JP4999495B2 - Fermented food and drink and method for producing the same - Google Patents
Fermented food and drink and method for producing the same Download PDFInfo
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Description
本発明は、新規乳酸菌株、該乳酸菌株を含有する飲食品および発酵飲食品、並びに該乳酸菌株を用いる発酵飲食品の製造方法に関する。 The present invention relates to a novel lactic acid strain, a food and drink and a fermented food and drink containing the lactic acid strain, and a method for producing a fermented food and drink using the lactic acid strain.
ラクトバチルス ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）に属する乳酸菌株は、乳酸菌の中でも特に耐ストレス性が強く、極めて広範な優れた生理作用を有することが知られており、これまでに、例えば、抗アレルギー剤、抗胃炎剤、抗潰瘍剤、肝炎治療・予防剤、腫瘍増殖抑制剤、抗腫瘍活性剤、 γ−アミノ酪酸の生産への利用について報告されている。
なかでも、ラクトバチルス ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）ＦＥＲＭ ＢＰ−４６９３株はインターフェロン産生能向上剤として利用可能な事が知られており（特許文献１参照）、極めて有用な乳酸菌である。また、生きた状態で摂取すれば、容易に腸に到達して長く生存できるため、健康増進を志向した飲食品として、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株を生きた状態で含有する発酵飲食品が望まれている。
Especially, it is known that Lactobacillus brevis ( Lactobacillus brevis ) FERM BP-4693 can be utilized as an interferon production ability improving agent (refer patent document 1), and is an extremely useful lactic acid bacterium. In addition, since it can easily reach the intestines and survive for a long time if ingested in a live state, a fermented food or drink containing Lactobacillus brevis FERM BP-4693 in a live state as a food or drink aimed at improving health. It is desired.
しかしながら、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株は、発酵中に菌体外多糖を産出するため、十分な生菌数を含有する発酵物を得ようとすると、菌が凝集してしまい、発酵物中の菌数の分布が不均一になったり、発酵物の粘度が変化したり、製品の外観が悪くなる等の問題点があった。 However, since the Lactobacillus brevis FERM BP-4693 strain produces exopolysaccharide during fermentation, the bacteria will aggregate when attempting to obtain a fermented product containing a sufficient number of viable cells. There are problems such as non-uniform distribution of the number of bacteria, changes in the viscosity of the fermented product, and poor product appearance.
本発明は上記事情に鑑みてなされたものであり、凝集性を示さないなど取り扱い性に優れ、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株と同様の生理作用を有し、飲食品や医薬品の製造に好適な新規乳酸菌株、該乳酸菌株を含有する飲食品および発酵飲食品、並びに該乳酸菌株を用いる発酵飲食品の製造方法を提供することを課題とする。 The present invention has been made in view of the above circumstances, has excellent handling properties such as not exhibiting aggregability, has the same physiological action as Lactobacillus brevis FERM BP-4693 strain, and is suitable for the production of foods and drinks and pharmaceuticals. It is an object of the present invention to provide a novel novel lactic acid strain, a food and drink and a fermented food and drink containing the lactic acid strain, and a method for producing a fermented food and drink using the lactic acid strain.
上記課題を解決するため、
請求項１に記載の発明は、ラクトバチルス ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）ＦＥＲＭ Ｐ−２１２２２株を用いて、野菜、果実、穀類および豆類からなる群から選択される一種以上を含む原料の発酵を行う工程を有する発酵飲食品の製造方法である。
請求項２に記載の発明は、請求項１に記載の製造方法で得られた発酵飲食品である。
To solve the above problem,
The invention according to
Invention of Claim 2 is the fermented food / beverage products obtained by the manufacturing method of
本発明の乳酸菌株は、凝集性を示さないため、取り扱い性に優れ、発酵物の品質を均一に保持でき、発酵物の製造に好適である。また、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株と同様の優れた生理作用を有する。したがって、飲食品や医薬品の製造に好適である。 Since the lactic acid strain of the present invention does not exhibit aggregability, it is excellent in handleability, can maintain the quality of the fermented product uniformly, and is suitable for producing the fermented product. Moreover, it has the same outstanding physiological effect as the Lactobacillus brevis FERM BP-4693 strain. Therefore, it is suitable for the production of food and drink and pharmaceutical products.
