JP2022130013A - Novel halophilic lactic acid bacteria and methods for producing recovering amino acid and food composition using the same - Google Patents
Novel halophilic lactic acid bacteria and methods for producing recovering amino acid and food composition using the same Download PDFInfo
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Abstract
Description
本発明は、回復系アミノ酸を高濃度で生産する新規なテトラゲノコッカス・ハロフィルス菌株およびこれを用いる味噌などの食品組成物の製造方法に関する。 TECHNICAL FIELD The present invention relates to a novel Tetragenococcus halophilus strain that produces recovery amino acids at high concentration and a method for producing food compositions such as miso using the same.
味噌は、発酵食品の１つであり、大豆、米、麦などの穀物と塩と麹を混ぜて発酵させることにより製造される。味噌の発酵は微生物と密接な関係があり、微生物の生育環境は、その土地の気候、水などによって変化することが知られている。 Miso is one of fermented foods, and is produced by fermenting a mixture of grains such as soybeans, rice and barley with salt and koji. Fermentation of miso is closely related to microorganisms, and it is known that the growth environment of microorganisms changes depending on the climate and water of the land.
まず、米、麦、大豆などを蒸したものに麹菌を繁殖させ、発酵させることにより、タンパク質やデンプンを分解し、アミノ酸や糖を生成させる。塩分により麹菌は不活化されるが、麹菌の酵素や、熟成のために添加した乳酸菌や酵母の作用により乳酸発酵、アルコール発酵が生じ、味噌に風味、香りが加わる。 First, steamed rice, barley, soybeans, etc. are propagated with Aspergillus oryzae and fermented to decompose proteins and starches and produce amino acids and sugars. The salt content inactivates the koji mold, but the enzymes of the koji mold and the lactic acid bacteria and yeast added for maturation cause lactic acid fermentation and alcoholic fermentation, adding flavor and aroma to the miso.
味噌には高濃度の食塩が加えられているので、ほとんどの微生物は活動できないが、例えば、耐塩性酵母Zygosaccharomyces rouxiiと耐塩性（好塩性）乳酸菌テトラゲノコッカス・ハロフィルス（Tetragenococcus halophilus）が味噌の熟成に用いられることが知られている。 Miso contains a high concentration of salt, so most microorganisms cannot be active. It is known to be used for aging.
特許文献１には、オルニチンを含有する味噌の製造方法が開示されており、キャベツの葉から発見されたラクトバチルス・ブレビス（Lactobacillus brevis）９Ｅ５３を用いて、オルチニンを含有する味噌が製造されている。しかしながら、特許文献１では、塩濃度が７重量％以下、すなわち減塩味噌の製造が記載されており、通常の味噌の塩濃度（１１～１２重量％）ではラクトバチルス・ブレビス９Ｅ５３は生育できず、塩と酵母を添加する前の段階での使用に限られているという問題があった。また、シトルリンに関しては何ら記載されていない。 Patent Document 1 discloses a method for producing miso containing ornithine, and ornithine-containing miso is produced using Lactobacillus brevis 9E53 found in cabbage leaves. . However, Patent Document 1 describes the production of low-salt miso with a salt concentration of 7% by weight or less, and Lactobacillus brevis 9E53 cannot grow at a normal miso salt concentration (11 to 12% by weight). The problem was that it was limited to use at a stage before the addition of salt and yeast. In addition, there is no description regarding citrulline.
本発明は、通常の塩濃度の味噌においても、回復系アミノ酸を大量に生産することができる菌株を提供することを課題とする。 An object of the present invention is to provide a strain capable of producing a large amount of recovery amino acids even in miso with a normal salt concentration.
本発明の１つの側面において、通常の塩濃度下で、アルギニンをシトルリンに変換することができる新たな好塩性細菌が提供される。
本発明の他の側面において、アルギニンをシトルリンに変換することができる新たな好塩性細菌を用いる、シトルリンを高含有する味噌などの食品組成物の製造方法が提供される。
In one aspect of the invention, new halophilic bacteria are provided that are capable of converting arginine to citrulline under normal salt concentrations.
In another aspect of the present invention, there is provided a method for producing food compositions such as miso containing high citrulline using novel halophilic bacteria capable of converting arginine to citrulline.
本発明の別の側面において、アルギニンをシトルリンに変換することができる菌株を用いることを特徴とする、アルギニンの存在下で、シトルリンを生産する方法が提供される。
本発明のさらに別の側面において、上記好塩性細菌によって生産されたシトルリンを含む、味噌などの食品組成物が提供される。
In another aspect of the present invention, a method for producing citrulline in the presence of arginine is provided, characterized by using a strain capable of converting arginine to citrulline.
