JP2017118868A - Novel lactic acid bacteria for feed addition - Google Patents
Novel lactic acid bacteria for feed addition Download PDFInfo
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Description
本発明は、新規乳酸菌株、それを含有する飼料またはサイレージ、飼料調製用添加剤またはサイレージ調製用添加剤および当該添加剤を用いるサイレージの調製方法に関する。 The present invention relates to a novel lactic acid strain, a feed or silage containing it, an additive for feed preparation or an additive for silage preparation, and a silage preparation method using the additive.
我が国の畜産は輸入飼料への依存度が高く、不安定な国際飼料相場や市場供給量に影響を受けやすいことから、畜産物を安定供給するために、飼料自給率の向上が大きな課題となっている。そこで、飼料自給率向上と遊休水田の有効活用の両者を見据え、トウモロコシを中心とした輸入飼料の代替としてイネ発酵粗飼料や飼料用米サイレージの利用拡大への取り組みが積極的に推進されている。具体的には、飼料用イネ専用品種において、高糖含量・高消化性・極晩生を特徴とする「たちすずか」等の新たな品種の普及が急速に進んでおり、飼料用イネの収穫適期も多様化している。また、国産自給飼料や地域食品副産物を活用した完全混合飼料（Ｔｏｔａｌ Ｍｉｘｅｄ Ｒａｔｉｏｎ、以下「ＴＭＲ」という。）の利用促進が図られており、寒冷地でも通年にわたり高品質な発酵飼料を安定して貯蔵できる技術が求められている。さらに、これらの通年安定貯蔵技術は、国外の飼料生産現場でも要望されている。 Japan's livestock production is highly dependent on imported feed, and is susceptible to unstable international feed prices and market supply, so raising feed self-sufficiency is a major issue in order to stably supply livestock products. ing. In view of both the feed self-sufficiency ratio and the effective use of idle paddy fields, efforts to expand the use of rice fermented roughage and rice silage for feed as an alternative to imported feed, mainly corn, are being actively promoted. Specifically, new varieties such as Tachi Suzuka, which is characterized by high sugar content, high digestibility, and extremely late growth, are rapidly spreading in feed-only rice varieties. Are also diversifying. In addition, the use of completely mixed feed ("TMR") using domestic self-contained feed and local food by-products is promoted, and stable high-quality fermented feed can be maintained throughout the year even in cold regions. There is a need for technology that can be stored. In addition, these year-round stable storage technologies are also demanded at overseas feed production sites.
サイレージは、乳酸菌の力を巧妙に利用して調製される家畜の貯蔵飼料である。乳酸菌はその発酵品質のみならず、栄養価値や反芻家畜の生理代謝に影響を及ぼす決め手となるが、サイレージ材料の草に共生する酪酸菌、好気性細菌、糸状菌および酵母等の微生物は、乳酸菌の発酵を競合的に阻害し、サイレージの品質の劣化や栄養損失を招く原因となる。寒冷期や寒冷・高標地域において調製されたサイレージは、乳酸発酵が緩慢で低乳酸高ｐＨ型となる場合が多く、このようなサイレージや発酵ＴＭＲは、大腸菌群およびカビ等の有害微生物が高レベルで生息し、カビ毒等の有害物質による家畜の生産性低下や、衛生管理面から解決すべき重要な課題となっている。
牧草を円柱上に成形しラップしてサイレージ化する手段であるロールベールは、その開封後、フレッシュＴＭＲや発酵ＴＭＲとして二次利用することや、小規模農家において、開封後に一定期間好気条件下で保管せざるを得ない場合も多いことから、開封後の二次発酵による変敗防止の技術への要望が高い。特に、発酵後にも比較的に高い糖含量を有する飼料用イネ「たちすずか」については、極晩生の収穫適期であることから、冬季貯蔵において発酵が緩慢となる場合が知られており、カビ発生や二次発酵の原因微生物の１つと考えられる酵母の増殖が顕著となる問題が生じている。
Silage is a livestock storage feed prepared by skillfully utilizing the power of lactic acid bacteria. Lactic acid bacteria are decisive factors that affect not only their fermentation quality but also nutritional value and physiological metabolism of ruminant livestock. Microorganisms such as butyric acid bacteria, aerobic bacteria, filamentous fungi and yeasts that coexist with the grass of silage materials are lactic acid bacteria. It competitively inhibits the fermentation of the silage, causing silage quality degradation and nutrient loss. Silage prepared in the cold season or in cold / high altitude areas often has a slow lactic acid fermentation and becomes a low lactic acid / high pH type, and such silage and fermentation TMR have a high level of harmful microorganisms such as coliforms and fungi. It has become an important issue to be solved from the aspect of sanitary management and the decline in livestock productivity due to harmful substances such as mold poisons.
Roll bale, which is a means to form silage on a cylinder and wrap it into silage, can be used as fresh TMR or fermentation TMR after opening, or in small-scale farmers for a certain period of time after opening. In many cases, there is a strong demand for technology for preventing deterioration by secondary fermentation after opening. In particular, the rice for feed “Tachisuka”, which has a relatively high sugar content even after fermentation, is known to be mildly fermented during winter storage because it is a suitable harvest time for extremely late harvesting. In addition, there has been a problem that the growth of yeast, which is considered to be one of the causative microorganisms of secondary fermentation, becomes remarkable.
従来、これら課題に対する報告がいくつかなされている。具体的には、特許文献１には、イネ発酵粗飼料の発酵促進に貢献するラクトバチルス・プランタルム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）畜草１号株（ＦＥＲＭ Ｐ−１８９３０）が、特許文献２には、ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）（ＮＣＩＭＢ−４０７８８）の、畜産飼料の二次発酵抑制効果が、さらに、非特許文献１には、ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）のある供試菌株が、イネ発酵飼料におけるアルコール産生を抑制することが報告されている。しかしながら、これらは必ずしも満足できるものではなく、低温条件下での有効性については全く言及されていない。
特に、サイレージに従来使用されてきた乳酸菌ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）において、低温増殖性能を有する菌株については、今まで全く報告例がなかった。
Several reports on these issues have been made. Specifically, Patent Document 1 discloses Lactobacillus plantarum No. 1 strain (FERM P-18930) that contributes to the promotion of fermentation of rice fermentation roughage, and Patent Document 2 discloses Lactobacillus plantarum. The secondary fermentation inhibitory effect of livestock feed of Lactobacillus buchneri (NCIMB-40788) is further described in Non-Patent Document 1, in which a test strain having Lactobacillus buchneri in Lactobacillus buchneri It has been reported to suppress alcohol production. However, these are not always satisfactory and no mention is made of their effectiveness under low temperature conditions.
In particular, in the lactic acid bacterium Lactobacillus buchneri that has been conventionally used for silage, there has been no report on a strain having a low temperature growth performance.
ラクトバチルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属乳酸菌の一般的な至適生育温度は３０〜４０℃であり、一部の菌種の中の菌株で４℃等の生育例が示されているものの、一般的特徴とはいいがたい。また、家畜向け発酵飼料調製に活用する乳酸菌において、低温増殖能があることと、低温環境で十分な乳酸等の有機酸生成能をもつことは同一性質ではなく、容易に飼料調製分野に応用できるかを判別することはできない。さらに、低温環境条件に着目をして、低温増殖性乳酸菌種をイネ発酵粗飼料等のサイレージ調製に応用した報告は未だなされていない。
そこで、本発明は、低温（概略５℃程度）において増殖性に優れた乳酸菌、当該乳酸菌を含有するサイレージ等の飼料やその調製用添加剤および、当該添加剤を添加するサイレージの調製方法を提供することを課題としている。
The general optimum growth temperature of Lactobacillus genus lactic acid bacteria is 30-40 ° C. Although some examples of growth are shown in 4 strains of some bacterial species, Is not good. In addition, lactic acid bacteria used for the preparation of fermented feed for livestock have the ability to grow at low temperatures and have sufficient ability to produce organic acids such as lactic acid in a low temperature environment, and can be easily applied to the field of feed preparation. Cannot be determined. Furthermore, no report has been made on the application of the low-temperature proliferating lactic acid bacteria species to silage preparation such as rice fermented roughage, paying attention to low-temperature environmental conditions.
Therefore, the present invention provides a lactic acid bacterium excellent in growth at a low temperature (approximately 5 ° C.), a feed such as silage containing the lactic acid bacterium, an additive for its preparation, and a method for preparing a silage to which the additive is added. The challenge is to do.
本発明者は上記課題を解決するために鋭意研究を重ねた結果、飼料調製用優良乳酸菌のスクリーニングの中から、低温増殖能に優れた乳酸菌株を見出し、上記課題を解決するに至ったものである。 As a result of intensive studies to solve the above problems, the present inventor found a lactic acid strain excellent in low-temperature growth ability from screening excellent lactic acid bacteria for feed preparation, and has led to the solution of the above problems. is there.
本発明は、具体的には次の事項を要旨とする。
１．ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）ＩＷＴ１９２株（ＮＩＴＥ Ｐ−０２１６８）。
２．１．記載の乳酸菌を含有する飼料。
３．１．記載の乳酸菌を含有するサイレージ。
４．１．記載の乳酸菌を含有する飼料調製用添加剤。
５．１．記載の乳酸菌を含有するサイレージ調製用添加剤。
６．５．記載のサイレージ調製用添加剤をサイレージ材料に添加することを特徴とするサイレージの調製方法。
７．６．記載のサイレージ材料が飼料用イネであるサイレージの調製方法。
The gist of the present invention is specifically as follows.
1. Lactobacillus buchneri IWT192 strain (NITE P-02168).
2.1. Feed containing the lactic acid bacteria described.
3.1. Silage containing the lactic acid bacteria described.
4.1. Additives for feed preparation containing the lactic acid bacteria described.
5.1. Silage preparation additive containing the lactic acid bacteria described.
6.5. A silage preparation method comprising adding the additive for silage preparation described above to a silage material.
7.6. A method for preparing silage, wherein the described silage material is rice for feed.
本発明の乳酸菌ＩＷＴ１９２株は、４℃環境下において分離・選抜された特徴ある菌株であり、低温増殖能に優れる。これにより、低温環境下においても乳酸や酢酸等の有機酸産生増加による発酵促進効果が得られ、良質な畜産飼料を生産することが可能となる。また、飼料栄養価の品質が高いので、牛等の家畜の摂取量が安定化し健康を維持することができる。さらに、サイレージを開封し好気下においた時の二次発酵や、飼料として供給後に生じるＴＭＲの二次発酵等、飼料の二次発酵やカビの発生も抑制できるので、家畜の飼養ならびに衛生管理に大きく寄与するという新たな効果も得られる。 The lactic acid bacterium IWT192 strain of the present invention is a characteristic strain isolated and selected in a 4 ° C. environment, and is excellent in low-temperature growth ability. Thereby, even in a low-temperature environment, a fermentation promoting effect can be obtained by increasing the production of organic acids such as lactic acid and acetic acid, and it becomes possible to produce a high quality livestock feed. Moreover, since the quality of feed nutrition value is high, the intake of livestock such as cattle can be stabilized and health can be maintained. In addition, secondary fermentation of the feed and mold generation, such as secondary fermentation when the silage is opened and kept in an aerobic condition, and secondary fermentation of TMR that occurs after feeding as feed, can be suppressed. There is also a new effect that contributes greatly.
本発明は、優れた低温増殖能を有し、かつ、良質な発酵飼料の製造に適した性質を有する新規のラクトバチルス属の菌株に関する。
また本発明は、前記菌株を含有してなる飼料調製用添加剤、前記菌株を添加することを特徴とするサイレージ等の飼料やその調製方法に関する。
以下、本発明について詳細に説明する。
（新規な乳酸菌の菌株）
本発明における新規の乳酸菌の菌株とは、優れた低温増殖能を有し、かつ、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。この乳酸菌ＩＷＴ１９２株は、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に寄託されており、その受託番号は、ＮＩＴＥ Ｐ−０２１６８である。
The present invention relates to a novel strain of the genus Lactobacillus having excellent cold-growing ability and properties suitable for producing a high-quality fermented feed.
The present invention also relates to a feed preparation additive comprising the strain, a silage feed characterized by adding the strain, and a method for preparing the same.
Hereinafter, the present invention will be described in detail.
(New strain of lactic acid bacteria)
The novel strain of lactic acid bacteria in the present invention has an excellent low-temperature growth ability and a property suitable for producing a high-quality fermented feed. This lactic acid bacterium IWT192 strain has been deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology, an independent administrative agency, and its deposit number is NITE P-02168.
乳酸菌ＩＷＴ１９２株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、優れた低温増殖能を挙げることができる。ここで、優れた低温増殖能とは、酸産生能と生残性であり、乳酸や有機酸を多く含む良質な発酵飼料を、低温環境下で製造する上で必須な性質である。なお、低温とは概略５℃程度を意味する。また、優れた酸産生能としては、効率の良い乳酸発酵能、代謝性の高いＬ型乳酸生成能を有するものである。さらに、乳酸菌ＩＷＴ１９２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等の有害微生物の増殖を抑制するものである。
本発明における乳酸菌ＩＷＴ１９２株は、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等の分離源の希釈液を寒天培地に塗布し、概略４℃、嫌気条件で菌株を分離培養した後、低温増殖能や生理生化学的性質等を調べることで、選抜したものである。
Examples of the bacteriological properties of the lactic acid bacterium IWT192 strain include “properties suitable for production of a high-quality fermented feed”, and specifically, excellent low-temperature growth ability. Here, the excellent low-temperature growth ability is acid production ability and survival, and is an essential property for producing a high-quality fermented feed containing a large amount of lactic acid and organic acid in a low-temperature environment. In addition, low temperature means about 5 degreeC. Further, as an excellent acid producing ability, it has an efficient lactic acid fermentation ability and an L-type lactic acid producing ability with high metabolism. Furthermore, lactic acid bacteria IWT192 strain | stump | stock suppresses the proliferation of harmful microorganisms, such as aerobic bacteria and coliform bacteria, in the fermentation process of fermented feed.
The lactic acid bacterium IWT192 strain in the present invention is applied to agar medium by applying a dilution source of a fermented feed raw material or fermented feed, specifically, a grass or feed crop silage or the like to an agar medium, and the strain is separated at approximately 4 ° C. under anaerobic conditions. After culturing, the cells were selected by examining their ability to grow at low temperatures, physiological biochemical properties, and the like.
