JPH05168467A - Phage-resistant lactic acid bacterium and production of soy sauce using the same - Google Patents
Phage-resistant lactic acid bacterium and production of soy sauce using the sameInfo
- Publication number
- JPH05168467A JPH05168467A JP3353939A JP35393991A JPH05168467A JP H05168467 A JPH05168467 A JP H05168467A JP 3353939 A JP3353939 A JP 3353939A JP 35393991 A JP35393991 A JP 35393991A JP H05168467 A JPH05168467 A JP H05168467A
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- Japan
- Prior art keywords
- phage
- lactic acid
- soy sauce
- plate
- colonies
- Prior art date
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- Soy Sauces And Products Related Thereto (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明はファージ耐性乳酸菌、並
びに麹及び／又は諸味にファージ耐性醤油乳酸菌を添加
する醤油の製造法の改良に関し、特に信頼性の高いファ
ージ耐性乳酸菌及びこれを用いて高品質の醤油を安定し
て得る醤油の製造法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for producing phage-resistant lactic acid bacteria, and soy sauce by adding phage-resistant soy sauce lactic acid bacteria to koji and / or moromi. The present invention relates to a method for producing soy sauce that stably obtains high quality soy sauce.
【０００２】[0002]
【従来の技術】醤油諸味の発酵において醤油乳酸菌［ペ
ディオコッカス・ハロフィルス（Pediococcus halophil
us）］は、糖から乳酸を生成して、諸味のｐＨを好まし
い値にまで低下させる他、アミノ酸類の分解変換、有機
酸の分解生成および還元作用など醤油の品質にとって重
要な働きをなす。従って香味共にバランスのとれた高品
質の醤油を製造するには、優れた性質を持つ醤油乳酸菌
による乳酸発酵を安定して確実に行うことが必要であ
る。2. Description of the Related Art In the fermentation of soy sauce moromi, soy sauce lactic acid bacteria [Pediococcus halophil
[us]] produces lactic acid from sugar, lowers the pH of moromi to a preferable value, and also plays an important role in the quality of soy sauce by decomposing and converting amino acids, decomposing and producing organic acids, and reducing action. Therefore, in order to produce a high-quality soy sauce having a well-balanced flavor, it is necessary to stably and surely perform lactic acid fermentation with soy sauce lactic acid bacteria having excellent properties.
【０００３】伝統的な醤油の製造法においては、諸味発
酵は通常開放系で行われており、乳酸菌等の微生物の人
為的添加は殆ど行われず、諸味中で活動する醤油乳酸菌
は総てその醸造場、施設、器具等に自然に住み着いてい
る菌群（ナチュラル・フローラ）の自然混入に委ねられ
ていた。このナチュラルフローラは非常に多種多様であ
り自然混入した乳酸菌の中には醤油の品質上、必ずしも
好ましくない性質の菌が含まれることも有る。また地域
により、あるいは時と共にその内容も変化するので、醸
造場所により製品品質に差がついたり、年間を通じて品
質の一定した醤油が得られない等の欠点があった。In the traditional method of producing soy sauce, moromi fermentation is usually carried out in an open system, and artificial addition of microorganisms such as lactic acid bacteria is rarely carried out, and all lactic acid bacteria of soy sauce active in moromi are brewed. It was entrusted to the natural mixture of bacteria (natural flora) that naturally settle in places, facilities and equipment. This natural flora is extremely diverse, and naturally mixed lactic acid bacteria may include bacteria having properties that are not always desirable in terms of soy sauce quality. In addition, since the contents change depending on the region or with time, there are drawbacks such as differences in product quality depending on the brewing place, and the fact that soy sauce of constant quality cannot be obtained throughout the year.
【０００４】この様な見地から、地域的或いは時間的な
制限に囚われずに常に品質の一定した醸造醤油を製造す
ることを目指して、予め選択された、または特に育種し
た、性質の優秀な醤油乳酸菌を人為的に、麹及び／また
は諸味に添加し、仕込み工程における乳酸発酵を安定し
て行わせようとすることが広く行われるようになった。From this point of view, soy sauce of excellent quality, preselected or particularly bred, with the aim of producing brewed soy sauce of consistent quality regardless of regional or time restrictions It has become widespread to artificially add lactic acid bacteria to koji and / or moromi to attempt stable lactic acid fermentation in the preparation step.
【０００５】しかしながら、通常の醤油の製造法におい
ては、無殺菌の麹を使用し、微生物学的に開放系の容器
で諸味管理を行っているために醤油諸味の乳酸発酵には
特定不能の複雑なトラブル要因が入り込みやすく、添加
した乳酸菌を長期にわたり安定して生育させ、好ましい
乳酸発酵を続けることは非常に難しい状況にあった。However, in the usual method for producing soy sauce, unsterilized koji is used and moromi is controlled in a microbiologically open container. Therefore, lactic acid fermentation of soy sauce moromi is complicated and cannot be specified. However, it was very difficult to grow the added lactic acid bacterium stably for a long period of time and to continue preferable lactic acid fermentation.
