JP6363326B2 - Yeast / bacteria common culture method and yeast / bacteria common culture medium used therefor - Google Patents
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Description
本発明は、酵母と細菌を同一培地中で増殖させることのできる培養方法に関し、更に詳細には、医薬品・化粧品等の酵母、細菌が存在することが問題となる検体について、これらの存在が極めて少ないか、あるいは存在しないことを保証するための試験における、酵母と細菌を同一培地中で一緒に増殖可能な培養方法およびそのために使用される共通培養培地に関する。 The present invention relates to a culture method in which yeast and bacteria can be grown in the same medium. More specifically, the presence of yeast and bacteria such as pharmaceuticals and cosmetics, which are problematic, is extremely present. The present invention relates to a culture method capable of growing yeast and bacteria together in the same medium in a test to ensure that they are low or absent, and a common culture medium used therefor.
酵母は、真核生物であるのに対し、細菌は原核生物であるため、共に培地中での培養は可能であっても、従来、そのために使用する培地としては別のものが使用されていた。 Yeast is a eukaryote, whereas bacteria are prokaryotes, so even if both can be cultured in a medium, conventionally, a different medium has been used for that purpose. .
例えば、日本薬局方に収載されている微生物限度試験法は、医薬品・化粧品等の微生物汚染の有無を確認する検査法であり、製品の出荷判定として用いられることが多いが、近年の改訂（非特許文献１）により、検査対象菌株として、従来の細菌の他、酵母であるカンジダ・アルビカンス（Candida albicans）が追加された。
For example, the microbial limit test method listed in the Japanese Pharmacopoeia is an inspection method for confirming the presence or absence of microbial contamination of pharmaceuticals and cosmetics, and is often used as a product shipment judgment. According to Patent Document 1), Candida albicans , which is a yeast, was added as a test target strain in addition to conventional bacteria.
そしてこの結果、同一検体について、酵母用培地と細菌用培地で別個にこれらを増殖させるための培養を行い、その後、各検査対象菌株の存在を個別に検出、確認することが実施されている。そして、この従来法では、原核生物であり、増殖速度の速い細菌では、増殖培養の時間が比較的短くてすむため、最終的な検査、判定までの時間は、２日程度と短いのに対し、真核生物に属する酵母であるカンジダ・アルビカンスは、増殖培養時間が長いため、判定終了までには、５日間程度かかっていた。 As a result, the same specimen is cultured in a yeast medium and a bacterial medium separately for growing them, and then the presence of each test target strain is individually detected and confirmed. In this conventional method, a prokaryotic organism with a fast growth rate requires a relatively short growth time, so that the time until the final inspection and determination is as short as 2 days. Since Candida albicans, a yeast belonging to eukaryotes, has a long growth and culture time, it took about 5 days to complete the determination.
このように、同じ検体の判定であっても、細菌用と酵母用の別個の培地による培養が必要であるため、培養の終了時間が異なり、検査の手続が煩雑になると共に、最終的に検査が終了し、製品の出荷が行えるようになるまでに時間がかかるという問題があった。 In this way, even if the same specimen is judged, culture using separate culture media for bacteria and yeast is necessary, so the end time of culture is different, the examination procedure becomes complicated, and finally the examination is performed. There is a problem that it takes time before the product is finished and the product can be shipped.
このようなことから、酵母と細菌を同一培地中、同一時間で培養した培養物中から、次の微生物存在確認試験に使用できる試料を得ることが求められるが、現在に至るまで、そのような条件を満たすような培地は提供されていない。 For this reason, it is required to obtain a sample that can be used for the next microorganism presence confirmation test from a culture in which yeast and bacteria are cultured in the same medium at the same time. A medium that satisfies the conditions is not provided.
本発明は、上記実情に鑑みなされたものであり、仮に細菌・酵母が検体中に混入していた場合に、それらを共に検出可能にまで培養可能な、酵母・細菌共通培養方法およびこれに用いる酵母・細菌共通培養培地を提供するものである。 The present invention has been made in view of the above circumstances, and in the case where bacteria / yeast are mixed in a specimen, the yeast / bacteria common culture method capable of cultivating them together so that they can be detected is used. It provides a common culture medium for yeast and bacteria.
本発明者らは、上記課題を解決すべく、微生物の培養方法およびそのための培地に関し鋭意検討を行った。そしてその結果、細菌用培地において、細菌の増殖を抑制し、かつ酵母の増殖を向上させることにより、同一の培地中で検体中の酵母と細菌を共に検出可能な数まで増殖でき、これらが検出可能であることを見出し、本発明を完成した。 In order to solve the above-mentioned problems, the present inventors diligently studied a microorganism culturing method and a culture medium therefor. As a result, by suppressing the growth of bacteria and improving the growth of yeast in the culture medium for bacteria, the yeast and bacteria in the specimen can be grown to a number that can be detected in the same medium. As a result, the present invention was completed.
