JP2005000160A - Method for improving survivability of microorganism in living body and the microorganism improved in the viability - Google Patents
Method for improving survivability of microorganism in living body and the microorganism improved in the viability Download PDFInfo
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- JP2005000160A JP2005000160A JP2003435583A JP2003435583A JP2005000160A JP 2005000160 A JP2005000160 A JP 2005000160A JP 2003435583 A JP2003435583 A JP 2003435583A JP 2003435583 A JP2003435583 A JP 2003435583A JP 2005000160 A JP2005000160 A JP 2005000160A
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Abstract
Description
本発明は微生物のhrcA遺伝子を破壊することにより、ヒト及び動物の生体内での微生物の生残性を向上させる方法に関する。
また、微生物のhrcA遺伝子を破壊することにより、ヒト及び動物の消化管内での微生物の胃酸及び／または胆汁酸耐性を向上させる方法に関する。
更に、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の生体内での生残性が向上した微生物に関する。また、hrcA遺伝子を破壊することにより、胃酸及び／または胆汁酸等のストレスに対して耐性が向上した微生物に関する。
また、本発明の微生物は、胃酸及び／または胆汁酸耐性が向上していることにより、腸管への到達率が上がり、生きている微生物が有する宿主への整腸作用等の有益な保健効果を有し、いわゆるプロバイオティクス機能が向上した微生物を含有する飲食品及び飼料を提供することができる。
The present invention relates to a method for improving the survival of microorganisms in humans and animals by destroying the hrcA gene of the microorganisms.
The present invention also relates to a method for improving gastric acid and / or bile acid resistance of microorganisms in the digestive tract of humans and animals by destroying the hrcA gene of microorganisms.
Furthermore, the present invention relates to a microorganism having improved survival in humans and animals by destroying the hrcA gene. The present invention also relates to a microorganism having improved resistance to stress such as gastric acid and / or bile acid by disrupting hrcA gene.
In addition, the microorganism of the present invention has improved gastric acid and / or bile acid resistance, so that the rate of reaching the intestinal tract is increased, and beneficial health effects such as intestinal regulation to the host of living microorganisms are obtained. It is possible to provide food and drink and feed containing microorganisms having so-called probiotic functions.
近年、プロバイオティクス菌を用いたヨーグルトやタブレット、ペットフード、家畜用飼料等が注目されている。プロバイオティクス菌とは、宿主の腸内菌叢のバランスを改善することにより宿主にとって有益な作用をもたらす生きた微生物のことであり、ヒトに用いられる微生物としては、Lactobacillus acidophilus、 Lb.casei、Lb.gasseri等のラクトバチルス属、Enterococcus faecium、Ec. faecalis 等のエンテロコッカス属、Bifidobacterium longum、Bif. breve等のビフィドバクテリウム属等の細菌が利用されている。また、これらの乳酸産生菌以外でも、Clostridium butyricum 等が使用されている。 In recent years, yogurt, tablets, pet food, livestock feed, etc. using probiotic bacteria have attracted attention. Probiotic bacteria are living microorganisms that have a beneficial effect on the host by improving the balance of the intestinal flora of the host, and microorganisms used in humans include Lactobacillus acidophilus, Lb.casei, Lactobacillus genera such as Lb.gasseri, Enterococcus faecium, Enterococcus genera such as Ec.faecalis, Bifidobacterium genus such as Bifidobacterium longum, Bif. In addition to these lactic acid-producing bacteria, Clostridium butyricum and the like are also used.
動物に対して用いるプロバイオティクス菌としては、畜産領域において利用が拡大しており、 Lb. acidophilus、 Lb. salivarius、 Lb. plantarum、 Ec. faecium、Ec. faecalis、Bif. thermophilum、Bif. pseudolongum 等の乳酸菌が利用されている。また、乳酸菌以外でも、Clostridium butyricum、Bacillus subtilis、B. coagulans、酵母が生菌剤として使用されている（例えば、非特許文献１参照。）。 As a probiotic bacterium used for animals, its use is expanding in the livestock field, such as Lb. acidophilus, Lb. salivarius, Lb. plantarum, Ec. Faecium, Ec. Faecalis, Bif. Thermophilum, Bif. Pseudolongum, etc. Lactic acid bacteria are used. In addition to lactic acid bacteria, Clostridium butyricum, Bacillus subtilis, B. coagulans, and yeast are used as viable agents (for example, see Non-patent Document 1).
ヒトの膣内にも多くの乳酸桿菌が常在しており、Lactobacillus acidophilus、 Lb. paracasei、Lb. rhamnosus等の検出が報告されている。また、膣内の菌叢は糞便中の菌叢に影響を受け、糞便から膣へ菌が移行することを示唆する報告もある。更に健康人の膣内に乳酸桿菌等の共生細菌がいることと、泌尿器感染症の患者の膣内にはこれらの微生物がいないということの間には強い相関関係があると言われている。そこで、プロバイオティクス菌の投与による泌尿器疾患の防止が試みられており、乳酸桿菌の経口投与及び膣内投与による、細菌性膣症や尿路感染症等の症状の軽減や再発の予防効果についてすでに報告されている。 Many lactobacilli are resident in the human vagina, and detection of Lactobacillus acidophilus, Lb. paracasei, Lb. rhamnosus, etc. has been reported. In addition, there are reports suggesting that the bacterial flora in the vagina is affected by the flora in the stool and that the bacteria migrate from the stool to the vagina. Furthermore, it is said that there is a strong correlation between the presence of symbiotic bacteria such as lactobacilli in the vagina of healthy people and the absence of these microorganisms in the vagina of patients with urinary tract infections. Therefore, the prevention of urological diseases by the administration of probiotic bacteria has been attempted, and the effect of reducing the symptoms of bacterial vaginosis and urinary tract infections and the prevention of recurrence by oral administration and intravaginal administration of lactobacilli It has already been reported.
これらのプロバイオティクス菌がその機能を発揮するためには、腸内等へ生きて到達することが重要である。しかし摂取された菌が腸内に到達するまでには、胃液や腸液がその生残性に影響を及ぼす。また、これらの菌は主に腸内細菌であり、製品中で酸素や低pH等にさらされることで、生菌数が減少する。 In order for these probiotic bacteria to exert their functions, it is important to live in the intestines and the like. However, by the time the ingested bacteria reach the intestine, gastric juice and intestinal juice affect their survival. In addition, these bacteria are mainly enteric bacteria, and the number of viable bacteria decreases when exposed to oxygen, low pH, etc. in the product.
ヒトの空腹時における胃の内容は、胃酸によってpH 1.0〜2.0に保たれており殺菌作用がある。動物においても種によって異なるが、胃（後部）のpHがウサギで1.9、サルで2.8といった報告もあり、一般にpHは低い。このような状態でプロバイオティクス菌を摂取した場合は、短時間のうちに死滅してしまうと予想される。しかし、実際の食生活を考えると食事によって胃内容のpHは上昇すると考えられ、例えば醗酵乳のpHは一般的に4.0〜4.5であることから、ヒトが空腹時に醗酵乳を摂取した場合には、胃内容のpHは3.0〜4.0程度まで上昇すると考えられる。しかし低いpHにより摂取した菌体が死滅してしまうため、十分な効果を得るためには多量の菌体を摂取しなければならないといった問題がある。 The stomach content when fasting in humans is kept at pH 1.0 to 2.0 by gastric acid and has a bactericidal action. There are reports that the pH of the stomach (rear part) is 1.9 for rabbits and 2.8 for monkeys, but the pH is generally low. Ingestion of probiotic bacteria in such a state is expected to die within a short time. However, considering the actual eating habits, the pH of the stomach content is thought to increase with meals, for example, the pH of fermented milk is generally 4.0 to 4.5, so when humans ingest fermented milk on an empty stomach The pH of stomach contents is considered to rise to about 3.0-4.0. However, there is a problem that a large amount of cells must be ingested in order to obtain a sufficient effect because the cells ingested by the low pH are killed.
そこで、プロバイオティクス菌を含む食品において、プロバイオティクス菌の生残性を高めるための様々な技術が、これまでに数多く報告されている。 例えば、醗酵乳製品中のプロバイオティクス菌の生残性を高める方法として、パーオキシダーゼを添加して保存中の酸度上昇を抑制する方法（例えば、特許文献１参照。）、カテキン類及び／またはトコフェロール類を添加する方法（例えば、特許文献２参照。）、ビフィズス菌と共生関係を持つ乳酸菌を添加する方法（例えば、特許文献３参照。）、着色フィルムにより遮光性を高める方法（例えば、特許文献４参照。）等がある。また、摂取後の生体内での生残性を高める方法については、乳酸菌を腸溶性カプセルに封入することで胃酸の影響を抑えて腸内に到達させる方法（例えば、特許文献５参照。）があるが、微生物そのものの耐性機構を利用した技術は報告されていない。 Thus, various techniques for improving the survival of probiotic bacteria in foods containing probiotic bacteria have been reported so far. For example, as a method for increasing the viability of probiotic bacteria in fermented milk products, a method for suppressing increase in acidity during storage by adding peroxidase (see, for example, Patent Document 1), catechins and / or A method of adding tocopherols (for example, refer to Patent Document 2), a method of adding lactic acid bacteria having a symbiotic relationship with bifidobacteria (for example, refer to Patent Document 3), a method for enhancing light-shielding properties with a colored film (for example, Patent Reference 4). In addition, as a method for increasing the survival in the living body after ingestion, a method of suppressing the influence of gastric acid by enclosing lactic acid bacteria in an enteric capsule (see, for example, Patent Document 5). However, no technology that utilizes the resistance mechanism of the microorganism itself has been reported.
さて、微生物にとって環境の変化に対応できる能力は、生存のために必須であり、この根幹をなす機構はストレスタンパク質の生産であると考えられている。ここでストレスタンパク質とは、外界からのストレス、例えば低pH、酸素、高温、飢餓、塩、有機酸等の刺激により誘導合成されるタンパク質の総称であり、各々の機構は異なるが、微生物体内の安定化に寄与していると考えられている。 また、分子シャペロンと呼ばれる一連のタンパク質（DnaK、GroEL、GroES、DnaJ等）もストレスタンパク質の一種であり、ストレス時に菌体内において、タンパク質の変性を抑制する働きや、変性したタンパク質の修復機能があるとされている。 Now, the ability of microorganisms to cope with environmental changes is essential for survival, and the underlying mechanism is considered to be production of stress proteins. Here, stress protein is a general term for proteins induced and synthesized by external stress such as low pH, oxygen, high temperature, starvation, salt, organic acid, etc., and each mechanism is different. It is thought to contribute to stabilization. In addition, a series of proteins called molecular chaperones (DnaK, GroEL, GroES, DnaJ, etc.) are also a type of stress protein, and they have a function of suppressing protein denaturation and a function of repairing denatured proteins in cells during stress. It is said that.
