JP2007189947A - Inhibitor of hydrogen sulfide-producing bacterium and method for inhibiting hydrogen sulfide-producing bacterium - Google Patents

Inhibitor of hydrogen sulfide-producing bacterium and method for inhibiting hydrogen sulfide-producing bacterium Download PDF

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JP2007189947A
JP2007189947A JP2006011250A JP2006011250A JP2007189947A JP 2007189947 A JP2007189947 A JP 2007189947A JP 2006011250 A JP2006011250 A JP 2006011250A JP 2006011250 A JP2006011250 A JP 2006011250A JP 2007189947 A JP2007189947 A JP 2007189947A
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hydrogen sulfide
bacteria
butyric acid
producing bacteria
producing
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Kunitomo Watanabe
邦友 渡邉
Wataru Takizawa
渉 滝澤
So Takizawa
爽 滝澤
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ACE BIO PRODUCT KK
Gifu University NUC
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ACE BIO PRODUCT KK
Gifu University NUC
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Abstract

<P>PROBLEM TO BE SOLVED: To provide an inhibitor of hydrogen sulfide-producing bacteria safe even by administration thereof to humans or livestock and effectively inhibiting the hydrogen sulfide-producing bacteria without being accompanied with adverse effects and to provide a method for inhibiting the hydrogen sulfide-producing bacteria. <P>SOLUTION: The inhibitor of the hydrogen sulfide-producing bacteria is characterized as comprising Clostridium butyricum or adding the Clostridium butyricum to a cultured extract of the Clostridium butyricum. The method for inhibiting the hydrogen sulfide-producing bacteria is characterized as comprising a step of adding the Clostridium butyricum or adding the cultured extract of the Clostridium butyricum to which the Clostridium butyricum is added to a site where the hydrogen sulfide-producing bacteria exist. Thereby, the inhibitor is safe even by administration thereof to the humans or livestock and effectively inhibits the hydrogen sulfide-producing bacteria without being accompanied with the adverse effects. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、硫化水素産生菌を効果的に抑制する硫化水素産生菌抑制剤及び硫化水素産生菌抑制方法に関する。   The present invention relates to a hydrogen sulfide-producing bacteria inhibitor and a method for inhibiting hydrogen sulfide-producing bacteria that effectively inhibit hydrogen sulfide-producing bacteria.

硫化水素は、腐敗した卵に似た特徴的な強い刺激臭があり、目、皮膚、粘膜を刺激する有毒な物質である。このため、悪臭防止法に基づく特定悪臭物質のひとつに指定されていて、問題視されている。硫化水素は、石油化学工業の過程において産生するほか、硫酸還元細菌によって下水処理場、ごみ処理場などでも発生する。このとき、硫酸還元細菌は、水環境中の底泥などのような嫌気的な条件下で、硫酸イオンが還元して、硫化水素を産生する。   Hydrogen sulfide has a characteristic strong pungent odor similar to spoiled eggs and is a toxic substance that irritate eyes, skin and mucous membranes. For this reason, it is designated as one of the specific malodorous substances based on the Malodor Control Law and is regarded as a problem. Hydrogen sulfide is produced in the process of the petrochemical industry, and is also generated in sewage treatment plants and waste treatment plants by sulfate-reducing bacteria. At this time, sulfate-reducing bacteria produce hydrogen sulfide by reducing sulfate ions under anaerobic conditions such as bottom mud in the water environment.

近年、このような硫酸還元細菌による悪臭や腐食などのトラブルを抑制する方法として、微生物を用いる方法が開発されている。特許文献１では、微生物の培養物を硫化水素が発生する汚泥等に投入して増殖させることにより、硫酸還元細菌による硫化水素の発生を防止することが開示されている。   In recent years, a method using microorganisms has been developed as a method for suppressing troubles such as malodor and corrosion caused by sulfate-reducing bacteria. Patent Document 1 discloses that the generation of hydrogen sulfide by sulfate-reducing bacteria is prevented by introducing a microorganism culture into sludge or the like that generates hydrogen sulfide and allowing it to proliferate.

ところで、硫化水素を産生する菌（硫化水素産生菌）は、硫酸還元細菌とアミノ酸発酵細菌からなる。硫酸還元細菌は、上記の下水処理場、ごみ処理場といった産業施設ばかりでなく、人や家畜の腸内にもその存在が確認されている。一方、腸内では、含硫アミノ酸から硫化水素を産生するアミノ酸発酵細菌が存在することが知られている。   By the way, bacteria producing hydrogen sulfide (hydrogen sulfide producing bacteria) are composed of sulfate-reducing bacteria and amino acid-fermenting bacteria. The presence of sulfate-reducing bacteria has been confirmed not only in industrial facilities such as the above-mentioned sewage treatment plants and waste treatment plants, but also in the intestines of humans and livestock. On the other hand, it is known that there are amino acid-fermenting bacteria that produce hydrogen sulfide from sulfur-containing amino acids in the intestine.