【０００６】そこで、本発明者らは、添加した醤油乳酸
菌を常に安定して生育させ、内容の一定した乳酸発酵を
確実に行うことを目的として、鋭意研究を重ねた結果、
醤油の諸味から、醤油乳酸菌に感染し、これを溶菌する
多種類のファージを検出し、このファージによって添加
した乳酸菌が溶菌あるいは増殖阻害を受けることが乳酸
発酵トラブルの一大要因となっていることを見出し、こ
の要因を解消すれば上記目的が達成できることを知り、
先に、麹及び／又は諸味に醤油乳酸菌を添加する醤油の
製造法において、醤油乳酸菌としてファージ耐性醤油乳
酸菌を使用することを特徴とする醤油の製造法（特開昭
６３ー２１６４４９号公報参照）を開発した。[0006] Therefore, as a result of intensive studies, the present inventors have conducted intensive studies for the purpose of constantly growing the added soy sauce lactic acid bacteria in a stable manner to ensure lactic acid fermentation with a constant content.
One of the major causes of lactic acid fermentation trouble is that from the moromi of soy sauce, many types of phages that infect and lyse soy sauce lactic acid bacteria are detected, and the lactic acid bacteria added by these phages undergo lysis or growth inhibition. And knowing that the above objective can be achieved by eliminating this factor,
First, in a method for producing soy sauce in which soy sauce lactic acid bacteria are added to koji and / or moromi, a method for producing soy sauce characterized in that phage-resistant soy sauce lactic acid bacteria are used as the soy sauce lactic acid bacteria (see JP-A-63-216449). Was developed.
【０００７】[0007]
【発明が解決しようとする課題】この方法において、フ
ァージ耐性醤油乳酸菌の造成は、醤油乳酸菌とファー
ジを接触処理し、自然突然変異によるファージ耐性菌を
分離するか、または醤油乳酸菌に突然変異処理を行
い、これとファージを接触処理し、得られた菌株の中か
ら他の性質は親株と同じでファージ耐性のみ強い［すな
わちファージに感受性の醤油乳酸菌（親株）が全く生育
できないくらいの高濃度のファージ存在下でも寒天平板
培養すると生育し、コロニーを形成する］耐性株を分離
するものである。In this method, the phage-resistant soy sauce lactic acid bacterium is constructed by contacting the soy sauce lactic acid bacterium with the phage and separating the phage-resistant bacterium by natural mutation, or mutating the soy sauce lactic acid bacterium. The resulting strains were contacted with each other, and other properties were the same as those of the parent strain, and only the phage resistance was strong [ie, soy sauce lactic acid bacteria (parent strain) sensitive to phages had a high concentration of phages that could not grow at all). It grows when agar-plated in the presence of agar and forms colonies.] It isolates resistant strains.
【０００８】しかしながら、こうして得たファージ耐性
醤油乳酸菌は、該乳酸菌の遺伝子の中にファージ遺伝子
を組込んだ、いわゆる溶原菌である可能性があり、継代
培養の繰返しで組込まれたファージ遺伝子が発現し、培
養液中にファージを放出することがあり、この様な場合
この後の乳酸醗酵を続けることが難しくなる。即ち、こ
の方法によって造成されたファージ耐性醤油乳酸菌は信
頼性に欠ける欠点を有していた。However, the thus obtained phage-resistant soy sauce lactic acid bacterium may be a so-called lysogen, in which the phage gene is incorporated into the gene of the lactic acid bacterium, and the phage gene incorporated by repeated subculture is used. May be expressed and the phage may be released into the culture medium, and in such a case, it becomes difficult to continue the lactic acid fermentation thereafter. That is, the phage-resistant soy sauce lactic acid bacterium produced by this method had a drawback of lacking reliability.
【０００９】[0009]
【発明を解決するための手段】そこで 本発明者らは、
信頼性の高いファージ耐性醤油乳酸菌を造成し、この乳
酸菌を使用して高品質の醤油を安定して得る醤油の製造
法を開発すべく鋭意研究を重ねた結果、ついに本発明を
完成した。[Means for Solving the Invention]
The present invention was finally completed as a result of earnestly researching to produce a highly reliable phage-resistant soy sauce lactic acid bacterium, and to develop a method for producing soy sauce by which the high-quality soy sauce is stably obtained using this lactic acid bacterium.