ストレスタンパク質の制御には、大腸菌等ではσ32因子が関与しているとされており、σ32因子が各種分子シャペロン遺伝子の5’上流に存在する共通プロモーター配列に結合し、一斉にその転写を増加させると考えられている。σ32因子自体の制御は、そのmRNAの二次構造が熱により変化して翻訳が開始されることにより増加し、分子シャペロンにより分解を受けることで減少する（例えば、非特許文献２参照。）。 このσ因子またはストレスタンパク質そのものを高発現することで、ストレスに対する耐性を付与した微生物による物質生産に関する報告がなされている。しかし、この方法は、培地中にアミノ酸等の醗酵生産物を蓄積させる製造法において、微生物自体が培地中に産生する高濃度のアミノ酸等の醗酵生産物による産生抑制ストレスに対し、ストレスタンパク質の発現量を増強し、醗酵生産物の生産性を改善するものであり、有益な微生物を摂取した後の生体内での生残性を高める方法ではない。 To control the stress proteins, in E. coli or the like are that the sigma 32 factor are involved, binds a common promoter sequences sigma 32 factor is present in 5 'upstream of various molecular chaperon gene, simultaneously to the transfer It is thought to increase. Control of sigma 32 factor itself is increased by the secondary structure of the mRNA is initiated translation changed by heat, reduced by subject to degradation by molecular chaperones (e.g., Non-Patent Document 2 referred to.) . There have been reports on substance production by microorganisms imparting resistance to stress by highly expressing this sigma factor or stress protein itself. However, this method is a production method in which fermentation products such as amino acids are accumulated in the medium, and the expression of stress proteins is suppressed against production-suppressing stress caused by fermentation products such as high concentrations of amino acids produced by microorganisms in the medium. The method is to increase the amount and improve the productivity of the fermentation product, and is not a method for increasing the survival in vivo after ingesting beneficial microorganisms.
一方、バチルス・スブティリス(例えば、非特許文献３参照。)、ストレプトコッカス・ミュータンス(例えば、非特許文献４参照。)、ラクトバチルス・サケ(例えば、非特許文献５参照。)等の菌種においては、分子シャペロンの制御に、その遺伝子の5’上流に存在する共通CIRCE配列とhrcAタンパク質が関与していることが近年明らかとなっている。hrcAは各種分子シャペロン遺伝子の5’上流に存在するCIRCE配列に結合し、その下流に位置する遺伝子の発現を抑制する働きがあり、上述した株においてはその遺伝子がDnaK、GrpE遺伝子とオペロンを形成している。上流にCIRCE配列を持ち、hrcAで制御されているものとしては、DnaKオペロン、GroELSオペロン等が知られている。しかし、これまでに、プロバイオティクス菌の機能とストレスタンパク質とを関連付けた研究は行われていない。
本発明は、消化管内でプロバイオティクス菌が受けると予想されるストレスと菌の耐性機構に着目し、それらの耐性を菌に付与することで生体内での生残性を向上させる方法を見出すことを課題とする。また、プロバイオティクスとしての機能が向上した微生物の作製を課題とする。
更に、胃酸及び／または胆汁酸等の消化液耐性が弱かったためにプロバイオティクスとしての効果をこれまで期待されていなかった微生物についても、消化液耐性を付与してその汎用性を広げることを課題とする。
The present invention focuses on the stress expected to be received by probiotic bacteria in the gastrointestinal tract and the resistance mechanism of the bacteria, and finds a method for improving survival in vivo by imparting such resistance to the bacteria. This is the issue. Another object is to produce microorganisms with improved functions as probiotics.
Furthermore, it is a problem to provide digestive juice resistance and broaden its versatility even for microorganisms that have not been expected to have probiotic effects due to weak resistance to digestive juice such as gastric acid and / or bile acids. And
本発明者らは、上記課題を解決するために鋭意検討を行った結果、各種分子シャペロン遺伝子の5’上流に存在するCIRCE配列に結合し、その下流に位置する遺伝子の発現を抑制する働きがあり、ストレスタンパク質の発現を抑制している、hrcAの遺伝子を破壊した微生物を作成し、この菌株においてプロバイオティクスとして必要とされる胃酸耐性や胆汁酸耐性等の機能が向上していることを見出し、本発明を完成させるに至った。
すなわち、本発明は、微生物のhrcA遺伝子を破壊することにより、該微生物のヒト及び動物の生体内での生残性を向上させる方法を提供することにある。
また、本発明は、微生物のhrcA遺伝子を破壊することにより、該微生物のヒト及び動物の消化管内での胃酸及び／または胆汁酸耐性を向上させる方法を提供することにある。
更に、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の生体内での生残性が向上した微生物を提供することにある。
また、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の消化管内での胃酸及び／または胆汁酸耐性が向上した微生物を提供することにある。
更にまた、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の生体内、特に、ヒト及び動物の消化管内での胃酸及び／または胆汁酸耐性が向上した食用または飼料に供されるラクトバチルス属、ビフィドバクテリウム属、エンテロコッカス属、ラクトコッカス属、ストレプトコッカス属及びクロストリジウム属の微生物を提供することにある。
更にまた、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の生体内、特に、ヒト及び動物の消化管内での胃酸及び／または胆汁酸耐性が向上したラクトバチルス・アシドフィルスまたはラクトバチルス・ガセリを提供することにある。
更にまた、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の生体内、特に、ヒト及び動物の消化管内での胃酸及び／または胆汁酸耐性が向上したラクトバチルス・アシドフィルスＳＢＴ２０６２hrcA遺伝子破壊株（ＦＥＲＭ Ｐ−１９２２４またはＦＥＲＭ Ｐ−１９３３７）またはラクトバチルス・ガセリＳＢＴ２０５５hrcA遺伝子破壊株を提供することにある。
更にまた、本発明は、hrcA遺伝子を破壊することにより、ヒト及び動物の生体内、特に、ヒト及び動物の消化管内での胃酸及び／または胆汁酸耐性が向上した微生物を含有することを特徴とする飲食品または飼料を提供することにある。
As a result of diligent studies to solve the above problems, the present inventors have a function of binding to a CIRCE sequence existing 5 ′ upstream of various molecular chaperone genes and suppressing the expression of genes located downstream thereof. Yes, we have created a microorganism that has disrupted the hrcA gene that suppresses the expression of stress proteins and has improved functions such as gastric acid resistance and bile acid resistance required as probiotics in this strain. The headline and the present invention have been completed.
That is, the present invention is to provide a method for improving the survival of microorganisms in humans and animals by destroying the hrcA gene of the microorganisms.
Another object of the present invention is to provide a method for improving gastric acid and / or bile acid resistance in the digestive tract of humans and animals by destroying the hrcA gene of the microorganism.
Furthermore, this invention is providing the microorganisms which the survival property in the living body of a human and an animal improved by destroying hrcA gene.
Another object of the present invention is to provide a microorganism having improved resistance to gastric acid and / or bile acid in the digestive tract of humans and animals by disrupting the hrcA gene.
Furthermore, the present invention provides a lactate for use in foods or feeds with improved gastric acid and / or bile acid tolerance in humans and animals, particularly in the digestive tract of humans and animals, by disrupting the hrcA gene. The object is to provide microorganisms of the genera Bacillus, Bifidobacterium, Enterococcus, Lactococcus, Streptococcus and Clostridium.
Furthermore, the present invention relates to Lactobacillus acidophilus or Lactobacillus lactobacillus having improved gastric acid and / or bile acid resistance in humans and animals, particularly in the digestive tract of humans and animals, by disrupting the hrcA gene. To provide gasseri.
Furthermore, the present invention relates to a Lactobacillus acidophilus SBT2062 hrcA gene-disrupted strain that has improved resistance to gastric acid and / or bile acids in humans and animals, particularly in the digestive tract of humans and animals, by disrupting the hrcA gene. (FERM P-19224 or FERM P-19337) or Lactobacillus gasseri SBT2055hrcA gene disruption strain.
Furthermore, the present invention is characterized in that it contains a microorganism having improved resistance to gastric acid and / or bile acid in the living body of humans and animals, particularly in the digestive tract of humans and animals, by disrupting the hrcA gene. It is to provide food and drink or feed.
本発明による、ヒト及び動物の生体内での微生物の生残性を向上させる方法、中でも、ヒト及び動物の消化管内での微生物の胃酸及び／または胆汁酸耐性を向上させる方法によって得られた微生物は胆汁酸や胃酸等に対する耐性が向上し、それを添加した飲食品・飼料・錠剤等をヒトまたは動物が摂取した際に体内での生残率が高く、より多くの菌体が腸内へ生きて到達することができる。その結果、微生物が有する宿主への整腸作用等の保健効果を高めることができる。
更には、本発明による、ヒト及び動物の生体内での微生物の生残性を向上させる方法は、消化管だけでなく他の体内の器官内への生きたままの到達や生残性を向上することができる。
Microorganisms obtained by the method according to the present invention for improving the viability of microorganisms in humans and animals in vivo, especially the method for improving the gastric acid and / or bile acid resistance of microorganisms in the digestive tract of humans and animals Has improved resistance to bile acids, gastric acids, etc., and when humans or animals consume foods, feeds, tablets, etc. to which it has been added, the survival rate in the body is high, and more bacterial cells enter the intestines Can be reached alive. As a result, health effects such as intestinal regulation on the host of the microorganism can be enhanced.
Furthermore, the method of improving the survival of microorganisms in human and animal organisms according to the present invention improves the survival and survival of not only the digestive tract but also other organs in the body. can do.
本発明の微生物としては、宿主の腸内等の菌叢バランスを改善することにより宿主にとって有益な作用をもたらすラクトバチルス属細菌、ビフィドバクテリウム属細菌、エンテロコッカス属細菌、ラクトコッカス属細菌、ストレプトコッカス属細菌、クロストリジウム属細菌が挙げられる。 The microorganisms of the present invention include Lactobacillus bacteria, Bifidobacterium bacteria, Enterococcus bacteria, Lactococcus bacteria, Streptococcus bacteria that have beneficial effects on the host by improving the bacterial flora balance of the host intestines and the like. Genus bacteria, Clostridium bacteria.
具体的にはBacillus acidophilus、Lb. gasseri、Lb. casei、Lb. salivarius、Lb. plantarum、Lb. johnsonii、Lb. reuteri、Lb. rhamnosus、Lb. fermentum、Lb. murinus 等のラクトバチルス属細菌、Bifidobacterium longum、Bif. bifidum、Bif. breve、Bif. animalis、Bif. thermophilum、Bif. pseudolongum等のビフィドバクテリウム属細菌、Enterococcus faecium、Ec. faecalis 等のエンテロコッカス属細菌、Clostridium butyricum等のクロストリジウム属細菌が挙げられる。
また、胃酸及び／または胆汁酸等の消化液耐性がもともと弱いと考えられるStreptococcus diacetylactis、Sc.cremoris、 Sc.thermophilus等のストレプトコッカス属細菌、Lactococcus lactis等のラクトコッカス属細菌やLb.delbrueckii subsp.bulgaricus等のラクトバチルス属細菌といったいわゆる酪農乳酸菌も挙げられる。
Specifically, Bacillus acidophilus, Lb. gasseri, Lb. casei, Lb. salivarius, Lb. plantarum, Lb. johnsonii, Lb. reuteri, Lb. rhamnosus, Lb. fermentum, Lb. longum, Bif. bifidum, Bif. breve, Bif. animalis, Bif. thermophilum, Bif. pseudolongum and other Bifidobacterium bacteria, Enterococcus faecium, Ec. faecalis and other Enterococcus bacteria, Clostridium butyricum Can be mentioned.
Furthermore, digestive juice resistance such as gastric acid and / or bile acid is considered to be weak originally, Streptococcus bacteria such as Streptococcus diacetylactis, Sc.cremoris, Sc.thermophilus, Lactococcus bacteria such as Lactococcus lactis, and Lb. And so-called dairy lactic acid bacteria such as Lactobacillus bacteria.
