JP2011193730A - Method for obtaining lactic acid bacterium having polysaccharide-peptidoglycan complex - Google Patents
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本発明は、多糖−ペプチドグリカン複合体１（ＰＳ−ＰＧ１）保有乳酸菌の取得方法に関し、更に詳細には、乳酸菌群中から、ＰＳ−ＰＧ１を保有する乳酸菌を分離し、これを取得する方法に関する。 The present invention relates to a method for obtaining lactic acid bacteria possessing polysaccharide-peptidoglycan complex 1 (PS-PG1), and more particularly, to a method for separating lactic acid bacteria retaining PS-PG1 from a group of lactic acid bacteria and obtaining the same.
乳酸菌は、免疫の賦活作用や抗炎症作用を持つことが知られている。その中でも、ラクトバチルス・カゼイＹＩＴ ９０２９（ＦＥＲＭ ＢＰ−１３６６）は、多くの生理効果が明らかとなっている。このラクトバチルス・カゼイ ＹＩＴ ９０２９は、胃液や胆汁等の強い消化液に耐えて生きたまま腸内に到達し、腸内に常在性のビフィドバクテリウム属細菌を増加させ、大腸菌群を減少させて腸内環境を改善させることが知られている。 Lactic acid bacteria are known to have immune activation and anti-inflammatory effects. Among them, Lactobacillus casei YIT 9029 (FERM BP-1366) has revealed many physiological effects. This Lactobacillus casei YIT 9029 is able to withstand strong digestive juices such as gastric juice and bile and reaches the intestine alive, increasing the number of resident Bifidobacterium in the intestine and reducing the coliform group It is known to improve the intestinal environment.
また、ラクトバチルス・カゼイ ＹＩＴ ９０２９は、腸内の有害菌が産生する有害物質の生成を抑制する。更に、ラクトバチルス・カゼイ ＹＩＴ ９０２９の飲用効果としては、便性改善、病原性大腸菌Ｏ−１５７の増殖抑制効果、ベロ毒素産生抑制効果、尿路感染症予防効果等の感染防御、ＮＫ細胞活性化作用等の免疫力の上昇作用、抗アレルギー効果、大腸がんや表在性膀胱がん等のがん抑制効果等が報告されている。更にまた、近年の研究の結果、ラクトバチルス・カゼイ ＹＩＴ ９０２９あるいはその菌体由来多糖画分にＩＬ−６産生抑制作用及び炎症性腸疾患に対する予防治療効果があることが見出されている（特許文献１）。 Lactobacillus casei YIT 9029 suppresses the production of harmful substances produced by harmful bacteria in the intestines. Furthermore, as a drinking effect of Lactobacillus casei YIT 9029, stool improvement, infection inhibition effect of pathogenic E. coli O-157, verotoxin production inhibition effect, urinary tract infection prevention effect, etc., NK cell activation It has been reported to increase immunity such as action, antiallergic effect, and cancer suppressive effect such as colon cancer and superficial bladder cancer. Furthermore, as a result of recent research, it has been found that Lactobacillus casei YIT 9029 or its cell-derived polysaccharide fraction has an IL-6 production inhibitory effect and a preventive and therapeutic effect against inflammatory bowel disease (patents). Reference 1).
上記の作用を奏する菌体由来多糖画分として、多糖−ペプチドグリカン複合体（polysaccharide-peptideglycan complex、以下「ＰＳ−ＰＧ」と略称する）が明らかにされている。ＰＳ−ＰＧには分子量の異なるＰＳ−ＰＧ１、ＰＳ−ＰＧ２の２種類が知られている（非特許文献１）。このうち、ＰＳ−ＰＧ１は、分子量が１００ｋＤａ以上、ＰＳ−ＰＧ２は分子量約３０ｋＤａと推定されており、ラクトバチルス・カゼイ ＹＩＴ ９０２９における含量の比率は、約１：８である（非特許文献1）。 A polysaccharide-peptide glycan complex (hereinafter abbreviated as “PS-PG”) has been clarified as a microbial cell-derived polysaccharide fraction exhibiting the above-described action. Two types of PS-PG, PS-PG1 and PS-PG2, having different molecular weights are known (Non-Patent Document 1). Among these, PS-PG1 is estimated to have a molecular weight of 100 kDa or more, PS-PG2 is estimated to have a molecular weight of about 30 kDa, and the content ratio in Lactobacillus casei YIT 9029 is about 1: 8 (Non-patent Document 1). .
上記ＰＳ−ＰＧ１の化学組成は、非特許文献１に開示されており、ラムノース、グルコース、ガラクトース、グルコサミン、ムラミン酸、アスパラギン酸、グルタミン酸、アラニン、リジンを含むことが報告されている。 The chemical composition of PS-PG1 is disclosed in Non-Patent Document 1, and it is reported that it contains rhamnose, glucose, galactose, glucosamine, muramic acid, aspartic acid, glutamic acid, alanine, and lysine.
さらに近年の研究の結果、ラクトバチルス・カゼイ ＹＩＴ ９０２９の炎症性腸疾患や腸炎随伴性ガン予防効果の活性本体が、ＰＳ−ＰＧ１であることが報告されている（非特許文献２）。 Furthermore, as a result of recent studies, it has been reported that Lactobacillus casei YIT 9029 has an active body for preventing inflammatory bowel disease and enteritis-associated cancer as a non-patent document 2 (Non-patent Document 2).
このようにＰＳ−ＰＧ１を持つ乳酸菌の生理効果については多くの報告があるものの、ＰＳ−ＰＧ１を持つ乳酸菌の簡便な分離方法、判別方法等については検討されていない。 Thus, although there are many reports on the physiological effects of lactic acid bacteria having PS-PG1, simple separation methods, discrimination methods, and the like of lactic acid bacteria having PS-PG1 have not been studied.