（発酵飼料およびその調製方法）
本発明のサイレージ等の発酵飼料は、この乳酸菌ＩＷＴ１９２株を含有するものであり、そのサイレージ等の発酵飼料の調製方法について説明する。
本発明では、サイレージ等の発酵飼料を製造する際に、発酵飼料用原料に乳酸菌ＩＷＴ１９２株を添加することによって、低温環境下においても高品質の発酵飼料を得ることができる。
本発明においては、乳酸菌ＩＷＴ１９２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、均一になるように行うことが望ましい。例えば、乳酸菌ＩＷＴ１９２株を水に懸濁し噴霧する方法、混合撹拌する方法等で行うことができる。添加する乳酸菌ＩＷＴ１９２株の量としては、原料１ｋｇに対して１０４〜１０７菌数レベルで添加することが望ましい。具体的には１０５〜１０６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。
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、嫌気条件で、外気温において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、フレコンバック法等で行うことができる。
本発明に用いることができる発酵飼料原料としては、サイレージ原料である牧草や飼料作物（例えば、イネ、イタリアンライグラス、スーダングラス、オーチャード、アルファルファ、ソルゴー、コメ、トウモロコシ、ソルガム等）、発酵ＴＭＲ飼料原料（例えば、濃厚飼料、乾草、ビートパルプ、ビタミン・ミネラル製剤、作物副産物、食品副産物等を混合した原料）等を挙げることができる。飼料用イネとしては、高糖分・高消化性能を有する「たちすずか」、「たちあやか」、「つきすずか」等を好適に使用することができる。
乳酸菌ＩＷＴ１９２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、低ｐＨであり、乳酸含量が多い良質の発酵飼料であり、発酵飼料の発酵過程における大腸菌群、酵母及びカビ等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、サイレージ、ＴＭＲ発酵飼料等を挙げることができる。
(Fermented feed and its preparation method)
The fermented feed such as silage of the present invention contains the lactic acid bacteria IWT192 strain, and a method for preparing the fermented feed such as silage will be described.
In the present invention, when producing a fermented feed such as silage, a high-quality fermented feed can be obtained even in a low temperature environment by adding the lactic acid bacteria IWT192 strain to the fermented feed raw material.
In the present invention, it is essential to add the lactic acid bacterium IWT192 strain to the fermented feed material. As a method of addition, it is desirable to carry out the addition so as not to be biased in the raw material. For example, lactic acid bacteria IWT192 strain can be suspended in water and sprayed, mixed and stirred, or the like. As the amount of lactic acid bacteria IWT192 strain added, it is desirable to add at a level of 10 4 to 10 7 bacteria per 1 kg of the raw material. Specifically, it is desirable to add at a level of 10 5 to 10 6 bacteria. 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.
As said fermentation, it can carry out on the conditions similar to normal silage fermentation, and it is anaerobic conditions and is performed in 30 days or more in external temperature. Specifically, it can be performed by a normal method using a silo, a roll bale silage method, a flexible container back method, or the like.
Examples of fermented feed materials that can be used in the present invention include silage materials such as grass and feed crops (eg, rice, Italian ryegrass, Sudan grass, orchard, alfalfa, solgo, rice, corn, sorghum, etc.), fermented TMR feed materials (For example, raw materials in which concentrated feed, hay, beet pulp, vitamin / mineral preparations, crop by-products, food by-products, etc.) are mixed. As feed rice, “Tachi Suzuka”, “Tatsu Ayaka”, “Tsuku Suzuka” and the like having high sugar content and high digestibility can be preferably used.
Fermented feed obtained by silage fermentation to which lactic acid bacteria IWT192 strain is added is a high-quality fermented feed having a low pH and a high lactic acid content, and growth of harmful microorganisms such as coliforms, yeasts and molds in the fermentation process of the fermented feed It has been suppressed. Specific examples of the fermented feed produced include silage and TMR fermented feed.
（発酵飼料調製用添加剤）
本発明においては、乳酸菌ＩＷＴ１９２株を含有してなるサイレージ等の発酵飼料調製用添加剤の形態にして提供することができる。発酵飼料調製用添加剤の形態としては、乳酸菌ＩＷＴ１９２株を凍結乾燥状態にして粉末状の形態、賦形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等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。
発酵飼料調製用添加剤の使用形態としては、直接発酵飼料の原料に添加することもできるが、水等に溶解したものを用いることが望ましい。
また、本発明の発酵飼料調製用添加剤の使用量としては、原料１ｋｇに対して、乳酸菌ＩＷＴ１９２株を１０４〜１０７菌数レベルで添加することが望ましい。具体的には、乳酸菌ＩＷＴ１９２株を１０５〜１０６菌数レベルで添加することが望ましい。
(Additive for fermented feed preparation)
In the present invention, it can be provided in the form of an additive for preparing fermented feed such as silage containing lactic acid bacteria IWT192 strain. Examples of the additive for preparing fermented feed include a powdered form obtained by freeze-drying lactic acid bacteria IWT192 strain, a solid form mixed with excipients, a capsule filling form, a liquid ampoule form, etc. be able to. Preferably, the form of a lyophilized formulation is suitable.
As a form of use of the additive for preparing fermented feed, it can be added directly to the raw material of fermented feed, but it is desirable to use a material dissolved in water or the like.
Moreover, as the usage-amount of the additive for fermented feed preparation of this invention, it is desirable to add the lactic acid bacteria IWT192 strain | stump | stock at the level of 10 < 4 > -10 < 7 > bacteria count with respect to 1 kg of raw materials. Specifically, it is desirable to add lactic acid bacteria IWT192 strain at a level of 10 5 to 10 6 bacteria.
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＞乳酸菌ＩＷＴ１９２株の分離と同定
（１）菌株の分離
完全混合飼料（ＴＭＲ）サンプル１０ｇを滅菌蒸留水１００ｍＬに懸濁後、ホモジナイザ（Pro・mediaSH-2M、株式会社エルメックス製）で1分間均質化処理し、ＴＭＲサイレージ抽出液を得た。本抽出液を乳酸桿菌向けＭＲＳ培地（BD、Difco、Lactobacilli MRS Broth）に塗布し、アネロパック・ケンキ（三菱ガス化学株式会社製）による嫌気条件下において４℃で１７日間培養し、形成した単一コロニーを菌株として分離した。得られたコロニーを３回培養純化した。また、各コロニーから得た２７菌株をバージーズマニュアル（Bergey's Manual）に従って同定したところ、ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）は２７菌株中２株であった。得られた２７菌株を、ＭＲＳ（de Man、Rogosa、Sharpe）液体培地において４℃で７日間培養し、培養液の６００ｎｍの吸光度やｐＨを測定することにより、各菌体の増殖性を評価し、最も増殖性に優れたＩＷＴ１９２株を選抜し、これを−８０℃で保存した。
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> Isolation and identification of lactic acid bacteria IWT192 strain (1) Separation of strain 10 g of a complete mixed feed (TMR) sample was suspended in 100 mL of sterile distilled water, and then homogenizer (Pro · mediaSH-2M, manufactured by Elmex Corporation) For 1 minute to obtain a TMR silage extract. This extract was applied to MRS medium (BD, Difco, Lactobacilli MRS Broth) for Lactobacillus and cultured for 17 days at 4 ° C. under anaerobic conditions with Aneropac Kenki (Mitsubishi Gas Chemical Co., Ltd.). Colonies were isolated as strains. The obtained colonies were purified by culture three times. Moreover, when 27 strains obtained from each colony were identified according to the Bergey's Manual, Lactobacillus buchneri was 2 strains out of 27 strains. The obtained 27 strains were cultured in MRS (de Man, Rogosa, Sharpe) liquid medium at 4 ° C. for 7 days, and the proliferation of each cell was evaluated by measuring the absorbance and pH at 600 nm of the culture. The IWT192 strain having the best growth was selected and stored at -80 ° C.
