JP2021122242A - Method for producing feed high-containing gaba and ornithine - Google Patents
Method for producing feed high-containing gaba and ornithine Download PDFInfo
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- JP2021122242A JP2021122242A JP2020018564A JP2020018564A JP2021122242A JP 2021122242 A JP2021122242 A JP 2021122242A JP 2020018564 A JP2020018564 A JP 2020018564A JP 2020018564 A JP2020018564 A JP 2020018564A JP 2021122242 A JP2021122242 A JP 2021122242A
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- Prior art keywords
- lactic acid
- feed
- ornithine
- culture
- gaba
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Images
Abstract
Description
本発明は飼料の製造方法に関する。好ましくは、γ−アミノ酪酸（以下、「ＧＡＢＡ」とも称する）及びオルニチンを含有する飼料を製造する方法に関する。 The present invention relates to a method for producing feed. Preferably, it relates to a method for producing a feed containing γ-aminobutyric acid (hereinafter, also referred to as “GABA”) and ornithine.
我が国の家畜飼料自給率は約２７％の低い水準で推移しており、大部分を輸入飼料に依存している。輸入飼料は為替相場や原油価格、社会情勢により価格が大きく変動するため、畜産経営の不安定化の原因の１つになっている。また、輸入飼料は家畜防疫の面からもリスクがあり、国産飼料やエコフィードの利用推進の重要性が高まっている。 Japan's livestock feed self-sufficiency rate has remained at a low level of about 27%, and most of it depends on imported feed. The price of imported feed fluctuates greatly depending on the exchange rate, crude oil price, and social conditions, which is one of the causes of destabilization of livestock farming. In addition, imported feed poses a risk in terms of livestock epidemics, and the importance of promoting the use of domestic feed and eco-feed is increasing.
一方、畜産農家においては、過密環境での飼育による家畜への過度のストレスや飼養管理不備による家畜の肝機能低下が問題となっており、抗ストレス、肝機能改善効果を有する飼料が求められている。 On the other hand, in livestock farmers, excessive stress on livestock due to breeding in an overcrowded environment and deterioration of liver function of livestock due to inadequate feeding management have become problems, and feeds having anti-stress and liver function improving effects are required. There is.
家畜のストレス低減については、非特許文献１、２のようにγ−アミノ酪酸（ＧＡＢＡ）が有効であることが知られており、特許文献１及び２のようにＧＡＢＡ含有飼料の製造方法が多数報告されている。また、ヒトなどに対しては、シジミなどに含まれるオルニチンが肝機能改善効果を持つことが知られており、特許文献３のようにオルニチンを含んだ飼料製造法が報告されている。
It is known that γ-aminobutyric acid (GABA) is effective for reducing stress in livestock as in
本発明は、ストレス低減効果があることが知られているＧＡＢＡ、及び肝機能改善効果が報告されているオルニチンを含有する飼料を製造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 producing a feed containing GABA, which is known to have a stress-reducing effect, and ornithine, which has been reported to have an effect of improving liver function.
本発明者らは、前記課題を解決すべく、鋭意検討を重ねていたところ、宮崎県内焼酎製造場の焼酎もろみの中に、ＧＡＢＡ及びオルニチンを生成する乳酸菌が存在することを見出し、当該乳酸菌を発酵スターターとして用いることで、ＧＡＢＡ及びオルニチンを高含有する飼料が製造できることを確認した。さらに、本発明者らは、当該乳酸菌は、ヒスチジン及びチロシンの存在下でもヒスタミン及びチラミンといったアレルギー性物質を生成しない特性を有し、１６Ｓ ｒＲＮＡ遺伝子の配列に基づく同定によりラクトバチルス・ブフネリに属する新規な乳酸菌であることを確認した。 As a result of diligent studies to solve the above problems, the present inventors have found that lactic acid bacteria that produce GABA and ornithine exist in the shochu mash of the Miyazaki prefecture shochu factory, and found that the lactic acid bacteria are present. It was confirmed that a feed containing a high content of GABA and ornithine could be produced by using it as a fermentation starter. Furthermore, the present inventors have the property that the lactic acid bacterium does not produce allergic substances such as histamine and tyramine even in the presence of histidine and tyrosine, and by identification based on the sequence of the 16S rRNA gene, the lactic acid bacterium belongs to Lactobacillus buchneri. It was confirmed that it was a lactic acid bacterium.
本発明は、これらの一連の知見をもとに完成したものであり、下記の実施様態を包含する。 The present invention has been completed based on these series of findings, and includes the following embodiments.
（Ｉ）飼料の製造方法
（Ｉ−１） ラクトバチルス・ブフネリに属する乳酸菌ＭＬ５３０株（受領番号：ＮＩＴＥ ＡＰ−０３１１１）、前記乳酸菌ＭＬ５３０株の子孫株、またはそれらの培養物を用いて、飼料原料を培養する工程を有する、飼料の製造方法。
（Ｉ−２）前記飼料原料が、グルタミン酸及びアルギニンを含有するか、または加水分解によりグルタミン酸及びアルギニンを生成し得る成分を含有するものであり、
後者の場合は、当該成分を加水分解してグルタミン酸及びアルギニンを生成する作用を有する物質とともに培養する工程を有する、（Ｉ−１）に記載する製造方法。
（Ｉ−３）γ−アミノ酪酸（ＧＡＢＡ）及びオルニチン高含有の飼料を製造する方法である、（Ｉ−２）に記載する製造方法。
(I) Method for producing feed (I-1) A feed raw material using a lactic acid bacterium ML530 strain belonging to Lactobacillus buchneri (receipt number: NITE AP-03111), a progeny strain of the lactic acid bacterium ML530 strain, or a culture thereof. A method for producing a feed, which comprises a step of culturing.
(I-2) The feed raw material contains glutamic acid and arginine, or contains a component capable of producing glutamic acid and arginine by hydrolysis.
In the latter case, the production method according to (I-1), which comprises a step of culturing the component together with a substance having an action of hydrolyzing the component to produce glutamic acid and arginine.
(I-3) The production method according to (I-2), which is a method for producing a feed containing a high content of γ-aminobutyric acid (GABA) and ornithine.
（ＩＩ）飼料
（ＩＩ−１）ラクトバチルス・ブフネリに属する乳酸菌ＭＬ５３０株（受領番号：ＮＩＴＥ ＡＰ−０３１１１）、前記乳酸菌ＭＬ５３０株の子孫株、またはそれらの培養物を含有する、飼料。
（ＩＩ−１）（Ｉ−１）〜（Ｉ−３）のいずれかに記載する製造方法によって得られる（ＩＩ−１）に記載する飼料。
(II) Feed (II-1) A feed containing a lactic acid bacterium ML530 strain belonging to Lactobacillus buchneri (receipt number: NITE AP-03111), a progeny strain of the lactic acid bacterium ML530 strain, or a culture thereof.
(II-1) The feed according to (II-1) obtained by the production method according to any one of (I-1) to (I-3).
（ＩＩＩ）非ヒト動物の飼育方法
（ＩＩＩ−１）（ＩＩ−１）または（ＩＩ−２）に記載する飼料を用いて非ヒト動物を飼育することを特徴とする、非ヒト動物の飼育方法。
（ＩＩＩ−２）前記非ヒト動物が、ペットまたは家畜である、（ＩＩＩ−１）に記載する飼育方法。
(III) Method for breeding non-human animals A method for breeding non-human animals, which comprises breeding non-human animals using the feed according to (III-1) (II-1) or (II-2). ..
(III-2) The breeding method according to (III-1), wherein the non-human animal is a pet or livestock.