その中でも、ラクトバチルス属細菌が好ましく、特に、ラクトバチルス・アシドフィルス及びラクトバチルス・ガセリに属する乳酸菌が好ましい。ラクトバチルス・アシドフィルス及びラクトバチルス・ガセリはアシドフィルス複合菌種を構成する６菌種の一つで、ヒト腸管から頻繁に分離されるアシドフィルス複合菌種である。 Among them, bacteria belonging to the genus Lactobacillus are preferable, and lactic acid bacteria belonging to Lactobacillus acidophilus and Lactobacillus gasseri are particularly preferable. Lactobacillus acidophilus and Lactobacillus gasseri are one of six species constituting the acidophilus complex species, and are acidophilus complex species that are frequently separated from the human intestinal tract.
次に、hrcA遺伝子を破壊する方法を記す。従来から微生物の育種に用いられてきた方法として、自然育種による方法、変異剤添加、紫外線照射、放射線照射等により突然変異を誘発させる方法等が挙げられるが、hrcA遺伝子の破壊にもこれらの方法を使用することが可能であると考えられる。また、Insertion Sequence (IS)、トランスポゾン（Tn）等によるランダムな突然変異による方法、 またはシングル及びダブルクロスオーバー法による部位特異的な遺伝子破壊法等も可能である。以上の中でも、部位特異的な遺伝子破壊法が効率良く安全である。特に、ダブルクロスオーバー法により遺伝子破壊を行った株は安定であり、食品等の最終産物には望ましくない抗生物質耐性やベクター部分が切除できる点が優れている。 Next, a method for disrupting the hrcA gene will be described. Methods that have been conventionally used for microbial breeding include natural breeding methods, mutagen addition methods, methods of inducing mutations by ultraviolet irradiation, radiation irradiation, etc., but these methods are also used to disrupt the hrcA gene. Can be used. In addition, methods such as random mutation using Insertion Sequence (IS), transposon (Tn), etc., or site-specific gene disruption using single and double crossover methods are also possible. Among these, site-specific gene disruption methods are efficient and safe. In particular, strains that have undergone gene disruption by the double crossover method are stable, and are excellent in that they can excise undesirable antibiotic resistance and vector parts in end products such as foods.
hrcA遺伝子のシングル及びダブルクロスオーバーによる破壊を行うためには、目的とする菌株のhrcA内部または周辺の遺伝子を取得する必要がある。hrcA遺伝子については、Lb. acidophilus、 Lb. sakei、Lb. sanfrancisco、Lactococcus lactis、 B. subtilis、 Clostridium acetobutylicum やStreptococcus mutans 等でその配列が決定されており、これらの配列の一部からプライマーを作成し、染色体DNAを鋳型としてＰＣＲ（ポリメラーゼ・チェーン・リアクション）を行い、hrcA周辺配列を単離する。シングルクロスオーバーの場合はhrcA内部配列を含む断片、ダブルクロスオーバーの場合はhrcAの上流及び下流配列をそれぞれ含む２つの断片（内部配列を欠損）を増幅する。 In order to disrupt the hrcA gene by single and double crossover, it is necessary to obtain genes in or around the target strain of hrcA. The sequence of the hrcA gene has been determined by Lb. acidophilus, Lb. sakei, Lb. sanfrancisco, Lactococcus lactis, B. subtilis, Clostridium acetobutylicum, Streptococcus mutans, etc., and primers were prepared from some of these sequences. Then, PCR (polymerase chain reaction) is performed using the chromosomal DNA as a template, and the sequence around hrcA is isolated. In the case of single crossover, a fragment containing hrcA internal sequence is amplified. In the case of double crossover, two fragments (internal sequence is deleted) each containing upstream and downstream sequences of hrcA are amplified.
ＰＣＲ法により得られた断片を、温度感受性レプリコンを含むシャトルベクターに組み込み、目的の菌株に導入して形質転換を行う。温度感受性ベクターは低温では複製できるが高温では複製できないといった特徴があり、例えば乳酸菌に対して使用可能であり、広い宿主域を持つpG+hostベクター等が適当である。もちろん、それぞれの宿主に有効なプラスミドを作製して使用することも可能である。
遺伝子導入の方法としては、一般的な接合伝達法や形質転換法等が挙げられるが、特に、エレクトロポレーション法が適当である。
The fragment obtained by the PCR method is incorporated into a shuttle vector containing a temperature-sensitive replicon and introduced into the target strain for transformation. A temperature-sensitive vector has a feature that it can replicate at low temperatures but cannot replicate at high temperatures. For example, it can be used for lactic acid bacteria, and a pG + host vector having a wide host range is suitable. Of course, it is also possible to prepare and use a plasmid effective for each host.
Examples of gene transfer methods include general conjugation transfer methods and transformation methods, and the electroporation method is particularly suitable.
まず、低温条件下にて形質転換体を取得し、この菌株を高温条件下、選択圧をかけて培養すると、プラスミドは複製されずに、染色体上のhrcA遺伝子内部に相同組換えにより組み込まれる。シングルクロスオーバーにより取得されたhrcA破壊株について、更に形質を安定させるためダブルクロスオーバーを行うことも有効な方法である。具体的には、低温条件下、選択圧をかけずに培養することでプラスミドを脱落させ、プラスミド由来のhrcA’遺伝子（内部配列欠損）と野生株のhrcA遺伝子を入れ替えることで、外来遺伝子を含まないhrcA破壊株を取得することができる。
本発明の微生物は、菌体及び／または培養物をそのままの状態で利用することができ、また乾燥させて粉末状態にしても利用可能である。なお、これらの乾燥は、凍結乾燥で行うことが菌体等を変質・死滅させることなく乾燥することができるため好ましい。
本発明の微生物は、飲食品や飼料の製造工程中に原材料として添加することもできる。
更に、本発明の微生物は、賦形剤、結合剤、崩壊剤、界面活性剤、流動性促進剤、滑沢剤等を使用して、糖衣錠やタブレット等の錠剤、顆粒剤、液剤、カプセル等の形態に製剤化して利用可能である。
更にまた、本発明の微生物は、紅茶、緑茶、果汁飲料、野菜飲料、乳製品、食肉製品、水産練製品、デザート類、ドレッシング類、健康食品類、麺類等の様々な飲食品に利用可能である。
更にまた、本発明の微生物のうち、乳酸菌は、醗酵乳やチーズ等の醗酵乳製品のスターター菌種として利用することができる。これらの乳酸菌を利用して乳酸醗酵させて調製したものや、醗酵ミックスにこれらの粉末等を添加することもできる。また、寒天やゼラチン等で固めたもの、醗酵後固まったカードを攪拌して液状にしたもの、更にアイスクリームと同様に凍結したものを含む。
更にまた、本発明の微生物は、牛、豚、鶏等の家畜用飼料に利用することができる。また、サイレージの醗酵を促進させるためのスターターとして利用することも可能である。
本発明の微生物は、乾燥菌体重量として通常飲食品中に0.01〜20(w/w)％、好ましくは0.1〜2(w/w)％添加すると良い。
以下に、実施例及び試験例を挙げ、本発明を更に詳しく説明する。しかし、本発明はこの記載内容に限定されるものではない。
First, when a transformant is obtained under low temperature conditions and this strain is cultured under high temperature conditions under selective pressure, the plasmid is not replicated and is integrated by homologous recombination inside the hrcA gene on the chromosome. For the hrcA-disrupted strain obtained by single crossover, it is also effective to perform double crossover to further stabilize the character. Specifically, plasmids are removed by culturing under low temperature conditions without applying selective pressure, and foreign genes are included by replacing the plasmid-derived hrcA 'gene (internal sequence deletion) with the wild-type hrcA gene. No hrcA disruption strain can be obtained.
The microorganism of the present invention can be used as it is in the state of bacterial cells and / or cultures, or can be used after drying to a powder state. In addition, it is preferable to perform these drying by freeze-drying, since the cells can be dried without deteriorating or killing the cells.
The microorganism of the present invention can also be added as a raw material during the production process of food and drink and feed.
Furthermore, the microorganism of the present invention uses excipients, binders, disintegrants, surfactants, fluidity promoters, lubricants, etc., and tablets such as sugar-coated tablets and tablets, granules, liquids, capsules, etc. It can be formulated and used in the form of
Furthermore, the microorganism of the present invention can be used in various foods and beverages such as black tea, green tea, fruit juice beverages, vegetable beverages, dairy products, meat products, marine products, desserts, dressings, health foods, and noodles. is there.
Furthermore, among the microorganisms of the present invention, lactic acid bacteria can be used as a starter species of fermented milk products such as fermented milk and cheese. These powders and the like can also be added to those prepared by lactic acid fermentation using these lactic acid bacteria and fermentation mix. Moreover, the thing hardened with agar, gelatin, etc., the thing which stirs the card | curd hardened after fermentation and made it liquid, and also the thing frozen like the ice cream are included.
Furthermore, the microorganism of the present invention can be used for feed for livestock such as cattle, pigs and chickens. It can also be used as a starter for promoting silage fermentation.
The microorganism of the present invention may be added in an amount of 0.01 to 20 (w / w)%, preferably 0.1 to 2 (w / w)% in a normal food or drink as a dry cell weight.
Hereinafter, the present invention will be described in more detail with reference to examples and test examples. However, the present invention is not limited to this description.
（ラクトバチルス・アシドフィルスＳＢＴ２０６２hrcA遺伝子破壊株の取得
（hrcA遺伝子の取得と導入プラスミドの構築））
プロバイオティクス菌として整腸作用等の生理効果が報告されているラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）株の染色体から、hrcA遺伝子周辺配列をＰＣＲ法により増幅した。ＰＣＲに用いたプライマーは、すでに決定しているラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）株のシーケンス情報を元に作製した。このラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）株のシーケンス情報は、本発明の出願人がシーケンスを行い決定したものである（Gene Bank Accession no. AB059359）。すなわち、以下に示すプライマーを合成して用いた（配列表参照）。
(Acquisition of Lactobacillus acidophilus SBT2062 hrcA gene disruption strain (acquisition of hrcA gene and construction of introduced plasmid))
The sequence around the hrcA gene was amplified by PCR from the chromosome of Lactobacillus acidophilus SBT2062 (FERM P-10730) strain, which has been reported to have physiological effects such as intestinal regulation as a probiotic bacterium. Primers used for PCR were prepared based on the sequence information of the already determined Lactobacillus acidophilus SBT2062 (FERM P-10730) strain. The sequence information of the Lactobacillus acidophilus SBT2062 (FERM P-10730) strain is determined by sequencing by the applicant of the present invention (Gene Bank Accession no. AB059359). That is, the following primers were synthesized and used (see Sequence Listing).
