JP6330237B2 - Lactobacillus lactic acid bacteria culture-grade culture medium, Lactobacillus lactic acid bacteria culture method, and Lactobacillus lactic acid bacteria culture production method - Google Patents
Lactobacillus lactic acid bacteria culture-grade culture medium, Lactobacillus lactic acid bacteria culture method, and Lactobacillus lactic acid bacteria culture production method Download PDFInfo
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- 241000186660 Lactobacillus Species 0.000 title claims description 70
- 239000004310 lactic acid Substances 0.000 title claims description 68
- 235000014655 lactic acid Nutrition 0.000 title claims description 68
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- 238000004519 manufacturing process Methods 0.000 title claims description 11
- 239000001963 growth medium Substances 0.000 title description 19
- 238000012136 culture method Methods 0.000 title description 2
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- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 20
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 4
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 3
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Images
Description
本発明は、ラクトバチルス属乳酸菌培養用食品グレード培地、ラクトバチルス属乳酸菌の培養方法及びラクトバチルス属乳酸菌培養物の製造方法に関する。 The present invention relates to a food grade medium for culturing Lactobacillus lactic acid bacteria, a method for culturing Lactobacillus lactic acid bacteria, and a method for producing a Lactobacillus lactic acid bacteria culture.
ラクトバチルス属乳酸菌は、ヒトや家畜の健康に有益な効果を示す生菌（プロバイオティクス）として食品や飼料への応用が期待されている。 Lactobacillus lactic acid bacteria are expected to be applied to food and feed as live bacteria (probiotics) that have beneficial effects on human and livestock health.
しかしながら、ラクトバチルス属乳酸菌の培養は困難であり、特殊な培地で培養する必要がある。 However, it is difficult to culture Lactobacillus lactic acid bacteria, and it is necessary to culture in a special medium.
例えば、世界的に汎用されている人工合成培地（ＭＲＳ培地）は、ラクトバチルス属乳酸菌を良好に培養することができる（例えば、非特許文献１を参照）。 For example, an artificial synthetic medium (MRS medium) that is widely used worldwide can cultivate Lactobacillus lactic acid bacteria well (see, for example, Non-Patent Document 1).
また、特許文献１には、ホエーに、ａ）グルコース、ｂ）カゼイン酵素分解物、ｃ）Ｔｗｅｅｎ（登録商標）８０、オレイン酸ナトリウムまたはデカグリセリンモノオレエート、ｄ）酵母エキス、を添加することを特徴とする乳酸桿菌用培地が開示されている。 Further, in Patent Document 1, a) glucose, b) casein enzymatic degradation product, c) Tween (registered trademark) 80, sodium oleate or decaglycerin monooleate, and d) yeast extract are added to whey. A medium for lactobacilli is disclosed.
しかしながら、ＭＲＳ培地は、食品に添加することが認められていない硫酸マンガン四水和物等の成分を含有しているため、ＭＲＳ培地で培養したラクトバチルス属乳酸菌の培養物は、そのまま食品に添加することができない。 However, since the MRS medium contains components such as manganese sulfate tetrahydrate that are not allowed to be added to foods, the Lactobacillus lactic acid bacteria culture cultured in the MRS medium is added to the food as it is. Can not do it.
一方、ホエー等の乳由来成分は、栄養成分が豊富なため、他の微生物による汚染を受けやすく、取り扱いが困難な場合がある。また、乳由来成分を含有する培地は、アレルギー源を含有するため、使用制限が生じる場合がある。 On the other hand, since milk-derived components such as whey are rich in nutritional components, they are easily contaminated by other microorganisms and may be difficult to handle. Moreover, since the culture medium containing a milk-derived component contains an allergy source, use restrictions may arise.
そこで、本発明は、硫酸マンガン四水和物や乳由来成分を実質的に含有しない、ラクトバチルス属乳酸菌培養用食品グレード培地を提供することを目的とする。本発明はまた、上記培地を用いたラクトバチルス属乳酸菌の培養方法及びラクトバチルス属乳酸菌培養物の製造方法を提供することを目的とする。 Then, an object of this invention is to provide the food grade culture medium for Lactobacillus genus lactic acid bacteria culture | cultivation which does not contain manganese sulfate tetrahydrate and milk-derived components substantially. Another object of the present invention is to provide a method for culturing Lactobacillus lactic acid bacteria and a method for producing a Lactobacillus lactic acid bacteria culture using the medium.
本発明は以下の通りである。
［１］ラクトバチルス属乳酸菌培養用食品グレード培地であって、ペプトン、牛肉エキス（ラブ・レムコ粉末）、工業用酵母エキス、硫酸マンガン四水和物を実質的に含有せず、食品添加用酵母エキスを含有する、培地。
［２］ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物を実質的に含有せず、食品添加用酵母エキスを含有する食品グレード培地中で、ラクトバチルス属乳酸菌を培養する工程を含む、ラクトバチルス属乳酸菌の培養方法。
［３］ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物を実質的に含有せず、食品添加用酵母エキスを含有する食品グレード培地中で、ラクトバチルス属乳酸菌を培養する工程を含む、ラクトバチルス属乳酸菌培養物の製造方法。
The present invention is as follows.
