JP5470637B2 - Microorganisms that reduce nitrate nitrogen and nitrite nitrogen in fermented feed - Google Patents
Microorganisms that reduce nitrate nitrogen and nitrite nitrogen in fermented feed Download PDFInfo
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- JP5470637B2 JP5470637B2 JP2009157011A JP2009157011A JP5470637B2 JP 5470637 B2 JP5470637 B2 JP 5470637B2 JP 2009157011 A JP2009157011 A JP 2009157011A JP 2009157011 A JP2009157011 A JP 2009157011A JP 5470637 B2 JP5470637 B2 JP 5470637B2
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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- Y02P60/87—Re-use of by-products of food processing for fodder production
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Description
本発明は、新規の硝酸態窒素低減能もしくは亜硝酸態窒素低減能を有する微生物菌株に関し、詳しくは、優れた硝酸態窒素低減能および良質な発酵飼料の製造に適した性質を有する新規のバチルス属の菌株、および、優れた亜硝酸態窒素低減能および良質な発酵飼料の製造に適した性質を有する新規の乳酸菌の菌株に関する。
また本発明は、前記菌株を含有してなる発酵飼料製造用の微生物製剤、；前記菌株を添加することを特徴とする硝酸態窒素および亜硝酸態窒素が低減された発酵飼料（詳しくは、野菜残さ発酵飼料、飼料作物・牧草サイレージ、ＴＭＲ発酵飼料）の製造方法、；に関する。
The present invention relates to a novel bacterial strain having nitrate nitrogen reducing ability or nitrite nitrogen reducing ability, and more particularly, a novel bacillus having excellent nitrate nitrogen reducing ability and properties suitable for producing a high-quality fermented feed. The present invention relates to a genus strain and a novel strain of lactic acid bacteria having excellent nitrite nitrogen reducing ability and properties suitable for producing a high-quality fermented feed.
The present invention also relates to a microorganism preparation for producing a fermented feed comprising the above strain; a fermented feed with reduced nitrate nitrogen and nitrite nitrogen characterized by adding the strain (specifically, vegetables Manufacturing method of residual fermented feed, feed crop / grass silage, TMR fermented feed).
野菜豊作時の余剰野菜カット野菜工場より排出される食品残さ（野菜残さ）は、年間排出量が多く、タンパク質やビタミン類は豊富に含まれる。
しかしながら、このような野菜残さ、特に、キャベツ、白菜などの葉菜類などには、硝酸態窒素含量が高い（非特許文献１参照）。また、このような野菜残さには、水分含量が高いものは多い。
そのため、これらを家畜飼料としては利用するためには、硝酸態窒素含量の低減および水分含量の調整などの良質の貯蔵法に工夫が必要であり、一部は圃場還元や堆肥化して処理されるか、そのほとんどは産業廃棄物として処理され、家畜飼料として有効に利用されてないのが現状である。
なお、このような水分含量が高い野菜残さは、長期にわたって貯蔵することはできないため、そのまま放置すると腐敗による悪臭や、蝿、蛆などの害虫の発生により、環境汚染に進展する恐れもあるため、排出後に早急に適切な処理が必要である。そのため、食品製造工場から排出された野菜残さは、排出後の迅速乾燥が必要となるが、高水分の食品副産物を乾燥するには、大型乾燥機の導入や多くの熱エネルギーを必要とする。したがって、製造コストが上がり、事実上、家畜飼料としての活用は極めて困難である。
Food residue (vegetable residue) discharged from the surplus vegetable cut vegetable factory at the time of rich vegetable harvesting has a large amount of annual discharge and is rich in proteins and vitamins.
However, such vegetable residues, particularly leaf vegetables such as cabbage and Chinese cabbage, have a high nitrate nitrogen content (see Non-Patent Document 1). Moreover, many of such vegetable residues have a high water content.
Therefore, in order to use them as livestock feed, it is necessary to devise high-quality storage methods such as reduction of nitrate nitrogen content and adjustment of water content, and some of them are processed by field reduction or composting. However, most of them are treated as industrial waste and are not effectively used as livestock feed.
In addition, since the vegetable residue with such a high water content cannot be stored for a long time, if left as it is, there is a possibility that it may progress to environmental pollution due to the generation of bad odors due to rot, pests such as moths, moths, etc. Appropriate treatment is required immediately after discharge. Therefore, although the vegetable residue discharged | emitted from the food manufacturing factory needs quick drying after discharge | emission, in order to dry a high moisture food by-product, introduction of a large sized dryer and much heat energy are required. Therefore, the manufacturing cost is increased, and practical use as a livestock feed is extremely difficult.
また、発酵飼料の原料に用いる飼料作物の畑には、堆厩肥が多く施用され、土壌中に窒素や塩類が高濃度に集積した飼料畑が多い。この様な圃場で栽培される‘野菜’、‘牧草’および‘飼料作物’中に硝酸態窒素（ＮＯ３−Ｎ）が多く蓄積されている。
また、ＴＭＲ発酵飼料の原料の中にも、硝酸態窒素（ＮＯ３−Ｎ）を多く含むものがある。
In addition, forage crop fields used as raw materials for fermented feeds are often applied with manure, and there are many forage fields in which nitrogen and salts are accumulated at high concentrations in the soil. A large amount of nitrate nitrogen (NO 3 -N) is accumulated in “vegetables”, “grass” and “forage crops” cultivated in such fields.
Some raw materials of TMR fermented feed contain a large amount of nitrate nitrogen (NO 3 -N).
従って、これらを原料として発酵飼料を調製する場合、硝酸態窒素含量が高いため、家畜に給与する場合、硝酸塩中毒を発生する場合があり、安全対策が求められる。
例えば、サイレージ発酵過程において、硝酸還元菌の働きによって、硝酸態窒素を減少させる方法（非特許文献２参照）や、触媒と還元剤を用いた化学反応により、硝酸態窒素を無害な窒素ガスに変換する方法（非特許文献３参照）、が報告されている。
しかしながら、硝酸還元菌の働きは不明であり、硝酸態窒素低減効果も不十分であり、安全な発酵飼料を調製・確保できる程度に技術が確立していない。また、触媒や還元剤を用いた方法で調製したものは、安全性の点で飼料として適さない。さらに、これらの還元過程で生じる亜硝酸態窒素も毒性がある物質であり、飼料中に残存すると好ましくない。
Therefore, when preparing fermented feed using these as raw materials, since nitrate nitrogen content is high, when feeding to livestock, nitrate poisoning may occur, and safety measures are required.
For example, in the silage fermentation process, nitrate nitrogen is converted into harmless nitrogen gas by a method of reducing nitrate nitrogen by the action of nitrate reducing bacteria (see Non-Patent Document 2) or a chemical reaction using a catalyst and a reducing agent. A conversion method (see Non-Patent Document 3) has been reported.
However, the action of nitrate-reducing bacteria is unknown, the effect of reducing nitrate nitrogen is insufficient, and the technology has not been established to the extent that a safe fermented feed can be prepared and secured. Moreover, what was prepared by the method using a catalyst or a reducing agent is not suitable as a feed in terms of safety. Furthermore, nitrite nitrogen generated in these reduction processes is also a toxic substance and is not preferable if it remains in the feed.