ラクトバチルス・ブフネリに属する乳酸菌ＭＬ５３０株を使用することで、機能性成分であるＧＡＢＡ及びオルニチンを高含有する飼料を製造することができる。また、当該乳酸菌ＭＬ５３０株によれば、ヒスチジン及びチロシンの存在下でもヒスタミン及びチラミンといったアレルギー性物質を生成しないため、アレルギー性物質を含まないか、含んでいても低量であるＧＡＢＡ及びオルニチン含有飼料を製造することができる。
斯くして製造される本発明の飼料を用いてペットや家畜等の非ヒト動物を飼育することで、非ヒト動物（飼育動物）のストレスや肝機能低下の問題を改善することが期待される。
By using the lactic acid bacterium ML530 strain belonging to Lactobacillus buchneri, a feed containing a high content of GABA and ornithine, which are functional components, can be produced. Further, according to the lactic acid bacterium ML530 strain, since allergic substances such as histamine and tyramine are not produced even in the presence of histidine and tyrosine, GABA and ornithine-containing feeds containing no or low amounts of allergic substances are contained. Can be manufactured.
By breeding non-human animals such as pets and livestock using the feed of the present invention thus produced, it is expected to improve the problems of stress and liver function deterioration of non-human animals (domestic animals). ..
（Ｉ）乳酸菌、その子孫株及びその培養物
本発明で用いる乳酸菌ＭＬ５３０株及びその子孫株は、焼酎もろみに由来するラクトバチルス・ブフネリ（Lactobacillus buchneri）に属する乳酸菌であって、下記（ａ）〜（ｄ）の特性を有することを特徴とする。
（ａ）グルタミン酸を代謝し、γ−アミノ酪酸（ＧＡＢＡ）を生成する。
（ｂ）アルギニンを代謝し、オルニチンを生成する。
（ｃ）ヒスチジン存在下でヒスタミンを生成しない。
（ｄ）チロシン存在下でチラミンを生成しない。
(I) Lactic acid bacteria, their progeny strains and their cultures The lactic acid bacteria ML530 strain and its progeny strains used in the present invention are lactic acid bacteria belonging to Lactobacillus buchneri derived from shochu mash, and are described in (a) to (a) to the following. It is characterized by having the characteristics of (d).
(A) Metabolize glutamic acid to produce γ-aminobutyric acid (GABA).
(B) Metabolize arginine to produce ornithine.
(C) Does not produce histamine in the presence of histidine.
(D) Does not produce tyramine in the presence of tyrosine.
これらの特性は、以下の試験により評価・確認することができる。
（ａ）グルタミン酸を代謝してＧＡＢＡを生成する
ＧＡＢＡ生成能は、乳酸菌が生育可能なグルタミン酸含有培地を用いて、乳酸菌を培養し、得られた培養物について、培養前（発酵前）と培養後（発酵後）のグルタミン酸濃度及びＧＡＢＡ濃度を求めることで確認することができる。培養前と比較して培養後の培養物中のグルタミン酸濃度が低下し、それに対応してＧＡＢＡ濃度が上昇している場合、当該乳酸菌は、前記（ａ）の特性を有すると判断することができる。
These characteristics can be evaluated and confirmed by the following tests.
(A) The GABA-producing ability of metabolizing glutamic acid to produce GABA is such that lactic acid bacteria are cultivated using a glutamic acid-containing medium in which lactic acid bacteria can grow, and the obtained culture is subjected to pre-culture (pre-fermentation) and post-culture. It can be confirmed by determining the glutamic acid concentration and GABA concentration (after fermentation). When the glutamic acid concentration in the culture after culturing is lower than that before culturing and the GABA concentration is correspondingly increased, it can be determined that the lactic acid bacterium has the property (a). ..
（ｂ）アルギニンを代謝してオルニチンを生成する
オルニチン生成能は、乳酸菌が生育可能なアルギニン含有培地を用いて、乳酸菌を培養し、得られた培養物について、培養前（発酵前）と培養後（発酵後）のアルギニン濃度及びオルニチン濃度を求めることで確認することができる。培養前と比較して培養後の培養物中のアルギニン濃度が低下し、それに対応してオルニチン濃度が上昇している場合、当該乳酸菌は、前記（ｂ）の特性を有すると判断することができる。
(B) The ornithine-producing ability to metabolize arginine to produce ornithine is such that arginine-containing medium in which arginine can grow is used to cultivate lactic acid bacteria, and the obtained culture is subjected to pre-culture (pre-fermentation) and post-culture. It can be confirmed by determining the arginine concentration and the ornithine concentration (after fermentation). When the arginine concentration in the culture after culturing is lower than that before culturing and the ornithine concentration is correspondingly increased, it can be determined that the lactic acid bacterium has the property (b). ..
（ｃ）ヒスチジン存在下でヒスタミンを生成しない
ヒスタミン生成能は、乳酸菌が生育可能なヒスチジン含有培地を用いて、乳酸菌を培養し、得られた培養物について、培養前（発酵前）と培養後（発酵後）のヒスチジン濃度及びヒスタミン濃度を求めることで確認することができる。培養前と比較して培養後の培養物のヒスタミン濃度が上昇していない場合、特に培養前後で培養物のヒスチジン濃度に大きな変動がなく、培養後の培養物のヒスタミン濃度が上昇していない場合、当該乳酸菌は、前記（ｃ）の特性を有すると判断することができる。
(C) The histamine-producing ability that does not produce histamine in the presence of histidine is such that the lactic acid bacteria are cultured using a histidine-containing medium in which the lactic acid bacteria can grow, and the obtained culture is subjected to pre-culture (pre-fermentation) and post-culture (pre-culture). It can be confirmed by determining the histidine concentration and histamine concentration (after fermentation). When the histamine concentration of the culture after culturing does not increase as compared with that before culturing, especially when the histidine concentration of the culture does not change significantly before and after culturing and the histamine concentration of the culture after culturing does not increase. , The lactic acid bacterium can be determined to have the property of (c) above.
（ｄ）チロシン存在下でチラミンを生成しない
チラミン生成能は、乳酸菌が生育可能なチロシン含有培地を用いて、乳酸菌を培養し、得られた培養物について、培養前（発酵前）と培養後（発酵後）のチロシン濃度及びチラミン濃度を求めることで実施することができる。培養前と比較して培養後の培養物のチラミン濃度が上昇していない場合、特に培養前後で培養物のチロシン濃度に大きな変動がなく、培養後の培養物のチラミン濃度が上昇していない場合、当該乳酸菌は、前記（ｄ）の特性を有すると判断することができる。
(D) The ability to produce tyramine, which does not produce tyramine in the presence of tyrosine , is such that lactic acid bacteria are cultured in a tyrosine-containing medium in which lactic acid bacteria can grow, and the obtained culture is subjected to pre-culture (pre-fermentation) and post-culture (pre-culture). It can be carried out by determining the tyrosine concentration and the tyramine concentration (after fermentation). When the tyramine concentration of the culture after culturing does not increase as compared with that before culturing, especially when the tyrosine concentration of the culture does not change significantly before and after culturing and the tyramine concentration of the culture after culturing does not increase. , The lactic acid bacterium can be determined to have the property (d).
また、乳酸菌の属種（ラクトバチルス・ブフネリ）は、１６Ｓ ｒＲＮＡ遺伝子の塩基配列により、定法に従い遺伝子データベースを用いてＢＬＡＳＴホモロジー検索を行うことで同定することができる。１６Ｓ ｒＲＮＡ遺伝子の塩基配列が、Ｍｉｃｒｏ ＳＥＱ微生物同定ソフトウェア Ｖ３．０（Applied Biosystems, U.S.）に登録された公知のラクトバチルス・ブフネリ（Lactobacillus buchneri）の当該塩基配列と、相同性が９８％以上、好ましくは９９％以上、より好ましくは９９．５％以上であれば、当該乳酸菌はラクトバチルス・ブフネリに属する乳酸菌であると判断することができる。 In addition, the genus species of lactic acid bacteria (Lactobacillus bufuneri) can be identified by performing a BLAST homology search using a gene database according to a conventional method based on the base sequence of the 16S rRNA gene. The base sequence of the 16S rRNA gene has a homology of 98% or more, preferably 98% or more, with the base sequence of known Lactobacillus buchneri registered in Micro SEQ Microbial Identification Software V3.0 (Applied Biosystems, US). If is 99% or more, more preferably 99.5% or more, it can be determined that the lactic acid bacterium is a lactic acid bacterium belonging to Lactobacillus buchneri.