[1] A food grade medium for cultivating Lactobacillus lactic acid bacteria, which is substantially free of peptone, beef extract (Love Lemco powder), industrial yeast extract, manganese sulfate tetrahydrate, and yeast for food addition A medium containing an extract.
[2] A step of cultivating Lactobacillus lactic acid bacteria in a food grade medium substantially free of peptone, beef extract, industrial yeast extract, manganese sulfate tetrahydrate and containing yeast extract for food addition A method for culturing Lactobacillus lactic acid bacteria.
[3] A step of cultivating Lactobacillus lactic acid bacteria in a food grade medium containing substantially no peptone, beef extract, industrial yeast extract, manganese sulfate tetrahydrate, and containing a yeast extract for food addition A method for producing a Lactobacillus lactic acid bacteria culture.
本発明によれば、硫酸マンガン四水和物や乳由来成分を実質的に含有しないにもかかわらず、ラクトバチルス属乳酸菌を良好に培養することができる、ラクトバチルス属乳酸菌培養用食品グレード培地、上記培地を用いたラクトバチルス属乳酸菌の培養方法及びラクトバチルス属乳酸菌培養物の製造方法を提供することができる。 According to the present invention, a food grade medium for cultivating Lactobacillus lactic acid bacteria, which can satisfactorily culture Lactobacillus lactic acid bacteria, despite containing substantially no manganese sulfate tetrahydrate or milk-derived components, A method for culturing Lactobacillus lactic acid bacteria using the medium and a method for producing a Lactobacillus lactic acid bacteria culture can be provided.
［ラクトバチルス属乳酸菌培養用食品グレード培地］
１実施形態において、本発明は、ラクトバチルス属乳酸菌培養用食品グレード培地であって、ペプトン、牛肉エキス（ラブ・レムコ粉末）、工業用酵母エキス、硫酸マンガン四水和物を含有せず、食品添加用酵母エキスを含有する、培地を提供する。
[Food-grade medium for culturing Lactobacillus lactic acid bacteria]
In one embodiment, the present invention is a food grade medium for cultivating Lactobacillus lactic acid bacteria, which does not contain peptone, beef extract (lab remco powder), industrial yeast extract, manganese sulfate tetrahydrate, A medium containing a yeast extract for addition is provided.
上記培地は、ラクトバチルス属乳酸菌を良好に増殖させることができる。ラクトバチルス属乳酸菌としては、ラクトバチルス・ガセリ菌株、ラクトバチルス・アシドフィルス菌株、ラクトバチルス・カゼイ菌株、ラクトバチルス・ヘルベティカス菌株等が挙げられる。 The above medium can favorably grow Lactobacillus lactic acid bacteria. Examples of Lactobacillus lactic acid bacteria include Lactobacillus gasseri strain, Lactobacillus acidophilus strain, Lactobacillus casei strain, Lactobacillus helveticus strain, and the like.
「食品グレード」とは、上記培地が食用に適することを意味する。より具体的には、上記培地に含まれる全ての成分が、食品に添加することを認められていることを意味する。したがって、上記培地をヒトが摂取しても人体に悪影響を及ぼすことがない。培地が食品グレードであるため、上記培地で培養したラクトバチルス属乳酸菌の培養物は、そのまま簡便かつ安全に各種食品に添加することができる。 “Food grade” means that the medium is edible. More specifically, it means that all the components contained in the medium are allowed to be added to food. Therefore, even if a human ingests the above medium, the human body is not adversely affected. Since the medium is food grade, the culture of Lactobacillus lactic acid bacteria cultured in the above medium can be simply and safely added to various foods as it is.
＜ＭＲＳ培地＞
世界的に汎用されている、ラクトバチルス属乳酸菌培養用人工合成培地である、ＭＲＳ培地の成分を表１に示す。
<MRS medium>
Table 1 shows the components of the MRS medium, which is an artificially synthesized medium for culturing Lactobacillus lactic acid bacteria widely used worldwide.
表１に示す成分のうち、ペプトン、牛肉エキス（ラブ・レムコ粉末）、工業用酵母エキス及び硫酸マンガン四水和物は、食品に添加することが認められていないものである。 Among the components shown in Table 1, peptone, beef extract (Love-Remco powder), industrial yeast extract and manganese sulfate tetrahydrate are not allowed to be added to food.
＜ペプトン＞
ペプトンとは、タンパク質を加水分解、酵素分解又は発酵することによって得られる、アミノ酸が複数結合した物質をいう。由来によって様々なペプトンが存在し、例えば、肉エキス、カゼインペプトン、魚肉ペプトン、大豆ペプトン、エンドウ豆ペプトン、小麦ペプトン、大麦ペプトン、綿実ペプトン等が挙げられる。
<Peptone>
Peptone refers to a substance obtained by hydrolyzing, enzymatically degrading or fermenting a protein and having a plurality of amino acids linked thereto. There are various peptones depending on the origin, and examples include meat extract, casein peptone, fish peptone, soybean peptone, pea peptone, wheat peptone, barley peptone, cottonseed peptone, and the like.
＜ラブ・レムコ粉末＞
ラブ・レムコ粉末とは、牛肉エキスである。
<Love Remco Powder>
Love Lemco powder is a beef extract.
