JP2005021156A - Lactobacterium, lactic acid fermentation product, fodder - Google Patents
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Abstract
Description
本発明は発酵アルコール製造工業から副生する、アルコール発酵残渣の有効利用に関し、アルコール発酵残渣に極めて良く増殖する乳酸菌の提供、前記乳酸菌の乳酸発酵によるアルコール発酵残渣の乳酸発酵生成物の付加価値の高い飼料原料および飼料としての提供を目的とするものである。 The present invention relates to the effective use of alcohol fermentation residue by-produced from the fermented alcohol production industry, providing lactic acid bacteria that grow extremely well on the alcohol fermentation residue, and the added value of the lactic acid fermentation product of the alcohol fermentation residue by lactic acid fermentation of the lactic acid bacterium. The purpose is to provide a high feed material and feed.
発酵アルコールは、サトウキビから得られる糖蜜、砂糖大根（シュガービート）から得られる廃液濃縮物、あるいはトウモロコシや甘蔗の加水分解にて得られる糖類を発酵し、発酵もろみを蒸留して製造される。アルコール発酵にて排出される蒸留残渣は所謂廃液であり、環境問題から海洋投棄が禁止されることとなり、有効利用が望まれている。
アルコール発酵後の蒸留残渣は、一部肥料として利用されているが、付加価値が低く、有効な廃棄物処理法とはなっていない。海外では海洋投棄により環境問題を引き起こしており、その有効利用法の開発は世界的に望まれている。飼料としての利用は、アルコール発酵原料となる糖蜜については、飼料の賦形剤（バインダー）としての利用や糖蜜飼料として、飼料に配合するなどの応用が知られているが、アルコール発酵廃液を飼料化することは知られていない。
Fermented alcohol is produced by fermenting molasses obtained from sugar cane, waste liquid concentrate obtained from sugar radish (sugar beet), or sugars obtained by hydrolysis of corn or sweet potato, and distilling the fermented mash. Distillation residue discharged by alcohol fermentation is so-called waste liquid, and ocean dumping is prohibited due to environmental problems, and effective utilization is desired.
Although the distillation residue after alcohol fermentation is partially used as fertilizer, it has low added value and is not an effective waste treatment method. Overseas, abandonment of the ocean has caused environmental problems, and the development of effective utilization methods is desired worldwide. As for feed, molasses, which is a raw material for alcohol fermentation, has been known to be used as an excipient (binder) for feed and blended into feed as molasses feed. It is not known to become.
我々は、アルコール発酵後に排出される残渣（もろみ濃縮液と称す）の有効利用について、鋭意研究を重ねた結果、もろみ濃縮液に極めて良好な増殖を示す乳酸菌を見出した。
乳酸菌は生菌体および死菌体共に、生体の免疫力を高めたり、腸内細菌叢を調節し、便秘を軽減し、整腸作用を示すなど、種々の有用性が示されている（細野朗、上野川修一：腸管免疫とプロバイオティクス、Ｆｏｏｄ
Ｓｔｙｌｅ ２１、ｖｏｌ．６、ｐｐ．４５−４９、２００２年；ＭＲ．Ｈｅｒｉｃｈ
ａｎｄ Ｍ．Ｌｅｖｋｕｔ：Ｌａｃｔｉｃ
ａｃｉｄ ｂａｃｔｅｒｉａ、ｐｒｏｂｉｏｔｉｃｓ
ａｎｄ ｉｍｍｕｎｅ
ｓｙｓｔｅｍ、Ｖｅｔ．Ｍｅｄ．−Czech、ｖｏｌ．４７、ｐｐ．１６９−１８０、２００２年）。
また、もろみ濃縮液を発見した乳酸菌にて乳酸発酵し、乳酸菌の菌体濃度が高く、タンパクやビタミンを含み、最近注目されているプロバイオティックスとなる飼料原料を開発することに成功し、本発明を完成した。
As a result of intensive research on the effective use of the residue discharged after alcoholic fermentation (referred to as mash concentrate), we found lactic acid bacteria exhibiting extremely good growth in the mash concentrate.
Lactic acid bacteria have various usefulness, such as enhancing the immunity of living organisms, regulating intestinal bacterial flora, reducing constipation, and showing intestinal regulation, both in living and dead cells (Hosono) Akira, Shuichi Uenogawa: Intestinal immunity and probiotics, Food
Style 21, vol. 6, pp. 45-49, 2002; MR. Herich
and M.M. Levkut: Lactic
acid bacteriaia, probiotics
and imune
system, Vet. Med. -Czech, vol. 47, pp. 169-180, 2002).
In addition, lactic acid fermentation was performed with the lactic acid bacteria found in the moromi concentrate, and it succeeded in developing feed materials that are probiotics that have recently been attracting attention, with high bacterial cell concentrations of lactic acid bacteria, including proteins and vitamins. Was completed.
発酵アルコールは甲類焼酎原料、食酢の発酵原料、味噌や醤油の保存性を高める為の添加剤、あるいは食品の保存性を高める為のアルコール製剤の原料として、消費量が増加傾向にある。アルコール発酵原料として最も多いのが、砂糖を採取した残液である糖蜜である。
従来、アルコール発酵・蒸留廃液は、色は黒く問題はあるが、毒性を示すものは含まないので、多くは海洋投棄されていた。
しかし、環境汚染問題から、多くの自治体で海洋投棄を禁止する方向にあり、また、海外でも生産量が多く、大量の蒸留廃液の不法投棄が環境問題を引き起こしており、コストの低い処分法あるいは付加価値の高い有効利用法が求められている。
Fermented alcohol tends to increase in consumption as a raw material for shell shochu, a fermented raw material for vinegar, an additive for enhancing the preservability of miso and soy sauce, or a raw material for alcohol preparations for enhancing the preservability of food. The most common alcohol fermentation raw material is molasses, which is a residual liquid obtained by collecting sugar.
Conventionally, alcohol fermentation / distillation wastewater has a problem of being black in color, but since it does not contain any toxic substances, many were dumped into the ocean.
However, due to environmental pollution problems, many local governments are banning ocean dumping, and there is a lot of production overseas, and illegal dumping of a large amount of distilled waste liquid is causing environmental problems. There is a need for effective use with high added value.
アルコール発酵・蒸留廃液の一部は肥料原料として利用されている。また、焼酎業界では共同で燃焼処理設備を導入し、廃液を燃焼して処理している。
然るに、肥料原料としての利用は付加価値が低く、運送費用も出ない現状がある。燃焼処理については、必ずしも十分な処理量でなく、また、燃焼する為には費用がかかり、コスト的に負担が大きい。本発明の課題は、アルコール発酵残液に付加価値をつけ、有効利用を図ることである。
A part of alcohol fermentation / distillation waste liquid is used as a fertilizer raw material. The shochu industry has jointly introduced combustion treatment equipment to burn and treat waste liquid.
However, the use as a fertilizer raw material has a low added value and there is no transportation cost. The combustion treatment is not necessarily a sufficient amount of processing, and it is expensive to burn, and the cost is high. An object of the present invention is to add value to an alcohol fermentation residue and to make effective use.