本発明で用いる乳酸菌は、宮崎県の焼酎製造場から入手した焼酎もろみから単離された乳酸菌（グラム陽性、桿菌）の中から、上記（ａ）〜（ｄ）に記載する方法に従ってスクリーニングし、またその１６Ｓ ｒＲＮＡ遺伝子の塩基配列を分析することで選抜したものである。当該乳酸菌株を、ラクトバチルス・ブフネリＭＬ５３０と命名し、宮崎県宮崎市佐土原町東上那珂１６５００−２に所在する宮崎県食品開発センターに分譲可能な状態で保存するとともに、その継代株の一部を、日本国千葉県木更津市かずさ鎌足２−５−８に住所を有する独立行政法人製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）に「微生物の識別の表示：ＭＬ５３０」として、２０２０年１月２２日（寄託日）に国内寄託した（受領番号：ＮＩＴＥ ＡＰ−０３１１１）。 The lactic acid bacteria used in the present invention are screened from among the lactic acid bacteria (gram-positive, bacilli) isolated from the shochu mash obtained from the shochu mash in Miyazaki Prefecture according to the methods described in (a) to (d) above. Further, it was selected by analyzing the base sequence of the 16S rRNA gene. The lactic acid bacterium strain was named Lactobacillus bufuneri ML530, and was stored in a state where it could be sold at the Miyazaki Prefectural Food Development Center located at 16500-2 Higashikaminaka, Sadowara-cho, Miyazaki City, Miyazaki Prefecture. 2020 as "Indication of Microbial Identification: ML530" at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD), which has an address in 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan. It was deposited domestically on the 22nd of March (deposit date) (receipt number: NITE AP-03111).
後述する本発明の飼料の製造には、焼酎もろみから単離された乳酸菌株（初代菌株）に限らず、その特性（ａ）〜（ｄ）を有する子孫株、及びそれらの培養物を用いることもできる。なお、当該子孫株には、前記初代菌株を、培地を用いて継代培養することによって得られる乳酸菌株（継代株）、並びに、初代菌株または継代株を、乳酸菌が生育可能な成分を含む組成物中で培養することで得られる乳酸菌株（培養株）が含まれる。本明細書では、こうした初代の乳酸菌ＭＬ５３０株、及びその子孫株を、総称して「本発明の乳酸菌」または「本乳酸菌」と記載する場合がある。 In the production of the feed of the present invention described later, not only the lactic acid bacterium strain (primary strain) isolated from the shochu mash, but also the progeny strains having the characteristics (a) to (d) and their cultures are used. You can also. The progeny strain includes a lactic acid bacterium strain (passage strain) obtained by subculturing the primary bacterium strain in a medium, and a primary bacterium strain or a subculture strain capable of growing lactic acid bacteria. A lactic acid bacterium strain (cultured strain) obtained by culturing in the containing composition is included. In the present specification, such a primary lactic acid bacterium ML530 strain and its progeny strain may be collectively referred to as "the lactic acid bacterium of the present invention" or "the present lactic acid bacterium".
本発明の乳酸菌の培養は、それが生育可能な培地であれば、いかなる培地を使用して行うことができる。また、培養方法も特に制限されず、例えば、試験管培養、フラスコ培養、発酵槽による培養などにより実施することができる。具体的には、制限されないものの、例えば、乳酸菌培養に一般的に使用されるＭＲＳ培地を用い、乳酸菌が生育可能な条件（例えば、２５〜４０℃、ｐＨ４〜８）で培養する方法を挙げることができる。また本発明の乳酸菌の培養は、培地に限らず、乳酸菌が生育可能な成分を含む組成物中で行うこともできる。当該組成物には、乳酸菌が生育するうえで必要な成分に加えて、ＧＡＢＡ生成基質であるグルタミン酸、及び／又は、オルニチン生成基質であるアルギニンを配合することもできる。本発明の乳酸菌の培養物は、こうした培養によって得られる培養物が含まれる。 The culture of the lactic acid bacterium of the present invention can be carried out using any medium as long as it can grow. Further, the culture method is not particularly limited, and for example, it can be carried out by in vitro culture, flask culture, culture in a fermenter, or the like. Specifically, although not limited, for example, a method of culturing under conditions in which lactic acid bacteria can grow (for example, 25 to 40 ° C., pH 4 to 8) using an MRS medium generally used for culturing lactic acid bacteria can be mentioned. Can be done. Further, the culture of the lactic acid bacterium of the present invention is not limited to the medium, and can be carried out in a composition containing a component capable of growing the lactic acid bacterium. In addition to the components necessary for the growth of lactic acid bacteria, the composition may contain glutamic acid, which is a GABA-producing substrate, and / or arginine, which is an ornithine-producing substrate. The culture of lactic acid bacteria of the present invention includes the culture obtained by such culture.
本発明が対象とする培養物には、本発明の乳酸菌を生菌状態で含む培養物、及び死菌状態で含む培養物の両方が含まれる。前者の培養物は、飼料の製造に際して、例えば発酵スターターとして好適に使用することができる。後者の培養物は、本発明の乳酸菌を生菌状態で含む培養物を、物理的（例えば、加熱、加圧、紫外線処理など）または化学的（例えば薬物処理など）に殺菌処理されることで調製することができ、主として飼料として用いることができる。 The cultures targeted by the present invention include both cultures containing the lactic acid bacteria of the present invention in a viable state and cultures containing the lactic acid bacteria in a killed state. The former culture can be suitably used, for example, as a fermentation starter in the production of feed. The latter culture is obtained by physically (for example, heating, pressurizing, ultraviolet treatment, etc.) or chemically (for example, drug treatment, etc.) sterilizing a culture containing the lactic acid bacterium of the present invention in a viable state. It can be prepared and can be used mainly as a feed.
（II）飼料、及び飼料の製造方法
本発明が対象とする飼料は、前述する乳酸菌またはその培養物を含有することを特徴とする。また、後述する実験例に示すように、本発明の乳酸菌の作用を妨げないことを限度として、乳酸菌を始めとする他の微生物を含んでいてもよい。かかる微生物には、発酵能を有する他の乳酸菌が含まれる。他の乳酸菌としては、制限されないが、例えばラクトコッカス・ラクティス（（Lactococcus lactis）、ラクトバチルス・パラカゼイ（Lactobacillus paracasei）、ラクトバチルス・ディオリボランス（Lactobacillus diolivorans）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、ラクトコッカス・ラクティス・サブスピーシーズ・クレモリス（Lactococcus lactis subsp. cremoris）、ラクトコッカス・ラクティス・サブスピーシーズ・ラクティス・バイオバラエティ・ディアセチラクティス（Lactococcus lactis subsp. lactis biovar. diacetylactis）、ラクトバチルス・カゼイ・サブスピーシーズ・カゼイ（Lactobacillus casei subsp. casei）などを挙げることができる。
(II) Feed and method for producing feed The feed targeted by the present invention is characterized by containing the above-mentioned lactic acid bacteria or a culture thereof. Further, as shown in an experimental example described later, other microorganisms such as lactic acid bacteria may be contained as long as the action of the lactic acid bacteria of the present invention is not hindered. Such microorganisms include other lactic acid bacteria capable of fermenting. Other lactic acid bacteria include, but are not limited to, for example, Lactococcus lactis, Lactobacillus paracasei, Lactobacillus diolivorans, Lactobacillus plantarum. , Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Lactococcus lactis biovar. Diacetylactis, Lactococcus lactis subsp. Lactobacillus casei Examples include Lactobacillus casei subsp. Casei.