ある乳酸菌がＰＳ−ＰＧ１を持つかどうかは、ストレプトマイセス・グロビスポラス（Streptomyces globisporus）由来のＮ−アセチルムラミダーゼ処理により可溶化した後、ゲル濾過法により分離し、さらにその生化学的分析を行うことによって判別する。このように、従来の方法では、乳酸菌がＰＳ−ＰＧ１を持つかどうか判断するのに煩雑な操作と時間、費用がかかるのが実情であった。 Whether a certain lactic acid bacterium has PS-PG1 is solubilized by treatment with N-acetylmuramidase derived from Streptomyces globisporus, then separated by gel filtration, and further subjected to biochemical analysis. To determine. As described above, in the conventional method, it is actually a complicated operation, time and cost to determine whether or not lactic acid bacteria have PS-PG1.
さらに、ＰＳ−ＰＧ１を持つ乳酸菌と持たない乳酸菌が培地中に混在した場合、この２つを効率的に分離する方法は、これまでに存在しなかった。 Furthermore, when lactic acid bacteria having PS-PG1 and lactic acid bacteria not having PS-PG1 coexist in the medium, there has been no method for efficiently separating the two.
本発明は、細菌中のＰＳ−ＰＧ１の生化学的分析や細菌のＲＮＡやＤＮＡの分離操作、あるいは遺伝学的分析操作を必要とせず、ＰＳ−ＰＧ１を持つ乳酸菌を簡便で迅速に分離、取得および判別する方法を提供することを課題とする。 The present invention does not require biochemical analysis of PS-PG1 in bacteria, separation of bacterial RNA or DNA, or genetic analysis, and allows easy and rapid separation and acquisition of lactic acid bacteria having PS-PG1. It is another object of the present invention to provide a discrimination method.
本発明者らは、上記課題を解決するため鋭意検討していた結果、カゼイン存在下でＰＳ−ＰＧ１を有する乳酸菌は、遠心分離で極めて沈殿しにくいという他の乳酸菌と異なる挙動を示すこと、そしてこの挙動を利用することにより、他の乳酸菌と簡単に分離できることを見出し、本発明を完成した。 As a result of intensive studies to solve the above problems, the inventors of the present invention show that lactic acid bacteria having PS-PG1 in the presence of casein exhibit a behavior different from that of other lactic acid bacteria that are extremely difficult to precipitate by centrifugation, and By utilizing this behavior, it was found that it could be easily separated from other lactic acid bacteria, and the present invention was completed.
すなわち本発明は、乳酸菌を含有する培養物を、カゼイン存在下で遠心分離し、その非沈殿画分を回収することを特徴とする多糖−ペプチドグリカン複合体１（ＰＳ−ＰＧ１）を持つ乳酸菌の取得方法である。 That is, the present invention obtains a lactic acid bacterium having a polysaccharide-peptidoglycan complex 1 (PS-PG1), wherein a culture containing lactic acid bacterium is centrifuged in the presence of casein and the non-precipitated fraction is collected. Is the method.
また本発明は、乳酸菌を含有する培養物を、カゼイン存在下で遠心分離し、その非沈殿画分を回収することを特徴とするＰＳ−ＰＧ１を持つ乳酸菌の分離方法である。 The present invention is also a method for separating lactic acid bacteria having PS-PG1, which comprises centrifuging a culture containing lactic acid bacteria in the presence of casein and collecting the non-precipitated fraction.
更に本発明は、上記のＰＳ−ＰＧ１を持つ乳酸菌の性質を用いたＰＳ−ＰＧ１を持つ乳酸菌の判別方法である。 Furthermore, the present invention is a method for discriminating lactic acid bacteria having PS-PG1 using the properties of lactic acid bacteria having PS-PG1 described above.
本発明方法によれば、ＰＳ−ＰＧ１を持つ乳酸菌と持たない乳酸菌が混在した状態から、簡便かつ迅速にＰＳ−ＰＧ１を持つ乳酸菌を分離、取得することができる。 According to the method of the present invention, lactic acid bacteria having PS-PG1 can be separated and obtained easily and rapidly from a state where lactic acid bacteria having PS-PG1 and lactic acid bacteria not having PS-PG1 coexist.
また、本発明方法を利用すれば、簡単にＰＳ−ＰＧ１を持つ乳酸菌を分離、取得することが可能であるし、また、ある乳酸菌がＰＳ−ＰＧ１を保有しているかどうかも容易に判別可能である。 Moreover, if the method of the present invention is used, it is possible to easily isolate and obtain lactic acid bacteria having PS-PG1, and it is also possible to easily determine whether a certain lactic acid bacterium has PS-PG1. is there.
本発明のＰＳ−ＰＧ１を持つ乳酸菌の分離、取得方法は、カゼイン存在下で遠心分離し、その非沈殿画分である上清を回収することによってＰＳ−ＰＧ１を持つ乳酸菌を分離、取得するものである。 The method for separating and obtaining lactic acid bacteria having PS-PG1 according to the present invention is a method for separating and obtaining lactic acid bacteria having PS-PG1 by centrifuging in the presence of casein and collecting the supernatant which is a non-precipitated fraction. It is.