我々は、アルコール発酵にて副生の残渣、例えばアルコール発酵残液即ちもろみ濃縮液の有効利用につき鋭意研究を重ねた結果、意外にも、このもろみ濃縮液に良好に生育する乳酸菌を見出した。また、発見した乳酸菌を用い、乳酸発酵飼料および乳酸菌高濃度液の開発に成功し発明を完成した。
即ち、本発明は、アルコール発酵・蒸留残渣に良好に生育する乳酸菌株、およびアルコール発酵蒸留残渣濃縮物（もろみ濃縮液）の乳酸菌発酵により、乳酸菌体、ビタミン類、タンパクなどを多く含み、飼料としても好適な乳酸発酵生成物としての乳酸発酵もろみ濃縮物の提供に関する。
As a result of intensive studies on the effective use of by-product residues such as alcohol fermentation residue, ie, mash concentrate, in alcohol fermentation, we have surprisingly found lactic acid bacteria that grow well in this mash concentrate. In addition, using the discovered lactic acid bacteria, we succeeded in developing a lactic acid fermented feed and a high concentration liquid of lactic acid bacteria, and completed the invention.
That is, the present invention contains a large amount of lactic acid bacteria, vitamins, proteins, and the like by lactic acid bacteria fermentation of alcohol fermentation / distillation residue and lactic acid bacteria fermentation of alcohol fermentation distillation residue concentrate (moromi concentrate) as feed. Also relates to the provision of a lactic acid fermentation moromi concentrate as a suitable lactic acid fermentation product.
本発明は、アルコール発酵にて副生の残渣に生育するラクトバチルス・パラカセイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ）に属する乳酸菌である。 The present invention is a lactic acid bacterium belonging to Lactobacillus paracasei that grows as a by-product residue by alcohol fermentation.
また本発明は、アルコール発酵物を蒸留し、得られる残渣に増殖するラクトバチルス・パラカセイ・サブスピシズ・パラカセイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ ｓｕｂｓｐ．ｐａｒａｃａｓｅｉ）に属する乳酸菌ＦＥＲＭ Ｐ−１９１６９である。 Further, the present invention is a lactic acid bacterium FERM P-19169 belonging to Lactobacillus paracasei subsp. Paracasei that is obtained by distilling an alcohol fermented product and growing it into a residue.
また本発明は、温度耐性およびエタノール耐性を有する乳酸菌ＦＥＲＭ Ｐ−１９１６９である。 Moreover, this invention is the lactic acid bacteria FERM P-19169 which has temperature tolerance and ethanol tolerance.
また本発明は、動物体内の病原菌に対し免疫増強作用を有する乳酸菌ＦＥＲＭ Ｐ−１９１６９である。 Moreover, this invention is the lactic acid bacterium FERM P-19169 which has an immunopotentiating effect | action with respect to the pathogenic microbe in an animal body.
また本発明の乳酸発酵生成物は、アルコール発酵にて副生の残渣と当該残渣に生育する乳酸菌ＦＥＲＭ Ｐ−１９１６９を含むことを特徴とする。 Moreover, the lactic acid fermentation product of the present invention is characterized by containing by-product residues by alcohol fermentation and lactic acid bacteria FERM P-19169 grown on the residues.
また本発明の乳酸発酵生成物は、アルコール発酵後に蒸留し得られる残渣と当該残渣を乳酸発酵して得られる乳酸菌ＦＥＲＭ Ｐ−１９１６９を含むことを特徴とする。 The lactic acid fermentation product of the present invention is characterized by containing a residue obtained by distillation after alcohol fermentation and a lactic acid bacterium FERM P-19169 obtained by lactic acid fermentation of the residue.
また本発明の飼料は、乳酸菌ＦＥＲＭ Ｐ−１９１６９を含む乳酸発酵生成物を原料若しくは少なくとも原料の一つとすることを特徴とする。 The feed of the present invention is characterized by using a lactic acid fermentation product containing lactic acid bacteria FERM P-19169 as a raw material or at least one of the raw materials.
また本発明の飼料は、動物体内の病原菌に対し免疫増強作用を有する乳酸菌ＦＥＲＭ Ｐ−１９１６９を含む乳酸発酵生成物を原料若しくは少なくとも原料の一つとすることを特徴とする。 The feed of the present invention is characterized in that a lactic acid fermentation product containing a lactic acid bacterium FERM P-19169 having an immunopotentiating action against pathogenic bacteria in an animal body is used as a raw material or at least one of the raw materials.
また本発明の乳酸発酵生成物は、発酵原料がサトウキビから砂糖を分離した糖蜜および/または柑橘果汁窄汁残渣より回収する果汁蜜であることを特徴とする。 The lactic acid fermentation product of the present invention is characterized in that the fermentation raw material is molasses obtained by separating sugar from sugarcane and / or fruit juice nectar collected from citrus juice juice residue.
また本発明の飼料は、乳酸発酵生成物は、発酵原料がサトウキビから砂糖を分離した糖蜜および/または柑橘果汁窄汁残渣より回収する果汁蜜であることを特徴とする。 In the feed of the present invention, the lactic acid fermentation product is characterized in that the fermentation raw material is molasses obtained by separating sugar from sugarcane and / or fruit juice obtained from citrus juice juice residue.
また本発明の乳酸発酵生成物は、乳酸菌ＦＥＲＭ Ｐ−１９１６９の生菌濃度が１０８ｃｅｌｌｓ／ｍＬ以上であることを特徴とする。 Moreover, the lactic acid fermentation product of the present invention is characterized in that the concentration of lactic acid bacteria FERM P-19169 is 10 8 cells / mL or more.
また本発明は、アルコール発酵にて副生の残渣に生育するラクトバチルス・パラカセイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ）に属する乳酸菌（Ｉ−５株、Ｉ−８株、Ｉ−９株、Ｉ−１３株、
Ｉ−１９株、Ｉ−２８株）であることを特徴とする。
The present invention also provides a lactic acid bacterium (I-5 strain, I-8 strain, I-9 strain, I-13 strain) belonging to Lactobacillus paracasei that grows as a by-product residue by alcohol fermentation.
I-19 strain, I-28 strain).
本発明により、従来低付加価値の利用しかされていなかった、アルコール発酵にて副生する残渣例えば蜜原料アルコール発酵後の蒸留残渣を有効利用することが出来、この残渣に良好に増殖する乳酸菌を発見したことで、発見した乳酸菌をプロバイオティクスとして利用できると共に、発酵生成物は付加価値のある乳酸発酵生成物および飼料或いは飼料原料として利用可能となった。 According to the present invention, it has been possible to effectively use a residue produced as a by-product in alcoholic fermentation, for example, a distillation residue after fermentation of nectar raw material alcohol, which has been used only for low added value in the past, As a result of the discovery, the discovered lactic acid bacteria can be used as probiotics, and the fermentation product can be used as a lactic acid fermentation product with added value and feed or feed raw material.
