cells produced in Example 1, the fermented wheat extract at an amount of 20 μg / kg of fish body weight per day, and the Bacillus cell mass per day. In the amount of 0.01 mg to 1 mg / kg body weight of fish, the daily continuous administration group and the second group were the same amount as the first group, and the intermittent administration group was administered for 4 days and was not administered for 3 days. During the non-administration period, the same wheat fermented extract and the mixed feed without the addition of Bacillus cells as in the control group were administered. Thereafter, the number of deaths during the test period was measured to determine the survival rate.
試験結果：本発明区及び対照区における投与２ヶ月後の生残率および主死因を表３に示した。試験期間中の主な死因は、レンサ球菌症であり、本発明区の生残率は対照区と比較して有意（ｐ＜0.05、0.01）に高く、間欠投与区で最も優れた成績が得られた。 Test results: Table 3 shows the survival rate and main cause of death 2 months after administration in the present invention group and the control group. The main cause of death during the study period is streptococci, and the survival rate of the present invention group is significantly higher (p <0.05, 0.01) than the control group, and the best results were obtained in the intermittent administration group. It was.
この結果から、ブリに対する本発明の疾病予防効果を有することが明らかとなった。
小麦発酵抽出物添加飼料(ペレット)中の有効成分の一つである、低分子リポ多糖（IP-PA1）含量を指標として、ELISA（Enzyme-Linked ImmunoSorbent Assay）により測定した。
試験方法
IP-PA1の水抽出法：飼料2.00gに水20gを加えて懸濁し、ボルテックスミキサーで撹拌後に、５分間の超音波処理を行い、これを遠心分離（2000g 、5分）し、上清を得た。IP-PA1の特異的測定：ELISA法を用いた。
Measurement was performed by ELISA (Enzyme-Linked ImmunoSorbent Assay) using the low-molecular-weight lipopolysaccharide (IP-PA1) content, which is one of the active ingredients in the wheat fermented extract-added feed (pellet), as an index.
Test method
IP-PA1 water extraction method: Add 2.00 g of water to 20 g of water and suspend it, stir it with a vortex mixer, perform ultrasonic treatment for 5 minutes, centrifuge it (2000 g, 5 minutes), and remove the supernatant. Obtained. Specific measurement of IP-PA1: ELISA method was used.
試験結果：加工前のIP-PA1添加飼料では、推定値(5μg/g)の約50%のIP-PA1（2.47μg/g）が回収できることがELISA法により確認された。ペレット加工後（加熱工程を含む）飼料では、推定IP-PA1量(5μg/g)の約10%のIP-PA1量がELISA法により測定された（表４）。以上のことから、ペレット飼料中への小麦発酵抽出物の添加は困難であることが確認された。
小麦発酵抽出物とバチルス菌体の併用によるマクロファージ系細胞(RAW264.7細胞)を用いてNO産生能を測定した。小麦発酵抽出物 (Lot No.070423, 有効成分としてIP-PA1含量（1％）)、バチルス菌体としてBacillus cereusを使用した。バチルス菌体は、重量濃度で取り扱った。小麦発酵抽出物はIP-PA1含量を基準に試験した。 NO production ability was measured using macrophage cells (RAW264.7 cells) by combined use of wheat extract and Bacillus cells. Fermented wheat extract (Lot No. 070423, IP-PA1 content (1%) as an active ingredient) and Bacillus cereus as Bacillus cells were used. Bacillus cells were handled at a weight concentration. Fermented wheat extract was tested based on IP-PA1 content.
試験方法
マウスマクロファージ系の培養細胞であるRAW264.7細胞（1.6×105個/100μl/ウエル）を９６穴平底プレートの播種し、6時間、37℃の5%炭酸ガス培養器で培養した。その後に各被検体の希釈液を各用量、穴当たり100μlずつ加えた。各被検体溶液は、細胞培養液中で終濃度が小麦発酵抽出物がIP-PA1量として0.1ng/mlから10ng/mlになるように、バチルス菌体が1μg/mlから100μg /mlになるように被検体を培養液（10%牛胎児血清、60μg/ml アンピシリンナトリウム、50μg/ml硫酸カナマイシンを含むRPMI 1640培地）にて系列希釈して調製した。試験の陰性対照には培養液を用いた。各検体を添加し、20時間培養した後、培養上清を一部(50μl)回収し、グリエス氏の方法（NOから生じる亜硝酸を酸性条件下でスルファニルアミドとのジアゾニウム塩化合物に変化させ、それとナフチルエチレンジアミンのアゾカップリングによって生成する赤色色素を検出する方法）により、一酸化窒素の代謝物である亜硝酸濃度を測定した。
Test Method RAW264.7 cells (1.6 × 10 5 cells / 100 μl / well), a cultured cell of mouse macrophage line, were seeded in a 96-well flat bottom plate and cultured for 6 hours in a 5% carbon dioxide incubator at 37 ° C. Thereafter, a dilution of each subject was added at 100 μl per well for each dose. Each analyte solution has a final concentration of 1 to 100 μg / ml of Bacillus cells so that the final concentration of the fermented wheat extract is 0.1 ng / ml to 10 ng / ml of IP-PA1 in the cell culture medium. Thus, the specimens were prepared by serial dilution in a culture solution (RPMI 1640 medium containing 10% fetal bovine serum, 60 μg / ml ampicillin sodium, 50 μg / ml kanamycin sulfate). The culture medium was used as a negative control for the test. After each sample was added and cultured for 20 hours, a portion (50 μl) of the culture supernatant was collected, and Gries method (nitrous acid generated from NO was changed to a diazonium salt compound with sulfanilamide under acidic conditions. The concentration of nitrous acid, which is a metabolite of nitric oxide, was measured by a method for detecting a red pigment produced by azo coupling of naphthylethylenediamine with that.
試験結果
バチルス菌体の用量が1μg/ml又は小麦発酵抽出物の用量が0.1ng/mlの時、それぞれ単独では、一酸化窒素の誘導が見られなかった。ところが、両者を併用したところ一酸化窒素が有意に誘導され、両者の併用により一酸化窒素が産生されることが見出された。一酸化窒素は自然免疫の中心的な細胞であるマクロファージの活性化を誘導する指標である。また、一酸化窒素はバクテリアやウイルスを直接傷害する実行分子である。
本発明によれば、ペット、畜産動物、甲殻類及び魚類の免疫機能を、ごく微量で的確に活性化して感染症を予防し、また、ペット、畜産動物、甲殻類及び魚類の疾病を予防し、薬物残留などの公衆衛生上の問題のない、安全なペット、畜産動物、甲殻類及び魚類を育成するための飼料添加剤、プレミックス及び飼料を製造する技術を提供することができる。
According to the present invention, the immune functions of pets, livestock animals, crustaceans and fish are accurately activated in very small amounts to prevent infectious diseases, and diseases of pets, livestock animals, crustaceans and fish are prevented. It is possible to provide a technology for producing feed additives, premixes, and feeds for growing safe pets, livestock animals, crustaceans and fish without public health problems such as drug residues.
Claims (8)
The feed according to claim 2, which is used for crustaceans or fish.
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