JP6811985B2 - Probiotic products containing natto spores - Google Patents
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本発明は、オロット酸産生能を有する納豆菌の胞子を含む、飼料、食品、サプリメント等のプロバイオティクス製品に関する。 The present invention relates to probiotic products such as feeds, foods and supplements containing spores of Bacillus natto having the ability to produce ollotic acid.
オロット酸は乳清から発見された化合物で、オロト酸、オロチン酸、ウラシル６−カルボン酸、ビタミンＢ１３とも呼ばれ、ピリミジンヌクレオチド生合成系における主要中間物質であり、ジヒドロオロト酸からジヒドロオロト酸デヒドロゲナーゼによって誘導され、オロト酸ホスホリボシルトランスフェラーゼ（ＰＲＰＰ）によってオロチジル酸となる。オロチジル酸は、さらに速やかにウリジン一リン酸(ＵＭＰ)に変換され、その後ウリジン三リン酸、シチジン三リン酸などのピリミジンヌクレオチドが合成される。このように、オロット酸はピリミジンの前駆体で分裂が盛んな組織や成長が盛んな状態では重要な栄養素である。オロット酸は、根菜、小麦胚芽、ビール酵母などの食品に含まれており、肝臓病の治療に用いられる外、葉酸やビタミンＢ１２の代謝に関与し、抗酸化作用や老化予防にも注目され、オロット酸含有飲料（特許文献１）も提案されている。 Orotic acid is a compound found from whey, orotic acid, orotic acid, uracil 6-carboxylic acid, also known as Vitamin B 13, a primary intermediates in pyrimidine nucleotide biosynthesis, by dihydroorotate dehydrogenase from dihydroorotate It is induced to orotic acid by orotic acid phosphoribosyl transferase (PRPP). Orotidilic acid is more rapidly converted to uridine monophosphate (UMP), after which pyrimidine nucleotides such as uridine triphosphate and cytidine triphosphate are synthesized. Thus, ollotic acid is a precursor of pyrimidine and is an important nutrient in tissues with active division and in a state of active growth. Orotic acid, root vegetables, wheat germ, are included in food products such as brewer's yeast, the outer used to treat liver disease, involved in the metabolism of folic acid and vitamin B 12, also it is noted antioxidant and anti-aging , Orot acid-containing beverage (Patent Document 1) has also been proposed.
他方、枯草菌(Bacillus subtilis)に関して、バチルス・ズブチリスSTA−17Eを培養し、培養物中にオロチン酸を生成蓄積させ、これを採取するオロチン酸の製造法（特許文献２）や、枯草菌を培養し、該微生物の菌体内で産生されたビタミンＫが菌体外に放出される前に該微生物菌体を回収することからなる枯草菌の培養方法（特許文献３）や、口内炎の痛みを軽減若しくは解消、又は傷口を矮小化、又は傷口を消失するバチルス・サブチルスＤＢ９０１１菌株の使用（特許文献４）が報告されている。 On the other hand, regarding Bacillus subtilis, a method for producing Bacillus subtilis (Patent Document 2) in which Bacillus subtilis STA-17E is cultured to generate and accumulate orotic acid in the culture, and the Bacillus subtilis is collected. A method for culturing Bacillus subtilis (Patent Document 3), which comprises culturing and recovering the Bacillus subtilis before the vitamin K produced in the cells of the microorganism is released to the outside of the cells, and the pain of stomatitis. The use of Bacillus subtilis DB9011 strain that reduces or eliminates, diminishes the wound, or eliminates the wound has been reported (Patent Document 4).
また、乳酸菌、ビフィズス菌、糖化菌、納豆菌、酵母菌のいずれか、もしくはそれらの混合物であるプロバイオティクス（特許文献５）や、ビィフィズス菌、乳酸菌、枯草菌、酪酸生成菌、乳酸利用菌、プロピオン酸生成菌の一種又は二種以上からなるプロバイオティクス（特許文献６）や、乳酸菌、酪酸菌、納豆菌、有胞子性乳酸菌等のプロバイオティクスを有効成分とする成長ホルモン分泌促進剤（特許文献７）が知られている。 In addition, probiotics (Patent Document 5), which is any one of lactic acid bacteria, bifidobacteria, saccharifying bacteria, natto bacteria, and yeast bacteria, or a mixture thereof, bifidobacteria, lactic acid bacteria, Bacillus subtilis, butyric acid-producing bacteria, and lactic acid-utilizing bacteria. , Probiotics consisting of one or more types of propionic acid-producing bacteria (Patent Document 6), and growth hormone secretion promoters containing probiotics such as lactic acid bacteria, butyric acid bacteria, natto bacteria, and spore-forming lactic acid bacteria as active ingredients. (Patent Document 7) is known.
