JP2018042507A - That can be easily raised to vegetable raw material genus enterococcus lactic acid bacterium and proliferation method of the same, and production method of bean miso using the same and bean miso - Google Patents
That can be easily raised to vegetable raw material genus enterococcus lactic acid bacterium and proliferation method of the same, and production method of bean miso using the same and bean miso Download PDFInfo
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Abstract
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本発明は、植物性原料に生育しやすいエンテロコッカス属乳酸菌及びその増殖方法、ならびに、それを使用した豆味噌の製造方法および豆味噌に関するものである。 The present invention relates to an Enterococcus lactic acid bacterium that easily grows on plant raw materials, a method for growing the same, a method for producing a bean miso using the same, and a bean miso.
乳酸菌は、古くから整腸効果などが知られており、飲食品や医薬品として広く利用されている。しかしながら、スーパー等に出回る普及品に利用されている乳酸菌の多くがラクトバチルス（Lactobacillus）属、ラクトコッカス（Lactococcus）属及びストレプトコッカス（Streptococcus）属に属し、そのほとんどは発酵乳（ヨーグルト）や乳酸菌飲料などの生菌を含む発酵物である（特許文献１、特許文献２）。発酵乳製品には、０．１億〜６億個／ｍｌの乳酸菌が含まれている。 Lactic acid bacteria have long been known for their intestinal regulating effects and are widely used as foods and drinks and pharmaceuticals. However, most of the lactic acid bacteria used in popular products that are available in supermarkets belong to the genus Lactobacillus, Lactococcus and Streptococcus, most of which are fermented milk (yogurt) and lactic acid bacteria beverages It is a fermented product containing live bacteria such as (Patent Document 1, Patent Document 2). Fermented dairy products contain 10-100 million / ml lactic acid bacteria.
また近年、乳酸菌の死菌体に免疫調節機能などが発見され、他工程を汚染しないといったハンドリングの良さもあって、乳酸菌死菌体の活用が広がっている。一般的に、乳酸菌死菌体に効果を期待するには、上述の生菌の場合と比較して、よりたくさんの菌体を摂取する必要があると言われている。例えば、現在最も利用が進んでいるエンテロコッカス・フェカリス菌の場合、1日に少なくとも１０００億個程度必要とされている。したがって、乳酸菌を安価に大量増殖させる技術が望まれている。 In recent years, dead cells of lactic acid bacteria have been found to have an immunoregulatory function and the like, and due to good handling that does not contaminate other processes, the utilization of dead cells of lactic acid bacteria has expanded. Generally, it is said that in order to expect an effect on dead lactic acid bacteria, it is necessary to ingest more microbial cells than in the case of the above-mentioned live bacteria. For example, in the case of Enterococcus faecalis, which is currently most used, at least about 100 billion are required per day. Therefore, a technique for mass-producing lactic acid bacteria at low cost is desired.
プロバイオティクス素材としてあまり注目されていないが、味噌にもヨーグルトに匹敵する乳酸菌が数億個／ｇレベルで含まれている。例えば、上述した属の乳酸菌を蒸煮大豆、蒸し米、豆麹または米麹に添加して培養したものや最終製品に添加したものが挙げられるが（特許文献３、特許文献４）、これらは最終製品において喫食１回あたり１００億個（１ｇ当たりたかだか数億〜１０億個）程度の乳酸菌を含むに過ぎない。また味噌においては、製造工程で乳酸菌を大量に増殖させようとすると、最終製品の酸味がどうしても強くなり最終製品として適さなくなるという発酵乳製品にない制約も存在している。 Although not attracting much attention as a probiotic material, miso also contains lactic acid bacteria comparable to yogurt at a level of several hundred million / g. For example, lactic acid bacteria belonging to the above genus can be added to steamed soybeans, steamed rice, soybean cake or rice bran and cultured or added to the final product (Patent Document 3, Patent Document 4). The product contains only about 10 billion lactic acid bacteria per meal (at most several hundred billion to 1 billion per gram). In addition, in miso, when trying to grow lactic acid bacteria in large quantities in the manufacturing process, there is a restriction that fermented milk products do not have as the final product sourness becomes so strong that it becomes unsuitable as a final product.
エンテロコッカス属乳酸菌は、味噌玉を使った従来の豆味噌の製麹過程において、味噌玉内部で１×１０８〜１０９個／ｇ程度まで増殖し、ｐＨを下げることで雑菌の混入抑制に寄与することが知られている（非特許文献１）。しかしながら、味噌業界において、エンテロコッカス属乳酸菌は、通常、ヒトの糞便や腸内に生息するものと認識されているため、工場の衛生管理の観点から衛生指標菌として取り扱われ（非特許文献２）、検出されれば、洗浄・殺菌して工程から排除してきた。従って、その特性についてはこれまで詳細に検討されておらず、活用しようとは考えてこなかった。 Enterococcus lactic acid bacteria grow to about 1 × 10 8 to 10 9 cells / g in the miso-dama during the process of making conventional bean miso using miso-dama and contribute to the suppression of contamination by reducing the pH. It is known to do (Non-Patent Document 1). However, in the miso industry, Enterococcus lactic acid bacteria are normally recognized as living in human feces and intestines, and are therefore treated as hygienic indicator bacteria from the viewpoint of factory hygiene management (Non-patent Document 2). If detected, it has been cleaned and sterilized and removed from the process. Therefore, the characteristics have not been studied in detail so far and have not been considered to be utilized.
これまでエンテロコッカス属乳酸菌の培養技術としては、単独で液体培養する方法（特許文献５）、ＭＲＳ培地にアルカリ剤を添加してｐＨ調整しながら中和培養する方法（特許文献６）、米麹に混ぜて１２℃で発酵させて得る方法（特許文献７）、麹菌を用いて大豆を発酵させた培養物から分離して得る方法（特許文献８）等が提案されている。 Until now, enterococcus lactic acid bacteria have been cultivated by liquid culture alone (Patent Document 5), neutralization culture while adding an alkaline agent to MRS medium and adjusting pH (Patent Document 6), A method obtained by mixing and fermenting at 12 ° C. (Patent Document 7), a method obtained by separating from a culture obtained by fermenting soybeans using Aspergillus oryzae (Patent Document 8), and the like have been proposed.
特許文献５や特許文献６で培養増殖されているエンテロコッカス属乳酸菌は、腸球菌といわれる消化管内常在菌のことであり、ヒトの糞便や腸内に棲息する、いわゆる動物性フェカリス菌である。このような動物性フェカリス菌の利用にあたっては、高価な培地上で培養する必要があるうえ、濃縮・水洗後に添加するか、もしくは適宜乾燥した粉末を添加する工程を含むことが一般的であり、安価で簡便な増殖法とはいえない。 The Enterococcus lactic acid bacteria cultured and proliferated in Patent Document 5 and Patent Document 6 are resident bacteria in the digestive tract called enterococci, and are so-called animal fecalis bacteria that inhabit human feces and intestines. In utilizing such animal faecalis, it is necessary to culture on an expensive medium, and it is common to include a step of adding after concentration and washing, or adding a dried powder as appropriate. It cannot be said to be an inexpensive and simple growth method.