以下、本発明を実施例１乃至６に基づいて説明する。 Hereinafter, the present invention will be described based on Examples 1 to 6.
乳酸菌分離
食品用に使用される工業用アルコールは、主として基質としてサトウキビ窄汁液の蔗糖採取後の廃液である、糖蜜を発酵して行う。通常、アルコール発酵の糖蜜培地は加熱殺菌を施さないので、微量であるが種々の雑菌が含まれる。アルコール発酵時には、多量の酵母菌を種母として接種するので、短時間でアルコール濃度が上昇し、雑菌汚染の頻度は低い。
我々は、微量含まれる雑菌の中から乳酸菌に着目し、アルコール発酵もろみからの乳酸菌分離を試みた。
Industrial alcohol used for foods isolated from lactic acid bacteria is mainly produced by fermentation of molasses, which is a waste liquid after sucrose collection of sugarcane squeeze juice as a substrate. Usually, since the molasses medium of alcohol fermentation is not heat-sterilized, it contains a small amount of various bacteria. At the time of alcoholic fermentation, a large amount of yeast is inoculated as a seed mother, so that the alcohol concentration rises in a short time and the frequency of contamination is low.
We focused on lactic acid bacteria from various microbes, and tried to isolate lactic acid bacteria from alcoholic fermentation.
アルコール発酵は、凝集性アルコール酵母 Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ
Ｆ−５をグルコース１.０％、酵母エキス０.３％、麦芽エキス０.３％、ペプトン０.３％を含む培地（以下ＹＭ培地と略記する）に接種し、３２℃で一晩培養したものを一次種母とし、これを糖蜜を希釈し、全糖１６％（ｗ／ｖ）とし、Ｎ源として硫酸アンモニウムを０.０３％添加した培地に５％添加し、３２℃で一晩培養し、２次種母とした。
アルコール発酵は糖蜜を希釈し、全糖１８％（ｗ／ｖ）とし、N源として硫酸アンモニウムを０.０３％添加した液に、２次種母を１０％添加し、３０℃にて４８時間発酵した（発酵１回目）。
以後、全糖濃度１８％とした糖蜜培地に発酵１回目の発酵もろみを２０％接種し、３０℃で２４時間発酵した（発酵２回目）。
発酵３回目は全糖濃度２０％、発酵４回目は全糖濃度２２％とし、種母量は２０％とした。
発酵５回目以降は、発酵４回目と同様の条件とし、８回から１０回繰り返し発酵を行った。
Alcohol fermentation is a flocculating alcohol yeast Saccharomyces cerevisiae
F-5 was inoculated into a medium containing glucose 1.0%, yeast extract 0.3%, malt extract 0.3% and peptone 0.3% (hereinafter abbreviated as YM medium) and cultured at 32 ° C. overnight. This was used as the primary seed mother, which was diluted with molasses to give 16% (w / v) total sugar, and 5% was added to the medium supplemented with 0.03% ammonium sulfate as an N source, and cultured at 32 ° C overnight. And a secondary seed mother.
In alcoholic fermentation, molasses is diluted to 18% (w / v) of total sugar, 10% of the secondary seed mother is added to a solution containing 0.03% of ammonium sulfate as the N source, and fermented at 30 ° C. for 48 hours. (1st fermentation).
Thereafter, 20% of the first fermentation mash was inoculated into a molasses medium with a total sugar concentration of 18% and fermented at 30 ° C. for 24 hours (second fermentation).
The third fermentation had a total sugar concentration of 20%, the fourth fermentation had a total sugar concentration of 22%, and the seed volume was 20%.
From the 5th fermentation, the same conditions as the 4th fermentation were applied, and the fermentation was repeated 8 to 10 times.
繰り返し回分発酵の最終回のもろみから、乳酸菌および酢酸菌分離用培地を用い、乳酸菌を分離した。
分離培地としては乳酸菌用にＧＹＰ−ＣａＣＯ３培地（Ｇｌｕｃｏｓｅ
２０ｇ／Ｌ、酵母エキス １０ｇ／Ｌ、ペプトン １０ｇ／Ｌ、Ｔｗｅｅｎ−８０
５ｇ／Ｌ、ＣａＣＯ３ １０ｇ／Ｌ、シクロヘキシミド ２μｇ／ｍＬ）、ＭＲＳ培地（Ｄｉｆｃｏ
ＭＲＳ ｂｒｏｔｈ）、酢酸菌用培地として、シクロヘキシミド
２μｇ／ｍＬ、ＣａＣＯ３ １０ｇ／Ｌ、エタノール ５ｇ／Ｌを添加したポテト−デキストロース寒天培地を使用した。
分離された菌は、桿菌、連鎖桿菌、球菌、連鎖球菌などであり、形態および乳酸の生成などから、多くはＬａｃｔｏｂａｃｉｌｌｕｓであり、Ｌｅｕｃｏｎｏｓｔｏｃと思われる乳酸菌も分離された。結果的に３６株の乳酸菌と考えられる菌株が単離・取得できた。
Lactic acid bacteria were separated from the mash of the final batch fermentation using lactic acid bacteria and acetic acid bacteria separation medium.
As a separation medium, GYP-CaCO 3 medium (Glucose) is used for lactic acid bacteria.
20 g / L, yeast extract 10 g / L, peptone 10 g / L, Tween-80
5 g / L, CaCO 3 10 g / L, cycloheximide 2 μg / mL), MRS medium (Difco
MRS broth), a potato-dextrose agar medium supplemented with 2 μg / mL of cycloheximide, 10 g / L of CaCO 3 and 5 g / L of ethanol was used as a medium for acetic acid bacteria.
The isolated bacteria are gonococci, streptococci, cocci, streptococci, and the like. From the form and production of lactic acid, many are Lactobacillus, and lactic acid bacteria that are considered to be Leuconostoc were also isolated. As a result, 36 strains considered to be lactic acid bacteria were isolated and obtained.
得られた３６株の乳酸菌より、アルコール耐性（８％エタノール以上に耐性）の観点から、６株（Ｉ−５、Ｉ−８、Ｉ−９、Ｉ−１３、
Ｉ−１９、Ｉ−２８）を選択した。
更に、選ばれた６株について、もろみ濃縮液での増殖を調べ、最終的に良好な増殖を示す株として、Ｉ−５株が選択された。
本株はアルコール発酵蒸留残液の２.５〜３倍濃縮液（もろみ濃縮液）の２倍希釈液にも、極めて良好な増殖を示した。
従来、アルコール発酵蒸留廃液で良好な増殖を示す乳酸菌は知られていなかった。
From the obtained 36 strains of lactic acid bacteria, from the viewpoint of alcohol resistance (resistance to 8% ethanol or more), 6 strains (I-5, I-8, I-9, I-13,
I-19, I-28) were selected.
Further, the selected 6 strains were examined for growth in the mash concentrate, and the I-5 strain was finally selected as a strain showing good growth.
This strain also showed extremely good growth even in a 2-fold dilution of a 2.5-3 fold concentrate (alcoholic concentrate) of the alcohol fermentation distillation residue.