＜hrcA上流配列＞
5’側：5’-GATCTCCTATGAAGAACTGCTC-3’（配列番号１）
3’側：5’-CACTGTCTTGGATCCAACTGGTTCA-3’（配列番号２）
＜hrcA下流配列＞
5’側：5’-CAACTGGATCCACCTTTTCATAGGGTAAAC-3’（配列番号３）
3’側：5’-AAATCATTTGGTAAGCTTGATCCTAATTT-3’（配列番号４）
すなわち、hrcA上流配列の5’側と3’側及びhrcA下流配列の5’側と3’側の配列からプライマーを合成した。これらのプライマーを用いたＰＣＲの増幅断片を制限酵素処理(HindIII及びBamH I)したのち、エリスロマイシン耐性遺伝子を含むpG+host6ベクターのマルチクローニングサイトに上流−下流の順に挿入した。こうして、内部配列が欠落したhrcA遺伝子（hrcA’遺伝子）を含むプラスミドpG+host6::hrcA’が得られ、これを大腸菌に導入し、大量調製、精製を行った。
<HrcA upstream sequence>
5 'side: 5'-GATCTCCTATGAAGAACTGCTC-3' (SEQ ID NO: 1)
3 'side: 5'-CACTGTCTTGGATCCAACTGGTTCA-3' (SEQ ID NO: 2)
<HrcA downstream sequence>
5 'side: 5'-CAACTGGATCCACCTTTTCATAGGGTAAAC-3' (SEQ ID NO: 3)
3 'side: 5'-AAATCATTTGGTAAGCTTGATCCTAATTT-3' (SEQ ID NO: 4)
That is, primers were synthesized from the 5 ′ side and 3 ′ side of the hrcA upstream sequence and the 5 ′ side and 3 ′ side of the hrcA downstream sequence. PCR amplified fragments using these primers were treated with restriction enzymes (HindIII and BamHI), and then inserted into the multiple cloning site of the pG + host6 vector containing the erythromycin resistance gene in the order of upstream-downstream. Thus, plasmid pG + host6 :: hrcA ′ containing hrcA gene lacking the internal sequence (hrcA ′ gene) was obtained and introduced into Escherichia coli for mass preparation and purification.
（プラスミドの導入と染色体への挿入（シングルクロスオーバー））
上記のようにして作成したプラスミドをWalkerらの方法(D.C.Walker,K.Aoyama and T.R.Klaenhammer, FEMS Microbiol.Lett. 138, 233-237(1996))に従い、電気パルス法を用いてラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）株に導入した。次に、導入したプラスミドを染色体へ挿入するため、プラスミド導入株をエリスロマイシン（シグマ社製）20mg/mlを含んだＭＲＳ培地（ディフコ社製）中、43℃で培養することにより相同組換えを誘発し、染色体上のhrcA遺伝子内部にプラスミドを挿入した。挿入株を選別するためエリスロマイシンを含んだ培地にて43℃で培養を継続し、染色体上に抗生物質耐性が付与されたhrcA遺伝子破壊株（シングルクロスオーバー）を取得した。本発明において得たラクトバチルス・アシドフィルスＳＢＴ２０６２hrcA遺伝子破壊株（シングルクロスオーバー）は、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ−１９２２４として寄託している。
(Introduction of plasmid and insertion into chromosome (single crossover))
The plasmid prepared as described above was subjected to the method of Walker et al. (DC Walker, K. Aoyama and TRKlaenhammer, FEMS Microbiol. Lett. 138, 233-237 (1996)) using the electric pulse method and Lactobacillus acidophilus SBT2062 ( FERM P-10730) strain. Next, in order to insert the introduced plasmid into the chromosome, homologous recombination is induced by culturing the plasmid-introduced strain at 43 ° C. in MRS medium (Difco) containing 20 mg / ml of erythromycin (Sigma). Then, a plasmid was inserted into the hrcA gene on the chromosome. In order to select the inserted strain, the culture was continued at 43 ° C. in a medium containing erythromycin to obtain an hrcA gene disrupted strain (single crossover) imparted with antibiotic resistance on the chromosome. The Lactobacillus acidophilus SBT2062hrcA gene disruption strain (single crossover) obtained in the present invention has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-19224.
（挿入遺伝子の脱落とhrcA遺伝子破壊株の取得（ダブルクロスオーバー））
更にこのhrcA遺伝子破壊株について、形質を安定させるために低温（28℃）条件下、エリスロマイシンを含まないＭＲＳ培地で培養することによりプラスミドを脱落させ、プラスミド由来のhrcA’遺伝子と野生株のhrcA遺伝子を入れ替えることで、hrcA遺伝子破壊株（ダブルクロスオーバー）を取得した。本発明において得たラクトバチルス・アシドフィルスＳＢＴ２０６２hrcA遺伝子破壊株（ダブルクロスオーバー）は、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ−１９３３７として寄託している。
(Deletion of inserted gene and acquisition of hrcA gene disruption strain (double crossover))
Further, for this hrcA gene disrupted strain, the plasmid was removed by culturing in an MRS medium not containing erythromycin under low temperature (28 ° C.) conditions in order to stabilize the trait, and the plasmid-derived hrcA ′ gene and the wild-type hrcA gene The hrcA gene disruption strain (double crossover) was obtained by replacing. The Lactobacillus acidophilus SBT2062hrcA gene disruption strain (double crossover) obtained in the present invention has been deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-19337.
（胃酸耐性試験）
実施例１で得られたhrcA遺伝子破壊株（シングルクロスオーバー）（以下、sΔhrcA株ということもある。）について、胃酸耐性試験を行った。対照として、ラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）野生株についても同様の試験を行った。
（方法）
鈴木らの方法（腸内細菌学雑誌,11,11-17（1997））を参考にして人工胃液試験を行った。ペプシン（和光純薬工業社製）0.4％を含む12.6％脱脂乳培地を塩酸でpH3.0及び2.5に調整したものを人工胃液混合物とし、これにＭＲＳ培地で培養後、洗浄した菌体を3％接種して、pH2.5のものは3時間、pH3.0のものは6時間、37℃で振とうした。振とう後の懸濁液をＭＲＳ寒天培地に塗布し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行った。培養後、生菌数を測定した。
（試験結果）
結果を表１及び図１に示した。sΔhrcA株は、野生株に比べてpH3.0・6時間処理後で約4倍、pH2.5・3時間処理後で約6倍と高い生残率を示した。
よって、hrcA遺伝子を破壊することにより、胃酸に対する耐性が向上することが明らかとなった。よって、ヒトまたは動物がこの菌体を摂取した際の腸内への到達率が高まると考えられる。
(Gastroacid tolerance test)
The gastric acid resistance test was performed on the hrcA gene disrupted strain (single crossover) obtained in Example 1 (hereinafter sometimes referred to as sΔhrcA strain). As a control, the same test was performed on the Lactobacillus acidophilus SBT2062 (FERM P-10730) wild type strain.
(Method)
An artificial gastric juice test was conducted with reference to the method of Suzuki et al. (Journal of Intestinal Bacteriology, 11, 11-17 (1997)). A 12.6% skim milk medium containing 0.4% pepsin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 3.0 and 2.5 with hydrochloric acid is used as an artificial gastric juice mixture. Inoculated at 37 ° C for 3 hours for pH 2.5 and 6 hours for pH 3.0. The suspension after shaking was applied to an MRS agar medium, and anaerobic culture was performed at 37 ° C. for 3 days using an anero pack (manufactured by Mitsubishi Gas Chemical Company). After cultivation, the number of viable bacteria was measured.
(Test results)
The results are shown in Table 1 and FIG. The sΔhrcA strain showed a high survival rate of about 4 times after pH 3.0 · 6 hours treatment and about 6 times after pH 2.5 · 3 hours treatment compared to the wild strain.
Thus, it was revealed that the resistance to gastric acid is improved by disrupting the hrcA gene. Therefore, it is considered that the rate of reaching the intestine when humans or animals ingest these cells is increased.
（胆汁酸耐性試験）
実施例１で得られたsΔhrcA株について、胆汁酸耐性試験を行った。対照として、ラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）野生株についても同様の試験を行った。
（方法）
ウシ胆汁末（OXOID社製）を3％含むＰＢＳ(-)緩衝液(日水製薬社製)に、ＭＲＳ培地で培養後、洗浄した各菌株を1％接種し、37℃で3時間及び6時間処理した。処理後の懸濁液をＭＲＳ寒天培地に塗布し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行い、培養後、生菌数測定を行った。
（試験結果）
結果を表２、図２に示した。sΔhrcA株は、野生株と比較して、3時間処理後では約100倍、6時間処理後では約160倍の高い生残率を示した。
(Bile acid tolerance test)
The sΔhrcA strain obtained in Example 1 was subjected to a bile acid resistance test. As a control, the same test was performed on the Lactobacillus acidophilus SBT2062 (FERM P-10730) wild type strain.
(Method)
PBS (-) buffer solution (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 3% bovine bile powder (manufactured by OXOID) was inoculated with 1% of each strain that had been cultured in MRS medium and washed at 37 ° C for 3 hours and 6 hours. Time processed. The suspension after the treatment was applied to an MRS agar medium, and anaerobic culture was performed at 37 ° C. for 3 days using an anero pack (manufactured by Mitsubishi Gas Chemical Company). After the culture, the viable cell count was measured.
(Test results)
The results are shown in Table 2 and FIG. The sΔhrcA strain showed a high survival rate of about 100 times after 3 hours treatment and about 160 times after 6 hours treatment compared to the wild strain.
以上の結果から、ラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）株において、hrcA遺伝子を破壊することで胆汁酸に対する耐性も向上し、ヒトまたは動物がこの菌体を摂取した際の腸内への到達率が高まると考えられる。 From the above results, in the Lactobacillus acidophilus SBT2062 (FERM P-10730) strain, the resistance to bile acids was also improved by disrupting the hrcA gene, and when humans or animals ingested this bacterial cell, The arrival rate is expected to increase.
（hrcA遺伝子破壊株を用いた醗酵乳の製造と人工消化試験）
プロバイオティクス菌をヒトが摂取する際の一つの形態として、栄養的にも優れている醗酵乳の形態が考えられる。そこで、sΔhrcA株を用いて醗酵乳を調製した。更に、ヒトが摂取した場合のsΔhrcA株の消化液耐性について調べるため、鈴木らの方法（腸内細菌学雑誌，11，11-17（1997））に従い、人工消化試験を行った。
（人工胃液試験方法）
殺菌した醗酵乳ベース（無脂乳固形分9.5％、脂肪3.2％）にL. delbrueckii subsp.bulgaricus ATCC11842株、S. thermophilusATCC19258株を各1％接種し、更にＭＲＳ培地で培養後、洗浄したラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）野生株、またはsΔhrcA株を3％接種し、38℃で3時間醗酵させ、醗酵乳を調製した。調製した醗酵乳、市販牛乳、及び4％ペプシン溶液（和光純薬工業社製）を50：49：1の割合で混合し、塩酸を用いてpHを2.5に調整し、人工胃液混合物を調製した。調製後、37℃で振とう保存し、経時的に生菌数を測定した。なお、生菌数は変法LBS寒天培地（LBS Agar(BBL社製) 84 g、LAB-LEMCO POWDER(OXOID社製) 8 g、酢酸ナトリウム・三水塩 7.5 g、及び水1Lを、115℃15分間滅菌後、酢酸1.8mlを添加して調製する） に塗抹し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行った。培養後、生菌数測定を行った。
(Fermented milk production and artificial digestion test using hrcA gene disruption strain)
One form of human intake of probiotic bacteria is considered to be fermented milk that is nutritionally superior. Therefore, fermented milk was prepared using the sΔhrcA strain. Furthermore, in order to investigate the digestive fluid resistance of the sΔhrcA strain when ingested by humans, an artificial digestion test was performed according to the method of Suzuki et al. (Intestinal Bacteriology, 11, 11-17 (1997)).