オロット酸高含有食品や飼料が望まれているが、牛乳の８０ｍｇ／Ｌ（ホエイ中でも０．１５％）が通常の食品では最大の高含有食品であり、オロット酸効果の体感には１日当たり２００ｍｇ程度が望ましいことから、牛乳摂取によりオロット酸補給の要求を満たすことはできない。本発明の課題は、ピリミジンの前駆体で分裂が盛んな組織や成長が盛んな状態では重要な栄養素であるオロット酸の必要量を、合成品としてではなく、いわゆる食品として安価かつ簡便に摂取しうるプロバイオティクス製品を提供することにある。 Foods and feeds high in olott acid are desired, but 80 mg / L of milk (0.15% even in whey) is the largest high-content food in ordinary foods, and the effect of olott acid is 200 mg per day. Milk intake cannot meet the requirements for ollotic acid supplementation, as the degree is desirable. The object of the present invention is to ingest the required amount of ollotic acid, which is a precursor of pyrimidine and is an important nutrient in a tissue with active division and a state with active growth, inexpensively and easily as a so-called food, not as a synthetic product. It is to provide Uru Probiotic products.
本発明者は、オロット酸を産生する納豆菌を可食性素材で増殖させた後、増殖した納豆菌を芽胞化させると、オロット酸産生能を有する納豆菌の胞子を含むオロット酸含有プロバイオティクス製品を提供できることを見いだした。 The present inventor grows natto bacteria that produce ollotic acid with an edible material, and then spores the grown natto bacteria. I found that I could offer a product.
すなわち、本発明は、以下の事項により特定されるとおりのものである。
（１）オロット酸産生能を有する納豆菌の胞子を含む可食性基材からなることを特徴とするプロバイオティクス製品。
（２）オロット酸産生能を有する納豆菌の培養乾燥物を含む可食性基材であることを特徴とする上記（１）記載のプロバイオティクス製品。
（３）１０６ＣＦＵ／ｇ以上の胞子を含むことを特徴とする上記（１）又は（２）記載のプロバイオティクス製品。
（４）飼料、食品又はサプリメントであることを特徴とする上記（１）〜（３）のいずれか記載のプロバイオティクス製品。
（５）オロット酸産生能を有する納豆菌を可食性基材に添加・発酵させた後、増殖した納豆菌（栄養細胞）を芽胞化させることを特徴とする、納豆菌の胞子を含むプロバイオティクス製品の製造方法。
（６）納豆菌が増殖した可食性基材を乾燥することにより、納豆菌を芽胞化させることを特徴とする上記（５）記載の製造方法。
（７）プロバイオティクス製品が、１０６ＣＦＵ／ｇ以上の胞子を含むことを特徴とする上記（５）又は（６）記載の製造方法。
（８）プロバイオティクス製品が、飼料、食品又はサプリメントであることを特徴とする上記（５）〜（７）のいずれか記載の製造方法。
That is, the present invention is as specified by the following matters.
(1) A probiotic product comprising an edible base material containing spores of Bacillus natto having an orot acid-producing ability.
(2) The probiotic product according to (1) above, which is an edible base material containing a dried culture of Bacillus natto having an orotic acid-producing ability.
(3) The probiotic product according to (1) or (2) above, which contains spores of 10 6 CFU / g or more.
(4) The probiotic product according to any one of (1) to (3) above, which is a feed, food or supplement.
(5) A probiotic containing spores of Bacillus natto, which is characterized by adding and fermenting Bacillus natto capable of producing ollotic acid to an edible substrate and then spore-forming the grown Bacillus natto (nutrient cells). How to make tics products.
(6) The production method according to (5) above, wherein the natto bacterium is spore-forming by drying the edible substrate on which the natto bacterium has grown.