すなわち本発明は、細菌培養用培地を用い、細菌の増殖を抑制し、かつ酵母の増殖を向上させる条件下で、細菌および酵母を含有する可能性のある検体を培養することを特徴とする酵母・細菌共通培養方法である。 That is, the present invention uses a culture medium for bacterial culture, cultivates a specimen that may contain bacteria and yeast under conditions that inhibit bacterial growth and improve yeast growth.・ Bacteria common culture method.
また本発明は、細菌培養用培地を用い、細菌の増殖を抑制し、かつ酵母の増殖を向上させる条件下で検体を培養した後、当該培養物中の細菌および酵母をそれぞれ検出することを特徴とする検体中の細菌および酵母の検出方法である。 Further, the present invention is characterized by using a culture medium for bacterial culture, detecting the bacteria and yeast in the culture after culturing the specimen under conditions that inhibit bacterial growth and improve yeast growth. And a method for detecting bacteria and yeast in a specimen.
更に本発明は、レシチンおよび非イオン性界面活性剤が加えられたトリプトソイ培地に、更に酸性成分または緩衝剤を加え、そのｐＨを、５〜６.５としてなることを特徴とする酵母・細菌共通培養培地である。
Furthermore, the present invention provides a common yeast / bacteria characterized in that an acidic component or a buffer is further added to a tryptic soy medium to which lecithin and a nonionic surfactant are added, and the pH is set to 5 to 6.5. Culture medium.
更にまた本発明は、上記酵母・細菌共通培養培地に、追加糖成分として１〜８ｇ／Ｌとなる量の、グルコース、マルトース、ガラクトース、フルクトース、スクロースおよびデキストリンから選ばれる糖成分を添加してなる、酵母・細菌共通培養培地である。 Furthermore, in the present invention, a sugar component selected from glucose, maltose, galactose, fructose, sucrose and dextrin in an amount of 1 to 8 g / L is added to the yeast / bacteria common culture medium as an additional sugar component. A common culture medium for yeast and bacteria.
本発明方法によれば、検体中に存在する可能性のある細菌と酵母とを同時に培養、増殖させることができるので、これらを別個の培地で、しかも異なる時間培養していた従来法と比べ、検査手続を簡素化することができる。また、酵母増殖のための培養が短縮するため、検査に要する全体の時間も短縮し、製品の検査、出荷のための時間も短くすることができる。 According to the method of the present invention, bacteria and yeast that may be present in a specimen can be cultured and propagated at the same time, so compared with the conventional method in which these are cultured in a separate medium and at different times, The inspection procedure can be simplified. In addition, since the culture for yeast growth is shortened, the total time required for inspection can be shortened, and the time for product inspection and shipping can be shortened.
本発明は、主に日本薬局方に収載されている微生物限度試験法の改良に関するものであり、医薬品や化粧品（以下、「製品」ということがある）中に存在する可能性のある細菌および酵母（以下、「検査微生物」と総称することがある）を増殖するための培養工程において、同一の培養培地を用い、同一時間で検査微生物の培養をおこなうものである。
The present invention relates to improvement of a microbial limit test method mainly listed in the Japanese Pharmacopoeia, and bacteria and yeast that may be present in pharmaceuticals and cosmetics (hereinafter sometimes referred to as “products”) In the culturing process for growing (hereinafter sometimes collectively referred to as “test microorganism”), the same culture medium is used and the test microorganism is cultured in the same time.
本発明方法では、基本的には細菌培養用培地を用いるが、これを細菌の増殖を抑制し、かつ酵母の増殖が向上する条件で使用するか、当該培地を細菌の増殖を抑制し、かつ酵母の増殖が向上するように変性するか、あるいはこの双方の技術を採用する。
In the method of the present invention, basically, a culture medium for bacterial culture is used, which is used under the condition that suppresses bacterial growth and yeast growth is improved, or suppresses bacterial growth, and Either denatured to improve yeast growth, or employ both techniques.