また、前記微生物は、タンパク質やペプチドを分解してグルタミン酸及び／又はアルギニンを生成する能力を有する微生物であってもよい。本発明の乳酸菌は、グルタミン酸を基質としてＧＡＢＡを産生する能力と、アルギニンを基質としてオルニチンを生産する能力を有することから、このような微生物と組み合わせて飼料原料を発酵させることで、有用アミノ酸であるＧＡＢＡやオルニチンを高濃度含有する飼料を製造することが可能になる。 Further, the microorganism may be a microorganism having an ability to decompose a protein or peptide to produce glutamic acid and / or arginine. Since the lactic acid bacterium of the present invention has the ability to produce GABA using glutamic acid as a substrate and the ability to produce ornithine using arginine as a substrate, it is a useful amino acid by fermenting a feed raw material in combination with such a microorganism. It will be possible to produce feeds containing high concentrations of GABA and ornithine.
本発明において、飼料とは非ヒト動物を飼育するために用いられる非ヒト動物用の飲食物であり、当該非ヒト動物には、ウシ、ウマ、ブタ、ヒツジなどの家畜類；ニワトリ（ブロイラー等の採肉鶏、及び採卵鶏の両方を含む。）、烏骨鶏、合鴨、七面鳥などの家禽類；マウス、ラット、モルモット、サル、イヌなどの実験動物；イヌやネコなどのペット（愛玩動物）が含まれる。 In the present invention, the feed is a food or drink for non-human animals used for breeding non-human animals, and the non-human animals include domestic animals such as cows, horses, pigs, and sheep; chickens (broilers, etc.). (Including both meat-laying chickens and egg-laying chickens), poultry such as crowbone chickens, duck, turkey; experimental animals such as mice, rats, guinea pigs, monkeys and dogs; pets such as dogs and cats (pet animals) ) Is included.
当該飼料は、前述する本発明の乳酸菌またはその培養物に加えて、通常の飼料と同様に、適当な可食性素材（飼料原料）を含有したものを挙げることができる。具体的には、当該飼料には、本発明の乳酸菌またはその培養物に加えて、通常の飼料原料を利用して飼料形態に調製したものが含まれる。 Examples of the feed include those containing an appropriate edible material (feed material) in addition to the above-mentioned lactic acid bacterium of the present invention or a culture thereof, as in the case of ordinary feed. Specifically, the feed includes, in addition to the lactic acid bacterium of the present invention or a culture thereof, a feed prepared in a feed form using a normal feed raw material.
また、本発明の乳酸菌は、前述するように、グルタミン酸を基質として有用アミノ酸であるＧＡＢＡを産生する作用、並びにアルギニンを基質として有用アミノ酸であるオルニチンを産生する作用を有する。このため、本発明の好適な飼料は、本発明の乳酸菌を利用してグルタミン酸及びアルギニンを含む飼料原料から製造される発酵組成物であって、本発明の乳酸菌の上記作用に基づいて産生されたＧＡＢＡ及びオルニチンを含有するものである。当該飼料は、制限されないが、本発明の乳酸菌またはその培養物を用いて、定法の発酵方法に従って製造することができる。例えば、本発明の乳酸菌が生育するのに必要な栄養源を含む飼料原料中で、本発明の乳酸菌を培養して、当該原料を発酵させることによって製造することができる。この場合、最終の飼料に含まれている乳酸菌の生死状態は特に制限されず、死菌状態であってもよい。 Further, as described above, the lactic acid bacterium of the present invention has an action of producing GABA which is a useful amino acid using glutamic acid as a substrate and an action of producing ornithine which is a useful amino acid using arginine as a substrate. Therefore, the suitable feed of the present invention is a fermentation composition produced from a feed raw material containing glutamic acid and arginine using the lactic acid bacterium of the present invention, and was produced based on the above-mentioned action of the lactic acid bacterium of the present invention. It contains GABA and ornithine. The feed can be produced, but not limited to, using the lactic acid bacterium of the present invention or a culture thereof according to a conventional fermentation method. For example, it can be produced by culturing the lactic acid bacterium of the present invention in a feed raw material containing a nutrient source necessary for the lactic acid bacterium of the present invention to grow and fermenting the raw material. In this case, the viable state of the lactic acid bacterium contained in the final feed is not particularly limited, and the lactic acid bacterium may be in a dead state.
飼料原料は、制限されないものの、例えば、穀物類または加工穀物類（とうもろこし、マイロ、大麦、小麦、ライ麦、燕麦、キビ、小麦粉、小麦胚芽粉等）、糟糠類（ふすま、米糠、コーングルテンフィード等）、植物性油粕類（大豆油粕、ごま油粕、綿実油粕、落花生粕、ヒマワリ粕、サフラワー粕等）、豆乳製造時にでる粕（おから等）、アルコール飲料製造時にでる粕（麦芽粕、酒粕、焼酎粕［麦焼酎粕、芋焼酎粕、蕎麦焼酎粕、米焼酎粕など］、動物性原料（脱脂粉乳、魚粉、肉骨粉等）、ミネラル類（炭酸カルシウム、リン酸カルシウム、食塩、無水ケイ酸等）、ビタミン類（ビタミンＡ、ビタミンＤ、ビタミンＥ、ビタミンＫ、ビタミンＢ１、ビタミンＢ２、ビタミンＢ６ 、ビタミンＢ１２、パントテン酸カルシウム、ニコチン酸アミド、葉酸等）、アミノ酸（グリシン、メチオニン等）、ビール酵母などの酵母類、無機物質の微粉末（結晶性セルロース、タルク、シリカ、白雲母、ゼオライト等）などが挙げられる。 Feed raw materials are not limited, but for example, grains or processed grains (corn, mylo, barley, wheat, rye, swallow, millet, wheat flour, wheat germ flour, etc.), bran (bran, rice bran, corn gluten feed, etc.) ), Vegetable oil lees (soybean oil lees, sesame oil lees, cottonseed oil lees, peanut lees, sunflower lees, saflower lees, etc.) , Shochu lees [wheat roast lees, potato roast lees, soba roast lees, rice roast lees, etc.], animal raw materials (defatted milk powder, fish flour, meat bone powder, etc.), minerals (calcium carbonate, calcium phosphate, salt, silicic anhydride, etc.) ), Vitamins (vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, calcium pantothenate, nicotinic acid amide, folic acid, etc.), amino acids (glycine, methionine, etc.), beer Examples thereof include yeasts such as yeast and fine powders of inorganic substances (crystalline cellulose, talc, silica, white mica, zeolite, etc.).
本発明で使用する飼料原料は、好ましくは、ＧＡＢＡ及びオルニチンの生成基質であるグルタミン酸及びアルギニンを含むものである。グルタミン酸及びアルギニンに代えて、グルタミン酸及びアルギニンを生成し得る成分であってもよい。かかる成分としては、グルタミン酸又は／及びアルギニンを一部に有するペプチドまたはタンパク質を例示することができる。この場合、飼料原料には、当該ペプチドまたはタンパク質を加水分解する作用を有する成分が含まれていることが好ましい。かかる成分としては、前述するタンパク質やペプチドを分解してグルタミン酸及び／又はアルギニンを生成する能力を有する微生物、およびペプチターゼやプロテアーゼ等の酵素を例示することができる。 The feed raw material used in the present invention preferably contains glutamic acid and arginine, which are substrates for producing GABA and ornithine. Instead of glutamic acid and arginine, it may be a component capable of producing glutamic acid and arginine. Examples of such components include peptides or proteins having glutamic acid and / and arginine in part. In this case, it is preferable that the feed raw material contains a component having an action of hydrolyzing the peptide or protein. Examples of such components include microorganisms having the ability to decompose the above-mentioned proteins and peptides to produce glutamic acid and / or arginine, and enzymes such as peptidase and protease.