(7) probiotic product The production method of the above-mentioned (5) or (6), wherein the containing 10 6 CFU / g or more spores.
(8) The production method according to any one of (5) to (7) above, wherein the probiotic product is a feed, food or supplement.
本発明によると、オロット酸を飼料、食品、サプリメント等のプロバイオティクス製品として、安全かつ簡易に摂取することができる。また本発明によると、納豆菌を用いて、納豆以外の飼料、食品、サプリメント等のプロバイオティクス製品を提供することができる。 According to the present invention, ollotic acid can be safely and easily ingested as a probiotic product such as feed, food and supplement. Further, according to the present invention, natto bacteria can be used to provide probiotic products such as feeds, foods, and supplements other than natto.
本発明のプロバイオティクス製品としては、オロット酸産生能を有する納豆菌の胞子を含む可食性基材であれば特に制限されず、また本発明の納豆菌の胞子を含むプロバイオティクス製品の製造方法としては、オロット酸産生能を有する納豆菌を可食性基材に添加・発酵（増殖）させた後、乾燥（芽胞化）させる方法であれば特に制限されず、本発明においてプロバイオティクス製品とは人体に良い影響を与える納豆菌の胞子を含む食品や飼料等の可食性基材を意味する。 The probiotic product of the present invention is not particularly limited as long as it is an edible base material containing spores of Bacillus natto having an orotic acid-producing ability, and production of a probiotic product containing spores of Bacillus natto of the present invention. The method is not particularly limited as long as it is a method in which Bacillus natto having an orotic acid-producing ability is added to an edible substrate, fermented (proliferated), and then dried (spore-forming), and the probiotic product in the present invention. Means an edible base material such as foods and feeds containing spores of Bacillus natto, which have a positive effect on the human body.
上記オロット酸産生能を有する納豆菌としては、分類学上納豆菌(Bacillus subtilis var. natto)に分類される細菌であって、かつオロット酸の産生能力を有する細菌をいい、かかるオロット酸産生能を有する納豆菌は、例えば前記特許文献２に開示されているように、親株に紫外線照射等の公知の変異処理を施し、ウリジンヌクレオシドフォスフォリラーゼを欠損しかつピリミジンアナログ耐性を示しウリジン生産能を有する納豆菌として誘導し、次いでかかる納豆菌にニトロソグアニジン（ＮＴＧ）処理等の公知の変異処理を施すことにより、オロチン酸フォスフォリボシルトランスフェラーゼ欠損株か、又はオロチジン５’−モノフォスフェートデカルボキシラーゼ欠損株を誘導することにより、作製することができる。また、納豆菌の親株に、ＮＴＧ処理等の公知の変異処理を施すことにより、６アザウラシルの耐性株を取得し、次いでウリジンとウラシル生産株を選択してＮＴＧ処理等の公知の変異処理を施すことによりウラシル要求性を誘導することにより、オロット酸産生能を有する納豆菌を作製することができる。 The natto bacterium having the above-mentioned ollotic acid-producing ability refers to a bacterium that is taxonomically classified as natto bacterium (Bacillus subtilis var. Natto) and has an ollotic acid-producing ability. As disclosed in Patent Document 2, for example, the parent strain of Bacillus natto is subjected to a known mutation treatment such as irradiation with ultraviolet rays, and is deficient in uridine nucleoside phosphorylase and exhibits pyrimidine analog resistance to exhibit uridine-producing ability. By inducing the natto bacterium to have and then subjecting the natto bacterium to a known mutation treatment such as nitrosoguanidine (NTG) treatment, an orotidine phosphoribosyl transferase deficient strain or orotidine 5'-monophosphate decarboxylase deficiency It can be produced by inducing a strain. Further, the parent strain of Bacillus natto is subjected to a known mutation treatment such as NTG treatment to obtain a resistant strain of 6 azauracil, and then uridine and a uracil-producing strain are selected and subjected to a known mutation treatment such as NTG treatment. By inducing uracil requirement, natto bacteria having an orotic acid-producing ability can be produced.
