JP2002000261A - Antibacterial agent - Google Patents
Antibacterial agentInfo
- Publication number
- JP2002000261A JP2002000261A JP2000194327A JP2000194327A JP2002000261A JP 2002000261 A JP2002000261 A JP 2002000261A JP 2000194327 A JP2000194327 A JP 2000194327A JP 2000194327 A JP2000194327 A JP 2000194327A JP 2002000261 A JP2002000261 A JP 2002000261A
- Authority
- JP
- Japan
- Prior art keywords
- present
- bacteria
- product
- growth
- antibacterial agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)
- Cosmetics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、広範な微生物、特
に有害細菌に対して作用してその生育を阻害する安全な
防菌剤に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a safe bactericidal agent which acts on a wide variety of microorganisms, especially harmful bacteria, and inhibits the growth thereof.
【０００２】[0002]
【従来の技術】食品および飲料の腐敗、変質やヒトの感
染症等には多くの微生物が関与しており、その防止に
は、これらの微生物を殺菌するか、あるいはその増殖を
抑制する方法が用いられている。殺菌は、加熱、放射線
・電子線照射、殺菌作用を持つ化学合成物質の添加等に
よって行われるが、食品やヒトに対するこれらの方法
は、変質や安全性の問題から必ずしも適しているとは限
らない。また、微生物の増殖を抑制する抗菌性物質とし
て、化学合成物質、植物から抽出される物質・成分、微
生物が産生する物質・成分等多くのものが知られてお
り、抗菌性を示す化学合成物質は、多量にかつ安価に製
造できることが可能であり、しかも多くの微生物に対し
て有効に作用するという利点がある。しかし、人体およ
び自然環境への安全性の観点から必ずしも適していると
は限らない。一方、植物や微生物から得られる物質・成
分は、特殊なものを除いて安全なものが多く、しかも時
間の経過と共に、自然環境下で分解され、生態系に与え
る影響も少ないことが知られている。例えば、微生物が
産生する抗菌性物質としてよく知られているものに、チ
ーズから分離された特殊な乳酸菌が産生するといわれて
いるナイシンがある。このナイシンは、ペプチドであ
り、欧州では実際に食品保存料として利用されているも
のである。しかしながら、その効果はグラム陽性菌にの
み抗菌性を示すといわれている。さらに、微生物が産生
する抗菌性物質の一つとして、細胞壁溶解酵素が注目さ
れている。それは、細胞壁溶解酵素も化学的防菌剤や保
存料と異なりタンパク質であるので、これ自身毒性がな
く、非常に安全な防菌剤であることや、細胞壁のみを特
異的に溶解し他の物質には作用しないという利点がある
ためである。しかし、細胞壁溶解酵素の欠点としては、
酵素であるため特異性が高く、特定の微生物はよく溶菌
するが、ごく近縁であっても別の構造の細胞壁を持つ微
生物には作用しないというようなことが挙げられる。例
えば、これまでに卵白リゾチームによる食品に対する防
菌の研究が数多くなされてきているが、その溶菌スペク
トルの狭さによって、対象菌がかなり限定されてしまっ
ているのが現状である。特にグラム陰性細菌に対して
は、効果が少なく、グリシンを併用してその効果を高め
ているという報告もある。つまり、これまでに発見され
た微生物が産生する抗菌物質の多くは、人体や環境への
安全性は高いものの、効果としては十分とはいえない。2. Description of the Related Art Many microorganisms are involved in spoilage and deterioration of foods and beverages and in human infectious diseases. To prevent such microorganisms, there is a method of killing these microorganisms or suppressing their growth. Used. Sterilization is carried out by heating, radiation / electron beam irradiation, addition of chemically synthesized substances having a bactericidal action, etc., but these methods for food and humans are not always suitable due to deterioration and safety issues. . In addition, many antibacterial substances that suppress the growth of microorganisms are known, such as chemically synthesized substances, substances and components extracted from plants, and substances and components produced by microorganisms. Has the advantage that it can be produced in large quantities and at low cost, and that it effectively acts on many microorganisms. However, it is not always suitable from the viewpoint of safety to the human body and the natural environment. On the other hand, it is known that many substances and components obtained from plants and microorganisms are safe except for special ones, and that they are degraded in the natural environment over time and have little effect on ecosystems. I have. For example, a well-known antibacterial substance produced by microorganisms is nisin, which is said to produce a special lactic acid bacterium isolated from cheese. This nisin is a peptide and is actually used as a food preservative in Europe. However, the effect is said to be antibacterial only for Gram-positive bacteria. Further, as one of the antibacterial substances produced by microorganisms, a cell wall lysing enzyme has attracted attention. Because cell wall lysing enzymes are also proteins, unlike chemical antibacterial agents and preservatives, they are not toxic by themselves and are very safe antibacterial agents. Has the advantage of not acting. However, disadvantages of cell wall lysing enzymes include:
Specificity is high because it is an enzyme, and specific microorganisms can be lysed well. However, even if they are closely related, they do not act on microorganisms having a cell wall of another structure. For example, many studies have been made on the antibacterial activity of foods with egg white lysozyme, but the target bacteria have been considerably limited due to the narrow lysis spectrum. Particularly, there is a report that the effect is small for gram-negative bacteria, and the effect is enhanced by using glycine in combination. In other words, many of the antibacterial substances produced by microorganisms that have been discovered so far have high safety for the human body and the environment, but are not sufficiently effective.
【０００３】[0003]
【発明が解決しようとする課題】かかる事情に鑑み、本
発明は、広範な微生物、特に有害細菌に対して作用する
安全な防菌剤を提供することを目的とする。In view of such circumstances, an object of the present invention is to provide a safe antibacterial agent which acts against a wide range of microorganisms, particularly harmful bacteria.