In yet another aspect of the present invention, food compositions, such as miso, are provided that contain citrulline produced by the halophilic bacteria.
すなわち、本発明は、以下に関するものである。
（１）好塩性乳酸菌テトラゲノコッカス・ハロフィルス（Tetragenococcus
halophilus）KA03033株（NITE P-03264）。
（２）食品の製造工程に際し、アルギニンをシトルリンに変換する乳酸菌を用いて乳酸発酵させる、回復系アミノ酸を含有する食品組成物の製造方法。
（３）前記の食品組成物が味噌である、（２）に記載の方法。
（４）前記の乳酸菌がテトラゲノコッカス・ハロフィルスKA03033株である、（２）または（３）に記載の方法。
（５）（２）～（４）のいずれかに記載の方法によって製造された食品組成物。
（６）アルギニンの存在下で、好塩性乳酸菌テトラゲノコッカス・ハロフィルスKA03033株を増殖させる、回復系アミノ酸を生産する方法。
（７）回復系アミノ酸がシトルリンである、（６）に記載の方法。
（８）（７）に記載の方法により生産されたシトルリンを有効成分とする、食品組成物。
（９）前記食品組成物が味噌である、（８）に記載の食品組成物。
That is, the present invention relates to the following.
(1) Halophilic lactic acid bacterium Tetragenococcus halophilus KA03033 strain (NITE P-03264).
(2) A method for producing a food composition containing a restorative amino acid, wherein lactic acid fermentation is performed using lactic acid bacteria that convert arginine to citrulline in the food production process.
(3) The method according to (2), wherein the food composition is miso.
(4) The method according to (2) or (3), wherein the lactic acid bacterium is Tetragenococcus halophilus KA03033 strain.
(5) A food composition produced by the method according to any one of (2) to (4).
(6) A method for producing a recovery system amino acid, comprising growing a halophilic lactic acid bacterium Tetragenococcus halophilus KA03033 strain in the presence of arginine.
(7) The method according to (6), wherein the recovery system amino acid is citrulline.
(8) A food composition containing the citrulline produced by the method according to (7) as an active ingredient.
(9) The food composition according to (8), wherein the food composition is miso.
本発明の新規な好塩性テトラゲノコッカス・ハロフィルス菌株は、回復系アミノ酸、特にシトルリンを高濃度で生産することができる。 The novel halophilic Tetragenococcus halophilus strains of the present invention are capable of producing high levels of recovery system amino acids, particularly citrulline.
本発明の好塩性テトラゲノコッカス・ハロフィルス菌株は、回復系アミノ酸、特にシトルリンを高濃度で生産できる。また、シトルリンだけでなく、オルニチンも併せて生産することができる。 The halophilic Tetragenococcus halophilus strain of the present invention can produce high concentrations of recovery amino acids, particularly citrulline. Moreover, not only citrulline but also ornithine can be produced together.
本発明の好塩性テトラゲノコッカス・ハロフィルス菌株は、例えばKA03033株であり、NITE P-03264の寄託番号で、2020年9月11日に独立行政法人製品評価技術基盤機構バイオテクノロジーセンター特許微生物寄託センター（ＮＰＭＤ）に寄託されている。
本発明のKA03033株は、グラム陽性球菌で、10～20％程度のNaClを含む培地で培養可能という微生物学的特性を有する。
The halophilic Tetragenococcus halophilus strain of the present invention is, for example, the KA03033 strain, deposited under the deposit number of NITE P-03264 on September 11, 2020 at the National Institute of Technology and Evaluation, Biotechnology Center Patent Microorganisms Deposit. It has been deposited with the Center (NPMD).
The KA03033 strain of the present invention is a Gram-positive coccus and has microbiological characteristics such that it can be cultured in a medium containing about 10 to 20% NaCl.
本発明において、回復系アミノ酸とは、シトルリンまたはオルニチンを指す。シトルリンは、アミノ酸の１種であり、機能性食品として、冷える時の手の体温の維持などが期待されており、血流改善、動脈硬化緩和、筋肉増強、疲労回復などを目的としたサプリメントや医薬品として販売されている。また、オルニチンも、アミノ酸の１種であり、尿素回路（またはオルニチン回路）を構成する物質の１つである。オルニチンの摂取により、肝機能の改善されることが知られており、機能性食品として、精神的ストレスによる疲労感の緩和、起床時の疲労感や眠気の軽減なども期待されている。 In the present invention, the recovery system amino acid refers to citrulline or ornithine. Citrulline is a kind of amino acid, and as a functional food, it is expected to maintain the body temperature of hands when it gets cold. It is marketed as a medicinal product. Ornithine is also one of amino acids and one of the substances constituting the urea cycle (or ornithine cycle). Ingestion of ornithine is known to improve liver function, and as a functional food, it is expected to alleviate fatigue caused by mental stress and reduce fatigue and drowsiness upon awakening.