特許文献７で利用されているエンテロコッカス属乳酸菌は、いか麹漬けから分離されたものであるが、その由来は必ずしも明らかではなく、その大豆または豆麹における増殖性や糖資化性、乳酸濃度についての言及はない。特許文献８で利用されているエンテロコッカス属乳酸菌は、発酵大豆由来であるとされるが、その発酵大豆における増殖性や糖資化性、乳酸濃度について記載や示唆はない。 The Enterococcus lactic acid bacterium used in Patent Document 7 is isolated from pickled squid, but its origin is not necessarily clear, and its growth property, sugar utilization, and lactic acid concentration in soybean or soybean cake There is no mention. The Enterococcus lactic acid bacterium used in Patent Document 8 is said to be derived from fermented soybean, but there is no description or suggestion about the growth property, sugar utilization, and lactic acid concentration in the fermented soybean.
本発明者らは、従来の豆味噌製造において製麹中に検出されるエンテロコッカス属乳酸菌の混入経路を調査してみたところ、ヒト由来の感染ではなく、豆味噌製造工程に古くから定着している菌であることを見いだした。この乳酸菌は、シュクロース、ガラクトースなど大豆由来の糖に対する資化性がヒト由来のエンテロコッカス属乳酸菌とは明らかに異なることから、非ヒト由来と考えられる。更に、本発明の非ヒト由来のエンテロコッカス・フェカリス菌は、豆麹中の菌数が高いにもかかわらず、ヒト由来エンテロコッカス属や他の乳酸菌種と比較して豆麹中の乳酸生成が少ないことが分かった。 When the present inventors investigated the mixing route of Enterococcus lactic acid bacteria detected during koji making in conventional bean miso production, it was not established as a human-derived infection but has long been established in the bean miso production process. I found it to be a fungus. This lactic acid bacterium is considered to be derived from non-humans because the assimilability to soybean-derived sugars such as sucrose and galactose is clearly different from human-derived Enterococcus lactic acid bacteria. Furthermore, the non-human-derived Enterococcus faecalis of the present invention has less lactic acid production in legumes compared to human-derived Enterococcus spp. I understood.
本発明の目的は、上記知見に基づき、植物性原料に生育しやすい非ヒト由来のエンテロコッカス属乳酸菌とその増殖方法、ならびに、単位質量あたりの乳酸菌の菌数が多いにもかかわらず酸味が強すぎない豆味噌およびその製造方法を提供することを目的とする。 The object of the present invention is based on the above findings, non-human-derived Enterococcus lactic acid bacteria that tend to grow on plant raw materials, and their growth methods, and the acidity is too strong despite the large number of lactic acid bacteria per unit mass It is an object to provide a non-bean miso and its production method.
上記目的を達成するためになされた本発明の１つの側面は、最少培地にシュクロースまたはガラクトースから選択される糖源を添加した培地上で初発生菌数を１０７ｃｆｕ／ｍｌ〜３×１０７ｃｆｕ／ｍｌの範囲に設定して３０℃で培養したときに、６．５時間後の増殖速度がヒト由来の基準菌株（エンテロコッカス・フェカリスＭＡＦＦ４００５０５）と比較して１．４倍以上増加したエンテロコッカス属乳酸菌である。 One aspect of the present invention made to achieve the above object is that the initial bacterial count is 10 7 cfu / ml to 3 × 10 6 on a medium obtained by adding a sugar source selected from sucrose or galactose to a minimal medium. Enterococcus whose growth rate after 6.5 hours increased by 1.4 times or more as compared with a human-derived reference strain (Enterococcus faecalis MAFF400505) when cultured at 30 ° C. in the range of 7 cfu / ml It is a genus lactic acid bacterium.
上記エンテロコッカス属乳酸菌は、シュクロースでの増殖速度が４倍以上増加したものであることが好ましい。 The Enterococcus lactic acid bacterium is preferably one in which the growth rate in sucrose is increased by 4 times or more.
上記エンテロコッカス属乳酸菌は、エンテロコッカス・フェカリス菌であることが好ましい。 The Enterococcus lactic acid bacterium is preferably Enterococcus faecalis.
上記目的を達成するためになされた本発明の別の側面は、エンテロコッカス・フェカリス（Enterococcus faecalis）ＮＩＴＥ Ｐ−０２３３６、エンテロコッカス・フェカリスＮＩＴＥ Ｐ−０２３１６、および／または、エンテロコッカス・フェカリス（Enterococcus faecalis）ＮＩＴＥ Ｐ−０２３１７である。これらの菌は、大豆由来培地での生育に適しており、特に豆味噌の製造工程において、豆麹中の菌数が高いにもかかわらず、ヒト由来のエンテロコッカス属や他の乳酸菌種と比較して乳酸生成が少ないものである。 Another aspect of the present invention made to achieve the above objects is that Enterococcus faecalis NITE P-02336, Enterococcus faecalis NITE P-02316, and / or Enterococcus faecalis NITE P -02317. These fungi are suitable for growth on soybean-derived media, especially in the process of producing bean miso, even though the number of fungi in bean paste is high, compared to human-derived Enterococcus spp. And other lactic acid bacteria species. Therefore, lactic acid production is low.
上記目的を達成するためになされた本発明の別の側面は、製造工程において、非ヒト由来のエンテロコッカス属乳酸菌を培養することを含む豆味噌の製造方法である。豆味噌の製麹工程を培養に利用することで、高価な培地を要することなく、乳酸菌の大量生産が可能になる。 Another aspect of the present invention made to achieve the above object is a method for producing a bean miso, which comprises culturing non-human-derived Enterococcus lactic acid bacteria in the production process. By utilizing the bean-miso making process for cultivation, mass production of lactic acid bacteria is possible without requiring an expensive medium.
上記製造方法において、さらに、エンテロコッカス属に属しない乳酸菌を培養する工程を含んでいてもよい。 The above production method may further include a step of culturing lactic acid bacteria that do not belong to the genus Enterococcus.
本発明の別の側面は、乳酸菌および／またはその死菌体を１ｇあたり２×１０９個以上含む豆味噌である。 Another aspect of the present invention is a bean miso containing 2 × 10 9 or more lactic acid bacteria and / or dead cells thereof per gram.
上記豆味噌において乳酸菌は、標準寒天培地から分離される細菌の中に、エンテロコッカス属乳酸菌を８０％以上含むものであることが好ましい。本発明のエンテロコッカス属乳酸菌は豆味噌の製造工程での増殖性に優れ、単位質量あたりの菌数も多くなりうるからである。 In the bean miso, the lactic acid bacteria preferably contain 80% or more of Enterococcus lactic acid bacteria in the bacteria isolated from the standard agar medium. This is because the Lactobacillus of the genus Enterococcus of the present invention is excellent in proliferating ability in the production process of bean miso and can increase the number of bacteria per unit mass.
上記豆味噌において乳酸菌および／またはその死菌体は、エンテロコッカス属に属しない乳酸菌を含むものであってもよい。 In the bean miso, the lactic acid bacteria and / or dead cells thereof may contain lactic acid bacteria that do not belong to the genus Enterococcus.
上記豆味噌は、さらに喫食時の乳酸量が０．０５５ｗ／ｗ％以下であることが好ましい。喫食時の乳酸量が上記範囲内であると、従来の豆味噌に比較するものがないほど乳酸菌量が多いにも関わらず食味も酸っぱくなりすぎないため、便通改善効果や免疫調節作用を高める効果が期待できる一方、クセがなく飽きない豆味噌になる。 The bean miso preferably has a lactic acid content of 0.055 w / w% or less during eating. If the amount of lactic acid at the time of eating is within the above range, the taste does not become sour despite the amount of lactic acid bacteria being so large that there is nothing to compare with conventional bean miso, and the effect of improving bowel movements and improving immune regulation On the other hand, you can expect a bean miso that doesn't get tired without habit.