【０００４】[0004]
【課題を解決するための手段】本発明者らは、上記目的
を達成すべく鋭意検討を行った結果、偶然にも、本出願
人の出願に係る特開平１１−５６３８号に開示されるス
トレプトマイセス・フルビシムス（Streptomyces fulvi
ssimus）ＴＵ−６株（ＦＥＲＭ Ｐ−１６３４７）によ
り生産される細胞壁溶解酵素を含む培養物が非常に広範
な微生物に対して生育阻害効果を有し、安全性の高いも
のであり、目的とする防菌剤として極めて好適であるこ
とを見出し、本発明を完成するに至った。すなわち、本
発明は、Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result, by accident, the streptotype disclosed in Japanese Patent Application Laid-Open No. H11-5638 filed by the present applicant has been discovered. Streptomyces fulvi
ssimus) The culture containing the cell wall lytic enzyme produced by the TU-6 strain (FERM P-16347) has a growth inhibitory effect on a very wide range of microorganisms, is highly safe, and is the object of the present invention. They have found that they are extremely suitable as antibacterial agents, and have completed the present invention. That is, the present invention
【０００５】ストレプトマイセス・フルビシムス（Stre
ptmyces fulvissimus）ＴＵ−６株（ＦＥＲＭ Ｐ−１
６３４７）またはその変異体もしくは遺伝子組換体の培
養物を有効成分とすることを特徴とする防菌剤、とりわ
け、食品用保存料、特に、アルコール飲料の腐敗防止剤
および虫歯予防剤として有用な防菌剤を提供するもので
ある。本発明によれば、非常に広範な微生物、特に有害
細菌に対して、生育阻害効果を有する安全な防菌剤を提
供できる。[0005] Streptomyces flubisimus (Stre
ptmyces fulvissimus) TU-6 strain (FERM P-1)
6347) or a bactericidal agent characterized by comprising a culture of a mutant or a genetically modified product thereof as an active ingredient, in particular, an antibacterial agent useful as a preservative for foods, especially an antiseptic and an anti-caries agent of alcoholic beverages. A fungicide is provided. According to the present invention, it is possible to provide a safe antibacterial agent having a growth inhibitory effect against a very wide range of microorganisms, particularly harmful bacteria.
【０００６】[0006]
【発明の実施の形態】本発明の防菌剤の有効成分を生産
する菌株は、ストレプトマイセス・フルビシムス ＴＵ
−６株である。この菌株は、平成９年７月２４日より工
業技術院生命工学工業技術研究所に、受託番号ＦＥＲＭ
Ｐ−１６３４７の下に寄託されている。以後、本明細
書において本菌をＴＵ−６株と略記する。このＴＵ−６
株は、上記特開平１１−５６３４８号記載の細胞壁溶解
酵素（以下、ＴＵ−６酵素と略記する）を生産する菌株
である。本発明においては、所望により、本菌株のみな
らず、自体公知の方法により、ＴＵ−６株に変異処理お
よび遺伝子組換え処理を施してそのＴＵ−６酵素の生産
性を修飾した変異体または遺伝子組換体を使用できる。BEST MODE FOR CARRYING OUT THE INVENTION A strain producing an active ingredient of the antibacterial agent of the present invention is Streptomyces fluvicimus TU.
-6 strains. This strain has been accredited by the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology on July 24, 1997, under the accession number FERM.
Deposited under P-16347. Hereinafter, this bacterium is abbreviated as TU-6 strain in this specification. This TU-6
The strain is a strain that produces the cell wall lytic enzyme (hereinafter abbreviated as TU-6 enzyme) described in JP-A-11-56348. In the present invention, if desired, not only this strain but also a mutant or gene obtained by subjecting the TU-6 strain to a mutation treatment and a gene recombination treatment to modify the productivity of the TU-6 enzyme by a method known per se. Recombinants can be used.
【０００７】本発明の防菌剤の有効成分は、このＴＵ−
６株またはその変異体もしくは遺伝子組換体を、生産培
地、例えば、０．５％ポリペプトン、０．１％キチン粉
末、０．１％硫酸マグネシウム、０．１％リン酸１カリ
ウム、０．１％ ３−（Ｎ−モルホリノ）プロパンスル
ホン酸からなるｐＨ７．０〜８．０、好ましくはｐＨ
８．０の液体培地を用い、常法により、振盪培養または
通気攪拌培養を行って得られた培養液や、それから菌体
を除去した液体等の培養物自体およびその処理物であ
る。このような処理物としては、例えば、培養液から、
遠心分離またはろ過等の公知の方法により上清を分離
し、この上清を、例えば、排除分子量５，０００から１
０，０００の限外ろ過膜を用いた限外ろ過または硫安に
より濃縮し、あるいは濃縮せずに、凍結乾燥等の公知の
乾燥方法を用いて乾燥した粉末が挙げられる。また、乾
燥前に濃縮液を透析しておいてもよく、乾燥せず、液体
として使用してもよい。本発明の防菌剤はこの有効成分
単独でもよく、さらに、所望により、本発明の目的に反
しない範囲で適宜の添加剤や、他の溶菌酵素、抗菌剤等
と合し、自体公知の方法により、液体や固体の適宜の剤
形にしてもよい。[0007] The active ingredient of the antibacterial agent of the present invention comprises the TU-
Six strains or mutants or recombinants thereof were transformed into a production medium, for example, 0.5% polypeptone, 0.1% chitin powder, 0.1% magnesium sulfate, 0.1% monopotassium phosphate, 0.1% PH 7.0 to 8.0 consisting of 3- (N-morpholino) propanesulfonic acid, preferably pH
A culture medium obtained by shaking culture or aeration and stirring culture using a liquid medium of 8.0 by a conventional method, and a culture itself such as a liquid from which cells have been removed, and a processed product thereof. As such a processed product, for example, from a culture solution,
The supernatant is separated by a known method such as centrifugation or filtration, and the supernatant is separated, for example, from an excluded molecular weight of 5,000 to 1
A powder obtained by concentrating by ultrafiltration using a 000 ultrafiltration membrane or ammonium sulfate, or by using a known drying method such as freeze-drying without concentrating, may be used. The concentrated solution may be dialyzed before drying, and may be used as a liquid without drying. The bactericidal agent of the present invention may be the active ingredient alone, or, if desired, combined with an appropriate additive or other lytic enzyme, an antibacterial agent and the like within a range not contrary to the object of the present invention, and a method known per se. Thus, an appropriate liquid or solid dosage form may be used.