すなわち、従来提供されていた細菌培養用培地は、細菌の速やかな増殖を追求するものであったため、この中で細菌と共に酵母を培養した場合、酵母の培養が遅れ、その同定に必要な程度にまで増殖させることはできない。しかし、単に培養期間を延長すると、培養液中の栄養素が枯渇してしまうため、酵母が十分に増殖ができなくなり、また、細菌は増殖しすぎて増殖曲線上の死滅期に入ってしまい、いずれも正しく同定できないことがある。一方、微生物限度試験は製品検査に一般的に行われる試験方法であるが、製品製造日程の都合に応じて長時間の培養となる場合があるため、培養時間が長期に亘っても増殖曲線上の対数増殖期を維持することが必要である。そこで本発明では、培養終了時に細菌も酵母も、増殖曲線上で同じ対数増殖期に存在し、かつ酵母を同定可能な程度にまで増殖させるために、培地中での細菌の増殖を抑制し、かつ酵母の増殖を高めるものである。 That is, the bacterial culture medium that has been provided in the past was for pursuing rapid growth of bacteria, so when yeast was cultured with bacteria in this, the yeast culture was delayed, and to the extent necessary for its identification. Can not be propagated to. However, if the culture period is simply extended, the nutrients in the culture medium will be depleted, so the yeast will not be able to grow sufficiently, and the bacteria will grow too much to enter the death phase on the growth curve. May not be identified correctly. On the other hand, the microbial limit test is a test method that is generally performed for product inspection. However, since the culture may be performed for a long time according to the convenience of the product manufacturing schedule, the microbial limit test is not performed on the growth curve even if the culture time is long. It is necessary to maintain the logarithmic growth phase. Therefore, in the present invention, at the end of the culture, both bacteria and yeast are present in the same logarithmic growth phase on the growth curve, and in order to grow the yeast to an identifiable level, the growth of bacteria in the medium is suppressed, It also enhances yeast growth.
いいかえると、本発明の細菌の増殖を抑制し、かつ酵母の増殖を向上させる条件下で、細菌および酵母を含有する可能性のある検体を培養するとは、培養終了時に細菌も酵母も、増殖曲線上で同じ対数増殖期に存在し、かつ酵母を同定可能な程度にまで増殖させることを意味する。 In other words, culturing a specimen that may contain bacteria and yeast under conditions that inhibit the growth of the bacteria of the present invention and improve the growth of yeast means that both the bacteria and yeast will grow at the end of the culture. It means to grow yeasts to the extent that they exist in the same logarithmic growth phase and can be identified.
具体的に、本発明の培養方法を実施するには、次の手段の何れかを採用できる。
（１）細菌培養用培地のｐＨを、５〜６.５程度、好ましくは、５〜６に調整し、温度を２０〜３０℃程度、好ましくは２０〜２５℃として培養する。
（２）細菌培養用培地に、追加糖成分として１〜８ｇ／Ｌの、グルコース、マルトース、ガラクトース、フルクトース、スクロースおよびデキストリンから選ばれる糖成分を添加した培地を使用し、ｐＨを、５〜６.８程度、好ましくは、５〜６.５、温度を２０〜３０℃程度、好ましくは２０〜２５℃として培養する。
Specifically, in order to carry out the culture method of the present invention, any of the following means can be employed.
(1) The pH of the culture medium for bacterial culture is adjusted to about 5 to 6.5, preferably 5 to 6, and the temperature is about 20 to 30 ° C, preferably 20 to 25 ° C.
(2) A medium in which a sugar component selected from glucose, maltose, galactose, fructose, sucrose, and dextrin at 1 to 8 g / L is added as an additional sugar component to a bacterial culture medium, and the pH is adjusted to 5-6 The culture is performed at about 0.8, preferably 5 to 6.5, and at a temperature of about 20 to 30 ° C, preferably 20 to 25 ° C.
本発明において使用される培地は、日本薬局方に収載されている微生物限度試験法で用いられる細菌培養用培地であれば特段制約はないが、好ましいものとしては、レシチンおよび非イオン性界面活性剤が加えられたトリプトソイ培地（ＳＣＤＬＰ培地）が挙げられる。
The medium used in the present invention is not particular limited as long a medium for bacterial culture for use in microbial limits test methods which are listed in the Japanese Pharmacopoeia, preferred are, lecithin and non-ionic surfactant Tryptosoy medium (SCDLP medium) to which is added.