Conventionally, no lactic acid bacteria showing good growth in alcoholic fermentation distillation waste liquid have been known.
菌学的性質
本発明の前記６株のうちＩ−５株の菌学性質を調べた結果を以下に示す。
本株（Ｉ−５株）をＤｉｆｃｏ社製ＭＲＳ培地に寒天を１.５％添加した培地でガスパック嫌気システム（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ Ｍｉｃｒｏｂｉｏｌｏｇｙ
Ｓｙｓｔｅｍｓ、ＭＤ、ＵＳＡ）にて、３７℃、１〜２日間培し、形態を観察した。Ｉ−５株は短径０.９〜１.１μｍ、長径１.９〜３.３の桿菌であり、単一の細胞でも存在したが、多くは２ヶの菌が連鎖状となり、それ以上の鎖状を示すものも観察された。本株は胞子を形成しなかった。本株をＭＲＳ培地で液体培養し、有機酸の生成を調べたところ、乳酸のほかに酢酸の生成もあり、ヘテロ型乳酸発酵を行う乳酸菌と考えられた。
Mycological properties The results of examining the mycological properties of the I-5 strain among the above 6 strains of the present invention are shown below.
Gas pack anaerobic system (Becton Dickinson Microbiology) with medium containing 1.5% agar added to Difco's MRS medium.
(Systems, MD, USA) at 37 ° C. for 1-2 days and observed for morphology. The I-5 strain is a koji mold with a minor axis of 0.9 to 1.1 μm and a major axis of 1.9 to 3.3, and even a single cell was present. A chain-like shape was also observed. This strain did not form spores. When this strain was liquid-cultured in MRS medium and the production of organic acid was examined, acetic acid was produced in addition to lactic acid, which was considered to be a lactic acid bacterium performing hetero-type lactic acid fermentation.
Ｉ−５株の同定を行う為、炭水化物代謝試験にて同定を行う、Ｂｉｏｍｅｒｉｅｕｘ社（フランス、輸入販売元：日本ビオメリュー（株））のＡＰＩ
５０ＣＨを用い、３０℃、４８時間培養し、同定を行った。その結果、本株はラクトバチルス・パラカセイ・サブスピシズ・パラカセイ（Ｌａｃｔｏｂａｃｉｌｌｕｓ
ｐａｒａｃａｓｅｉ ｓｕｂｓｐ．ｐａｒａｃａｓｅｉ）と同定された。そこで、ラクトバチルス・パラカセイ・サブスピシズ・パラカセイのタイプ・カルチャーであるＬ．ｐａｒａｃａｓｅｉ
ｓｕｂｓｐ．ｐａｒａｃａｓｅｉ ＮＢＲＣ １５８８９を取り寄せ、種々の性質を比較検討した。
In order to identify the I-5 strain, API of Biomerieux (France, import sales agency: Nihon Biomeryu Co., Ltd.), which is identified in a carbohydrate metabolism test, is identified.
Identification was performed by culturing at 50 ° C. for 48 hours using 50CH. As a result, the strain was found to be Lactobacillus subspices paracasei (Lactobacillus).
paracasei subsp. paracasei). Therefore, L., which is a type culture of Lactobacillus paracatheis, subspices and paracathesia. paracasei
subsp.
ＡＰＩ ５０ＣＨによる各種糖の発酵性について調べた結果、多くの糖質でＩ−５株とＮＢＲＣ １５８８９は類似の資化パターンを示したが、表１に示す種々の糖質について、異なる結果を示した。また、図１に各種培養温度における生育についての結果を、図２に各種エタノール濃度におけるエタノールの生育阻害についての結果を示す。
As a result of examining the fermentability of various sugars by API 50CH, the I-5 strain and
表１よりＩ−５株はソルボース、ソルビトール、イヌリン、グルコン酸の発酵性がタイプカルチャーであるＮＢＲＣ
１５８８９と異なった。温度特性は、図１よりＮＢＲＣ １５８８９が４０℃までの増殖温度であるのに対し、Ｉ−５株は４５℃でも生育し、増殖温度特性が異なった。また、図２に示すように、エタノールに対する耐性もＩ−５はＮＢＲＣ
１５８８９より高く、タイプカルチャーでは８％（ｖ／ｖ）でほぼ完全に増殖が阻害するのに対し、Ｉ−５株はエタノール８％（ｖ／ｖ）で生育し、１６％（ｖ／ｖ）でも若干の増殖が認められた。以上より、Ｉ−５株はＡＰＩ
５０ＣＨを用いる同定キットではＬ．ｐａｒａｃａｓｅｉ ｓｕｂｓｐ．ｐａｒａｃａｓｅｉと同定されるが、タイプカルチャーであるＮＢＲＣ １５８８９とは、生理的に種々の点で異なり、Ｉ−５株はＬ．ｐａｒａｃａｓｅｉ ｓｕｂｓｐ．ｐａｒａｃａｓｅｉの生理的変種であり、ラクトバチルス・パラカセイ・サブスピシズ・パラカセイＩ−５株（Ｌ．ｐａｒａｃａｓｅｉ sｕｂｓｐ．ｐａｒａｃａｓｅｉ Ｉ−５）と命名された。Ｌａｃｔｏｂａｃｉｌｌｕｓ ｐａｒａｃａｓｅｉ ｓｕｂｓｐ．ｐａｒａｃａｓｅｉ
Ｉ−５株はＦＥＲＭ Ｐ−１９１６９として、独立行政法人産業技術総合研究所・特許生物寄託センターに寄託されている。
From Table 1, the I-5 strain is NBRC in which the fermentability of sorbose, sorbitol, inulin and gluconic acid is a type culture.
Different from 15889. From FIG. 1,
It is higher than 15889, and growth is almost completely inhibited at 8% (v / v) in type culture, whereas strain I-5 grows at 8% (v / v) ethanol and 16% (v / v) But some growth was observed. Based on the above, the I-5 strain is API
In the identification kit using 50CH, L.P. paracasei subsp. Although identified as paracasei, it is physiologically different from
The I-5 strain has been deposited as FERM P-19169 at the National Institute of Advanced Industrial Science and Technology (AIST).
本発明のもろみ濃縮液での乳酸菌増殖試験について示す。
まず、もろみ濃縮液の各希釈液で乳酸菌（Ｉ−５株）の増殖を調べた。
純粋分離したＩ−５株を５ｍＬのＭＲＳに接種し、ガスパック中で３７℃一晩培養した。試験管にて調製した、各試験区のもろみ濃縮液１０ｍＬに本種母を５％接種し、スクリューキャップをし、３７℃で増殖試験を行った。
その結果、もろみ濃縮液原液では、乳酸菌の増殖は認められず、
２倍希釈以上の希釈液にて増殖が認められた。
また、各希釈液にブドウ糖２％を添加した培地も検討したが、４培希釈液まではブドウ糖の添加は増殖に影響を与えなかった。
もろみ濃縮液はＮＥＤＯアルコール事業本部出水アルコール工場にて得られた、もろみ２.５倍濃縮のもろみ濃縮液を使用した。
分析データを以下に示す。
アルコール発酵原料としては糖蜜と果汁蜜の混合物を使用した。試験に供したもろみ濃縮液のｐＨは４.６９、全糖（％、ｗｔ／ｖ）は７.００％、直接還元糖（％、ｗｔ／ｖ）は６.０８％、ケットの赤外線水分計による水分（％、ｗｔ／ｖ）は６８.９％であった。表２に液体クロマトグラフィー（ＨＰＬＣ）による糖および有機酸分析値を示す。
It shows about the lactic acid bacteria growth test in the mash concentrate of this invention.