近年の研究により、硫化水素は、人や家畜に対して毒性が強く、口腔内及び消化管内で発生した硫化水素が消化管粘膜細胞の代謝を阻害するなどして傷害する可能性が示唆されている。近年増加傾向にある難病である炎症性腸疾患の患者は、健康人や炎症性腸疾患以外の腸疾患患者と比較して、ある種の硫化水素産生菌が有意に分離されることが報告されている。また、硫酸還元菌が産生する硫化水素は、潰瘍性大腸炎の一因であることが示唆されている（非特許文献１）。腸管内でこれらの硫化水素産生菌が産生した硫化水素は、腸管のクリプトに存在する細胞のロダナーゼ（ｒｈｏｄａｎａｓｅ）により解毒されることが明らかとなっている。そのため、通常の場合にはその有害作用が抑制されていると考えられる。しかし、食生活などの変化により腸管内で過剰に産生された硫化水素は、人の腸管粘膜に障害作用、有害作用を示すと考えられる。また、なんらかの原因で、人の粘膜、粘膜下における硫化水素の解毒作用が低下した個体においては、通常量の硫化水素によっても人に有害作用を示すようになると考えられる。   Recent research suggests that hydrogen sulfide is highly toxic to humans and livestock, and that hydrogen sulfide generated in the oral cavity and gastrointestinal tract may be damaged by inhibiting the metabolism of gastrointestinal mucosal cells. Yes. It has been reported that patients with inflammatory bowel disease, an intractable disease that has been increasing in recent years, can significantly isolate certain types of hydrogen sulfide-producing bacteria compared to healthy people and patients with bowel diseases other than inflammatory bowel disease. ing. Moreover, it has been suggested that hydrogen sulfide produced by sulfate-reducing bacteria contributes to ulcerative colitis (Non-patent Document 1). It has been clarified that the hydrogen sulfide produced by these hydrogen sulfide-producing bacteria in the intestinal tract is detoxified by the cellular rhodanase present in the intestinal crypt. Therefore, it is considered that the harmful effect is suppressed in normal cases. However, hydrogen sulfide produced excessively in the intestinal tract due to changes in eating habits and the like is considered to have a damaging or harmful effect on the human intestinal mucosa. In addition, in an individual whose detoxification action of hydrogen sulfide under human mucous membrane or submucosa has been reduced for some reason, it is considered that even a normal amount of hydrogen sulfide may cause harmful effects on human beings.

これらのことより、消化管内の環境を良好に保つため又は炎症性粘膜疾患の治療をするため、または、硫化水素産生菌起因の口内炎の粘膜を修復するため、生体内の硫化水素産生菌の硫化水素産生を抑制する方法が望まれている。
特開２００４−３４４７５７ William et al., Gastroenterology, 104, 802-809 (1993) Based on these facts, sulfurization of hydrogen sulfide-producing bacteria in vivo is necessary to maintain a good environment in the digestive tract, to treat inflammatory mucosal diseases, or to repair the mucosa of stomatitis caused by hydrogen sulfide-producing bacteria. A method for suppressing hydrogen production is desired.
JP 2004-344757 A William et al., Gastroenterology, 104, 802-809 (1993)

上述したように、生体内の硫化水素の産生を抑制することは、消化管内の環境を良好に保つため、又は炎症性粘膜疾患の治療のため、または、硫化水素産生菌起因の口内炎の粘膜を修復するため、有効な手段の一つである。しかし、この抑制のために抗生物質を用いた場合、長期投与時の副作用など安全性や耐性菌の出現の問題がある。また、特許文献１に記載されている硫化水素の発生を抑制する微生物は、有害菌から構成されており、動物体内において用いることはできない。このため、生体内の硫化水素産生菌を安全かつ効果的に抑制できる方法は未だ知られていない。そこで本発明は、人や家畜に投与しても安全であるとともに、副作用をともなうことなく、生体内の硫化水素産生菌を効果的に抑制できる方法を提供することを目的とする。   As described above, suppressing the production of hydrogen sulfide in the living body is effective for maintaining the environment in the digestive tract, for treating inflammatory mucosal diseases, or for the mucosa of stomatitis caused by hydrogen sulfide producing bacteria. It is one of the effective means for repairing. However, when antibiotics are used for this suppression, there are problems of safety and appearance of resistant bacteria such as side effects during long-term administration. Moreover, the microorganisms which suppress generation | occurrence | production of hydrogen sulfide described in patent document 1 are comprised from harmful bacteria, and cannot be used in an animal body. For this reason, the method which can suppress the hydrogen sulfide producing microbe in the living body safely and effectively is not yet known. Therefore, an object of the present invention is to provide a method capable of effectively suppressing hydrogen sulfide-producing bacteria in a living body without causing side effects while being safe even if administered to humans and livestock.

上記課題を鑑みて鋭意検討した結果、酪酸菌（Clostridium butyricum）は、腸内細菌の中の種々の硫化水素産生菌を抑制する働きを有することが明らかになった。また、酪酸菌は、すでに整腸剤として人や動物に利用され、永年にわたり、安全に利用されている実績があるプロバイオテイックスの一つであり、本発明の目的にかなう。このことより、酪酸菌を用いて、腸内細菌の中の種々の硫化水素産生菌の硫化水素産生を効率よく抑制するという本発明を完成するに至った。すなわち、本発明は、以下の通りである。   As a result of intensive studies in view of the above problems, it has been clarified that butyric acid bacteria (Clostridium butyricum) have a function of suppressing various hydrogen sulfide-producing bacteria in intestinal bacteria. In addition, butyric acid bacteria are one of the probiotics that have been used in humans and animals as intestinal regulating agents and have been used safely for many years, and meet the purpose of the present invention. This has led to the completion of the present invention using butyric acid bacteria to efficiently suppress the production of hydrogen sulfide by various hydrogen sulfide-producing bacteria in the intestinal bacteria. That is, the present invention is as follows.

本発明の請求項１は、酪酸菌（Clostridium butyricum）を含有することを特徴とする硫化水素産生菌抑制剤である。   Claim 1 of the present invention is a hydrogen sulfide-producing bacterium inhibitor comprising a butyric acid bacterium (Clostridium butyricum).

本発明の請求項２は、酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたことを特徴とする硫化水素産生菌抑制剤である。   Claim 2 of the present invention is a hydrogen sulfide-producing bacterium inhibitor characterized by adding butyric acid bacteria to a culture extract of butyric acid bacteria (Clostridium butyricum).

本発明の請求項３は、酪酸菌（Clostridium butyricum）を含有することを特徴とする生体内硫化水素産生菌抑制剤である。   Claim 3 of the present invention is an in vivo hydrogen sulfide-producing bacterium inhibitor comprising a butyric acid bacterium (Clostridium butyricum).

本発明の請求項４は、酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたことを特徴とする生体内硫化水素産生菌抑制剤である。   Claim 4 of the present invention is an in vivo suppressor for hydrogen sulfide-producing bacteria characterized by adding butyric acid bacteria to a culture extract of butyric acid bacteria (Clostridium butyricum).