このＳＣＤＬＰ培地は、ペプトン ２０ｇ／Ｌ、Ｋ２ＨＰＯ４２.５、グルコース ２.５ｇ／Ｌ、ＮａＣｌ ５ｇ／Ｌの組成に、微生物の生長に影響を与えない量のレシチンおよび界面活性剤（ポリソルベート８０等）を添加したもので、その使用時のｐＨは、７.１〜７.５である。この培地は、含まれているレシチンと界面活性剤（ポリソルベート８０）により、医薬品や化粧品中に含まれる各種防腐剤や殺菌剤例えば四級アンモニウム化合物やパラオキシ安息香酸エステル類、ビスービグアニド類などを不活性化させるため、これら製品中の細菌や酵母を防腐剤等の影響なく培養することができ、正確にそれら菌を同定できる。なお、市販されているものとしては、「ＳＣＤＬＰブイヨン培地‘栄研’」（栄研化学株式会社製；レシチン１ｇ／Ｌ、ポリソルベ−ト８０ ７ｇ／Ｌ含有）が例示できる。 This SCDLP medium has a composition of peptone 20 g / L, K 2 HPO 4 2.5, glucose 2.5 g / L, NaCl 5 g / L in an amount that does not affect the growth of microorganisms and a surfactant (polysorbate). 80 etc.), and the pH at the time of use is 7.1 to 7.5. This medium is free from various preservatives and fungicides such as quaternary ammonium compounds, paraoxybenzoates, and bis-biguanides contained in pharmaceuticals and cosmetics due to the contained lecithin and surfactant (polysorbate 80). Since they are activated, the bacteria and yeast in these products can be cultured without the influence of preservatives and the like, and these bacteria can be accurately identified. In addition, as what is marketed, "SCDLP bouillon culture medium" Eiken "" (Eiken Chemical Co., Ltd .; lecithin 1g / L, polysorbate 807g / L containing) can be illustrated.
本発明方法の第一の態様は、上記のように細菌培養用培地のｐＨを、５〜６.５程度、好ましくは、５〜６に調整し、温度を２０〜３０℃程度、好ましくは２０〜２５℃として培養するというものであるが、このｐＨは、酵母（真菌）についての至適ｐＨに近いものである。同様に、上記温度も酵母の至適温度に近い温度である。
A first aspect of the present invention method, the pH of the medium for bacterial culture as described above, about 5 to 6.5, preferably, adjusted to 5-6, the temperature 20 to 30 ° C. approximately, preferably 20 Although culturing at ˜25 ° C., this pH is close to the optimum pH for yeast (fungi). Similarly, the above temperature is also close to the optimum temperature of yeast.
このように、培地組成自体は、細菌用のものを使用しながらも、培養条件を酵母（真菌）に適したものとすることで、細菌と酵母の同時培養が可能となるのである。 As described above, the culture medium composition itself can be used for bacteria, but the culture conditions are suitable for yeast (fungi), so that the bacteria and yeast can be cultured simultaneously.
なお、本発明の第一の態様の実施に有利に利用できる培地としては、ＳＣＤＬＰ培地のような細菌培養用培地組成中に、そのｐＨを、５〜６.５程度とすることのできる塩酸、酢酸、乳酸、乳酸ナトリウム、クエン酸、クエン酸ナトリウム、リン酸二水素カリウム、リン酸水素一カリウム、リン酸二水素ナトリウム、リン酸水素一ナトリウム、コハク酸、コハク酸ナトリウムなどの公知の無機酸あるいは有機酸等の酸性剤または緩衝剤を配合したものを使用すればよい。この中で、特に塩酸、クエン酸、クエン酸ナトリウム、リン酸二水素カリウム、リン酸水素一カリウムが好適に使用できる。
In addition, as a medium that can be advantageously used for carrying out the first aspect of the present invention, hydrochloric acid whose pH can be adjusted to about 5 to 6.5 in a medium composition for bacterial culture such as SCDLP medium, Known inorganic acids such as acetic acid, lactic acid, sodium lactate, citric acid, sodium citrate, potassium dihydrogen phosphate, monopotassium hydrogen phosphate, sodium dihydrogen phosphate, monosodium hydrogen phosphate, succinic acid, sodium succinate Or what mix | blended acidic agents, such as an organic acid, or a buffer should just be used. Among these, hydrochloric acid, citric acid, sodium citrate, potassium dihydrogen phosphate, and monopotassium hydrogen phosphate can be preferably used.
以上説明した、この方法では、製品から採取した検体を約２４〜９６時間培養することで、仮にこの製品中に検査微生物が存在した場合、十分に検出可能なレベルまで増殖させることができる。 In this method described above, a specimen collected from a product is cultured for about 24 to 96 hours, and if a test microorganism is present in the product, it can be grown to a sufficiently detectable level.