また飼料原料には、必要に応じて本発明の乳酸菌の良好な生育のための発酵促進物質、例えばグルコース、澱粉、蔗糖、乳糖、糖蜜、デキストリン、ソルビトール、フラクトースなどの炭素源；酵母エキス、ペプトンなどの窒素源；ビタミン類、ミネラル類、脂肪酸類などを加えることができる。 Further, as a feed raw material, if necessary, a fermentation promoting substance for good growth of the lactic acid bacterium of the present invention, for example, a carbon source such as glucose, starch, sucrose, lactose, molasses, dextrin, sorbitol, fructose; yeast extract, peptone Nitrogen sources such as; vitamins, minerals, fatty acids, etc. can be added.
飼料原料を発酵する際に使用される乳酸菌の接種量は、制限されないものの、一般には飼料原料１ｇ中に菌体濃度が約１×１０4 ＣＦＵ／ｇ以上、好ましくは１０５ＣＦＵ／ｇ以上の割合で含まれるように調整されることが好ましい。発酵（培養）条件及び方法は、製造する飼料の種類に応じて、適宜選択調整することができる。発酵（培養）条件として、制限されないものの、例えば、培養温度２５〜４０℃、好ましくは３５℃程度；培養ｐＨ条件４〜８程度、好ましくはｐＨ６程度；培養時間３〜２４時間程度、好ましくは５〜７時間程度を挙げることができる。 Inoculum of lactic acid bacteria used to ferment feedstuff, although not limited, generally bacterial cell concentration in the feed raw material 1g in approximately 1 × 10 4 CFU / g or more, preferably more than 10 5 CFU / g It is preferably adjusted to be included in proportion. Fermentation (culture) conditions and methods can be appropriately selected and adjusted according to the type of feed to be produced. The fermentation (culture) conditions are not limited, but for example, the culture temperature is 25 to 40 ° C., preferably about 35 ° C.; the culture pH condition is about 4 to 8, preferably about pH 6, and the culture time is about 3 to 24 hours, preferably about 5. It can be mentioned for about 7 hours.
本発明の飼料には、配合飼料に通常使用される賦形剤、増量剤、結合剤、増粘剤、乳化剤、着色料、香料、食品添加物、調味料等の飼料用添加剤、所望によりその他の成分（抗生物質や殺菌剤、駆虫剤、防腐剤等）が配合されていてもよい。 The feed of the present invention includes excipients, bulking agents, binders, thickeners, emulsifiers, colorants, fragrances, food additives, seasonings and other feed additives commonly used in compound feeds, if desired. Other ingredients (antibiotics, bactericides, insecticides, preservatives, etc.) may be blended.
本発明の飼料の形態は特に限定されるものではなく、例えば、粉末状、顆粒状、ペースト状、ペレット状、カプセル剤（ハードカプセル，ソフトカプセル）、錠剤等が挙げられる。 The form of the feed of the present invention is not particularly limited, and examples thereof include powder, granule, paste, pellet, capsule (hard capsule, soft capsule), and tablet.
本発明の乳酸菌またはその培養物を接種して培養（発酵）されることで製造される飼料は、本発明の乳酸菌のＧＡＢＡ産生能及びオルニチン産生能に基づいて、ＧＡＢＡ及びオルニチンを含有する。このため、当該飼料を非ヒト動物に飼料として摂取させることによってＧＡＢＡ及びオルニチン及びに基づく有用な生体作用を得ることができる。具体的には、本発明の飼料は、成長ホルモンの分泌促進作用、ストレス緩和作用、精神安定作用、腎・肝機能活性作用、睡眠の質改善作用などの主としてＧＡＢＡの作用、並びに、肝臓保護作用、腎臓保護作用、体臭予防、成長ホルモン分泌促進作用などの主としてオルニチン作用に基づく種々の効能・効果を有する機能性飼料として有効に使用することができる。 The feed produced by inoculating (fermenting) the lactic acid bacterium of the present invention or a culture thereof contains GABA and ornithine based on the GABA-producing ability and the ornithine-producing ability of the lactic acid bacterium of the present invention. Therefore, by ingesting the feed to non-human animals as feed, useful biological actions based on GABA and ornithine can be obtained. Specifically, the feed of the present invention mainly has GABA's action such as growth hormone secretion promoting action, stress relieving action, tranquilizing action, renal / hepatic function activating action, and sleep quality improving action, and liver protective action. It can be effectively used as a functional feed having various effects and effects mainly based on the ornithine action such as renal protection action, body odor prevention action, and growth hormone secretion promoting action.
また、本発明の乳酸菌は、ヒスチジンやチロシンの存在下で培養しても、アレルギー様症状を引き起こすヒスタミン及びチラミンを生成しないことを特徴とする。このため、本発明の乳酸菌を用いて製造される飼料は、前述の作用を有することに加えて、ヒスタミン及びチラミンを含まないため、これらの成分に対してアレルギー様症状を示す非ヒト動物も安心して飲食できる、安全性の高い飼料として提供することができる。 In addition, the lactic acid bacterium of the present invention is characterized in that it does not produce histamine and tyramine that cause allergic-like symptoms even when cultured in the presence of histidine or tyrosine. Therefore, since the feed produced by using the lactic acid bacterium of the present invention does not contain histamine and tyramine in addition to having the above-mentioned effects, non-human animals showing allergic-like symptoms to these components are also safe. It can be provided as a highly safe feed that can be eaten and eaten with care.
（III）非ヒト動物の飼育方法
本発明の非ヒト動物の飼育方法は、非ヒト動物に前述する本発明の飼料を、飼料の少なくとも一部として供餌することにより、非ヒト動物を飼育することを特徴とする。本発明の飼料を飼料として供餌する以外は、特に制限されることなく、非ヒト動物の種類に応じて、通常の方法や条件で飼育を行うことができる。
(III) Non-Human Animal Breeding Method The non-human animal breeding method of the present invention breeds a non-human animal by feeding the non-human animal with the feed of the present invention described above as at least a part of the feed. It is characterized by that. Except for feeding the feed of the present invention as a feed, the animals can be bred by ordinary methods and conditions according to the type of non-human animal without any particular limitation.
以下に実験例を用いて本発明の構成及び効果を説明する。但し、本発明はこれらの実験例に何ら影響されるものではない。なお、特に言及しない場合、下記の実験は大気圧及び室温（２５±５℃）条件下で実施した。また特に言及しない場合、「％」は「質量％」を意味するものとする。 The configuration and effect of the present invention will be described below using experimental examples. However, the present invention is not influenced by these experimental examples. Unless otherwise specified, the following experiments were carried out under atmospheric pressure and room temperature (25 ± 5 ° C.). Unless otherwise specified, "%" shall mean "mass%".
以下の実験に使用した乳酸菌は、宮崎県宮崎市佐土原町東上那珂１６５００−２に所在する宮崎県食品開発センターに分譲可能な状態で保存している「ラクトバチルス・ブフネリＭＬ５３０株」の継代株である。前述するように、その一部を、日本国千葉県木更津市かずさ鎌足２−５−８に住所を有する独立行政法人製品評価技術基盤機構 特許微生物寄託センター（ＮＰＭＤ）に「微生物の識別の表示：ＭＬ５３０」として、２０２０年１月２２日（寄託日）に国内寄託している（受領番号：ＮＩＴＥ ＡＰ−０３１１１）。 The lactic acid bacteria used in the following experiments are the successor strains of "Lactobacillus buchneri ML530 strain" stored in a state where they can be sold at the Miyazaki Prefectural Food Development Center located at 16500-2 Higashikaminaka, Sadowara-cho, Miyazaki City, Miyazaki Prefecture. Is. As mentioned above, a part of it is displayed on the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation, which has an address at 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan. : ML530 ”was deposited domestically on January 22, 2020 (deposit date) (receipt number: NITE AP-03111).
