JP2021126048A - Method for detecting microorganisms, and medium - Google Patents
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は、炭酸飲料についての微生物の検出方法、および該微生物の検出に用いることができる培地に関する。 The present invention relates to a method for detecting a microorganism in a carbonated beverage and a medium that can be used for detecting the microorganism.
近年、炭酸飲料であるビールテイストノンアルコール飲料や所謂チューハイの需要が高まっている。 In recent years, demand for beer-taste non-alcoholic beverages, which are carbonated beverages, and so-called chu-hi has been increasing.
これらは工場などで製造されるが、一般に工場などで製造される飲料に対しては、微生物の検出試験が行われている（例えば非特許文献１）。例えば上記のような炭酸飲料は加熱殺菌などの殺菌工程を経て製造されることが多いが、その殺菌処理の確認などのために上記微生物の検出試験が行われている。
具体的に説明すると、加熱殺菌等を経て製造される炭酸飲料においては、芽胞形成菌は飲料中に存在していても増殖しない。そのため、該炭酸飲料に対しては加熱殺菌を経ても死滅しなかった非芽胞形成菌が存在するか否かを判定するために上記検出が行われている。
These are manufactured in factories and the like, but in general, microorganism detection tests are conducted on beverages manufactured in factories and the like (for example, Non-Patent Document 1). For example, the above-mentioned carbonated beverages are often produced through a sterilization step such as heat sterilization, and a detection test of the above-mentioned microorganisms is carried out to confirm the sterilization treatment.
Specifically, in a carbonated beverage produced through heat sterilization or the like, spore-forming bacteria do not grow even if they are present in the beverage. Therefore, the above detection is performed to determine whether or not non-spore-forming bacteria that have not been killed even after heat sterilization are present in the carbonated beverage.
本発明は、より容易に非芽胞形成菌を検出できる技術を提供することを目的とする。 An object of the present invention is to provide a technique capable of detecting non-spore-forming bacteria more easily.
本発明者は炭酸飲料についての、メンブレンフィルタ（ＭＦ）法に基づく非芽胞形成菌（以下、非芽胞菌ともいう）の検出方法の確立を試みた。具体的には、メンブレンフィルタを用いてのろ過処理を行い、次いで培養処理を行うことによる非芽胞形成菌（以下、非芽胞菌ともいう）の検出を行った。
しかしながら、上記炭酸飲料中に芽胞形成菌（以下、芽胞菌ともいう）が存在する場合、炭酸飲料中では芽胞菌は増殖しないものの、芽胞のままで飲料中にあり、培地を用いた培養では芽胞菌が増殖してしまった。その結果、増殖した芽胞菌が非芽胞菌検出にあたってのノイズとなり、炭酸飲料中に非芽胞菌が存在するかどうかの判定が困難となることが明らかとなった。
The present inventor has attempted to establish a method for detecting non-spore-forming bacteria (hereinafter, also referred to as non-spore-forming bacteria) based on the membrane filter (MF) method for carbonated beverages. Specifically, non-spore-forming bacteria (hereinafter, also referred to as non-spore-forming bacteria) were detected by performing a filtration treatment using a membrane filter and then performing a culture treatment.
However, when spore-forming bacteria (hereinafter, also referred to as spore-forming bacteria) are present in the above-mentioned carbonated drink, the spore-forming bacteria do not grow in the carbonated drink, but the spores remain in the drink and are spores in the culture using a medium. The bacterium has grown. As a result, it was clarified that the grown spore-forming bacteria became noise when detecting the non-spore-forming bacteria, and it became difficult to determine whether or not the non-spore-forming bacteria were present in the carbonated drink.
本発明者は、鋭意研究の結果、ポリフッ化ビニリデン製のメンブレンフィルタを用いて炭酸飲料に対しろ過処理を行うとともに、該ろ過処理で分離された非芽胞菌を所定の培地を用いて培養を行うことで、芽胞菌の増殖を抑えての非芽胞菌の培養を行うことができ、より容易に非芽胞菌を検出できることを見出した。 As a result of diligent research, the present inventor filters a carbonated beverage using a membrane filter made of polyvinylidene fluoride, and cultivates the non-spore-forming bacteria isolated by the filtration treatment using a predetermined medium. Therefore, it was found that the non-spore-forming bacterium can be cultured while suppressing the growth of the spore-forming bacterium, and the non-spore-forming bacterium can be detected more easily.