【０００８】以下の実施例に示すごとく、本発明の防菌
剤は、ＴＵ−６酵素を含むものであり、その効果はＴＵ
−６酵素の溶菌活性によると考えられる。すなわち、本
発明の防菌剤を用いて、例えば、代表的なグラム陽性細
菌である乳酸桿菌、乳酸球菌特に虫歯の原因菌であるう
蝕細菌（ストレプトコッカス・ミュータンス(Streptoco
ccus mutans)）、清酒やビールをはじめとするアルコー
ル飲料の腐敗、腐造の原因となっている火落菌および乳
酸菌、食中毒の原因の一つとなっている黄色ブドウ球菌
（スタフィロコッカス・オウレウス(Staphylococcus au
reus)）およびバチルス・サブチルス(Bacillus subtili
s)や、代表的なグラム陰性細菌であるエシェリシア・コ
リ(Escherichia coli)やシュウドモナス・フルオレセン
ス（Pseudomonas fluorescens）を、ＴＵ−６酵素の酵
素反応至適条件下（４０℃、ｐＨ４．０（マキルベイン
緩衝液中））で溶菌させたところ、グラム陽性、陰性に
関わらず上記の菌をよく溶解した。また、本発明品の溶
菌活性をアルコール（エタノール）が１５％存在すると
いう条件下で検討したところ、アルコールが存在してい
ない条件で測定した結果と、全く遜色のない結果であっ
た。したがって、本発明の防菌剤の溶菌活性は、比較的
アルコールに対する耐性度が高く、清酒やビール等のア
ルコール飲料へ応用が可能であるということが判明し
た。さらに、本発明の防菌剤を、例えば、上記細菌の生
育用培地に添加したものと、添加していないものとの生
育度合いを比較したところ、本発明の防菌剤を添加した
培地では明らかに菌の生育が阻害されていた。特に、火
落菌や、う蝕細菌(Streptococcus mutans)に関しては完
全に増殖を阻害した。また、その結果を、代表的な細胞
壁溶解酵素であり、既に食品の防腐剤として利用されて
いる卵白リゾチームと比較したところ、卵白リゾチーム
ではほとんど効果がなく、本発明の防菌剤の方が明らか
に増殖阻害活性において優れていた。As shown in the following examples, the antibacterial agent of the present invention contains TU-6 enzyme, and its effect is
This is probably due to the lytic activity of the -6 enzyme. That is, using the antibacterial agent of the present invention, for example, typical gram-positive bacteria such as lactobacilli and lactococci, in particular, cariogenic bacteria (Streptococcus mutans (Streptococcus mutans)
ccus mutans), spoilage of alcoholic beverages such as sake and beer, fire-killing bacteria and lactic acid bacteria that cause rotting, and Staphylococcus aureus (Staphylococcus that causes food poisoning) au
reus)) and Bacillus subtili
s) and Escherichia coli and Pseudomonas fluorescens, which are typical gram-negative bacteria, were oxidized under optimal conditions (40 ° C., pH 4.0 (40 ° C., pH 4.0) for the TU-6 enzyme. When the bacterium was lysed with a McIlvain buffer)), the above bacterium was well dissolved regardless of whether it was gram positive or negative. Further, when the bacteriolytic activity of the product of the present invention was examined under the condition that 15% of alcohol (ethanol) was present, the result was not inferior to the result measured under the condition where no alcohol was present. Therefore, it was found that the bacteriolytic activity of the antibacterial agent of the present invention has relatively high resistance to alcohol, and can be applied to alcoholic beverages such as sake and beer. Furthermore, when the bactericidal agent of the present invention was added to, for example, the above-mentioned bacterium growth medium, the growth degree of the bactericidal agent was compared with that of the bacterium not added. The growth of the fungus was inhibited. In particular, the growth of fire-killed bacteria and carious bacteria (Streptococcus mutans) was completely inhibited. In addition, when the results were compared with egg white lysozyme, which is a typical cell wall lysing enzyme and is already used as a food preservative, egg white lysozyme has almost no effect, and the antibacterial agent of the present invention is clearer. It was excellent in growth inhibitory activity.
【０００９】一方、本発明の防菌剤を加熱処理（沸騰水
中、１５分）して、本発明の防菌剤に含まれるＴＵ−６
酵素を完全に失活させた後、同様に増殖阻害効果の検討
を行ったところ、増殖阻害効果の低下は見られたものの
完全に消失しなかった。このことより、本発明の防菌剤
の増殖阻害効果は、細胞壁溶解酵素の溶菌活性のみが働
いているのではなく、ＴＵ−６株の培養液中に含まれる
未知の物質との相乗効果よるものであることが判明し
た。また、本発明の防菌剤は熱に対して、安定であると
いえる。さらに、本発明の防菌剤に対して、公知の方法
であるサルモネラ・チフィムリウム（Salmonella typhi
murium） ＴＡ９８株を用いた変異原性試験（エイムス
テスト）を実施したところ、少なくとも０．１％濃度に
おいては変異原性がないという結果であり、本発明品は
安全であるということが判明した。On the other hand, the bactericide of the present invention is subjected to heat treatment (for 15 minutes in boiling water) to obtain TU-6 contained in the bactericide of the present invention.