本発明の別の側面は、エンテロコッカス属乳酸菌を大豆由来培地上で培養して、培地１ｇあたり２×１０９個以上に達するまで増殖させる方法である。 Another aspect of the present invention is a method of culturing Enterococcus lactic acid bacteria on a soybean-derived medium and growing them until reaching 2 × 10 9 or more per 1 g of the medium.
上記増殖方法において、エンテロコッカス属乳酸菌は非ヒト由来であり、シュクロースでの増殖速度が基準菌株と比較して著しく速いことが好ましい。このようなエンテロコッカス属乳酸菌であれば、菌数が高いにもかかわらず、酸味が少ないので得られる培養物に対して濃縮・水洗・乾燥等の工程は不要になる。 In the above growth method, it is preferable that the Enterococcus lactic acid bacterium is derived from non-humans and the growth rate in sucrose is significantly faster than that of the reference strain. With such an Enterococcus lactic acid bacterium, although the number of bacteria is high, the acidity is low, and therefore the steps of concentration, washing with water, drying and the like are not required for the obtained culture.
本発明の別の側面は、エンテロコッカス属乳酸菌を含む乳酸菌群および／またはその死菌体を１ｇあたり２×１０９個以上含む培養物である。斯かる培養物は、単位質量あたりの菌量が多いことから、その形態によっては、乳酸菌含有量を増やしたい飲食品に対して食味に影響を与えることなくそのまま添加することも可能である。 Another aspect of the present invention is a culture containing 2 × 10 9 or more of lactic acid bacteria group including Enterococcus lactic acid bacteria and / or dead cells thereof. Since such a culture has a large amount of bacteria per unit mass, depending on the form, it can be added as it is without affecting the taste to foods and beverages for which the content of lactic acid bacteria is to be increased.
上記培養物は、乳酸菌群がさらにアミノ酸分解能を有する乳酸菌および／またはその死菌体を含んでいてもよい。 The culture may contain a lactic acid bacterium and / or dead cells of which the lactic acid bacteria group further has amino acid resolution.
本発明の別の側面は、上記エンテロコッカス属乳酸菌および／またはその死菌体を含む食品組成物である。上記エンテロコッカス属乳酸菌および／またはその死菌体は、便通改善作用、免疫調節作用、生活習慣病予防を謳う食品組成物にも利用可能である。 Another aspect of the present invention is a food composition comprising the Enterococcus lactic acid bacterium and / or dead cells thereof. The enterococcus lactic acid bacteria and / or dead cells thereof can also be used in food compositions that have a bowel movement improving effect, an immunomodulatory effect, and prevention of lifestyle-related diseases.
上記食品組成物は、便通改善、免疫調節作用、整腸作用または生活習慣病予防効果がある旨の表示が付されたものであってもよい。上記エンテロコッカス属乳酸菌および／またはその死菌体は、上記表示を付した特定保健用食品や機能性表示食品での利用に好適である。 The food composition may be labeled with an indication that it has a bowel movement improvement, an immunoregulatory effect, an intestinal regulating effect, or a lifestyle-related disease preventive effect. The enterococcus lactic acid bacteria and / or dead cells thereof are suitable for use in foods for specified health and functional foods with the above-mentioned indications.
本発明によれば、乳酸菌含有量をこれまでに比べて安価かつ飛躍的に増やしつつも酸味が強くなりすぎず、従来の乳酸菌添加豆味噌の欠点を改善した豆味噌が得られる。また、培養物に対して濃縮・水洗・乾燥等の工程は不要であり、乳酸菌含有量を増やしたい飲食品に対して培養物をそのまま添加することも可能である。 ADVANTAGE OF THE INVENTION According to this invention, the bean miso which improved the fault of the conventional lactic acid bacteria addition bean miso is obtained, without increasing sour taste, while increasing lactic acid bacteria content cheaply and dramatically compared with the past. In addition, steps such as concentration, washing with water, and drying are not required for the culture, and the culture can be added as it is to a food or drink for which the content of lactic acid bacteria is to be increased.
以下に本発明の実施態様を説明する。
本発明の１つは、定法に従って、１６ＳｒＤＮＡの塩基配列を決定し、既知の塩基配列と比較して、９９％以上の相同性を示すため、エンテロコッカス属乳酸菌に分類される、シュクロースおよび／またはガラクトースでの増殖速度が速い乳酸菌株である。その取得方法としては、市販菌株、または、蔵もしくは工程に定着している菌株に対するスクリーニング、物理的な紫外線（ＵＶ）照射や化学的な変異剤の使用等の変異作出手段の作用が挙げられる。なかでも簡便性の観点から、蔵または工程に定着している菌株に対するスクリーニングやＵＶ照射が好ましい。具体的には、例えば、乳酸菌株にＵＶ照射を施した後、シュクロースおよび／またはガラクトースを含む糖液を添加したプレート上で培養し、シュクロースおよび／またはガラクトースでの増殖速度が速い変異株を選抜することによって本発明のシュクロースおよび／またはガラクトース高資化性の変異株が得られる。
Embodiments of the present invention will be described below.
According to one aspect of the present invention, sucrose and / or categorized as Enterococcus lactic acid bacteria because the base sequence of 16S rDNA is determined according to a conventional method and exhibits 99% or more homology compared to a known base sequence. It is a lactic acid strain with a high growth rate in galactose. Examples of the acquisition method include screening of commercially available strains or strains that have been established in a storehouse or process, and the action of mutation creation means such as physical ultraviolet (UV) irradiation and use of chemical mutagens. Among these, from the viewpoint of simplicity, screening and UV irradiation for strains that have been established in the warehouse or process are preferred. Specifically, for example, a mutant strain having a high growth rate in sucrose and / or galactose after culturing on a plate to which a sugar solution containing sucrose and / or galactose is added after irradiating the lactic acid strain with UV irradiation The sucrose and / or galactose-utilizing mutant strain of the present invention is obtained by selecting.
上記方法で新規に分離した乳酸菌の３株は、後述する遺伝子解析によってエンテロコッカス属に属することが判明したものであるが、シュクロースおよび／またはガラクトースに対する資化性が著しく高くなっていることから、その変異株と同定し、エンテロコッカス・フェカリス（Enterococcus faecalis）ＩＣＫ−１と命名、表示し、特許微生物寄託センター（ＮＰＭＤ）にＮＩＴＥ Ｐ−０２３３６として寄託され、さらにもう１株は、エンテロコッカス・フェカリス（Enterococcus faecalis）ＩＣＫ−２と命名、表示し、特許微生物寄託センター（ＮＰＭＤ）にＮＩＴＥ Ｐ−０２３１６として寄託され、さらにもう１株は、エンテロコッカス・フェカリス（Enterococcus faecalis）ＡＰ１８と命名、表示し、特許微生物寄託センター（ＮＰＭＤ）にＮＩＴＥ Ｐ−０２３１７として寄託されている。 Three strains of lactic acid bacteria that were newly isolated by the above method were found to belong to the genus Enterococcus by the genetic analysis described later. However, since the assimilability to sucrose and / or galactose is remarkably high, It was identified as the mutant strain, named and displayed as Enterococcus faecalis ICK-1, and deposited as NITE P-02336 at the Patent Microorganism Depositary Center (NPMD), and another strain was Enterococcus faecalis (Enterococcus faecalis). faecalis) Named and labeled ICK-2, deposited as NITE P-02316 at the Patent Microorganism Depositary Center (NPMD), and another strain named and labeled Enterococcus faecalis AP18, deposited as a patented microorganism NIT at the Center (NPMD) Deposited as EP-02317.