（２）菌株の性質
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。主要な菌学的性質を、ラクトバチルス・ブクネリの基準株「ＪＣＭ１１１５Ｔ」と併せ表１に示す
(2) Properties of strains The bacterial strains isolated from the above culture were examined for mycological properties and physiological / biochemical properties. The main bacteriological properties are shown in Table 1 together with the reference strain “JCM1115 T ” of Lactobacillus bukuneri.
乳酸菌ＩＷＴ１９２株は、ラクトバチルス・ブクネリの基準株「ＪＣＭ１１１５Ｔ」と16S rRNA遺伝子と相同性が99.9%一致しており、0.1%の16S rRNA遺伝子配列が異なる。
また、乳酸菌ＩＷＴ１９２株は、「実施例２」において詳細に説明するように、基準株「ＪＣＭ１１１５Ｔ」と概略４℃の低温増殖能に大きな違いがある。
さらに、API 50CHキット（シスメックス・ビオメリュー株式会社製）による糖資化性解析において、乳酸菌ＩＷＴ１９２株はラクトバチルス・ブクネリの基準株「ＪＣＭ１１１５Ｔ」と、Methyl-β-D-xylopyranoside、D-Lactose、D-Turanoseの資化性が異なっており、乳酸菌ＩＷＴ１９２株にのみ顕著にこれらの３糖の利用性が認められた（30℃、48時間培養）。
以上のことから、本発明の乳酸菌ＩＷＴ１９２株は新株であると決定した。
Lactic acid bacteria IWT192 strain, the type strain of Lactobacillus Bukuneri as "JCM1115 T" 16S rRNA gene homology coincides 99.9%, 0.1% 16S rRNA gene sequence is different.
In addition, as described in detail in “Example 2”, the lactic acid bacterium IWT192 strain is greatly different from the reference strain “JCM1115 T ” in low temperature growth ability at about 4 ° C.
Furthermore, in the sugar utilization analysis by API 50CH kit (manufactured by Sysmex Biomelieu Co., Ltd.), lactic acid bacteria IWT192 strain is Lactobacillus bucumeri reference strain “JCM1115 T ”, Methyl-β-D-xylopyranoside, D-Lactose, Utilization of these trisaccharides was remarkably observed only in the lactic acid bacteria IWT192 strain (cultured at 30 ° C. for 48 hours).
From the above, it was determined that the lactic acid bacterium IWT192 strain of the present invention is a new strain.
＜実施例２＞乳酸菌ＩＷＴ１９２株の低温増殖能の確認
次いで、乳酸菌ＩＷＴ１９２株の低温増殖能について詳細に検討した。
乳酸菌は次の４つの乳酸菌を使用した。
「ＩＷＴ１９２」：ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）ＩＷＴ１９２株（ＮＩＴＥ Ｐ−０２１６８）
「ＦＧ１」：ラクトバチルス・プランタルム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）畜草１号株（ＦＥＲＭ Ｐ−１８９３０）
「ＪＣＭ１１１５Ｔ」：ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）ＪＣＭ１１１５Ｔ株
「ＳＰ」：ラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）ＮＫ０１株
上記４つの乳酸菌を、１０℃（Ａ）と４℃（Ｂ）の温度条件下、ＭＲＳ（de Man、Rogosa、Sharpe）液体培地において、全ての菌株を１０℃（Ａ）で３日、４日、５日、７日、１０日間、４℃（Ｂ）で７日、９日、１２日、１５日、２１日間培養した。菌体の増殖について、６００ｎｍの吸光度を測定することにより評価し、その結果を図１に示した。
<Example 2> Confirmation of low-temperature growth ability of lactic acid bacteria IWT192 strain Next, the low-temperature growth ability of lactic acid bacteria IWT192 strain was examined in detail.
The following four lactic acid bacteria were used.
"IWT192": Lactobacillus buchneri IWT192 strain (NITE P-02168)
“FG1”: Lactobacillus plantarum No. 1 stock (FERM P-18930)
“JCM1115 T ”: Lactobacillus buchneri JCM1115 T strain “SP”: Lactobacillus buchnerii NK01 strain The above four lactic acid bacteria were subjected to a temperature condition of 10 ° C. (A) and 4 ° C. (B). Below, in MRS (de Man, Rogosa, Sharpe) liquid medium, all strains were treated at 10 ° C. (A) for 3 days, 4 days, 5 days, 7 days, 10 days, 4 ° C. (B) for 7 days, 9 Cultured on days 12, 12 and 15 for 21 days. The growth of the bacterial cells was evaluated by measuring the absorbance at 600 nm, and the results are shown in FIG.
その結果、図１が示すように、ラクトバチルス・ブクネリの基準株であるＪＣＭ１１１５Ｔ株やＳＰ株には、低温（４℃）における増殖能は認められなかった。また、従来サイレージに使用されている、ラクトバチルス・プランタルム（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐｌａｎｔａｒｕｍ）畜草１号株も同様に、低温（４℃）における増殖能は認められなかった。
一方、本発明の新規乳酸菌ＩＷＴ１９２株は、極めて優れた低温増殖能を有するものであることが確認された。
As a result, as shown in FIG. 1, the JCM1115 T strain and the SP strain, which are reference strains of Lactobacillus bukuneri, did not show the ability to grow at a low temperature (4 ° C.). Similarly, Lactobacillus plantarum No. 1 strain conventionally used for silage did not show the ability to grow at low temperature (4 ° C.).
On the other hand, it was confirmed that the novel lactic acid bacterium IWT192 strain of the present invention has extremely excellent cold growth ability.
＜実施例３＞乳酸菌ＩＷＴ１９２株の発酵促進効果の確認
次いで、乳酸菌ＩＷＴ１９２株の発酵促進効果について詳細に検討した。
乳酸菌は「ＩＷＴ１９２株」と「ＦＧ１」（畜草１号株）を使用した。
各乳酸菌株をそれぞれ添加した飼料用イネ「たちすずか」のイネ発酵粗飼料を、パウチ法により５℃（Ａ）あるいは２０℃（Ｂ）の温度条件下で、３０日、６０日、９０日間貯蔵して調製した。貯蔵後の発酵飼料の品質について、イネ発酵粗飼料サンプル１００ｇを蒸留水５００ｍｌに２４時間冷蔵浸漬して得た抽出液を用いて調査した。ｐＨをｐＨメーター（Ｆ−１６、株式会社堀場製作所製）で、乳酸含量（ｇ／Ｋｇ ＦＭ）、酢酸含量（ｇ／Ｋｇ ＦＭ）、エタノール含量（ｇ／Ｋｇ ＦＭ）を高速液体クロマトグラフィー（Ｗａｔｅｒｓ社製）でそれぞれ測定し、その結果を図２に示した。なお、図２中の「ＦＭ」は得られた発酵飼料（乾燥していないもの）を意味する。
高速液体クロマトグラフィーの分析条件は以下のとおりである。
カラム：ＨＰＸ−８７Ｈ（ＢＩＯ ＲＡＤ社製）
カラム温度：４０℃
検出器：示差屈折検出器
移動相：０．０１規定硫酸
送液速度：０．６ｍｌ／分
<Example 3> Confirmation of Fermentation Promoting Effect of Lactic Acid Bacteria IWT192 Strain Next, the fermentation promoting effect of lactic acid bacteria IWT192 strain was examined in detail.