After the enzyme was completely inactivated, the growth inhibitory effect was examined in the same manner. As a result, the growth inhibitory effect was reduced but not completely eliminated. From this, the growth inhibitory effect of the bactericidal agent of the present invention is not only due to the lytic activity of the cell wall lysing enzyme, but also due to a synergistic effect with an unknown substance contained in the culture solution of the TU-6 strain. Turned out to be something. Further, it can be said that the antibacterial agent of the present invention is stable against heat. In addition, Salmonella typhimurium (Salmonella typhimurium), which is a known method, is applied to the antibacterial agent of the present invention.
murium) When a mutagenicity test (Ames test) was performed using the TA98 strain, it was found that there was no mutagenicity at least at a concentration of 0.1%, indicating that the product of the present invention was safe. .
【００１０】以上のことより、本発明の防菌剤を用いれ
ば、非常に広範囲の有害細菌の増殖を効果的に抑えるこ
とができ、本発明の防菌剤は、食品や、薬品の保存料と
して幅広く応用でき、特に、清酒醸造において問題とな
っている火落ちの防止や、ビールなどのアルコール飲料
の乳酸菌汚染防止剤としての用途ならびに、虫歯予防剤
としての用途に有用である。As described above, the use of the antibacterial agent of the present invention can effectively suppress the growth of a very wide range of harmful bacteria. In particular, it is useful for preventing burn-off, which is a problem in sake brewing, as an agent for preventing lactic acid bacteria contamination of alcoholic beverages such as beer, and as an agent for preventing tooth decay.
【００１１】[0011]
【実施例】つぎに、実施例を挙げて本発明をさらに具体
的に説明するが、本発明はこれらの実施例に限定される
ものではない。「％」は、アルコール濃度を除いて、い
ずれもｗ／ｖ％、アルコール濃度はｖ／ｖ％である。 実施例１ 本発明の防菌剤（以下、本発明品と称する）の溶菌スペ
クトルの検討 本発明品の溶菌スペクトルを、対象菌に対する溶菌活性
を測定することで検討した。溶菌活性を対象菌の菌体懸
濁液の、濁度の減少と見なし、以下の方法で濁度減少率
を測定した。対象菌菌体（対数増殖期）懸濁液の濁度
（ＯＤ660）が１．０、本発明の防菌剤が０．２５％に
最終的になるようにマキルベイン緩衝液（ｐＨ４．０）
で調製し、２時間反応後の反応液の濁度を測定した。コ
ントロールとして本発明品を除いて同様に調製したもの
の濁度も、同様にして測定した。溶解率は、以下の式に
より求めた。結果を表１に示す。EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. “%” Is w / v% and the alcohol concentration is v / v%, except for the alcohol concentration. Example 1 Examination of the lysis spectrum of the bactericidal agent of the present invention (hereinafter, referred to as the present product) The lytic spectrum of the present product was examined by measuring the lytic activity against the target bacterium. The lytic activity was regarded as a decrease in turbidity of the cell suspension of the target bacterium, and the turbidity decrease rate was measured by the following method. Macilbain buffer (pH 4.0) so that the turbidity (OD 660 ) of the target bacterial cell (logarithmic growth phase) suspension is 1.0 and the antibacterial agent of the present invention is finally 0.25%.
And the turbidity of the reaction solution after 2 hours of reaction was measured. As a control, turbidity of a sample similarly prepared except for the product of the present invention was measured in the same manner. The dissolution rate was determined by the following equation. Table 1 shows the results.
【００１２】[0012]
【数１】 (Equation 1)
【００１３】[0013]
【表１】 [Table 1]
【００１４】表１から明らかなように、本発明品は、含
有する細胞壁溶解酵素であるＴＵ−６酵素により、検討
した微生物を溶菌していた。また、グラム陽性細菌（乳
酸菌、火落菌等）のみならず、グラム陰性細菌（大腸
菌、シュウドモナス属等）もよく溶菌し、幅広い溶菌ス
ペクトルを持っていることがわかった。特に、清酒など
の火落ちの原因菌である火落菌、食中毒の原因菌の一つ
であるとされているスタフィロコッカス・オウレウス、
また、う蝕細菌であるストレプトコッカス・ミュータン
スをよく溶解し、火落ち予防、食中毒予防さらに虫歯予
防への有用性が示された。As is apparent from Table 1, the microorganism of the present invention lysed the microorganisms studied by TU-6 enzyme which is a cell wall lysing enzyme contained therein. In addition, it was found that not only gram-positive bacteria (lactic acid bacteria, fire-killed bacteria, etc.) but also gram-negative bacteria (Escherichia coli, Pseudomonas sp., Etc.) lysed well and had a broad lysis spectrum. In particular, Saccharomyces oleus, which is said to be one of the causative bacteria of food poisoning, which is the causative fungus of burns such as sake,
In addition, Streptococcus mutans, which is a cariogenic bacterium, was well dissolved, and was shown to be useful for preventing burnout, preventing food poisoning, and preventing tooth decay.
【００１５】実施例２ 本発明品のアルコール存在下における溶菌活性の検討 本発明品のアルコール存在下での溶菌活性を検討するた
めに、実施例１の反応系に、アルコール（エタノール）
を１５％存在させた場合と、存在させなかった場合の乳
酸菌および火落菌の溶解率を検討し、比較した。結果を
表２に示す。Example 2 Examination of the bacteriolytic activity of the product of the present invention in the presence of alcohol In order to examine the lytic activity of the product of the present invention in the presence of alcohol, the reaction system of Example 1 was added to alcohol (ethanol).