First, the growth of lactic acid bacteria (strain I-5) was examined with each dilution of the moromi concentrate.
Purely isolated strain I-5 was inoculated into 5 mL of MRS and cultured overnight at 37 ° C. in a gas pack. 5% of the seed mother was inoculated into 10 mL of the moromi concentrate of each test section prepared in a test tube, screw-capped, and subjected to a growth test at 37 ° C.
As a result, no growth of lactic acid bacteria was observed in the mash concentrate solution,
Proliferation was observed in dilute solutions more than 2-fold diluted.
Moreover, although the culture medium which added
The moromi concentrate used in the NEDO Alcohol Business Division Izumi Alcohol Factory was a 2.5-fold concentrated moromi concentrate.
Analytical data is shown below.
A mixture of molasses and fruit juice was used as an alcohol fermentation raw material. The mash concentrate used for the test had a pH of 4.69, a total sugar (%, wt / v) of 7.00%, a direct reducing sugar (%, wt / v) of 6.08%, and Kett's infrared moisture meter. The water content (%, wt / v) was 68.9%. Table 2 shows the sugar and organic acid analysis values by liquid chromatography (HPLC).
２倍希釈もろみ濃縮液で乳酸菌が良好な生育を示すことが判明したので、この培地を用い、アルコール耐性を示す６株の乳酸菌の増殖試験を行った。
２００ｍＬ容の三角フラスコに、２倍希釈もろみ濃縮液２００ｍＬを入れ、シリコ栓（株式会社三商販売）を施し、ＭＲＳ培地で前培養した乳酸菌株を接種し、３７℃で時々攪拌し、４日間培養を行った。ただし、初発ｐＨは菌の増殖を考慮し、６.５に調整した。培養試験の結果を表３に示す。
Since it was found that the lactic acid bacteria showed good growth in the 2-fold diluted mash concentrate, this medium was used to conduct a growth test of 6 strains of lactic acid bacteria exhibiting alcohol resistance.
In a 200 mL Erlenmeyer flask, add 200 mL of a 2-fold diluted mash concentrate, apply a silico stopper (sales by Sansho Co., Ltd.), inoculate with a lactic acid strain pre-cultured in MRS medium, and stir occasionally at 37 ° C for 4 days. Culture was performed. However, the initial pH was adjusted to 6.5 in consideration of bacterial growth. The results of the culture test are shown in Table 3.
表３の結果より、乳酸菌Ｉ−５株が優秀菌株として選択できた。
Ｉ−５株のもろみ濃縮液培養物には多量の乳酸菌生菌が含有され、プロバイオティックスとしての有用性が示唆された。
また、有機酸である乳酸が約２％も生成され、腸内細菌叢を整える作用が期待できる。
From the results shown in Table 3, the lactic acid bacteria I-5 strain could be selected as an excellent strain.
The moromi concentrate culture of the strain I-5 contained a large amount of viable lactic acid bacteria, suggesting its usefulness as a probiotic.
In addition, about 2% of lactic acid, which is an organic acid, is produced, and an effect of adjusting the intestinal bacterial flora can be expected.
ビタミンの生成
更に、本発明の効果を明かにするため、スケールアップし、もろみ濃縮液でＩ−５株を培養し、乳酸菌の生菌数や組成、乳酸菌の特徴であるビタミンの生成を調べた。
３０００ｍＬ容のジャーファーメンターに、２倍希釈もろみ濃縮液２０００ｍＬを入れ、５％アンモニア水で培地ｐＨを６.５に調整した。ＭＲＳ培地で前培養した乳酸菌培養液１００ｍＬを接種し、３７℃無通気で、時々攪拌し、４日間培養を行った。乳酸菌増殖の結果を図３に示す。図３から、乳酸菌のもろみ濃縮液での増殖は３日目で最大に達することが判明した。表４に培養３日目の乳酸菌培養もろみ濃縮液の組成を示す。
In order to clarify the effects of the present invention, the scale-up was performed, and the I-5 strain was cultured with the mash concentrate to examine the number and composition of lactic acid bacteria and the production of vitamins, which are characteristic of lactic acid bacteria. .
A 3000 mL jar fermenter was charged with 2000 mL of 2-fold diluted mash concentrate, and the medium pH was adjusted to 6.5 with 5% aqueous ammonia. 100 mL of a lactic acid bacteria culture solution pre-cultured in MRS medium was inoculated, and the culture was performed for 4 days with agitation at 37 ° C. without aeration. The results of lactic acid bacteria growth are shown in FIG. From FIG. 3, it was found that the growth of lactic acid bacteria in the mash concentrate reached a maximum on the third day. Table 4 shows the composition of the lactic acid bacteria culture mash concentrate on the third day of culture.
表４より、乳酸菌Ｉ−５株はもろみ濃縮液の２倍希釈液で良好な増殖を示し、培養３日目で生菌数が１０９
ｃｅｌｌｓ／ｍＬに達し、十分な乳酸生菌を提供できることが判明した。また、有機酸含有量も培養前と比べ、総量で約２倍となり、特に乳酸は約２％となった。十分に乳酸菌Ｉ−５株が増殖したので、ビタミン類も生成しているものと考え、（社）日本分析センターにビタミンの分析を依頼し、乳酸菌発酵によるビタミン生成に対する効果を調べた。結果を表５に示す。
From Table 4, lactic acid bacteria strain I-5 showed good growth in a 2-fold dilution of the mash concentrate, and the number of viable bacteria was 10 9 on the third day of culture.
cells / mL, and it was found that sufficient lactic acid bacteria can be provided. In addition, the total amount of organic acid was about twice that before the cultivation, and especially lactic acid was about 2%. Since the lactic acid bacteria strain I-5 had grown sufficiently, it was considered that vitamins were also produced, and the analysis of vitamins was requested from the Japan Analytical Center to investigate the effect of lactic acid bacteria fermentation on vitamin production. The results are shown in Table 5.
表５より明らかなように、乳酸菌Ｉ−５株にてもろみ濃縮液を発酵すると、多量のビタミンが生成されることが判明した。
中でも、葉酸はＩ−５株で発酵する前が５μｇ／ｍＬであったものが、４６μｇ／ｍＬと約１０倍に増加しており、本発明の効果は明らかである。
As is clear from Table 5, it was found that a large amount of vitamins was produced when the mash concentrate was fermented in lactic acid bacteria strain I-5.