Lactic acid bacteria used were “IWT192 strain” and “FG1” (livestock No. 1 strain).
Rice fermented roughage of feed rice “Tachisuzuka” to which each lactic acid strain is added is stored for 30 days, 60 days, and 90 days under the temperature condition of 5 ° C. (A) or 20 ° C. (B) by the pouch method. Prepared. The quality of the fermented feed after storage was investigated using an extract obtained by immersing 100 g of rice fermented rough feed sample in 500 ml of distilled water for 24 hours. The pH was measured with a pH meter (F-16, manufactured by Horiba, Ltd.), and the lactic acid content (g / Kg FM), acetic acid content (g / Kg FM), and ethanol content (g / Kg FM) were measured by high performance liquid chromatography (Waters). The results are shown in FIG. In addition, “FM” in FIG. 2 means the obtained fermented feed (not dried).
The analysis conditions of high performance liquid chromatography are as follows.
Column: HPX-87H (manufactured by BIO RAD)
Column temperature: 40 ° C
Detector: Differential refraction detector Mobile phase: 0.01 N sulfuric acid Liquid feed rate: 0.6 ml / min
その結果、図２が示すように、畜草１号株の添加により、２０℃貯蔵区では従来技術を反映した発酵促進効果が得られたものの、５℃貯蔵区では２０℃貯蔵区と同じ発酵促進効果は得られなかった。
一方、本発明の新規乳酸菌ＩＷＴ１９２株添加区では、５℃貯蔵条件下においても、乳酸や酢酸等の有機酸産生増加やｐＨの低下が認められ、極めて優れた発酵促進効果を有するものであることが確認された。
特に、５℃貯蔵区における乳酸産生量は、２０℃貯蔵区の畜草１号株と同等の産生量を示し、本発明の乳酸菌ＩＷＴ１９２株が低温条件で優れたサイレージ発酵性を有していることがわかった。
また、両温度貯蔵区において、ＩＷＴ１９２株添加区では、畜草１号株添加区よりもエタノール産生が抑制されていることが明らかとなり、二次発酵に関与する酵母類の増殖を制御し、酵母によるエタノール産生を抑制していることを示唆するものであった。
As a result, as shown in FIG. 2, the fermentation promotion effect reflecting the conventional technology was obtained in the 20 ° C. storage area by adding the No. 1 stock of livestock, but in the 5 ° C. storage area, the same fermentation as the 20 ° C. storage area was obtained. No promoting effect was obtained.
On the other hand, in the addition group of the novel lactic acid bacteria IWT192 strain of the present invention, an increase in production of organic acids such as lactic acid and acetic acid and a decrease in pH are observed even under 5 ° C. storage conditions, and it has a very excellent fermentation promoting effect. Was confirmed.
In particular, the amount of lactic acid produced in the 5 ° C. storage area is equivalent to that of the No. 1 stock in the 20 ° C. storage area, and the lactic acid bacterium IWT192 strain of the present invention has excellent silage fermentability under low temperature conditions. I understood it.
Moreover, in both temperature storage zones, it was revealed that ethanol production was suppressed in the IWT192 strain addition zone compared to the stock No. 1 addition zone, controlling the growth of yeasts involved in secondary fermentation, This suggests that ethanol production is suppressed.
＜実施例４＞乳酸菌ＩＷＴ１９２株を使用した実規模ロールベール実証試験
次いで、実規模ロールベール実証試験について説明する。
乳酸菌は「ＩＷＴ１９２株」と「ＳＰ株」を使用した。
各乳酸菌株をそれぞれ添加した飼料用イネ「たちすずか」のイネ発酵粗飼料および無添加処理したイネ発酵粗飼料をそれぞれ３検体、ロールベールサイレージ法により冬季寒冷期間を含む６ヶ月間貯蔵して、下記のサンプル１とサンプル２をそれぞれ調製した。
「サンプル１」：２０１３年１０月〜２０１４年４月貯蔵調製
「サンプル２」：２０１４年１０月〜２０１５年４月貯蔵調製
開封直後と５日後の発酵飼料の品質については、イネ発酵粗飼料「サンプル１」を１００ｇ使用して、これを蒸留水５００ｍｌに２４時間冷蔵浸漬して得た抽出液を用いて調査した。ｐＨはｐＨメーター（Ｆ−１６、株式会社堀場製作所製）で、乳酸、酢酸、プロピオン酸、酪酸、エタノール、１，２-プロパンジオールの含有量（ｇ／Ｋｇ、未乾燥物）を高速液体クロマトグラフィー（Ｗａｔｅｒｓ社製）で測定した。
また、サイレージに含まれる微生物数については、イネ発酵粗飼料「サンプル２」を使用して測定した。
それぞれ３検体の平均値を表２、３に示した。
また、開封後のイネ発酵粗飼料「サンプル１」の温度変化を、下記式で表される「Δ温度」により表し、図３に示した。
式：Δ温度＝サイレージ温度−環境温度（約２２℃）
なお、有機酸類、エタノール、１，２−プロパンジオールの高速液体クロマトグラフィーによる分析条件は以下のとおりである。
カラム：ＨＰＸ−８７Ｈ（ＢＩＯ ＲＡＤ社製）
カラム温度：４０℃
検出器：示差屈折検出器
移動相：０．０１規定硫酸
送液速度：０．６ｍｌ／分
<Example 4> Real scale roll bale demonstration test using lactic acid bacteria IWT192 strain Next, a real scale roll bale demonstration test will be described.
Lactic acid bacteria used were “IWT192 strain” and “SP strain”.
Rice fermented roughage of feed rice "Tasusuka" to which each lactic acid bacterial strain was added and rice fermented roughage treated without addition were stored for 6 months each including the winter cold period by the roll bale silage method. Sample 1 and sample 2 were prepared, respectively.
“Sample 1”: storage preparation from October 2013 to April 2014 “Sample 2”: storage preparation from October 2014 to April 2015 For the quality of the fermented feed immediately after opening and 5 days later, the rice fermented roughage “sample” 100 g of “1” was used, and this was investigated using an extract obtained by refrigerated immersion in 500 ml of distilled water for 24 hours. The pH was measured with a pH meter (F-16, manufactured by Horiba, Ltd.), and high-performance liquid chromatography was performed on the contents of lactic acid, acetic acid, propionic acid, butyric acid, ethanol, and 1,2-propanediol (g / Kg, undried product). Measured by graphy (manufactured by Waters).
The number of microorganisms contained in the silage was measured using a rice fermentation roughage “Sample 2”.
The average values of the three specimens are shown in Tables 2 and 3, respectively.
Moreover, the temperature change of the rice fermented roughage “sample 1” after opening was expressed by “Δ temperature” expressed by the following formula and shown in FIG.
Formula: Δ temperature = silage temperature−environment temperature (about 22 ° C.)
Analysis conditions for organic acids, ethanol, and 1,2-propanediol by high performance liquid chromatography are as follows.