本発明方法の第二の態様は、第一の態様で使用する細菌培養用培地に、更に追加成分として糖を加えるというものである。この培養培地に追加される糖成分としては、グルコース、マルトース、ガラクトース、フルクトース、スクロース、デキストリンが挙げられ、その量は、培養時の培地中で１〜８ｇ／Ｌとなる量である。このように糖が追加された培養培地では、酵母がこの糖を資化することでその増殖が盛んになる。
A second aspect of the present invention method for bacterial culture medium used in the first embodiment, is that further addition of sugar as an additional component. Examples of the sugar component added to the culture medium include glucose, maltose, galactose, fructose, sucrose, and dextrin, and the amount thereof is 1 to 8 g / L in the culture medium. In the culture medium to which sugar is added in this way, yeast grows as the sugar is assimilated.
なお、この第二の態様では、追加等のみならず、更に酵母に好ましい条件で培養することもできる。具体的には、ｐＨを、５〜６.８程度、好ましくは、５〜６.５、温度を２０〜３０℃程度、好ましくは２０〜２５℃として培養することができる。 In addition, in this 2nd aspect, not only addition etc. but it can also culture | cultivate on conditions preferable for yeast. Specifically, it can be cultured at a pH of about 5 to 6.8, preferably 5 to 6.5, and a temperature of about 20 to 30 ° C., preferably 20 to 25 ° C.
この第二の態様を実施するために使用される培地としては、細菌培養培地中に、１〜８ｇ／Ｌとなる量のグルコース、マルトース、ガラクトース、フルクトース、スクロースまたはデキストリンを配合した培地が挙げられる。また、より好ましい結果を得るには、更にこの培地中に、そのｐＨを、５〜６.８程度とすることのできる公知の酸性剤や、緩衝剤を加えたものを挙げることができる。 Examples of the medium used for carrying out this second embodiment include a medium in which glucose, maltose, galactose, fructose, sucrose, or dextrin is mixed in a bacterial culture medium in an amount of 1 to 8 g / L. . Moreover, in order to obtain a more preferable result, the thing which added the well-known acidic agent and buffer which can make the pH into about 5-6.8 further in this culture medium can be mentioned.
この第二の態様でも、約２４〜９６時間培養することで、仮に製品中に検査微生物が存在した場合、十分に検出可能なレベルまで増殖させることができる。 Also in this second aspect, by culturing for about 24 to 96 hours, if a test microorganism is present in the product, it can be grown to a sufficiently detectable level.
上記本発明の酵母・細菌共通培養方法で培養された、検査微生物の検出方法について説明する。この検出方法は、従来公知の検査微生物を、医薬品、化粧品についての日本薬局方による微生物限度試験では、細菌である緑膿菌、黄色ブドウ状球菌、大腸菌と、酵母であるカンジダ・アルビカンスを、それぞれの検出に適した培地（以下、「選択培地」という）を用い、これら選択培地に培養物の所定量を個別に塗布、培養し、一定時間後に選択培地に検査微生物の特徴的コロニーが認められるか否かを判定する。 A method for detecting a test microorganism cultivated by the yeast / bacteria common culture method of the present invention will be described. In this detection method, conventionally known test microorganisms, and in the microbial limit test by the Japanese Pharmacopoeia for pharmaceuticals and cosmetics, bacteria Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and yeast Candida albicans, Using a culture medium suitable for detection of urine (hereinafter referred to as “selective medium”), a predetermined amount of the culture is individually applied to the selective medium, cultured, and after a certain time, characteristic colonies of the test microorganism are observed in the selective medium. It is determined whether or not.
そして、この結果、特徴的コロニーが検出されなかった場合、製品中への微生物混入がないと判断され、製品として出荷が可能となるのである。 As a result, if a characteristic colony is not detected, it is determined that there is no microbial contamination in the product, and the product can be shipped.
この検査微生物の検出方法は、上記医薬品や化粧品はもとより、それ以外の防腐剤や抗菌剤を含む製品について適用でき、製品の安全性を保証することができるものである。 This method for detecting a test microorganism can be applied not only to the above-mentioned pharmaceuticals and cosmetics, but also to products containing other preservatives and antibacterial agents, and can guarantee the safety of the product.