テトラゲノコッカス・ハロフィルスは、アルギニンデイミナーゼ経路を有しており、以下の通り、アルギニンは、arcA酵素によりシトルリンに変換され、その後、arcB酵素によりオルニチンに変換されることが知られている（Jieting Lin et. al., Appl Microbiol Biotechnol. 2020 Oct;104(20):8775-8787. doi: 10.1007/s00253-020-10868-6.(https://pubmed.ncbi.nlm.nih.gov/32880693/)）。
本発明のKA03033株は、例えば、菌株の増殖速度の高さや、KA03033株が発現するarcA酵素の高発現に起因して、アルギニンをシトルリンに変換する能力が高いと考えられる。一方で、arcB酵素の発現が抑制もしくは欠損しており、シトルリンを菌体内に蓄積する能力が高いと考えられる。 The KA03033 strain of the present invention is considered to have a high ability to convert arginine to citrulline due to, for example, the high growth rate of the strain and the high expression of the arcA enzyme expressed by the KA03033 strain. On the other hand, the expression of the arcB enzyme is suppressed or deleted, and it is considered that the ability to accumulate citrulline in the cells is high.
本発明において、「通常の塩濃度」とは、味噌製造における食塩の濃度を指す。本発明において、味噌製造における通常の塩濃度は、例えば１０～１３重量％、１１～１２重量％であり、味噌製造における低い塩濃度とは、例えば７重量％以下を指す。 In the present invention, "ordinary salt concentration" refers to the salt concentration in manufacturing miso. In the present invention, the normal salt concentration in miso production is, for example, 10 to 13% by weight, 11 to 12% by weight, and the low salt concentration in miso production means, for example, 7% by weight or less.
本発明において、「高濃度で生産」および「高生産」とは、シトルリンまたはオルニチンなどの回復系アミノ酸について、生産率および生産量が高いことを指し、「高含有」とは、含有量が高いことを指す。
「高濃度で生産」および「高生産」とは、例えば、味噌製造時における回復系アミノ酸の生産は、味噌２０ｇ（味噌汁1杯）あたりの回復系アミノ酸の量が、例えば２０ｍｇ以上、３０～４０ｍｇである。
産生された回復系アミノ酸は、例えば、キャピラリー電気泳動－飛行時間型質量分析計、高速液体クロマトグラフ質量分析計によって検出することができる。
In the present invention, "produced at high concentration" and "high production" refer to high production rate and production amount for recovery amino acids such as citrulline or ornithine, and "high content" means that the content is high point to
"High concentration production" and "high production" mean that, for example, the production of recovery-type amino acids during the production of miso is such that the amount of recovery-type amino acids per 20 g of miso (1 cup of miso soup) is, for example, 20 mg or more, 30 to 40 mg. is.
The produced recovery system amino acids can be detected by, for example, capillary electrophoresis-time-of-flight mass spectrometer, high performance liquid chromatograph mass spectrometer.
本発明の味噌の製造方法は、大豆、米、麦などの原材料を蒸煮したものに麹菌を添加して培養し、麹を得る製麹工程；製麹工程で得られた麹と、大豆、米、麦などを蒸煮したものと、塩、水、その他任意の材料とを所望の割合で混合して麹混合材料を得る仕込み工程；及び、麹混合材料を発酵熟成させて醸造物を得る発酵熟成工程からなる。 The method for producing miso according to the present invention includes a koji-making step in which koji mold is added to steamed raw materials such as soybeans, rice, and barley and cultured to obtain koji; , steamed barley, etc., and salt, water, and other optional ingredients are mixed in a desired ratio to prepare a koji mixed material; consists of processes.
本発明の味噌の製造方法は、前記仕込み工程または発酵熟成工程において、好塩性テトラゲノコッカス・ハロフィルス菌株、例えばKA03033株を添加することを特徴とする。
本発明の味噌の製造方法において、仕込み工程における食塩濃度は、低濃度であってもよいし、通常の濃度であってもよい。好塩性テトラゲノコッカス・ハロフィルス菌株は、通常の味噌の塩濃度（例えば１１～１２重量％）で生育できるので、本発明の味噌の製造方法は、減塩味噌の製造または通常塩濃度の味噌の製造にかかわらず、実施することができ、回復系アミノ酸を高含量で含む味噌を製造することができる。
本発明の製造方法はまた、味噌以外の食品組成物の製造方法も含む。例えばKA03033株などの、好塩性テトラゲノコッカス・ハロフィルス菌株を使用して、嫌気性条件または好気性条件下で、乳酸発酵させることにより、食品組成物を製造することができる。食品組成物としては、乳酸発酵過程を経て生成されるものであれば限定されないが、例えば、しょうゆ、漬物などが挙げられる。
The method for producing miso of the present invention is characterized by adding a halophilic Tetragenococcus halophilus strain, for example, KA03033 strain, in the preparation step or the fermentation and ripening step.