本発明の要旨は以下のとおりである。
［１］ 炭酸飲料における微生物検出方法であって、
前記炭酸飲料をポリフッ化ビニリデン製メンブレンフィルタを用いてろ過し、
前記ろ過処理により前記炭酸飲料から分離された非芽胞形成菌をナイシンおよびチモールを含む培地を用いて培養し、検出することを含む、微生物検出方法。
［２］ 前記培地におけるナイシンの濃度が５０〜２００ｐｐｍである［１］に記載の微生物検出方法。
［３］ 前記培地におけるチモールの濃度が２０〜１５０ｐｐｍである［１］または［２］に記載の微生物検出方法。
［４］ 前記非芽胞形成菌がシトロバクター属、クレブシエラ属、セラチア属、メチロバクテリウム属、エンテロバクター属、スタフィロコッカス属およびラルストニア属からなる群から選択される１種または２種以上の微生物である、［１］から［３］のいずれか一つに記載の微生物検出方法。
［５］ 前記炭酸飲料のｐＨが４．０以下である[１]から[４]のいずれか一つに記載の微生物検出方法。
［６］ 前記炭酸飲料が加熱殺菌されている、[１]から[５]のいずれか一つに記載の微生物検出方法。
［７］ 非芽胞形成菌の培養が可能である培地であって、
ナイシンおよびチモールを含む培地。
［８］ 前記培地におけるナイシンの濃度が５０〜２００ｐｐｍである[７]に記載の培地。
［９］ 前記培地におけるチモールの濃度が２０〜１５０ｐｐｍである[７]または[８]に記載の培地。
The gist of the present invention is as follows.
[1] A method for detecting microorganisms in carbonated beverages.
The carbonated beverage was filtered using a polyvinylidene fluoride membrane filter.
A method for detecting a microorganism, which comprises culturing and detecting non-spore-forming bacteria separated from the carbonated beverage by the filtration treatment using a medium containing nisin and thymol.
[2] The method for detecting a microorganism according to [1], wherein the concentration of nisin in the medium is 50 to 200 ppm.
[3] The method for detecting a microorganism according to [1] or [2], wherein the concentration of thymol in the medium is 20 to 150 ppm.
[4] One or more species of the non-blastogenic bacterium selected from the group consisting of Citrobacter, Klebsiella, Serratia, Methylobacterium, Enterobacter, Staphylococcus and Larstonia. The method for detecting a genus according to any one of [1] to [3], which is a genus.
[5] The method for detecting a microorganism according to any one of [1] to [4], wherein the pH of the carbonated beverage is 4.0 or less.
[6] The method for detecting a microorganism according to any one of [1] to [5], wherein the carbonated beverage is sterilized by heating.
[7] A medium capable of culturing non-spore-forming bacteria.
Medium containing nisin and thymol.
[8] The medium according to [7], wherein the concentration of nisin in the medium is 50 to 200 ppm.
[9] The medium according to [7] or [8], wherein the concentration of thymol in the medium is 20 to 150 ppm.
本発明によれば、より容易に非芽胞形成菌を検出できる技術を提供することができる。 According to the present invention, it is possible to provide a technique capable of detecting non-spore-forming bacteria more easily.
以下、本発明の１つの実施形態について、詳細に説明する。
本実施形態は炭酸飲料における微生物検出方法に関する。本実施形態の微生物検出方法は、炭酸飲料をポリフッ化ビニリデン製メンブレンフィルタを用いてろ過し、ろ過処理により炭酸飲料から分離された非芽胞形成菌をナイシンおよびチモールを含む培地を用いて培養し、検出することを含む。
Hereinafter, one embodiment of the present invention will be described in detail.
The present embodiment relates to a method for detecting microorganisms in a carbonated beverage. In the method for detecting microorganisms of the present embodiment, a carbonated beverage is filtered using a membrane filter made of polyvinylidene fluoride, and non-blast-forming bacteria separated from the carbonated beverage by filtration are cultured in a medium containing nisin and timol. Including to detect.