上記納豆菌を添加して、発酵・増殖させる可食性基材としては、ヒトや家畜等の動物が食することができる材料であればよく、大豆、脱脂大豆粉、全脂大豆粉、大豆タンパク質、おから、大豆ミール、大豆煮汁、豆乳、籾殻、コーンコブ、豆殻、稲藁、バガス、ソバから、麦皮、モミガラ、米、米糠、小麦、小麦フスマ、麦芽絞り粕、グルテンフィード、小麦粉、トウモロコシ、コーンブラン、コーンミール、ビート、ビート粕、ミカン、カシスなどの果汁、青汁、野菜汁、落花生、落花生殻、油粕、魚粉、カニ殻、エビ殻、オキアミ微粉末、鋸屑、パルプ廃材、古紙、デンプン粕、可溶性デンプン、糖類、酵母エキス、脱脂粉乳、骨粉、ピートモス、食品カス等から選ばれる１種又は２種以上の固形物や液状物を挙げることができる。また、これら可食性基材には、納豆菌の培養に用いられる炭素原、窒素源、ミネラル等を必要に応じて添加・混合することができるが、通常の納豆菌の培養培地成分のみからなる培地は除かれる。 The edible base material to be fermented and propagated by adding the above-mentioned natto bacteria may be any material that can be eaten by animals such as humans and livestock, soybeans, defatted soybean flour, full-fat soybean flour, and soybean protein. , Okara, soybean meal, soybean broth, soymilk, rice husk, corn cob, bean husk, rice straw, bagus, buckwheat, wheat bark, fir, rice, rice bran, wheat, wheat bran, malt squeezed cake, gluten feed, flour, Fruit juice such as corn, corn blanc, corn meal, beet, beet cake, citrus, cassis, green juice, vegetable juice, peanuts, peanut shells, oil cake, fish flour, crab shells, shrimp shells, fine powder of oysters, sawdust, waste pulp, One or more solids or liquids selected from used paper, starch cake, soluble starch, sugars, yeast extract, non-fat dry milk, bone meal, peat moss, food waste and the like can be mentioned. In addition, carbon sources, nitrogen sources, minerals, etc. used for culturing Bacillus natto can be added and mixed to these edible substrates as needed, but they consist only of components of the culture medium of ordinary Bacillus natto. The medium is removed.
可食性基材上での納豆菌の培養条件としては、培養温度３０〜４５℃、好ましくは３５〜４０℃、培養ｐＨ６．０〜８．０、好ましくは６．５〜７．５を挙げることができる。また、培養時間は培養条件によって異なるが、通常３時間〜１０日間、好ましくは１２〜６０時間を挙げることができる。 Examples of the culture conditions for Bacillus natto on an edible substrate include a culture temperature of 30 to 45 ° C., preferably 35 to 40 ° C., a culture pH of 6.0 to 8.0, and preferably 6.5 to 7.5. Can be done. The culturing time varies depending on the culturing conditions, but is usually 3 to 10 days, preferably 12 to 60 hours.
可食性基材上で増殖させた、オロット酸産生能を有する納豆菌（栄養細胞）を芽胞化させて胞子を産生（形成）させる方法としては、公知の芽胞化技術であれば特に制限されず、栄養源の欠乏した状態で可食性基材上での培養を継続する方法や、可食性基材上で増殖した納豆菌（栄養細胞）を乾燥、例えば３５〜５０℃の温風によって風乾させる方法や、可食性基材上で増殖した納豆菌（栄養細胞）を高温処理する方法や、可食性基材上で増殖した納豆菌（栄養細胞）に酸又はアルカリ処理を施す方法等を挙げることができる。 The method for producing (forming) spores by spore-forming Bacillus natto (nutrient cells) having the ability to produce ollotic acid grown on an edible substrate is not particularly limited as long as it is a known spore-forming technique. , A method of continuing culture on an edible substrate in a state of lack of nutrient source, or drying natto bacteria (nutrient cells) grown on the edible substrate, for example, air-drying with warm air at 35 to 50 ° C. Examples include a method of treating natto bacteria (nutrient cells) grown on an edible base material at a high temperature, and a method of applying acid or alkali treatment to natto bacteria (nutrient cells) grown on an edible base material. Can be done.