このように、従来技術では、発酵飼料中の硝酸態窒素の有効な低減技術が開発されておらず、上記のような野菜残さは産業廃棄物として処分され、硝酸態窒素含有量の高い飼料作物も安全に利用できないのが現状である。
従って、家畜生産において、これら野菜残さや未利用の飼料作物などの低・未利用資源を有効に利用することは、飼料自給率を向上することだけでなく、環境の保全や資源循環型の畜産を進めるためにも極めて有用な技術であり、極めて重要な課題である。
Thus, in the prior art, an effective technology for reducing nitrate nitrogen in fermented feed has not been developed, and vegetable residues such as those described above are disposed of as industrial waste, and forage crops with a high nitrate nitrogen content. However, the current situation is that it cannot be used safely.
Therefore, in livestock production, the effective use of low and unused resources such as vegetable residues and unused feed crops not only improves the feed self-sufficiency rate, but also preserves the environment and recycles livestock. This is an extremely useful technology for promoting the process, and is an extremely important issue.
本発明は、上記課題を解決し、硝酸態窒素含有量の高い発酵飼料原料（特に野菜残さ、サイレージ原料である牧草や飼料作物、ＴＭＲ発酵飼料原料）を用いて発酵飼料を調製した際に、発酵飼料中の硝酸態窒素および亜硝酸態窒素を‘安全性が高く’且つ‘顕著に’低減する方法、を提供することを目的とする。
また、本発明は、良質な発酵飼料（特に野菜残さ発酵飼料、サイレージ、ＴＭＲ発酵飼料）を製造することを目的とする。
When the present invention solves the above-mentioned problems and prepares a fermented feed using a fermented feed material having a high nitrate nitrogen content (particularly vegetable residue, silage raw material grass or feed crop, TMR fermented feed material), It is an object of the present invention to provide a method for reducing nitrate nitrogen and nitrite nitrogen in fermented feeds 'highly safe' and 'significantly'.
Another object of the present invention is to produce a high-quality fermented feed (particularly vegetable residue fermented feed, silage, TMR fermented feed).
（１）本発明者は、発酵飼料原料およびサイレージから、バチルスと乳酸菌を分離し、硝酸態窒素と亜硝酸態窒素の低減能を示す微生物のスクリーニングを行った。そして、細胞形態、生理生化学性状、１６ＳｒＤＮＡ塩基配列の解析により菌種を同定した。
その結果、優れた硝酸態窒素低減能を示すバチルス菌株、並びに、優れた亜硝酸態窒素低減能を示す乳酸菌の菌株を見出した。
（２）また本発明者は、硝酸態窒素含有量の高い発酵飼料原料（特に野菜残さ、サイレージ原料である牧草や飼料作物、ＴＭＲ発酵飼料原料）に、これら菌株を添加してサイレージ発酵を行うことで、発酵飼料中の硝酸態窒素と亜硝酸態窒素を低減できることを見出した。
(1) The present inventor separated Bacillus and lactic acid bacteria from fermented feed raw material and silage, and screened for microorganisms having the ability to reduce nitrate nitrogen and nitrite nitrogen. Then, bacterial species were identified by analysis of cell morphology, physiological and biochemical properties, and 16S rDNA base sequence.
As a result, a Bacillus strain exhibiting excellent nitrate nitrogen reducing ability and a lactic acid bacteria strain exhibiting excellent nitrite nitrogen reducing ability were found.
(2) Further, the present inventor performs silage fermentation by adding these strains to fermented feed raw materials having a high nitrate nitrogen content (particularly vegetable residues, silage raw grass and feed crops, TMR fermented feed raw materials). It was found that nitrate nitrogen and nitrite nitrogen in the fermented feed can be reduced.
サイレージ発酵過程において、これらの微生物を添加して増殖させることで、硝酸態窒素低減能に優れた微生物によって硝酸塩を亜硝酸塩まで還元し、そして亜硝酸態窒素低減能に優れた微生物によって、亜硝酸塩が資化される（もしくは、一部がアンモニアに還元されて除去される）と考えられる。
すなわち、本発明者は、これら微生物の働きによって、‘硝酸態窒素’を‘菌体成分’に変換することで発酵飼料中から除去し、硝酸塩中毒の発生の危険性を低減された発酵飼料（サイレージ）を調製できる技術を開発した。
本発明は、これらの知見に基づいて完成したものである。
In the silage fermentation process, by adding these microorganisms and growing them, nitrate is reduced to nitrite by microorganisms with excellent nitrate nitrogen reduction ability, and nitrite is reduced by microorganisms with excellent nitrite nitrogen reduction ability. Is thought to be assimilated (or partially reduced to ammonia and removed).
That is, the present inventor has removed the fermented feed by converting 'nitrate nitrogen' into 'bacterial component' by the action of these microorganisms, and reduced the risk of occurrence of nitrate poisoning ( A technology that can prepare silage was developed.
The present invention has been completed based on these findings.
即ち、請求項１に係る本発明は、嫌気条件および低ｐＨ条件で生育可能であり、酸耐性、胞子形成能、高温耐性、並びに、優れた硝酸態窒素低減能を有する「バチルス・サブティリス（Bacillus subtilis）ＮＡＳ１菌株（NITE P-753）」、に関するものである。
請求項２に係る発明は、嫌気条件および低ｐＨ条件で生育可能であり、酸耐性、優れた乳酸生成能、並びに、優れた亜硝酸態窒素低減能を有する「ラクトコカス・ラクティス（Lactococcus lactis）ＮＡＳ２菌株（NITE P-754）」、に関するものである。
請求項３に係る発明は、発酵飼料用原料に、請求項１に記載の菌株および請求項２に記載の菌株、を添加することを特徴とする、；硝酸態窒素および亜硝酸態窒素が低減された発酵飼料の製造方法、に関するものである。
請求項４に係る発明は、前記発酵飼料用原料が、野菜残さ、サイレージ原料である牧草又は飼料作物、およびＴＭＲ発酵飼料用原料、のいずれか１以上のものである、請求項３に記載の発酵飼料の製造方法、に関するものである。
請求項５に係る発明は、請求項１に記載の菌株および請求項２に記載の菌株、を含有してなる発酵飼料製造用の微生物製剤、に関するものである。
請求項６に係る発明は、請求項１に記載の菌株を含有してなる微生物製剤、および、請求項２に記載の菌株を含有してなる微生物製剤、を含む発酵飼料製造用の微生物製剤キット、に関するものである。
That is, the present invention according to claim 1 is capable of growing under anaerobic conditions and low pH conditions, and has acid resistance, spore formation ability, high temperature resistance, and excellent nitrate nitrogen reduction ability. Bacillus subtilis) NAS1 strain (NITE P-753) " .
The invention according to claim 2 is capable of growing under anaerobic conditions and low pH conditions, and has “Lactococcus lactis NAS2” having acid resistance, excellent ability to produce lactic acid, and excellent ability to reduce nitrite nitrogen. strain (NITE P-754) ", the present invention relates.
The invention according to claim 3 is characterized in that the strain according to claim 1 and the strain according to claim 2 are added to the raw material for fermented feed; nitrate nitrogen and nitrite nitrogen are reduced. The present invention relates to a method for producing a fermented feed.