Column: HPX-87H (manufactured by BIO RAD)
Column temperature: 40 ° C
Detector: Differential refraction detector Mobile phase: 0.01 N sulfuric acid Liquid feed rate: 0.6 ml / min
その結果、表２が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加したロールベールサイレージは、開封直後の乳酸、酢酸、１，２-プロパンジオール含量が無添加区や「ＳＰ株」添加区より有意に高く、エタノール含量が低かったことから、本発明の新規乳酸菌ＩＷＴ１９２株が、極めて優れた発酵促進効果と有害微生物制御効果を有するものであることが確認された。
また、表２及び３が示すように、乳酸菌ＩＷＴ１９２株添加区は、開封０日において、大腸菌群、カビ及び酵母が未検出であった。また、他区と比べて乳酸菌数が多かった。開封５日後でも、低ｐＨと高有機酸含量が維持されており、カビ、酵母、一般好気性細菌、バチルス属細菌及びクロストリジウム属細菌等の微生物の増加を抑制し、その結果、二次発酵やカビ発生が抑制され発酵飼料栄養価の品質維持効果に優れていることも明らかとなった。乳酸菌ＩＷＴ１９２株添加区は、開封０日及び５日後において、プロピオン酸に加えて酪酸も未検出であったことから、検出されたクロストリジウム属細菌は酪酸型発酵による品質悪化レベルには至らない極めて低レベルであると考えられ、十分に良好な品質であると言える。
さらに、乳酸菌ＩＷＴ１９２株添加区は開封直後と開封５日後において、二次発酵の原因微生物とされる酵母数が、無添加区や「ＳＰ株」添加区より顕著に少ないことが確認された。この結果、図３が示すように二次発酵による発熱も認められず、本発明の新規乳酸菌ＩＷＴ１９２株は、二次発酵抑制機能にも優れた発酵飼料生産菌であることが示された。
As a result, as shown in Table 2, the roll bale silage to which the novel lactic acid bacterium IWT192 strain of the present invention was added had no lactic acid, acetic acid, and 1,2-propanediol content added immediately after opening or the “SP strain” added group. Since the ethanol content was significantly higher and the ethanol content was low, it was confirmed that the novel lactic acid bacterium IWT192 strain of the present invention has an extremely excellent fermentation promoting effect and harmful microorganism controlling effect.
In addition, as shown in Tables 2 and 3, in the lactic acid bacteria IWT192 strain addition group, coliform group, mold and yeast were not detected on the opening day 0. In addition, the number of lactic acid bacteria was higher than other areas. Even after 5 days of opening, the low pH and high organic acid content are maintained, and the increase of microorganisms such as mold, yeast, general aerobic bacteria, Bacillus bacteria and Clostridium bacteria is suppressed. It was also revealed that mold generation was suppressed and the quality maintenance effect of fermented feed nutritional value was excellent. In the lactic acid bacteria IWT192 addition group, butyric acid was not detected in addition to propionic acid on the 0th and 5th days after opening, the detected Clostridium genus bacteria was extremely low, not reaching the quality deterioration level due to butyric acid type fermentation. It can be said that the quality is sufficiently good.
Further, it was confirmed that the number of yeasts that are the causative microorganisms of secondary fermentation in the lactic acid bacteria IWT192 added group was significantly smaller immediately after opening and 5 days after opening than in the non-added group and the “SP strain” added group. As a result, as shown in FIG. 3, no fever due to secondary fermentation was observed, indicating that the novel lactic acid bacterium IWT192 strain of the present invention is a fermented feed-producing bacterium having an excellent secondary fermentation inhibiting function.
＜実施例５＞実規模ロールベールにおけるカビ廃棄率低減効果の検証試験
次いで、乳酸菌ＩＷＴ１９２株を使用した実規模ロールベールにおける、カビ廃棄率低減効果の検証試験について説明する。
乳酸菌は「ＩＷＴ１９２株」と「ＳＰ株」を使用した。
各乳酸菌株をそれぞれ添加した飼料用イネ「たちすずか」のイネ発酵粗飼料および無添加処理したイネ発酵粗飼料をそれぞれ３検体、ロールベールサイレージ法により冬季寒冷期間を含む８ヶ月間貯蔵した。開封後、１検体から４５Ｋｇのサンプルを採取し、当該サンプルの目視によるカビ発生確認部位の重量を計測し、カビ廃棄率（％）を算出した。
それぞれ３検体のカビ廃棄率（％）の平均値を表４に示した。
<Example 5> Verification test of fungus disposal rate reduction effect in actual scale roll bale Next, a verification test of fungus disposal rate reduction effect in actual scale roll veil using lactic acid bacteria IWT192 strain will be described.
Lactic acid bacteria used were “IWT192 strain” and “SP strain”.
Three rice fermented roughage of feed rice “Tachisuka” to which each lactic acid strain was added and rice fermented roughage treated without addition were each stored for 8 months by the roll bale silage method including the winter cold period. After opening, a 45 kg sample was taken from one specimen, the weight of the mold confirmed by visual inspection of the sample was measured, and the mold discard rate (%) was calculated.
Table 4 shows the average value of mold waste rate (%) for three specimens.
その結果、表４が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加したロールベールサイレージはカビ廃棄率が０％であり、無添加区や「ＳＰ株」添加区より有意に高く、ＩＷＴ１９２株添加ロールベールに比べてカビ発生による廃棄率が有意に低いことが確認された。これは、本発明の新規乳酸菌ＩＷＴ１９２株は、カビ発生によるロールベールのロス削減効果に優れていることが示された。 As a result, as shown in Table 4, the roll bale silage to which the novel lactic acid bacterium IWT192 strain of the present invention was added had a mold waste rate of 0%, which was significantly higher than the non-addition group and the “SP strain” addition group, and the IWT192 strain It was confirmed that the waste rate due to mold generation was significantly lower than that of the added roll bale. This indicates that the novel lactic acid bacterium IWT192 strain of the present invention is excellent in the effect of reducing roll bale loss due to mold generation.
＜実施例６＞乳酸菌ＩＷＴ１９２株を使用した実規模バンカーサイロ貯蔵の実証試験
次いで、実規模バンカーサイロ貯蔵の実証試験について説明する。
乳酸菌は「ＩＷＴ１９２株」を使用した。
乳酸菌株を添加した飼料用イネ「たちすずか」のイネ発酵粗飼料および無添加処理したイネ発酵粗飼料を、バンカーサイロに１年間貯蔵した。貯蔵後開封直後の発酵飼料の品質について、バンカーサイロの中央上部、中央下部、横サイド上部、横サイド下部の４箇所で採取したサンプルを、実施例４と同様の手法により測定した。平均値を表５に示した。なお、表５中の「1,2-ＰＤ」は１，２-プロパンジオールを、「nd」は未検出を意味する。
また、開封時から約１ヶ月間、毎日幅２０ｃｍ分の発酵飼料を取り出し、バンカーサイロ内の発酵飼料の温度変化を調査した。その結果を図４に示した。
<Example 6> Demonstration test of actual scale bunker silo storage using lactic acid bacterium IWT192 strain Next, an actual test of actual scale bunker silo storage will be described.
As the lactic acid bacteria, “IWT192 strain” was used.
Rice fermented roughage of feed rice “Tachisuzuka” to which a lactic acid strain was added and rice fermented roughage treated without addition were stored in a bunker silo for one year. About the quality of the fermented feed immediately after opening after storage, samples collected at four locations of the central upper part, central lower part, lateral side upper part, and lateral side lower part of the bunker silo were measured in the same manner as in Example 4. The average values are shown in Table 5. In Table 5, “1,2-PD” means 1,2-propanediol, and “nd” means not detected.