＜工業用酵母エキス＞
工業用酵母エキスとは、培地用酵母エキスとも呼ばれて流通しているものであり、食品に添加することが認められていない酵母エキスである。
<Industrial yeast extract>
The industrial yeast extract is also called a yeast extract for culture medium, and is a yeast extract that is not allowed to be added to food.
＜硫酸マンガン四水和物＞
上述の非特許文献１に記載されているように、硫酸マンガン四水和物は、ラクトバチルス属乳酸菌の培養に必須の培地成分であると考えられている。しかしながら、硫酸マンガン四水和物には毒性があり、食品に添加することが認められていない。
<Manganese sulfate tetrahydrate>
As described in Non-Patent Document 1 described above, manganese sulfate tetrahydrate is considered to be an essential medium component for culturing Lactobacillus lactic acid bacteria. However, manganese sulfate tetrahydrate is toxic and is not allowed to be added to food.
本実施形態に係る培地は、ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物を実質的に含有せず、食品添加用酵母エキスを含有する。ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物を実質的に含有しないとは、これらの成分を、全く含有しないか、又は人体に悪影響を及ぼさない量しか含有しないことを意味する。あるいは、これらの成分を、本実施形態に係る培地を食用に適するものと管轄当局が判断する量しか含有しないことを意味する。 The culture medium according to this embodiment contains substantially no peptone, beef extract, industrial yeast extract, or manganese sulfate tetrahydrate, and contains a yeast additive for food addition. The fact that peptone, beef extract, industrial yeast extract, and manganese sulfate tetrahydrate are substantially free means that these components are not contained at all or contained in an amount that does not adversely affect the human body. . Alternatively, it means that these components are contained only in amounts determined by the competent authority that the medium according to this embodiment is suitable for food.
＜食品添加用酵母エキス＞
食品添加用酵母エキスとしては、食品用（食品添加用）に市販されているものであれば特に制限なく用いることができる。酵母エキスは、酵母の有効成分を自己消化法、酵素分解法、熱水抽出法等の方法により抽出したものである。自己消化法とは、生酵母を原料に用い、酵母自身が持つ酵素を利用して酵母の成分を分解し抽出する方法である。酵素分解法とは、酵母の持つ酵素群を、熱処理や乾燥処理等により不活化させておき、改めてタンパク質分解酵素、核酸分解酵素等の酵素を添加して酵母の成分を分解し抽出する方法である。酵母エキスの原料の酵母としては、パン酵母、ビール酵母、トルラ酵母等が挙げられる。
<Yeast extract for food addition>
The yeast extract for food addition can be used without particular limitation as long as it is commercially available for food (for food addition). Yeast extract is obtained by extracting an active ingredient of yeast by a method such as an autolysis method, an enzymatic decomposition method, or a hot water extraction method. The self-digestion method is a method in which raw yeast is used as a raw material, and yeast components are decomposed and extracted using an enzyme possessed by the yeast. Enzymatic degradation is a method in which the yeast enzymes are inactivated by heat treatment, drying treatment, etc., and enzymes such as proteolytic enzymes and nucleolytic enzymes are added again to decompose and extract yeast components. is there. Examples of yeast as a raw material for yeast extract include baker's yeast, brewer's yeast, and Torula yeast.
食品添加用酵母エキスの具体例としては、酵母エキスＳＡ−Ｒ、酵母エキスＳＡ−Ｍ、酵母エキスＳ、酵母エキスＦＲ、酵味（ペースト）、酵味（粉末）、酵母エキスＳＬ−Ｗ、酵母エキスＢＹ−Ｇ、酵母エキスＨＲ（以上、ＭＣフードスペシャリティーズ株式会社製）；酵母エキスＨ（協和発酵キリン株式会社製）；酵母エキスサンライク、酵母エキスＲＮ−１（以上、三栄源エフ・エフ・アイ株式会社製）等が挙げられる。 Specific examples of the yeast extract for food addition include yeast extract SA-R, yeast extract SA-M, yeast extract S, yeast extract FR, fermentation taste (paste), fermentation taste (powder), yeast extract SL-W, yeast Extract BY-G, yeast extract HR (above, manufactured by MC Food Specialties Co., Ltd.); yeast extract H (produced by Kyowa Hakko Kirin Co., Ltd.);・ Made by Eye Co., Ltd.)
これらの食品添加用酵母エキスのうち、ラクトバチルス属乳酸菌の増殖性の観点からは、酵母エキスＳＡ−Ｍ、酵母エキスＳ、酵母エキスＦＲ、酵味（ペースト）、酵母エキスＨ、酵母エキスＳＬ−Ｗ、酵母エキスＨＲが好ましく、酵母エキスＳ、酵母エキスＦＲ、酵味（ペースト）、酵母エキスＨ、酵母エキスＳＬ−Ｗがより好ましく、酵母エキスＦＲ、酵母エキスＨがさらに好ましい。 Among these yeast extracts for food addition, from the viewpoint of the growth of Lactobacillus lactic acid bacteria, yeast extract SA-M, yeast extract S, yeast extract FR, fermentation taste (paste), yeast extract H, yeast extract SL- W and yeast extract HR are preferable, yeast extract S, yeast extract FR, fermentation taste (paste), yeast extract H, and yeast extract SL-W are more preferable, and yeast extract FR and yeast extract H are more preferable.