ここで、本明細書において炭酸飲料とは、飲料中に溶存している二酸化炭素（炭酸ガス）を含有する飲料をいう。二酸化炭素が溶存していることで飲料のｐＨは中性より下がり、例えばｐＨ４．０以下となる（ｐＨの測定は公知の方法により行うことができ、例えばガラス電極ｐＨメーターを用いた方法が挙げられる（ＪＩＳ−Ｚ８８０２や、「最新・ソフトドリンクス」（社）全国清涼飲料工業会 監修 p910-911, 2003.9.30.）。なお、他の炭酸飲料と比較してより検出を容易に行うことができるため、ｐＨ４．０以下の炭酸飲料であることが好ましい。
また、本実施形態の炭酸飲料における微生物検出方法は、加熱殺菌される工程を経て製造される炭酸飲料を対象にして行うことができる（加熱殺菌を行っていない炭酸飲料と比較してより検出を容易に行うことができるため、好ましい）。具体的な加熱殺菌の方法としては、特に限定されないが、含有成分の混合などを経て調製された飲料を缶等の容器に充填した後に６０〜７０℃程度の熱水によるシャワー殺菌に供する方法や、容器充填前の飲料を配管中などで８０℃程度まで加熱し、加熱された飲料を容器に充填しながら容器ごと殺菌する方法（ホットパック）などを挙げることができる。
炭酸飲料に対して加熱殺菌を行うときの温度、時間などは特に限定されず、当業者が適宜設定することができる。
Here, the carbonated beverage in the present specification means a beverage containing carbon dioxide (carbon dioxide gas) dissolved in the beverage. Due to the dissolved carbon dioxide, the pH of the beverage is lower than neutral, for example, pH 4.0 or less (pH can be measured by a known method, for example, a method using a glass electrode pH meter can be mentioned. (JIS-Z8802 and "Latest Soft Drinks" (company) National Soft Drink Industry Association supervised p910-911, 2003.9.30.). It should be noted that detection is easier than other carbonated beverages. Therefore, it is preferable to use a carbonated beverage having a pH of 4.0 or less.
Further, the method for detecting microorganisms in a carbonated beverage of the present embodiment can be performed on a carbonated beverage produced through a step of heat sterilization (more detection than a carbonated beverage not subjected to heat sterilization). It is preferable because it can be easily performed). The specific method of heat sterilization is not particularly limited, but a method of filling a container such as a can with a beverage prepared by mixing the contained components and then subjecting it to shower sterilization with hot water at about 60 to 70 ° C. Examples thereof include a method (hot pack) in which a beverage before filling in a container is heated to about 80 ° C. in a pipe or the like, and the heated beverage is sterilized together with the container while being filled in the container.
The temperature, time, and the like when heat sterilizing a carbonated beverage are not particularly limited, and can be appropriately set by those skilled in the art.
具体的な本実施形態に係る炭酸飲料としては、ノンアルコール炭酸飲料やアルコール炭酸飲料などを挙げることができる。ノンアルコール炭酸飲料としては、ビールテイストノンアルコール飲料、ノンアルコールチューハイ、ノンアルコールカクテル、そのほか炭酸飲料に分類される清涼飲料水などが例示される。また、アルコール炭酸飲料としては、RTD（Ready to Drink）飲料と称される、所謂チューハイ飲料、カクテル飲料などが例示される。 Specific examples of the carbonated drink according to the present embodiment include non-alcoholic carbonated drinks and alcoholic carbonated drinks. Examples of non-alcoholic carbonated beverages include beer-taste non-alcoholic beverages, non-alcoholic chu-hi, non-alcoholic cocktails, and soft drinks classified as carbonated beverages. Examples of alcoholic carbonated beverages include so-called chu-hi beverages and cocktail beverages, which are called RTD (Ready to Drink) beverages.
また、本実施形態において検出対象となる微生物とは上記の炭酸飲料中に含まれる微生物をいい、具体的には非芽胞菌を意味する。本実施形態においては培地を用いての検出対象である非芽胞菌の培養のときに炭酸飲料中に非芽胞菌と共に存在し得る芽胞菌の増殖が抑えられるため、非芽胞菌を検出しやすい。 Further, the microorganism to be detected in the present embodiment means a microorganism contained in the above-mentioned carbonated drink, and specifically means a non-spore-forming bacterium. In the present embodiment, when the non-spore-forming bacterium to be detected using the medium is cultured, the growth of the spore-forming bacterium that may exist together with the non-spore-forming bacterium in the carbonated drink is suppressed, so that the non-spore-forming bacterium can be easily detected.