The invention according to claim 4 is the method according to claim 3, wherein the raw material for fermented feed is any one or more of vegetable residue, pasture or feed crop as silage raw material, and raw material for TMR fermented feed. The present invention relates to a method for producing fermented feed.
The invention according to claim 5 relates to a microbial preparation for producing a fermented feed comprising the strain according to claim 1 and the strain according to claim 2.
The invention according to claim 6 is a microorganism preparation kit for producing fermented feed, comprising a microorganism preparation containing the strain of claim 1 and a microorganism preparation containing the strain of claim 2. , About.
本発明は、硝酸態窒素含有量の高い発酵飼料原料（特に野菜残さ、サイレージ原料である牧草や飼料作物、ＴＭＲ発酵飼料原料）を用いて発酵飼料を調製した際に、発酵飼料中の硝酸態窒素および亜硝酸態窒素を‘安全性が高く’且つ‘顕著に’低減することを可能とする。
また、本発明は、‘硝酸態窒素および亜硝酸態窒素が顕著に低減され’、且つ、‘良質な発酵飼料’（低ｐＨであり、乳酸含量が多く、アンモニア態窒素含量が低い発酵飼料）、を製造することを可能とする。
さらに本発明は、前記発酵飼料の製造に用いる微生物製剤、微生物製剤キットを提供することを可能とする。
The present invention relates to a fermented feed material having a high nitrate nitrogen content (particularly vegetable residue, silage raw material such as grass and feed crops, and TMR fermented feed material). It is possible to reduce nitrogen and nitrite nitrogen 'highly safe' and 'significantly'.
In addition, the present invention provides that “nitrate nitrogen and nitrite nitrogen are remarkably reduced” and “good quality fermented feed” (fermented feed having a low pH, high lactic acid content, and low ammonia nitrogen content). , Can be manufactured.
Furthermore, the present invention makes it possible to provide a microbial preparation and a microbial preparation kit for use in the production of the fermented feed.
さらに、本発明は、硝酸態窒素含量が高い野菜残さや飼料作物などの未利用飼料資源の有効利用に図れるばかりでなく、野菜工場の生産効率の向上や廃棄物等の発生抑制・有効利用など、より一層の環境負荷の少ない生産システムの構築を可能とするものである。
従って、本発明では、多種多様な食品残さの飼料化を図るために、栄養価や機能性成分を有効に活用し、資源の特性に応じた発酵飼料の製造を可能とする。
Furthermore, the present invention can be used not only for the effective use of unused feed resources such as vegetable residues and feed crops with a high nitrate nitrogen content, but also for improving the production efficiency of vegetable factories, suppressing the generation and use of waste, etc. Therefore, it is possible to construct a production system with less environmental load.
Therefore, in the present invention, in order to make a wide variety of food residues into feed, it is possible to produce fermented feed according to the characteristics of resources by effectively using nutritional values and functional ingredients.
本発明は、優れた硝酸態窒素低減能および良質な発酵飼料の製造に適した性質を有する新規のバチルス属の菌株、および、優れた亜硝酸態窒素低減能および良質な発酵飼料の製造に適した性質を有する新規の乳酸菌の菌株、に関する。
また本発明は、前記菌株を含有してなる発酵飼料製造用の微生物製剤、；前記菌株を添加することを特徴とする硝酸態窒素および亜硝酸態窒素が低減された発酵飼料（詳しくは、野菜残さ発酵飼料、飼料作物・牧草サイレージ、ＴＭＲ発酵飼料）の製造方法、；に関する。
The present invention is suitable for the production of a novel strain of the genus Bacillus having excellent nitrate nitrogen reducing ability and properties suitable for producing a high-quality fermented feed, and excellent nitrite nitrogen reducing ability and good-quality fermented feed. It relates to a novel lactic acid bacteria strain having the above properties.
The present invention also relates to a microorganism preparation for producing a fermented feed comprising the above strain; a fermented feed with reduced nitrate nitrogen and nitrite nitrogen characterized by adding the strain (specifically, vegetables Manufacturing method of residual fermented feed, feed crop / grass silage, TMR fermented feed).
＜新規のバチルス属の菌株＞
本発明における前記新規のバチルス属の菌株とは、‘優れた硝酸態窒素低減能’を有し且つ‘良質な発酵飼料の製造に適した性質’を有するものである。
当該菌株としては、特に「バチルス・サブティリス（Bacillus subtilis） ＮＡＳ１菌株（NITE P-753）」を挙げることができる。
<New strains of Bacillus>
The novel strain of the genus Bacillus in the present invention has 'excellent nitrate nitrogen reducing ability' and 'property suitable for producing a high-quality fermented feed'.
Examples of such strains include “Bacillus subtilis NAS1 strain (NITE P-753)”.
ＮＡＳ１菌株の菌学的性質の特徴としては、まず「優れた硝酸態窒素低減能」を挙げることができる。
ここで、優れた硝酸態窒素低減能とは、発酵飼料中における硝酸態窒素のほとんどを除去できる能力を有するものである。なお、当該菌株による硝酸態窒素の低減は、発酵飼料中に存在する硝酸態窒素を、亜硝酸態窒素に‘還元’することによって行われるものである。
As a feature of the mycological properties of the NAS1 strain, first, “excellent ability to reduce nitrate nitrogen” can be mentioned.
Here, the excellent nitrate nitrogen reducing ability has the ability to remove most of the nitrate nitrogen in the fermented feed. The reduction of nitrate nitrogen by the bacterial strain is performed by 'reducing' nitrate nitrogen present in the fermented feed to nitrite nitrogen.
また、ＮＡＳ１の菌学的性質として、「良質な発酵飼料の製造に適した性質」を挙げることができ、具体的には、‘嫌気性’‘低ｐＨ耐性’‘酸耐性’‘胞子形成能’‘高温耐性’を挙げることができる。
ここで、‘嫌気性’は、嫌気条件での生育能を指すものであり、通常の発酵飼料の発酵が嫌気条件で行われることから必須の性質である。
また、‘低ｐＨ耐性’‘酸耐性は’、良質な発酵飼料（低ｐＨであり、乳酸や有機酸を多く含む）を製造する上で必須な性質である。なお、低ｐＨ耐性として具体的には、ｐＨ３．５までの生育能を示すものである。
また、‘胞子形成能’‘高温耐性’は、厳しい環境でも生育可能となる性質であり、発酵熱による温度の上昇等にも耐えうる性質である。なお、高温耐性としては、具体的には５０℃までの生育能、７５℃までの生存耐性を示すものである。
In addition, the bacteriological properties of NAS1 can include “property suitable for the production of high-quality fermented feed”, specifically, “anaerobic” “low pH tolerance” “acid tolerance” spore formation ability. '' High temperature resistance '' can be mentioned.
Here, 'anaerobic' refers to the ability to grow under anaerobic conditions, and is an essential property because normal fermented feed is fermented under anaerobic conditions.
Also, “low pH tolerance” acid resistance is an essential property for producing a high-quality fermented feed (low pH and high in lactic acid and organic acids). In addition, specifically, the low pH tolerance indicates a growth ability up to pH 3.5.