以下、本発明について、詳しく説明する。
＜ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の取得＞
ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株（以下、「ＦＥＲＭ ＢＰ−４６９３株」と略記することがある）を、市販のＭＲＳ液体培地（ＯＸＯＩＤ社製）で、３０℃、１８時間培養した。次に、その培養液を、トマト果汁１０ｍＬに1容量％植菌し、３０℃で１８時間培養し、この培養物を、ＭＲＳ寒天培地（ＯＸＯＩＤ社製）を用いて混釈培養した。
ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株をＭＲＳ寒天培地で培養した時に通常観察されるコンペイトウ状の形態とは異なる円盤状のコロニーを、イノキュレーティングニードル（Ｎｕｎｃ社製）で穿刺し、付着した菌体をＭＲＳ液体培地に接種して、３０℃で１８時間培養した。
さらに純化するため、この培養液を、ＭＲＳ寒天培地を用いて混釈培養し、３０℃で２日間培養後に形成した円盤状のコロニーを同様に分離、培養し、これを４回繰り返した。
その結果、ＭＲＳ寒天培地で混釈培養した時のプレート中でのコロニー形態が、ラクトバチルス ブレビスＦＥＲＭ ＢＰ−４６９３株とは明らかに異なる変異株として、ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株（以下、「本菌株」と略記することがある）を得た。この時のコロニーの撮影画像を図１に示す。図１（ａ）は、ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の撮影画像、図１（ｂ）は、ラクトバチルス ブレビスＦＥＲＭ ＢＰ−４６９３株の撮影画像である。
なお、ここで培養に用いたラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株は、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より分譲可能である。
Hereinafter, the present invention will be described in detail.
<Acquisition of Lactobacillus brevis FERM P-21222 strain>
Lactobacillus brevis FERM BP-4693 strain (hereinafter may be abbreviated as “FERM BP-4693 strain”) was cultured in a commercially available MRS liquid medium (manufactured by OXOID) at 30 ° C. for 18 hours. Next, the culture solution was inoculated into 10 mL of tomato juice at 1% by volume, cultured at 30 ° C. for 18 hours, and this culture was subjected to pour culture using MRS agar medium (manufactured by OXOID).
Lactobacillus brevis FERM BP-4693 strain was punctured with an innoculating needle (manufactured by Nunc), and adhered to a discoid colony different from the complex-like shape normally observed when cultured on MRS agar medium. The body was inoculated into MRS liquid medium and cultured at 30 ° C. for 18 hours.
In order to further purify, this culture broth was mixed-cultured using MRS agar medium, and the discoid colonies formed after culturing at 30 ° C. for 2 days were similarly separated and cultured, and this was repeated four times.
As a result, the Lactobacillus brevis FERM P-21222 strain (hereinafter referred to as the “Lactobacillus brevis FERM P-21222 strain”) is a mutant that clearly differs from the Lactobacillus brevis FERM BP-4693 strain in the plate when mixed with MRS agar. This strain may be abbreviated as “this strain”. A captured image of the colony at this time is shown in FIG. FIG. 1 (a) is a photographed image of Lactobacillus brevis FERM P-21222 strain, and FIG. 1 (b) is a photographed image of Lactobacillus brevis FERM BP-4693 strain.
In addition, the Lactobacillus brevis FERM BP-4693 strain | stump | stock used for culture | cultivation here can be sold from an independent administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center.
＜ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の同定＞
（形態的性質）
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の形態的性質は、前記各培地で培養した時のコロニーを顕微鏡観察することにより確認した。その結果、細胞形態は桿菌であり、ラクトバチルス ブレビスＦＥＲＭ ＢＰ−４６９３株およびラクトバチルス ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）ＪＣＭ１０５９Ｔ株（以下、「基準株」と略記することがある）同様、胞子を有さず、運動性も有していなかった。
<Identification of Lactobacillus brevis FERM P-21222 strain>
(Morphological properties)
The morphological properties of Lactobacillus brevis FERM P-21222 strain were confirmed by microscopic observation of colonies when cultured in each of the above media. As a result, the cell morphology was Neisseria gonorrhoeae, and it did not have spores, like Lactobacillus brevis FERM BP-4693 and Lactobacillus brevis ( Lactobacillus brevis ) JCM1059 T strain (hereinafter sometimes abbreviated as “reference strain”). It also had no motility.
（培養的性質）
前記ＭＲＳ寒天培地で培養した時のコロニー形態は、円形、半球形、スムース、白色であった。
また、ＢＬ寒天培地（栄研化学株式会社製）で培養した時のコロニー形態は、円形、半レンズ状、スムース、白色であった。
なお、ラクトバチルス ブレビスＦＥＲＭ ＢＰ−４６９３株も、これら培地で培養した場合は、いずれも本菌株と同様の性質を示し、前記混釈培養の場合とは、異なるコロニー形態であった。
(Culture characteristics)
The colony morphology when cultured on the MRS agar medium was circular, hemispherical, smooth, and white.
Moreover, the colony form when cultured on BL agar medium (manufactured by Eiken Chemical Co., Ltd.) was circular, semi-lens, smooth, and white.
The Lactobacillus brevis FERM BP-4693 strain also showed the same properties as this strain when cultured in these media, and had a different colony form from that of the pour culture.
（生理学的性質）
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の生理学的性質を公知の方法により評価し、ラクトバチルス ブレビスＦＥＲＭ ＢＰ−４６９３株およびラクトバチルス ブレビス ＪＣＭ１０５９Ｔ株と比較した。結果を表１に示す。
表１に示すように、本菌株は、ＦＥＲＭ ＢＰ−４６９３株および基準株と同じ生理学的性質を示した。
(Physiological properties)
Physiological properties of Lactobacillus brevis FERM P-21222 strain were evaluated by a known method and compared with Lactobacillus brevis FERM BP-4693 strain and Lactobacillus brevis JCM1059 T strain. The results are shown in Table 1.