本発明において、カゼイン存在下での遠心分離とは、α−カゼイン、β−カゼインあるいはκ−カゼインを含む培地あるいはこれらカゼインの水溶液中において乳酸菌の遠心分離操作を行うことをいう。この場合、使用されるカゼイン濃度としては、０．０１〜２０％が好ましく、さらに好ましくは０．１〜１０％であり、この量を含有する培地あるいは水溶液を好適に使用する事が出来る。また、本発明のカゼイン存在下での遠心分離とは、乳酸菌をカゼイン含有培地で培養後、培養液をそのまま遠心分離する場合だけでなく、乳酸菌を培養後、カゼイン含有培地あるいは水溶液に懸濁して遠心分離する場合も含む。 In the present invention, the centrifugation in the presence of casein means that a lactic acid bacterium is centrifuged in a medium containing α-casein, β-casein or κ-casein or an aqueous solution of these caseins. In this case, the concentration of casein used is preferably 0.01 to 20%, more preferably 0.1 to 10%, and a medium or an aqueous solution containing this amount can be suitably used. The centrifugation in the presence of casein of the present invention is not only the case where the lactic acid bacterium is cultured in a casein-containing medium and then the culture solution is centrifuged as it is, but the lactic acid bacterium is cultured and then suspended in a casein-containing medium or an aqueous solution. This includes the case of centrifugation.
使用されるカゼイン源としては、市販の牛乳由来カゼイン、牛乳、全粉乳、脱脂粉乳等を使用する事が出来るが、その中でも脱脂粉乳が好ましい。カゼイン源として脱脂粉乳を使用する場合、その培地中の濃度は、０．１〜２０％が好ましく、さらに１０％程度が好適である。 As a casein source to be used, commercially available milk-derived casein, milk, whole milk powder, skim milk powder or the like can be used, and among these, skim milk powder is preferable. When skim milk powder is used as a casein source, the concentration in the medium is preferably 0.1 to 20%, and more preferably about 10%.
乳酸菌を含有する培養物を遠心分離する際に使用される上記カゼイン含有培地の温度は、１〜４０℃の間であれば良く、好ましくは、４℃〜３７℃の間である。さらにｐＨについては、カゼインタンパク質の立体構造が大きく変化しない範囲において特に限定されないが、好ましくはｐＨ３.７〜７、さらに好適にはｐＨ５〜７である。 The temperature of the casein-containing medium used when centrifuging a culture containing lactic acid bacteria may be between 1 and 40 ° C, and preferably between 4 and 37 ° C. Further, the pH is not particularly limited as long as the three-dimensional structure of the casein protein does not change greatly, but is preferably pH 3.7 to 7, more preferably pH 5 to 7.
本発明における、遠心分離の遠心力（相対遠心加速度）としては、８００〜２０４００×ｇの間が好ましく、さらに２３００〜９１００×ｇが好適である。なお、この相対遠心加速度は以下の式で計算する事が出来る。 In the present invention, the centrifugal force (relative centrifugal acceleration) of the centrifugal separation is preferably between 800 and 20400 × g, and more preferably between 2300 and 9100 × g. This relative centrifugal acceleration can be calculated by the following formula.
相対遠心加速度（×ｇ）＝１１１８×Ａ×Ｂ２×１０−８
Ａ：回転半径（ｃｍ）
Ｂ：回転数（ｒｐｍ）
Relative centrifugal acceleration (× g) = 1118 × A × B 2 × 10 −8
A: Turning radius (cm)
B: Number of revolutions (rpm)
上記、遠心分離に用いる遠心分離装置は、適当な遠心力がかかるものであれば、特に限定されないが、一般的にはスイング型あるいはローター型のものが使用できる。 The centrifuge used for the centrifuge is not particularly limited as long as an appropriate centrifugal force is applied. Generally, a swing type or a rotor type can be used.
なお、本発明のＰＳ−ＰＧ１を持つ乳酸菌の分離、取得方法は、カゼインの他、更にカルシウム塩を加えることにより、効率良く実施することができる。すなわち、ＰＳ−ＰＧ１を持つ乳酸菌の遠心分離による沈殿をカゼイン単独より効率よく防止することができ、ＰＳ−ＰＧ１を持たない乳酸菌との分離効率を上げることが可能である。 In addition, the isolation | separation and acquisition method of lactic acid bacteria with PS-PG1 of this invention can be efficiently implemented by adding a calcium salt other than casein. That is, precipitation due to centrifugation of lactic acid bacteria having PS-PG1 can be prevented more efficiently than casein alone, and separation efficiency from lactic acid bacteria not having PS-PG1 can be increased.
使用されるカルシウム塩としては、塩化カルシウム、硝酸カルシウム等が挙げられ、遠心分離の際の使用量は、カルシウムとして、１ｍＭ〜４０ｍＭ程度が好ましい。 Examples of calcium salts used include calcium chloride and calcium nitrate, and the amount used during centrifugation is preferably about 1 mM to 40 mM as calcium.
本発明のＰＳ−ＰＧ１を持つ乳酸菌の分離、取得方法は、上述のカゼイン存在下で遠心分離し、非沈殿画分である上清を回収する方法である。ＰＳ−ＰＧ１を持つ乳酸菌と持たない乳酸菌が混在しており、しかもＰＳ−ＰＧ１を持つ乳酸菌が少ない場合は、遠心分離操作を複数回繰り返すことにより、ＰＳ−ＰＧ１を持つ乳酸菌を分離、取得することが出来る。 The method for separating and obtaining lactic acid bacteria having PS-PG1 according to the present invention is a method of collecting the supernatant which is a non-precipitated fraction by centrifugation in the presence of casein. When lactic acid bacteria having PS-PG1 and lactic acid bacteria not having PS-PG1 are mixed and there are few lactic acid bacteria having PS-PG1, the lactic acid bacteria having PS-PG1 are separated and obtained by repeating the centrifugation operation several times. I can do it.