本発明のプロバイオティクス製品には、製品１ｇ当たり１０６ＣＦＵ（Colony Forming Unit）以上、好ましくは１０８ＣＦＵ以上の納豆菌の胞子を含むことが好ましい。本発明のプロバイオティクス製品を食すると、納豆菌の栄養細胞は胃酸で死にやすいのに対して、納豆菌胞子は酸に強く、多くは生きたままで腸に到達すると考えられる。腸に到達した後に胞子は発芽し、栄養細胞となって、オロット酸を産生する可能性がある。 The probiotic product of the present invention, per product 1g 10 6 CFU (Colony Forming Unit ) or, preferably contain spores or Bacillus natto 10 8 CFU. When the probiotic product of the present invention is eaten, the vegetative cells of Bacillus natto are easily killed by gastric acid, whereas the spores of Bacillus natto are resistant to acid, and most of them are considered to reach the intestine alive. After reaching the intestine, spores can germinate and become vegetative cells, producing ollotic acid.
本発明のプロバイオティクス製品の態様・形態としては特に制限されないが、顆粒状、粉末状、バー（棒）状、ブロック状、カプセル状、錠剤状等の種々の態様、形態を挙げることができる。また、本発明のプロバイオティクス製品は包装された態様、形態で提供することができる。 The aspect and form of the probiotic product of the present invention are not particularly limited, and various aspects and forms such as granule, powder, bar, block, capsule, and tablet can be mentioned. .. In addition, the probiotic product of the present invention can be provided in a packaged form and form.
以下、実施例等により本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例等により制限されない。 Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the technical scope of the present invention is not limited by these Examples and the like.
Bacillus subtilis ver Natto NBRC-3335株をBacillus用培地（Tryptone １％, Yeast Extract ０．３％、ＮａＣｌ ０．５％、ｐＨ７．０）で３０℃にて一夜培養後、同培地に１％（ｖ／ｖ）接種し３０℃にて振盪培養した。対数期（ＯＤ６６０≒０．５）に達した菌体を遠心分離（３，０００ｒｐｍ、１０分）で集菌し、５０ｍＭトリスマレイン酸緩衝液（ｐＨ６．５）で洗浄後、１００μｇ／Ｌのニトロソグアニジン（ＮＴＧ）を含む同緩衝液中で３０分処理を行った。洗浄後に、６アザウラシル１ｍｇ／ｍＬを添加した最小寒天培地に塗布し、３７℃にて４日間培養し、６アザウラシルの耐性株を５０株取得した。上記最小寒天培地の組成は、以下の［表１］に示すとおりである。 Bacillus subtilis ver Natto NBRC-3335 strain was cultured overnight at 30 ° C. in Bacillus medium (Tryptone 1%, Yeast Extract 0.3%, NaCl 0.5%, pH 7.0), and then 1% (v) in the same medium. / V) Inoculated and cultured with shaking at 30 ° C. Bacteria that have reached the logarithmic phase (OD 660 ≈0.5) are collected by centrifugation (3,000 rpm, 10 minutes), washed with 50 mM Trismalate buffer (pH 6.5), and then 100 μg / L. The treatment was carried out in the same buffer solution containing nitrosoguanidine (NTG) for 30 minutes. After washing, the cells were applied to a minimum agar medium containing 1 mg / mL of 6 azauracil and cultured at 37 ° C. for 4 days to obtain 50 resistant strains of 6 azauracil. The composition of the minimum agar medium is as shown in [Table 1] below.
得られた６アザウラシルの耐性株５０株を以下の［表２］に示す種培養培地で培養後、生産培地で４日間、３０℃にて培養を行った結果、Ｎｏ．３９株において、ウリジン３ｇ／Ｌ、ウラシル７ｇ／Ｌの蓄積を認めた。 The obtained 50 resistant strains of 6 azauracil were cultured in the seed culture medium shown in the following [Table 2], and then cultured in the production medium for 4 days at 30 ° C. Accumulation of uridine 3 g / L and uracil 7 g / L was observed in 39 strains.