As shown in Table 1, this strain showed the same physiological properties as the FERM BP-4693 strain and the reference strain.
（炭水化物の分解性）
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の各種炭水化物の分解性を、細菌検査同定用キットＡｐｉ ５０ ＣＨ（ｂｉｏ Ｍｅｒｉｅｕｘ社製）を使用して評価し、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株およびラクトバチルス ブレビス ＪＣＭ１０５９Ｔ株と比較した。その結果を表２および３に示す。
表２および３から明らかなように、本菌株と基準株とは、リボース、β−メチル−Ｄ−キシロース、ガラクトース、Ｄ−フラクトース、α−メチル−Ｄ−グルコシドの資化性に相違が認められた（表中、「＊」で示した）。一方、本菌株とＦＥＲＭ ＢＰ−４６９３株との間に資化性の相違は認められなかった。
(Carbohydrate degradability)
The degradability of various carbohydrates of Lactobacillus brevis FERM P-21222 strain was evaluated using a bacterial test identification kit Api 50 CH (manufactured by bio Merieux), and Lactobacillus brevis FERM BP-4893 strain and Lactobacillus brevis JCM1059 Comparison with T strain. The results are shown in Tables 2 and 3.
As is apparent from Tables 2 and 3, there are differences in the utilization of ribose, β-methyl-D-xylose, galactose, D-fructose, and α-methyl-D-glucoside between this strain and the reference strain. (Indicated by “*” in the table). On the other hand, there was no difference in assimilation between the present strain and the FERM BP-4693 strain.
（塩基配列）
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の１６Ｓ ｒＤＮＡの塩基配列（部分配列）を公知の方法で決定した。決定した塩基配列のうち、５’末端（１番目）から４９０番目までの塩基配列を配列番号１に示し、９１１番目から１４１０番目までの塩基配列を配列番号２に示す。この決定した塩基配列について、大学共同利用機関法人 情報・システム研究機構 国立遺伝学研究所 生命情報・ＤＤＢＪ研究センターが運営している日本ＤＮＡデータバンクの相同性検索（ＢＬＡＳＴ）を行った。
その結果、本菌株の配列番号１に示す塩基配列は、ＦＥＲＭ ＢＰ−４６９３株と同じであり、８１番目の塩基が基準株とは異なっていた。また本菌株の配列番号２に示す塩基配列は、３７６番目の塩基が基準株とは異なっており、４７０、４９１、４９２、４９６、４９８〜５００番目の塩基が、ＦＥＲＭ ＢＰ−４６９３株および基準株と異なっていた（ＦＥＲＭ ＢＰ−４６９３株と基準株とでは同じであった）。そして、本菌株とＦＥＲＭ ＢＰ−４６９３株、本菌株と基準株とは、いずれも９９％以上の相同性を示したが、異なる菌である事が示唆された。
(Base sequence)
The base sequence (partial sequence) of 16S rDNA of Lactobacillus brevis FERM P-21222 strain was determined by a known method. Among the determined base sequences, the base sequence from the 5 ′ end (first) to the 490th is shown in SEQ ID NO: 1, and the base sequence from the 911st to 1410th is shown in SEQ ID NO: 2. The determined nucleotide sequence was subjected to a homology search (BLAST) of the Japan DNA data bank operated by the National Institute of Genetics, National Institute of Genetics, Bioinformatics / DDBJ Research Center.
As a result, the base sequence shown in SEQ ID NO: 1 of the present strain was the same as the FERM BP-4693 strain, and the 81st base was different from the reference strain. The base sequence shown in SEQ ID NO: 2 of this strain is different from the reference strain in the 376th base, and the 470, 491, 492, 496, and 498-500th bases are the FERM BP-4693 strain and the reference strain. (FERM BP-4693 and the reference strain were the same). And although this strain and the FERM BP-4693 strain and this strain and the reference strain all showed 99% or more homology, it was suggested that they are different bacteria.
以上より、本菌株は、ＦＥＲＭ ＢＰ−４６９３株と比較すると、前記生理学的性質および炭水化物の分解性が同じであり、１６Ｓ ｒＤＮＡの前記塩基配列が９９％以上の相同性を有し、前記混釈培養時のコロニー形態が異なっていた。そして、後記実施例に示すように、培養液の凝集性が全く異なっており、本菌株は凝集性を示さなかった。
以上の結果より、本菌株は、ラクトバチルス ブレビスに属する新規の乳酸菌株であることが示された。
From the above, this strain has the same physiological properties and carbohydrate degradability as the FERM BP-4893 strain, the base sequence of 16S rDNA has 99% or more homology, The colony morphology at the time of culture was different. And as shown in the below-mentioned Example, the aggregation property of the culture solution was completely different, and this strain did not show the aggregation property.
From the above results, this strain was shown to be a novel lactic acid strain belonging to Lactobacillus brevis.