更に、ＰＳ−ＰＧ１を持つ乳酸菌の判別方法は、乳酸菌を上述のカゼイン存在下で遠心分離して沈殿画分と非沈殿画分に分け、それぞれの画分中の乳酸菌の存在を調べることにより実施することができる。そして、約２３００ないし１４２００×ｇ程度の遠心分離により、非沈殿画分中に存在する乳酸菌が沈殿画分中に存在する乳酸菌の０．１倍以上、好ましくは１．０倍以上、さらに好ましくは１０倍以上となる場合、当該乳酸菌はＰＳ−ＰＧ１を持つと判別することができる。 Furthermore, the method for discriminating lactic acid bacteria having PS-PG1 is carried out by centrifuging the lactic acid bacteria in the presence of casein described above to separate the precipitated fraction and the non-precipitated fraction, and examining the presence of the lactic acid bacteria in each fraction. can do. Then, by centrifuging about 2300 to 14200 × g, lactic acid bacteria present in the non-precipitated fraction are 0.1 times or more, preferably 1.0 times or more of the lactic acid bacteria present in the precipitated fraction, more preferably When it becomes 10 times or more, it can be determined that the lactic acid bacterium has PS-PG1.
以上、ＰＳ−ＰＧ１を持つ乳酸菌の取得方法、分離方法、判別方法について説明してきたが、ここでいうＰＳ−ＰＧ１を持つ乳酸菌としては、特に属、種に限定されるものでなく、いわゆる乳酸菌と呼ばれるものが含まれる。しかしながら、ＰＳ−ＰＧ１を持つ乳酸菌の代表的なものとしては、ラクトバチルス・カゼイ ＹＩＴ ９０２９（ＦＥＲＭ ＢＰ−１３６６）やＹＩＴ ９０１８（ＦＥＲＭ ＢＰ−６６５）を挙げることができる。 As described above, the method for obtaining, separating and discriminating lactic acid bacteria having PS-PG1 has been described. However, the lactic acid bacteria having PS-PG1 here are not particularly limited to genus and species, and so-called lactic acid bacteria Includes what is called. However, representative examples of lactic acid bacteria having PS-PG1 include Lactobacillus casei YIT 9029 (FERM BP-1366) and YIT 9018 (FERM BP-665).
なお、本発明方法により、ＰＳ−ＰＧ１を持つ乳酸菌が取得でき、また、分離、判別できる理由は、不明な点もあるが、次のように考えられている。すなわち、カゼイン分子とＰＳ−ＰＧ１は結合性が高く、このＰＳ−ＰＧ１を有する乳酸菌は、カゼインを含有する培地中で、カゼインミセルに接着した状態にある。この結果、遠心分離処理によってもＰＳ−ＰＧ１を有する乳酸菌は沈殿せず、カゼインミセルと共に上清に残存し、沈殿したＰＳ−ＰＧ１を有さない乳酸菌と分離できるものと考えられる。このことは、ＰＳ−ＰＧ１を有さないラクトバチルス・カゼイは、５１００×ｇ、５分間の遠心で、ほぼすべてが沈殿することからも確認される。 The reason why lactic acid bacteria having PS-PG1 can be obtained and separated and discriminated by the method of the present invention is unclear, but is considered as follows. That is, the casein molecule and PS-PG1 have high binding properties, and the lactic acid bacterium having PS-PG1 is in a state of adhering to casein micelles in a medium containing casein. As a result, it is considered that lactic acid bacteria having PS-PG1 are not precipitated even by centrifugation, remain in the supernatant together with casein micelles, and can be separated from lactic acid bacteria having no precipitated PS-PG1. This is also confirmed from the fact that almost all Lactobacillus casei without PS-PG1 precipitates at 5100 × g for 5 minutes.
次に実施例を挙げ、本発明を更に詳しく説明するが、本発明はこれら実施例に何ら制約されるものではない。 EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.
実 施 例 １
ＰＳ−ＰＧ１を持つ乳酸菌の分離試験（１）：
被験菌株として、同じ種に属するラクトバチルス・カゼイ ＹＩＴ０１８０Ｔ（上付Ｔは、その菌株がタイプストレインであることを示す。以下同じ。ＡＴＣＣ ３３４４）（ＰＳ−ＰＧ１を持たない）、およびラクトバチルス・カゼイ ＹＩＴ９０２９（ＰＳ−ＰＧ１を持つ）を利用し、ＰＳ−ＰＧ１を持つ乳酸菌の分離が可能であるかどうか試験した。
Example 1
Isolation test of lactic acid bacteria having PS-PG1 (1):
As a test strain, Lactobacillus casei YIT0180 T belonging to the same species (superscript T indicates that the strain is a type strain. The same applies hereinafter. ATCC 3344) (without PS-PG1), and Lactobacillus Casei YIT9029 (with PS-PG1) was used to test whether it was possible to isolate lactic acid bacteria with PS-PG1.
まず、乳酸菌の培養培地として、ＭＲＳ培地（ＤＩＦＣＯ）を用い、各被験菌株を種菌として、３７℃で、１５．５時間前培養した。得られた前培養２０μｌを、それぞれＭＲＳ培地１ｍｌに加え、３７℃で４時間本培養を行った。 First, MRS medium (DIFCO) was used as a culture medium for lactic acid bacteria, and each test strain was inoculated at 37 ° C. for 15.5 hours. 20 μl of the obtained preculture was added to 1 ml of MRS medium, respectively, and main culture was performed at 37 ° C. for 4 hours.