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
以下に示す試験培地を使用し、種々の条件で、細菌・酵母複合菌試料を培養して、細菌と酵母がそれぞれ検出可能な程度まで増殖するかどうかを確認した。増殖は、細菌および酵母について、試験培地と同じ培地を用い、ｐＨ６.８、温度３２.５℃で培養（標準培養）した場合と比較し、下記増殖スコアを求め、このスコアを総合した増殖性判定基準により評価した。また、増殖時間は、酵母（カンジダ・アルビカンス）が、初期菌数（１〜９ＣＦＵ／ｍＬ）から１０３ＣＦＵ／ｍＬにまでかかった時間により下記増殖時間判定基準で評価した。この結果を表１に示す。
Example 1
Using the test medium shown below, the bacteria / yeast complex sample was cultured under various conditions, and it was confirmed whether the bacteria and yeast were grown to a detectable level. Growth is the same as the test medium for bacteria and yeast, and the following growth score is obtained in comparison with the case where culture is performed at a pH of 6.8 and a temperature of 32.5 ° C. (standard culture). Evaluation was made according to the criteria. The growth time was evaluated according to the following growth time criteria based on the time taken for yeast (Candida albicans) from the initial number of bacteria (1 to 9 CFU / mL) to 10 3 CFU / mL. The results are shown in Table 1.
［ 試験培地組成 ］
ペプトン ２０ｇ／Ｌ
リン酸一水素二カリウム ２.５ｇ／Ｌ
グルコース ２.５ｇ／Ｌ
塩化ナトリウム ５ｇ／Ｌ
レシチン １ｇ／Ｌ
ポリソルベート８０ ７ｇ／Ｌ
脱イオン水 １０００ ｍＬ
[Test medium composition]
Peptone 20g / L
Dipotassium monohydrogen phosphate 2.5 g / L
Glucose 2.5g / L
Sodium chloride 5g / L
Lecithin 1g / L
Polysorbate 80 7g / L
Deionized water 1000 mL
［ 細菌・酵母複合菌試料 ］
緑膿菌として、シュードモナス・エルギノーザ（Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ）ＩＦＯ １３２７５株、黄色ブドウ球菌として、スタフィロコッカス・アウレウス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ）ＩＦＯ １３２７６株、大腸菌として、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＩＦＯ ３９７２株、酵母として、カンジダ・アルビカンス（Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ）ＩＦＯ１５９４株を使用した。これらを、試験培地中に、それぞれ、１〜９ＣＦＵ／ｍＬ、１〜９ＣＦＵ／ｍＬ、１〜９ＣＦＵ／ｍＬ、１〜９ＣＦＵ／ｍＬとなるように添加し、混合して細菌・酵母複合菌試料とした。
[Bacteria / yeast complex sample]
As Pseudomonas aeruginosa, Pseudomonas aeruginosa (Pseudomonas aeruginosa) IFO 13275 strain, as Staphylococcus aureus, Staphylococcus aureus (Staphylococcus aureus) IFO 13276 strain, as E. coli, Escherichia coli (Escherichia coli) IFO 3972 strain, as yeast Candida albicans IFO1594 strain was used. These were added to the test medium so as to be 1 to 9 CFU / mL, 1 to 9 CFU / mL, 1 to 9 CFU / mL, and 1 to 9 CFU / mL, respectively, and mixed with the bacterial / yeast complex sample. did.
［ 増殖スコア ］
酵母（カンジダ・アルビカンス）
評 点 ： 内 容
３ ： 標準培養に比べ、培養菌数が２桁（１００倍）以上多い。
２ ： 標準培養に比べ、培養菌数が１桁（１０倍）以上、２桁（１００倍）
未満である。
１ ： 標準培養に比べ、培養菌数がほぼ１桁（５〜１０倍）多い。
０ ： 標準培養とほぼ同じ培養菌数（０.７〜５倍）である。
−１ ： 標準培養に比べ、培養菌数がほぼ１桁（０.７倍）少ない。
−２ ： 培養菌が検出されない。
注）上記酵母の菌数は、クロモアガーカンジダ寒天培地（ＣＨＲＯＭａｇａｒ社製）を用い、混釈培養後、検出した緑青色コロニーをカンジダ・アルビカンスとして計測した。
[Proliferation score]
Yeast (Candida albicans)
Score: Contents 3: Compared with standard culture, the number of cultured bacteria is two digits (100 times) or more.
2: Compared to standard culture, the number of cultured cells is 1 digit (10 times) or more, 2 digits (100 times)
Is less than.
1: The number of cultured bacteria is almost one digit (5 to 10 times) larger than that of the standard culture.
0: Approximately the same number of cultures (0.7 to 5 times) as in the standard culture.
-1: The number of cultured bacteria is almost one digit (0.7 times) smaller than that of standard culture.
-2: Culture is not detected.
Note) The number of yeast cells was measured using Cromoagar Candida agar medium (manufactured by CHROMagar), and after the pour culture, the detected green-blue colony was counted as Candida albicans.