また、食品添加用酵母エキスとしては、核酸分解物を含有する食品添加用酵母エキスが好ましく、グアノシンを含有する食品添加用酵母エキスがより好ましい。 Moreover, as the yeast extract for food addition, the yeast extract for food addition containing a nucleic acid degradation product is preferable, and the yeast extract for food addition containing guanosine is more preferable.
ラクトバチルス属乳酸菌培養用食品グレード培地中の食品添加用酵母エキスの含有量は、例えば０．３〜５ｗ／ｖ％が例示できる。 Examples of the content of the yeast extract for food addition in the food grade medium for culturing Lactobacillus lactic acid bacteria include, for example, 0.3 to 5 w / v%.
１実施形態において、ラクトバチルス属乳酸菌培養用食品グレード培地は、食品添加用酵母エキスを唯一の窒素源として含有する。このため、食品添加用酵母エキスの濃度を制御することにより、ラクトバチルス属乳酸菌の増殖を容易に調節することができる。 In one embodiment, the food grade medium for culturing Lactobacillus lactic acid bacteria contains a yeast extract for food addition as the sole nitrogen source. For this reason, the growth of Lactobacillus genus lactic acid bacteria can be easily controlled by controlling the concentration of the yeast extract for food addition.
ラクトバチルス属乳酸菌培養用食品グレード培地は、食品に添加することが認められている成分である限り、微生物用培地に通常含有される他の成分を含有していてもよい。このような成分として、例えば、グルコース、モノオレイン酸ソルビタン（Ｔｗｅｅｎ（登録商標）８０）、リン酸水素二カリウム、酢酸ナトリウム三水和物、クエン酸三アンモニウム、硫酸マグネシウム七水和物等が挙げられる。 The food grade medium for culturing Lactobacillus lactic acid bacteria may contain other components usually contained in the microorganism medium as long as it is a component that is permitted to be added to food. Examples of such components include glucose, sorbitan monooleate (Tween (registered trademark) 80), dipotassium hydrogen phosphate, sodium acetate trihydrate, triammonium citrate, magnesium sulfate heptahydrate, and the like. It is done.
＜乳由来成分＞
ラクトバチルス属乳酸菌培養用食品グレード培地は、乳由来成分を実質的に含有しないことが好ましい。乳由来成分を含有する培地は不透明な場合があり、濁度測定によるラクトバチルス属乳酸菌の生育性判定が困難な場合がある。しかしながら、乳由来成分を実質的に含有しないことにより、培地が透明となり、濁度測定により乳酸菌の生育性判定が容易となりやすい。
<Milk-derived ingredients>
The food grade medium for culturing Lactobacillus lactic acid bacteria preferably contains substantially no milk-derived components. A medium containing milk-derived components may be opaque, and it may be difficult to determine the viability of Lactobacillus lactic acid bacteria by turbidity measurement. However, by substantially not containing milk-derived components, the medium becomes transparent, and it is easy to determine the viability of lactic acid bacteria by measuring turbidity.
また、乳由来成分を含有する培地は、ｐＨの変化や加熱によって性状が変化しやすく、例えば遠心分離により培養後の菌体を回収するときに、凝固したカゼイン等が混入し、純粋な菌体を得ることが困難な場合がある。しかしながら、乳由来成分を実質的に含有しないことにより、遠心分離等により培養後の菌体を回収するときに、菌体以外の成分が混入することが少なく、純粋な菌体を得ることが容易である。 In addition, the medium containing milk-derived components is likely to change its properties due to changes in pH or heating. For example, when collecting cultured cells by centrifugation, coagulated casein and the like are mixed into pure cells. It may be difficult to get. However, by substantially not containing milk-derived components, it is easy to obtain pure cells by collecting components other than the cells when collecting the cells after culturing by centrifugation or the like. It is.
また、乳由来成分を含有すると、アレルギーの原因となる場合がある。しかしながら、乳由来成分を実質的に含有しないことにより、アレルギーの原因になりにくい培地を得ることができる。 Moreover, if milk-derived components are contained, it may cause allergies. However, a medium that does not cause allergies can be obtained by substantially not containing milk-derived components.
乳由来成分を実質的に含有しないとは、乳由来成分を全く含有しないか、上述の問題が生じない程度しか含有しないことを意味する。乳由来成分としては、ホエー、カゼイン、乳清タンパク質濃縮物、これらを酵素処理等により加工したもの等が挙げられる。 “Contains substantially no milk-derived component” means that it does not contain any milk-derived component or contains only a degree that does not cause the above-mentioned problems. Examples of the milk-derived component include whey, casein, whey protein concentrate, and those processed by enzyme treatment.
また、本実施形態に係る培地は、従来広く用いられてきたＭＲＳ培地の約１／１０のコストで製造することができるため、食品工業等に広く応用することができる。 Moreover, since the culture medium according to the present embodiment can be produced at a cost of about 1/10 of the MRS medium that has been widely used heretofore, it can be widely applied to the food industry and the like.