Among them, folic acid, which was 5 μg / mL before fermenting with I-5 strain, increased by about 10 times to 46 μg / mL, and the effect of the present invention is clear.
免疫増強効果
乳酸菌Ｉ−５株の免疫増強効果を調べる為、マウスを使用した動物試験を行った。乳酸菌の効果を明瞭にするため、被検液として実施例２で培養した乳酸菌発酵もろみ濃縮液を遠心し、含まれる乳酸菌量を約１０倍多くした発酵もろみ濃縮液を調製した。
即ち、実施例２で培養した乳酸菌発酵液２Ｌを遠心チューブにとり、６０００回転で２０分間遠心し、乳酸菌体を含む沈殿を得た。沈殿を先の遠心上清液で懸濁し、乳酸菌を含む液量を２００ｍＬとした。この液の乳酸菌の生菌数は１.４７×１０１０ｃｅｌｌｓ／ｍＬであった。
Immunity enhancing effect In order to examine the immune enhancing effect of lactic acid bacteria strain I-5, an animal test using mice was conducted. In order to clarify the effect of lactic acid bacteria, the lactic acid bacteria fermentation mash concentrated solution cultured in Example 2 was centrifuged as a test solution to prepare a fermentation mash concentrated liquid in which the amount of lactic acid bacteria contained was increased about 10 times.
That is, 2 L of lactic acid bacteria fermentation broth cultured in Example 2 was placed in a centrifuge tube and centrifuged at 6000 rpm for 20 minutes to obtain a precipitate containing lactic acid bacteria. The precipitate was suspended in the previous centrifugal supernatant, and the amount of liquid containing lactic acid bacteria was 200 mL. The viable count of lactic acid bacteria in this solution was 1.47 × 10 10 cells / mL.
実験動物は日本エスエルシー（株）より３．５週齢で購入したＩＣＲ系雄性マウス（ＳＰＦ）を４日間予備飼育して実験に供した。マウスは予備飼育期間および実験期間を通して室温２４±３℃、相対湿度５５±１５％の感染動物飼育室（照明時間８時〜１８時、換気回数１８回／時）で飼育した。なお、マウスの大腸菌感染時の週齢は５．５週齢とした。
マウスは５匹／ケージとし、滅菌蒸留水を給水瓶にて、またマウス用放射線滅菌固形飼料（ＣＭＦ、オリエンタル酵母工業）を給餌器にて、それぞれ自由に与えた。これらの飼育器具は全て滅菌したものを使用した。なお、マウスの個体識別はピクリン酸を被毛に塗布した。
As experimental animals, ICR male mice (SPF) purchased from Japan SLC Co., Ltd. at 3.5 weeks of age were preliminarily raised for 4 days and used for experiments. Mice were raised in an infected animal breeding room (lighting time: 8:00 to 18:00, ventilation rate: 18 times / hour) at a room temperature of 24 ± 3 ° C. and a relative humidity of 55 ± 15% throughout the preliminary breeding period and experimental period. Note that the age of the mouse at the time of infection with E. coli was 5.5 weeks.
The mice were 5 mice / cage, and sterilized distilled water was freely given in a water supply bottle and radiation sterilized solid feed for mice (CMF, Oriental Yeast Industry) was freely given in a feeder. All of these breeding instruments were sterilized. For identification of mice, picric acid was applied to the hair.
被験物質として、先に調製した生菌数１.４７×１０１０ｃｅｌｌｓ／ｍＬである乳酸菌培養もろみ濃縮液を用いた。
この溶液は冷暗所（４℃）に保存し、感染前１１日間（１日１回）および感染後２時間の合計１２回、それぞれマウス用経口ゾンデを用いて強制経口投与した。投与容量はマウスの体重３０ｇ当り０.５ｍＬとした。
As the test substance, the previously prepared lactic acid bacteria culture mash mash concentrate having a viable cell count of 1.47 × 10 10 cells / mL was used.
This solution was stored in a cool dark place (4 ° C.) and was orally administered by gavage using an oral sonde for mice for 11 days before infection (once a day) and 2 hours after infection for a total of 12 times. The administration volume was 0.5 mL per 30 g body weight of the mouse.
感染菌株として病原性大腸菌であるＥｓｃｈｅｒｉｃｈｉａ
ｃｏｌｉ Ｊｕｈｌ（メルシャン株式会社生物資源研究所保存菌株）を使用した。
凍結乾燥保存菌にブドウ糖リン酸ペプトン水を加え復元し、ＳＣＤ寒天平板培地（Lｏｔ Ｎｏ． ０５５０１２、日水製薬）に塗抹した。３１℃で２０時間培養し、再度ＳＣＤ寒天平板培地に継代培養した（３１℃、２０時間）。生育した菌体をブドウ糖リン酸ペプトン水に懸濁して６×１０７ＣＦＵ（＝Ｃｏｌｏｎｙ Ｆｏｒｍｉｎｇ ｕｎｉｔ）／ｍＬおよび２×１０８ＣＦＵ／ｍＬに調製した（Ｍａｃ
Ｆａｒｌａｎｄ ｓｔａｎｄａｒｄ使用）。５．５週齢のマウスに各濃度の大腸菌０．５ｍＬを腹腔内投与した（３×１０７ＣＦＵ／マウスおよび１×１０８ＣＦＵ／マウス）。動物数は一群１０匹とした。群構成は表６に示した。
Escherichia, a pathogenic Escherichia coli as an infectious strain
E. coli Juhl (stored strain of Bioresource Research Institute, Mercian Co., Ltd.) was used.
Glucose phosphate peptone water was added to the freeze-dried preserved bacteria to reconstitute it and smeared on a SCD agar plate medium (Lot No. 055012, Nissui Pharmaceutical). The cells were cultured at 31 ° C. for 20 hours, and subcultured again on SCD agar plate medium (31 ° C., 20 hours). The grown cells were suspended in dextrose phosphate peptone water to prepare 6 × 10 7 CFU (= Colony Forming unit) / mL and 2 × 10 8 CFU / mL (Mac
Use Farland standard). 5.5-week-old mice were intraperitoneally administered with 0.5 mL of each concentration of E. coli (3 × 10 7 CFU / mouse and 1 × 10 8 CFU / mouse). The number of animals was 10 per group. The group composition is shown in Table 6.
検査項目は以下の通りである。
体重：大腸菌の接種日をｄａｙ０とし、ｄａｙ−１１，−８，−４，０，１，３，５および７に体重計にて測定した。ｄａｙ０における体重に群間のバラツキが生じた場合には、ｄａｙ０を基準とした体重増減で表した。一般症状：大腸菌接種日のｄａｙ０よりｄａｙ７まで毎日観察した。症状は個体別に記録し、顕著な変化がみられた症状項目を発現個体数で表した。死亡：マウスの死亡をｄａｙ０よりｄａｙ７まで毎日朝夕の２回調べた。マウスの死亡日より平均生存日数および死亡率（％）を計算した。採血および血漿保存：試験最終日に生存マウスをエーテル吸入麻酔下で心臓より採血し、遠心分離後の上清（血漿）部分を−８０℃に凍結保存した。
The inspection items are as follows.