本発明の請求項５は、酪酸菌（Clostridium butyricum）を含有することを特徴とする生体内硫化水素産生菌抑制飲料である。   Claim 5 of the present invention is an in-vivo hydrogen sulfide-producing bacteria-suppressing beverage characterized in that it contains butyric acid bacteria (Clostridium butyricum).

本発明の請求項６は、酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたことを特徴とする生体内硫化水素産生菌抑制飲料である。   Claim 6 of the present invention is an in-vivo hydrogen sulfide-producing bacteria-suppressing beverage characterized in that butyric acid bacteria are added to a cultured extract of butyric acid bacteria (Clostridium butyricum).

本発明の請求項７は、酪酸菌（Clostridium butyricum）を硫化水素産生菌が存在する部位へ添加する工程を含むことを特徴とする硫化水素産生菌抑制方法である。   Claim 7 of the present invention is a method for inhibiting hydrogen sulfide-producing bacteria, comprising a step of adding butyric acid bacteria (Clostridium butyricum) to a site where hydrogen sulfide-producing bacteria are present.

本発明の請求項８は、酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたものを、硫化水素産生菌が存在する部位へ添加する工程を含むことを特徴とする硫化水素産生菌抑制方法である。   Claim 8 of the present invention comprises a step of adding butyric acid bacteria to a culture extract of butyric acid bacteria (Clostridium butyricum) to a site where hydrogen sulfide-producing bacteria are present, and a method for inhibiting hydrogen sulfide-producing bacteria It is.

本発明の請求項９は、酪酸菌（Clostridium butyricum）を人以外の動物へ投与する工程を含むことを特徴とする生体内硫化水素産生菌抑制方法である。   Claim 9 of the present invention is a method for suppressing in vivo hydrogen sulfide-producing bacteria, comprising a step of administering a butyric acid bacterium (Clostridium butyricum) to an animal other than a human.

本発明の請求項１０は、酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたものを、人以外の動物へ投与する工程を含むことを特徴とする生体内硫化水素産生菌抑制方法である。   Claim 10 of the present invention is a method for inhibiting in vivo hydrogen sulfide-producing bacteria, comprising a step of administering butyric acid bacteria to a cultured extract of Clostridium butyricum to an animal other than a human. .

本発明によれば、人や家畜に投与しても安全であるとともに、副作用をともなうことなく、硫化水素産生菌を効果的に抑制できる。安全であるという特性より、人を含めた動物に投与することが可能であり、生体内硫化水素産生菌を効果的に抑制できる。   According to the present invention, it is safe to administer to humans and livestock, and hydrogen sulfide-producing bacteria can be effectively suppressed without causing side effects. Because of its safety characteristics, it can be administered to animals including humans and can effectively suppress in vivo hydrogen sulfide-producing bacteria.

以下、本発明の実施形態について詳細に説明する。   Hereinafter, embodiments of the present invention will be described in detail.

本発明に用いられる酪酸菌（Clostridium butyricum）（以下、かっこ内は省略する）は、偏性嫌気菌で芽胞を形成するグラム陽性の桿菌である。芽胞の形成により、耐熱性、耐酸性をもち、生菌としての安定性に富んでいる。これらの種に属する菌株としては、一般に市販されているものを容易に入手できる。さらに、これらの種に属する菌株としては、様々な寄託機関や研究機関等で維持されている各種の菌株を使用することもできる。例えば、理化学研究所 微生物系統保存施設（Ｊａｐａｎ Ｃｏｌｌｅｃｔｉｏｎ ｏｆ Ｍｉｃｒｏｏｒｇａｎｉｓｍｓ； ＪＣＭ）からそれらの菌株を入手することができる。本発明の酪酸菌株としては、例えば、Clostridium butyricum NCIB 7423株、Clostridium butyricum M588株またはATCC 19398T株のような菌株を好適に用いることができる。 The butyric acid bacterium (Clostridium butyricum) used in the present invention (hereinafter omitted in parentheses) is a Gram-positive gonococcus that forms spores with obligate anaerobes. Due to the formation of spores, it has heat resistance and acid resistance, and is highly stable as a living microbe. As strains belonging to these species, those generally available on the market can be easily obtained. Furthermore, as strains belonging to these species, various strains maintained in various depository institutions and research institutions can be used. For example, these strains can be obtained from the Japan Collection of Microorganisms (JCM). As the butyric acid strain of the present invention, for example, a strain such as Clostridium butyricum NCIB 7423 strain, Clostridium butyricum M588 strain or ATCC 19398 T strain can be preferably used.

本発明において酪酸培養液とは、酪酸菌を通常、培養する培地例えば、アミノ酸，バレイショデンプン，水等を含む培地で２０〜４２℃、２０〜７２時間培養して得られる培養液のことをいう。   In the present invention, the butyric acid culture solution refers to a culture solution obtained by culturing 20 to 42 ° C. for 20 to 72 hours in a medium usually cultivating butyric acid bacteria, for example, a medium containing amino acids, potato starch, water and the like. .

また、本発明において培養エキスとは、上記培養液を遠心分離等により固液分離して、得られる液部のことをいう。   In the present invention, the culture extract refers to a liquid part obtained by solid-liquid separation of the culture solution by centrifugation or the like.

なお、酪酸菌培養液又は酪酸菌培養エキス中には、代謝産物である酪酸，酢酸，プロピオン酸等の有機酸、アミラーゼ、各種アミノ酸、ビタミンＢ２、ビタミンＢ６、ビタミンＢ１２、ビタミンＣ、及び自己融解した菌体成分等が含まれている。また、酪酸菌培養液又は酪酸菌培養エキスには有機酸として酪酸や酢酸等を含有しているため、特異臭を有する欠点があるが、この臭いを例えば香料でマスキングしてもよい。香料としては、酪酸菌臭のマスキングに有効なものであれば特に制限されないが、食品添加物として用いられるものが好ましい。   In the butyric acid bacteria culture solution or butyric acid bacteria culture extract, metabolites such as organic acids such as butyric acid, acetic acid and propionic acid, amylase, various amino acids, vitamin B2, vitamin B6, vitamin B12, vitamin C, and autolysis Contained microbial cell components and the like. Moreover, since the butyric acid bacteria culture solution or the butyric acid bacteria culture extract contains butyric acid, acetic acid, or the like as an organic acid, there is a drawback of having a specific odor, but this odor may be masked with a fragrance, for example. The fragrance is not particularly limited as long as it is effective for masking the butyric acid odor, but is preferably used as a food additive.