The lysis rates of lactic acid bacteria and burnt bacteria when 15% was present and when they were not present were examined and compared. Table 2 shows the results.
【００１６】[0016]
【表２】 [Table 2]
【００１７】表２より明らかなように、本発明品の溶菌
活性は、エタノールが少なくても、１５％存在していて
も存在していない時と全く遜色がなかった。つまり、本
発明品は、アルコール濃度１５％以下である清酒や、ビ
ールなどのアルコール飲料に直接作用させても、溶菌活
性は十分に効果が有るということが分かった。As is evident from Table 2, the lytic activity of the product of the present invention was not inferior to the case where ethanol was present in a small amount, even in the presence of 15%. In other words, it was found that the bacteriolytic activity of the product of the present invention was sufficiently effective even when applied directly to alcoholic beverages such as sake or beer having an alcohol concentration of 15% or less.
【００１８】実施例３ 本発明品を用いた黄色ブドウ球菌（スタフィロコッカス
・オウレウス）の増殖阻害試験 濾過滅菌した本発明品の水溶液を、オートクレーブ済み
の一般細菌用培地であるブレインハートインヒュージョ
ンブイヨン「ニッスイ」（日水製薬（株）製）に最終濃
度０．２５％になるように添加したものと、本発明品の
水溶液の代わりに滅菌した蒸留水を加えたものに、１白
金耳の食中毒の原因菌である黄色ブドウ球菌（スタフィ
ロコッカス・オウレウス）を植菌し、３７℃で２４時
間、振盪培養したときの濁度（ＯＤ660）の経時変化を
比較した。比較として卵白リゾチーム（シグマ社製）を
最終濃度０．２５％となるように、同様に添加した場合
も行った。結果を図１に示す。例えば、吸光度変化が起
こるまでは、静菌効果があったとすると、図１から明ら
かなように、無添加区分と比較して、卵白リゾチームを
培地に０．２５％になるように添加した場合、約２時間
の静菌効果がみられ、本発明品を同量添加した場合にお
いては、約６時間の静菌効果があった。したがって、本
発明品を食品などに作用させれば、食中毒の原因である
細菌の増殖を抑えることができる。またその効果は、防
腐剤として以前から用いられている卵白リゾチームと比
較してはるかに高かった。Example 3 Growth Inhibition Test of Staphylococcus aureus Using the Product of the Invention The aqueous solution of the product of the present invention, which was sterilized by filtration, was used as an autoclaved Brain Heart Infusion Bouillon, a medium for general bacteria. "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) was added to a final concentration of 0.25%, and one obtained by adding sterilized distilled water in place of the aqueous solution of the product of the present invention was used. Staphylococcus aureus, the causative bacterium of food poisoning, was inoculated, and the changes over time in turbidity (OD 660 ) when cultured with shaking at 37 ° C. for 24 hours were compared. For comparison, a case where egg white lysozyme (manufactured by Sigma) was similarly added to a final concentration of 0.25% was performed. The results are shown in FIG. For example, assuming that there is a bacteriostatic effect until the change in absorbance occurs, as apparent from FIG. 1, when egg white lysozyme is added to the medium at 0.25% as compared to the non-added group, A bacteriostatic effect was observed for about 2 hours. When the same amount of the product of the present invention was added, a bacteriostatic effect was observed for about 6 hours. Therefore, when the product of the present invention is applied to foods and the like, the growth of bacteria that cause food poisoning can be suppressed. The effect was much higher than that of lysozyme, which has been used as a preservative.
【００１９】実施例４ 本発明品を用いた火落菌増殖阻害試験 濾過滅菌した本発明品の水溶液を、オートクレーブ済み
のＳＩ培地（１％酵母エキス、０．５％ポリペプトン、
２．５％グルコース、１％酢酸ナトリウム、０．０１％
硫酸マグネシウム、０．０００２５％硫酸マンガン、
０．０００２５％硫酸第一鉄、０．００５％メバロン
酸、１０％エタノール、ｐＨ５．１）に最終濃度０．２
５％、０．１２５％および、０．０２５％になるように
添加したものと、本発明品の水溶液の代わりに滅菌した
蒸留水を加えたものに、１白金耳の火落菌（Ｓ２４）を
植菌し、３０℃で１２０時間、静置培養したときの濁度
（ＯＤ660）の経時変化を比較した。比較として卵白リ
ゾチーム（シグマ社製）を最終濃度０．２５％となるよ
うに、同様に添加した場合も行った。結果を図２に示
す。図２から明らかなように、無添加区分と比較して、
本発明品を培地に添加した区分はいずれの場合において
も火落菌Ｓ２４の増殖が全く見られず、顕著な火落菌に
対する増殖阻害効果があった。一方、卵白リゾチームに
は、ほとんど火落菌への増殖阻害効果はみられなかっ
た。また、他の火落菌に関しても同様の検討を行った
が、いずれの場合においても同様の結果であった。この
本検討は、１０％エタノールの存在下で行っており、本
発明品の増殖阻害効果は少なくとも１０％エタノールの
存在下においても有効であるということが判明した。し
たがって、本発明品を作用させることによって、清酒や
ビールをはじめとするアルコール飲料の火落ちおよび乳
酸菌による雑菌汚染を防止できる。Example 4 Bacterial growth inhibition test using the product of the present invention An aqueous solution of the product of the present invention, which was sterilized by filtration, was mixed with an autoclaved SI medium (1% yeast extract, 0.5% polypeptone,
2.5% glucose, 1% sodium acetate, 0.01%
Magnesium sulfate, 0.00025% manganese sulfate,
0.00025% ferrous sulfate, 0.005% mevalonic acid, 10% ethanol, pH 5.1) to a final concentration of 0.2
5%, 0.125%, and 0.025% were added to the solution, and sterilized distilled water was added instead of the aqueous solution of the product of the present invention. The time-dependent changes in turbidity (OD 660 ) when the cells were inoculated and cultured at 30 ° C. for 120 hours were compared. For comparison, a case where egg white lysozyme (manufactured by Sigma) was similarly added to a final concentration of 0.25% was performed. The results are shown in FIG. As is clear from FIG. 2, as compared with the non-added category,
In all the cases where the product of the present invention was added to the culture medium, no growth of H24 was observed, and there was a remarkable growth inhibitory effect on H12. On the other hand, egg white lysozyme had almost no growth inhibitory effect on Hiochi bacteria. In addition, the same examination was performed on other fire-killed bacteria, and the same result was obtained in each case. This study was performed in the presence of 10% ethanol, and it was found that the growth inhibitory effect of the product of the present invention was effective even in the presence of at least 10% ethanol. Therefore, by applying the product of the present invention, it is possible to prevent burnout of alcoholic beverages such as sake and beer and contamination of various bacteria by lactic acid bacteria.