Further, the “spore-forming ability” “high-temperature resistance” is a property that enables growth even in harsh environments, and is a property that can withstand an increase in temperature caused by fermentation heat. In addition, specifically as high temperature tolerance, the growth ability to 50 degreeC and the survival tolerance to 75 degreeC are shown.
また、ＮＡＳ１菌株以外の菌株であっても、ＮＡＳ１菌株と前記した菌学的性質の点で同一の性質を有する「バチルス・サブティリス（Bacillus subtilis）に属する分離菌株であれば、本発明の菌株として用いることができる。
また、特には、配列表の配列番号１に記載の１６ＳｒＤＮＡと同一の塩基配列を有するものを用いることができる。
Moreover, even if it is a strain other than the NAS1 strain, the strain of the present invention can be used as long as it is an isolated strain belonging to “Bacillus subtilis” having the same properties as the above-mentioned mycological properties. Can be used as
In particular, those having the same base sequence as 16S rDNA described in SEQ ID NO: 1 in the sequence listing can be used.
なお、当該菌株の分離方法としては、発酵飼料の原料や発酵飼料（具体的には、野菜残さ）などの分離源の希釈液を寒天培地に塗布し、嫌気条件で菌株を分離培養した後、硝酸態窒素の低減能、生理生化学的性質などを調べることで、選抜することができる。 In addition, as a method for separating the strain, after applying a diluted solution of a separation source such as a raw material of fermented feed or fermented feed (specifically, vegetable residue) to an agar medium, the strain is separated and cultured under anaerobic conditions, Selection can be made by examining the ability to reduce nitrate nitrogen and physiological and biochemical properties.
＜新規の乳酸菌の菌株＞
本発明における前記新規の乳酸菌の菌株とは、‘優れた亜硝酸態窒素低減能’を有し且つ‘良質な発酵飼料の製造に適した性質’を有するものである。
当該菌株としては、特に「ラクトコカス・ラクティス（Lactococcus lactis）ＮＡＳ２株（NITE P-754）」を挙げることができる。
<New strain of lactic acid bacteria>
The novel strain of lactic acid bacteria in the present invention has 'excellent ability to reduce nitrite nitrogen' and 'property suitable for producing a high-quality fermented feed'.
Examples of the strain include “Lactococcus lactis NAS2 strain (NITE P-754)”.
ＮＡＳ２菌株の菌学的性質の特徴としては、まず「優れた亜硝酸態窒素低減能」を挙げることができる。
ここで、優れた亜硝酸態窒素低減能とは、発酵飼料中における亜硝酸態窒素（前記バチルス属の菌株によって硝酸態窒素から還元されて生成されたもの）、を除去できる能力を有するものである。なお、ＮＡＳ２菌株の亜硝酸態窒素低減能としては、他の乳酸菌と比べて２倍以上の硝酸態窒素低減能を有するものであると推定される。
なお、当該菌株による亜硝酸態窒素の低減は、発酵飼料中に存在する亜硝酸態窒素を菌体内に取り込んで‘資化’することによって行われるものである。なお一部は、アンモニアに還元されて除去されると考えられる。
As the characteristics of the mycological properties of the NAS2 strain, first, “excellent ability to reduce nitrite nitrogen” can be mentioned.
Here, the excellent ability to reduce nitrite nitrogen has the ability to remove nitrite nitrogen (produced by reduction from nitrate nitrogen by the Bacillus strain) in the fermented feed. is there. In addition, as nitrite nitrogen reduction ability of NAS2 strain, it is estimated that it has the nitrate nitrogen reduction ability of 2 times or more compared with other lactic acid bacteria.
In addition, the reduction of nitrite nitrogen by the strain is performed by taking nitrite nitrogen present in the fermented feed into the cells and 'assimilating' it. A part is considered to be reduced to ammonia and removed.
また、ＮＡＳ２の菌学的性質として、「良質な発酵飼料の製造に適した性質」を挙げることができ、具体的には、‘嫌気性’‘低ｐＨ耐性’‘酸耐性’‘優れた乳酸生成能’を挙げることができる。
ここで、‘嫌気性’は、嫌気条件での生育能を指すものであり、通常の発酵飼料の発酵が嫌気条件で行われることから必須の性質である。
また、‘低ｐＨ耐性’‘酸耐性’‘優れた乳酸生成能’は、良質な発酵飼料（低ｐＨであり、乳酸や有機酸を多く含む）を製造する上で必須な性質である。なお、低ｐＨ性として具体的には、ｐＨ３．５までの生育能を示すものである。また、優れた乳酸生成能としては、効率の良いホモ発酵型乳酸発酵能、代謝性の高いＬ型乳酸生成能を有するものである。
また、当該菌株は、発酵飼料の発酵過程における好気性細菌や大腸菌群などの有害微生物の増殖を抑制するものである。
In addition, the bacteriological properties of NAS2 include “property suitable for the production of high-quality fermented feed”, specifically, “anaerobic” “low pH resistance” “acid resistance” excellent lactic acid Can be mentioned.
Here, 'anaerobic' refers to the ability to grow under anaerobic conditions, and is an essential property because normal fermented feed is fermented under anaerobic conditions.
Further, “low pH tolerance” acid tolerance ”excellent lactic acid production ability” is an essential property for producing a high-quality fermented feed (low pH and containing a large amount of lactic acid and organic acid). In addition, specifically, the low pH property indicates a growth ability up to pH 3.5. Moreover, as an excellent lactic acid-producing ability, it has an efficient homofermentation-type lactic acid fermentation ability and an L-type lactic acid production ability with high metabolism.
Moreover, the said strain suppresses the growth of harmful microorganisms such as aerobic bacteria and coliform bacteria in the fermentation process of fermented feed.
また、ＮＡＳ２菌株以外の菌株であっても、ＮＡＳ２菌株と前記した菌学的性質の点で同一の性質を有する「ラクトコカス・ラクティス（Lactococcus lactis）に属する分離菌株」であれば、本発明の菌株として用いることができる。
また、特には、配列表の配列番号２に記載の１６ＳｒＤＮＡと同一の塩基配列を有するものを用いることができる。
Moreover, even if it is a strain other than the NAS2 strain, the strain of the present invention can be used as long as it is an “isolated strain belonging to Lactococcus lactis” having the same properties as the NAS2 strain in terms of the above-mentioned mycological properties. Can be used as
In particular, those having the same base sequence as 16S rDNA described in SEQ ID NO: 2 in the sequence listing can be used.
なお、当該菌株の分離方法としては、発酵飼料の原料や発酵飼料（具体的には、牧草・飼料作物サイレージ）などの分離源の希釈液を寒天培地に塗布し、嫌気条件で菌株を分離培養した後、亜硝酸態窒素の低減能、生理生化学的性質などを調べることで、選抜することができる。 As a method for separating the strain, a diluted solution of a separation source such as fermented feed material or fermented feed (specifically, grass or feed crop silage) is applied to an agar medium, and the strain is separated and cultured under anaerobic conditions. Then, selection can be made by examining the ability to reduce nitrite nitrogen, physiological biochemical properties, and the like.