Ｎｏ．３９株について実施例１と同様のＮＴＧ処理を行い、５フルオロウラシル１ｍｇ／ｍＬ、ウラシル５０μｇ／ｍＬを添加した最小寒天培地に塗布し、３７℃にて４日間培養し、得られたコロニーを前記最小寒天培地とウラシル５０μｇ／ｍＬを添加した最小培地にレプリカし、ウラシル要求性を示す株１０株を取得した。これらウラシル要求性株１０株を実施例１の生産培地で培養を行った結果、すべての菌株でオロット酸の生産性が確認され、６株がオロット酸単独、４株がオロット酸とオロチジンの併産株であった。ＴＧ−７株がオロット酸１６ｇ／Ｌと最も高い生産性を示した。この際、保存培養液中にオロット酸の結晶の析出も認められた。 No. The 39 strains were subjected to the same NTG treatment as in Example 1, applied to a minimum agar medium supplemented with 5fluorouracil 1 mg / mL and uracil 50 μg / mL, cultured at 37 ° C. for 4 days, and the obtained colonies were obtained as the minimum. 10 strains showing uracil requirement were obtained by replicating to agar medium and a minimum medium supplemented with 50 μg / mL of uracil. As a result of culturing these 10 uracil-requiring strains in the production medium of Example 1, the productivity of ollotic acid was confirmed in all the strains, 6 strains were ollotic acid alone, and 4 strains were a combination of ollotic acid and orotidine. It was a stock. The TG-7 strain showed the highest productivity of 16 g / L of ollotic acid. At this time, precipitation of ollotic acid crystals was also observed in the preservation culture solution.
大豆１００ｇをフライパンで１５分炒った後すり鉢中で軽く粉砕し、１８時間水に浸漬後、圧力鍋で８０分加熱後に自然冷却した。寒天培地で１日生育させたＴＧ−７株を１白金耳でかきとって２ｍＬの殺菌水に懸濁後、蒸した大豆とよく混ぜ、煮沸殺菌したタッパーに入れて４０℃で１８時間醗酵後、冷蔵庫で１日保存し、オロット酸含有ひきわり納豆を調製した。得られた納豆は、やや糸引きが親株よりも弱い感があるものの、十分納豆としての形状や風味を有し、納豆質量１ｇあたり２．５〜３．５ｍｇのオロット酸を含有していた。 After roasting 100 g of soybeans in a frying pan for 15 minutes, they were lightly crushed in a mortar, soaked in water for 18 hours, heated in a pressure cooker for 80 minutes, and then naturally cooled. The TG-7 strain grown on an agar medium for 1 day is scraped with a loop loop, suspended in 2 mL of sterilized water, mixed well with steamed soybeans, placed in a boiled sterilized tapper, and fermented at 40 ° C for 18 hours. , Stored in a refrigerator for 1 day to prepare natto containing ollotic acid. The obtained natto had a sufficient shape and flavor as natto, although the stringiness was slightly weaker than that of the parent strain, and contained 2.5 to 3.5 mg of ollotic acid per 1 g of natto mass.
青汁粉末５％、グルコース１０％、ソイビーンミール２％、尿素１％、ウラシル５０ｍｇ／Ｌを含む培地溶液３０ｍＬを２００ｍＬのひだ付きフラスコに入れ、ＴＧ−７株のタネ培養液１ｍＬを添加し、３７℃にて４日間、２２０ｒｐｍで浸透培養を行った。得られた培養液を加熱乾燥した結果、オロット酸を１５質量％含有し、オロット酸生産性納豆菌胞子を５×１０９ＣＦＵ／ｇ含有する粉末が得られた。 30 mL of a medium solution containing 5% green juice powder, 10% glucose, 2% soybean meal, 1% urea, and 50 mg / L uracil was placed in a 200 mL pleated flask, and 1 mL of TG-7 strain seed culture solution was added. Osmotic culture was performed at 220 rpm for 4 days at 37 ° C. As a result of heating and drying the obtained culture solution, a powder containing 15% by mass of ollotic acid and 5 × 10 9 CFU / g of natto spores producing ollotic acid was obtained.