ここで、本明細書において、芽胞形成菌とは、芽胞（ｓｐｏｒｅ）を形成する微生物を意味する。また、芽胞とは、該微生物が形成する強固な殻構造を意味する。
一方、非芽胞形成菌とは上述の芽胞を形成しない微生物を意味する。
Here, in the present specification, the spore-forming bacterium means a microorganism that forms a spore. Further, the spore means a strong shell structure formed by the microorganism.
On the other hand, the non-spore-forming bacterium means the above-mentioned microorganism that does not form spores.
増殖が抑制される芽胞菌としては特に限定されないが、より抑制される芽胞菌としてバチルス属（genus Bacillus）、ブレビバチルス属（genus Brevibacillus）、またはパニバチルス属（genus Paenibacillus）に属する微生物を挙げることができる。さらにより抑制される芽胞菌としては、以下の微生物を挙げることができる。
バチルス リケニフォルミス（Bacillus licheniformis）
ブレビバチルス ラテロスポラス（Brevibacillus laterosporus）
バチルス ギンセンギフミ（Bacillus ginsengihumi）
バチルス コアグランス（Bacillus coagulans）
ブレビバチルス パラブレビス（Brevibacillus parabrevis）
バチルス サブティリス（Bacillus subtilis）
パエニバチルス アルギノリティカス(Paenibacillus alginolyticus)
バチルス プミルス（Bacillus pumilus）
バチルス アミロリクエファシエンス（Bacillus amyloliquefaciens）
パエニバチルス バリダス（Paenibacillus validus）
パエニバチルス ポリミザ（Paenibacillus polymyxa）
The spore-forming bacterium whose growth is suppressed is not particularly limited, and examples of the spore-forming bacterium whose growth is suppressed include microorganisms belonging to the genus Bacillus, the genus Brevibacillus, or the genus Paenibacillus. can. Examples of the spore-forming bacterium that is further suppressed include the following microorganisms.
Bacillus licheniformis
Brevibacillus laterosporus
Bacillus ginsengihumi
Bacillus coagulans
Brevibacillus parabrevis
Bacillus subtilis
Paenibacillus alginolyticus
Bacillus pumilus
Bacillus amyloliquefaciens
Paenibacillus validus
Paenibacillus polymyxa
また、非芽胞菌として、特に限定されないが、本実施形態の方法を適用したときに他の菌と比較してより検出しやすいため、シトロバクター属（genus Citrobacter）、クレブシエラ属（genus Klebsiella）、セラチア属（genus Serratia）、メチロバクテリウム属（genus Methylobacterium）、エンテロバクター属（genus Enterobacter）、スタフィロコッカス属（genus Citrobacter）、またはラルストニア属（genus Citrobacter）に属する微生物が好ましく、以下の非芽胞菌のうち１種または２種以上を検出対象とすることがより好ましい。
シトロバクター フロインデイ（Citrobacter freundii）
クレブシエラ オキシトカ（Klebsiella oxytoca）
セラチア マルセッセンス（Serratia marcescens）
メチロバクテリウム エキストロクエンス（Methylobacterium extorquens）
エンテロバクター クロアカ（Enterobactor cloacae）
クレブシエラ ニューモニエ（Klebsiella pneumoniae）
スタフィロコッカス エピデルミデス（Staphylococcus epidermidis）
ラルストニア ピッケテ（Ralstonia pickettii）
スフィンゴモナス トゥルーペリ（Sphingomonas trueperi）
パントエア アグロメランス（Pantoea agglomerans）
バークホルデリア ベトナミエンシス（Burkholderia vietnamiensis）
シュードモナス エルギノーザ（Pseudomonas aeruginosa）
シュードモナス フルオレッセンス（Pseudomonas fluorescens）
ステノトロホモナス マルトフィリア（Stenotrophomonas maltophilia）
Further, the non-blastic bacterium is not particularly limited, but when the method of the present embodiment is applied, it is easier to detect as compared with other bacterium. Therefore, Citrobacter, Klebsiella, etc. Microorganisms belonging to the genus Serratia, the genus Methylobacterium, the genus Enterobacter, the genus Citrobacter, or the genus Citrobacter are preferred, and the following non-genus It is more preferable to detect one or more of Citrobacter.