また、以下の実験例で使用したＭＲＳ液体培地の組成、ＭＬ５３０培養液、アミノ酸分析、及び高速液体クロマトグラフ質量分析は下記の通りである。
１ ＭＲＳ液体培地（１Ｌ）の組成（ｐＨ６．５±０．２）
Proteose Pepton No.3 10.0 g
Beef Extract 10.0 g
Yeast Extract 5.0 g
Dextrose 20.0 g
Polysorbate 80 1.0 g
Ammonium Citrate 2.0 g
Sodium Acetate 5.0 g
Magnesium Sulfate 0.1 g
Manganese Sulfate 0.05 g
Dipotassium Phosphate 2.0 g
The composition of the MRS liquid medium, the ML530 culture medium, the amino acid analysis, and the high performance liquid chromatograph mass spectrometry used in the following experimental examples are as follows.
Composition of 1 MRS liquid medium (1 L) (pH 6.5 ± 0.2)
Proteose Pepton No.3 10.0 g
Beef Extract 10.0 g
Yeast Extract 5.0 g
Dextrose 20.0 g
Polysorbate 80 1.0 g
Ammonium Citrate 2.0 g
Sodium Acetate 5.0 g
Magnesium Sulfate 0.1 g
Manganese Sulfate 0.05 g
Dipotassium Phosphate 2.0 g
２ アミノ酸分析
アミノ酸分析は、ニンヒドリンを試薬とするポストカラム誘導体化法による高速アミノ酸分析計Ｌ−８９００（株式会社日立ハイテクノロジーズ製）を用いて行った。具体的には、被験試料中に含まれているグルタミン酸、ＧＡＢＡ、アルギニン、及びオルニチンの量を当該分析計推奨のマニュアルに従って分析した。
2 Amino acid analysis Amino acid analysis was performed using a high-speed amino acid analyzer L-8900 (manufactured by Hitachi High-Technologies Corporation) by a post-column derivatization method using ninhydrin as a reagent. Specifically, the amounts of glutamic acid, GABA, arginine, and ornithine contained in the test sample were analyzed according to the manual recommended by the analyzer.
３ 高速液体クロマトグラフ質量分析（ＬＣＭＳＭＳ分析）
ヒスタミン及びチラミンの定量は、下記条件に設定した高速液体クロマトグラフ質量分析計（ＬＣＭＳＭＳ）（株式会社エービー・サイエックス、ＡＰＩ３２００）を用いて行った。
3 High Performance Liquid Chromatograph Mass Spectrometry (LCMSMS Analysis)
The quantification of histamine and tyramine was performed using a high performance liquid chromatograph mass spectrometer (LCMSMS) (AB SIX Co., Ltd., API3200) set under the following conditions.
実験例１ 麦芽粕の乳酸発酵による飼料製造
（１）乳酸発酵による飼料の製造
麦汁の絞り粕（麦芽粕）に、ＭＬ５３０培養液、または／および、市販乳酸菌製剤（サイマスターＡＣ、サイマスターＬＰ、サイマスターＳＰ：すべて雪印種苗（株））を表２に示す配合割合でそれぞれ添加してよく攪拌した。なお、乳酸菌ＭＬ５３０株としては、オートクレーブ滅菌（１２１℃、２０分）した前記ＭＲＳ液体培地に乳酸菌ＭＬ５３０株を接種し、嫌気条件下で３０℃で２日間培養して調製したものを使用した。また、「サイマスターＡＣ」及び「サイマスターＬＰ」は、ともにラクトコッカス・ラクティスＳＢＳ０００１−Ｓ株とラクトバチルス・パラカゼイＳＢＳ０００３株を含有する乳酸菌製剤であり、前者の「サイマスターＡＣ」には前記乳酸菌に加えて繊維分解酵素が含まれている。「サイマスターＳＰ」は、ラクトバチルス・ディオリボランスＳＢＳ０００７株を含有する乳酸菌製剤である。いずれの乳酸菌も牧草やサイレージから分離・選抜された乳酸菌である。
Experimental Example 1 Feed production by lactic acid fermentation of malt lees (1) Manufacture of feed by lactic acid fermentation For wort squeezed lees (malt lees), ML530 culture solution and / and commercially available lactic acid bacteria preparations (Cymaster AC, Cymaster LP) , Cymaster SP: All Snow Brand Seedlings Co., Ltd. were added in the blending ratios shown in Table 2 and stirred well. As the lactic acid bacterium ML530 strain, a lactic acid bacterium ML530 strain was inoculated into the MRS liquid medium sterilized by autoclave (121 ° C., 20 minutes) and cultured at 30 ° C. for 2 days under anaerobic conditions. Further, "Cymaster AC" and "Cymaster LP" are lactic acid bacteria preparations containing both Lactococcus lactis SBS0001-S strain and Lactobacillus paracasei SBS0003 strain, and the former "Cymaster AC" is the lactic acid bacterium. In addition, it contains fibrodegrading enzymes. "Cymaster SP" is a lactic acid bacterium preparation containing the Lactobacillus diolivolance SBS0007 strain. All lactic acid bacteria are lactic acid bacteria isolated and selected from grass and silage.
その後、袋につめて真空パックし、２５℃インキュベータで１０日間、発酵させた。 Then, it was packed in a bag, vacuum packed, and fermented in a 25 ° C. incubator for 10 days.
（２）飼料の評価
１０日後、２５℃インキュベータから培養した各被験飼料を取り出し、被験飼料に対して２倍容量の蒸留水を用いて２４時間抽出を行い、ろ紙でろ過した抽出液のｐＨを測定するとともに、それに含まれているグルタミン酸（Ｇｌｕ）、ＧＡＢＡ、アルギニン（Ａｒｇ）、オルニチン（Ｏｒｎ）の濃度（mg/L）をアミノ酸分析により測定した。
(2) Evaluation of feed After 10 days, each test feed cultured at 25 ° C. was taken out, extracted with double volume of distilled water for 24 hours, and the pH of the extract filtered with filter paper was adjusted. In addition to the measurement, the concentrations (mg / L) of glutamic acid (Glu), GABA, arginine (Arg), and ornithine (Orn) contained therein were measured by amino acid analysis.
ｐＨの測定結果を表３に、また、前記被験飼料の水抽出液中のアミノ酸濃度（mg/L）を図１に示す。 The pH measurement results are shown in Table 3, and the amino acid concentration (mg / L) in the water extract of the test feed is shown in FIG.
乳酸菌が増殖すると、乳酸菌が乳酸や酢酸等を産生するためにｐＨが下がる。表３に示すように、いずれの被験飼料も、培養によりｐＨが低下しており、これから乳酸発酵が良好に進んだことが確認された。一方、図１に示すように、ＭＬ５３０培養液を添加した区（ＭＬ）は、オルニチン量の増加に加えて、市販乳酸菌製剤を添加した区（ＡＣ、ＬＰ、ＳＰ）と比較してＧＡＢＡ量が著しく増加していることが確認された。ＧＡＢＡ及びオルニチンの両方の増加は、ＭＬ５３０培養液に加えて、市販乳酸菌製剤を添加した区（ＭＬ＋ＡＣ、ＭＬ＋ＬＰ、ＭＬ＋ＳＰ）でも同様に認められた。このことから、乳酸菌ＭＬ５３０株またはその培養液を用いることで、ＧＡＢＡ及びオルニチンの量が豊富な飼料が製造できることが確認された。 When lactic acid bacteria grow, the pH drops because the lactic acid bacteria produce lactic acid, acetic acid, and the like. As shown in Table 3, the pH of each of the test feeds was lowered by culturing, and it was confirmed that the lactic acid fermentation proceeded satisfactorily from this. On the other hand, as shown in FIG. 1, in the group to which the ML530 culture solution was added (ML), in addition to the increase in the amount of ornithine, the amount of GABA was higher than that in the group to which the commercially available lactic acid bacterium preparation was added (AC, LP, SP). It was confirmed that there was a significant increase. An increase in both GABA and ornithine was also observed in the groups (ML + AC, ML + LP, ML + SP) to which the commercially available lactobacillus preparation was added in addition to the ML530 culture medium. From this, it was confirmed that a feed rich in GABA and ornithine can be produced by using the lactic acid bacterium ML530 strain or its culture solution.