本発明に用いられる酪酸菌は、芽胞形成期のものでも栄養体期のものでもよく、特に限定はされない。酪酸菌は、栄養体、芽胞を含む。栄養体を豊富に含有する酪酸菌は、例えばペプトン，イーストエクストラクト，グルコース，水等を含有するＰＹＧ培地などで、２０〜４２℃、２０〜７２時間培養し、遠心分離等の方法で集菌して得ることができる。また、芽胞の形成に最適な培養液の組成を検討することが主に行われており、そのような最適な炭素源、窒素源等の各種の栄養素を含む液体培地で培養増殖させ芽胞を形成させた後、遠心分離等により芽胞を培養液より分離回収することができる。   The butyric acid bacterium used in the present invention may be in the spore formation stage or in the vegetative stage, and is not particularly limited. Butyric acid bacteria include nutrients and spores. For example, butyric acid bacteria containing abundant nutrients are cultured in a PYG medium containing peptone, yeast extract, glucose, water, etc., and cultured at 20 to 42 ° C. for 20 to 72 hours, and collected by a method such as centrifugation. Can be obtained. In addition, the composition of the optimal culture solution for spore formation is mainly studied, and the spore is formed by culturing and growing in a liquid medium containing various nutrients such as the optimal carbon source and nitrogen source. Then, the spore can be separated and recovered from the culture solution by centrifugation or the like.

本発明の硫化水素産生菌は、硫化水素を産生する菌をさす。硫化水素産生菌は、硫酸還元細菌とアミノ酸発酵細菌からなる。硫酸還元細菌が硫酸塩から硫化水素を産生するのに対して、アミノ酸発酵細菌は、含硫アミノ酸から硫化水素を産生する。硫酸還元細菌としては、Desulfovibrio pigerやDesulfovibrio fairfieldensisを中心とするデスルホビブリオ（Desulfovibrio）属が挙げられる。アミノ酸発酵細菌としては、クロストリジウム（Clostridum）属といった嫌気性細菌が挙げられる。後述の実施例で述べるように、本発明者らにより、生体内の硫化水素産生能の高い細菌として、以下の細菌が確認された。
Clostridium sporogenes (図1.)、Clostridium sordellii (図2.)、Clostridium difficile (図3.)、Clostridium perfringens(図4.)、E.coil(図5.)、Bilophila wadsworthia(図6.)、Sutterella wadsworthensis(図7.)、Desulfovibrio desulfuricans 、Desulfovibrio piger(図8.)、Desulfovibrio fairfieldensis、Campylobacter gracilis(図9.)、Campylobacter rectus、Dialister pneumosintes、Fusobacterium varium、Fusobacterium nucleatum、Fusobacterium necrophorum
実施例では、酪酸菌を含んでいる酪酸菌培養エキスがこれらの各硫化水素産生菌に対して抑制作用を有することを示している。 The hydrogen sulfide-producing bacterium of the present invention refers to a bacterium that produces hydrogen sulfide. Hydrogen sulfide-producing bacteria are composed of sulfate-reducing bacteria and amino acid-fermenting bacteria. While sulfate-reducing bacteria produce hydrogen sulfide from sulfates, amino acid-fermenting bacteria produce hydrogen sulfide from sulfur-containing amino acids. Examples of sulfate-reducing bacteria include the genus Desulfovibrio centered on Desulfovibrio piger and Desulfovibrio fairfieldensis. Examples of amino acid-fermenting bacteria include anaerobic bacteria such as the genus Clostridium. As described in Examples below, the present inventors have confirmed the following bacteria as bacteria having a high ability to produce hydrogen sulfide in vivo.
Clostridium sporogenes (Figure 1.), Clostridium sordellii (Figure 2.), Clostridium difficile (Figure 3.), Clostridium perfringens (Figure 4.), E.coil (Figure 5.), Bilophila wadsworthia (Figure 6.), Sutterella wadsworthensis (Figure 7.), Desulfovibrio desulfuricans, Desulfovibrio piger (Figure 8.), Desulfovibrio fairfieldensis, Campylobacter gracilis (Figure 9.), Campylobacter rectus, Dialister pneumosintes, Fusobacterium varium, Fusobacterium nuclecro, Fusobacterium nuclecro,
In an Example, it has shown that the butyric-acid-bacteria culture extract containing a butyric acid bacterium has an inhibitory action with respect to each of these hydrogen sulfide producing bacteria.