【００２０】実施例５ 本発明品を用いたう蝕細菌（ストレプトコッカス・ミュ
ータンス）の増殖阻害試験 濾過滅菌した本発明品の水溶液を、オートクレーブ済み
の一般細菌用培地であるブレインハートインヒュージョ
ンブイヨン「ニッスイ」（日水製薬（株）製）に最終濃
度０．２５％、０．１２５％および、０．０２５％にな
るように添加したものと、本発明品の水溶液の代わりに
滅菌した蒸留水を加えたものに、１白金耳の虫歯の原因
菌であるう蝕細菌（ストレプトコッカス・ミュータン
ス）を植菌し、３７℃で２４時間、静置培養したときの
濁度（ＯＤ660）の経時変化を比較した。比較として卵
白リゾチーム（シグマ社製）を最終濃度０．２５％とな
るように、同様に添加した場合も行った。結果を図３に
示す。図３から明らかなように、無添加区分と比較し
て、本発明品を培地に添加した区分はいずれの場合にお
いても、う蝕細菌（ストレプトコッカス・ミュータン
ス）の増殖が全く見られず、顕著なう蝕細菌に対する増
殖阻害効果があった。一方、卵白リゾチームには、全く
う蝕細菌への増殖阻害効果はみられなかった。したがっ
て、本発明品を、例えば、ねり歯磨き、粉状歯磨き、マ
ウスウオッシュ、入れ歯洗浄剤等に混合することによっ
て、虫歯の原因の一つであるう蝕細菌の増殖を抑え、虫
歯予防剤として利用できるということが分かった。Example 5 Growth Inhibition Test of Dental Caries Bacteria (Streptococcus mutans) Using the Product of the Present Invention An aqueous solution of the product of the present invention, which had been sterilized by filtration, was used as an autoclaved Brain Heart Infusion Bouillon, a medium for general bacteria. "Nissui" (manufactured by Nissui Pharmaceutical Co., Ltd.) added to final concentrations of 0.25%, 0.125% and 0.025%, and sterilized distilled water instead of the aqueous solution of the product of the present invention. To which decayed caries bacteria (Streptococcus mutans), which is the causative agent of the caries of one platinum loop, were inoculated and incubated for 24 hours at 37 ° C. for 24 hours, followed by turbidity (OD 660 ). The changes were compared. For comparison, a case where egg white lysozyme (manufactured by Sigma) was similarly added to a final concentration of 0.25% was performed. The results are shown in FIG. As is clear from FIG. 3, the growth of the carious bacteria (Streptococcus mutans) was not observed at all in the case where the product of the present invention was added to the medium, and the growth was remarkable in any case as compared with the non-added case. It had a growth inhibitory effect on cariogenic bacteria. On the other hand, egg white lysozyme did not show any growth inhibitory effect on cariogenic bacteria. Therefore, by mixing the product of the present invention with toothpaste, powdered toothpaste, mouthwash, denture cleanser, etc., it suppresses the growth of dental caries, which is one of the causes of tooth decay, and is used as an anti-caries agent. I knew that I could do it.
【００２１】実施例６ 本発明品の熱に対する感受性の検討 実施例３、実施例４および実施例５と同様にして、本発
明品の最終濃度が０．２５％になる場合において、培地
に添加する前に加熱処理（沸騰水中、１５分間）したも
のと、していないものとのそれぞれの菌体に対する増殖
抑制効果を検討し、比較した。結果を図４、図５および
図６に示す。本実施例は、本発明品の中に存在するＴＵ
−６酵素の溶菌活性を熱によって完全に失活させて、本
発明品の微生物の増殖阻害効果がＴＵ−６酵素単独によ
るものか、もしくはＴＵ−６酵素とは全く別の微生物に
対する増殖阻害活性を有する物質によるものかを明らか
にするために行ったものである。図４から明らかなよう
に、加熱処理を行って酵素を失活させても３時間程度の
静菌効果があったことより、本発明品の黄色ブドウ球菌
に対する増殖阻害効果は、ＴＵ−６酵素だけでなく他の
増殖阻害効果を持った物質も効いているということが分
かった。また、図５の火落菌（Ｓ２４）の場合において
は、加熱処理の有無に関わらず、少なくとも１２０時間
は完全に火落菌の増殖を阻害しており、本発明品にＴＵ
−６酵素以外の微生物の増殖阻害物質が存在することが
分かった。さらに、図６のう蝕細菌の場合においても、
加熱によってある程度の増殖阻害効果の低下が見られる
ものの、完全に効果を失わないという結果を得た。以上
の結果により、本発明品の増殖阻害効果は、その対象と
なる菌株によって多少異なるが、細胞壁溶解酵素の溶菌
活性のみが働いているのではなく、ＴＵ−６株の培養液
中に含まれる未知の物質との相乗効果よるものであるこ
とが判明した。また、この結果から、本発明の防菌剤は
熱に対して、安定であるといえる。Example 6 Examination of heat sensitivity of the product of the present invention In the same manner as in Examples 3, 4 and 5, when the final concentration of the product of the present invention was 0.25%, it was added to the medium. Before and after the heat treatment (in boiling water for 15 minutes), the effect of inhibiting the growth of the respective cells was examined and compared. The results are shown in FIGS. 4, 5 and 6. This embodiment is based on the TU that exists in the product of the present invention.