［ラクトバチルス属乳酸菌の培養方法］
１実施形態において、本発明は、ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物を含有せず、食品添加用酵母エキスを含有する食品グレード培地中で、ラクトバチルス属乳酸菌を培養する工程を含む、ラクトバチルス属乳酸菌の培養方法を提供する。
[Lactobacillus lactic acid bacteria culture method]
In one embodiment, the present invention cultivates Lactobacillus lactic acid bacteria in a food grade medium that does not contain peptone, beef extract, industrial yeast extract, manganese sulfate tetrahydrate, and contains yeast extract for food addition A method for culturing Lactobacillus lactic acid bacteria, comprising the step of:
本実施形態に係る方法により、ラクトバチルス属乳酸菌を低コストで良好に増殖することができる。 By the method according to this embodiment, Lactobacillus lactic acid bacteria can be successfully grown at low cost.
［ラクトバチルス属乳酸菌培養物の製造方法］
１実施形態において、本発明は、ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物を含有せず、食品添加用酵母エキスを含有する食品グレード培地中で、ラクトバチルス属乳酸菌を培養する工程を含む、ラクトバチルス属乳酸菌培養物の製造方法を提供する。
[Method for producing Lactobacillus lactic acid bacteria culture]
In one embodiment, the present invention cultivates Lactobacillus lactic acid bacteria in a food grade medium that does not contain peptone, beef extract, industrial yeast extract, manganese sulfate tetrahydrate, and contains yeast extract for food addition The manufacturing method of the Lactobacillus genus lactic acid bacteria culture | cultivation including the process to do is provided.
本実施形態に係る製造方法により、ラクトバチルス属乳酸菌培養物を低コストで製造することができる。ラクトバチルス属乳酸菌培養物とは、ラクトバチルス属乳酸菌の培養液、分離した菌体、菌体外生産物を含む培養上清等を意味する。このラクトバチルス属乳酸菌培養物は、そのまま簡便かつ安全に各種食品に添加することができる。例えば、発酵乳等の食品にスターターとして添加する、プロバイオティクスとして各種食品に添加する等の用途が挙げられる。 By the production method according to this embodiment, a Lactobacillus lactic acid bacteria culture can be produced at low cost. The Lactobacillus lactic acid bacteria culture means a culture solution of Lactobacillus lactic acid bacteria, isolated bacterial cells, a culture supernatant containing extracellular products, and the like. This Lactobacillus lactic acid bacteria culture can be simply and safely added to various foods as it is. For example, it can be used as a starter for foods such as fermented milk, or as a probiotic for various foods.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
（実施例１〜１０）
＜ラクトバチルス属乳酸菌培養用食品グレード培地の作製＞
表２に示す組成の材料に蒸留水を加えて溶解し、１Ｌになるように調整した。続いて、１Ｎ−ＮａＯＨを用いてｐＨを６．５に調整し、１２１℃、１５分の条件で高圧蒸気滅菌を行い、実施例１~１０の培地を調製した。材料としては、全て食品添加物として認可されているものを用いた。また、酵母エキスとしては、表３に示す食品添加用酵母エキスを用いた。実施例１〜１０の培地の材料のコストは、後述する比較例１の培地の約１／１０であった。
(Examples 1 to 10)
<Production of food grade medium for culturing Lactobacillus lactic acid bacteria>
Distilled water was added to the material having the composition shown in Table 2 and dissolved to adjust to 1 L. Subsequently, the pH was adjusted to 6.5 using 1N-NaOH, and autoclaving was performed at 121 ° C. for 15 minutes to prepare the media of Examples 1 to 10. As materials, all those approved as food additives were used. Moreover, the yeast extract for food addition shown in Table 3 was used as a yeast extract. The cost of the material of the culture medium of Examples 1-10 was about 1/10 of the culture medium of the comparative example 1 mentioned later.
（比較例１）
世界的に汎用されているＭＲＳ培地（ＯＸＯＩＤ社製）を比較例１の培地とした。より具体的には、上述の表１に示す組成の材料に蒸留水を加えて溶解し、１Ｌになるように調整した。続いて、１Ｎ−ＮａＯＨを用いてｐＨを６．５に調整し、１２１℃、１５分の条件で高圧蒸気滅菌を行い、比較例１の培地とした。
(Comparative Example 1)
The MRS medium (manufactured by OXOID), which is widely used worldwide, was used as the medium of Comparative Example 1. More specifically, distilled water was added to the material having the composition shown in Table 1 and dissolved to adjust to 1 L. Subsequently, the pH was adjusted to 6.5 using 1N-NaOH, and autoclaving was performed at 121 ° C. for 15 minutes to obtain the medium of Comparative Example 1.
（比較例２）
酵母エキスとして工業用酵母エキスであるＤ−３（日本製薬株式会社製）を使用した点以外は、実施例１と同様にして培地を調製し、比較例２の培地とした。
(Comparative Example 2)
A medium was prepared in the same manner as in Example 1 except that D-3 (manufactured by Nippon Pharmaceutical Co., Ltd.), which is an industrial yeast extract, was used as the yeast extract.