本発明の硫化水素産生菌抑制剤は、酪酸菌を含有することを特徴とする。また、本発明の硫化水素産生菌抑制剤は、酪酸菌培養エキスに酪酸菌を加えることを特徴とする。このとき、酪酸菌培養エキスに、酪酸菌が１０3個／ｍｌ以上含まれていることが好ましい。本発明の硫化水素産生菌抑制剤により、硫化水素産生菌を抑制することができる。上記のように、酪酸菌は、すでに整腸剤として人や動物に利用され、永年にわたり、安全に利用されている実績があるプロバイオテイックスの一つである。さらに、本発明者らは、酪酸菌は、同時に硫化水素を産生しない有用菌として知られているビフィドバクテリア（Bifidobacterium）属や食物繊維の利用に関わるバクテロイド（Bacteroides）属の増殖を抑制しないことを明らかにした（図10）。このため、本発明の硫化水素産生菌抑制剤は、生体内硫化水素産生菌にも効果的に適用することができる。本発明の硫化水素産生菌抑制剤の用途は限定されず、下水処理場、ごみ処理場等の硫化水素産生菌を抑制するために用いてもよい。また、特許文献１に記載されている硫化水素の発生を抑制する微生物は、硫化水素産生菌のうち硫酸還元細菌だけに対する抑制作用を有するのに対して、本発明の硫化水素産生菌抑制剤は、硫酸還元細菌とアミノ酸発酵細菌の両方の硫化水素産生菌に対して抑制作用を有する。 The inhibitor of hydrogen sulfide-producing bacteria of the present invention is characterized by containing butyric acid bacteria. Moreover, the hydrogen sulfide producing bacteria inhibitor of the present invention is characterized by adding butyric acid bacteria to a butyric acid bacteria culture extract. At this time, it is preferable that butyric acid bacteria are contained in the butyric acid bacteria culture extract in an amount of 10 3 / ml or more. Hydrogen sulfide-producing bacteria can be suppressed by the hydrogen sulfide-producing bacteria inhibitor of the present invention. As described above, butyric acid bacteria are one of the probiotics that have been used in humans and animals as intestinal regulating agents and have been used safely for many years. Furthermore, the present inventors have found that butyric acid bacteria do not suppress the growth of the genus Bifidobacterium and the genus Bacteroides that are involved in the use of dietary fiber, which are known as useful bacteria that do not simultaneously produce hydrogen sulfide. (Figure 10). For this reason, the hydrogen sulfide-producing bacteria inhibitor of the present invention can be effectively applied to in vivo hydrogen sulfide-producing bacteria. The use of the hydrogen sulfide-producing bacterium inhibitor of the present invention is not limited, and may be used to suppress hydrogen sulfide-producing bacteria such as sewage treatment plants and waste treatment plants. In addition, the microorganisms described in Patent Document 1 that suppress the generation of hydrogen sulfide have an inhibitory action on only sulfate-reducing bacteria among hydrogen sulfide-producing bacteria, whereas the hydrogen sulfide-producing bacteria inhibitor of the present invention is It has an inhibitory action against hydrogen sulfide-producing bacteria, both sulfate-reducing bacteria and amino acid-fermenting bacteria.

本発明の硫化水素産生菌抑制剤を製剤するには、目的、投与形態、投与対象、最終形態等に応じて、上記のようにして得られた酪酸菌又は酪酸菌培養エキスに酪酸菌を加えたものに、任意成分としてその他の添加剤を配合し、例えば錠剤，散剤，顆粒剤，カプセル剤，丸剤，トローチ剤，液剤，エキス剤等の任意の形状に調製することができる。これらは当業者の通常の方法に従って調製することができる。例えば、硫化水素産生菌抑制剤が錠剤である場合には、酪酸菌又は酪酸菌培養エキスに酪酸菌を加えたものに所定の配合物をよく混合し、これを打錠機のような圧縮成形手段によって圧縮成形することにより製造することができる。   In order to formulate the hydrogen sulfide producing bacteria inhibitor of the present invention, butyric acid bacteria are added to the butyric acid bacteria or butyric acid culture extract obtained as described above, depending on the purpose, administration form, administration target, final form, etc. Other additives can be blended with these as optional components, and can be prepared in any shape such as tablets, powders, granules, capsules, pills, troches, liquids, extracts and the like. These can be prepared according to the usual methods of one skilled in the art. For example, when the hydrogen sulfide-producing bacteria inhibitor is a tablet, the prescribed compound is mixed well with butyric acid bacteria or butyric acid bacteria culture extract added with butyric acid bacteria, and this is compression molded like a tableting machine. It can be manufactured by compression molding by means.

本発明の硫化水素産生菌抑制飲料は、酪酸菌を含有することを特徴とする。また、本発明の硫化水素産生菌飲料は、酪酸菌培養エキスに酪酸菌を加えることを特徴とする。このとき、酪酸菌培養エキスに、酪酸菌が１０3個／ｍｌ以上含まれていることが好ましい。上記の酪酸菌又は酪酸菌培養エキスに酪酸菌を加えたものを飲料に加えて、一般の製造法により、本発明の硫化水素産生菌抑制飲料を加工製造することができる。飲料としては、炭酸飲料、清涼飲料、乳飲料、コーヒー飲料、ミネラルウォーター、アルコール飲料、果汁飲料、茶類，ゼリー、栄養飲料等が挙げられる。また、上記の酪酸菌培養液又は酪酸菌培養エキスを上記飲料以外の飲食物に加え、一般の製造法により、あめ、せんべい、ガム、クッキー、調味料、米穀類、パン、麺類等の飲食物を加工製造することができる。本発明の硫化水素産生菌抑制飲料は、生体内硫化水素産生菌にも適用できる。 The hydrogen sulfide-producing bacteria-suppressing beverage of the present invention is characterized by containing butyric acid bacteria. In addition, the hydrogen sulfide-producing bacterial beverage of the present invention is characterized by adding butyric acid bacteria to a butyric acid bacteria culture extract. At this time, it is preferable that butyric acid bacteria are contained in the butyric acid bacteria culture extract in an amount of 10 3 / ml or more. The above-mentioned butyric acid bacteria or butyric acid bacteria culture extract added with butyric acid bacteria can be added to the beverage, and the hydrogen sulfide-producing bacteria-suppressed beverage of the present invention can be processed and produced by a general production method. Examples of the beverage include carbonated beverages, soft drinks, milk beverages, coffee beverages, mineral water, alcoholic beverages, fruit juice beverages, teas, jelly, and nutritional beverages. In addition, the above-mentioned butyric acid bacteria culture solution or butyric acid bacteria culture extract is added to foods and drinks other than the above-mentioned beverages, and foods and beverages such as candy, rice crackers, gums, cookies, seasonings, rice grains, bread, noodles, etc. Can be processed and manufactured. The beverage for suppressing hydrogen sulfide-producing bacteria of the present invention can also be applied to in vivo hydrogen sulfide-producing bacteria.