Citrobacter freundii
Klebsiella oxytoca
Serratia marcescens
Methylobacterium extorquens
Enterobactor cloacae
Klebsiella pneumoniae
Staphylococcus epidermidis
Ralstonia pickettii
Sphingomonas trueperi
Pantoea agglomerans
Burkholderia vietnamiensis
Pseudomonas aeruginosa
Pseudomonas fluorescens
Stenotrophomonas maltophilia
本実施形態の微生物検出方法は、メンブレンフィルタ法（ＭＦ法）に基づき行うことができる。具体的には、試料となる炭酸飲料をメンブレンフィルタを用いたろ過処理に供し、次にメンブレンフィルタを培地に貼り付けて炭酸飲料から分離された微生物の培養を試み、形成されたコロニーの検出、形態観察などにより微生物を検出する。 The microorganism detection method of the present embodiment can be performed based on the membrane filter method (MF method). Specifically, the carbonated beverage as a sample was subjected to a filtration treatment using a membrane filter, and then the membrane filter was attached to the medium to attempt to cultivate the microorganisms separated from the carbonated beverage, and the formed colonies were detected. Microorganisms are detected by morphological observation.
ここで、本実施形態においては、メンブレンフィルタとして、ポリフッ化ビニリデン（Polyvinylidene difluoride，ＰＶＤＦ）製メンブレンフィルタを用いる。
なお、メンブレンフィルタの孔径などは特に限定されず、当業者が適宜設定できるが、例えば０．２μｍ〜１．０μｍとすることができる。ＰＶＤＦ製メンブレンフィルタとして市販のものを用いればよく、例えばメルクミリポア社製MSP001026を挙げることができる。
また、ろ過処理におけるその他の条件（例えば、ろ過処理を吸引ろ過とする場合のろ過圧力など）についても特に限定されず、当業者が適宜設定できる。
Here, in the present embodiment, a membrane filter made of polyvinylidene difluoride (PVDF) is used as the membrane filter.
The pore size of the membrane filter is not particularly limited and can be appropriately set by those skilled in the art, but can be set to, for example, 0.2 μm to 1.0 μm. A commercially available PVDF membrane filter may be used, and examples thereof include MSP001026 manufactured by Merck Millipore.
Further, other conditions in the filtration process (for example, filtration pressure when the filtration process is suction filtration) are not particularly limited and can be appropriately set by those skilled in the art.
本実施形態において、培地はナイシンおよびチモールを含む培地を用いる。
ナイシンは、乳酸菌由来の３４アミノ酸残基からなる水溶性の多環式ペプチドである。ナイシンは、乳酸菌を公知の方法により培養し精製することによって得ることができるほか、市販のものを用いてもよい。
また、チモールはＣ１０Ｈ１４Ｏで表されるモノテルペン誘導体である。チモールも例えば市販のものを用いることができ、特に限定されない。
In this embodiment, a medium containing nisin and thymol is used as the medium.
Nisin is a water-soluble polycyclic peptide consisting of 34 amino acid residues derived from lactic acid bacteria. Nisin can be obtained by culturing and purifying lactic acid bacteria by a known method, or commercially available ones may be used.
Thymol is a monoterpene derivative represented by C 10 H 14 O. As thymol, for example, a commercially available product can be used, and the thymol is not particularly limited.
ナイシン、チモールの含有量は当業者が適宜設定でき、特に限定されないが、より芽胞菌の増殖を抑制できるため、培地におけるナイシンの濃度が５０〜２００ｐｐｍであることが好ましい。また、同様の理由から培地におけるチモールの濃度が２０〜１５０ｐｐｍであることが好ましい。
なお、ナイシン、チモールの含有量は、例えば培地成分への添加量に基づき算出することができる。また、他に、ナイシンについては、液体クロマトグラフ・質量分析計（ＬＣ/ＭＳ）により検出する方法、チモールについては、ガスクロマトグラフ・質量分析計（ＧＣ/ＭＳ）で検出する方法がある。
The contents of nisin and thymol can be appropriately set by those skilled in the art and are not particularly limited, but the concentration of nisin in the medium is preferably 50 to 200 ppm because the growth of spore-forming bacteria can be further suppressed. Further, for the same reason, the concentration of thymol in the medium is preferably 20 to 150 ppm.