＜乳酸菌株＞
本発明の乳酸菌株は、１６Ｓ ｒＤＮＡの、５’末端（１番目）〜４９０番目の塩基配列が配列番号１で表わされ、９１１番目〜１４１０番目の塩基配列が配列番号２で表わされるラクトバチルス ブレビスに属するものである。
そして、該乳酸菌株に、ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株は包含される。
<Lactic acid strain>
In the lactic acid strain of the present invention, the 5S end (first) to 490th base sequence of 16S rDNA is represented by SEQ ID NO: 1, and the 911st to 1410th base sequence is represented by SEQ ID NO: 2. It belongs to Brevis.
And the Lactobacillus brevis FERM P-21222 strain is included in this lactic acid strain.
本発明のラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株は、平成１９年２月１５日）付けで受託番号ＦＥＲＭ Ｐ−２１２２２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。 The Lactobacillus brevis FERM P-21222 strain of the present invention is deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-21222 on February 15, 2007).
本発明の乳酸菌株は、抗菌活性を有する。特に食品危害微生物であるバチルス コアギュランス（Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ）やリステリア モノサイトジェネス（Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓ）等に優れた活性を示すため、飲食品や医薬品の成分として好適である。 The lactic acid strain of the present invention has antibacterial activity. In particular, it exhibits excellent activity against food-hazardous microorganisms such as Bacillus coagulans and Listeria monocytogenes , and therefore, it is suitable as a component of foods and drinks and pharmaceuticals.
また、本発明の乳酸菌株は、培養時および保存時に凝集性がない。したがって、本菌株を用いた発酵飲食品は、製造適性および保存適性に優れる。 In addition, the lactic acid strain of the present invention does not aggregate when cultured and stored. Therefore, the fermented food / beverage products using this strain are excellent in manufacturing aptitude and storage aptitude.
本発明の乳酸菌株は、特に植物性原料の発酵に好適である。使用可能な植物性原料としては、例えば、野菜、果実、穀類および豆類が挙げられる。
野菜としては、例えば、トマト、赤ピーマン、ニンジン、キャベツ、はくさい、レタス、大根、ほうれん草、ケール、玉葱、なす、プチヴェール（登録商標）（ケールと芽キャベツの交雑種）、しいたけ、シメジ等が挙げられる。
果実としては、例えば、グレープフルーツ、オレンジ、リンゴ、ブドウ、イチゴ、パイナップル、キウイ、グアバ、マンゴー、アセロラ、ブルーベリー、ザクロ、モモ、洋なし、パパイヤ、メロン、スイカ、バナナ、イチジク等が挙げられる。
穀類としては、例えば、麦（麦芽）、米等を、豆類としてはダイズ、エンドウ等が挙げられる。
本発明においては、これら植物性原料を単独で用いても良く、二種以上を併用しても良い。目的に応じて、適宜最適な組み合わせを選択できる。
The lactic acid strain of the present invention is particularly suitable for fermentation of plant raw materials. Examples of plant raw materials that can be used include vegetables, fruits, cereals, and beans.
Examples of vegetables include tomatoes, red peppers, carrots, cabbage, cabbage, lettuce, radish, spinach, kale, onion, eggplant, petit ver (registered trademark) (a hybrid of kale and brussels sprouts), shiitake mushrooms, and shimeji mushrooms. It is done.
Examples of the fruit include grapefruit, orange, apple, grape, strawberry, pineapple, kiwi, guava, mango, acerola, blueberry, pomegranate, peach, pear, papaya, melon, watermelon, banana, fig and the like.
Examples of cereals include wheat (malt) and rice, and examples of beans include soybeans and peas.
In the present invention, these plant raw materials may be used alone or in combination of two or more. An optimal combination can be selected as appropriate according to the purpose.
本発明の乳酸菌株は、前培養してから発酵に用いることが好ましい。前培養は公知の方法で行えば良く、例えば、市販の乳酸菌用培地を、必要に応じて所定の濃度となるように蒸留水に溶解させ、オートクレーブ滅菌した後、本発明の乳酸菌株を植菌して、所定時間培養する方法が挙げられる。前培養時に乳酸菌株は、固体培地に植菌しても良いが、上記のように液体培地に植菌することが好ましい。 The lactic acid strain of the present invention is preferably used for fermentation after pre-culture. The preculture may be performed by a known method. For example, a commercially available medium for lactic acid bacteria is dissolved in distilled water so as to have a predetermined concentration as necessary, sterilized by autoclave, and then the lactic acid strain of the present invention is inoculated. Thus, a method of culturing for a predetermined time can be mentioned. The lactic acid strain may be inoculated in a solid medium during pre-culture, but it is preferable to inoculate in a liquid medium as described above.
本発明の乳酸菌株を用いた場合の発酵条件は、用いる原料および目的に応じて適宜選択すれば良いが、本発明の乳酸菌株の植菌量は０．１〜１０容量％であることが好ましく、発酵温度は２０〜４０℃であることが好ましく、発酵時間は１２〜７２時間であることが好ましい。 Fermentation conditions when the lactic acid strain of the present invention is used may be appropriately selected according to the raw material to be used and the purpose, but the inoculated amount of the lactic acid strain of the present invention is preferably 0.1 to 10% by volume. The fermentation temperature is preferably 20 to 40 ° C., and the fermentation time is preferably 12 to 72 hours.