本発明の硫化水素産生菌抑制方法は、酪酸菌を硫化水素産生菌が存在する部位へ添加する工程を含むことを特徴とする。また、本発明の硫化水素産生菌抑制方法は、酪酸菌培養エキスに酪酸菌を加えたものを、硫化水素産生菌が存在する部位へ添加する工程を含むことを特徴とする。このとき、酪酸菌培養エキスに、酪酸菌が１０3個／ｍｌ以上含まれていることが好ましい。各種工場廃水処理あるいは公共汚水処理等の硫化水素の発生が問題となる環境や施設において、酪酸菌又は酪酸菌培養エキスに酪酸菌を加えたものを硫化水素産生菌が存在する部位へ添加することにより、硫化水素が発生するのを抑制することができる。 The method for suppressing hydrogen sulfide-producing bacteria of the present invention includes a step of adding butyric acid bacteria to a site where hydrogen sulfide-producing bacteria are present. The method for inhibiting hydrogen sulfide-producing bacteria of the present invention is characterized by including a step of adding butyric acid bacteria to a butyric acid culture extract to a site where hydrogen sulfide-producing bacteria are present. At this time, it is preferable that butyric acid bacteria are contained in the butyric acid bacteria culture extract in an amount of 10 3 / ml or more. In environments and facilities where generation of hydrogen sulfide is a problem, such as in various factory wastewater treatment or public sewage treatment, add butyric acid bacteria or butyric acid culture extract plus butyric acid bacteria to the site where hydrogen sulfide producing bacteria exist Thus, generation of hydrogen sulfide can be suppressed.

本発明の生体内硫化水素産生菌抑制方法は、酪酸菌を人以外の動物へ投与する工程を含むことを特徴とする。また、本発明の生体内硫化水素産生菌抑制方法は、酪酸菌培養エキスに酪酸菌を加えたものを、人以外の動物へ投与する工程を含むことを特徴とする。このとき、酪酸菌培養エキスに、酪酸菌が１０3個／ｍｌ以上含まれていることが好ましい。本発明の酪酸菌又は酪酸菌培養エキスに酪酸菌を加えたものを配合飼料と配合することにより、食餌として人以外の動物に投与できる。また、上記のように、硫化水素産生菌抑制剤又は硫化水素産生菌抑制飲料として加工したものを人以外の動物に投与してもよい。以上のように硫化水素産生菌抑制剤の投与方法としては、経口投与が例示されるが、他にも肛門注入等が挙げられ、その投与方法は特に限定されない。 The method for inhibiting in vivo hydrogen sulfide-producing bacteria of the present invention includes a step of administering butyric acid bacteria to animals other than humans. In addition, the method for suppressing in vivo hydrogen sulfide-producing bacteria of the present invention includes a step of administering a butyric acid bacteria culture extract to which butyric acid bacteria are added to an animal other than a human. At this time, it is preferable that butyric acid bacteria are contained in the butyric acid bacteria culture extract in an amount of 10 3 / ml or more. By mixing the butyric acid bacteria or butyric acid bacteria culture extract of the present invention with butyric acid bacteria and the mixed feed, it can be administered as a diet to animals other than humans. Moreover, you may administer what was processed as a hydrogen sulfide production microbe inhibitor or a hydrogen sulfide production microbe suppression drink to animals other than a human as mentioned above. As described above, the administration method of the hydrogen sulfide-producing bacterium inhibitory agent is exemplified by oral administration, but other examples include anal injection and the administration method is not particularly limited.

以下、具体的な実施例について説明するが、本発明は下記の実施例に限定されるものではない。   Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.

［培地］
培地として、以下の培地１又は培地２を用いた。 [Culture medium]
As the medium, the following medium 1 or medium 2 was used.

１．培地１：Ｗａｔａｎａｂｅ'ｓ ＳＲＢ培地
１５ｇの混合ペプトン、７．５ｇのダイズペプトン、７．５ｇの酵母エキス、７．５ｇの肉エキス、１．０ｇのクエン酸鉄アンモニウム、０．７５ｇのLーシステイン塩酸塩、５〜１０ｇのデキストラン硫酸ナトリウム、３０ｇの寒天及び１Ｌの蒸留水、ｐＨ７．６からなる培地。硫酸還元細菌はこの培地で硫化鉄を形成し、黒色集落を形成する。 1. Medium 1: Watanabe's SRB medium 15 g mixed peptone, 7.5 g soybean peptone, 7.5 g yeast extract, 7.5 g meat extract, 1.0 g ammonium iron citrate, 0.75 g L-cysteine hydrochloride Medium consisting of salt, 5-10 g dextran sulfate sodium, 30 g agar and 1 L distilled water, pH 7.6. Sulfate-reducing bacteria form iron sulfide in this medium, forming a black colony.

２．培地２：クロストリジア寒天（日水製薬株式会社）
１５ｇの混合ペプトン、７．５ｇのダイズペプトン、７．５ｇの酵母エキス、７．５ｇの肉エキス、１．０ｇのクエン酸鉄アンモニウム、１．０ｇのメタ重亜硫酸ナトリウム、０．７５ｇのLーシステイン塩酸塩、３０ｇの寒天、及び１Ｌの蒸留水、ｐＨ７．６からなる培地。アミノ酸発酵菌と硫酸還元細菌が、この培地で硫化鉄を形成し、黒色集落を形成する。 2. Medium 2: Clostridia agar (Nissui Pharmaceutical Co., Ltd.)
15 g mixed peptone, 7.5 g soybean peptone, 7.5 g yeast extract, 7.5 g meat extract, 1.0 g ammonium iron citrate, 1.0 g sodium metabisulfite, 0.75 g L-cysteine A medium consisting of hydrochloride, 30 g agar, and 1 L distilled water, pH 7.6. Amino acid-fermenting bacteria and sulfate-reducing bacteria form iron sulfide in this medium, forming black colonies.