得られた培養液１μｌを、ミルク培地（脱脂粉乳（四つ葉乳業製）１０％溶液）５０μｌに懸濁し、３７℃で３０分間保温した後、懸濁液を５１００×ｇで５分間遠心分離した。その後、遠心分離後の上清および沈殿について、それぞれの菌数をＭＲＳ寒天培地で菌数計測を行った。なお、比較として、ミルク培地に代え、ＭＲＳ培地を用いて上記と同様に懸濁させ、遠心分離したものについても、菌数計測を行った。この結果を表１に示す。 1 μl of the obtained culture solution is suspended in 50 μl of a milk medium (10% solution of skim milk powder (manufactured by Yotsuba Dairy)), incubated at 37 ° C. for 30 minutes, and then the suspension is centrifuged at 5100 × g for 5 minutes. did. Thereafter, the number of bacteria was counted on the MRS agar medium for the supernatant and the precipitate after centrifugation. For comparison, the number of bacteria was also measured for those suspended and centrifuged in the same manner as described above using MRS medium instead of milk medium. The results are shown in Table 1.
表１に示すように、カゼインを含有するミルク培地で懸濁、遠心分離した場合には、ＰＳ−ＰＧ１を持つＹＩＴ９０２９は、上清に１０５ＣＦＵ存在するのに対し、ＹＩＴ ０１８０Ｔは上清にほとんど検出されなかった。これに対し、ＭＲＳ培地に懸濁、遠心分離した場合には、ＹＩＴ９０２９およびＹＩＴ ０１８０Ｔはいずれも沈殿、上清のどちらの部分にも存在していた。 As shown in Table 1, when suspended in a milk medium containing casein and centrifuged, YIT9029 with PS-PG1 is present in 10 5 CFU in the supernatant, whereas YIT 0180 T is in the supernatant Was hardly detected. In contrast, suspended in MRS medium, when centrifugation, YIT9029 and YIT 0180 T Any precipitation in either portion of the supernatant was present.
このように、ミルク培地を用いて遠心分離し、上清中の生菌数や沈殿中の生菌数を測定することにより、ＰＳ−ＰＧ１を持つ乳酸菌かどうか判別することが可能であることが示された。 Thus, by centrifuging using a milk medium and measuring the number of viable bacteria in the supernatant and the number of viable bacteria in the precipitate, it is possible to determine whether the lactic acid bacteria have PS-PG1. Indicated.
実 施 例 ２
ＰＳ−ＰＧ１を持つ乳酸菌の分離試験（２）：
被験菌株として、同じ属に属し、ＰＳ−ＰＧ１を持たないことが知られている、ラクトバチルス・アシドフィラス ＹＩＴ００７０Ｔ（ＡＴＣＣ ４３５６）、ラクトバチルス・カゼイ ＹＩＴ ０１８０Ｔおよびラクトコッカス・ラクティス・サブスピーシーズ・ラクティス ＹＩＴ２００８Ｔ（ＡＴＣＣ １９４３５）を用い、これらとＰＳ−ＰＧ１を持っていることが知られているラクトバチルス・カゼイ ＹＩＴ ９０２９が分離、判別可能であるかどうかを試験した。
Example 2
Isolation test of lactic acid bacteria having PS-PG1 (2):
Lactobacillus acidophilus YIT0070 T (ATCC 4356), Lactobacillus casei YIT 0180 T and Lactococcus lactis subspecies lactis belonging to the same genus as test strains and known to have no PS-PG1 YIT2008 T (ATCC 19435) was used to test whether these and Lactobacillus casei YIT 9029, which are known to have PS-PG1, can be separated and discriminated.
各被験菌株を、ミルク培地（脱脂粉乳（四つ葉乳業）１０％溶液）中、３７℃で４時間培養した（但し、ＹＩＴ２００８Ｔは、ミルク培地に０．２％ブドウ糖を添加したものを使用し、培養温度は３０℃とした）。 Each test strain was cultured for 4 hours at 37 ° C. in milk medium (10% solution of skim milk powder (four-leaf dairy industry)) (YIT2008 T uses milk medium supplemented with 0.2% glucose) The culture temperature was 30 ° C.).
培養後、５０ｍｌの各培養液を２３００×ｇで５分間遠心し、上清と沈殿に分け、更に沈殿は５０ｍｌの精製水に懸濁した。上清および懸濁液それぞれを精製水で１／１０希釈したもの２．５ｍｌをＭＲＳ平板培地に置き、３７℃で２日間培養し、菌数を測定し、上清と沈殿でどちらに微生物が多いかどうか検討した。この結果を表２に示す。 After culturing, 50 ml of each culture solution was centrifuged at 2300 × g for 5 minutes, divided into a supernatant and a precipitate, and the precipitate was suspended in 50 ml of purified water. 2.5 ml of each supernatant / suspension diluted 1/10 with purified water is placed on MRS plate medium, cultured at 37 ° C. for 2 days, the number of bacteria is measured, We examined whether there were many. The results are shown in Table 2.
この結果、ＰＳ−ＰＧ１を持たないラクトバチルス・アシドフィラスＹＩＴ ００７０Ｔ、ラクトバチルス・カゼイ ＹＩＴ ０１８０Ｔおよびラクトコッカス・ラクティス・サブスピーシーズ・ラクティス ＹＩＴ２００８Ｔは、ミルク培地で培養後、遠心分離による上清での分離菌数は沈殿よりも極めて少なく、難沈殿性を示さなかった。一方、ＰＳ−ＰＧ１を持つラクトバチルス・カゼイＹＩＴ ９０２９は、遠心分離によってもあまり沈殿せず、上清で分離する事が可能であることが示された。 As a result, Lactobacillus acidophilus YIT 0070 T , Lactobacillus casei YIT 0180 T and Lactococcus lactis subspices lactis YIT2008 T without PS-PG1 were cultured in a milk medium and then centrifuged in a supernatant. The number of isolated bacteria was significantly less than that of precipitation, and did not show difficult precipitation. On the other hand, Lactobacillus casei YIT 9029 with PS-PG1 did not precipitate much even by centrifugation and was shown to be separable with the supernatant.