(Artificial gastric juice test method)
Inoculated with 1% each of L. delbrueckii subsp. Bulgaricus ATCC11842 strain and S. thermophilus ATCC19258 strain on a sterilized fermented milk base (non-fat milk solid content 9.5%, fat 3.2%), further cultured in MRS medium, washed Lactobacillus -Acidophilus SBT2062 (FERM P-10730) wild strain or s (DELTA) hrcA strain | stump | stock was inoculated 3%, it was fermented at 38 degreeC for 3 hours, and fermented milk was prepared. The prepared fermented milk, commercial milk, and 4% pepsin solution (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed at a ratio of 50: 49: 1, and the pH was adjusted to 2.5 using hydrochloric acid to prepare an artificial gastric juice mixture. . After preparation, it was stored with shaking at 37 ° C., and the viable cell count was measured over time. The number of viable bacteria was 115g at a modified LBS agar medium (LBS Agar (BBL) 84 g, LAB-LEMCO POWDER (OXOID) 8 g, sodium acetate / trihydrate 7.5 g, and 1 L of water) Sterilized for 15 minutes and then added with 1.8 ml of acetic acid), and anaerobic culture was carried out at 37 ° C. for 3 days using Aneropack (Mitsubishi Gas Chemical Co., Ltd.). After culturing, the viable cell count was measured.
（人工腸液試験方法）
牛乳97.6 g、脱脂粉乳1.5 g 、酵母エキス（アサヒビール社製）0.5 g 及びウシ胆汁末（OXOID社製）0.4 gを混合溶解し、95℃で30分間加熱殺菌し、人工腸液基礎培地を調製した。人工腸液基礎培地、25%パンクレアチン（「パンクレアチンF」：天野エンザイム社製）溶液、及び人工胃液試験方法で使用したものと同じpH2.5で１時間処理後の人工胃液混合物（醗酵乳：市販牛乳：4％ペプシン溶液＝50：49：1）を、96：1：3の割合で混合し、人工腸液混合物を調製した。この人工腸液混合物を37℃で嫌気的に保持し、経時的に生菌数を測定した。生菌数測定用培地は、人工胃液試験と同様の変法LBS寒天培地を使用し、人工胃液試験と同様の培養条件にて試験を行った。
(Artificial intestinal fluid test method)
97.6 g of milk, 1.5 g of skim milk powder, 0.5 g of yeast extract (Asahi Breweries) and 0.4 g of bovine bile powder (OXOID) are mixed and dissolved, and heat-sterilized at 95 ° C for 30 minutes to prepare an artificial intestinal juice basic medium did. Artificial intestinal juice basal medium, 25% pancreatin (“Pancreatine F”: Amano Enzyme) solution, and artificial gastric juice mixture (fermented milk: treated with artificial gastric fluid test method for 1 hour at the same pH 2.5) Commercial milk: 4% pepsin solution = 50: 49: 1) was mixed at a ratio of 96: 1: 3 to prepare an artificial intestinal juice mixture. This artificial intestinal juice mixture was kept anaerobically at 37 ° C., and the viable cell count was measured over time. For the viable cell count medium, a modified LBS agar medium similar to the artificial gastric juice test was used, and the test was performed under the same culture conditions as the artificial gastric juice test.
（試験結果）
人工胃液試験の結果を表３、図３に、人工腸液試験の結果を図４に示した。人工胃液試験は、sΔhrcA株は野生株と比較して、3時間処理後では約28倍、6時間処理後では約13倍の生残率を示した。一方、人工腸液試験では、sΔhrcA株と野生株の両株とも減少することなく、菌数を維持していた。
(Test results)
The results of the artificial gastric juice test are shown in Table 3 and FIG. 3, and the results of the artificial intestinal juice test are shown in FIG. In the artificial gastric juice test, the survival rate of the sΔhrcA strain was about 28 times after treatment for 3 hours and about 13 times after treatment for 6 hours compared to the wild strain. On the other hand, in the artificial intestinal fluid test, both the sΔhrcA strain and the wild strain maintained the number of bacteria without decreasing.
以上の結果より、醗酵乳中でもhrcA遺伝子破壊株の消化液耐性が高まることが明らかであり、ヒトまたは動物が本発明の微生物を摂取した場合に、腸内への到達率は高まるといえる。 From the above results, it is clear that the digestive juice resistance of the hrcA gene disruption strain is increased even in fermented milk, and it can be said that when the human or animal ingests the microorganism of the present invention, the arrival rate in the intestine is increased.
実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株を10％還元脱脂乳培地（121℃、10分加熱）で37℃、16時間にて培養し、この培養物を凍結乾燥して粉末化した。 The sΔhrcA (FERM P-19224) strain obtained in Example 1 was cultured in a 10% reduced skim milk medium (121 ° C., heated for 10 minutes) at 37 ° C. for 16 hours, and this culture was lyophilized to a powder. Turned into.
実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株をヨーグルトミックス（生乳に脱脂粉乳を2％添加し、100℃、10分間加熱した）に接種し、紙カップに充填後、41℃で6時間培養し、醗酵乳を調製した。 The sΔhrcA (FERM P-19224) strain obtained in Example 1 was inoculated into a yogurt mix (2% non-fat dry milk was added to raw milk and heated at 100 ° C. for 10 minutes), filled into a paper cup, and then added at 41 ° C. at 6 ° C. Cultured for hours, fermented milk was prepared.
乳脂肪分を2.8％に調整した生乳を75℃15秒間殺菌後、30℃に冷却し、これに塩化カルシウム0.01％、チーズ用スターターCHN-11 Frozen DVS(クリスチャン・ハンセン社製)5g/100L、及び実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株の脱脂乳培養物１％を添加した。1時間静置した後、レンネットSTANDARD PLUS 900(クリスチャン・ハンセン社製)を0.005％添加して攪拌し、25分程度静置してカードを凝固させた。カードナイフでカッティングを行い、ゆっくり攪拌しつつ、38℃まで徐々に加温した。pHが6.1になるまで攪拌したのち、乳清を除き、2.0％の食塩を加えてよく混ぜ合わせ、木綿布を敷いた型に詰めて、1時間予備圧搾を行った。更に反転して、圧搾を2時間行い、完成したチーズを10℃の冷蔵庫で冷却・保存した。 Raw milk adjusted to 2.8% milk fat is sterilized at 75 ° C for 15 seconds, cooled to 30 ° C, calcium chloride 0.01%, cheese starter CHN-11 Frozen DVS (manufactured by Christian Hansen) 5g / 100L, And 1% of skim milk culture of sΔhrcA (FERM P-19224) strain obtained in Example 1 was added. After standing for 1 hour, 0.005% of Rennet STANDARD PLUS 900 (Christian Hansen) was added and stirred, and allowed to stand for about 25 minutes to coagulate the curd. Cutting was performed with a card knife, and the mixture was gradually warmed to 38 ° C. while slowly stirring. After stirring until the pH reached 6.1, whey was removed, 2.0% sodium chloride was added and mixed well, packed in a mold with cotton cloth, and pre-pressed for 1 hour. Further, it was inverted and pressed for 2 hours, and the finished cheese was cooled and stored in a refrigerator at 10 ° C.
実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株をＭＲＳ液体培地（ディフコ社製）５Lに接種後、37℃、18時間静置培養を行った。培養終了後、遠心分離を行い、培養液の1／50量の濃縮菌体を得た。次いで、この濃縮菌体を脱脂粉乳10％、グルタミン酸ソーダ1％を含む分散媒と同量混合し、pH7.0に調整後、凍結乾燥を行った。得られた凍結乾燥物を60メッシュのふるいを通し、凍結乾燥菌末を得た。 The sΔhrcA (FERM P-19224) strain obtained in Example 1 was inoculated into 5 L of MRS liquid medium (manufactured by Difco), and then static culture was performed at 37 ° C. for 18 hours. After completion of the culture, centrifugation was performed to obtain concentrated bacterial cells of 1/50 volume of the culture solution. Next, the same amount of the concentrated cells was mixed with a dispersion medium containing 10% skim milk powder and 1% sodium glutamate, adjusted to pH 7.0, and then lyophilized. The obtained lyophilized product was passed through a 60-mesh sieve to obtain a lyophilized bacterial powder.
10％脱脂乳溶液を90℃で30分間殺菌した後、ホモジナイズし、冷却した。これにスターターとして、実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株の純培養物を3.5％加え、38℃で16時間醗酵させた。別に、蔗糖15％のほかに適量の酸味料、香料、色素を含有する糖液を調合してホモジナイズし、75℃で25分間殺菌後、5℃に冷却し、糖液とした。このようにして得た糖液70部に対して酸乳30部の割合で混合して酸乳飲料を得た。 The 10% skimmed milk solution was sterilized at 90 ° C. for 30 minutes, and then homogenized and cooled. As a starter, 3.5% of a pure culture of the sΔhrcA (FERM P-19224) strain obtained in Example 1 was added and fermented at 38 ° C. for 16 hours. Separately, in addition to 15% sucrose, a sugar solution containing appropriate amounts of acidulant, flavor, and pigment was prepared and homogenized, sterilized at 75 ° C. for 25 minutes, cooled to 5 ° C., and used as a sugar solution. The soy milk beverage was obtained by mixing with 70 parts of the sugar solution thus obtained at a ratio of 30 parts of sour milk.
ビタミンＣ40 g 、ビタミンＣとクエン酸の等量混合物40 g、グラニュー糖45 g、コーンスターチと乳糖の等量混合物60 gに、実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株の脱脂乳培養物の凍結乾燥物を40 g加えて混合した。混合物を袋に詰め、1袋1.5 g のスティック状栄養健康食品を150袋製造した。 Non-fat milk of sΔhrcA (FERM P-19224) strain obtained in Example 1 to 40 g of vitamin C, 40 g of an equal mixture of vitamin C and citric acid, 45 g of granulated sugar, and 60 g of an equal mixture of corn starch and lactose 40 g of the freeze-dried culture was added and mixed. The mixture was packed in a bag to produce 150 bags of 1.5 g of stick-shaped nutritional health food.
収穫したアルファルファ材料草を軽く乾燥し、マウントカッターで15〜25ｍｍに切断し、実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株の純培養物を材料草1 gあたり105 cfuとなるように接種した。ポリサイロを30℃で10日間貯蔵して、サイレージを調製した。 The harvested alfalfa material grass is lightly dried, cut to 15 to 25 mm with a mount cutter, and the pure culture of sΔhrcA (FERM P-19224) strain obtained in Example 1 becomes 10 5 cfu per 1 g of material grass. Inoculated as follows. Polysilos were stored at 30 ° C. for 10 days to prepare silage.
実施例１で得られたsΔhrcA（ＦＥＲＭ Ｐ−１９２２４）株を50 g、ラクトース140g、シュガーエステル8 g、カルボキシメチルセルロース2 gを混合し、圧縮錠剤器（y-5010-Q、富士薬品機械社製）により圧縮(条件1〜4ton)して、1錠1 gの錠剤200個を製造した。 50 g of sΔhrcA (FERM P-19224) strain obtained in Example 1 was mixed with 140 g of lactose, 8 g of sugar ester, and 2 g of carboxymethylcellulose, and a compressed tablet device (y-5010-Q, manufactured by Fuji Pharmaceutical Co., Ltd.). ) (Conditions 1 to 4 tons) to produce 200 1 g 1 g tablets.