細 菌（緑膿菌、黄色ブドウ球菌、大腸菌）
評 点 ： 内 容
３ ： 細菌数が１０６ＣＦＵ／ｍＬ以上である。
２ ： 細菌数が１０５ＣＦＵ／ｍＬ以上である。
１ ： 細菌数が１０４ＣＦＵ／ｍＬ以上である。
０ ： 細菌数が１０３ＣＦＵ／ｍＬ以上である。
−１ ： 細菌数が１０２ＣＦＵ／ｍＬ以上である。
−２ ： 細菌数が１０２ＣＦＵ／ｍＬ以下である。
注）上記細菌についての数は、緑膿菌はＮＡＣ寒天培地（栄研化学株式会社製）、黄色ブドウ球菌はＭＳ寒天培地（栄研化学株式会社製）、大腸菌はＥＭＢ寒天培地（栄研化学株式会社製）をそれぞれ選択培地として用い、混釈培養後、ＮＡＣ寒天培地では緑色〜青色、ＭＳ寒天培地では黄色、ＥＭＢ寒天培地では黒色のコロニーを、各々の対象細菌数として計測した。緑膿菌、黄色ブドウ球菌、大腸菌の菌数のうち、最も菌数が少ない菌種の評点を、その培養条件における評点とした。
Bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli)
Score: Content 3: Bacterial count is 10 6 CFU / mL or more.
2: The number of bacteria is 10 5 CFU / mL or more.
1: The number of bacteria is 10 4 CFU / mL or more.
0: The number of bacteria is 10 3 CFU / mL or more.
-1: The number of bacteria is 10 2 CFU / mL or more.
-2: The number of bacteria is 10 2 CFU / mL or less.
Note) The numbers of the above-mentioned bacteria are as follows: Pseudomonas aeruginosa is NAC agar medium (Eiken Chemical Co., Ltd.), S. aureus is MS agar medium (Eiken Chemical Co., Ltd.), E. coli is EMB agar medium (Eiken Chemical) (Manufactured by Co., Ltd.) was used as the selective medium, and after pour culture, green to blue in the NAC agar medium, yellow in the MS agar medium, and black colony in the EMB agar medium were counted as the number of target bacteria. Among the numbers of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, the score of the bacterial species with the smallest number was used as the score under the culture conditions.
［ 増殖性判定基準 ］
評 価 ： 内 容
◎ ： 細菌の増殖スコアが０〜３で、酵母の増殖スコアが２〜３
○ ： 細菌の増殖スコアが０〜３で、酵母の増殖スコアが１
△ ： 細菌の増殖スコアが０〜３で、酵母の増殖スコアが０
× ： 細菌の増殖スコアおよび酵母の増殖スコアの少なくとも一方が、−２〜
−１
[Proliferation criteria]
Evaluation: Contents ◎: Bacterial growth score is 0-3, yeast growth score is 2-3
○: Bacterial growth score is 0-3, yeast growth score is 1
Δ: Bacterial growth score 0-3, yeast growth score 0
X: At least one of bacterial growth score and yeast growth score is -2 to
-1
［ 増殖時間判定基準 ］
評 価 ： 内 容
◎ ： ２４時間以内
○ ： ２４〜４８時間
△ ： ４８〜９６時間
× ： ９６時間以上
注）上記評価の「×」には、一旦増殖したが、その後９６時間以内に減少が見
られた場合も含む。
[Growth time criteria]
Evaluation: Contents ◎: Within 24 hours ○: 24-48 hours △: 48-96 hours ×: 96 hours or more Note) “×” in the above evaluation was once proliferated, but decreased within 96 hours thereafter You see
This includes cases where
［ 結 果 ］
この結果、標準培養条件から温度を下げただけでも「菌増殖性」及び「増殖時間」の向上が認められたが、更にｐＨを低く調整した場合に顕著な「菌増殖性」及び「増殖時間」改善効果が得られ、培養温度２０〜３０℃、ｐＨ５.０〜６.５の組み合わせ範囲内であれば細菌と酵母の増殖が良好であることが明らかとなった。 As a result, even when the temperature was lowered from the standard culture conditions, an improvement in “bacterial growth” and “growth time” was observed. The improvement effect was obtained, and it was revealed that the bacteria and yeast grew well if the culture temperature was within the combined range of 20-30 ° C. and pH 5.0-6.5.