＜飲食品、発酵飲食品の製造方法＞
本発明の飲食品は、上記本発明の乳酸菌株を含有するものである。具体的には、例えば、別途調製した飲食品に本発明の乳酸菌株を添加したものでも良いし、本発明の乳酸菌株を用いて原料を発酵させて得られた発酵飲食品でも良い。
別途調製した飲食品に本発明の乳酸菌株を添加する場合の添加量は、特に限定されない。
本発明の乳酸菌株を用いて原料を発酵させる場合は、発酵物をそのまま発酵飲食品としても良いし、適宜後処理や加工を行って発酵飲食品としても良い。発酵条件は先に述べた条件と同様である。
<Manufacturing method of food and drink, fermented food and drink>
The food-drinks of this invention contain the lactic acid strain of the said invention. Specifically, for example, a product prepared by adding the lactic acid strain of the present invention to a separately prepared food or beverage, or a fermented food or beverage obtained by fermenting a raw material using the lactic acid strain of the present invention may be used.
The addition amount in the case of adding the lactic acid strain of this invention to the food-drinks prepared separately is not specifically limited.
When fermenting a raw material using the lactic acid strain of the present invention, the fermented product may be used as it is as a fermented food or drink, or may be appropriately subjected to post-treatment or processing to obtain a fermented food or drink. Fermentation conditions are the same as those described above.
発酵時の原料は、乳酸菌発酵に使用可能なものであれば特に限定されないが、前記植物性原料が好適である。そして所望の品質に応じて、適宜組み合わせて用いても良い。
また、発酵時の原料は、目的に応じて適宜加工した状態のものを用いることができる。例えば、植物性原料は、搾汁液、磨砕物または破砕物、あるいはこれらを濃縮物、希釈物または乾燥物等に加工した形態で用いることができる。具体的には、ダイズであれば、豆乳の形態で用いることもできる。本発明においては、発酵原料として液状のものが特に好適である。
Although the raw material at the time of fermentation will not be specifically limited if it can be used for lactic acid bacteria fermentation, the said vegetable raw material is suitable. And according to desired quality, you may use it combining suitably.
Moreover, the raw material at the time of fermentation can use the thing processed suitably according to the objective. For example, the vegetable raw material can be used in a form obtained by processing a juice, a ground product or a crushed product, or a concentrate, a diluted product or a dried product thereof. Specifically, if it is soybean, it can also be used with the form of soymilk. In the present invention, a liquid material is particularly suitable as a fermentation raw material.
本発明の飲食品には、本発明の効果を損なわない範囲で、食品用として一般的に用いられているその他の原料、ｐＨ調整剤、香料、糖液等の副資材を添加しても良い。 To the food and drink of the present invention, other raw materials generally used for foods, pH adjusters, fragrances, sugar solutions and the like may be added within the range not impairing the effects of the present invention. .
以下、具体的実施例により、本発明についてさらに詳しく説明する。ただし、本発明は、以下に示す実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
＜ＭＲＳ培地の発酵特性＞
［実施例１］
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液を、ＭＲＳ液体培地に１容量％接種し、３０℃で１８時間培養して賦活した後、同様の条件で継代したものを前培養液とした。得られた前培養液をＭＲＳ液体培地１０ｍＬに1容量％接種し、３０℃で培養して、下記の方法により経時的に生菌数、並びに培養液の濁度、ｐＨおよび酸度を測定した。結果を図２〜５に示す。図２は生菌数、図３は培養液の濁度、図４は培養液のｐＨ、図５は培養液の酸度の経時変化をそれぞれ示すグラフである。
（測定方法）
生菌数；段階希釈した培養液１ｍＬを分注したシャーレに、ＭＲＳ寒天培地約２０ｍＬを加えて混釈プレートを作成し、３０℃で２日間培養した後、形成したコロニーをカウントして算出した。
培養液の濁度；分光光度計Ｕ−３３１０（株式会社日立ハイテクノロジーズ製）を用いて、測定波長６６０ｎｍで測定した。
培養液のｐＨ；ｐＨメーターＦ−５２（株式会社堀場製作所製）を用いて測定した。
培養液の酸度；滴定法により算出した。すなわち、試料を約５ｇ量り採り、フェノールフタレイン溶液を約５００μＬ加えて、０．１Ｎ水酸化ナトリウム水溶液を滴下し、微赤色が呈する点を終点とした。そのときの滴定量から下記式を用いて酸度を算出した。
（算出式）
酸度（％）＝（水酸化ナトリウム水溶液の規定数）×（水酸化ナトリウム水溶液の滴定量（Ｌ））×（乳酸１ｇ当量）÷（試料採取量）×１００
<Fermentation characteristics of MRS medium>
[Example 1]
A cryopreserved bacterial solution of Lactobacillus brevis FERM P-21222 strain was inoculated into 1% by volume of MRS liquid medium, cultured at 30 ° C. for 18 hours and activated, and then subcultured under the same conditions as the precultured solution. did. The obtained preculture solution was inoculated into 10 mL of MRS liquid medium at 1 volume%, cultured at 30 ° C., and the number of viable bacteria and the turbidity, pH, and acidity of the culture solution were measured over time by the following method. The results are shown in FIGS. FIG. 2 is a graph showing the number of viable bacteria, FIG. 3 is a graph showing the turbidity of the culture solution, FIG. 4 is a pH of the culture solution, and FIG.