実験例２ おからの乳酸発酵による飼料製造
おから５０ｇに水１００ｍＬを添加し、オートクレーブ滅菌（１２１℃、２０分）したものに、乳酸菌ＭＬ５３０を１白金耳、市販乳酸菌製剤（サイマスターＡＣ：雪印種苗（株））０．１ｇ／ｍＬ水溶液を１ｍＬ、酸性プロテアーゼ（ＹＰ−ＳＳ：ヤクルト薬品工業（株））０．０１ｇ／ｍＬ水溶液を１ｍＬ添加し、３０℃で４日間培養した。また、対照区として、前記おからの滅菌処理物に乳酸菌ＭＬ５３０、市販乳酸菌製剤、及び酸性プロテアーゼのいずれも添加しない区を設定した。
Experimental Example 2 Feed production by lactic acid fermentation of
発酵後の被験飼料（ＭＬ５３０等添加区、対照区）は、ｐＨを測定した後に、ろ過を行い、アミノ酸分析によりグルタミン酸（Ｇｌｕ）、ＧＡＢＡ、アルギニン（Ａｒｇ）、オルニチン（Ｏｒｎ）濃度を測定した。 The test feed after fermentation (ML530 etc. addition group, control group) was filtered after measuring the pH, and the concentrations of glutamic acid (Glu), GABA, arginine (Arg), and ornithine (Orn) were measured by amino acid analysis.
ｐＨ測定結果を表４、アミノ酸濃度測定結果を図２に示す。
実験例３ 芋焼酎粕とおからの乳酸発酵による飼料製造
表５に示すように、７０Ｌペール缶に、飼料原料として芋焼酎粕またはおからを入れ、４５℃以下に下がったところで、乳酸菌ＭＬ５３０培養液、市販乳酸菌製剤（サイマスターＡＣ：雪印種苗（株））、酸性プロテアーゼ（ＹＰ−ＳＳ：ヤクルト薬品工業（株））、及び糖蜜をそれぞれ添加して、各試験区（３−１〜３−３）の試料を調製した。なお、乳酸菌ＭＬ５３０培養液としては、芋焼酎粕をろ過して得られたろ液に１％のグルコースを添加し、オートクレーブ滅菌（１２１℃、２０分）したものを培地として、これに乳酸菌ＭＬ５３０株を接種して、３０℃で２日間培養して調製したものを用いた。
Experimental Example 3 Feed production by lactic acid fermentation of potato molasses and okara As shown in Table 5, lactic acid bacteria ML530 culture solution was placed in a 70 L pail as a feed raw material and when the temperature dropped to 45 ° C or lower. , Commercially available lactic acid bacteria preparation (Cymaster AC: Snow Brand Seedling Co., Ltd.), acidic protease (YP-SS: Yakult Yakuhin Kogyo Co., Ltd.), and molasses were added to each test group (3-1 to 3-3). ) Was prepared. As the lactic acid bacterium ML530 culture solution, 1% glucose was added to the filtrate obtained by filtering the sweet potato shochu lees, and autoclave sterilized (121 ° C., 20 minutes) was used as a medium, and the lactic acid bacterium ML530 strain was added thereto. Those prepared by inoculating and culturing at 30 ° C. for 2 days were used.
７０Ｌ容量のペール缶は屋内（室温）に静置し、発酵開始後１、２、３、４、及び７日後に、攪拌とサンプリングを行った。得られた被験飼料は、ｐＨを測定した後にろ過を行い、アミノ酸分析によりＧＡＢＡ、及びオルニチン濃度を測定した。ｐＨの経時変化を図３に、ＧＡＢＡ濃度の経時変化を図４に、オルニチン濃度の経時変化を図５に示す。 The 70 L capacity pail can was allowed to stand indoors (room temperature), and stirred and sampled 1, 2, 3, 4, and 7 days after the start of fermentation. The obtained test feed was filtered after measuring the pH, and the GABA and ornithine concentrations were measured by amino acid analysis. The time course of pH is shown in FIG. 3, the time course of GABA concentration is shown in FIG. 4, and the time course of ornithine concentration is shown in FIG.
図３に示すように、いずれの試験区とも、発酵開始１日後からｐＨが低下し、乳酸菌（乳酸菌ＭＬ５３０、または市販乳酸菌製剤中の乳酸菌）が増殖したことがわかる。また、図４及び図５に示すように、乳酸菌が増殖した発酵開始１日後から、ＭＬ５３０を添加した試験区３−１及び３−２においてのみＧＡＢＡとオルニチンの双方が増加することが確認された。また、その増加の程度は試験区３−１で著しく、飼料原料としておからを用いることでＧＡＢＡ及びオルニチンの産生量が著しく増加することが確認された。これは、おからに含まれる大豆由来のタンパク質が酸性プロテアーゼの作用により分解され、飼料原料中に、ＧＡＢＡ、及びオルニチンを生成する基質となるグルタミン酸及びアルギニンが多く生成されたことによるものと考えられる。 As shown in FIG. 3, it can be seen that the pH of each test group decreased from 1 day after the start of fermentation, and lactic acid bacteria (lactic acid bacteria ML530 or lactic acid bacteria in a commercially available lactic acid bacteria preparation) grew. Further, as shown in FIGS. 4 and 5, it was confirmed that both GABA and ornithine increased only in the test groups 3-1 and 3-2 to which ML530 was added from 1 day after the start of fermentation in which lactic acid bacteria grew. .. In addition, the degree of increase was remarkable in Test Group 3-1 and it was confirmed that the production amounts of GABA and ornithine were significantly increased by using okara as a feed raw material. It is considered that this is because the soybean-derived protein contained in okara was decomposed by the action of acidic protease, and a large amount of glutamic acid and arginine, which are substrates for producing GABA and ornithine, were produced in the feed raw material. ..
実験例４ 麦焼酎粕と大豆粕の乳酸発酵による飼料製造
表６に示すように、５００Ｌ容量の農業用ポリタンクに、飼料原料として、麦焼酎粕または大豆粕を入れ、４５℃以下に下がったところで乳酸菌ＭＬ５３０培養液、市販乳酸菌製剤（サイマスターＡＣ：雪印種苗（株））、酸性プロテアーゼ（ＹＰ−ＳＳ：ヤクルト薬品工業（株））、及び糖蜜をそれぞれ添加して、各試験区（４−１〜４−３）の試料を調製した。なお、乳酸菌ＭＬ５３０培養液として、麦焼酎粕をろ過して得られたろ液に１％のグルコースを添加し、オートクレーブ滅菌（１２１℃、２０分）したものを培地として、これに乳酸菌ＭＬ５３０株を接種し、３０℃で２日間培養して調製したものを使用した。
Experimental Example 4 Feed production by lactic acid fermentation of barley molasses and soybean meal As shown in Table 6, barley molasses or soybean meal was placed as a feed raw material in a 500 L capacity agricultural plastic tank, and the temperature was lowered to 45 ° C or lower. Lactic acid bacteria ML530 culture solution, commercially available lactic acid bacteria preparation (Cymaster AC: Snow Brand Seedling Co., Ltd.), acidic protease (YP-SS: Yakult Yakuhin Kogyo Co., Ltd.), and molasses were added to each test group (4-1). Samples of ~ 4-3) were prepared. As a lactic acid bacterium ML530 culture solution, 1% glucose was added to a filtrate obtained by filtering barley shochu lees, and autoclave sterilized (121 ° C., 20 minutes) was used as a medium, and the lactic acid bacterium ML530 strain was inoculated thereto. Then, the one prepared by culturing at 30 ° C. for 2 days was used.