乳酸菌の選抜、同定および特性評価の方法及び結果、遺伝子解析の方法及び結果ならびに糖資化性試験の方法及び結果については後述する。 Methods and results of selection, identification and characterization of lactic acid bacteria, methods and results of gene analysis, and methods and results of a glucose utilization test will be described later.
本発明の１つは、豆味噌の常法に従った製造工程において、エンテロコッカス属乳酸菌を培養することを含む豆味噌の製造方法である。
豆味噌の常法に従った製造工程は、典型例としては、図１に示すように、原料大豆への撒水、浸漬、蒸煮を経て、味噌玉を成形し、ここに種麹と香煎との混合物を種付けし製麹、食塩および食塩水を加えて仕込み、熟成へと続く一連の工程のことである。培養は、最終的な乳酸菌数が所望の範囲に達するのであれば、上記工程のいずれの段階でなされてもよいが、なかでも、仕込み時の食塩添加で乳酸菌が増殖しないかもしくは死滅することから、蒸煮後仕込みに至るまでの工程でなされることが好ましく、蒸煮後出麹までの工程でなされることがより好ましい。
One aspect of the present invention is a method for producing bean miso, comprising culturing Enterococcus lactic acid bacteria in a production process according to a conventional method for bean miso.
As shown in FIG. 1, a typical process for producing soybean miso is to form a miso ball through soaking, soaking, and steaming into raw soybeans, and then adding the soy sauce It is a series of steps of seeding a mixture, adding koji, salt and saline, and continuing to ripening. Cultivation may be performed at any stage of the above process as long as the final number of lactic acid bacteria reaches the desired range, especially because lactic acid bacteria do not grow or die due to the addition of salt at the time of preparation. It is preferable to be performed in a process up to the preparation after cooking, and more preferably in a process up to brewing after cooking.
味噌玉を作る際の蒸煮大豆の温度としては、麹菌の破精込みより乳酸菌の増殖を優先する観点では味噌玉が若干固い方が好ましいため、比較的高温にすることが好ましく、例えば、５０℃〜６５℃で行うことができるが、これより低温（３０℃〜５０℃）にすることも許容される。 As the temperature of the steamed soybean when making miso balls, it is preferable that the miso balls are a little harder from the viewpoint of giving priority to the growth of lactic acid bacteria over the rupture of koji molds, and therefore, it is preferable to use a relatively high temperature. Although it can be carried out at ˜65 ° C., lower temperatures (30 ° C. to 50 ° C.) are also acceptable.
味噌玉の大きさとしては、乳酸菌培養の観点からは、直径３０〜６５ｍｍが好適であるが、これに限定されず、直径１５ｍｍ〜３０ｍｍの一般的な味噌玉であってもよく、直径１５ｍｍ〜６５ｍｍの範囲内で小径玉と大径玉との混合物としてもよい。 The size of the miso ball is preferably 30 to 65 mm in diameter from the viewpoint of lactic acid bacteria culture, but is not limited thereto, and may be a general miso ball with a diameter of 15 mm to 30 mm. It is good also as a mixture of a small diameter ball and a large diameter ball within the range of 65 mm.
培養するエンテロコッカス属乳酸菌は、少なくとも非ヒト由来であり、好ましくは植物性と判別されるものである。「非ヒト由来」であることは、ヒト由来のエンテロコッカス属乳酸菌に比べてシュクロースおよびガラクトースの資化性が異なることによって判別しうる。また「植物性」とは、非ヒト由来であって、植物や植物由来原料中でヒト由来のエンテロコッカス属乳酸菌に比べて有意に多く増殖しうることを意味し、典型的には大豆の主要糖分であるシュクロースを資化できる。 The Enterococcus lactic acid bacterium to be cultured is at least non-human and preferably discriminated as plant. “Non-human origin” can be identified by the difference in utilization of sucrose and galactose compared to human-derived Enterococcus lactic acid bacteria. “Plant” means that it is non-human and can grow significantly more in plants and plant-derived materials than human-derived Enterococcus lactic acid bacteria. Can assimilate sucrose.
エンテロコッカス属乳酸菌としては、エンテロコッカス属に分類されるものであれば特に限定されず、例えば、エンテロコッカス・フェカリス、エンテロコッカス・フェシウム等が含まれる。 Enterococcus lactic acid bacteria are not particularly limited as long as they are classified into the genus Enterococcus, and include, for example, Enterococcus faecalis, Enterococcus faecium, and the like.
培養するエンテロコッカス属乳酸菌としては、蔵または工程に定着している菌を活用してもよいし、外部から添加してもよい。外部から添加する場合の添加量としては、味噌玉１ｇ当たり、１０５ＣＦＵ未満、具体的には１０３〜１０４ＣＦＵに設定することができる。外部から添加する場合は、後述する大豆由来培地上で培養して得られた培養物を添加してもよい。 As the Enterococcus lactic acid bacteria to be cultured, bacteria that have been established in the warehouse or process may be utilized, or may be added from the outside. The amount added from the outside can be set to less than 10 5 CFU, specifically 10 3 to 10 4 CFU per gram of miso ball. When adding from the outside, you may add the culture obtained by culture | cultivating on the soybean origin culture medium mentioned later.
培養するエンテロコッカス属乳酸菌は、最少培地にシュクロース又はガラクトースから選択される糖源を添加した培地での増殖速度が基準菌株と比較して１．４倍以上、２倍以上、４倍以上、８倍以上増加したものであることが好ましい。本明細書において、増殖速度の増加割合は、最少培地に各種糖源を添加した培地上で初発生菌数を１０７ｃｆｕ／ｍｌ〜３×１０７ｃｆｕ／ｍｌの範囲に設定して３０℃で培養した時の、培養開始６．５時間後における対象となるエンテロコッカス属乳酸菌株の総菌数を初発菌数で除した値と培養開始６．５時間後における基準菌株（エンテロコッカス・フェカリスＭＡＦＦ４００５０５）の総菌数を初発菌数で除した値との比率によって評価している。総菌数は、培養液を適宜希釈した後、トーマ血球計を用いて顕微鏡下で乳酸菌をカウントして得られた値である。 Enterococcus lactic acid bacteria to be cultured have a growth rate in a medium in which a sugar source selected from sucrose or galactose is added to a minimal medium at least 1.4 times, 2 times or more, 4 times or more, 8 It is preferable that it is increased by a factor of two or more. As used herein, increasing rate of growth rate, set the first occurrence number of bacteria on a medium obtained by adding various sugars source minimal medium in the range of 10 7 cfu / ml~3 × 10 7 cfu / ml to 30 ° C. The value obtained by dividing the total number of Enterococcus lactic acid bacterial strains of the subject genus Enterococcus 6.5 hours after the start of culture by the initial number of bacteria and the reference strain 6.5 hours after the start of culture (Enterococcus faecalis MAFF400505) It is evaluated by the ratio with the value obtained by dividing the total number of bacteria by the initial number of bacteria. The total number of bacteria is a value obtained by appropriately diluting the culture solution and counting lactic acid bacteria under a microscope using a Toma hemocytometer.