(Measuring method)
Number of viable cells: About 20 mL of MRS agar medium was added to a petri dish containing 1 mL of serially diluted culture solution, and after culturing at 30 ° C. for 2 days, the formed colonies were counted and calculated. .
Turbidity of culture solution: Measured at a measurement wavelength of 660 nm using a spectrophotometer U-3310 (manufactured by Hitachi High-Technologies Corporation).
The pH of the culture solution was measured using a pH meter F-52 (manufactured by Horiba, Ltd.).
Acidity of the culture solution; calculated by titration method. That is, about 5 g of a sample was weighed, about 500 μL of a phenolphthalein solution was added, a 0.1N sodium hydroxide aqueous solution was dropped, and the point where a faint red color was exhibited was defined as the end point. The acidity was calculated from the titration at that time using the following formula.
(Calculation formula)
Acidity (%) = (Regular number of sodium hydroxide aqueous solution) × (Titration amount of sodium hydroxide aqueous solution (L)) × (1 g equivalent of lactic acid) ÷ (sample amount) × 100
［比較例１］
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液に代わり、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株の凍結保存菌液を用いたこと以外は、実施例１と同様に測定を行った。結果を図２〜５に示す。
[Comparative Example 1]
The measurement was carried out in the same manner as in Example 1 except that the cryopreserved bacterial solution of Lactobacillus brevis FERM BP-4693 was used instead of the cryopreserved bacterial solution of Lactobacillus brevis FERM P-21222 strain. The results are shown in FIGS.
生菌数および培養液の濁度からみた増殖速度や到達菌数について、本菌株とＦＥＲＭ ＢＰ−４６９３株との間に大きな違いはなかった。
一方、長時間培養すると、本菌株の方がやや培養液の酸度が高く、それに伴い培養液のｐＨの低下がやや大きい傾向が認められた。
There was no significant difference between the present strain and the FERM BP-4693 strain in terms of the growth rate and the number of reached bacteria as seen from the viable cell count and the turbidity of the culture solution.
On the other hand, when cultivated for a long time, the acidity of the culture broth was slightly higher in this strain, and accordingly, a decrease in the pH of the culture broth tended to be somewhat large.
＜透明ニンジン果汁の発酵特性および凝集性＞
［実施例２］
Ｂｒｉｘ．６０％の透明ニンジン濃縮果汁を蒸留水で希釈して、Ｂｒｉｘ．１２％に調整し、３００ｍＬの三角フラスコに１００ｍＬ入れて、１１５℃、１５分間の条件でオートクレーブ滅菌したものを発酵培地とした。
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液を、ＭＲＳ液体培地に１容量％接種し、３０℃で１８時間培養して賦活した後、同様の条件で継代したものを前培養液とした。
次いで、調製した発酵培地に、５容量％の前培養液を接種し、３２℃で１８時間静置培養し、下記の方法により培養前後における生菌数、並びに培養液の酸度およびｐＨを測定し、培養後の培養液の凝集性の有無を確認した。結果を表４に示す。
（測定および確認方法）
生菌数；段階希釈した培養液１ｍＬを分注したシャーレに、ＭＲＳ寒天培地約２０ｍＬを加えて混釈プレートを作成し、３０℃で２日間培養した後、形成したコロニーをカウントして算出した。
培養液の酸度およびｐＨ；実施例１と同様の方法で測定した。
培養液の凝集性；培養液を試験管に移し、試験管ミキサーで軽く攪拌後、目視することにより凝集性の有無を確認した。
<Fermentation characteristics and cohesiveness of transparent carrot juice>
[Example 2]
Brix. 60% clear carrot concentrated fruit juice was diluted with distilled water, and Brix. The fermentation medium was adjusted to 12%, placed in a 300 mL Erlenmeyer flask and autoclaved at 115 ° C. for 15 minutes.
A cryopreserved bacterial solution of Lactobacillus brevis FERM P-21222 strain was inoculated into 1% by volume of MRS liquid medium, cultured at 30 ° C. for 18 hours and activated, and then subcultured under the same conditions as the precultured solution. did.
Next, the prepared fermentation medium is inoculated with 5% by volume of the pre-culture solution, and left to stand at 32 ° C. for 18 hours. The number of viable cells before and after the culture, and the acidity and pH of the culture solution are measured by the following methods. Then, the presence or absence of cohesiveness of the culture solution after the culture was confirmed. The results are shown in Table 4.
(Measurement and confirmation method)
Number of viable cells: About 20 mL of MRS agar medium was added to a petri dish containing 1 mL of serially diluted culture solution, and after culturing at 30 ° C. for 2 days, the formed colonies were counted and calculated. .