The bacteriolytic activity of the -6 enzyme is completely inactivated by heat, and the growth inhibitory effect of the microorganism of the present invention is due to the TU-6 enzyme alone or the growth inhibitory activity against a microorganism completely different from the TU-6 enzyme. The purpose of this study was to clarify whether or not the substance had the following. As is clear from FIG. 4, even when the enzyme was inactivated by heat treatment, there was a bacteriostatic effect for about 3 hours. Not only that, but other substances having a growth inhibitory effect were also found to be effective. In addition, in the case of the fire-killed bacteria (S24) in FIG. 5, regardless of the presence or absence of the heat treatment, the growth of the fire-killed bacteria was completely inhibited for at least 120 hours.
It was found that there were microorganism growth inhibitors other than the -6 enzyme. Furthermore, in the case of the carious bacteria of FIG.
Although the growth inhibition effect was reduced to some extent by heating, the result was obtained that the effect was not completely lost. From the above results, the growth inhibitory effect of the product of the present invention is slightly different depending on the target strain, but not only the lytic activity of the cell wall lytic enzyme works but is contained in the culture solution of the TU-6 strain. It turned out to be due to a synergistic effect with unknown substances. From the results, it can be said that the antibacterial agent of the present invention is stable against heat.
【００２２】[0022]
【発明の効果】本発明によれば、従来の卵白リゾチーム
を用いた防菌剤とは異なり、一つの防菌剤で、火落菌の
増殖を抑えたり、食中毒の原因菌である黄色ブドウ球菌
（スタフィロコッカス・オウレウス）の増殖を抑えた
り、またう蝕細菌であるストレプトコッカス・ミュータ
ンスの増殖を抑えたりと幅広い用途に対応した安全か
つ、熱に安定な防菌剤を提供できる。According to the present invention, unlike a conventional antibacterial agent using egg white lysozyme, a single antibacterial agent can suppress the growth of fire-killed bacteria and can be used as a causative agent of food poisoning. Thus, it is possible to provide a safe and heat-stable antibacterial agent suitable for a wide range of uses, such as suppressing the growth of Staphylococcus aureus) and suppressing the growth of Streptococcus mutans, which is a cariogenic bacterium.
【図１】 本発明品の黄色ブドウ球菌（スタフィロコッ
カス・オウレウス）に対する増殖阻害効果を検討した結
果を示す図である。FIG. 1 is a view showing the results of examining the growth inhibitory effect of the product of the present invention on Staphylococcus aureus (Staphylococcus aureus).
【図２】 本発明品の火落菌（Ｓ２４）に対する増殖阻
害効果を検討した結果を示す図である。FIG. 2 is a diagram showing the results of examining the growth inhibitory effect of the product of the present invention on fire-killed bacteria (S24).
【図３】 本発明品のう蝕細菌（ストレプトコッカス・
ミュータンス）に対する増殖阻害効果を検討した結果を
示す図である。FIG. 3 shows carious bacteria (Streptococcus and
FIG. 9 is a view showing the results of examining the growth inhibitory effect on C. mutans).
【図４】 本発明品の熱に対する感受性を黄色ブドウ球
菌（スタフィロコッカス・オウレウス）について検討し
た結果を示す図である。FIG. 4 is a diagram showing the results of examining the sensitivity of the product of the present invention to heat with respect to Staphylococcus aureus (Staphylococcus aureus).
【図５】 本発明品の熱に対する感受性を火落菌（Ｓ２
４）について検討した結果を示す図である。FIG. 5 shows that the sensitivity of the product of the present invention to heat is determined by the fire-killed bacteria (S2
It is a figure showing the result of having examined about 4).
【図６】 本発明品の熱に対する感受性をう蝕細菌（ス
トレプトコッカス・ミュータンス）について検討した結
果を示す図である。FIG. 6 is a graph showing the results of an investigation of the sensitivity of the product of the present invention to heat for carious bacteria (Streptococcus mutans).