（実験例１）
＜ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株の培養＞
実施例１〜１０、比較例１及び比較例２の培地を用いて、ラクトバチルス属乳酸菌である、ラクトバチルス・ガセリ株の基準株である、ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を培養し、増殖性を検討した。
(Experimental example 1)
<Cultivation of Lactobacillus gasseri JCM1131 T strain>
Lactobacillus gasseri JCM1131 T strain, which is a reference strain of Lactobacillus gasseri strain, which is a lactic acid bacterium of the genus Lactobacillus, is cultured using the media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2, It was investigated.
＜前培養＞
（１）１５ｍＬ容遠沈管にＭＲＳ培地４．５ｍＬと供試菌液０．５ｍＬを接種した。
（２）別の遠沈管にＭＲＳ培地５．０ｍＬを加え、これをブランクとした。
（３）（１）、（２）を３７℃で２４時間静置培養した。
<Pre-culture>
(1) A 15 mL centrifuge tube was inoculated with 4.5 mL of MRS medium and 0.5 mL of the test bacteria solution.
(2) 5.0 mL of MRS medium was added to another centrifuge tube, and this was used as a blank.
(3) (1) and (2) were incubated at 37 ° C. for 24 hours.
＜本培養＞
（１）１５ｍＬ容遠沈管を遠心分離機（ＣＰ１６ＲＸ型ローター：Ｔ９Ａ３１０４３６）を用いて、３５００×ｇ、４℃、１０分の条件で遠心分離した。
（２）遠心分離後、クリーンベンチ内で上清を捨て、５ｍＬの０．８５％リン酸緩衝生理食塩水を加え、ボルテックスミキサーで撹拌した。その後３５００×ｇ、４℃、１０分の条件で遠心分離を行い、菌体を洗浄した。この作業をもう一度繰り返した。
（３）遠心分離後、クリーンベンチ内で上清を捨て、培地を５ｍＬ加え、ボルテックスミキサーでよく撹拌した。この前培養菌液を、実施例１〜１０、比較例１及び２の培地を用いた培養用にそれぞれ調製した。
（４）滅菌試験管に、実施例１〜１０、比較例１及び比較例２の培地をそれぞれ４．８５ｍＬずつ入れ、（３）で調製した各前培養菌液を０．１５ｍＬ（３容量％）ずつ接種した。これを培養開始直後及び２４時間培養後のｐＨ及び濁度測定用に各１本ずつの計４本作製し、同じ本数分のブランクも作製した。
（５）３７℃で２４時間静置培養した。
（６）培養開始直後及び２４時間培養後のｐＨ及び濁度（波長６２０ｎｍ）を培地ごとに測定し、その値を記録した。
<Main culture>
(1) A 15 mL centrifuge tube was centrifuged using a centrifuge (CP16RX type rotor: T9A310436) under conditions of 3500 × g, 4 ° C., and 10 minutes.
(2) After centrifugation, the supernatant was discarded in a clean bench, 5 mL of 0.85% phosphate buffered saline was added, and the mixture was stirred with a vortex mixer. Thereafter, centrifugation was performed under conditions of 3500 × g, 4 ° C. and 10 minutes to wash the cells. This process was repeated once more.
(3) After centrifugation, the supernatant was discarded in a clean bench, 5 mL of medium was added, and the mixture was well stirred with a vortex mixer. This precultured bacterial solution was prepared for culture using the media of Examples 1 to 10 and Comparative Examples 1 and 2, respectively.
(4) 4.85 mL each of the culture media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2 were placed in a sterile test tube, and 0.15 mL (3% by volume) of each precultured bacterial solution prepared in (3) ) Inoculated one by one. A total of four of these were prepared for measurement of pH and turbidity immediately after the start of culture and after 24 hours of culture, and blanks for the same number were also prepared.
(5) Static culture was performed at 37 ° C. for 24 hours.
(6) The pH and turbidity (wavelength 620 nm) immediately after the start of culture and after 24 hours of culture were measured for each medium, and the values were recorded.
図１は、実施例１〜１０、比較例１及び比較例２の培地でラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を２４時間培養後のｐＨの値を示したグラフである。菌が増殖するとｐＨが下がるため、ｐＨが低いほど菌が良好に増殖したことを示す。 FIG. 1 is a graph showing the pH values after culturing Lactobacillus gasseri JCM1131 T strain in the media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2 for 24 hours. Since the pH decreases as the bacteria grow, the lower the pH, the better the bacteria grew.
図２は、実施例１〜１０、比較例１及び比較例２の培地でラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を２４時間培養後の濁度の値を示したグラフである。濁度が高いほど菌が良好に増殖したことを示す。ただし、実施例７の培地はもともと濁っていたため、濁度の測定が不能であった。 FIG. 2 is a graph showing turbidity values after culturing Lactobacillus gasseri JCM1131 T strain in the culture media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2 for 24 hours. The higher the turbidity, the better the bacteria grew. However, since the medium of Example 7 was originally turbid, turbidity could not be measured.
図１、２の結果から、実施例１〜１０の培地は、比較例１の培地と同程度にラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を増殖できることが示された。また、実施例１〜１０の培地でラクトバチルス属乳酸菌の増殖が可能であることから、ラクトバチルス属乳酸菌の増殖には、硫酸マンガン四水和物は必須ではないと考えられる。 The results of FIGS. 1 and 2 show that the culture media of Examples 1 to 10 can grow the Lactobacillus gasseri JCM1131 T strain to the same extent as the culture media of Comparative Example 1. Moreover, since the growth of Lactobacillus lactic acid bacteria is possible in the culture medium of Examples 1-10, it is thought that manganese sulfate tetrahydrate is not essential for the growth of Lactobacillus lactic acid bacteria.