（胃酸耐性試験）
実施例１で得られたhrcA遺伝子破壊株（ダブルクロスオーバー）（以下、dΔhrcA株ということもある。）について、胃酸耐性試験を行った。対照として、ラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）野生株についても同様の試験を行った。
（方法）
鈴木らの方法（腸内細菌学雑誌,11,11-17（1997））を参考にして人工胃液試験を行った。ペプシン（和光純薬工業社製）0.4％を含むPBS緩衝液を塩酸でpH3.2に調整したものを人工胃液混合物とし、これにＭＲＳ培地で培養後、洗浄した菌体を3％接種して、3又は6時間、37℃で振とうした。振とう後の懸濁液をＭＲＳ寒天培地に塗布し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行った。培養後、生菌数を測定した。
（試験結果）
結果を表４及び図５に示した。dΔhrcA株は、野生株に比べて3時間処理後では約17倍、6時間処理後では約190倍の高い生残率を示した。
(Gastroacid tolerance test)
The gastric acid resistance test was performed on the hrcA gene disrupted strain (double crossover) obtained in Example 1 (hereinafter sometimes referred to as dΔhrcA strain). As a control, the same test was performed on the Lactobacillus acidophilus SBT2062 (FERM P-10730) wild type strain.
(Method)
An artificial gastric juice test was conducted with reference to the method of Suzuki et al. (Journal of Intestinal Bacteriology, 11, 11-17 (1997)). PBS buffer containing 0.4% pepsin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 3.2 with hydrochloric acid is used as an artificial gastric juice mixture, and this is cultured in MRS medium and then inoculated with 3% of washed cells. Shake at 37 ° C. for 3 or 6 hours. The suspension after shaking was applied to an MRS agar medium, and anaerobic culture was performed at 37 ° C. for 3 days using an anero pack (manufactured by Mitsubishi Gas Chemical Company). After cultivation, the number of viable bacteria was measured.
(Test results)
The results are shown in Table 4 and FIG. The dΔhrcA strain showed a high survival rate of about 17 times after 3 hours treatment and about 190 times after 6 hours treatment compared to the wild strain.
よって、hrcA遺伝子を破壊することにより胃酸に対する耐性が向上することが明らかとなった。よって、ヒトまたは動物がこの菌体を摂取した際の腸内への到達率が高まると考えられる。 Therefore, it was revealed that the resistance to gastric acid is improved by disrupting the hrcA gene. Therefore, it is considered that the rate of reaching the intestine when humans or animals ingest these cells is increased.
（胆汁酸耐性試験）
実施例１で得られたdΔhrcA株について、胆汁酸耐性試験を行った。対照として、ラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）野生株についても同様の試験を行った。
(Bile acid tolerance test)
The dΔhrcA strain obtained in Example 1 was subjected to a bile acid resistance test. As a control, the same test was performed on the Lactobacillus acidophilus SBT2062 (FERM P-10730) wild type strain.
（方法）
ウシ胆汁末（OXOID社製）を6％含むＰＢＳ(-)緩衝液(日水製薬社製)に、ＭＲＳ培地で培養後、洗浄した各菌株を1％接種し、37℃で3時間及び6時間処理した。処理後の懸濁液をＭＲＳ寒天培地に塗布し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行い、培養後、生菌数測定を行った。
(Method)
PBS (-) buffer solution (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 6% bovine bile powder (manufactured by OXOID) was inoculated with 1% of each strain that had been cultured in MRS medium and washed at 37 ° C for 3 hours and 6 hours. Time processed. The suspension after the treatment was applied to an MRS agar medium, and anaerobic culture was performed at 37 ° C. for 3 days using an anero pack (manufactured by Mitsubishi Gas Chemical Company). After the culture, the viable cell count was measured.
（試験結果）
結果を表５、図６に示した。dΔhrcA株は、野生株と比較して、3時間処理後では約158倍、6時間処理後では約46倍の高い生残率を示した。
(Test results)
The results are shown in Table 5 and FIG. The dΔhrcA strain showed a high survival rate of about 158 times after 3 hours treatment and about 46 times after 6 hours treatment compared to the wild strain.
以上の結果から、ラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）株において、hrcA遺伝子を破壊することで胆汁酸に対する耐性も向上し、ヒトまたは動物がこの菌体を摂取した際の腸内への到達率が高まると考えられる。 From the above results, in the Lactobacillus acidophilus SBT2062 (FERM P-10730) strain, the resistance to bile acids was also improved by disrupting the hrcA gene, and when humans or animals ingested this bacterial cell, The arrival rate is expected to increase.
（hrcA遺伝子破壊株を用いた醗酵乳の製造と人工消化試験）
プロバイオティクス菌をヒトが摂取する際の一つの形態として、栄養的にも優れている醗酵乳の形態が考えられる。そこで、dΔhrcA株を用いて醗酵乳を調製した。更に、ヒトが摂取した場合のdΔhrcA株の消化液耐性について調べるため、鈴木らの方法（腸内細菌学雑誌，11，11-17（1997））に従い、人工消化試験を行った。
(Fermented milk production and artificial digestion test using hrcA gene disruption strain)
One form of human intake of probiotic bacteria is considered to be fermented milk that is nutritionally superior. Therefore, fermented milk was prepared using the dΔhrcA strain. Furthermore, in order to investigate the digestive fluid resistance of the dΔhrcA strain when ingested by humans, an artificial digestion test was performed according to the method of Suzuki et al. (Intestinal Bacteriology, 11, 11-17 (1997)).
（人工胃液試験方法）
殺菌した醗酵乳ベース（無脂乳固形分9.5％、脂肪3.2％）にL. delbrueckii subsp.bulgaricus ATCC11842株、S. thermophilusATCC19258株を各1％接種し、更にＭＲＳ培地で培養後、洗浄したラクトバチルス・アシドフィルスＳＢＴ２０６２（ＦＥＲＭ Ｐ−１０７３０）野生株、またはdΔhrcA株を3％接種し、38℃で3時間醗酵させ、醗酵乳を調製した。調製した醗酵乳、市販牛乳、及び4％ペプシン溶液（和光純薬工業社製）を50：49：1の割合で混合し、塩酸を用いてpHを2.5に調整し、人工胃液混合物を調製した。調製後、37℃で振とう保存し、経時的に生菌数を測定した。なお、生菌数は変法LBS寒天培地（LBS Agar(BBL社製) 84 g、LAB-LEMCO POWDER(OXOID社製) 8 g、酢酸ナトリウム・三水塩 7.5 g、及び水1Lを、115℃15分間滅菌後、酢酸1.8mlを添加して調製する） に塗抹し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行った。培養後、生菌数測定を行った。
(Artificial gastric juice test method)
Inoculated with 1% each of L. delbrueckii subsp. Bulgaricus ATCC11842 strain and S. thermophilus ATCC19258 strain on a sterilized fermented milk base (non-fat milk solid content 9.5%, fat 3.2%), further cultured in MRS medium, washed Lactobacillus -Acidophilus SBT2062 (FERM P-10730) wild strain or dΔhrcA strain was inoculated 3%, fermented at 38 ° C. for 3 hours to prepare fermented milk. The prepared fermented milk, commercial milk, and 4% pepsin solution (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed at a ratio of 50: 49: 1, and the pH was adjusted to 2.5 using hydrochloric acid to prepare an artificial gastric juice mixture. . After preparation, it was stored with shaking at 37 ° C., and the viable cell count was measured over time. The number of viable bacteria was 115g at a modified LBS agar medium (LBS Agar (BBL) 84 g, LAB-LEMCO POWDER (OXOID) 8 g, sodium acetate / trihydrate 7.5 g, and 1 L of water) Sterilized for 15 minutes and then added with 1.8 ml of acetic acid), and anaerobic culture was carried out at 37 ° C. for 3 days using Aneropack (Mitsubishi Gas Chemical Co., Ltd.). After culturing, the viable cell count was measured.
（人工腸液試験方法）
牛乳97.6 g、脱脂粉乳1.5 g 、酵母エキス（アサヒビール社製）0.5 g 及びウシ胆汁末（OXOID社製）0.4 gを混合溶解し、95℃で30分間加熱殺菌し、人工腸液基礎培地を調製した。人工腸液基礎培地、25%パンクレアチン（「パンクレアチンF」：天野エンザイム社製）溶液、及び人工胃液試験方法で使用したものと同じpH2.5で1時間処理後の人工胃液混合物（醗酵乳：市販牛乳：4％ペプシン溶液＝50：49：1）を、96：1：3の割合で混合し、人工腸液混合物を調製した。この人工腸液混合物を37℃で嫌気的に保持し、経時的に生菌数を測定した。生菌数測定培地は、人工胃液試験と同様の変法LBS寒天培地を使用し、人工胃液試験と同様の培養条件にて試験を行った。
(Artificial intestinal fluid test method)
97.6 g of milk, 1.5 g of skim milk powder, 0.5 g of yeast extract (Asahi Breweries) and 0.4 g of bovine bile powder (OXOID) are mixed and dissolved, and heat-sterilized at 95 ° C for 30 minutes to prepare an artificial intestinal juice basic medium did. Artificial intestinal juice basal medium, 25% pancreatin (“Pancreatine F”: Amano Enzyme) solution, and artificial gastric juice mixture (fermented milk: after treatment with the same pH 2.5 used in the artificial gastric fluid test method) Commercial milk: 4% pepsin solution = 50: 49: 1) was mixed at a ratio of 96: 1: 3 to prepare an artificial intestinal juice mixture. This artificial intestinal juice mixture was kept anaerobically at 37 ° C., and the viable cell count was measured over time. As the viable cell count medium, a modified LBS agar medium similar to the artificial gastric juice test was used, and the test was performed under the same culture conditions as the artificial gastric juice test.
（試験結果）
人工胃液試験の結果を表６、図７に、人工腸液試験の結果を図８に示した。人工胃液試験では、dΔhrcA株は野生株と比較して、3時間処理後では約45倍、6時間処理後では約16倍の生残率を示した。一方、人工腸液試験では、dΔhrcA株と野生株の両株とも減少することなく、菌数を維持していた。
(Test results)
The results of the artificial gastric juice test are shown in Table 6 and FIG. 7, and the results of the artificial intestinal juice test are shown in FIG. In the artificial gastric juice test, the dΔhrcA strain showed about 45 times survival rate after 3 hours treatment and about 16 times survival rate after 6 hours treatment compared to the wild strain. On the other hand, in the artificial intestinal fluid test, both the dΔhrcA strain and the wild strain maintained the number of bacteria without decreasing.
実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株を10％還元脱脂乳培地（120℃、10分加熱）で37℃、16時間にて培養し、この培養物を凍結乾燥して粉末化した。 The dΔhrcA (FERM P-19337) strain obtained in Example 1 was cultured at 37 ° C. for 16 hours in a 10% reduced skim milk medium (120 ° C., heated for 10 minutes), and this culture was freeze-dried and powdered Turned into.
実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株をヨーグルトミックス（生乳に脱脂粉乳を2％添加し、100℃、10分間加熱した）に接種し、紙カップに充填後、42℃で6時間培養し、醗酵乳を調製した。 The dΔhrcA (FERM P-19337) strain obtained in Example 1 was inoculated into a yogurt mix (2% non-fat dry milk was added to raw milk and heated at 100 ° C. for 10 minutes), filled in a paper cup, and then put into 6 at 42 ° C. Cultured for hours, fermented milk was prepared.