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ａ６１Ｐ 1/02 Ａ６１Ｐ 1/02 ４Ｃ０８７ 31/04 31/04 Ｃ１２Ｈ 1/00 Ｃ１２Ｈ 1/00 Ｃ１２Ｐ 21/00 Ｃ１２Ｐ 21/00 Ａ //(Ｃ１２Ｎ 1/20 (Ｃ１２Ｎ 1/20 Ａ Ｃ１２Ｒ 1:465） Ｃ１２Ｒ 1:465） (Ｃ１２Ｐ 21/00 (Ｃ１２Ｐ 21/00 Ａ Ｃ１２Ｒ 1:465） Ｃ１２Ｒ 1:465） (72)発明者 尾関 健二 兵庫県西宮市今津出在家町４番９号 大関 株式会社総合研究所内 (72)発明者 濱地 正昭 兵庫県西宮市今津出在家町４番９号 大関 株式会社総合研究所内 (72)発明者 熊谷 知栄子 兵庫県西宮市今津出在家町４番９号 大関 株式会社総合研究所内 Ｆターム(参考） 4B021 MC01 MK06 MK07 MP01 MP02 4B028 AC10 AG04 AP29 AS18 4B064 AH19 CA04 CA19 CC24 DA01 DA10 4B065 AA50X AB01 AC14 BD14 CA27 CA41 CA42 CA44 4C083 AA031 AA032 CC41 EE32 FF01 4C087 BC23 ZA67 ZB35 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) A61P 1/02 A61P 1/02 4C087 31/04 31/04 C12H 1/00 C12H 1/00 C12P 21/00 C12P 21/00 A // (C12N 1/20 (C12N 1/20 A C12R 1: 465) C12R 1: 465) (C12P 21/00 (C12P 21/00 A C12R 1: 465) C12R 1: 465) ( 72) Inventor Kenji Ozeki 4-9, Imazu, Iizazu-cho, Nishinomiya City, Hyogo Prefecture, Ozeki Research Institute (72) Inventor Masaaki Hamachi 4-9, Imazu, Iizazu, Iizazu-cho, Nishinomiya City, Hyogo Prefecture, Ozeki Co., Ltd. (72) Inventor Chieko Kumagai 4-9, Imazu, Iizazu-cho, Nishinomiya-shi, Hyogo F-term in the Ozeki Research Institute (reference) 4B021 MC01 MK06 MK07 MP01 MP02 4B028 AC10 AG04 AP29 AS1 8 4B064 AH19 CA04 CA19 CC24 DA01 DA10 4B065 AA50X AB01 AC14 BD14 CA27 CA41 CA42 CA44 4C083 AA031 AA032 CC41 EE32 FF01 4C087 BC23 ZA67 ZB35
Claims (4)
reptmyces fulvissimus）ＴＵ−６株（ＦＥＲＭ Ｐ−
１６３４７）またはその変異体もしくは遺伝子組換体の
培養物を有効成分とすることを特徴とする防菌剤。[Claim 1] Streptomyces fulvicimus (St
reptmyces fulvissimus) TU-6 strain (FERM P-
16347) or a bactericidal agent comprising, as an active ingredient, a culture of a mutant or a recombinant thereof.
剤。2. The antibacterial agent according to claim 1, which is a food preservative.
項１記載の防菌剤。3. The antibacterial agent according to claim 1, which is an antiseptic for alcoholic beverages.
剤。4. The antibacterial agent according to claim 1, which is a caries preventive agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000194327A JP2002000261A (en) | 2000-06-28 | 2000-06-28 | Antibacterial agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000194327A JP2002000261A (en) | 2000-06-28 | 2000-06-28 | Antibacterial agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2002000261A true JP2002000261A (en) | 2002-01-08 |
Family
ID=18693176
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2000194327A Pending JP2002000261A (en) | 2000-06-28 | 2000-06-28 | Antibacterial agent |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012028759A3 (en) * | 2010-08-31 | 2012-08-02 | Centro Superior De Investigación En Salud Pública (Csisp) | Anticaries compositions and probiotics/prebiotics |
| CN102978144A (en) * | 2012-12-24 | 2013-03-20 | 湖南农业大学 | Streptomyces fulvissimus |
| CN107619803A (en) * | 2017-09-19 | 2018-01-23 | 浙江大学 | A kind of preparation of the sour and dark yellow strepto- ketone of dark yellow strepto- and its medical usage |
| KR102291267B1 (en) * | 2020-03-04 | 2021-08-19 | 임형엽 | An air purification device using the inhabitation body of the microorganism for inhibiting growth of Staphylococcus aureus |
-
2000
- 2000-06-28 JP JP2000194327A patent/JP2002000261A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012028759A3 (en) * | 2010-08-31 | 2012-08-02 | Centro Superior De Investigación En Salud Pública (Csisp) | Anticaries compositions and probiotics/prebiotics |
| CN103119153A (en) * | 2010-08-31 | 2013-05-22 | 公共健康高级研究中心 | Anticaries compositions and probiotics/prebiotics |
| JP2013543374A (en) * | 2010-08-31 | 2013-12-05 | セントロ スペリオール デ インベスティガシオン エン サルード プブリカ （セ・エセ・イ・エセ・ペ） | Anti-caries composition and probiotic / prebiotic |
| CN103119153B (en) * | 2010-08-31 | 2016-03-23 | 公共健康高级研究中心 | Anti-dental caries composition and probiotic bacterium/probiotics |
| US9629883B2 (en) | 2010-08-31 | 2017-04-25 | Centro Superior De Investigación En Salud Pública (Csisp) | Anticaries compositions and probiotics/prebiotics |
| KR101739802B1 (en) | 2010-08-31 | 2017-05-25 | 센트로 수페리오 드 인베스티가시온 엔 살루드 퍼블리카 (씨에스아이에스피) | Anti-caries compositions and probiotics/prebiotics |
| CN102978144A (en) * | 2012-12-24 | 2013-03-20 | 湖南农业大学 | Streptomyces fulvissimus |
| CN102978144B (en) * | 2012-12-24 | 2013-12-04 | 湖南农业大学 | Streptomyces fulvissimus |
| CN107619803A (en) * | 2017-09-19 | 2018-01-23 | 浙江大学 | A kind of preparation of the sour and dark yellow strepto- ketone of dark yellow strepto- and its medical usage |
| CN107619803B (en) * | 2017-09-19 | 2020-03-03 | 浙江大学 | Preparation and medical application of streptomyces xanthioides acid and streptomyces xanthione |
| KR102291267B1 (en) * | 2020-03-04 | 2021-08-19 | 임형엽 | An air purification device using the inhabitation body of the microorganism for inhibiting growth of Staphylococcus aureus |
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