＜発酵飼料の製造方法＞
本発明では、発酵飼料を製造する際に、発酵飼料用原料に前記新規のバチルス菌株と新規の乳酸菌の菌株を添加することによって、製造される発酵飼料中の硝酸態窒素および亜硝酸態窒素を顕著に低減することができる。
<Method for producing fermented feed>
In the present invention, when fermented feed is produced, nitrate nitrogen and nitrite nitrogen in the produced fermented feed are obtained by adding the novel Bacillus strain and a novel lactic acid bacteria strain to the fermented feed raw material. It can be significantly reduced.
本発明においては、前記新規のバチルス菌株と新規の乳酸菌の菌株の‘２種類の菌株’を、発酵飼料原料に添加することが必須である。
添加の方法としては、原料中に偏りなく、好ましくは均一になるように行うことが望ましい。例えば、菌株を水に懸濁し噴霧する方法、混合攪拌する方法、などで行うことができる。
２種類の菌株は、両者を混合して形態で添加してもよく、別々に１種類ずつ添加してもよい。
添加する各菌株の量としては、原料１ｋｇに対して、前記新規のバチルス菌株１０５〜１０７菌数レベル、前記新規の乳酸菌の菌株１０５〜１０７菌数レベルで添加することが望ましい。具体的には、前記新規のバチルス菌株１０６菌数レベル、前記新規の乳酸菌の菌株１０６菌数レベルで添加することが望ましい。
なお、菌株の添加時期は、サイレージ発酵を開始する前に行うことが望ましいが、サイレージ発酵の途中の段階で添加を行ってもよい。
In the present invention, it is essential to add “two types of strains” of the novel Bacillus strain and the novel strain of lactic acid bacteria to the fermented feed material.
As a method of addition, it is desirable that the addition is carried out so as not to be biased in the raw material and preferably uniform. For example, it can be carried out by a method of suspending and spraying the strain in water, a method of mixing and stirring, or the like.
Two types of strains may be mixed and added in the form, or may be added separately one by one.
As for the amount of each strain to be added, it is desirable to add at a level of 10 5 to 10 7 bacteria of the new Bacillus strain and 10 5 to 10 7 strains of the novel lactic acid bacteria per 1 kg of the raw material. Specifically, the novel Bacillus strain 10 6 bacteria count level, it is desirable to add in the new lactic acid bacteria strain 10 6 bacteria count level.
In addition, although it is desirable to perform the addition time of a strain before starting silage fermentation, you may add at the stage in the middle of silage fermentation.
本発明においては、原料中にこの２種類の菌株を添加してサイレージ発酵中に増殖させることによって、硝酸態窒素低減能に優れた前記新規のバチルス菌株によって硝酸塩を亜硝酸塩まで還元し、そして亜硝酸態窒素低減能に優れた前記新規の乳酸菌株によって、亜硝酸塩を資化（もしくは、一部がアンモニアに還元されて除去）することができる。
即ち、本発明では、これら２種類の菌株の働きによって、‘硝酸態窒素’を‘菌体成分’に変換することで発酵飼料中から除去することができるため、発酵飼料原料として、硝酸態窒素含量が高いものを用いた場合においても、‘硝酸態窒素および亜硝酸態窒素が低減された発酵飼料’を製造することができる。
In the present invention, by adding these two strains to the raw material and growing them during silage fermentation, nitrate is reduced to nitrite by the novel Bacillus strain excellent in nitrate nitrogen reduction ability, Nitrite can be assimilated (or partially reduced to ammonia and removed) by the novel lactic acid strain excellent in nitrate nitrogen reducing ability.
That is, in the present invention, the nitrate nitrate can be removed from the fermented feed by converting the “nitrate nitrogen” into the “bacterial component” by the action of these two strains. Even when a high content is used, a 'fermented feed with reduced nitrate nitrogen and nitrite nitrogen' can be produced.
従って、本発明における発酵飼料原料としては、硝酸態窒素含量が高く、従来は発酵飼料原料として用いることが難しかったものでも用いることができる。具体的には、食品残さである野菜残さ（例えば、キャベツ、レタス、ハクサイ、などの葉菜類）、サイレージ原料である牧草や飼料作物（例えば、イタリアンライグラス、スーダングラス）、ＴＭＲ発酵飼料原料、などを挙げることができる。 Therefore, as the fermented feed material in the present invention, even a material that has a high nitrate nitrogen content and has conventionally been difficult to use as a fermented feed material can be used. Specifically, vegetable residues (for example, leaf vegetables such as cabbage, lettuce, Chinese cabbage) such as food residues, grass and feed crops (for example, Italian ryegrass, Sudan grass), TMR fermented feed materials, etc. Can be mentioned.
上記発酵としては、通常のサイレージ発酵と同様の条件で行うことができる。嫌気条件で、外気温で３０日以上で行うものである。
具体的には、サイロを用いる通常の方法、ロールベールサイレージ法、フレコンバック法、などで行うことができる。
As said fermentation, it can carry out on the conditions similar to normal silage fermentation. It is performed in an anaerobic condition at an outside temperature in 30 days or more.
Specifically, it can be performed by a normal method using a silo, a roll bale silage method, a flexible container back method, and the like.
上記２種類の菌株を添加したサイレージ発酵によって得られる発酵飼料は、‘良質の発酵飼料’（低ｐＨであり、乳酸含量が多く、アンモニア態窒素含量が低い）と認められるものである。また、発酵飼料の発酵過程における好気性細菌や大腸菌群などの有害微生物の増殖が抑制されたものである。
なお、具体的に、製造される発酵飼料としては、野菜残さ発酵飼料、サイレージ、ＴＭＲ発酵飼料を挙げることができる。
The fermented feed obtained by silage fermentation to which the above two types of strains are added is recognized as 'good quality fermented feed' (low pH, high lactic acid content, low ammonia nitrogen content). In addition, the growth of harmful microorganisms such as aerobic bacteria and coliforms in the fermentation process of the fermented feed is suppressed.
Specific examples of the fermented feed to be produced include vegetable residue fermented feed, silage, and TMR fermented feed.
＜微生物製剤＞
本発明においては、上記菌株を含有してなる微生物製剤の形態にして提供することができる。
微生物製剤の形態としては、上記菌株を凍結乾燥状態にして粉末状の形態、賦型剤等と混ぜて固形にした形態、カプセルに充填する形態、液体アンプルの形態、などを挙げることができる。好ましくは、凍結乾燥製剤の形態が好適である。
また、本発明では、‘２種類の菌株を混合して含有する剤’の形態とすることもできるが、‘１種類の各菌株ずつを含有する剤を２種類含むキット’の形態とすることもできる。
なお、２種類の菌株の含有比（もしくは使用時における混合比）は、特に制限はなく１：９〜９：１程度の範囲であればよいが、特には１：１とすることが望ましい。
<Microbial preparation>
In this invention, it can provide in the form of the microorganism formulation formed by containing the said strain.
Examples of the form of the microbial preparation include a powdered form obtained by freeze-drying the above strain, a form obtained by mixing with an excipient and the like, a form filled with a capsule, a form of a liquid ampule, and the like. Preferably, the form of a lyophilized formulation is suitable.
Moreover, in this invention, although it can also be set as the form of 'the agent which mixes and contains two types of strains', it is set as the form of 'the kit which contains two types of agents containing each one type of each strain'. You can also.