乳脂肪分を3.0％に調整した生乳を75℃、20分間殺菌後、30℃に冷却し、これに塩化カルシウム0.01％、チーズ用スターターCHN-11 Frozen DVS(クリスチャン・ハンセン社製)5g/100L、及び実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株の脱脂乳培養物１％を添加した。1時間静置した後、レンネットSTANDARD PLUS 900(クリスチャン・ハンセン社製)を0.005％添加して攪拌し、25分程度静置してカードを凝固させた。カードナイフでカッティングを行い、ゆっくり攪拌しつつ、38℃まで徐々に加温した。pHが6.0になるまで攪拌したのち、乳清を除き、2.0％の食塩を加えてよく混ぜ合わせ、木綿布を敷いた型に詰めて、1時間予備圧搾を行った。更に反転して、圧搾を2時間行い、完成したチーズを10℃の冷蔵庫で冷却・保存した。 Raw milk adjusted to 3.0% milk fat is sterilized at 75 ° C for 20 minutes, cooled to 30 ° C, calcium chloride 0.01%, cheese starter CHN-11 Frozen DVS (manufactured by Christian Hansen) 5g / 100L And 1% of skim milk culture of dΔhrcA (FERM P-19337) strain obtained in Example 1 was added. After standing for 1 hour, 0.005% of Rennet STANDARD PLUS 900 (Christian Hansen) was added and stirred, and allowed to stand for about 25 minutes to coagulate the curd. Cutting was performed with a card knife, and the mixture was gradually warmed to 38 ° C. while slowly stirring. After stirring until pH was 6.0, whey was removed, 2.0% salt was added and mixed well, packed in a mold laid with cotton cloth, and pre-pressed for 1 hour. Further, it was inverted and pressed for 2 hours, and the finished cheese was cooled and stored in a refrigerator at 10 ° C.
実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株をＭＲＳ液体培地（ディフコ社製）５Lに接種後、36℃、19時間静置培養を行った。培養終了後、遠心分離を行い、培養液の1／50量の濃縮菌体を得た。次いで、この濃縮菌体を脱脂粉乳10％、グルタミン酸ソーダ1％を含む分散媒と同量混合し、pH7.0に調整後、凍結乾燥を行った。得られた凍結乾燥物を60メッシュのふるいを通し、凍結乾燥菌末を得た。 The dΔhrcA (FERM P-19337) strain obtained in Example 1 was inoculated into 5 L of an MRS liquid medium (manufactured by Difco), followed by stationary culture at 36 ° C. for 19 hours. After completion of the culture, centrifugation was performed to obtain concentrated bacterial cells of 1/50 volume of the culture solution. Next, the same amount of the concentrated cells was mixed with a dispersion medium containing 10% skim milk powder and 1% sodium glutamate, adjusted to pH 7.0, and then lyophilized. The obtained lyophilized product was passed through a 60-mesh sieve to obtain a lyophilized bacterial powder.
10％脱脂乳溶液を90℃で28分間殺菌した後、ホモジナイズし、冷却した。これにスターターとして実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株の純培養物を3.5％加え、38℃で18時間醗酵し、酸乳とした。別に、蔗糖15％のほかに適量の酸味料、香料、色素を含有する糖液を調合してホモジナイズし、75℃で28分間殺菌後、5℃に冷却し、糖液とした。このようにして得た糖液70部に対して酸乳30部の割合で混合して酸乳飲料を得た。 The 10% skimmed milk solution was sterilized at 90 ° C. for 28 minutes, and then homogenized and cooled. To this, 3.5% of a pure culture of the dΔhrcA (FERM P-19337) strain obtained in Example 1 was added as a starter and fermented at 38 ° C. for 18 hours to obtain sour milk. Separately, in addition to 15% sucrose, a sugar solution containing appropriate amounts of acidulant, flavor, and pigment was prepared and homogenized, sterilized at 75 ° C. for 28 minutes, cooled to 5 ° C., and used as a sugar solution. The soy milk beverage was obtained by mixing with 70 parts of the sugar solution thus obtained at a ratio of 30 parts of sour milk.
ビタミンＣ40 g 、ビタミンＣとクエン酸の等量混合物40 g、グラニュー糖45 g、コーンスターチと乳糖の等量混合物60 gに、実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株の脱脂乳培養物の凍結乾燥物を40 g加えて混合した。混合物を袋に詰め、1袋1.5 g のスティック状栄養健康食品を150袋製造した。 Non-fat milk of dΔhrcA (FERM P-19337) strain obtained in Example 1 to 40 g of vitamin C, 40 g of an equal mixture of vitamin C and citric acid, 45 g of granulated sugar, and 60 g of an equal mixture of corn starch and lactose 40 g of the freeze-dried culture was added and mixed. The mixture was packed in a bag to produce 150 bags of 1.5 g of stick-shaped nutritional health food.
収穫したアルファルファ材料草を軽く乾燥し、マウントカッターで15〜25ｍｍに切断し、実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株の純培養物を材料草1 gあたり105 cfuとなるように接種した。ポリサイロを30℃で10日間貯蔵して、サイレージを調製した。 The harvested alfalfa material grass is lightly dried, cut to 15 to 25 mm with a mount cutter, and the pure culture of dΔhrcA (FERM P-19337) strain obtained in Example 1 becomes 10 5 cfu per 1 g of material grass. Inoculated as follows. Polysilos were stored at 30 ° C. for 10 days to prepare silage.
実施例１で得られたdΔhrcA（ＦＥＲＭ Ｐ−１９３３７）株を50 g、ラクトース140 g、シュガーエステル8 g、カルボキシメチルセルロース2 gを混合し、圧縮錠剤器（y-5010-Q、富士薬品機械社製）により圧縮(条件1〜4ton)して、1錠1gの錠剤200個を製造した。 50 g of dΔhrcA (FERM P-19337) strain obtained in Example 1 was mixed with 140 g of lactose, 8 g of sugar ester, and 2 g of carboxymethylcellulose, and a compressed tablet device (y-5010-Q, Fuji Pharmaceutical Machinery Co., Ltd.). The product was compressed (conditions 1 to 4 tons) to produce 200 tablets of 1 g per tablet.
（ラクトバチルス・ガセリＳＢＴ２０５５hrcA遺伝子破壊株の取得
（hrcA遺伝子の取得と導入プラスミドの構築））
プロバイオティクス菌として整腸作用等の生理効果が報告されているラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）の染色体から、hrcA遺伝子周辺配列をＰＣＲ法により増幅した。ＰＣＲに用いたプライマーは、現在解析が進められていて、その大部分がすでに一般公開されているラクトバチルス・ガセリのシーケンス情報（NCBI Microbial Genomes Annotation Projectによる）を元に作製した。すなわち、以下に示すプライマーを合成して用いた（配列表参照）。
＜hrcA上流配列＞
5’側：5’-GGAGACCCTAATCAGGAAGATGGG-3’（配列番号5）
3’側：5’-GCACTTGAAAGCTTGACAGGCAGTTAATTC -3’（配列番号6）
＜hrcA下流配列＞
5’側：5’-GATGCTATTTCAAGCTTGATTGGTTTTAATCC-3’（配列番号7）
3’側：5’-GGCAACTACAGAAGGAGTTGTACG-3’（配列番号8）
すなわち、hrcA上流配列の5’側と3’側及びhrcA下流配列の5’側と3’側の配列からプライマーを合成した。これらのプライマーを用いたＰＣＲの増幅断片を制限酵素(HindIII)処理したのち、エリスロマイシン耐性遺伝子を含むpG+host6ベクターのマルチクローニングサイトに上流−下流の順に挿入した。こうして、内部配列が欠落したhrcA遺伝子（hrcA’遺伝子）を含むプラスミドpG+host6::hrcA’が得られ、これを大腸菌に導入し、大量調製、精製を行った。
(Acquisition of Lactobacillus gasseri SBT2055hrcA gene disruption strain (Acquisition of hrcA gene and construction of introduced plasmid))
The sequence around the hrcA gene was amplified by PCR from the chromosome of Lactobacillus gasseri SBT2055 (FERM P-15535), which has been reported to have physiological effects such as intestinal regulation as a probiotic bacterium. Primers used for PCR were currently analyzed, and most of them were prepared based on Lactobacillus gasseri sequence information (according to NCBI Microbial Genomes Annotation Project) that has already been made public. That is, the following primers were synthesized and used (see Sequence Listing).
<HrcA upstream sequence>
5 'side: 5'-GGAGACCCTAATCAGGAAGATGGG-3' (SEQ ID NO: 5)
3 'side: 5'-GCACTTGAAAGCTTGACAGGCAGTTAATTC-3' (SEQ ID NO: 6)
<HrcA downstream sequence>
5 'side: 5'-GATGCTATTTCAAGCTTGATTGGTTTTAATCC-3' (SEQ ID NO: 7)
3 'side: 5'-GGCAACTACAGAAGGAGTTGTACG-3' (SEQ ID NO: 8)
That is, primers were synthesized from the 5 ′ side and 3 ′ side of the hrcA upstream sequence and the 5 ′ side and 3 ′ side of the hrcA downstream sequence. PCR amplified fragments using these primers were treated with a restriction enzyme (HindIII), and then inserted into the multiple cloning site of the pG + host6 vector containing the erythromycin resistance gene in the order of upstream-downstream. Thus, plasmid pG + host6 :: hrcA ′ containing hrcA gene lacking the internal sequence (hrcA ′ gene) was obtained and introduced into Escherichia coli for mass preparation and purification.
（プラスミドの導入と染色体への挿入（シングルクロスオーバー））
上記のようにして作成したプラスミドを実施例１で行った方法と同様にして、ラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）株に導入した。次に実施例１で行った方法と同様にして相同組換えを誘発し、染色体上に抗生物質耐性が付与されたhrcA遺伝子破壊株（シングルクロスオーバー）を取得した。
(Introduction of plasmid and insertion into chromosome (single crossover))
The plasmid prepared as described above was introduced into the Lactobacillus gasseri SBT2055 (FERM P-15535) strain in the same manner as in Example 1. Next, homologous recombination was induced in the same manner as in Example 1, and an hrcA gene disrupted strain (single crossover) imparted with antibiotic resistance on the chromosome was obtained.
（挿入遺伝子の脱落とhrcA遺伝子破壊株の取得（ダブルクロスオーバー））
更にこのhrcA遺伝子破壊株について、形質を安定させるために実施例１で行った方法と同様にしてプラスミドを脱落させ、プラスミド由来のhrcA’遺伝子と野生株のhrcA遺伝子を入れ替えることで、hrcA遺伝子破壊株（ダブルクロスオーバー）を取得した。本発明において得たラクトバチルス・ガセリＳＢＴ２０５５hrcA遺伝子破壊株（ダブルクロスオーバー）は、ラクトバチルス・ガセリＳＢＴ１０８４２と命名して、独立行政法人産業技術総合研究所特許生物寄託センターに寄託したところ、バイオセーフティーレベル２に該当するとして受託拒否となっているが、受託拒否証明を得ている。本微生物株は本出願人の研究所に保管されており、独立行政法人産業技術総合研究所特許生物寄託センターの管理と同様に、出願時に第三者に分譲する用意があったし、引き続き分譲する用意がある。
(Deletion of inserted gene and acquisition of hrcA gene disruption strain (double crossover))
Further, for this hrcA gene disrupted strain, the plasmid was removed in the same manner as in Example 1 in order to stabilize the trait, and the hrcA gene disruption was performed by replacing the plasmid-derived hrcA 'gene with the wild-type hrcA gene. Acquired stock (double crossover). The Lactobacillus gasseri SBT2055hrcA gene disruption strain (double crossover) obtained in the present invention was named Lactobacillus gasseri SBT10842 and deposited at the National Institute of Advanced Industrial Science and Technology Patent Biodeposition Center. Although it has been rejected as a categorized as No. 2, the refusal of acceptance has been obtained. This microbial strain is stored in the applicant's research institute and, like the management of the National Institute of Advanced Industrial Science and Technology, the Patent Organism Depositary Center was prepared to be distributed to a third party at the time of filing. Ready to do.