Body weight: The day of inoculation of E. coli was
統計処理は以下の通りに行った。
体重、体重増減および平均生存日数は群毎の平均値±標準誤差を算出した。また、これらのデータの対照群（２群および４群）に対する各群の統計的有意を検定するため、解析ソフト（Ｓｔａｔ
Ｖｉｅｗ， Ａｂａｃｕｓ
Ｉｎｃ．，ＵＳＡ）を用いて有意差の検定を行った（Ｓｔｕｄｅｎｔのｔ検定、２群間の検定）。統計的有意差はＰ＜０．０５の場合を有意であるとした。試験結果を以下に示す。死亡マウスの死亡状況および生存日数を表７および図４に示す。
Statistical processing was performed as follows.
The body weight, weight gain and loss, and average survival time were calculated as the mean value ± standard error for each group. In addition, in order to test the statistical significance of each group with respect to the control group (
View, Abacus
Inc. , USA) was used to test for significant difference (Student t test, test between two groups). Statistical significance was significant when P <0.05. The test results are shown below. Table 7 and FIG. 4 show the death status and survival days of the dead mice.
表７および図４より、３×１０７ＣＦＵ／マウスの大腸菌を接種したグループにおいて、対照群（感染）は１／１０例の死亡を示した。これに対し、乳酸菌培養もろみ濃縮液投与群は０／１０例の死亡を示した。平均生存日数は対照群（感染）が６．５±０．５以上を示したのに対し、乳酸菌培養もろみ濃縮液投与群は７．０±０．０以上を示し、両群間に大差は認められなかった。１×１０８ＣＦＵ／マウスの大腸菌を接種したグループでは、対照群（感染）が９／１０例（９０％）の死亡を示した。これに対し、乳酸菌培養もろみ濃縮液投与群は３／１０例（３０％）の死亡を示し、乳酸菌培養もろみ濃縮液投与群で顕著な死亡マウス数の減少がみられた。また、平均生存日数は対照群（感染）が２．５±０．５以上を示したのに対し、乳酸菌培養もろみ濃縮液投与群は５．５±０．８以上を示し、乳酸菌培養もろみ濃縮液が有意な生存日数の延長を示した（ｐ＜０．０１）。乳酸菌培養もろみ濃縮液の大腸菌感染防御効果が明らかとなった。 From Table 7 and FIG. 4, in the group inoculated with 3 × 10 7 CFU / mouse of E. coli, the control group (infection) showed 1/10 deaths. On the other hand, the lactic acid bacteria culture moromi concentrate administration group showed 0/10 deaths. The mean survival time of the control group (infection) was 6.5 ± 0.5 or more, while the lactic acid bacteria culture mash concentrate treatment group was 7.0 ± 0.0 or more. I was not able to admit. In the group inoculated with 1 × 10 8 CFU / mouse of E. coli, the control group (infection) showed 9/10 deaths (90%). In contrast, 3/10 cases (30%) died in the lactic acid bacteria culture mash concentrate administration group, and a marked decrease in the number of dead mice was observed in the lactic acid bacteria culture mash concentrate administration group. The mean survival time was 2.5 ± 0.5 or more in the control group (infection), whereas the lactic acid bacteria culture mash concentrate group was 5.5 ± 0.8 or more. The fluid showed a significant increase in survival days (p <0.01). The effect of lactic acid bacteria culture mash concentrate on E. coli infection was clarified.
体重測定結果を表８、表９および図５、図６に示す。
表８、表９、図５、図６より、感染前の体重推移でみた場合、３×１０７ＣＦＵ／マウスの大腸菌接種および１×１０８ＣＦＵ／マウスの大腸菌接種のいずれのグループにおいても、乳酸菌培養もろみ濃縮液群は対照群（非感染）および対照群（感染）との間に大きな体重差を示さなかった。感染後の体重推移では、３×１０７ＣＦＵ／マウスの大腸菌接種グループにおいて、乳酸菌培養もろみ濃縮液群は対照群（感染）との間に大きな体重差を示さなかった。１×１０８ＣＦＵ／マウスの大腸菌接種グループでは、ｄａｙ３以降の対照群（感染）の生存動物数が１例のため、乳酸菌培養もろみ濃縮液群との差を判断できなかった。
From Table 8, Table 9, FIG. 5 and FIG. 6, in terms of body weight transition before infection, in any group of 3 × 10 7 CFU / mouse inoculation and 1 × 10 8 CFU / mouse inoculation, The lactic acid bacteria culture mash concentrate group showed no significant difference in body weight between the control group (non-infected) and the control group (infected). In the body weight transition after infection, in the 3 × 10 7 CFU / mouse E. coli-inoculated group, the lactic acid bacteria culture mash concentrate group did not show a large difference in body weight from the control group (infection). In the E. coli inoculation group of 1 × 10 8 CFU / mouse, since the number of surviving animals in the control group (infected) after
一般症状の観察結果を表１０に示した。
表１０より、３×１０７ＣＦＵ／マウスの大腸菌接種グループにおいて、対照群（感染）に比べて乳酸菌培養もろみ濃縮液群が軽度の軟便および肛門部の便付着を示した動物数の増加と肛門部の充血を示す動物数の増加を示した。また、１×１０８ＣＦＵ／マウスの大腸菌接種グループにおいて、ｄａｙ１までの変化では対照群（感染）と乳酸菌培養もろみ濃縮液群との間に一般症状の差が認められなかった。ｄａｙ３以降では、対照群（感染）の生存動物数が１例のため、乳酸菌培養もろみ濃縮液群との間の一般症状の差を判断できなかった。
From Table 10, in the 3 × 10 7 CFU / mouse E. coli-inoculated group, the lactic acid bacteria culture mash concentrate concentrate showed mild loose stool and anus stool attachment and anus compared to the control group (infection) An increase in the number of animals showing partial hyperemia was shown. Further, in the 1 × 10 8 CFU / mouse E. coli inoculated group, no change in general symptoms was observed between the control group (infection) and the lactic acid bacteria culture mash and concentrate group in the change up to
一群10匹のマウスに３×１０７ＣＦＵ／マウスまたは１×１０８ＣＦＵ／マウスの大腸菌を接種し、死亡動物数、生存日数、体重および一般症状の変化から乳酸菌培養もろみ濃縮液の防御効果を検討した。
その結果、３×１０７ＣＦＵ／マウスの大腸菌を接種したグループにおいて、乳酸菌培養もろみ濃縮液群は対照群（感染）と比べてマウスの死亡匹数、平均生存日数および体重に顕著な差を示さなかったが、一般症状観察において軽度の消化器症状発現動物数の増加を示した。一方、１×１０８ＣＦＵ／マウスの大腸菌を接種したグループにおいて、乳酸菌培養もろみ濃縮液群は対照群（感染）と比べて一般症状に差を示さないものの、顕著なマウスの死亡匹数の減少と有意な平均生存日数の延長を示した。以上の結果から、乳酸菌培養もろみ濃縮液はマウスの大腸菌感染に対する強い感染防御作用を有することが判明した。
したがって、本発明の乳酸菌および発酵もろみ濃縮液の動物における免疫増強作用が証明された。
A group of 10 mice was inoculated with 3 × 10 7 CFU / mouse or 1 × 10 8 CFU / mouse of E. coli, and the protective effect of lactic acid bacteria culture mash concentrate was changed from changes in the number of dead animals, survival days, body weight and general symptoms. investigated.