Further, for about one month from the time of opening, a fermented feed having a width of 20 cm was taken out every day, and the temperature change of the fermented feed in the bunker silo was investigated. The results are shown in FIG.
その結果、表５が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加したバンカーサイロによる発酵飼料は、開封直後の酢酸、１，２-プロパンジオール含量が無添加区より有意に高く、エタノール含量が低かったことから、本発明の新規乳酸菌ＩＷＴ１９２株は、バンカーサイロにおいても、極めて優れた有害微生物制御効果を奏することが確認された。
また、図４が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加したバンカーサイロにおいては、二次発酵による発熱も認められず、最後まで良好な品質の発酵飼料を取り出すことができた。一方、無添加区のバンカーサイロにおいては、開封後７日目頃から品温が徐々に上昇し、３週間後にはバンカーサイロ内の発酵飼料の温度が約４０℃となり、発酵飼料の品質が著しく低下し、大量に廃棄せざるを得ない状況となった。
バンカーサイロ貯蔵法は、ロールベールサイレージ法に比べると、気密性が低く、開封後の取出し期間が長いため、発酵飼料の品質が低下しやすい貯蔵方法であり、しかも、発酵飼料原料がイネの場合は、茎部が強固な中空構造であるため詰込み密度を高くすることが難しく、イネは糖含量が少なく貯蔵発酵が進みにくいことから、イネ発酵粗飼料をバンカーサイロで調製することは困難とされてきた。しかしながら、高糖分である極短穂系イネ品種に本発明の新規乳酸菌ＩＷＴ１９２株を使用することにより、バンカーサイロにおいても、二次発酵が抑制された、高品質のイネ発酵飼料を調製できることが明らかとなった。
As a result, as shown in Table 5, the fermented feed by the bunker silo to which the novel lactic acid bacterium IWT192 strain of the present invention was added had a significantly higher content of acetic acid and 1,2-propanediol immediately after opening than the non-added group, and the ethanol content Therefore, it was confirmed that the novel lactic acid bacterium IWT192 strain of the present invention has an extremely excellent harmful microorganism control effect even in a bunker silo.
Moreover, as FIG. 4 shows, in the bunker silo to which the novel lactic acid bacterium IWT192 strain of the present invention was added, no fever due to secondary fermentation was observed, and a fermented feed of good quality could be taken out to the end. On the other hand, in the bunker silo in the additive-free zone, the temperature of the product gradually rises from the seventh day after opening, and the temperature of the fermented feed in the bunker silo becomes about 40 ° C after three weeks, and the quality of the fermented feed is remarkably high The situation declined and a large amount of waste had to be discarded.
The bunker silo storage method is less airtight than the roll bale silage method, and the take-out period after opening is long, so the quality of the fermented feed is likely to deteriorate, and when the fermented feed material is rice Is difficult to increase the packing density due to the solid stem structure, and rice has low sugar content and storage fermentation is difficult to proceed, so it is difficult to prepare rice fermented roughage in a bunker silo. I came. However, it is clear that by using the novel lactic acid bacterium IWT192 strain of the present invention for extremely short ear rice varieties having a high sugar content, it is possible to prepare a high-quality rice fermented feed in which secondary fermentation is suppressed even in bunker silos. It became.
＜実施例７＞乳酸菌ＩＷＴ１９２株を使用したＴＭＲ製造への活用検討
次いで、ＴＭＲ製造への活用検討について説明する。
乳酸菌は「ＩＷＴ１９２株」と「ＳＰ株」を使用した。
各乳酸菌株をそれぞれ添加した飼料用イネ「たちすずか」のイネ発酵粗飼料および無添加処理したイネ発酵粗飼料を、ロールベールサイレージ法により冬季寒冷期間を含む８ヶ月間貯蔵した。開封後、各発酵飼料を使用してフレッシュＴＭＲ（1/3たちすずかイネ発酵粗飼料 + 2/3配合飼料 + 水、水分５０%）を調製し、２５℃の恒温器にて貯蔵した。調製後のフレッシュＴＭＲの温度変化を図５に示した。
<Example 7> Examination of utilization for TMR production using lactic acid bacteria IWT192 strain Next, examination of utilization for TMR production will be described.
Lactic acid bacteria used were “IWT192 strain” and “SP strain”.
Rice fermented roughage of feed rice “Tasusuzuka” to which each lactic acid strain was added and rice fermented roughage treated without addition were stored for 8 months by the roll bale silage method including the winter cold period. After opening, fresh TMR (1/3 Tatsusuka rice fermented rough feed + 2/3 mixed feed + water, water 50%) was prepared using each fermented feed, and stored in a thermostat at 25 ° C. The temperature change of fresh TMR after preparation is shown in FIG.
その結果、図５が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加したイネ発酵粗飼料を使用したフレッシュＴＭＲは、明確な温度上昇が認められず、フレッシュＴＭＲとして「たちすずか」イネ発酵粗飼料を二次利用した場合においても、ＩＷＴ１９２株の二次発酵抑制機能は持続することが明らかとなった。
混合調製したＴＭＲをそのまま当日牛に給与する形態であるフレッシュＴＭＲは、発熱を伴った変敗が生じやすいという短所があり、特に気温の高い夏季において、当日の朝に給与したＴＭＲが夕方には飼槽の中で発熱していることも珍しくない。フレッシュＴＭＲの発熱は栄養価の低下や、嗜好性の悪化による飼料摂取量低下の原因になり、生産性を低下させるため、飼育現場で問題となる。
しかしながら、本発明の新規乳酸菌ＩＷＴ１９２株を添加したイネ発酵粗飼料を使用したフレッシュＴＭＲは、無添加区やＳＰ株添加区のイネ発酵粗飼料を二次利用したフレッシュＴＭＲに比べて、温度上昇が顕著に抑制されており、二次発酵抑制機能に優れていることが確認されたので、上記問題解決に有用であると思われる。
As a result, as shown in FIG. 5, the fresh TMR using the rice fermented roughage to which the novel lactic acid bacterium IWT192 strain of the present invention was added did not show a clear increase in temperature, and the fresh TMR was “Tatsusuka” rice fermented roughage. It has been clarified that the secondary fermentation inhibiting function of the IWT192 strain is sustained even when it is secondarily used.
Fresh TMR, which is a form of feeding mixed TMR as it is to cattle on the day, is prone to deterioration with fever, especially in the summer when the temperature is high, It is not uncommon to have fever in the tank. The fever of fresh TMR causes a decrease in nutritional value and a decrease in feed intake due to a deterioration in palatability, which lowers productivity and becomes a problem at breeding sites.
However, the fresh TMR using the rice fermented roughage to which the novel lactic acid bacterium IWT192 strain of the present invention is added has a marked increase in temperature compared to the fresh TMR using the rice fermented roughage in the non-addition zone and the SP strain addition zone. Since it was confirmed that it was suppressed and excellent in the secondary fermentation inhibiting function, it seems to be useful for solving the above problems.