The content ratio (or mixing ratio at the time of use) of the two types of strains is not particularly limited and may be in the range of about 1: 9 to 9: 1, but is preferably 1: 1.
剤の使用形態としては、直接原料に添加することもできるが、好ましくは、水等に溶けて用いることが望ましい。
また、本発明の微生物製剤の使用量としては、原料１ｋｇに対して、前記新規のバチルス菌株１０５〜１０７菌数レベル、前記新規の乳酸菌の菌株１０５〜１０７菌数レベルで添加することが望ましい。具体的には、前記新規のバチルス菌株１０６菌数レベル、前記新規の乳酸菌の菌株１０６菌数レベルで添加することが望ましい。
As a usage form of the agent, it can be added directly to the raw material, but it is desirable to dissolve it in water or the like.
Moreover, as the usage-amount of the microorganism preparation of this invention, it adds with the said new Bacillus strain 10 < 5 > -10 < 7 > bacteria count level with respect to 1 kg of raw materials, and the said novel lactic acid bacteria strain 10 < 5 > -10 < 7 > bacteria count level. It is desirable. Specifically, the novel Bacillus strain 10 6 bacteria count level, it is desirable to add in the new lactic acid bacteria strain 10 6 bacteria count level.
以下、実施例を挙げて本発明を説明するが、本発明の技術範囲はこれらにより限定されるものではない。
＜実施例１＞ ＮＡＳ１，２菌株の分離と同定
（１）菌株の分離
各種野菜残さ、もしくは、牧草・飼料作物サイレージから、発酵飼料中の硝酸態窒素、亜硝酸態窒素の低減に優れた菌株の分離を行った。
まず、これら試料各１０gをストマッカ−用ビニール袋（飛竜KN208、旭化成（株）製）に採取し、滅菌した生理食塩水９０ｍｌを加えて１０倍希釈液とした後、この液をさらに1０８倍まで希釈した。
これらの希釈液をLactobacilli ＭＲＳ寒天培地（DIFCO Laboratories, Detroit, USA）、普通寒天培地（日水製薬（株）、日本）及びＧＹＰ白亜寒天培地（小崎ら、乳酸菌実験マニュアル、1992）に塗布して、恒温培養装置を使って嫌気条件にて３０℃で２日間培養するか、もしくは、乳酸菌用嫌気培養装置を使って嫌気条件にて３０℃で２日間培養した。
得られた培養物から分離した菌株（バチルス３０株、酵母１０株、乳酸菌５０株について）、硝酸態窒素、亜硝酸態窒素の低減能に優れたものをスクリーニングした。分析には、ＨＰＬＣ硝酸態窒素分析システム（日本分光社製）を用いて行った。
その結果、上記野菜残さのうちのキャベツ由来の培養物から、硝酸態窒素低減能に優れたバチルス属の‘ＮＡＳ１菌株’を選抜した。また、牧草サイレージ由来の菌株から亜硝酸態窒素低減能に優れた乳酸菌の‘ＮＡＳ２菌株’を選抜した。
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the technical scope of this invention is not limited by these.
<Example 1> Separation and identification of NAS 1 and 2 strains (1) Separation of strains From various vegetable residues, or grass and feed crop silage, strains excellent in reducing nitrate nitrogen and nitrite nitrogen in fermented feed Separation.
First, stomacher these samples each 10 g - for plastic bags were taken (wyvern KN208, manufactured by Asahi Kasei Corporation), after a 10-fold dilutions by adding a sterile physiological saline 90 ml, the solution further 10 8 Dilute to double.
Apply these diluted solutions to Lactobacilli MRS agar medium (DIFCO Laboratories, Detroit, USA), normal agar medium (Nissui Pharmaceutical Co., Ltd., Japan) and GYP white agar medium (Ozaki et al., Lactobacillus Experiment Manual, 1992). They were cultured for 2 days at 30 ° C. under anaerobic conditions using a constant temperature culture apparatus, or were cultured at 30 ° C. for 2 days under anaerobic conditions using an anaerobic culture apparatus for lactic acid bacteria.
Strains isolated from the obtained culture (30 strains of Bacillus, 10 strains of yeast, 50 strains of lactic acid bacteria), and those having excellent ability to reduce nitrate nitrogen and nitrite nitrogen were screened. The analysis was performed using an HPLC nitrate nitrogen analysis system (manufactured by JASCO Corporation).
As a result, a “NAS1 strain” belonging to the genus Bacillus excellent in nitrate nitrogen reduction ability was selected from the cabbage-derived culture of the above vegetable residue. In addition, “NAS2 strain” of lactic acid bacteria having excellent ability to reduce nitrite nitrogen was selected from strains derived from grass silage.
（２）菌学的性質
上記の培養物から分離した菌株について、菌学的諸性質や生理的・生化学的性質等を調べた。菌学的性質を表１に示す。そして、これら分離した菌株の顕微鏡写真を、図１（Ａ：ＮＡＳ１菌株、Ｂ：ＮＡＳ２菌株）に示す。
(2) Mycological properties The bacterial strains isolated from the above culture were examined for mycological properties and physiological / biochemical properties. The mycological properties are shown in Table 1. And the micrograph of these isolate | separated strains is shown in FIG. 1 (A: NAS1 strain, B: NAS2 strain).
図１（Ａ）が示すように、ＮＡＳ１菌株は、短桿菌であることが示された。そして表１が示すように、ｐＨ３．５まで生育可能な菌株であり、酸耐性を有することが示された。さらに、高温耐性をもち、芽胞（胞子）を形成する菌株であることが示された。
これらのことから、ＮＡＳ１菌株は、‘良質の発酵飼料の製造に適した性質’（嫌気性、低ｐＨ耐性、耐酸性、高温耐性、胞子形成能）を有するバチルス属の菌株であることが示された。なお、高温耐性、胞子形成能を有することで、発酵熱による温度の上昇等の厳しい環境でも生育可能であることが示された。
As shown in FIG. 1 (A), the NAS1 strain was shown to be a short koji mold. And as Table 1 shows, it was shown that it is a strain which can grow to pH 3.5 and has acid tolerance. Furthermore, it was shown that the strain has high temperature resistance and forms spores (spores).
From these facts, it is shown that NAS1 strain is a strain of the genus Bacillus having 'property suitable for the production of high-quality fermented feed' (anaerobic, low pH resistance, acid resistance, high temperature resistance, sporulation ability). It was done. In addition, it was shown that by having high-temperature tolerance and spore-forming ability, it can grow even in severe environments such as an increase in temperature due to fermentation heat.
また、図１（Ｂ）が示すように、ＮＡＳ２菌株は、ラクトコッカス属（乳酸菌）に属する球菌であることが示された。そして表１が示すように、ｐＨ３．５まで生育可能な菌株であり、酸耐性を有することが示された。さらに、ホモ発酵型乳酸発酵をし、Ｌ型乳酸を生成することが示された。
これらのことから、ＮＡＳ２菌株は、‘良質の発酵飼料の製造に適した性質’（嫌気性、低ｐＨ耐性、耐酸性、優れた乳酸生成能）を有する、ラクトコッカス属（乳酸菌）の菌株であることが示された。なお、優れた乳酸生成能として、効率の良いホモ発酵型乳酸発酵能、代謝性の高いＬ型乳酸生成能を有することが示された。
また、ＮＡＳ２菌株は、発酵飼料の発酵過程における好気性細菌や大腸菌群などの有害微生物の増殖を抑制する乳酸菌株であることも示された。
Moreover, as FIG. 1 (B) shows, it was shown that NAS2 strain is a cocci belonging to the genus Lactococcus (lactic acid bacterium). And as Table 1 shows, it was shown that it is a strain which can grow to pH 3.5 and has acid tolerance. Furthermore, it was shown that homo-fermentative lactic acid fermentation was performed to produce L-type lactic acid.