【００１０】即ち本発明は、多数の乳酸菌コロニーを形
成したマスター平板を、滅菌した担体表面に軽く押しつ
け、該マスター平板上のコロニーをプリントし、次いで
この面に予めファージ懸濁液を塗布した平板培地を軽く
押しつけ、前記担体表面上にプリントされたコロニーを
写し取ってレプリカ平板を得、これを培養した後、前記
マスター平板と該レプリカ平板に形成したコロニーを対
比し、該レプリカ平板に形成したコロニーに対応する前
記マスター平板上のコロニーを分離することにより得ら
れたファージ耐性乳酸菌であり、また本発明は、麹及び
／又は諸味にファージ耐性醤油乳酸菌を添加する醤油の
製造法において、該ファージ耐性醤油乳酸菌として、上
記ファージ耐性乳酸菌を使用することを特徴とする醤油
の製造法である。That is, according to the present invention, a master plate on which a large number of lactic acid bacterium colonies are formed is lightly pressed against the surface of a sterilized carrier, the colonies on the master plate are printed, and then the plate is preliminarily coated with a phage suspension. Lightly press the medium, copy the colonies printed on the carrier surface to obtain a replica plate, and after culturing this, compare the master plate and the colonies formed on the replica plate, colonies formed on the replica plate Which is a phage-resistant lactic acid bacterium obtained by separating colonies on the master plate corresponding to, and the present invention is a method for producing soy sauce in which phage-resistant soy sauce lactic acid bacterium is added to koji and / or moromi, A method for producing soy sauce, which comprises using the phage-resistant lactic acid bacterium as a soy sauce lactic acid bacterium.
【００１１】以下、本発明を詳細に説明する。本発明に
於けるファージ耐性醤油乳酸菌は、醤油乳酸菌に感染
し、これを溶菌させるファージに対して抵抗性のある乳
酸菌で以下の方法によって得ることが出来る。The present invention will be described in detail below. The phage-resistant soy sauce lactic acid bacterium of the present invention is a lactic acid bacterium that is resistant to phages that infect and lyse soy sauce lactic acid bacterium and can be obtained by the following method.
【００１２】１．［ファージの分離］ 醤油乳酸菌のファージは、食塩を１０〜２０％含有させ
る以外は通常のファージの検出、分離に用いられる培地
及び方法に準じて分離することが出来る（昭和４５年１
０月１０日、朝倉書店発行、植村定次郎、相田洗 編集
「発酵と微生物１１」第７５〜９２頁「ファージ実験
法」参照）。例えば、任意の醤油乳酸菌に感染するファ
ージを検索するには、ファージの存在が推定される試料
あるいは野性乳酸菌が豊富に生育していると思われる天
然仕込み醤油諸味を濾過して諸味液汁を得、これを孔径
０．２〜０．４μｍのメンブランフィルターで濾過して
微生物を完全に除去し、ファージ含有無菌濾過液を得
る。これを１０〜２０％食塩水で多段に希釈し、その一
部、例えば０．５ｍｌと特定の醤油乳酸菌の培養液０．
５ｍｌとを混ぜ、この混合液の一部、例えば１００μｌ
を、予め調製しておいた寒天平板培地上に一様に塗布
し、通常の乳酸菌の寒天平板培養法に従い、嫌気的条件
下［Gas Pak(Becton-Dickson社製）法または重層培養
法］で、２０〜３０℃で２〜６日間培養する。塗布した
醤油乳酸菌（指示菌）に感染するファージが存在する場
合には、希釈の段階に応じて溶菌斑［プラーク（Plaqu
e)］が現れるので、これを釣菌し、これを再度同一指示
菌と混ぜて寒天平板培養すれば、再び生じたプラークか
ら前記醤油乳酸菌に感染するファージを分離することが
出来る。総ての醤油乳酸菌に対して常にファージが検出
されるとは言い切れないが、分離源に適当な試料を選ん
で実験を繰返せば、その菌株に感染するファージを取得
することができる。1. [Separation of Phages] Phage of soy sauce lactic acid bacteria can be separated according to a medium and a method used for ordinary detection and separation of phages, except that sodium chloride is contained in an amount of 10 to 20% (1965).
(October 10, published by Asakura Shoten, Sadajiro Uemura, edited by Aidarai "Fermentation and Microorganisms 11", pp. 75-92, "Phage Experimental Method"). For example, to search for a phage that infects any soy sauce lactic acid bacterium, a sample presumed to contain the phage or a naturally-prepared soy sauce moromi that seems to be rich in wild lactic acid bacteria is filtered to obtain a moromi juice. This is filtered through a membrane filter having a pore size of 0.2 to 0.4 μm to completely remove microorganisms, and a phage-containing sterile filtrate is obtained. This is diluted with 10 to 20% saline in multiple stages, and a part of it, for example, 0.5 ml, and a culture solution of a specific soy sauce lactic acid bacterium.
Mix with 5 ml and a portion of this mixture, eg 100 μl
Is evenly spread on an agar plate medium prepared in advance, and according to the usual agar plate culture method of lactic acid bacteria, under anaerobic conditions [Gas Pak (Becton-Dickson) method or multilayer culture method] Incubate at 20-30 ° C for 2-6 days. When there is a phage that infects the applied soy sauce lactic acid bacterium (indicator bacterium), the lytic plaque [Plaqu (Plaqu
e)] appears, and if this is picked up and mixed again with the same indicator bacteria and agar plating is carried out, the phage infecting the soy sauce lactic acid bacterium can be separated from the regenerated plaque. Although it cannot be said that phages are always detected in all the soy sauce lactic acid bacteria, phages that infect the strain can be obtained by selecting an appropriate sample as a separation source and repeating the experiment.