（実験例２）
＜ラクトバチルス・ガセリＪＣＭ１１３０菌株の培養＞
実施例１〜１０、比較例１及び比較例２の培地を用いて、ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株以外のラクトバチルス・ガセリ菌株を培養し、増殖性を検討した。ラクトバチルス・ガセリ菌株として、ラクトバチルス・ガセリＪＣＭ１１３０菌株を培養した。培養の手順は実験例１と同様にして行った。
(Experimental example 2)
<Cultivation of Lactobacillus gasseri JCM1130 strain>
Lactobacillus gasseri strains other than the Lactobacillus gasseri JCM1131 T strain were cultured using the culture media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2, and the growth was examined. As the Lactobacillus gasseri strain, the Lactobacillus gasseri JCM1130 strain was cultured. The culture procedure was the same as in Experimental Example 1.
図３は、実施例１〜１０、比較例１及び比較例２の培地でラクトバチルス・ガセリＪＣＭ１１３０菌株を２４時間培養後のｐＨの値を示したグラフである。 FIG. 3 is a graph showing pH values after culturing the Lactobacillus gasseri JCM1130 strain for 24 hours in the media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2.
図４は、実施例１〜１０、比較例１及び比較例２の培地でラクトバチルス・ガセリＪＣＭ１１３０菌株を２４時間培養後の濁度の値を示したグラフである。ただし、実施例７の培地はもともと濁っていたため、濁度の測定が不能であった。 FIG. 4 is a graph showing turbidity values after culturing Lactobacillus gasseri JCM1130 strain for 24 hours in the media of Examples 1 to 10, Comparative Example 1 and Comparative Example 2. However, since the medium of Example 7 was originally turbid, turbidity could not be measured.
図３、４の結果から、実施例１〜１０の培地は、比較例１の培地と同程度にラクトバチルス・ガセリＪＣＭ１１３０菌株を増殖できることが示された。 The results of FIGS. 3 and 4 show that the culture media of Examples 1 to 10 can grow the Lactobacillus gasseri JCM1130 strain to the same extent as the culture media of Comparative Example 1.
（実験例３）
＜各種ラクトバチルス・ガセリ菌株の培養＞
ラクトバチルス・ガセリ菌株の増殖性が良好であった、実施例４の培地（酵母エキスとして酵母エキスＦＲ、キリン協和フーズ株式会社製を用いたもの）を使用して各種ラクトバチルス・ガセリ菌株を培養し、増殖性を検討した。比較例１の培地（ＭＲＳ培地）を対照に用いた。ラクトバチルス・ガセリ菌株として、ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株、１１３０菌株、５３４３菌株、１１６５７菌株、８７９０菌株、１１０４６菌株を培養した。培養の手順は実験例１と同様にして行った。
(Experimental example 3)
<Culture of various Lactobacillus gasseri strains>
Cultivation of various Lactobacillus gasseri strains using the medium of Example 4 (yeast extract FR, manufactured by Kirin Kyowa Foods Co., Ltd.), which had good growth of Lactobacillus gasseri strains Then, proliferation was examined. The medium of Comparative Example 1 (MRS medium) was used as a control. As the Lactobacillus gasseri strain, Lactobacillus gasseri JCM1131 T strain, 1130 strain, 5343 strain, 11657 strain, 8790 strain, 11046 strain were cultured. The culture procedure was the same as in Experimental Example 1.
図５は、実施例４及び比較例１の培地で各種ラクトバチルス・ガセリ菌株を２４時間培養後のｐＨの値を示したグラフである。図６は、実施例４及び比較例１の培地で各種ラクトバチルス・ガセリ菌株を２４時間培養後の濁度の値を示したグラフである。 FIG. 5 is a graph showing pH values after culturing various Lactobacillus gasseri strains for 24 hours in the media of Example 4 and Comparative Example 1. FIG. 6 is a graph showing turbidity values after culturing various Lactobacillus gasseri strains for 24 hours in the media of Example 4 and Comparative Example 1.
図５、６の結果から、実施例４の培地は、比較例１の培地と同程度に各種ラクトバチルス・ガセリ菌株を増殖できることが示された。 The results of FIGS. 5 and 6 showed that the medium of Example 4 can grow various Lactobacillus gasseri strains to the same extent as the medium of Comparative Example 1.
（実験例４）
＜酵母エキス濃度の検討＞
実施例４の培地において、酵母エキスの濃度を変化させた培地を調製し、ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を培養し、増殖性を検討した。具体的には、表５に示す量の酵母エキスを含有する点以外は実施例４の培地と同様にして、実施例１１〜１４の培地を調製した。実施例４、実施例１１〜１４及び比較例１の培地を用いて、実験例１と同様にしてラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を培養し、増殖性を検討した。
(Experimental example 4)
<Examination of yeast extract concentration>
In the medium of Example 4, a medium in which the concentration of the yeast extract was changed was prepared, and the Lactobacillus gasseri JCM1131 T strain was cultured to examine the growth ability. Specifically, the media of Examples 11 to 14 were prepared in the same manner as the media of Example 4 except that the yeast extract was contained in an amount shown in Table 5. Lactobacillus gasseri JCM1131 T strain was cultured in the same manner as in Experimental Example 1 using the culture media of Example 4, Examples 11-14, and Comparative Example 1, and the growth was examined.