From these facts, the NAS2 strain is a strain of the genus Lactococcus (lactic acid bacterium) having 'property suitable for the production of good quality fermented feed' (anaerobic, low pH tolerance, acid resistance, excellent lactic acid producing ability). It was shown that there is. In addition, it was shown that it has an efficient homo-fermentation type | mold lactic-acid fermentation ability and L-type lactic acid production ability with high metabolism as the outstanding lactic acid production ability.
It was also shown that the NAS2 strain is a lactic acid strain that suppresses the growth of harmful microorganisms such as aerobic bacteria and coliforms in the fermentation process of fermented feed.
（３）硝酸態窒素および亜硝酸態窒素の低減能
次いで、ＮＡＳ１菌株およびＮＡＳ２菌株における、硝酸態窒素と亜硝酸態窒素の低減能を詳細に調べた。
まず、硝酸カリウムを０．２％、もしくは、亜硝酸カリウムを０．２％、を添加した各液体培地（ＮＡ，ＭＲＳ，ＹＭ培地）を調製した。
各種菌株として、ＮＡＳ１菌株、ＮＡＳ２菌株、酵母（ＣＯ１１９）、乳酸菌（畜草１号）、バチルス（ＬＱ１３）を植菌し、３０日間培養した。
その後、ＨＰＬＣ硝酸態窒素分析システム（日本分光社製）を用いて、培地における硝酸態窒素の濃度と亜硝酸態窒素の濃度を測定し、培養前と後でのこれらの低減率を算出した。結果を図２に示す。
(3) Reduction ability of nitrate nitrogen and nitrite nitrogen Next, the reduction ability of nitrate nitrogen and nitrite nitrogen in NAS1 strain and NAS2 strain was examined in detail.
First, each liquid medium (NA, MRS, YM medium) supplemented with 0.2% potassium nitrate or 0.2% potassium nitrite was prepared.
As various strains, NAS1 strain, NAS2 strain, yeast (CO119), lactic acid bacteria (livestock No. 1), and Bacillus (LQ13) were inoculated and cultured for 30 days.
Thereafter, using a HPLC nitrate nitrogen analysis system (manufactured by JASCO Corporation), the concentration of nitrate nitrogen and the concentration of nitrite nitrogen in the medium were measured, and the reduction rates before and after the culture were calculated. The results are shown in FIG.
その結果、図２（左）が示すように、ＮＡＳ１菌株を、硝酸カリウム０．２％を添加した培地で３０日間培養することによって、ほぼ１００％の硝酸態窒素を培地中から低減（除去）できることが示された（なお、培養途中である１週間後の低減率は約５０％であり、１０日間の培養では約９０％であった）。
一方、ＮＡＳ１菌株と同じバチルス属である「ＬＱ１３」でも、約１８％しか低減することができず、ＮＡＳ２菌株、乳酸菌、酵母では１０％以下であった。
このことから、「ＮＡＳ１菌株」は、‘極めて優れた硝酸態窒素低減能’を有するものであることが示された。
As a result, as shown in FIG. 2 (left), almost 100% nitrate nitrogen can be reduced (removed) from the medium by culturing NAS1 strain in a medium supplemented with 0.2% potassium nitrate for 30 days. (The reduction rate after one week during the culture was about 50%, and about 90% for the 10-day culture).
On the other hand, “LQ13”, which is the same genus Bacillus as the NAS1 strain, could reduce only about 18%, and it was 10% or less for the NAS2 strain, lactic acid bacteria, and yeast.
From this, it was shown that the “NAS1 strain” has “very excellent nitrate nitrogen reducing ability”.
また、図２（右）が示すように、ＮＡＳ２菌株を、亜硝酸カリウム０．２％を添加した培地で３０日間培養することによって、６２％の亜硝酸態窒素を培地中から低減できることが示された。
一方、ＮＡＳ２菌株と同じ乳酸菌である「畜草１号」でも、約２０％しか低減することができず、ＮＡＳ１菌株、バチルス、酵母では５％以下であった。
このことから、「ＮＡＳ２菌株」は、‘極めて優れた亜硝酸態窒素低減能’を有するものであることが示された。
Further, as shown in FIG. 2 (right), it is shown that 62% of nitrite nitrogen can be reduced from the medium by culturing NAS2 strain in a medium supplemented with 0.2% potassium nitrite for 30 days. It was.
On the other hand, “Livestock No. 1”, which is the same lactic acid bacterium as the NAS2 strain, could only reduce about 20%, and the NAS1 strain, Bacillus, and yeast had 5% or less.
From this, it was shown that the “NAS2 strain” has “excellent ability to reduce nitrite nitrogen”.
（４）分子系統解析
選抜したＮＡＳ１菌株、ＮＡＳ２菌株について、１６ＳｒＲＮＡ遺伝子の全領域塩基配列を決定して分子系統解析を行った。なお、決定した１６ＳｒＤＮＡの塩基配列を配列表（配列番号１：ＮＡＳ１菌株、配列番号２：ＮＡＳ２菌株）に示す。
(4) Molecular phylogenetic analysis The selected NAS1 strain and NAS2 strain were subjected to molecular phylogenetic analysis by determining the entire region base sequence of 16S rRNA gene. The determined base sequence of 16S rDNA is shown in the sequence listing (SEQ ID NO: 1: NAS1 strain, SEQ ID NO: 2: NAS2 strain).
分子系統解析の結果、ＮＡＳ１菌株の分子系統位置はBacillus属のクラスターにあり、Bacillus subtilisとの基準株と最も近縁な系統関係を示した。さらにＤＮＡ−ＤＮＡ相同性試験の結果でも、Bacillus subtilisと同定された。
また、ＮＡＳ２菌株の分子系統位置はLactococcus属のクラスターにあり、Lactococcus lactisとの基準株と最も近縁な系統関係を示した。さらにＤＮＡ−ＤＮＡ相同性試験の結果でも、Lactococcus lactisと同定された。
これらの菌株は、基準株と最も近縁な系統関係にあるが、糖類発酵特性等において基準株と異なることから、本発明者らは新規菌株種であると判定した。そして、それぞれを「バチルス・サブティリス（Bacillus subtilis） ＮＡＳ１」、「バチルス・サブティリス（Lactococcus lactis） ＮＡＳ２」と命名した。
なお、これら菌株は、独立行政法人 製品評価技術基盤機構 特許生物寄託センターに寄託されており、その受託番号はバチルス・サブティリス（Bacillus subtilis）ＮＡＳ１株が「NITE P-753」であり、ラクトコカス・ラクティス（Lactococcus lactis）ＮＡＳ２株が「NITE P-754」である。
As a result of molecular phylogenetic analysis, the position of the molecular strain of NAS1 strain was in the cluster of the genus Bacillus, and the phylogenetic relationship closest to the reference strain with Bacillus subtilis was shown. Furthermore, it was identified as Bacillus subtilis as a result of DNA-DNA homology test.