エンテロコッカス属乳酸菌を培養する際に、エンテロコッカス属に属しない乳酸菌と共に乳酸菌叢として培養し、また結果として培養されることになってもよい。エンテロコッカス属に属しない乳酸菌としては特に限定されず、ラクトバチルス（Lactobacillus）属、ペディオコッカス(Pediococcus)属、テトラジェノコッカス（Tetragenococcus）属、ロイコノストック(Leuconostoc)属、ラクトコッカス（Lactococcus）属、ストレプトコッカス（Streptococcus）属、ワイセラ（Weissella）属等が挙げられる。 When culturing Enterococcus lactic acid bacteria, it may be cultured as a lactic acid bacterial flora together with lactic acid bacteria that do not belong to the Enterococcus genus, and may be cultured as a result. Lactic acid bacteria that do not belong to the genus Enterococcus are not particularly limited, and the genus Lactobacillus, Pediococcus, Tetragenococcus, Leuconostoc, and Lactococcus , Streptococcus genus, Weissella genus and the like.
エンテロコッカス属に属しない乳酸菌は、アミノ酸分解能を有する乳酸菌であってもよい。斯かる乳酸菌としては、従来公知のものを使用することができ、テトラジェノコッカスハロフィラスＤＡ−３５３株（イチビキ社）等のアルギニンをオルニチンに変換する乳酸菌；ラクトバチルス・プランタラムＩＦＯ３０７０（特開2011-004723号公報）等のグルタミン酸をＧＡＢＡに変換する乳酸菌；テトラジェノコッカスハロフィラスＳ−２株（キッコーマン社、特開2003-079363号公報）等のアスパラギン酸をβ−アラニンに変換する乳酸菌等が挙げられる。 The lactic acid bacterium not belonging to the genus Enterococcus may be a lactic acid bacterium having an amino acid resolution. As such a lactic acid bacterium, a conventionally known lactic acid bacterium can be used. A lactic acid bacterium that converts arginine such as Tetragenococcus halophyllus DA-353 strain (Ichibiki) into ornithine; Lactobacillus plantarum IFO3070 Lactic acid bacteria that convert glutamic acid to GABA such as 2011-004723; Lactic acid bacteria that convert aspartic acid to β-alanine such as Tetragenococcus halophyllus S-2 strain (Kikkoman, JP 2003-079363) Etc.
培養温度としては、ヒト由来のエンテロコッカス属乳酸菌の至適生育温度（３０〜３７℃）よりやや低めに設定するのが通常であり、例えば、１５℃〜３３℃、より好ましくは１５℃〜３０℃、さらに好ましくは１５℃〜２５℃に管理する。 The culture temperature is usually set slightly lower than the optimum growth temperature (30 to 37 ° C.) of human-derived Enterococcus lactic acid bacteria, for example, 15 to 33 ° C., more preferably 15 to 30 ° C. More preferably, the temperature is controlled at 15 ° C to 25 ° C.
仕込み時の塩分濃度は、通常の仕込み時の塩分濃度（１０．０〜１３．０％）であってもよいが、適宜塩化カリウム、グルコン酸塩、塩基性アミノ酸、オルニチル−β−アラニン等の塩味増強剤と併用することにより、例えば、６．０％〜１０．０％となるように調整することができる。塩分濃度は、培養菌の乳酸産生の状態（速度、収率）やアミノ酸分解の状態を確認したうえで調整することができる。 The salinity at the time of charging may be the normal salinity (10.0 to 13.0%) at the time of charging, but as appropriate, such as potassium chloride, gluconate, basic amino acid, ornithyl-β-alanine, etc. By using together with a salty taste enhancer, for example, it can be adjusted to 6.0% to 10.0%. The salinity concentration can be adjusted after confirming the state of lactic acid production (rate, yield) and the state of amino acid degradation of the cultured bacteria.
上記豆味噌製造の工程を経て得られた豆味噌は、乳酸菌および／またはその死菌体を好ましくは１ｇあたり２×１０９個以上、より好ましくは、３×１０９個以上、５×１０９個以上、８×１０９個以上、１×１０１０個以上含んだものとすることができる。豆味噌中の乳酸菌数は、最終製品中の乳酸菌数と死菌体数との合計であり、顕微鏡法、リアルタイムＰＣＲ法により測定された値である。ここで死菌体には、デブリ、遺伝子断片等も含まれる。 The bean miso obtained through the above-mentioned process for producing bean miso preferably contains 2 × 10 9 or more, more preferably 3 × 10 9 or more, 5 × 10 9 lactic acid bacteria and / or dead cells thereof per gram. One or more, 8 × 10 9 or more, 1 × 10 10 or more may be included. The number of lactic acid bacteria in the soybean paste is the sum of the number of lactic acid bacteria and the number of dead cells in the final product, and is a value measured by a microscopic method or a real-time PCR method. Here, dead cells include debris, gene fragments, and the like.
豆味噌は、同属もしくは異なる属に属する２種以上の乳酸菌を含むものであってよく、その場合、豆麹を適宜希釈し、標準寒天培地に塗沫し、生育したコロニーから無作為に２０株釣菌し、１６ＳｒＤＮＡの塩基配列を決定し、既知の塩基配列と比較して菌種を特定したとき、エンテロコッカス属乳酸菌を８０％以上、８５％以上、９０％以上または９５％以上含むものであることが好ましい。 The bean miso may contain two or more lactic acid bacteria belonging to the same or different genera. In that case, the bean paste is appropriately diluted, smeared on a standard agar medium, and randomly 20 strains from the grown colonies. When the fungus is fished, the base sequence of 16S rDNA is determined, and the bacterial species is specified by comparison with a known base sequence, it may contain 80% or more, 85% or more, 90% or more, or 95% or more of Enterococcus lactic acid bacteria preferable.
豆味噌に含まれる乳酸菌および／またはその死菌体は、アミノ酸分解能を有する乳酸菌および／またはその死菌体を含むものであってもよい。アミノ酸分解能を有する乳酸菌は、製法において上述の通りである。 The lactic acid bacteria and / or dead cells thereof contained in the bean paste may contain lactic acid bacteria having amino acid resolution and / or dead cells thereof. Lactic acid bacteria having amino acid resolution are as described above in the production method.
豆味噌は、喫食時の乳酸量が０．０５５ｗ／ｗ％以下、０．０４５ｗ／ｗ％以下、０．０３５ｗ／ｗ％以下、０．０２５ｗ／ｗ％以下または０．０１５ｗ／ｗ％以下である。本明細書において、「喫食時」とは、食品包装等で指示された標準的な方法に従って味噌を熱湯で溶いて（具材が別形態で同一包装内に添付されているかまたは味噌と混合されている場合は、味噌を溶きかつ具材を戻して）食する状態とした時点を意味する。乳酸の濃度は、喫食時の試料をイオン排除モードのＨＰＬＣ（移動相：4mM過塩素酸、カラム：shodex RSpak KC-LG+KC-811）にかけて検出、定量した値である。 Bean miso has a lactic acid content of 0.055 w / w% or less, 0.045 w / w% or less, 0.035 w / w% or less, 0.025 w / w% or less, or 0.015 w / w% or less when eating. is there. In the present specification, “when eating” means that miso is melted in hot water according to a standard method specified in food packaging or the like (the ingredients are attached in the same package in another form or mixed with miso). If it is, it means the time when the miso is melted and the ingredients are put back into a state of eating. The concentration of lactic acid is a value obtained by detecting and quantifying a sample at the time of eating by HPLC in an ion exclusion mode (mobile phase: 4 mM perchloric acid, column: shodex RSpak KC-LG + KC-811).