The acidity and pH of the culture solution were measured in the same manner as in Example 1.
Aggregability of the culture solution: The culture solution was transferred to a test tube, stirred gently with a test tube mixer, and visually checked for the presence or absence of agglutination.
［比較例２］
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液に代わり、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株の凍結保存菌液を用いたこと以外は、実施例２と同様に測定および確認を行った。結果を表４に示す。
[Comparative Example 2]
Measurement and confirmation were carried out in the same manner as in Example 2 except that the cryopreserved bacterial solution of Lactobacillus brevis FERM BP-4693 was used instead of the cryopreserved bacterial solution of Lactobacillus brevis FERM P-21222 . The results are shown in Table 4.
表４に示すように、いずれの菌株を用いた場合でも、生菌数、並びに培養液の酸度およびｐＨに大きな違いはなかった。しかし、凝集性に大きな違いが認められ、ＦＥＲＭ ＢＰ−４６９３株では凝集性があるのに対し、本菌株では凝集性がなかった。この時の培養液の撮影画像を図６に示す。図６（ａ）は本菌株の培養液の撮影画像、図６（ｂ）はＦＥＲＭ ＢＰ−４６９３株の培養液の撮影画像である。 As shown in Table 4, there was no significant difference in the number of viable bacteria and the acidity and pH of the culture solution even when any strain was used. However, a large difference was observed in the agglutinability, and the FERM BP-4693 strain was agglutinable, whereas this strain was not agglutinable. A photographed image of the culture solution at this time is shown in FIG. FIG. 6A is a photographed image of the culture solution of this strain, and FIG. 6B is a photographed image of the culture solution of FERM BP-4693 strain.
＜抗菌活性＞
［実施例３］
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液を、ＭＲＳ液体培地に１容量％接種し、３０℃で１８時間培養して賦活した後、さらに同条件で２回継代培養したものを培養液とした。
次いで、１３０００×ｇ、５分間の条件の遠心分離により、該培養液から菌体を除去し、さらに乳酸などの有機酸の影響を除去するために、得られた培養液上清をｐＨ６．０に調整した。これを滅菌フィルターＭＩＬＬＥＸＧＶ Ｆｉｌｔｅｒ Ｕｎｉｔ ０．２２μｍ（ミリポア社製）でろ過滅菌したものを被評価培養液とした。
また、抗菌活性を評価する際の対象菌として、食品危害微生物であるバチルス セレウス（Ｂａｃｉｌｌｕｓ ｃｅｒｅｕｓ）ＪＣＡ１５０１株、バチルス セレウス ＩＦＯ１３６９０株、バチルス ズブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＪＣＡ５２７株、バチルス ズブチリス ＩＦＯ１３７１９株、バチルス コアギュランス（Ｂａｃｉｌｌｕｓ ｃｏａｇｕｌａｎｓ）ＪＣＭ２２５７Ｔ株、リステリア モノサイトジェネス（Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓ）ＡＴＣＣ７６４４株、スタフィロコッカス アウレウス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ）ＲＣＳＡ１株を使用した。これら対象菌は、その凍結保存菌液をＳＣＤ培地（日水製薬株式会社製）に１容量％接種し、３０℃で１８時間、好気培養した。さらに同条件で２回継代培養を行って供試菌体とした。
<Antimicrobial activity>
[Example 3]
Lactobacillus brevis FERM P-21222 strain cryopreserved bacterial solution is inoculated into 1% by volume in MRS liquid medium, cultured at 30 ° C. for 18 hours, activated, and further subcultured twice under the same conditions. Liquid.
Subsequently, in order to remove cells from the culture solution by centrifugation under conditions of 13000 × g for 5 minutes and further remove the influence of an organic acid such as lactic acid, the obtained culture solution supernatant is adjusted to pH 6.0. Adjusted. This was sterilized by filtration with a sterilizing filter MILLEXGV Filter Unit 0.22 μm (manufactured by Millipore) and used as a culture for evaluation.
Further, as the target bacterium in assessing antimicrobial activity, Bacillus a food harm microorganisms cereus (Bacillus cereus) JCA1501 strain, Bacillus cereus IFO13690 strain, Bacillus subtilis (Bacillus subtilis) JCA527 strain, Bacillus subtilis IFO13719 strain, Bacillus coagulans (Bacillus Coagulans ) JCM2257 T strain, Listeria monocytogenes ATCC7644 strain, Staphylococcus aureus RCSA1 strain were used. These target bacteria were inoculated with 1% by volume of the cryopreserved bacterial solution in SCD medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and aerobically cultured at 30 ° C. for 18 hours. Further, subculture was performed twice under the same conditions to obtain test cells.