当該ポリタンクを屋根のある屋外に静置し、発酵開始後１週間は１〜２日ごとに、その後は３０日後まで１週間ごとに攪拌およびサンプリングを行った。得られた被験飼料をアミノ酸分析に供して、ＧＡＢＡ、及びオルニチンの各濃度を測定した。ＧＡＢＡ濃度の経時変化を図６に、オルニチン濃度の経時変化を図７に示す。 The plastic tank was allowed to stand outdoors with a roof, and stirring and sampling were performed every 1 to 2 days for 1 week after the start of fermentation, and every week thereafter until 30 days later. The obtained test feed was subjected to amino acid analysis, and the concentrations of GABA and ornithine were measured. The time course of GABA concentration is shown in FIG. 6, and the time course of ornithine concentration is shown in FIG. 7.
図６及び７に示すように、飼料原料として麦焼酎粕を用いて、乳酸菌ＭＬ５３０を用いて発酵することで、ＧＡＢＡ及びオルニチンの含有量が著しく増加すること、さらに飼料原料に大豆粕を加えることで、それらの含有量がさらに高まることが確認された。 As shown in FIGS. 6 and 7, the content of GABA and ornithine is significantly increased by fermenting with barley shochu lees as a feed raw material and using lactic acid bacterium ML530, and soybean meal is added to the feed raw material. It was confirmed that their content was further increased.
参考実験例 ヒスタミンおよびチラミン生成に関する本発明の乳酸菌の特性評価
本乳酸菌を用いて、豆乳を発酵し、発酵豆乳飲料を製造した。
（１）豆乳の乳酸発酵（発酵豆乳飲料の製造）
表７に示すように、滅菌した容器に豆乳１５０ｍＬを入れ、これに本乳酸菌試料とグルコース水溶液（試験区１）、または本乳酸菌試料とグルコース水溶液とプロテアーゼ水溶液（試験区２）を添加して、嫌気条件下、３０℃で３日間培養して発酵させた。なお、本乳酸菌試料として、本乳酸菌をＭＲＳ液体培地で嫌気条件下、３０℃で２日間培養した培養物を用いた。また、グルコース水溶液は、グルコースを滅菌水に０．５ｇ／ｍＬ濃度になるように溶解した水溶液を０．２μｍメンブランフィルターでろ過したものを使用した。プロテアーゼ水溶液は、酸性プロテアーゼ（ＹＰ−ＳＳ：ヤクルト薬品工業（株）製）を、滅菌水に０．１ｇ／ｍＬ濃度になるように溶解した水溶液を０．２μｍメンブランフィルターでろ過したものを使用した。
Reference Experimental Example Evaluation of Characteristics of Lactic Acid Bacteria of the Present Invention Regarding Histamine and Tyramine Production Using this lactic acid bacterium, soymilk was fermented to produce a fermented soymilk beverage.
(1) Lactic acid fermentation of soymilk (manufacturing of fermented soymilk beverage)
As shown in Table 7, 150 mL of soymilk was placed in a sterilized container, and the lactic acid bacterium sample and glucose aqueous solution (test group 1) or the lactic acid bacterium sample, glucose aqueous solution and protease aqueous solution (test group 2) were added thereto. It was fermented by culturing at 30 ° C. for 3 days under anaerobic conditions. As the lactic acid bacterium sample, a culture in which the lactic acid bacterium was cultured in an MRS liquid medium under anaerobic conditions at 30 ° C. for 2 days was used. Further, as the glucose aqueous solution, an aqueous solution obtained by dissolving glucose in sterilized water at a concentration of 0.5 g / mL and filtering it with a 0.2 μm membrane filter was used. As the protease aqueous solution, an aqueous solution prepared by dissolving acidic protease (YP-SS: manufactured by Yakult Yakuhin Kogyo Co., Ltd.) in sterilized water to a concentration of 0.1 g / mL was filtered with a 0.2 μm membrane filter. ..
グルコース水溶液は、グルコースが乳酸菌が好んで資化する栄養素であることから、乳酸菌の生育をよくするために添加した。また、プロテアーゼ水溶液は、プロテアーゼによって豆乳中に含まれるタンパク質が分解されて、ＧＡＢＡとオルニチン生成の基質となるグルタミン酸とアルギニンの含有量を増加させる効果を期待して添加した。 Since glucose is a nutrient that lactic acid bacteria prefer to assimilate, an aqueous glucose solution was added to improve the growth of lactic acid bacteria. In addition, the aqueous protease solution was added with the expectation that the protein contained in soymilk was decomposed by the protease to increase the contents of glutamic acid and arginine, which are substrates for GABA and ornithine production.
発酵前の豆乳、及び発酵後の試験区１、２について、実験例１に記載する方法でグルタミン酸（Glu）、ＧＡＢＡ、アルギニン（Arg）、及びオルニチン（Orn）のそれぞれの濃度を測定した。その結果、試験区１及び２とも発酵前よりＧＡＢＡ及びオルニチン量が増加していることが確認された。特にプロテアーゼを添加した試験区２で、ＧＡＢＡ及びオルニチンの著しい増加が確認された。
The concentrations of glutamic acid (Glu), GABA, arginine (Arg), and ornithine (Orn) were measured for the soymilk before fermentation and the
また上記で製造した発酵豆乳飲料について、０．２μｍメンブランフィルターでろ過した後、得られたろ液を高速アミノ酸分析計に供して、ヒスチジン及びチロシン濃度を測定した。また、同ろ液を高速液体クロマトグラフ質量分析計に供して、ヒスタミン及びチラミン濃度を測定した。測定結果を表８に示す。
表８から、ヒスチジン及びチロシンの濃度に関わらず、発酵前後でヒスタミン及びチラミンの濃度はほぼ一定であり、本発明の乳酸菌による発酵により、ヒスチジン及びチロシン存在下においてもヒスタミン及びチラミンは生成されないことが確認された。このことから、本乳酸菌を飼料の製造に使用しても、（ｃ）ヒスチジン存在下でヒスタミンを生成しない特性、及び（ｄ）チロシン存在下でチラミンを生成しないことが確認された。 From Table 8, the concentrations of histamine and tyramine are almost constant before and after fermentation regardless of the concentrations of histidine and tyrosine, and the fermentation by the lactic acid bacterium of the present invention does not produce histamine and tyramine even in the presence of histidine and tyrosine. confirmed. From this, it was confirmed that even if this lactic acid bacterium is used in the production of feed, (c) the property of not producing histamine in the presence of histidine and (d) the property of not producing tyramine in the presence of tyrosine.
本発明により、機能性成分であるＧＡＢＡ、及びオルニチンを高濃度で含有する飼料の製造が可能となる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce a feed containing a high concentration of GABA and ornithine, which are functional components.
Claims (5)
後者の場合は、当該成分を加水分解してグルタミン酸及びアルギニンを生成する作用を有する物質とともに培養する工程を有する、請求項１に記載する製造方法。 The feed raw material contains glutamic acid and arginine, or contains a component capable of producing glutamic acid and arginine by hydrolysis.
The production method according to claim 1, wherein in the latter case, there is a step of culturing the component together with a substance having an action of hydrolyzing the component to produce glutamic acid and arginine.
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