実 施 例 ３
混合乳酸菌中からのＰＳ−ＰＧ１を持つ乳酸菌の分離試験：
被験菌株として、ラクトバチルス・カゼイ ＹＩＴ ０１８０Ｔのリファンピシン耐性株であるラクトバチルス・カゼイ ＹＩＴ ０１８０ＴＲｉｆ（１００μｇ／ｍｌのリファンピシン（シグマ社製）を含むＭＲＳ寒天培地に１０８ＣＦＵ程度のＹＩＴ０１８０Ｔを塗沫し、３７℃で３日培養後出現した耐性コロニーを単コロニー分離し、ＭＲＳ培地で培養後さらに１００μｇ／ｍｌのリファンピシンに対する耐性を確認したもの。；ヤクルト本社中央研究所保存）およびラクトバチルス・カゼイ ＹＩＴ９０２９のストレプトマイシン耐性株であるラクトバチルス・カゼイ ＹＩＴ ９０２９Ｓｍ（１００μｇ／ｍｌの硫酸ストレプトマイシン（明治製菓社製）を含むＭＲＳ寒天培地に１０８ＣＦＵ程度のＹＩＴ９０２９を塗沫し、３７℃で３日培養後出現した耐性コロニーを単コロニー分離し、ＭＲＳ培地で培養後さらに１００μｇ／ｍｌの硫酸ストレプトマイシンに対する耐性を確認したもの。；ヤクルト本社中央研究所保存）を下記表３の割合で混合して培養し、培養物中からラクトバチルス・カゼイ ＹＩＴ９０２９Ｓｍを分離できるかどうか試験した。
Example 3
Separation test of lactic acid bacteria having PS-PG1 from mixed lactic acid bacteria:
As test strains, the YIT0180 T of about 10 8 CFU in MRS agar medium containing Lactobacillus casei YIT 0180 T of rifampicin resistant strains and is Lactobacillus casei YIT 0180 T Rif (100μg / ml of rifampicin (Sigma) Resistant colonies that appeared after smearing and culturing at 37 ° C. for 3 days were isolated, cultured in MRS medium, and further confirmed to be resistant to 100 μg / ml rifampicin; preserved at Yakult Central Research Laboratory) and Lactobacillus casei and smeared the YIT9029 of about 10 8 CFU in MRS agar medium containing streptomycin resistant strain is Lactobacillus casei YIT 9029Sm of YIT9029 (100μg / ml streptomycin sulfate (Meiji Seika Co., Ltd.), 3 at 37 ° C. Resistant colonies that emerged after culture were isolated, cultured in MRS medium, and further confirmed to be resistant to 100 μg / ml streptomycin sulfate (preserved at Yakult Central Research Laboratory) and mixed at the ratio shown in Table 3 below. Then, it was tested whether Lactobacillus casei YIT9029Sm could be separated from the culture.
まず、各混合被験菌をＭＲＳ培地で３７℃で、１５．５時間前培養し、次いで、この前培養液２０μｌをＭＲＳ培地１ｍｌに加え、３７℃で４時間培養した。得られた、それぞれの培養液１μｌを１００μｌミルク培地（脱脂粉乳１０％溶液）に懸濁し、３７℃で３０分間保温した後、この懸濁液を５１００×ｇで５分間遠心分離した。 First, each mixed test bacterium was precultured in MRS medium at 37 ° C. for 15.5 hours, and then 20 μl of this preculture was added to 1 ml of MRS medium and cultured at 37 ° C. for 4 hours. 1 μl of each of the obtained culture solutions was suspended in 100 μl milk medium (10% solution of skim milk powder) and incubated at 37 ° C. for 30 minutes, and then the suspension was centrifuged at 5100 × g for 5 minutes.
得られた上清または沈殿に含まれるそれぞれの菌数を、リファンピシン含有ＭＲＳ寒天培地及びストレプトマイシン含有ＭＲＳ培地でそれぞれの耐性菌の菌数計測を行うことにより求めた。この結果を表３に示す。 The number of each bacteria contained in the obtained supernatant or precipitate was determined by counting the number of resistant bacteria in a rifampicin-containing MRS agar medium and a streptomycin-containing MRS medium. The results are shown in Table 3.
表３から、ＹＩＴ ０１８０ＴＲｉｆ菌液とＹＩＴ ９０２９Ｓｍ菌液を１：１に混合した場合、遠心分離後の上清中のＹＩＴ９０２９Ｓｍ菌数はＹＩＴ ０１８０ＴＲｉｆ菌数の約１０００倍であり、ＰＳ−ＰＧ１を持つ乳酸菌を特異的に取得することができた。また、混合比が１：１０−３のデータに示されるように、ＹＩＴ ９０２９Ｓｍ菌数が全体の約１／１０００であってもこの分離、取得効果が確認された。 From Table 3, when YIT 0180 T Rif bacterial solution and YIT 9029Sm bacterial solution were mixed 1: 1, the number of YIT9029Sm bacteria in the supernatant after centrifugation was about 1000 times the number of YIT 0180 T Rif bacteria, and PS It was possible to specifically obtain lactic acid bacteria having -PG1. In addition, as shown in the data of the mixing ratio of 1:10 −3 , this separation and acquisition effect was confirmed even when the YIT 9029Sm bacteria count was about 1/1000 of the whole.