The contents of nisin and thymol can be calculated based on, for example, the amount added to the medium component. In addition, there is a method of detecting nisin by a liquid chromatograph / mass spectrometer (LC / MS), and a method of detecting timol by a gas chromatograph / mass spectrometer (GC / MS).
具体的には、チモールについて、試料となる培地を以下の条件で、ガスクロマトグラフィー質量分析（ＧＣ−ＭＳ）に供し、含有量を得ることができる。
ＧＣ装置：Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ ＧＣ−ＭＳＤ
ＧＣオーブン温度条件：４０℃（５分）− ６℃／ｍｉｎ − ２４０℃
質量分析（ＭＳ）条件
四重極設定値：１５０
イオン源設定値：２３０
面積値算出条件
トータルイオンモード
質量（ＬＯＷ）：３５
質量（ＨＩＧＨ）：５５０
カラム：ＤＢ−ＷＡＸＥＴＲ ６０ｍ、内径３２０μｍ、膜厚０．２５μｍ
試料前処理条件：試料８０μＬと内部標準物質（デカン酸メチルエステル２０ｐｐｍ
アルコール水溶液）２０μＬを２０ｍＬスクリューキャップバイアル瓶中で混合
ダイナミックヘッドスペース条件
装置：ゲステル社ＭＰＳ
吸着剤：ＴＥＮＡＸ
試料気化温度：８０℃
試料気化用ガス供給量：３０００ｍｌ
試料気化用ガス供給速度：１００ｍｌ／ｍｉｎ
試料気化用ガス種類：窒素
ピーク保持時間：ＭＳの解析によって成分および濃度の同定を行う。
Specifically, the content of thymol can be obtained by subjecting the sample medium to gas chromatography-mass spectrometry (GC-MS) under the following conditions.
GC device: Agilent Technologies GC-MSD
GC oven temperature conditions: 40 ° C (5 minutes) -6 ° C / min-240 ° C
Mass spectrometry (MS) conditions Quadrupole set value: 150
Ion source setting value: 230
Area value calculation conditions Total ion mode mass (LOW): 35
Mass (HIGH): 550
Column: DB-WAXETR 60m, inner diameter 320μm, film thickness 0.25μm
Sample pretreatment conditions: 80 μL of sample and internal standard substance (20 ppm of methyl decanoate)
Alcohol aqueous solution) 20 μL mixed in a 20 mL screw cap vial Dynamic headspace condition Device: Gesuteru MPS
Adsorbent: TENAX
Sample vaporization temperature: 80 ° C
Gas supply for sample vaporization: 3000 ml
Gas supply rate for sample vaporization: 100 ml / min
Gas type for sample vaporization: Nitrogen Peak retention time: Identification of components and concentration by analysis of MS.
また、ナイシンについては、厚生労働省ホームページ、食品中の食品添加物分析法 https://www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/syokuten/bunseki/index.html 「食品中の食品添加物分析法」の改正について（令和元年6月28日）（別添３）に従って分析できる。 For nisin, see the Ministry of Health, Labor and Welfare website, analysis method for food additives in food https://www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/shokuhin/syokuten/bunseki/index.html Regarding the revision of "Food Additive Analysis Method of Food Additives" (June 28, 1st year of Ordinance) (Attachment 3), it can be analyzed.
培地組成について、ナイシン、チモール以外の他の成分は非芽胞菌を培養できる限り特に限定されず、当業者が適宜設定することができる。例えば、標準寒天培地などを構成する成分にナイシン、チモールを添加して培地を調製するなどすればよい。
また、培地の形態についても限定されず、例えば平板培地とすることができる。
The medium composition is not particularly limited as long as non-spore-forming bacteria can be cultured, and components other than nisin and thymol can be appropriately set by those skilled in the art. For example, the medium may be prepared by adding nisin and thymol to the components constituting the standard agar medium and the like.
Further, the form of the medium is not limited, and for example, a plate medium can be used.
上記培地を用いての培養条件は特に限定されず当業者が適宜設定できる。
また、非芽胞菌の検出についても限定されず、例えば形成されたコロニーに対する目視での観察などにより行うことができる。
The culture conditions using the above medium are not particularly limited and can be appropriately set by those skilled in the art.