実 施 例 ２
表２に示す、所定量のグルコースを更に加えた試験培地を用い、同表に示す条件下で、実施例１で使用した細菌・酵母複合菌試料を培養し、細菌と酵母がそれぞれ検出可能な程度まで増殖するかどうかを確認した。増殖に関する確認は、実施例１に記載の方法で行った。この結果も表２に示す。
Example 2
Using the test medium further added with a predetermined amount of glucose shown in Table 2, the bacterial / yeast complex sample used in Example 1 was cultured under the conditions shown in the same table, and bacteria and yeast can be detected respectively. It was confirmed whether it grew to the extent. Confirmation of growth was performed by the method described in Example 1. The results are also shown in Table 2.
［ 結 果 ］
この結果から、グルコース追加添加により「菌増殖性」及び「増殖時間」が向上することが明らかとなった。その効果は標準培養条件でも一部傾向が認められたが、培養温度を２０〜３２.５℃、ｐＨを５.０〜６.５とした場合に、より顕著な効果が得られた。なお、表１の結果でもｐＨ５.０かつ温度２５℃、及びｐＨ６.０かつ温度２５℃で良好な「菌増殖性」及び「増殖時間」が示されているが、更にグルコース１〜７.５ｇ／Ｌを追加添加した表２の方が、より「菌増殖性」及び「増殖時間」に優れていた。しかし、グルコース追加添加量が１０ｇ／Ｌになると逆に酵母の増殖に悪影響が現れ、菌数が減少に転じて「増殖時間」が×となった。 From this result, it was clarified that “bacterial growth” and “growth time” were improved by addition of glucose. A part of the effect was observed even under standard culture conditions, but a more remarkable effect was obtained when the culture temperature was 20 to 32.5 ° C. and the pH was 5.0 to 6.5. The results in Table 1 also show good “fungal growth” and “growth time” at pH 5.0 and a temperature of 25 ° C., and pH 6.0 and a temperature of 25 ° C., but further 1 to 7.5 g of glucose. Table 2 to which / L was additionally added was more excellent in “bacterial growth” and “growth time”. However, when the additional amount of glucose added was 10 g / L, adversely affected the growth of the yeast, and the number of bacteria started to decrease, and the “growth time” became x.
実 施 例 ３
表３に示す種類および量の各種糖類を実施例１の試験培地に加え、これを用いて、同表に示す条件下で、実施例１で使用した細菌・酵母複合菌試料を培養し、細菌と酵母がそれぞれ検出可能な程度まで増殖するかどうかを確認した。増殖に関する確認は、実施例１に記載の方法で行った。この結果も表３に示す。
Example 3
Various saccharides of the types and amounts shown in Table 3 were added to the test medium of Example 1, and this was used to culture the bacteria / yeast complex sample used in Example 1 under the conditions shown in the same table. And yeast were grown to a detectable level. Confirmation of growth was performed by the method described in Example 1. The results are also shown in Table 3.
［ 結 果 ］
この結果から、培地に新たな糖成分としてマルトース、（Ｄ）−ガラクトース、（Ｄ）−フルクトース、スクロース、デキストリンを添加した場合にも、グルコース追加添加と同様に、「菌増殖性」及び「増殖時間」改善効果があることが明らかとなった。 From this result, when maltose, (D) -galactose, (D) -fructose, sucrose, and dextrin were added as new sugar components to the medium, “fungal growth” and “growth” were added as well as the addition of glucose. It became clear that there was an effect of "time" improvement.
本発明方法によれば、検体中に存在する可能性のある細菌と酵母とを同時に培養、増殖させることができ、別個の培地で、異なる時間培養していた従来法と比べ、検査手続を簡素化することができる。また、酵母増殖のための培養が短縮するため、検査に要する全体の時間も短縮し、製品の検査、出荷のための時間も短くすることができる。 According to the method of the present invention, bacteria and yeast that may be present in a specimen can be cultured and propagated at the same time, and the test procedure is simplified compared to the conventional method in which culture is performed in a separate medium for different times. Can be In addition, since the culture for yeast growth is shortened, the total time required for inspection can be shortened, and the time for product inspection and shipping can be shortened.
従って本発明方法は、医薬品・化粧品等の微生物汚染の有無を簡便かつ早期に確認することが可能であり、これら製品の安全性確保のために有用である。
Therefore, the method of the present invention can easily and quickly confirm the presence or absence of microbial contamination of pharmaceuticals and cosmetics, and is useful for ensuring the safety of these products.
Claims (12)
A group consisting of glucose, maltose, galactose, fructose, sucrose and dextrin in an amount of 1 to 8 g / L as an additional sugar component in the yeast / bacteria common culture medium for culturing yeast and bacteria simultaneously according to claim 11 A yeast-bacterial common culture medium for culturing yeast and bacteria at the same time, to which a sugar component selected from is added.
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