図７は、実施例４、実施例１１〜１４及び比較例１の培地で、ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を２４時間培養後のｐＨの値を示したグラフである。図８は、実施例４、実施例１１〜１４及び比較例１の培地で、ラクトバチルス・ガセリＪＣＭ１１３１Ｔ菌株を２４時間培養後の濁度の値を示したグラフである。 FIG. 7 is a graph showing the pH value after culturing the Lactobacillus gasseri JCM1131 T strain for 24 hours in the media of Example 4, Examples 11-14, and Comparative Example 1. FIG. 8 is a graph showing turbidity values after culturing Lactobacillus gasseri JCM1131 T strain for 24 hours in the media of Example 4, Examples 11-14, and Comparative Example 1.
図７、８の結果から、培地中の酵母エキスの含有量を変化させることによって、菌株の増殖性を調節できることが示された。 From the results of FIGS. 7 and 8, it was shown that the growth of the strain can be controlled by changing the content of the yeast extract in the medium.
（実験例５）
＜ラクトバチルス・ガセリ菌株以外のラクトバチルス属乳酸菌株の培養＞
実施例４及び比較例１の培地を用いて、ラクトバチルス・ガセリ菌株以外のラクトバチルス属乳酸菌株を培養し、増殖性を検討した。ラクトバチルス・ガセリ菌株以外のラクトバチルス属乳酸菌株として、ラクトバチルス・アシドフィルスＪＣＭ１１３２Ｔ菌株、ラクトバチルス・カゼイＪＣＭ１１３４Ｔ菌株、ラクトバチルス・ヘルベティカスＪＣＭ１１２０Ｔ菌株を培養した。培養の手順は実験例１と同様にして行った。
(Experimental example 5)
<Culture of Lactobacillus Lactobacillus strains other than Lactobacillus gasseri strain>
Using the culture media of Example 4 and Comparative Example 1, Lactobacillus genus lactic acid strains other than Lactobacillus gasseri strains were cultured and examined for growth. Lactobacillus acidophilus JCM1132 T strain, Lactobacillus casei JCM1134 T strain, and Lactobacillus helveticus JCM1120 T strain were cultured as Lactobacillus lactic acid strains other than Lactobacillus gasseri strain. The culture procedure was the same as in Experimental Example 1.
図９は、実施例４及び比較例１の培地で各ラクトバチルス属乳酸菌株を２４時間培養後のｐＨの値を示したグラフである。図１０は、実施例４及び比較例１の培地で各ラクトバチルス属乳酸菌株を２４時間培養後の濁度の値を示したグラフである。 FIG. 9 is a graph showing the pH value after culturing each Lactobacillus lactic acid strain for 24 hours in the medium of Example 4 and Comparative Example 1. FIG. 10 is a graph showing turbidity values after culturing each Lactobacillus lactic acid strain in the medium of Example 4 and Comparative Example 1 for 24 hours.
図９、１０の結果から、実施例４の培地は、比較例１の培地と同程度にラクトバチルス・ガセリ菌株以外のラクトバチルス属乳酸菌株を増殖できることが示された。 The results of FIGS. 9 and 10 indicate that the medium of Example 4 can grow Lactobacillus lactic acid bacterial strains other than the Lactobacillus gasseri strain to the same extent as the medium of Comparative Example 1.
本発明によれば、硫酸マンガン四水和物や乳由来成分を実質的に含有しないにもかかわらず、ラクトバチルス属乳酸菌を良好に培養することができる、ラクトバチルス属乳酸菌培養用食品グレード培地、上記培地を用いたラクトバチルス属乳酸菌の培養方法及びラクトバチルス属乳酸菌培養物の製造方法を提供することができる。 According to the present invention, a food grade medium for cultivating Lactobacillus lactic acid bacteria, which can satisfactorily culture Lactobacillus lactic acid bacteria, despite containing substantially no manganese sulfate tetrahydrate or milk-derived components, A method for culturing Lactobacillus lactic acid bacteria using the medium and a method for producing a Lactobacillus lactic acid bacteria culture can be provided.
Claims (3)
ペプトン、牛肉エキス、工業用酵母エキス、硫酸マンガン四水和物及び乳由来成分を実質的に含有せず、食品添加用酵母エキス、グルコース、モノオレイン酸ソルビタン、リン酸水素ニカリウム、酢酸ナトリウム三水和物、クエン酸三アンモニウム及び硫酸マグネシウム七水和物を含有する、培地。 A food grade medium for culturing Lactobacillus lactic acid bacteria,
Peptone, beef extract, industrial yeast extract, manganese sulfate tetrahydrate, and milk-derived components are not substantially contained, yeast extract for food addition , glucose, sorbitan monooleate, dipotassium hydrogen phosphate, sodium acetate trihydrate A medium containing a Japanese hydrate, triammonium citrate and magnesium sulfate heptahydrate .
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