抗菌活性の評価は、対象菌を含む寒天培地に被評価培養液を滴下するＳｐｏｔ−ｏｎ−ｌａｗｎ法により行った。ＭＲＳ寒天培地を分注し凝固させたプレートに、前記条件で培養した対象菌を１容量％接種したＳＣＤ寒天培地を重層した。そしてＳＣＤ寒天培地が凝固した後、蓋を開けてプレートを裏返した状態で保ち、寒天培地の表面を乾燥させた。次いで、滅菌水で被評価培養液の希釈物（１／２倍、１／４倍〜１／３２倍）を調製し、対象菌を重層した前記プレートに１０μＬずつ滴下した。これを、以下に示す各対象菌の至適温度で２４時間培養に供した後に、阻止円形成の有無を観察した。そして阻止円を形成する最大の希釈率から、抗菌活性（Ａｃｔｉｖｉｔｙ Ｕｎｉｔ（ＡＵ）／ｍｌ）を下記式に従って算出した。結果を表５に示す。
（至適温度）
バチルス セレウス ＪＣＡ１５０１株、バチルス セレウス ＩＦＯ１３６９０株、バチルス ズブチリス ＪＣＡ５２７株、バチルス ズブチリス ＩＦＯ１３７１９株；３０℃
バチルス コアギュランス ＪＣＭ２２５７Ｔ株、リステリア モノサイトジェネス ＡＴＣＣ７６４４株、スタフィロコッカス アウレウス ＲＣＳＡ１株；３５℃
（算出式）
抗菌活性（ＡＵ／ｍｌ）＝（最大希釈率（ＡＵ））÷０．０１
The antibacterial activity was evaluated by the spot-on-lawn method in which the culture medium to be evaluated was dropped onto an agar medium containing the target bacteria. A plate on which MRS agar medium was dispensed and solidified was overlaid with SCD agar medium inoculated with 1% by volume of the target bacteria cultured under the above conditions. After the SCD agar medium was solidified, the lid was opened and the plate was turned upside down, and the surface of the agar medium was dried. Next, a dilution (1/2 times, 1/4 times to 1/32 times) of a culture solution to be evaluated was prepared with sterilized water, and 10 μL was dropped on the plate on which the target bacteria were overlaid. This was subjected to culture for 24 hours at the optimum temperature of each target bacterium shown below, and the presence or absence of inhibition circle formation was observed. And the antibacterial activity (Activity Unit (AU) / ml) was computed from the maximum dilution rate which forms an inhibition circle according to a following formula. The results are shown in Table 5.
(Optimum temperature)
Bacillus cereus JCA1501 strain, Bacillus cereus IFO 13690 strain, Bacillus subtilis JCA527 strain, Bacillus subtilis IFO 13719 strain; 30 ° C
Bacillus coagulans JCM2257 T strain, Listeria monocytogenes ATCC7644 strain, Staphylococcus aureus RCSA1 strain; 35 ° C
(Calculation formula)
Antibacterial activity (AU / ml) = (maximum dilution rate (AU)) / 0.01
［比較例３］
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液に代わり、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株の凍結保存菌液を用いたこと以外は、実施例３と同様に評価を行った。結果を表５に示す。
[Comparative Example 3]
Evaluation was carried out in the same manner as in Example 3 except that the cryopreserved bacterial solution of Lactobacillus brevis FERM BP-4693 was used instead of the cryopreserved bacterial solution of Lactobacillus brevis FERM P-21222 strain. The results are shown in Table 5.
［比較例４］
ラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株の凍結保存菌液に代わり、ラクトバチルス ブレビス 基準株の凍結保存菌液を用いたこと以外は、実施例３と同様に評価を行った。結果を表５に示す。
[Comparative Example 4]
Evaluation was carried out in the same manner as in Example 3 except that the cryopreserved bacterial solution of Lactobacillus brevis reference strain was used instead of the cryopreserved bacterial solution of Lactobacillus brevis FERM P-21222 strain. The results are shown in Table 5.
表５から明らかなように、本菌株は、ＦＥＲＭ ＢＰ−４６９３株と同様に、バチルス コアギュランス ＪＣＭ２２５７Ｔ株、およびリステリア モノサイトジェネス ＡＴＣＣ７６４４株に対して特異的に抗菌活性を示した。 As is apparent from Table 5, this strain showed antibacterial activity specifically against the Bacillus coagulans JCM2257T strain and the Listeria monocytogenes ATCC7644 strain, as with the FERM BP-4693 strain.
以上のように、本発明のラクトバチルス ブレビス ＦＥＲＭ Ｐ−２１２２２株は、抗菌活性を有するなど、ラクトバチルス ブレビス ＦＥＲＭ ＢＰ−４６９３株と同様の優れた生理作用を有するので、飲食品や医薬品の製造に好適である。また、本発明の菌株は凝集性を示さないため、発酵物の製造を安定して行うことができ、発酵物の品質を均一に保持できる。 As described above, the Lactobacillus brevis FERM P-21222 strain of the present invention has the same excellent physiological action as the Lactobacillus brevis FERM BP-4693 strain, such as having antibacterial activity. Is preferred. In addition, since the strain of the present invention does not exhibit aggregability, the fermentation product can be stably produced, and the quality of the fermentation product can be maintained uniformly.
本発明は、飲食品や医薬品の製造に利用可能であり、特に、発酵飲食品の製造に好適である。 The present invention can be used for the production of foods and beverages and pharmaceuticals, and is particularly suitable for the production of fermented foods and beverages.
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