As a result, in the group inoculated with 3 × 10 7 CFU / mouse of Escherichia coli, the lactic acid bacteria culture mash concentrate group showed a significant difference in the number of dead mice, average survival days and body weight compared to the control group (infection). There was no increase in the number of animals with mild gastrointestinal symptoms. On the other hand, in the group inoculated with 1 × 10 8 CFU / mouse of Escherichia coli, the lactic acid bacteria culture mash concentrate group showed no difference in general symptoms compared with the control group (infection), but the marked decrease in the number of dead mice And showed a significant increase in mean survival days. From the above results, it was found that the lactic acid bacterium culture mash concentrate has a strong protective effect against E. coli infection in mice.
Therefore, the immunity enhancing action in animals of the lactic acid bacteria and fermentation mash concentrate of the present invention was proved.
飼料
本発明の乳酸菌は、上記のとおり、いわゆる熟成もろみ、この例では、繰り返し回分発酵なる発酵法により最終回に取得した熟成もろみであり、その後の蒸留工程で回収するエタノール分も含む中間工程品であって、当該熟成もろみの蒸留によりエタノール分ともろみ濃縮液とに分離された、当該濃縮液から分離されたものである。
飼料は、このもろみ濃縮液（アルコール発酵残渣）を増殖可能な濃度に希釈すると共にｐＨを例えば６〜６．５程度に調整した所要の育成条件の基で、当該もろみ濃縮液に本発明の乳酸菌を添加するという人為的方法によって当該もろみ濃縮液に本発明の乳酸菌を増殖（乳酸発酵）させたものであり、こうしてアルコール発酵によって副生する残渣（廃液）を乳酸発酵生成物化して飼料としたものである。
As described above, the lactic acid bacterium of the present invention is a so-called ripening mash, in this example, an aging mash obtained by the fermentation process that is repeated batch fermentation, and an intermediate process product that also contains ethanol recovered in the subsequent distillation step Then, it is separated from the concentrated liquid, which is separated into an ethanol content and a concentrated liquid by distillation of the ripened mash.
The feed is prepared by diluting the mash concentrate (alcohol fermentation residue) to a concentration capable of growth and adjusting the pH to about 6 to 6.5, for example, in the mash concentrate according to the present invention. The lactic acid bacterium of the present invention is grown (lactic acid fermentation) in the mash concentrate by an artificial method of adding lactic acid, and thus a residue (waste liquid) by-produced by alcohol fermentation is converted into a lactic acid fermentation product to obtain a feed Is.
この乳酸発酵生成物は、直接、主な飼料の配合物として混合することによって飼料原料として利用することもできるし、乳酸発酵生成物を一旦遠心分離後、沈殿、粉末化したものを主な飼料の配合物として混合することによって飼料原料として利用することもできる。また、乳酸発酵生成物を直接或いは遠心分離後、沈殿、粉末化したもののみを飼料とすることもできる。 This lactic acid fermentation product can be used as a feed raw material by directly mixing it as a main feed composition, or after centrifuging the lactic acid fermentation product once precipitated and powdered, the main feed It can also be used as a feed raw material by mixing as a blend. Moreover, only the lactic acid fermentation product directly or after centrifugation and then precipitated and powdered can be used as feed.
図７のフローに基づいて、飼料原料の製造方法の一例を説明する。
先ず、もろみ濃縮液（濃縮残渣および残渣濃縮液）の一部をスタータータンク（小規模タンク）に導入する。
次に、予め培養（前々培養→前培養）してあった本発明の乳酸菌を前記スタータータンクに導入して、所定の攪拌、ｐＨおよび温度条件の基で本発明の乳酸菌を培養する。
次に、所定の濃度まで達した本発明の乳酸菌を、もろみ濃縮液を導入（所定の攪拌、ｐＨおよび温度条件の基）した発酵タンク（大規模タンク）に添加し、多量の本発明乳酸菌を培養する。
次に、発酵タンクで所定濃度まで達した本発明の乳酸菌を、遠心分離にかけ、沈殿物（＝乳酸菌を含むもの）とする。
最後に、この沈殿物を粉末化手段例えば流動層造粒、スプレードライ、真空凍結（←粉砕工程要）等により粉末化し、当該粉末物を回収しそのままおよび所定の加工（＝配合）、包装を施し飼料とする。
Based on the flow of FIG. 7, an example of the feed raw material manufacturing method will be described.
First, a part of the mash concentrate (concentrated residue and residue concentrate) is introduced into a starter tank (small tank).
Next, the lactic acid bacteria of the present invention previously cultured (pre-culture → pre-culture) are introduced into the starter tank, and the lactic acid bacteria of the present invention are cultured under predetermined agitation, pH and temperature conditions.
Next, the lactic acid bacteria of the present invention having reached a predetermined concentration are added to a fermentation tank (large-scale tank) into which a mash concentrate is introduced (based on predetermined stirring, pH and temperature conditions), and a large amount of the lactic acid bacteria of the present invention is added. Incubate.
Next, the lactic acid bacterium of the present invention that has reached a predetermined concentration in the fermentation tank is subjected to centrifugation to obtain a precipitate (= containing lactic acid bacterium).
Finally, the precipitate is pulverized by pulverization means such as fluidized bed granulation, spray drying, vacuum freezing (← requiring pulverization process), and the powdered material is collected as it is and subjected to predetermined processing (= formulation) and packaging. Use as feed.
本発明の乳酸菌を原料若しくは少なくとも原料の一つとした飼料は、当該乳酸菌の免疫増強作用および整腸作用等が発揮される有用な飼料として提供できると共に、当該飼料が、従来では破棄されていたアルコール残渣の乳酸発酵生成物であるため、飼料或いは飼料原料としての生産コストを低廉化することができ、牛・鶏・豚等の家畜やペットの好適な飼料として提供することができる。 The feed using the lactic acid bacterium of the present invention as a raw material or at least one of the raw materials can be provided as a useful feed that exhibits the immunity enhancing action and intestinal regulating action of the lactic acid bacterium, and the feed is an alcohol that has been conventionally discarded Since it is a residual lactic acid fermentation product, the production cost as feed or feed material can be reduced, and it can be provided as a suitable feed for livestock such as cattle, chickens and pigs, and pets.
Claims (12)
The lactic acid fermentation product of claims 5 and 6, wherein the concentration of viable bacteria of lactic acid bacteria FERM P-19169 is 10 8 cells / mL or more.
A lactic acid bacterium belonging to Lactobacillus paracasei that grows as a by-product residue by alcohol fermentation.
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