エンテロコッカス属乳酸菌を大豆由来培地上で培養して、培地１ｇあたり２×１０９個以上に達するまで増殖させる方法もまた本発明に包含される。ここでの大豆由来培地には、おから、きなこ、豆腐、豆乳、納豆、軟化大豆、大豆麹等が含まれる。また軟化大豆は、含水処理により軟化したものであれば足り、味噌玉のような成型培地に限定されず、非成型培地も含まれる。エンテロコッカス属乳酸菌の性質および量は、豆味噌の製造方法において上述した通りであり、温度条件は、豆味噌の製造方法において上述した範囲を採用しうる。 A method of culturing Enterococcus lactic acid bacteria on a soybean-derived medium and growing them until reaching 2 × 10 9 or more per 1 g of the medium is also included in the present invention. The soybean-derived medium here includes okara, kinako, tofu, soy milk, natto, softened soybean, soybean meal, and the like. Further, the softened soybean is sufficient if it is softened by water-containing treatment, and is not limited to a molding medium such as miso ball, and includes a non-molding medium. The nature and amount of Enterococcus lactic acid bacteria are as described above in the method for producing bean miso, and the temperature condition may employ the range described above in the method for producing bean miso.
エンテロコッカス属乳酸菌、好ましくは非ヒト由来のエンテロコッカス属乳酸菌を含む乳酸菌群および／またはその死菌体を１ｇあたり２×１０９個以上含む培養物もまた、本発明の１つである。培養物の形態としては、特に限定されないが、例えば、ペトリフィルム等の専用培地上で培養された形態、米麹、豆麹、またはこれらを利用した食品（伝統発酵食品）の形態、乾燥体、フリーズドライ、液体培養物、懸濁培養物等を挙げることができる。これらの製造方法は常法によればよい。なお、培養物は、乳酸菌のみを分離抽出したものであっても、培地に乳酸菌や他の成分を含んだものであってもよい。エンテロコッカス属乳酸菌の性質および量、ならびに、エンテロコッカス属乳酸菌以外に含み得る乳酸菌の種類、性質および量は、豆味噌の製造方法において上述した通りである。ここでの乳酸菌数も、最終製品中の乳酸菌数と死菌体数との合計であり、トーマ血球計を用いた顕微鏡法により測定された値である。 A lactic acid bacterium group containing Enterococcus lactic acid bacteria, preferably a non-human Enterococcus lactic acid bacterium, and / or a culture containing 2 × 10 9 or more dead cells thereof per gram is also one aspect of the present invention. The form of the culture is not particularly limited. For example, the form cultured on a dedicated medium such as petri film, the rice bran, the soybean meal, or the form of food (traditional fermented food) using these, dried body, Freeze drying, liquid culture, suspension culture, etc. can be mentioned. These production methods may be based on conventional methods. Note that the culture may be a product obtained by separating and extracting only lactic acid bacteria, or a culture medium containing lactic acid bacteria and other components. The nature and amount of Enterococcus lactic acid bacteria, and the type, nature and amount of lactic acid bacteria that can be contained in addition to Enterococcus lactic acid bacteria are as described above in the method for producing bean miso. The number of lactic acid bacteria here is also the sum of the number of lactic acid bacteria and the number of dead cells in the final product, and is a value measured by a microscopic method using a Toma hemocytometer.
実施例１ 豆味噌製造工程からの乳酸菌の選抜、同定及び特性評価
イチビキの製造工程（蔵・室・桶に定着した乳酸菌群や豆麹など）から常法に従って乳酸菌を分離採取し、最少培地にシュクロースのみを含む糖液を添加したプレート上で培養し、シュクロースに対する増殖速度が速い菌株を選抜した。得られたエンテロコッカス属乳酸菌ＩＣＫ−１、ＩＣＫ−２、ＡＰ１８に対して以下の手順で遺伝子解析を行った。
（１）染色体ＤＮＡの取得
ＭＲＳ培地（メルク社製）２００ｍｌを５００ｍｌ容坂口フラスコに入れ、オートクレーブし、Enterococcus faecalis ＩＣＫ−１、Enterococcus faecalis ＩＣＫ−２、Enterococcus faecalis ＡＰ１８をそれぞれ別のフラスコに植菌した。３０℃で２４時間培養した後、ろ過・遠心分離して菌体を回収した。液体窒素で菌体を凍結し、乳鉢で微粉末になるまで粉砕し、ＤＮＡ抽出用緩衝液（５．０％ＳＤＳ、０．１Ｍ、ＮａＣｌ、５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）１５ｍｌを加えゆっくり振盪して溶解した。遠心分離（５，０００ｒｐｍ、６ｍｉｎ、ｒｔ．）により上清を得、フェノール／クロロホルム抽出を３回、エーテル抽出を２回行い、エーテルを蒸発させた後、３Ｍ酢酸ナトリウム１ｍｌ、エタノール２５ｍｌを加え、−３０℃で３０分放置した。遠心分離（１２，０００ｒｐｍ、１０ｍｉｎ、４℃）により染色体ＤＮＡを回収し、７０％エタノールにて洗浄した後、ＴＥ４００μｌに溶解した。次に、得られたＤＮＡ溶液にＲＮａｓｅ１０μｌ（０．１３２Ｕ）を加え、３７℃で１時間ＲＮａｓｅ処理した後、プロテイナーゼＫを５μｌ（０．６Ｕ）加え、５０℃で１時間処理した。フェノール／クロロホルム抽出（２回）、クロロホルム／イソアミルアルコール抽出（１回）を行った後、エタノール沈殿にてＤＮＡを回収した。７０％エタノール溶液により洗浄した後、エタノールを除去し、ＴＥ２００μｌに溶解した。
（２）１６ＳｒＤＮＡ遺伝子断片の増幅
Ｐ１ ５‘−ａｇａｇｔｔｔｇａｔｃｃｔｇｇｃｔｃａｇ−３’
Ｐ２ ５‘−ｇｇｔｔａｃｃｔｔｇｔｔａｃｇａｃｔｔ−３’
前述の方法で得られた染色体ＤＮＡを鋳型ＤＮＡとしてＰ１プライマーとＰ２プライマーの組み合わせでＰＣＲ反応を２５サイクル行った。その後、得られたＤＮＡ断片の塩基配列を決定し、既知の塩基配列データーベースと比較したところ、いずれもEnterococcus faecalis標準菌株ＪＣＭ５８０３の１６ＳｒＤＮＡ塩基配列と９９％以上の相同性を示したため（ＩＣＫ−１、ＩＣＫ−２、ＡＰ１８に対してそれぞれ配列番号１〜３）、本菌株をEnterococcus faecalisと同定した。
Example 1 Selection, identification and characterization of lactic acid bacteria from the bean miso manufacturing process Lactic acid bacteria are separated and collected from the production process of ibisibiki (a group of lactic acid bacteria and bean paste established in a brewery / room / koji) according to a conventional method and used as a minimal medium. The cells were cultured on a plate to which a sugar solution containing only sucrose was added, and a strain having a high growth rate against sucrose was selected. The obtained Enterococcus lactic acid bacteria ICK-1, ICK-2, and AP18 were subjected to gene analysis by the following procedure.