［実験方法］
嫌気培養は、嫌気性グローブボックス法で行った。嫌気性グローブボックス法は、高圧滅菌した培地を冷却後、すぐ嫌気性グローブボックスの中に入れて、その後のすべての作業をその中で行う方法である。以下、硫化水素産生菌による硫化水素が抑制されるかどうかについて硫化水素産生抑制試験を実施した。以下、硫化水素産生抑制試験の方法について述べる。この方法は、硫化水素産生菌が抑制されたとき、硫化水素の発生が抑えられ、硫化水素による上記培地の黒色の呈色が失われることを利用している。
１）適当な嫌気性菌用寒天培地（ブルセラＨＫ血液寒天培地）に試験菌の純培養菌を得る。試験菌としては、以下に示す硫化水素産生菌を用いた。
Clostridium sporogenes (図1.)、Clostridium sordellii (図2.)、Clostridium difficile (図3.)、Clostridium perfringens(図4.)、E.coil(図5.)、Bilophila wadsworthia(図6.)、Sutterella wadsworthensis(図7.)、Desulfovibrio piger(図8.)、Campylobacter gracilis(図9.)
２）培地上の集落を滅菌綿棒でかきとり、滅菌０．０５％酵母エキス水溶液２ｍｌ中に、マックファーランド＃３程度の濁度の菌液を調整する。
３）滅菌した培地２を５０℃の温浴中に準備する。
４）培地２の４０ｍｌに対し、試験菌液の４００μｌを加えて、よく混合する。
５）混合後、所定の滅菌ペトリ皿に培地の厚さが５ｍｍとなるように分注し、固化させる。固化後、すみやかに培地の表面に酪酸菌液又はコントロール液（約７０μｌ）などをしみ込ませたペーパーデイスクを置き、水素ガス炭酸ガス窒素ガスの三種混合ガスと触媒で嫌気的にされた嫌気性ワークステーション内（嫌気性環境）に移す。ここでのコントロール液は、酪酸菌培養液と同濃度に調整した酪酸と酢酸等を用いた。
６）３６度で１日〜７日培養し、デイスク周囲の阻止円の形成、デイスク周囲の培地黒色化の抑制の有無およびデイスク自体（培地接触面と培地非接触面）の黒色化の有無と程度を観察する。 [experimental method]
Anaerobic culture was performed by an anaerobic glove box method. The anaerobic glove box method is a method in which a medium sterilized by autoclaving is cooled and immediately put into an anaerobic glove box, and all subsequent operations are performed therein. Hereinafter, a hydrogen sulfide production suppression test was conducted to determine whether hydrogen sulfide produced by hydrogen sulfide-producing bacteria was suppressed. Hereinafter, the method of the hydrogen sulfide production suppression test is described. This method utilizes the fact that when hydrogen sulfide-producing bacteria are suppressed, the generation of hydrogen sulfide is suppressed and the black coloration of the medium by hydrogen sulfide is lost.
1) A pure culture of a test bacterium is obtained on an appropriate anaerobic agar medium (Brucera HK blood agar medium). As test bacteria, the following hydrogen sulfide-producing bacteria were used.
Clostridium sporogenes (Figure 1.), Clostridium sordellii (Figure 2.), Clostridium difficile (Figure 3.), Clostridium perfringens (Figure 4.), E.coil (Figure 5.), Bilophila wadsworthia (Figure 6.), Sutterella wadsworthensis (Figure 7.), Desulfovibrio piger (Figure 8.), Campylobacter gracilis (Figure 9.)
2) Scrape off the colonies on the medium with a sterile cotton swab, and adjust the turbidity of Mac Farland # 3 in 2 ml of sterile 0.05% yeast extract aqueous solution.
3) Prepare sterilized medium 2 in a 50 ° C. warm bath.
4) Add 400 μl of the test bacterial solution to 40 ml of medium 2 and mix well.
5) After mixing, dispense into a predetermined sterile Petri dish so that the thickness of the medium is 5 mm and solidify. After solidification, an anaerobic work that was immediately anaerobic by placing a paper disk soaked with butyric acid bacteria or a control solution (about 70 μl) on the surface of the medium and using a mixture of hydrogen gas, carbon dioxide, nitrogen gas and a catalyst. Move to the station (anaerobic environment). The control solution used here was butyric acid, acetic acid, etc. adjusted to the same concentration as the butyric acid bacteria culture solution.
6) Cultivation at 36 degrees for 1 to 7 days, formation of inhibition circle around the disk, presence or absence of suppression of medium blackening around the disk, and presence or absence of blackening of the disk itself (medium contact surface and medium non-contact surface) Observe the degree.

次に、酪酸菌が、硫化水素を産生しない有用菌として知られているBifidobacterium adolescentis（図10）の増殖を抑制しないこと確認試験を実施した。実験方法は、有用菌をBifidobacterium adolescentisとした以外は、上述の硫化水素産生抑制試験と同様である。   Next, a confirmation test was carried out to confirm that butyric acid bacteria do not suppress the growth of Bifidobacterium adolescentis (FIG. 10), which is known as a useful bacteria that does not produce hydrogen sulfide. The experimental method is the same as the hydrogen sulfide production suppression test described above except that the useful bacteria are Bifidobacterium adolescentis.

「実験結果」
図１〜図９に、以下に示す硫化水素産生菌を用いた場合の、硫化水素産生抑制試験結果（48時間後）を示す。
Clostridium sporogenes (図1.)、Clostridium sordellii (図2.)、Clostridium difficile (図3.)、Clostridium perfringens(図4.)、E.coil(図5.)、Bilophila wadsworthia(図6.)、Sutterella wadsworthensis(図7.)、Desulfovibrio piger(図8.)、Campylobacter gracilis(図9.)
硫化水素産生菌を用いた場合を用いた場合でも、ペトリ皿内は黒色となっており硫化水素が発生したことが確認された。また、いずれの硫化水素産生菌を用いた場合を用いた場合も、酪酸菌液をしみ込ませたペーパーデイスクの周囲には白色の阻止円が形成されており、酪酸菌液により、硫化水素産生菌の増殖が抑制された。 "Experimental result"
The hydrogen sulfide production suppression test result (after 48 hours) at the time of using the hydrogen sulfide producing microbe shown below in FIGS. 1-9 is shown.
Clostridium sporogenes (Figure 1.), Clostridium sordellii (Figure 2.), Clostridium difficile (Figure 3.), Clostridium perfringens (Figure 4.), E.coil (Figure 5.), Bilophila wadsworthia (Figure 6.), Sutterella wadsworthensis (Figure 7.), Desulfovibrio piger (Figure 8.), Campylobacter gracilis (Figure 9.)
Even when the case using hydrogen sulfide producing bacteria was used, the inside of the Petri dish was black and it was confirmed that hydrogen sulfide was generated. In addition, when using any of the hydrogen sulfide-producing bacteria, a white blocking circle is formed around the paper disk soaked with the butyric acid bacteria solution. Growth was suppressed.