（胃酸耐性試験）
実施例１８で得られたラクトバチルス・ガセリＳＢＴ２０５５hrcA遺伝子破壊株（ダブルクロスオーバー）（以下、dΔhrcA株ということもある。）について、胃酸耐性試験を行った。対照として、ラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）野生株についても同様の試験を行った。
（方法）
鈴木らの方法（腸内細菌学雑誌,11,11-17（1997））を参考にして人工胃液試験を行った。ペプシン（和光純薬工業社製）0.4％を含む12.6％脱脂乳培地を塩酸でpH2.5に調整したものを人工胃液混合物とし、これにＭＲＳ培地で培養後、洗浄した菌体を3％接種して、37℃で6時間振とうした。振とう後の懸濁液をＭＲＳ寒天培地に塗布し、37℃で3日間、アネロパック（三菱ガス化学社製）を用いて嫌気培養を行った。培養後、生菌数を測定した。
(Gastroacid tolerance test)
The Lactobacillus gasseri SBT2055hrcA gene disruption strain (double crossover) obtained in Example 18 (hereinafter sometimes referred to as dΔhrcA strain) was subjected to a gastric acid resistance test. As a control, the same test was performed for the Lactobacillus gasseri SBT2055 (FERM P-15535) wild type strain.
(Method)
An artificial gastric juice test was conducted with reference to the method of Suzuki et al. (Journal of Intestinal Bacteriology, 11, 11-17 (1997)). A 12.6% skim milk medium containing 0.4% pepsin (manufactured by Wako Pure Chemical Industries, Ltd.) adjusted to pH 2.5 with hydrochloric acid is used as an artificial gastric juice mixture, which is then inoculated with MRS medium and washed with 3% of washed cells. And shaken at 37 ° C. for 6 hours. The suspension after shaking was applied to an MRS agar medium, and anaerobic culture was performed at 37 ° C. for 3 days using an anero pack (manufactured by Mitsubishi Gas Chemical Company). After cultivation, the number of viable bacteria was measured.
（試験結果）
結果を表７及び図９に示した。dΔhrcA株は、野生株に比べて6時間処理後で約10倍の高い生残率を示した。
よって、hrcA遺伝子を破壊することにより、胃酸に対する耐性が向上することが明らかとなった。よって、ヒトまたは動物がこの菌体を摂取した際の腸内への到達率が高まると考えられる。
(Test results)
The results are shown in Table 7 and FIG. The dΔhrcA strain showed a survival rate about 10 times higher after treatment for 6 hours than the wild strain.
Thus, it was revealed that the resistance to gastric acid is improved by disrupting the hrcA gene. Therefore, it is considered that the rate of reaching the intestine when humans or animals ingest these cells is increased.
実施例１８で得られたラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）dΔhrcA株をヨーグルトミックス（生乳に脱脂粉乳を2％添加し、100℃、10分間加熱した）に接種し、紙カップに充填後、42℃で6時間培養し、醗酵乳を調製した。 Lactobacillus gasseri SBT2055 (FERM P-15535) dΔhrcA strain obtained in Example 18 was inoculated into yogurt mix (2% skim milk powder was added to raw milk and heated at 100 ° C. for 10 minutes), and after filling into a paper cup And cultured at 42 ° C. for 6 hours to prepare fermented milk.
乳脂肪分を3.0％に調整した生乳を75℃20分間殺菌後、30℃に冷却し、これに塩化カルシウム0.01％、チーズ用スターターCHN-11 Frozen DVS(クリスチャン・ハンセン社製)5g/100L、及び実施例１８で得られたラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）dΔhrcA株の脱脂乳培養物1％を添加した。1時間静置した後、レンネットSTANDARD PLUS 900(クリスチャン・ハンセン社製)を0.005％添加して攪拌し、25分程度静置してカードを凝固させた。カードナイフでカッティングを行い、ゆっくり攪拌しつつ、38℃まで徐々に加温した。pHが6.0になるまで攪拌したのち、乳清を除き、2.0％の食塩を加えてよく混ぜ合わせ、木綿布を敷いた型に詰めて、1時間予備圧搾を行った。更に反転して、圧搾を2時間行い、完成したチーズを10℃の冷蔵庫で冷却・保存した。 Raw milk adjusted to 3.0% milk fat is sterilized at 75 ° C for 20 minutes, cooled to 30 ° C, calcium chloride 0.01%, cheese starter CHN-11 Frozen DVS (manufactured by Christian Hansen) 5g / 100L, And 1% of skim milk culture of Lactobacillus gasseri SBT2055 (FERM P-15535) dΔhrcA strain obtained in Example 18 was added. After standing for 1 hour, 0.005% of Rennet STANDARD PLUS 900 (Christian Hansen) was added and stirred, and allowed to stand for about 25 minutes to coagulate the curd. Cutting was performed with a card knife, and the mixture was gradually warmed to 38 ° C. while slowly stirring. After stirring until pH was 6.0, whey was removed, 2.0% salt was added and mixed well, packed in a mold laid with cotton cloth, and pre-pressed for 1 hour. Further, it was inverted and pressed for 2 hours, and the finished cheese was cooled and stored in a refrigerator at 10 ° C.
実施例１８で得られたラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）dΔhrcA株をＭＲＳ液体培地（ディフコ社製）５Lに接種後、36℃、19時間静置培養を行った。培養終了後、遠心分離を行い、培養液の1／50量の濃縮菌体を得た。次いで、この濃縮菌体を脱脂粉乳10％、グルタミン酸ソーダ1％を含む分散媒と同量混合し、pH7.0に調整後、凍結乾燥を行った。得られた凍結乾燥物を60メッシュのふるいに通し、凍結乾燥菌末を得た。 The Lactobacillus gasseri SBT2055 (FERM P-15535) dΔhrcA strain obtained in Example 18 was inoculated into 5 L of MRS liquid medium (manufactured by Difco), and then static culture was performed at 36 ° C. for 19 hours. After completion of the culture, centrifugation was performed to obtain concentrated bacterial cells of 1/50 volume of the culture solution. Next, the same amount of the concentrated cells was mixed with a dispersion medium containing 10% skim milk powder and 1% sodium glutamate, adjusted to pH 7.0, and then lyophilized. The obtained lyophilized product was passed through a 60-mesh sieve to obtain a lyophilized bacterial powder.
10％脱脂乳溶液を90℃で28分間殺菌した後、ホモジナイズし、冷却した。これにスターターとして、実施例１８で得られたラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）dΔhrcA株の純培養物を3.5％加え、36℃で18時間醗酵し、酸乳とした。別に、蔗糖15％のほかに適量の酸味料、香料、色素を含有する糖液を調合してホモジナイズし、75℃で28分間殺菌後、5℃に冷却し、糖液とした。このようにして得た糖液70部に対して酸乳30部の割合で混合して酸乳飲料を得た。 The 10% skimmed milk solution was sterilized at 90 ° C. for 28 minutes, and then homogenized and cooled. As a starter, 3.5% of a pure culture of Lactobacillus gasseri SBT2055 (FERM P-15535) dΔhrcA strain obtained in Example 18 was added and fermented at 36 ° C. for 18 hours to obtain sour milk. Separately, in addition to 15% sucrose, a sugar solution containing appropriate amounts of acidulant, flavor, and pigment was prepared and homogenized, sterilized at 75 ° C. for 28 minutes, cooled to 5 ° C., and used as a sugar solution. The soy milk beverage was obtained by mixing with 70 parts of the sugar solution thus obtained at a ratio of 30 parts of sour milk.
実施例１８で得られたラクトバチルス・ガセリＳＢＴ２０５５（ＦＥＲＭ Ｐ−１５５３５）dΔhrcA株を50 g、ラクトース140 g、シュガーエステル8 g、カルボキシメチルセルロース2 gを混合し、圧縮錠剤器（y-5010-Q、富士薬品機械社製）により圧縮(条件1〜4ton)して、錠剤200個を製造した。 50 g of Lactobacillus gasseri SBT2055 (FERM P-15535) dΔhrcA strain obtained in Example 18 was mixed with 140 g of lactose, 8 g of sugar ester, and 2 g of carboxymethylcellulose, and a compressed tablet device (y-5010-Q , Manufactured by Fuji Pharmaceutical Machinery Co., Ltd.) (conditions 1 to 4 tons) to produce 200 tablets.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2007082468A (en) * | 2005-09-22 | 2007-04-05 | National Agriculture & Food Research Organization | Microbial preparations for feed preparation and their use |
| KR200448046Y1 (en) * | 2009-05-22 | 2010-03-10 | 라군란 | Advanced Automatic Paper Feeding Device of Flat Plate Launcher |
| JP2010057406A (en) * | 2008-09-03 | 2010-03-18 | National Agriculture & Food Research Organization | Gene-deficient bacterium strain not transgenic and having no exogenous gene introduced, method of production and selection thereof |
| JP2014121281A (en) * | 2012-12-20 | 2014-07-03 | Snow Brand Milk Products Co Ltd | Microorganism having improved resistance to oxidation stress and preparation method of it |
| WO2023163015A1 (en) | 2022-02-22 | 2023-08-31 | 株式会社明治 | Lactic acid bacteria, lactic acid bacteria composition, production method for lactic acid bacteria, method for improving acid resistance of lactic acid bacteria, screening method for lactic acid bacteria, and production method for fermented milk |
| KR20250110196A (en) | 2022-11-22 | 2025-07-18 | 가부시키가이샤 야쿠르트 혼샤 | Method for improving gastric acid bile acid resistance of Lacticazeibacillus paracasei |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007082468A (en) * | 2005-09-22 | 2007-04-05 | National Agriculture & Food Research Organization | Microbial preparations for feed preparation and their use |
| JP2010057406A (en) * | 2008-09-03 | 2010-03-18 | National Agriculture & Food Research Organization | Gene-deficient bacterium strain not transgenic and having no exogenous gene introduced, method of production and selection thereof |
| KR200448046Y1 (en) * | 2009-05-22 | 2010-03-10 | 라군란 | Advanced Automatic Paper Feeding Device of Flat Plate Launcher |
| JP2014121281A (en) * | 2012-12-20 | 2014-07-03 | Snow Brand Milk Products Co Ltd | Microorganism having improved resistance to oxidation stress and preparation method of it |
| WO2023163015A1 (en) | 2022-02-22 | 2023-08-31 | 株式会社明治 | Lactic acid bacteria, lactic acid bacteria composition, production method for lactic acid bacteria, method for improving acid resistance of lactic acid bacteria, screening method for lactic acid bacteria, and production method for fermented milk |
| KR20250110196A (en) | 2022-11-22 | 2025-07-18 | 가부시키가이샤 야쿠르트 혼샤 | Method for improving gastric acid bile acid resistance of Lacticazeibacillus paracasei |
| EP4624566A1 (en) | 2022-11-22 | 2025-10-01 | Kabushiki Kaisha Yakult Honsha | Method for improving gastric acid/bile acid-resisting properties of lacticaseibacillus paracasei |
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