(1) Acquisition of chromosomal DNA 200 ml of MRS medium (Merck) was placed in a 500 ml Sakaguchi flask, autoclaved, and Enterococcus faecalis ICK-1, Enterococcus faecalis ICK-2, Enterococcus faecalis AP18 were inoculated in separate flasks. . After culturing at 30 ° C. for 24 hours, the cells were collected by filtration and centrifugation. Freeze the cells with liquid nitrogen, pulverize them to a fine powder in a mortar, slowly add 15 ml of DNA extraction buffer (5.0% SDS, 0.1 M, NaCl, 50 mM Tris-HCl, pH 8.0). Dissolved by shaking. The supernatant was obtained by centrifugation (5,000 rpm, 6 min, rt.), Phenol / chloroform extraction was performed three times, ether extraction was performed twice, and the ether was evaporated. Then, 1 M of 3M sodium acetate and 25 ml of ethanol were added, It was left at -30 ° C for 30 minutes. Chromosomal DNA was recovered by centrifugation (12,000 rpm, 10 min, 4 ° C.), washed with 70% ethanol, and dissolved in 400 μl of TE. Next, 10 μl (0.132 U) of RNase was added to the obtained DNA solution, treated with RNase at 37 ° C. for 1 hour, then 5 μl (0.6 U) of proteinase K was added, and treated at 50 ° C. for 1 hour. After phenol / chloroform extraction (twice) and chloroform / isoamyl alcohol extraction (once), DNA was collected by ethanol precipitation. After washing with 70% ethanol solution, ethanol was removed and dissolved in 200 μl of TE.
(2) Amplification of 16S rDNA gene fragment P1 5′-agagtttgattccgtggctcag-3 ′
P2 5'-ggttactttgttacgactt-3 '
PCR reaction was performed for 25 cycles with the combination of P1 primer and P2 primer using the chromosomal DNA obtained by the above method as template DNA. Thereafter, the base sequence of the obtained DNA fragment was determined and compared with a known base sequence database. As a result, all showed 99% or more homology with the 16S rDNA base sequence of Enterococcus faecalis standard strain JCM5803 (ICK-1). The strains were identified as Enterococcus faecalis with respect to ICK-2 and AP18, respectively.
特性評価：糖資化性試験
上記選抜手法によって得られたエンテロコッカス属乳酸菌およびヒト由来の基準菌株であるエンテロコッカス・フェカリスＭＡＦＦ４００５０５に対して、非特許文献３で提案された手順を参考に下記表１に示す手順で糖資化性試験を行った。その結果、各種糖類に対して下記表２のような資化性を示した。
Characteristic evaluation: Sugar assimilation test For Enterococcus lactic acid bacteria obtained by the above selection method and Enterococcus faecalis MAFF400505, which is a human-derived reference strain, in the following Table 1 with reference to the procedure proposed in Non-Patent Document 3 The sugar utilization test was performed according to the procedure shown. As a result, the assimilation properties shown in Table 2 below were shown for various sugars.
表２から、エンテロコッカス属乳酸菌ＩＣＫ−１、ＩＣＫ−２、ＡＰ１８はいずれも、シュクロースまたはガラクトースを糖源とする場合の増殖速度がヒト由来のエンテロコッカス・フェカリス菌であるＭＡＦＦ４００５０５に比べて著しく高いことがわかった。このことから大豆由来原料中に主に含まれる糖源であるシュクロースまたはガラクトースを利用でき、ヒト由来のエンテロコッカス属乳酸菌に比べて有意に多く増殖することから、植物性でもあることがわかった。 From Table 2, the growth rate of Enterococcus lactic acid bacteria ICK-1, ICK-2, and AP18 when using sucrose or galactose as a sugar source is significantly higher than MAFF400505, which is Enterococcus faecalis derived from humans. I understood. From this, it was found that sucrose or galactose, which is a sugar source mainly contained in the soybean-derived raw material, can be used, and grows significantly more than human-derived Enterococcus lactic acid bacteria, so that it is also plant-based.
実施例３ エンテロコッカス乳酸菌高含有豆味噌の製造
原料大豆１０ｋｇを重量が１．５〜１．６倍になるまで浸漬し、水切り後、蒸し釜に入れ、０．７〜１．０ｋｇ／ｃｍ２で９０分間蒸し、蒸し大豆を得た。次いで冷却後、１種または２種の乳酸菌を蒸し大豆に対して１０４ＣＦＵ／ｇとなるように添加して、直径３０ｍｍの味噌玉を作成した。これに香煎及び種麹を常法に従い適量まぶし、これを２５〜３５°Ｃの品温で約４８時間かけて豆麹を製造した。この豆麹を生理食塩水で適宜希釈し、標準寒天培地（ニッスイ社製）に塗抹し、３０℃で２日間培養した。培地に生育したコロニーを、無作為に２０個選択し、実施例１と同様に染色体ＤＮＡを取得し、ＰＣＲ反応を行った。その後、得られたＤＮＡ断片の塩基配列を決定し、添加した乳酸菌（Enterococcus faecalis）の１６ＳｒＤＮＡ塩基配列と９９％以上の相同性を示すコロニーの割合を算出した。その結果、ＩＣＫ−１を添加した豆麹は８５％、ＩＣＫ−２を添加した豆麹は９０％、ＡＰ１８を添加した豆麹は９５％が、添加した乳酸菌の１６ＳｒＤＮＡ塩基配列と９９％以上の相同性を示した。このように用意した豆麹１０ｋｇを圧潰し、塩化ナトリウム１．４３ｋｇと種水２．０３ｋｇと共に仕込み容器内に仕込んだ。これに上蓋と重石を載せて２５〜３５°Ｃの範囲で発酵・熟成を開始し、最終的に得られた豆味噌中１ｇあたりの乳酸菌および／またはその死菌体数を顕微鏡下でトーマ血球計（サンリード硝子有限会社製）により測定した。また得られた豆味噌を希釈率１０倍（豆味噌２０ｇを２００ｍｌの熱湯で希釈）に希釈して、乳酸濃度をイオン排除モードのＨＰＬＣ（移動相：4mM過塩素酸、カラム：shodex RSpak KC-LG+KC-811）で乳酸量を測定した。クエン酸濃度および酢酸濃度も乳酸濃度と同様の方法で測定した。またそれぞれの豆味噌２０ｇを２００ｍｌの熱湯で希釈してよく溶かし、官能試験を行った。結果を表３に示す。なお、表３の乳酸濃度、クエン酸濃度および酢酸濃度は、味噌そのものの値に換算した値である。
Example 3 Production of Enterococcus lactic acid bacteria-rich bean miso Soak 10 kg of raw soybean until the weight is 1.5 to 1.6 times, drain it, put it in a steamer, and 0.7 to 1.0 kg / cm 2 Steamed for 90 minutes to obtain steamed soybeans. Next, after cooling, one or two kinds of lactic acid bacteria were added so as to be 10 4 CFU / g with respect to steamed soybeans, thereby producing miso balls having a diameter of 30 mm. An appropriate amount of perfume and seed meal were sprinkled with this according to a conventional method, and a soybean cake was produced at a product temperature of 25 to 35 ° C. over about 48 hours. The soybean cake was appropriately diluted with physiological saline, smeared on a standard agar medium (Nissui), and cultured at 30 ° C. for 2 days. Twenty colonies grown on the medium were selected at random, and chromosomal DNA was obtained in the same manner as in Example 1 to carry out PCR reaction. Thereafter, the base sequence of the obtained DNA fragment was determined, and the proportion of colonies having a homology of 99% or more with the 16S rDNA base sequence of the added lactic acid bacterium (Enterococcus faecalis) was calculated. As a result, 85% of the soybean cake with ICK-1 added, 90% of the soybean cake with ICK-2 added, and 95% of the soybean cake with AP18 added had more than 99% of the 16S rDNA base sequence of the added lactic acid bacteria. Showed homology. 10 kg of the soybean cake prepared in this way was crushed and charged into a charging container together with 1.43 kg of sodium chloride and 2.03 kg of seed water. Fermentation and ripening were started in the range of 25-35 ° C by placing an upper lid and a heavy stone on this, and the number of lactic acid bacteria and / or dead cells per gram in the finally obtained bean miso was measured under a microscope. It was measured by a meter (manufactured by Sunlead Glass Co., Ltd.). Further, the obtained bean miso was diluted to a dilution ratio of 10 times (20 g of bean miso was diluted with 200 ml of hot water), and the lactic acid concentration was adjusted to HPLC in an ion exclusion mode (mobile phase: 4 mM perchloric acid, column: shodex RSpak KC- The amount of lactic acid was measured with LG + KC-811). Citric acid concentration and acetic acid concentration were also measured by the same method as lactic acid concentration. Further, 20 g of each bean miso was diluted with 200 ml of hot water and dissolved well, and a sensory test was conducted. The results are shown in Table 3. In addition, the lactic acid concentration, the citric acid concentration, and the acetic acid concentration in Table 3 are values converted into the values of the miso itself.