乾燥おから（国産おからパウダー ヘルシーカンパニー社製）５０ｇに脱イオン水２００ｍＬを添加し、十分に混ぜて含水率８０％としたものを、１２１℃にて２０分殺菌した。４０℃まで自然冷却した後に、ＴＧ−７株の種培養液を２ｍＬ添加したおからと、ＴＧ−７株の親株のＮＢＲＣ−３３３５株の種培養液を２ｍＬ添加したおからについて、それぞれ十分に撹拌後、３７℃にて２日間培養した。乾燥後に得られたＴＧ−７株おから粉末は２×１０９ＣＦＵ／ｇの胞子を含有し、ＮＢＲＣ−３３３５株おから粉末は５×１０９ＣＦＵ／ｇの胞子を含有していた。 200 mL of deionized water was added to 50 g of dried okara (manufactured by Domestic Okara Powder Healthy Company), and the mixture was thoroughly mixed to have a water content of 80% and sterilized at 121 ° C. for 20 minutes. After naturally cooling to 40 ° C., 2 mL of the seed culture solution of the TG-7 strain was added to the okara and 2 mL of the seed culture solution of the parent strain of the TG-7 strain, NBRC-3335, was added to the okara. After stirring, the cells were cultured at 37 ° C. for 2 days. The TG-7 strain okara powder obtained after drying contained 2 × 10 9 CFU / g spores, and the NBRC-3335 strain okara powder contained 5 × 10 9 CFU / g spores.
２Ｌの２つのひだ付きフラスコに市販の豆乳２００ｍＬ、グルコース１０ｇ、尿素４ｇ、ウラシル１０ｍｇを添加し、１２１℃にて２０分殺菌した。４０℃まで自然冷却した後に、ＴＧ−７株とＮＢＲＣ−３３３５株の種培養液を２ｍＬ添加し、３７℃、２２０ｒｐｍで３日間培養した。ＮＢＲＣ−３３３５株ではオロット酸の蓄積は無かったが、ＴＧ−７株では１２ｇ／Ｌのオロット酸を含む発酵豆乳が得られた。 200 mL of commercially available soymilk, 10 g of glucose, 4 g of urea, and 10 mg of uracil were added to two 2 L pleated flasks, and the mixture was sterilized at 121 ° C. for 20 minutes. After air cooling to 40 ° C., 2 mL of seed culture solutions of TG-7 strain and NBRC-3335 strain were added, and the cells were cultured at 37 ° C. and 220 rpm for 3 days. There was no accumulation of ollotic acid in the NBRC-3335 strain, but fermented soymilk containing 12 g / L of ollotic acid was obtained in the TG-7 strain.
大ヨークシャー種２腹の新生豚１８頭を６頭ずつ３群に分け、実施例６で得た２種類の納豆菌発酵豆乳を凍結乾燥した粉末について、それぞれ納豆菌胞子が５×１０８個／ｍＬになるように生理食塩水で懸濁し、出産後から毎日１ｍＬを母乳摂取後に経口で投与した。コントロールには同量の乾燥おからを懸濁して投与した。
２週齢目での豚糞便中のBacillus群の菌は、コントロールの乾燥おから投与群では１０５ＣＦＵ／ｇ以下であったのに対し、上記納豆菌株おから粉末投与群のいずれにおいても１０６〜１０７ＣＦＵ／ｇと有意に高く、菌株間での差は認められなかった。大腸菌数は３群とも、約１０８ＣＦＵ／ｇで有意な差はなかった。また糞便の状態は、以下の［表３］に示す０から４の５段階で毎日評価した。糞便の平均スコアと２８日目の平均体重を［表４］に示す。
18 newborn pigs of Large White pig breed 2 litter were divided into 3 groups of 6 pigs each, and the powder obtained by lyophilizing the 2 types of fermented soymilk of Bacillus natto obtained in Example 6 had 5 × 10 8 spores of Bacillus natto. It was suspended in physiological saline so as to be mL, and 1 mL was orally administered after breastfeeding every day after delivery. The same amount of dried okara was suspended and administered to the control.
The Bacillus group bacteria pig feces in 2 weeks of age, whereas in the dry Okara administration group control was less than 10 5 CFU / g, 10 in any of the above Natto Okara powder administered group 6 ~10 7 CFU / g and significantly higher, the difference between the strains was observed. The number of E. coli All three groups did not differ significantly from about 10 8 CFU / g. The stool condition was evaluated daily on a scale of 0 to 4 shown in [Table 3] below. The average stool score and the average body weight on the 28th day are shown in [Table 4].
本発明によると、ピリミジンの前駆体で分裂が盛んな組織や成長が盛んな状態においては重要な栄養素であるオロット酸を提供することができるので、本発明は健康産業、飼育産業で有用である。 According to the present invention, the precursor of pyrimidine can provide ollotic acid, which is an important nutrient in tissues with active division and in a state of active growth, and thus the present invention is useful in the health industry and the breeding industry. ..
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