Further, the detection of non-spore-forming bacteria is not limited, and can be performed, for example, by visual observation of the formed colonies.
以上、本実施形態によれば、ＭＦ法に基づき炭酸飲料由来の非芽胞菌の検出を行うにあたり、ノイズとなる芽胞菌の増殖を抑えることができる。そのため、より容易に非芽胞菌の検出を行うことができるので、炭酸飲料の製造における作業性の改善などに寄与することができる。 As described above, according to the present embodiment, when detecting non-spore-forming bacteria derived from carbonated beverages based on the MF method, it is possible to suppress the growth of spore-forming bacteria that cause noise. Therefore, non-spore-forming bacteria can be detected more easily, which can contribute to improvement of workability in the production of carbonated beverages.
以下、本発明を実施例により更に詳細に説明するが、本発明はこれらに限定されない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
[試験例１]
１．培地の作製
標準寒天培地（日水製薬（株））を培地記載の方法に従って調製した。ナイシン溶液は８０℃で３０分の加熱、チモール水溶液は０．４５μｍのフィルタ除菌の処理それぞれを行い、分注前に無菌的に培地添加し、培地を作成した。
[Test Example 1]
1. 1. Preparation of medium A standard agar medium (Nissui Pharmaceutical Co., Ltd.) was prepared according to the method described in the medium. The nisin solution was heated at 80 ° C. for 30 minutes, and the Timor aqueous solution was subjected to a filter sterilization treatment of 0.45 μm, and the medium was aseptically added before dispensing to prepare a medium.
２．供試菌の前培養
飲料製造工場の環境頻出菌を実験に用いた。
いずれも前培養には、標準寒天培地（日水製薬（株））を用いた。
芽胞菌については、８０℃で３０分間加熱し、栄養細胞を死滅させたものを用いた。
2. Pre-culture of test bacteria Environmentally frequent bacteria in a beverage manufacturing plant were used in the experiment.
In both cases, a standard agar medium (Nissui Pharmaceutical Co., Ltd.) was used for the preculture.
As for the spore-forming bacteria, those obtained by heating at 80 ° C. for 30 minutes to kill the vegetative cells were used.
３．菌液の調整
菌数が２００−３００個程度となるよう、前培養した菌体をオートクレーブ処理済みの生理食塩水５０ｍＬに懸濁させ、菌液とした。
3. 3. Preparation of bacterial solution The pre-cultured bacterial cells were suspended in 50 mL of autoclaved physiological saline to prepare a bacterial solution so that the number of bacteria was about 200 to 300.
４．菌液のメンブレンろ過・培養
ファンネルを用いて、所定のメンブレンで調整した菌液をろ過した後、メンブレンを各培地へ移し、４８時間培養した。培養温度は、３５℃とした。
4. Membrane filtration and culture of bacterial solution After filtering the bacterial solution prepared with a predetermined membrane using a funnel, the membrane was transferred to each medium and cultured for 48 hours. The culture temperature was 35 ° C.
５．観察
培養後の継時的にコロニー数を確認し、各菌種の生育状況を観察した。
5. The number of colonies was confirmed over time after the observation culture, and the growth status of each bacterial species was observed.
試験に供した菌種、メンブレンフィルタの種類、培地への添加物（未添加、ナイシンのみ添加、またはナイシンおよびチモールを添加）については、結果と共に表１〜５に示す。 The bacterial species used in the test, the type of membrane filter, and the additives to the medium (not added, only nisin added, or nisin and thymol added) are shown in Tables 1 to 5 together with the results.
表１、２から理解できるように、メンブレンフィルタとしてＰＶＤＦ製メンブレンフィルタを用いることで芽胞菌の増殖が抑制された。さらに、表３から理解できるように、ナイシン、チモールを含有する培地を用いることで芽胞菌の増殖がさらに抑制された（表３の試験では芽胞菌は検出されなかった）。
一方、表４、５から理解できるように非芽胞菌についてはメンブレンフィルタとしてＰＶＤＦ製メンブレンフィルタを用い、ナイシン、チモールを含有する培地を用いる場合にもその増殖が確認できた。
As can be understood from Tables 1 and 2, the growth of spore-forming bacteria was suppressed by using a PVDF membrane filter as the membrane filter. Furthermore, as can be seen from Table 3, the growth of spore-forming bacteria was further suppressed by using a medium containing nisin and thymol (no spore-forming bacteria were detected in the test of Table 3).