＜実施例８＞乳酸菌ＩＷＴ１９２株を使用した飼料用米サイレージへの活用検討
次いで、飼料用米サイレージへの活用検討について説明する。
乳酸菌は「ＩＷＴ１９２株」を使用した。
破砕処理後、水分約３０％に加水調整した飼料用米「べこごのみ」を無添加およびＩＷＴ１９２株添加処理後にフレコンバックに冬季寒冷期間を含む４ヶ月間貯蔵した。貯蔵後のサンプルに含まれる成分と微生物について、実施例４と同様の手法により測定した。サンプルの平均値を表６に示した。なお、表６中の「nd」は未検出を意味する。
さらに、本飼料用米サイレージの開封後、下記式で表される「Δ温度」に到達する時間（ｈ）を、図６に示した。
式：Δ温度＝サイレージ温度−環境温度（約２１℃）
<Example 8> Examination of utilization to feed rice silage using lactic acid bacteria IWT192 strain Next, examination of utilization to feed rice silage will be described.
As the lactic acid bacteria, “IWT192 strain” was used.
After the crushing treatment, feed rice “bekogo only” adjusted to a water content of about 30% was added and stored in the flexible container bag for 4 months including the winter cold period after the addition of IWT192 strain. The components and microorganisms contained in the sample after storage were measured by the same method as in Example 4. The average value of the samples is shown in Table 6. Note that “nd” in Table 6 means not detected.
Furthermore, the time (h) to reach the “Δ temperature” expressed by the following formula after opening the rice silage for feed is shown in FIG.
Formula: Δ temperature = silage temperature−environment temperature (about 21 ° C.)
その結果、表６が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加した飼料用米サイレージは、開封直後の酢酸含量が無添加区よりも高く、良好な発酵品質を示した。また、酵母数が無添加区よりも少なく、乳酸菌数が多いことが認められた。さらに、開封８日後でも、低ｐＨと高有機酸含量が維持され、酵母数が低減していることにより、発酵飼料栄養価の品質維持効果に優れていることも明らかとなった。
また、図６が示すように、無添加区では約１日程度でΔ温度３℃の上昇が、開封後４０時間程度でΔ温度１５℃までの急激な発熱が認められた。一方、ＩＷＴ１９２株では開封後８日間の期間内において温度上昇は全く認められなかった。
以上のことから、飼料用米サイレージ調製においても、ＩＷＴ１９２株の優れた二次発酵抑制効果が確認された。
As a result, as Table 6 shows, the rice silage for feed to which the novel lactic acid bacterium IWT192 strain of the present invention was added had higher acetic acid content immediately after opening than the non-added section, and showed good fermentation quality. Moreover, it was recognized that the number of yeasts is smaller than that in the non-added group and the number of lactic acid bacteria is large. Furthermore, even after 8 days from opening, it was revealed that the low pH and high organic acid content were maintained, and the number of yeasts was reduced, so that the quality maintenance effect of fermented feed nutritional value was excellent.
In addition, as shown in FIG. 6, in the non-added zone, an increase in the Δ temperature of 3 ° C. was observed in about 1 day, and a rapid exotherm up to the Δ temperature of 15 ° C. was observed in about 40 hours after opening. On the other hand, in the IWT192 strain, no temperature increase was observed in the period of 8 days after opening.
From the above, also in the preparation of feed rice silage, the excellent secondary fermentation inhibitory effect of the IWT192 strain was confirmed.
＜実施例９＞乳酸菌ＩＷＴ１９２株を使用した高糖分・高消化性飼料用イネの活用検討
次いで、高糖分・高消化性飼料用イネ（つきすずか）の活用検討について説明する。
乳酸菌は「ＩＷＴ１９２株」を使用した。
高糖分・高消化性飼料用稲「つきすずか」を無添加、ＩＷＴ１９２株添加処理を行い、小規模パウチ法により調製後、約６週間、１５℃と２５℃の温度条件下で貯蔵した。貯蔵後のサンプルに含まれる成分と微生物について、実施例４と同様の手法により測定した。サンプルの平均値を表７に示した。なお、表７中の「nd」は未検出を意味する。
<Example 9> Examination of utilization of rice for high sugar content / high digestibility feed using lactic acid bacteria IWT192 strain Next, examination of utilization of rice for high sugar content / high digestibility feed (Tsukusuka) will be described.
As the lactic acid bacteria, “IWT192 strain” was used.
Rice with high sugar content and high digestibility for feed “Tsukusuka” was added, IWT192 strain was added, and after preparation by the small-scale pouch method, it was stored at 15 ° C. and 25 ° C. for about 6 weeks. The components and microorganisms contained in the sample after storage were measured by the same method as in Example 4. Table 7 shows the average value of the samples. Note that “nd” in Table 7 means not detected.
その結果、表７が示すように、本発明の新規乳酸菌ＩＷＴ１９２株を添加した発酵飼料は、両貯蔵温度条件において、無添加区よりも低いｐＨ値を示し、乳酸、酢酸含量が有意に高かった。また、ＩＷＴ１９２株添加区においてカビ、酵母、大腸菌は未検出であり、無添加区と比較して一般好気性細菌数、バチルス属細菌数が少なく、乳酸菌数が多いことが認められた。１５℃貯蔵において、無添加区で認められたクロストリジウム属細菌は、ＩＷＴ１９２株添加区においては未検出であった。
以上のことから、低ｐＨと高有機酸含量が維持されており、二次発酵の原因とされる酵母・カビ数の低減が認められ、発酵飼料栄養価の品質維持効果に優れていることが明らかとなった。
As a result, as shown in Table 7, the fermented feed to which the novel lactic acid bacterium IWT192 strain of the present invention was added showed a pH value lower than that in the non-added section under both storage temperature conditions, and the lactic acid and acetic acid contents were significantly higher. . In addition, mold, yeast, and Escherichia coli were not detected in the group added with IWT192 strain, and it was confirmed that the number of general aerobic bacteria and the number of bacteria belonging to the genus Bacillus were small and the number of lactic acid bacteria was large compared with the group without addition. The clostridium bacterium observed in the non-added group during storage at 15 ° C. was not detected in the group added with IWT192 strain.
From the above, low pH and high organic acid content are maintained, the reduction of the number of yeasts and molds that are the cause of secondary fermentation is recognized, and it is excellent in quality maintenance effect of fermented feed nutritional value It became clear.
本発明のラクトバチルス・ブクネリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｕｃｈｎｅｒｉ）ＩＷＴ１９２株（ＮＩＴＥ Ｐ−０２１６８）は、低温増殖能に優れるので、低温環境下においても高品質のサイレージ等の発酵飼料を生産でき、二次発酵やカビ発生の心配がないことから、畜産分野において広範囲な利用が期待される。 Since the Lactobacillus buchneri strain IWT192 (NITE P-02168) of the present invention is excellent in low-temperature growth ability, it can produce a high-quality fermented feed such as silage even in a low-temperature environment. Since there is no concern about the occurrence, it is expected to be used widely in the livestock field.
Claims (7)
A method for preparing silage, wherein the silage material according to claim 6 is rice for feed.
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|---|---|---|---|---|
| JP2020534856A (en) * | 2017-09-28 | 2020-12-03 | バイオツェーツェー オウ | Microbial strains Lactobacillus buchneri BioCC 203 DSM32650, Lactobacillus buchneri BioCC 228 DSM32651, and their use |
| JP7250783B2 (en) | 2017-09-28 | 2023-04-03 | バイオツェーツェー オウ | Microbial strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 and uses thereof |
| CN112410251A (en) * | 2020-11-13 | 2021-02-26 | 河北大学 | Lactobacillus plantarum with quick acid production and high acid production capacity and application thereof |
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