Moreover, the position of the molecular strain of the NAS2 strain was in a cluster of the genus Lactococcus, indicating a phylogenetic relationship closest to the reference strain with Lactococcus lactis. Furthermore, it was identified as Lactococcus lactis as a result of the DNA-DNA homology test.
Although these strains have the closest relationship with the reference strain, they differ from the reference strain in saccharide fermentation characteristics and the like, so the present inventors have determined that they are new strain species. And each was named "Bacillus subtilis (Bacillus subtilis) NAS1" and "Bacillus subtilis (Lactococcus lactis) NAS2".
These strains are deposited with the Patent Organism Depositary of the National Institute of Technology and Evaluation, the deposit number is “NITE P-753” for the Bacillus subtilis NAS1 strain, Lactococcus lactis NAS2 strain is “NITE P-754”.
＜実施例２＞ 野菜残さ発酵飼料の製造と発酵品質
野菜圃場や野菜工場で排出された野菜残さ（キャベツ）を用いて、小規模発酵試験法によって、発酵飼料を調製した。
まず、野菜残さを切断し、表２に記載の各微生物を液体培地で培養し、材料１ｋｇあたり培養液１ｍｌ（各菌１０６菌数レベル）を添加した後、サイロに詰め込んで密封し、温度２０〜３０℃、６０日間貯蔵してサイレージ発酵して得た発酵飼料の品質を分析した。
また、得られた各発酵飼料の発酵品質（ｐＨ、各種有機酸含量、アンモニア態窒素含量）について調べた。結果を表２に示す。
また、各発酵飼料中の発酵硝酸態窒素含量についてＨＰＬＣ硝酸態窒素分析システム（日本分光社製）を用いて測定した。結果を図３に示す。
<Example 2> Production and fermentation quality of vegetable residue fermented feed A fermented feed was prepared by a small-scale fermentation test method using vegetable residue (cabbage) discharged in a vegetable field or vegetable factory.
First, vegetable residue by cutting, each microorganism listed in Table 2 was cultured in a liquid medium, after addition of per material 1kg culture 1 ml (each bacteria 10 6 cell count levels), sealed packed in silos, temperature The quality of the fermented feed obtained by storage at 20-30 ° C. for 60 days and silage fermentation was analyzed.
Further, the fermentation quality (pH, various organic acid contents, ammonia nitrogen content) of each fermented feed obtained was examined. The results are shown in Table 2.
Moreover, the fermented nitrate nitrogen content in each fermented feed was measured using an HPLC nitrate nitrogen analysis system (manufactured by JASCO Corporation). The results are shown in FIG.
その結果、図３が示すように、ＮＡＳ１菌株の添加、ＮＡＳ１菌株とＮＡＳ２菌株の混合添加により、野菜残さ発酵飼料中の硝酸態窒素が顕著に低減されることが示された。
また、表２が示すように、ＮＡＳ１菌株とＮＡＳ２菌株の混合添加により調製した発酵飼料は、ｐＨが低く、乳酸が多く生産され、アンモニア態窒素含量が少ない、良質の発酵飼料であることが示された。
なお、微生物無添加（対照）や他の菌株の添加に比べ、ＮＡＳ２菌株、ＮＡＳ１菌株とＮＡＳ２菌株の混合添加した発酵飼料では、発酵飼料中の大腸菌数が減少し、乳酸菌数が増加した。即ち、ＮＡＳ２菌株は、発酵飼料の発酵過程における好気性細菌や大腸菌群などの有害微生物の増殖を抑制する乳酸菌株であることが分った。
As a result, as shown in FIG. 3, it was shown that nitrate nitrogen in the vegetable residue fermented feed was remarkably reduced by the addition of the NAS1 strain and the mixed addition of the NAS1 strain and the NAS2 strain.
Moreover, as Table 2 shows, the fermented feed prepared by the mixed addition of the NAS1 strain and the NAS2 strain is a high-quality fermented feed having a low pH, a large amount of lactic acid, and a low ammonia nitrogen content. It was done.
In addition, compared with the addition of no microorganism (control) and the addition of other strains, the number of E. coli in the fermented feed decreased and the number of lactic acid bacteria increased in the fermented feed in which NAS2 strain, NAS1 strain and NAS2 strain were mixed and added. That is, it was found that the NAS2 strain is a lactic acid strain that suppresses the growth of harmful microorganisms such as aerobic bacteria and coliform bacteria in the fermentation process of fermented feed.
＜実施例３＞ 牧草サイレージの製造
牧草（イタリアンライグラス）を用いて、ロールベールサイレージを調製した。
まず、牧草を切断し、実施例２の方法と同様にして上記ＮＡＳ１菌株とＮＡＳ２菌株を混合添加した後、ラップフィルムでラッピングして野外で６０日間貯蔵した。
得られた牧草サイレージは、硝酸態窒素が顕著に低減され、良質の発酵飼料であった。
<Example 3> Production of grass silage Roll bale silage was prepared using grass (Italian ryegrass).
First, the grass was cut and the NAS1 strain and the NAS2 strain were mixed and added in the same manner as in Example 2, then wrapped with a wrap film and stored in the field for 60 days.
The obtained grass silage was a high-quality fermented feed with significantly reduced nitrate nitrogen.
＜実施例４＞ ＴＭＲ発酵飼料の製造
ＴＭＲ発酵飼料原料である、牧草、作物・食品残さ、濃厚飼料を混合したものを用いて、実施例２の方法と同様にして上記ＮＡＳ１菌株とＮＡＳ２菌株を混合添加した後、フレコンバック法で発酵ＴＭＲ飼料を調製した。
得られたＴＭＲ発酵飼料は、硝酸態窒素が顕著に低減され、良質の発酵飼料であった。
<Example 4> Manufacture of TMR fermented feed The above-mentioned NAS1 strain and NAS2 strain were obtained in the same manner as in Example 2 using a mixture of grass, crops, food residue, and concentrated feed, which are TMR fermented feed materials. After mixed addition, fermented TMR feed was prepared by the flexible container back method.
The obtained TMR fermented feed was a high-quality fermented feed with significantly reduced nitrate nitrogen.
本発明は、食品残さ発酵飼料、サイレージ、ＴＭＲ発酵飼料の硝酸態窒素低減方法として、畜産分野において広範な利用が期待される。また、環境にやさしく、安全性にすぐれている。
また、新規サイレージ調製（製造）用添加物として、飼料メーカーへの技術移転が期待される。
The present invention is expected to be widely used in the field of livestock as a method for reducing nitrate nitrogen in food residue fermented feed, silage, and TMR fermented feed. It is also environmentally friendly and safe.
In addition, technology transfer to feed manufacturers is expected as an additive for new silage preparation (manufacturing).
ＮＩＴＥ Ｐ−７５３
ＮＩＴＥ Ｐ−７５４
NITE P-753
NITE P-754
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