【００１３】２．［ファージ耐性醤油乳酸菌の造成］ ファージ耐性醤油乳酸菌は、予め選択された、または特
に育種した、性質の優秀な醤油乳酸菌をそのまま、また
は人工変異処理して得られる多数の醤油乳酸菌コロニー
を形成したマスター平板を、滅菌したビロード、コロニ
ートランスファーパッド等の担体表面に軽く押しつけ、
該マスター平板上のコロニーをプリントし、次いでこの
面に予めファージ懸濁液を塗布した平板培地を軽く押し
つけ、前記担体表面上にプリントされたコロニーを写し
取ってレプリカ平板を得、これを培養した後、前記マス
ター平板と該レプリカ平板に形成したコロニーを対比
し、該レプリカ平板に形成したコロニーに対応する前記
マスター平板上のコロニーを分離することにより得られ
る。2. [Creation of Phage-Resistant Soy Sauce Lactic Acid Bacteria] Phage-resistant soy sauce lactic acid bacterium is a master that has formed many soy sauce lactic acid bacterium colonies obtained by pre-selected or particularly bred soy lactic acid bacterium with excellent properties as it is or by artificial mutation treatment. Lightly press the flat plate onto the surface of the carrier such as sterilized velvet or colony transfer pad,
After printing the colonies on the master plate, then lightly pressing a plate medium previously coated with a phage suspension on this surface, copying the colonies printed on the surface of the carrier to obtain a replica plate, and culturing this The colony formed on the master plate is compared with the colony formed on the replica plate, and the colony on the master plate corresponding to the colony formed on the replica plate is separated.
【００１４】そして、この中から特にファージ抵抗性の
強い菌株を取得したい場合は、更に次のような操作を行
えば良い。前記マスター平板より得られた菌株を、ファ
ージ液を１％、食塩１５％を添加したＭＲＳ液体培地２
００μｌに白金線で接種し、３０℃、４日培養した後、
菌体の増殖が肉眼ではっきりと認められたものを選抜し
た。更にここで選抜された菌株の培養液を、ファージ液
を１％、食塩１５％を添加したＭＲＳ液体培地、並びに
ファージ液を含まず、食塩１５％を添加したＭＲＳ液体
培地（対比培地）４００μｌにそれぞれ１０μｌづつ接
種し、３０℃で３日間培養し、菌体の増殖を５８０ｎｍ
の吸光度で測定し、対比培地に比べて菌体の生育が良好
で、ファージ添加の影響の無い菌株を選択する。If it is desired to obtain a strain having a particularly strong phage resistance from these, the following operation may be further performed. The strain obtained from the master plate was used as an MRS liquid medium 2 containing 1% phage solution and 15% sodium chloride.
After inoculating 00 μl with a platinum wire and culturing at 30 ° C. for 4 days,
Those in which the growth of bacterial cells was clearly recognized with the naked eye were selected. Furthermore, the culture solution of the strains selected here was added to 400 μl of MRS liquid medium containing 1% of phage solution and 15% of sodium chloride, and MRS liquid medium containing no phage solution and containing 15% of sodium chloride (contrast medium). 10 μl of each was inoculated and cultured at 30 ° C. for 3 days to grow the cells at 580 nm.
Select the strain that has better growth of the bacterial cells than the contrast medium and that is not affected by the addition of phage.
【００１５】以下、実施例を示して本発明をより具体的
に説明する。Hereinafter, the present invention will be described more specifically by showing examples.
【００１６】[0016]
【実施例 １】天然仕込み醤油諸味中より分離したペデ
ィオコッカス・ハロフィルス Ｄ１０（親株）について
Ｎ−メチル−Ｎ′−ニトロ−Ｎ−ニトロソグアニジンに
よる突然変異処理を行い、その希釈懸濁液（２，０００
個／ｍｌ）１００μｌを、食塩１５％及び寒天１．５％
を添加したＭＲＳ平板培地（Ｄｉｆｃｏ社製、０８８１
−０１−３）に接種し、３０℃で６日培養して、多数の
醤油乳酸菌が形成したコロニーを有するマスター平板を
得た。ついで、これを滅菌したコロニートランファーパ
ッド（レプリプレート、ＦＭＣ社製）表面に軽く押しつ
け、該マスター平板上のコロニーをレプリプレート表面
上にプリントした。次いで、後述する方法で調製したフ
ァージ懸濁液を塗布した平板培地を上記プリントしたレ
プリプレート表面に軽く押しつけ、コロニーを写し取っ
てレプリカ平板を得た。次いで、これを３０℃で６日培
養した後、前記マスター平板と前記レプリカ平板に形成
したコロニーを対比し、該レプリカ平板に形成したコロ
ニーに対応する該マスター平板上のコロニーを分離する
ことにより目的とする菌株を得た。Example 1 Pediococcus halophyllus D10 (parent strain) separated from naturally-mixed soy sauce moromi was subjected to mutation treatment with N-methyl-N′-nitro-N-nitrosoguanidine, and the diluted suspension (2 1,000
100 μl / ml), 15% salt and 1.5% agar
MRS plate medium (Difco, 0881)
-01-3) was inoculated and cultured at 30 ° C. for 6 days to obtain a master plate having a colony formed by many soy sauce lactic acid bacteria. Then, this was lightly pressed against the surface of a sterilized colony transfer pad (repli plate, manufactured by FMC), and the colony on the master plate was printed on the surface of the repli plate. Then, a plate medium coated with a phage suspension prepared by the method described below was gently pressed against the surface of the above-mentioned printed repliplate, and a colony was copied to obtain a replica plate. Then, after culturing this at 30 ° C. for 6 days, the colonies formed on the master plate and the replica plate are compared, and the colonies on the master plate corresponding to the colonies formed on the replica plate are separated. The strain was obtained.