表３から、本発明のエンテロコッカス属乳酸菌を添加した豆味噌は、乳酸菌数が顕著に高いにもかかわらず、乳酸量が低く、官能検査でも酸味、酸臭がなく、通常の味噌と遜色ないことが分かった。また、本発明のエンテロコッカス属乳酸菌とアルギニンをオルニチンに変換する乳酸菌ＤＡ−３５３株（イチビキ社）とを混ぜて添加した場合、乳酸発酵が過度に進行しｐＨが著しく低下したものの、充分な総菌数が得られ、高菌量の培養物を得るための大豆由来培地としては適していることがわかった。 From Table 3, the bean miso to which the Enterococcus lactic acid bacterium of the present invention is added has a low amount of lactic acid despite the remarkably high number of lactic acid bacteria, has no acidity or acid odor even in a sensory test, and is not inferior to normal miso I understood. In addition, when the Enterococcus lactic acid bacterium of the present invention and the lactic acid bacterium DA-353 strain (Ichibiki) which converts arginine to ornithine are mixed and added, the lactic acid fermentation proceeds excessively and the pH is remarkably lowered, but sufficient total bacteria The number was obtained and was found to be suitable as a soybean-derived medium for obtaining a high bacterial culture.
実施例４ 本発明のエンテロコッカス・フェカリス菌等の豆味噌以外の大豆由来培地における培養
１２１℃で１５分間オートクレーブ処理したＭＲＳ培地（メルク社製）に各種菌を接種し、３０℃で２４時間前培養し、無菌的に滅菌水で２回洗浄した菌液を本培養のスターターとした。次いで無調整豆乳（キッコーマン社製）に対して、各スターター１％を接種して、３０℃、２４時間培養し、ｐＨ、生菌数、乳酸量を測定した。なお、ｐＨ測定は、ガラス電極ｐＨメータ（東亜ＤＫＫ株式会社、HM-25R）で行い、生菌数測定は、培養物の菌数測定と同様の方法で行った。乳酸濃度はイオン排除モードのＨＰＬＣ（移動相：4mM過塩素酸、カラム：shodex RSpak KC-LG+KC-811）で測定した。クエン酸濃度および酢酸濃度も乳酸濃度と同様の方法で測定した。結果を表４に示す。
Example 4 Culture in soybean-derived medium other than bean miso such as Enterococcus faecalis of the present invention MRS medium (manufactured by Merck) that had been autoclaved at 121 ° C for 15 minutes was inoculated with various bacteria and pre-cultured at 30 ° C for 24 hours Then, the bacterial solution washed aseptically twice with sterilized water was used as a starter for main culture. Next, 1% of each starter was inoculated into unadjusted soymilk (manufactured by Kikkoman Corp.), cultured at 30 ° C. for 24 hours, and pH, viable cell count, and lactic acid content were measured. In addition, pH measurement was performed with the glass electrode pH meter (Toa DKK Co., Ltd., HM-25R), and viable cell count measurement was performed by the method similar to the cell count measurement of a culture. The lactic acid concentration was measured by HPLC in ion exclusion mode (mobile phase: 4 mM perchloric acid, column: shodex RSpak KC-LG + KC-811). Citric acid concentration and acetic acid concentration were also measured by the same method as lactic acid concentration. The results are shown in Table 4.
表４に示すように、本発明のエンテロコッカス属乳酸菌を使用することで、豆乳特有のオフフレーバーが抑えられ、１ｇあたり２×１０９個以上の高菌量の乳酸菌を含みながら酸っぱすぎない発酵豆乳が得られた。 As shown in Table 4, by using the Enterococcus lactic acid bacteria of the present invention, the off-flavor unique to soy milk is suppressed, and fermented soy milk that contains 2 × 10 9 or more high lactic acid bacteria per gram but is not too sour was gotten.
以上、本発明の実施例について説明したが、本発明はこれらの実施例に限られるものではなく、その要旨を逸脱しない範囲内においてさらに種々の形態で実施することができる。 As mentioned above, although the Example of this invention was described, this invention is not restricted to these Examples, In the range which does not deviate from the summary, it can implement with a various form further.
本発明の増殖方法は、乳酸発酵（減塩）豆味噌の製造、豆味噌以外の食品用途または医薬用途のための乳酸菌の培養に特に好適に利用することができる。 The growth method of the present invention can be particularly preferably used for the production of lactic acid fermentation (reduced salt) bean miso, the cultivation of lactic acid bacteria for food use or pharmaceutical use other than bean miso.
Claims (18)
18. The food composition according to claim 17, which is labeled as having fecal improvement, bowel regulation, immunomodulation, or lifestyle-related disease prevention.
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| CN112501105A (en) * | 2020-12-30 | 2021-03-16 | 天津科技大学 | Production strain for producing lactic acid monomer by using sucrose as raw material and obtaining method thereof |
| WO2024084851A1 (en) * | 2022-10-17 | 2024-04-25 | 株式会社 レオロジー機能食品研究所 | Plasmalogen production–promoting culture medium, production method for plasmalogen-containing composition, and production method for plasmalogen |
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| CN112501105A (en) * | 2020-12-30 | 2021-03-16 | 天津科技大学 | Production strain for producing lactic acid monomer by using sucrose as raw material and obtaining method thereof |
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