実 施 例 ４
乳酸菌の分離に対する保温時間の影響：
ミルク培地における懸濁時間を５〜６０分に変化させる以外は、実施例１と同様に培養、遠心分離を行い、ラクトバチルス・カゼイ ＹＩＴ ９０２９の上清の菌数を調べた。その結果、保温時間が５分〜６０分のいずれでも分離に影響が無いことが示された（表４）。
Example 4
Effect of incubation time on the isolation of lactic acid bacteria:
Cultivation and centrifugation were carried out in the same manner as in Example 1 except that the suspension time in the milk medium was changed to 5 to 60 minutes, and the number of bacteria in the supernatant of Lactobacillus casei YIT 9029 was examined. As a result, it was shown that there was no influence on the separation even if the heat retention time was 5 minutes to 60 minutes (Table 4).
実 施 例 ５
乳酸菌の分離に対する保温温度の影響：
ミルク培地における懸濁温度を４〜３７℃に変化させる以外は、実施例１と同様に培養、遠心分離を行い、ラクトバチルス・カゼイ ＹＩＴ９０２９の上清の菌数を調べた。その結果、懸濁温度は４〜３７℃のいずれでも分離に影響が無いことが示された（表５）。
Example 5
Effect of incubation temperature on the separation of lactic acid bacteria:
Cultivation and centrifugation were carried out in the same manner as in Example 1 except that the suspension temperature in the milk medium was changed to 4 to 37 ° C., and the number of bacteria in the supernatant of Lactobacillus casei YIT9029 was examined. As a result, it was shown that the suspension temperature had no influence on the separation at any of 4 to 37 ° C (Table 5).
実 施 例 ６
乳酸菌の分離に対する遠心分離条件の影響：
実施例１と同様に、ＭＲＳ培地あるいはミルク培地を用いてラクトバチルス・カゼイ ＹＩＴ ９０２９を３７℃で４時間培養した。次いで、各培養液５０μｌを、それぞれ２３００×ｇ、５１００×ｇ、９１００×ｇ、１４２００×ｇおよび２０４００×ｇの遠心分離条件で５分間遠心し、上清と沈殿に分けた。
Example 6
Effect of centrifugation conditions on the separation of lactic acid bacteria:
In the same manner as in Example 1, Lactobacillus casei YIT 9029 was cultured at 37 ° C. for 4 hours using MRS medium or milk medium. Next, 50 μl of each culture solution was centrifuged for 5 minutes under centrifugal conditions of 2300 × g, 5100 × g, 9100 × g, 14200 × g, and 20400 × g, respectively, and separated into a supernatant and a precipitate.
沈殿は、５０μｌの精製水に懸濁させた後、上清はそのままで、１０ｍＭ ＥＤＴＡで１／１０希釈し、それらの２．５μｌをＭＲＳ平板培地に置き、３７℃で２日間培養した。この結果を図１に示す。 After suspending the precipitate in 50 μl of purified water, the supernatant was left as it was and diluted 1/10 with 10 mM EDTA, and 2.5 μl of these was placed in MRS plate medium and cultured at 37 ° C. for 2 days. The result is shown in FIG.
この結果から明らかなように、ミルク培地では、１４２００×ｇから、沈殿中にもラクトバチルス・カゼイ ＹＩＴ ９０２９の存在が認められるものの、２３００〜２０４００×ｇで遠心分離を行っても、沈殿画分よりも上清画分に多くの菌が存在していた。これに対し、ＭＲＳ培地では、一貫して沈殿画分にほとんどの菌が存在していた。 As is clear from this result, in the milk medium, although the presence of Lactobacillus casei YIT 9029 is recognized during precipitation from 14200 × g, the precipitate fraction is not removed even after centrifugation at 2300-20400 × g. More bacteria were present in the supernatant fraction. On the other hand, in the MRS medium, most bacteria were consistently present in the precipitate fraction.
実 施 例 ７
乳酸菌の分離に対する培地の影響：
被験菌株として、ＰＳ−ＰＧ１を持たないラクトバチルス・カゼイ ＹＩＴ ０１８０Ｔと、ＰＳ−ＰＧ１を持つラクトバチルス・カゼイ ＹＩＴ ９０２９を用い、培地の含有成分と乳酸菌の分離の関係を検討した。
Example 7
Effect of medium on the isolation of lactic acid bacteria:
Lactobacillus casei YIT 0180 T without PS-PG1 and Lactobacillus casei YIT 9029 with PS-PG1 were used as test strains, and the relationship between the components contained in the medium and the separation of lactic acid bacteria was examined.
検討に用いた培地は、α−カゼイン、β−カゼインまたはＮ，Ｎ−ジメチルカゼイン（以上、いずれもＳｉｇｍａ社製）を１０ｍｇ／ｍｌ濃度で含有する水溶液、濃度１０ｗ／ｖ％脱脂粉乳（四つ葉乳業）水溶液、牛乳（明治乳業）および豆乳（ヤクルト本社）を用いた。 The medium used for the examination was an aqueous solution containing α-casein, β-casein or N, N-dimethylcasein (all of which were manufactured by Sigma) at a concentration of 10 mg / ml, a concentration of 10 w / v% nonfat dry milk (four Leaf milk industry) Aqueous solutions, milk (Meiji Dairies) and soy milk (Yakult Honsha) were used.
実験方法は、各被験菌株を実施例１と同様の方法で培養し、その培養液１μｌを、５０μｌの上記各培地に加え、３７℃で１０分保温した。次いで、５分の遠心後（５１００×ｇ）、沈殿と上清をとりわけ、上清を４５０μｌの、沈殿を５００μｌの精製水にそれぞれ加え、その１〜２μｌをＭＲＳ寒天平板培地上に塗沫して３７℃で培養し、菌数を判定した。この結果を表６に示す。 In the experimental method, each test strain was cultured in the same manner as in Example 1, 1 μl of the culture solution was added to 50 μl of each medium and incubated at 37 ° C. for 10 minutes. Then, after centrifugation for 5 minutes (5100 × g), the precipitate and the supernatant, in particular, 450 μl of the supernatant and 500 μl of the precipitate are added to the purified water, respectively, and 1 to 2 μl are smeared on the MRS agar plate medium. And cultured at 37 ° C., and the number of bacteria was determined. The results are shown in Table 6.