【００１７】次いで、ここで得られた菌株はファージ抵
抗性の弱い株を含んでいたので、更に次のようにしてフ
ァージ抵抗性の強い株を選択した。前記マスター平板よ
り得られた菌株を、ファージ液を１％、食塩１５％を添
加したＭＲＳ液体培地２００μｌに白金線で接種し、３
０℃で４日培養した後、菌体の増殖が肉眼ではっきりと
認められたものを選抜した。次いで、更にここで選抜さ
れた菌株の培養液を、ファージ液を１％、食塩１５％を
添加したＭＲＳ液体培地、並びにファージ液を含まず、
食塩１５％を添加したＭＲＳ液体培地（対比培地）４０
０μｌにそれぞれ１０μｌづつ接種して、３０℃で３日
培養し、菌体の増殖を５８０ｎｍの吸光度で測定し、対
比培地に比べて菌体の生育が良好な菌株を選択した。以
上のようにして親株と比べて菌学的性質は同じだがファ
ージ抵抗性の非常に強い醤油乳酸菌ペディオコッカス・
ハロフィルスＤ１０−Ｎｏ．１株を分離した。Next, since the strain obtained here contained a strain with weak phage resistance, a strain with strong phage resistance was selected as follows. The strain obtained from the master plate was inoculated with 200 μl of MRS liquid medium containing 1% of phage solution and 15% of sodium chloride with a platinum wire and 3
After culturing at 0 ° C. for 4 days, those in which the growth of bacterial cells was clearly visible were selected. Then, a culture solution of the strain selected here was further prepared without adding the MRS liquid medium containing 1% of phage solution and 15% of sodium chloride and the phage solution,
MRS liquid medium (comparative medium) 40 supplemented with 15% salt
10 μl of each was inoculated into 0 μl and cultured at 30 ° C. for 3 days, and the growth of the bacterial cells was measured by the absorbance at 580 nm. A bacterial strain in which the bacterial cells grew better than the contrast medium was selected. As described above, soy sauce lactic acid bacterium Pediococcus
Halofils D10-No. One strain was isolated.
【００１８】次に、上記ペディオコッカス・ハロフィル
ス Ｄ１０−Ｎｏ．１のファージ放出の有無について調
べた結果を示す。即ち、上記菌株の懸濁液５０μｌを１
５％食塩添加ＭＲＳ液体培地５ｍｌに接種し、３０℃で
４日間培養し、この培養液５０μｌを上記ＭＲＳ液体培
地に再び接種し、以下同様にして合計５回植え継いだ
後、この培養液を孔径０．２μｍのメンブランフィルタ
ーで濾過し、その透過液２μｌを、親株を塗布した食塩
１５％及び寒天１．５％を添加したＭＲＳ平板培地上に
滴下し３０℃で４日間培養して、溶菌斑の有無により、
培養液中のファージの有無を調べた。この結果、溶菌斑
は全く検出されなかった。Next, the Pediococcus halophilus D10-No. The result of having investigated about the presence or absence of phage release of No. 1 is shown. That is, 50 μl of a suspension of the above strain was added to 1
After inoculating 5 ml of MRS liquid medium containing 5% salt and culturing at 30 ° C. for 4 days, 50 μl of this culture solution was inoculated again into the above MRS liquid medium, and after subculturing a total of 5 times in the same manner, this culture solution was After filtration with a membrane filter having a pore size of 0.2 μm, 2 μl of the permeate was dropped onto an MRS plate medium supplemented with 15% of salt coated with the parent strain and 1.5% of agar, and cultured at 30 ° C. for 4 days to lyse cells. Depending on the presence or absence of spots,
The presence or absence of phage in the culture solution was examined. As a result, no lytic plaque was detected at all.