図１０に、有用菌をBifidobacterium adolescentisとした場合の試験結果を示す。酪酸菌が、Bifidobacterium adolescentisの増殖を抑制しないことが確認された。   FIG. 10 shows the test results when the useful bacteria are Bifidobacterium adolescentis. It was confirmed that butyric acid bacteria did not inhibit the growth of Bifidobacterium adolescentis.

硫化水素産生菌Clostridium sporogenesに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of butyric acid bacteria liquid with respect to hydrogen sulfide production bacteria Clostridium sporogenes is shown. 硫化水素産生菌Clostridium sordelliiに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of the butyric acid bacteria liquid with respect to hydrogen sulfide production bacteria Clostridium sordellii is shown. 硫化水素産生菌Clostridium difficileに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of the butyric acid bacteria liquid with respect to hydrogen sulfide production bacteria Clostridium difficile is shown. 硫化水素産生菌Clostridium perfringensに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of the butyric acid bacteria liquid with respect to hydrogen sulfide production bacteria Clostridium perfringens is shown. 硫化水素産生菌E.coilに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of a butyric acid bacteria liquid with respect to hydrogen sulfide producing bacteria E.coil is shown. 硫化水素産生菌Bilophila wadsworthiaに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of the butyric acid bacteria liquid with respect to hydrogen sulfide producing bacteria Bilophila wadsworthia is shown. 硫化水素産生菌Sutterella wadsworthensisに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of butyric acid bacteria liquid with respect to hydrogen sulfide production bacteria Sutterella wadsworthensis is shown. 硫化水素産生菌Desulfovibrio pigerに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of the butyric acid bacteria liquid with respect to hydrogen sulfide producing bacteria Desulfovibrio piger is shown. 硫化水素産生菌Campylobacter gracilisに対する、酪酸菌液の抑制試験結果を示す。The suppression test result of butyric acid bacteria liquid with respect to hydrogen sulfide production bacteria Campylobacter gracilis is shown. 有用菌Bifidobacterium adolescentisに対する、酪酸菌液の抑制試験結果を示す。The inhibition test result of butyric acid bacteria liquid against useful bacteria Bifidobacterium adolescentis is shown.

Claims (10)

酪酸菌（Clostridium butyricum）を含有することを特徴とする硫化水素産生菌抑制剤。   An inhibitor of hydrogen sulfide-producing bacteria characterized by containing butyric acid bacteria (Clostridium butyricum). 酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたことを特徴とする硫化水素産生菌抑制剤。   An inhibitor of hydrogen sulfide-producing bacteria, characterized in that butyric acid bacteria are added to a culture extract of butyric acid bacteria (Clostridium butyricum). 酪酸菌（Clostridium butyricum）を含有することを特徴とする生体内硫化水素産生菌抑制剤。   An in vivo hydrogen sulfide-producing bacterium inhibitor comprising a butyric acid bacterium (Clostridium butyricum). 酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたことを特徴とする生体内硫化水素産生菌抑制剤。   An in vivo suppressor for hydrogen sulfide-producing bacteria, characterized by adding butyric acid bacteria to a culture extract of butyric acid bacteria (Clostridium butyricum). 酪酸菌（Clostridium butyricum）を含有することを特徴とする生体内硫化水素産生菌抑制飲料。   An in-vivo hydrogen sulfide-producing bacteria-suppressing beverage characterized by containing butyric acid bacteria (Clostridium butyricum). 酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたことを特徴とする生体内硫化水素産生菌抑制飲料。   An in-vivo hydrogen sulfide-producing bacteria-suppressing beverage characterized by adding a butyric acid bacterium to a cultured extract of Clostridium butyricum. 酪酸菌（Clostridium butyricum）を硫化水素産生菌が存在する部位へ添加する工程を含むことを特徴とする硫化水素産生菌抑制方法。 A method for inhibiting hydrogen sulfide-producing bacteria, comprising a step of adding butyric acid bacteria (Clostridium butyricum) to a site where hydrogen sulfide-producing bacteria are present. 酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたものを、硫化水素産生菌が存在する部位へ添加する工程を含むことを特徴とする硫化水素産生菌抑制方法。   A method for suppressing hydrogen sulfide-producing bacteria, comprising a step of adding butyric acid bacteria to a culture extract of butyric acid bacteria (Clostridium butyricum) to a site where hydrogen sulfide-producing bacteria are present. 酪酸菌（Clostridium butyricum）を人以外の動物へ投与する工程を含むことを特徴とする生体内硫化水素産生菌抑制方法。   A method for suppressing in vivo hydrogen sulfide-producing bacteria, comprising a step of administering butyric acid bacteria (Clostridium butyricum) to animals other than humans. 酪酸菌（Clostridium butyricum）培養エキスに酪酸菌を加えたものを、人以外の動物へ投与する工程を含むことを特徴とする生体内硫化水素産生菌抑制方法。

A method for inhibiting in vivo hydrogen sulfide-producing bacteria, comprising a step of administering a butyric acid bacterium culture extract to a non-human animal.

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JP2019532665A (en) * 2016-10-28 2019-11-14 ヴェダンタ バイオサイエンシーズ インコーポレーテッドＶｅｄａｎｔａ Ｂｉｏｓｃｉｅｎｃｅｓ， Ｉｎｃ． Methods and compositions for preserving bacteria
JP7105768B2 (en) 2016-10-28 2022-07-25 ヴェダンタ バイオサイエンシーズ インコーポレーテッド Methods and compositions for preserving bacteria
KR20190123513A (en) * 2018-04-24 2019-11-01 울산과학기술원 Apparatus for controlling hydrogen sulfide and recovering sulfur
KR102126447B1 (en) 2018-04-24 2020-07-08 울산과학기술원 Apparatus for controlling hydrogen sulfide and recovering sulfur

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