この結果、培養した菌株を遠心する際の分散液として、α−カゼインまたはβ−カゼインを利用した培地や、脱脂粉乳または牛乳を培地とした時には、ＰＳ−ＰＧ１を持つＹＩＴ９０２９は難沈殿性を示したが、ＰＳ−ＰＧ１を持たないＹＩＴ ０１８０Ｔは沈殿することが明らかになった。一方、アミノ基がメチル化されたジメチルカゼインを利用した培地や、豆乳培地では、ＹＩＴ ９０２９には難沈殿性が認められなかった。以上より、ＰＳ−ＰＧ１を持つ乳酸菌の分離は、カゼインタンパクを含む培地で実施できる事が明らかとなった。 As a result, YIT9029 with PS-PG1 shows poor precipitation when α-casein or β-casein is used as a dispersion when culturing the cultured strain, or skim milk powder or cow milk is used as a culture medium. However, YIT 0180 T without PS-PG1 was found to precipitate. On the other hand, in the medium using dimethyl casein in which the amino group was methylated or in the soymilk medium, YIT 9029 was not found to be difficult to precipitate. From the above, it became clear that the separation of lactic acid bacteria having PS-PG1 can be carried out in a medium containing casein protein.
実 施 例 ８
ＣａＣｌ２添加によるＰＳ−ＰＧ１を持つ乳酸菌の難沈殿性の促進：
ラクトバチルス・カゼイ ＹＩＴ ９０２９をＭＲＳ培地で４時間培養後、遠心で集菌した。一方、１０ｍｇ／ｍｌの濃度でαーカゼインまたはβ−カゼインを含む水溶液並びにα−カゼインおよびβ−カゼインをそれぞれ５ｍｇ／ｍｌで含む水溶液と、更にこれら各水溶液にＣａＣｌ２を１ｍＭとなるよう加えた水溶液の６つの水溶液を調製した。
Example 8
Promotion of poor precipitation of lactic acid bacteria having PS-PG1 by addition of CaCl 2 :
Lactobacillus casei YIT 9029 was cultured in MRS medium for 4 hours and then collected by centrifugation. On the other hand, an aqueous solution containing α-casein or β-casein at a concentration of 10 mg / ml, an aqueous solution containing α-casein and β-casein at 5 mg / ml, and an aqueous solution obtained by adding CaCl 2 to each aqueous solution to 1 mM. 6 aqueous solutions were prepared.
これらの各水溶液に、前記の集菌した微生物を入れ、３７℃で３０分保温後、５１００×ｇで５分間遠心し、沈殿と上清を分離し、それぞれに精製水を加え等量とした。その後、ＭＲＳ平板培地を用い、沈殿と上清中の生菌の存在状況を調べた。この結果を図２に示す。 In each of these aqueous solutions, the collected microorganisms are placed, incubated at 37 ° C. for 30 minutes, then centrifuged at 5100 × g for 5 minutes to separate the precipitate and the supernatant, and purified water is added to each to make equal amounts. . Thereafter, the presence of viable bacteria in the precipitate and supernatant was examined using MRS plate medium. The result is shown in FIG.
図２から明らかなように、ＣａＣｌ２添加によってカゼインのＰＳ−ＰＧ１を持つ乳酸菌の難沈殿効果が促進されていた。 As can be seen from FIG. 2, the addition of CaCl 2 promoted the effect of difficult precipitation of lactic acid bacteria having casein PS-PG1.
本発明のＰＳ−ＰＧ１を持つ乳酸菌の取得方法によれば、乳酸菌群中から、ＰＳ−ＰＧ１を保有する乳酸菌のみを簡単に分離、取得することができ、種々の生理活性作用を有する新しいタイプのヨーグルトや乳酸菌飲料の開発に有用である。 According to the method for obtaining lactic acid bacteria having PS-PG1 of the present invention, only a lactic acid bacterium having PS-PG1 can be easily separated and obtained from a group of lactic acid bacteria, and a new type having various physiological activities. It is useful for the development of yogurt and lactic acid bacteria beverages.
また、ＰＳ−ＰＧ１を持つ乳酸菌の判別方法によれば、細菌中に存在するＰＳ−ＰＧ１の生化学的分析、細菌のＲＮＡやＤＮＡの分離操作や遺伝学的分析操作を必要とせず、乳酸菌がＰＳ−ＰＧ１を持つかどうかを簡便、迅速に判別することが可能となるので、乳酸菌の研究に大きく資するものである。 In addition, according to the method for discriminating lactic acid bacteria having PS-PG1, there is no need for biochemical analysis of PS-PG1 present in bacteria, RNA RNA or DNA separation operation or genetic analysis operation. Whether it has PS-PG1 or not can be easily and quickly determined, which greatly contributes to research on lactic acid bacteria.
Claims (8)
The culture containing lactic acid bacteria is centrifuged in the presence of casein to separate the precipitate and non-precipitated fractions, and the presence of lactic acid bacteria in each fraction is examined. The lactic acid bacteria present in the non-precipitated fraction are precipitated. A method for discriminating a lactic acid bacterium having a polysaccharide-peptidoglycan complex 1, wherein the lactic acid bacterium is identified as having a polysaccharide-peptidoglycan complex 1 when the lactic acid bacteria are 1.0 times or more of the lactic acid bacteria present in the minute.
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