【００１９】次に、比較の為従来のファージ耐性醤油乳
酸菌の造成法により得られた乳酸菌のファージ放出の有
無について同様に調べた結果を示す。即ち、親株ペディ
オコッカス・ハロフィルス Ｄ１０について、Ｎ−メチ
ル−Ｎ′−ニトロ−Ｎ−ニトロソグアニジンによる突然
変異処理を行う。次いで、後述する方法で調製したファ
ージ液０．５ｍｌと上記突然変異処理を行った親株培養
液０．５ｍｌとを混合し、この１００μｌを食塩１５％
及び寒天１．５％を添加したＭＲＳ平板培地（Ｄｉｆｃ
ｏ社製、０８８１−０１−３）上に塗布して、３０℃で
６日培養した後、形成してきたコロニーから、他の性質
は親株と同じでファージ耐性のみ強い［すなわちファー
ジに感受性の醤油乳酸菌（親株）が全く生育できないく
らいの高濃度のファージ存在下でも寒天平板培養すると
生育し、コロニーを形成する］耐性株１００株を分離し
た。次に上記で分離した耐性株のファージ放出の有無に
ついて示す。即ち、これらの菌株を１５％食塩を添加し
たＭＲＳ液体培地５ｍｌに接種し、３０℃で４日培養
し、得られた培養液のうち５０μｌを上記ＭＲＳ液体培
地に再び接種し、以下同様にして合計５回植え継いだ。
次いで、この培養液を孔径０．２μｍのメンブランフィ
ルターで濾過し、その透過液２μｌを、親株を塗布した
食塩１５％及び寒天１．５％を添加したＭＲＳ平板培地
上に滴下し、３０℃で４日培養して、溶菌斑の有無を調
べたところ、耐性株として得られた親株（１００株）の
中から１０株について溶菌斑が確認され、ファージが検
出された。この結果から、上記の方法で造成したファー
ジ耐性の親株にはファージ遺伝子を組込んだ菌株が混入
しており、信頼性に欠けることが判った。Next, for comparison, the results of the same examination as to the presence or absence of phage release of lactic acid bacteria obtained by the conventional method for producing phage-resistant soy sauce lactic acid bacteria will be shown. That is, the parent strain Pediococcus halophilus D10 is subjected to mutation treatment with N-methyl-N'-nitro-N-nitrosoguanidine. Then, 0.5 ml of the phage solution prepared by the method described below was mixed with 0.5 ml of the mutagenized parent strain culture solution, and 100 μl of this was added to 15% sodium chloride.
And MRS plate medium supplemented with 1.5% agar (Difc
o81-081-3), and cultured for 6 days at 30 ° C., and the colonies formed have the same other properties as the parent strain, but only strong phage resistance [ie, soy sauce sensitive to phages]. 100 resistant strains were isolated which grow and form colonies on agar plate culture even in the presence of a high concentration of phages such that lactic acid bacteria (parent strain) cannot grow at all. Next, the presence or absence of phage release of the resistant strains isolated above is shown. That is, these strains were inoculated into 5 ml of MRS liquid medium supplemented with 15% sodium chloride, cultured at 30 ° C. for 4 days, and 50 μl of the obtained culture solution was inoculated again into the above MRS liquid medium, and the like. It was replanted 5 times in total.
Then, this culture solution was filtered through a membrane filter having a pore size of 0.2 μm, and 2 μl of the permeate was dropped on an MRS plate medium supplemented with 15% salt coated with the parent strain and 1.5% agar, at 30 ° C. When cultured for 4 days and examined for the presence or absence of lytic spots, 10 of the parent strains (100 strains) obtained as resistant strains were confirmed to have lytic spots, and phages were detected. From this result, it was found that the phage-resistant parent strain produced by the above method was contaminated with the strain in which the phage gene had been incorporated, and thus lacked reliability.
【００２０】以上本発明と、比較例の結果から、従来法
によって得られたファージ耐性醤油乳酸菌は信頼性が低
いが、本発明により得られたそれはファージ放出の心配
が全く無く、安全性が非常に高いことが判る。From the above results of the present invention and the comparative example, the phage-resistant soy sauce lactic acid bacterium obtained by the conventional method has low reliability, but that obtained by the present invention has no fear of phage release and is extremely safe. It turns out to be very expensive.
【００２１】ファージ液の調製 天然仕込み醤油諸味を濾過して諸味液汁を得、これを孔
径０．２μｍのメンブランファイルター濾過し微生物を
完全に除去し、ファージ含有無菌濾過液を得る。これを
１５％食塩水で多段に希釈し、０．５ｍｌと親株の培養
液０．５ｍｌとを混ぜ、この混合液１００μｌを、あら
かじめ調整しておいた食塩１５％及び寒天１．５％を添
加したＭＲＳ平板培地上に一様に塗布し通常の乳酸菌の
寒天平板培養法に従い、嫌気条件下［Gas Pak(Becton-D
ickson社製）法］で３０℃で４日培養を行った。希釈の
段階に応じて溶菌斑［プラーク（Plaque)］が現れたの
で、釣菌し、これを再度同一親株と混ぜて液体培養する
と、溶菌が起り、溶菌液を孔径０．２μｍのメンブラン
ファイルター濾過し微生物を完全に除去し、ファージ含
有無菌濾過液を得、これをファージ液とした。Preparation of Phage Solution The soy sauce moromi, which is naturally charged, is filtered to obtain moromi broth, which is filtered with a membrane filter having a pore size of 0.2 μm to completely remove microorganisms to obtain a phage-containing sterile filtrate. This was diluted with 15% saline in multiple stages, 0.5 ml was mixed with 0.5 ml of the culture solution of the parent strain, and 100 μl of this mixed solution was added with 15% salt and 1.5% agar prepared beforehand. It was applied evenly on the prepared MRS plate medium according to the usual agar plate culture method of lactic acid bacteria, and under the anaerobic condition [Gas Pak (Becton-D
ickson company) method] and cultured at 30 ° C. for 4 days. Plaques appeared depending on the stage of dilution, so when bacillus was picked and mixed again with the same parent strain in liquid culture, lysis occurred and the lysate was a membrane filter with a pore size of 0.2 μm. The microorganisms were completely removed by filtration to obtain a sterile phage-containing filtrate, which was used as a phage solution.