On the other hand, as can be understood from Tables 4 and 5, the growth of non-spore-forming bacteria was confirmed even when a PVDF membrane filter was used as the membrane filter and a medium containing nisin and thymol was used.
[試験例２]
表６に示す非芽胞菌、表７に芽胞菌について、試験例１と同様のろ過処理および培養を行った。結果を表６、７に示す。
[Test Example 2]
The non-spore-forming bacteria shown in Table 6 and the spore-forming bacteria shown in Table 7 were subjected to the same filtration treatment and culture as in Test Example 1. The results are shown in Tables 6 and 7.
表６から理解できるように、これら非芽胞菌についてもメンブレンフィルタとしてＰＶＤＦ製メンブレンフィルタを用い、ナイシン、チモールを含有する培地を用いる場合でもその増殖が確認された。一方、表７に示す芽胞菌についてはその増殖が抑制されていることが確認された。 As can be understood from Table 6, the growth of these non-spore-forming bacteria was confirmed even when a PVDF membrane filter was used as the membrane filter and a medium containing nisin and thymol was used. On the other hand, it was confirmed that the growth of the spore-forming bacteria shown in Table 7 was suppressed.
[試験例３]
ＲＴＤ飲料（アサヒビール社製 チューハイ「もぎたて（グレープフルーツ）」）に菌液を添加し、試験例１と同様（具体的には表３、４と同様の条件）に試験した結果、ＰＶＤＦ製メンブレンフィルタ、およびナイシン、チモールを含有する培地を用いた実施例に該当する例においては、試験例１、２と同様に芽胞菌の増殖が抑制された一方で非芽胞菌の増殖が確認された。
[Test Example 3]
A PVDF membrane filter was obtained as a result of adding a bacterial solution to an RTD beverage (Asahi Breweries chu-hi "freshly picked (grapefruit)") and testing in the same manner as in Test Example 1 (specifically, the same conditions as in Tables 3 and 4). , And in the example corresponding to the example using the medium containing nisin and timol, the growth of spore-forming bacteria was suppressed while the growth of non-spore-forming bacteria was confirmed as in Test Examples 1 and 2.
Claims (9)
前記炭酸飲料をポリフッ化ビニリデン製メンブレンフィルタを用いてろ過し、
前記ろ過処理により前記炭酸飲料から分離された非芽胞形成菌をナイシンおよびチモールを含む培地を用いて培養し、検出することを含む、微生物検出方法。 A method for detecting microorganisms in carbonated beverages.
The carbonated beverage was filtered using a polyvinylidene fluoride membrane filter.
A method for detecting a microorganism, which comprises culturing and detecting non-spore-forming bacteria separated from the carbonated beverage by the filtration treatment using a medium containing nisin and thymol.
ナイシンおよびチモールを含む培地。 A medium capable of culturing non-spore-forming bacteria
Medium containing nisin and thymol.
The medium according to claim 7 or 8, wherein the concentration of thymol in the medium is 20 to 150 ppm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115404188A (en) * | 2022-09-21 | 2022-11-29 | 北京三药科技开发公司 | Culture medium additive for improving recovery rate and growth rate of ginseng bacillus terreus, improved culture medium and application |
| CN117568239A (en) * | 2024-01-05 | 2024-02-20 | 成都医学院 | Brevibacillus paraBrevibacillus and application thereof in degradation and decoloration of aniline blue dye |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115404188A (en) * | 2022-09-21 | 2022-11-29 | 北京三药科技开发公司 | Culture medium additive for improving recovery rate and growth rate of ginseng bacillus terreus, improved culture medium and application |
| CN115404188B (en) * | 2022-09-21 | 2024-03-22 | 北京三药科技有限公司 | Culture medium additive for improving recovery rate and growth rate of geobacillus ginseng, improved culture medium and application |
| CN117568239A (en) * | 2024-01-05 | 2024-02-20 | 成都医学院 | Brevibacillus paraBrevibacillus and application thereof in degradation and decoloration of aniline blue dye |
| CN117568239B (en) * | 2024-01-05 | 2024-03-26 | 成都医学院 | Brevibacillus paraBrevibacillus and application thereof in degradation and decoloration of aniline blue dye |
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