【００２２】[0022]
【実施例２】脱脂大豆１００ｋｇと小麦１００ｋｇとを
それぞれ変性処理し、種麹菌アスペルギルス・ソーエ
（Asp. sojae)を接種して、常法により醤油麹を造り、
これを３等分して、各々２５％食塩水１２０ｌと共に別
々のタンクＡ、Ｂ、Ｃに仕込みを行った。次いで、タン
クＡにはペディオコッカス・ハロフィルスＤ１０（ファ
ージ感受性菌）を、タンクＢには上記実施例１で得られ
たペディオコッカス・ハロフィルスＤ１０−Ｎｏ．１
（ファージ耐性菌）をそれぞれ生菌数が１×１０5／ｇ
となるように仕込み時に添加した。そして、タンクＣに
はコントロール（対照）として醤油乳酸菌は添加しなか
った。次に、このように調製した諸味へのファージの自
然出現は前以て予測できないので、ここでは本発明の効
果を確認するためＡ、Ｂ、Ｃの各タンクにペディオコッ
カス・ハロフィルスＤ１０に感染するファージ液を各々
２０ｍｌ接種した。そして、各タンクの諸味間で相互感
染が全く起らぬよう良く注意して攪拌し、通常の諸味管
理を行い、発酵、熟成させた。Example 2 100 kg of defatted soybeans and 100 kg of wheat were each modified, inoculated with Aspergillus sojae (Asp. Sojae), and soy sauce koji was prepared by a conventional method.
This was divided into three equal parts and charged into separate tanks A, B and C together with 120 l of 25% saline. Then, in tank A, Pediococcus halophilus D10 (phage-sensitive bacteria), and in tank B, Pediococcus halophilus D10-No. 1
(Phage resistant bacteria) each have a viable cell count of 1 × 10 5 / g
Was added at the time of preparation so that Then, soy sauce lactic acid bacteria were not added to tank C as a control. Next, since the spontaneous appearance of phages in the thus prepared moromi can not be predicted in advance, here, in order to confirm the effect of the present invention, tanks A, B, and C were infected with Pediococcus halophilus D10. 20 ml of each phage solution was inoculated. Then, the moromi mash was agitated with care so that mutual infection did not occur at all between the moromi mashes in each tank, and the moromi mash was managed normally, and fermented and aged.
【００２３】そして、仕込み後、５０日目までの諸味の
経日的なｐＨの変化を調べたところ、ファージ耐性株を
接種したＢのタンクでは、ファージの接種があっても乳
酸が次第に諸味中に生成蓄積してｐＨはほぼ正常に下が
ったが、ファージ感受性の醤油乳酸菌を接種したＡのタ
ンクでは乳酸が全く生成せず、ｐＨの経過は乳酸菌を添
加しないコントロールＣとほぼ同様であった。仕込み後
約１ヵ月目に主発酵酵母チゴサッカロミセス・ルーキシ
ーを適量添加し、アルコール発酵を行わせ、以後常法に
より熟成させた後、圧搾して生醤油を得た。Then, the change in the pH of the moromi mash with the 50th day after the preparation was examined. As a result, in the tank B in which the phage-resistant strain was inoculated, the lactic acid gradually increased in the moromi mash even when the phage was inoculated. However, the pH of the tank A inoculated with the phage-sensitive soy sauce lactic acid bacterium did not generate lactic acid at all, and the pH was almost the same as that of the control C to which no lactic acid bacterium was added. Approximately one month after the preparation, an appropriate amount of the main fermenting yeast, Tigosaccharomyces rouxii, was added, alcoholic fermentation was performed, and after aging by a conventional method, pressing was performed to obtain raw soy sauce.
【００２４】このようにして得られた生醤油のｐＨと乳
酸を分析し、また熟練したパネル２３名による官能検査
を実施したところ表１に示す如き結果が得られた。な
お、官能検査は風味について、次の基準で採点し、その
合計点で示した。 風味が優れている…１点、風味が劣る…０点。 表１の結果から、醤油乳酸菌としてファージ耐性菌を使
用すると、安定して乳酸発酵が行われ、高品質の醤油が
得られることが判る。The raw soy sauce thus obtained was analyzed for pH and lactic acid, and a sensory test was conducted by 23 trained panelists. The results shown in Table 1 were obtained. In the sensory test, the flavor was scored according to the following criteria, and the total score was shown. Excellent flavor ... 1 point, poor flavor ... 0 point. From the results of Table 1, it is understood that when a phage-resistant bacterium is used as the soy sauce lactic acid bacterium, lactic acid fermentation is stably performed, and high-quality soy sauce is obtained.
【００２５】 [0025]
───────────────────────────────────────────────────── フロントページの続き (72)発明者 中野 衛一 千葉県野田市野田339番地 キッコーマン 株式会社内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Eiichi Nakano 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation
Claims (2)
ー平板を、滅菌した担体表面に軽く押しつけ、該マスタ
ー平板上のコロニーをプリントし、次いでこの面に予め
ファージ懸濁液を塗布した平板培地を軽く押しつけ、前
記担体表面上にプリントされたコロニーを写し取ってレ
プリカ平板を得、これを培養した後、前記マスター平板
と該レプリカ平板に形成したコロニーを対比し、該レプ
リカ平板に形成したコロニーに対応する前記マスター平
板上のコロニーを分離することにより得られたファージ
耐性乳酸菌。1. A master plate on which a large number of lactic acid bacteria colonies are formed is lightly pressed against the surface of a sterilized carrier, colonies on the master plate are printed, and then a plate medium precoated with a phage suspension is lightly pressed on this surface. By pressing, a colony printed on the carrier surface is copied to obtain a replica plate, and after culturing this, the master plate and the colony formed on the replica plate are compared to correspond to the colony formed on the replica plate. A phage-resistant lactic acid bacterium obtained by separating colonies on the master plate.
酸菌を添加する醤油の製造法において、該ファージ耐性
醤油乳酸菌として、多数の醤油乳酸菌コロニーを形成し
たマスター平板を、滅菌した担体表面に軽く押しつけ、
該マスター平板上のコロニーをプリントし、次いでこの
面に予めファージ懸濁液を塗布した平板培地を軽く押し
つけ、前記担体表面上にプリントされたコロニーを写し
取ってレプリカ平板を得、これを培養した後前記マスタ
ー平板と該レプリカ平板に形成したコロニーを対比し、
該レプリカ平板に形成したコロニーに対応する前記マス
ター平板上のコロニーを分離することにより得られたフ
ァージ耐性醤油乳酸菌を使用することを特徴とする醤油
の製造法。2. In a method for producing soy sauce in which phage-resistant soy sauce lactic acid bacteria are added to koji and / or moromi, a master plate having a large number of soy sauce lactic acid bacterium colonies formed thereon is gently pressed against the sterilized carrier surface as the phage-resistant soy sauce lactic acid bacteria. ,
After printing the colonies on the master plate, then lightly pressing a plate medium previously coated with a phage suspension on this surface, copying the colonies printed on the surface of the carrier to obtain a replica plate, and culturing this Comparing the colony formed on the master plate and the replica plate,
A method for producing soy sauce, which comprises using a phage-resistant soy sauce lactic acid bacterium obtained by separating colonies on the master plate corresponding to colonies formed on the replica plate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP03353939A JP3027460B2 (en) | 1991-12-19 | 1991-12-19 | Phage-resistant lactic acid bacteria and method for producing soy sauce using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP03353939A JP3027460B2 (en) | 1991-12-19 | 1991-12-19 | Phage-resistant lactic acid bacteria and method for producing soy sauce using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05168467A true JPH05168467A (en) | 1993-07-02 |
| JP3027460B2 JP3027460B2 (en) | 2000-04-04 |
Family
ID=18434236
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP03353939A Expired - Fee Related JP3027460B2 (en) | 1991-12-19 | 1991-12-19 | Phage-resistant lactic acid bacteria and method for producing soy sauce using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3027460B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013517785A (en) * | 2010-01-28 | 2013-05-20 | セーホーエル．ハンセン アクティーゼルスカブ | Lactic acid bacteria for adding texture to foods selected based on phage resistance |
| US10392597B2 (en) | 2010-10-22 | 2019-08-27 | Chr. Hanse A/S | Texturizing lactic acid bacteria strains |
| CN115141724A (en) * | 2022-05-21 | 2022-10-04 | 吕欣怡 | Stamp type strain inoculation sac for substance bacteriostasis experiment |
-
1991
- 1991-12-19 JP JP03353939A patent/JP3027460B2/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013517785A (en) * | 2010-01-28 | 2013-05-20 | セーホーエル．ハンセン アクティーゼルスカブ | Lactic acid bacteria for adding texture to foods selected based on phage resistance |
| US9562221B2 (en) | 2010-01-28 | 2017-02-07 | Chr. Hansen A/S | Lactic bacterium for texturizing food products selected on the basis of phage resistance |
| US10392597B2 (en) | 2010-10-22 | 2019-08-27 | Chr. Hanse A/S | Texturizing lactic acid bacteria strains |
| CN115141724A (en) * | 2022-05-21 | 2022-10-04 | 吕欣怡 | Stamp type strain inoculation sac for substance bacteriostasis experiment |
| CN115141724B (en) * | 2022-05-21 | 2024-06-07 | 吕欣怡 | Stamp type strain inoculation bag for substance bacteriostasis experiment |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3027460B2 (en) | 2000-04-04 |
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