In the method for producing miso of the present invention, the salt concentration in the preparation step may be low or normal. The halophilic Tetragenococcus halophilus strain can grow at a normal salt concentration of miso (for example, 11 to 12% by weight). can be carried out regardless of the production method, and miso containing a high content of recovery system amino acids can be produced.
The manufacturing method of the present invention also includes methods for manufacturing food compositions other than miso. For example, a food composition can be produced by lactic acid fermentation using a halophilic Tetragenococcus halophilus strain, such as the KA03033 strain, under anaerobic or aerobic conditions. The food composition is not limited as long as it is produced through a lactic acid fermentation process, and examples thereof include soy sauce and pickles.
また、本発明の好塩性テトラゲノコッカス・ハロフィルス菌株は、アルギニンの存在下、回復系アミノ酸の生産に用いることができる。本発明の好塩性テトラゲノコッカス・ハロフィルス菌株の培養条件は、特に限定されないが、嫌気性条件または好気性条件下で、０℃～４０℃で培養することが好ましく、１０℃～３０℃で培養することがより好ましい。上述のとおり、KA03033株は、アルギニンの存在下、シトルリンを高濃度で生産できるので、得られたシトルリンを食品組成物に添加することができる。本発明のKA03033株は、菌株自体の増殖速度の高さ、または、KA03033株が発現するarcA酵素の高発現に起因して、アルギニンをシトルリンに変換する能力が高いと考えられる。食品組成物中の回復系アミノ酸の添加量は、例えば、１００ｍｇ～５００ｍｇとすることができる。また、本発明の好塩性テトラゲノコッカス・ハロフィルス菌株は、大豆などの穀物、食塩、麹菌をともに含む、味噌などの食品組成物の製造キットとしても提供することができる。 Also, the halophilic Tetragenococcus halophilus strain of the present invention can be used for the production of recovery system amino acids in the presence of arginine. The culture conditions for the halophilic Tetragenococcus halophilus strain of the present invention are not particularly limited, but it is preferably cultured at 0°C to 40°C under anaerobic or aerobic conditions, and at 10°C to 30°C. Culturing is more preferable. As described above, the KA03033 strain can produce citrulline at high concentrations in the presence of arginine, so the obtained citrulline can be added to food compositions. The KA03033 strain of the present invention is considered to have a high ability to convert arginine to citrulline due to the high growth rate of the strain itself or the high expression of the arcA enzyme expressed by the KA03033 strain. The amount of the recovery amino acid added to the food composition can be, for example, 100 mg to 500 mg. In addition, the halophilic Tetragenococcus halophilus strain of the present invention can be provided as a kit for producing a food composition such as miso containing grains such as soybean, salt, and koji mold.
本発明のテトラゲノコッカス・ハロフィルス菌株によって生成された回復系アミノ酸は、分離・精製後、食品組成物に含有させたり、サプリメントとして提供することができる。本発明の食品組成物は、例えば快眠をサポートする食品、冷える時の手の体温の維持する食品として利用できる。食品は、健康食品、機能性食品、栄養補助食品、保健機能食品（特定保健用食品、機能性表示食品、栄養機能食品）およびサプリメントを含む。食品は、味噌汁およびスープなどの加工食品、みそ、しょうゆ、漬物など、高濃度の食塩を含む食品を例示することができる。 After separation and purification, the recovery system amino acid produced by the Tetragenococcus halophilus strain of the present invention can be contained in a food composition or provided as a supplement. The food composition of the present invention can be used, for example, as a food that supports good sleep, or as a food that maintains the body temperature of hands when cold. Foods include health foods, functional foods, dietary supplements, foods with health claims (foods for specified health uses, foods with function claims, foods with nutrient claims), and supplements. Foods can be exemplified by processed foods such as miso soup and soup, foods containing high concentrations of salt such as miso, soy sauce and pickles.
本発明を下記の実施例によってさらに具体的に説明するが、本発明の技術的範囲は、これらの実施例に限定されないものとする。 The present invention will be described more specifically by the following examples, but the technical scope of the present invention shall not be limited to these examples.
例１：味噌メタボローム解析
味噌に存在する代謝産物を網羅的に解析するために、メタボローム解析を行った。検体に含まれる代謝産物を、メタノールを用いて抽出し、キャピラリー電気泳動―飛行時間型質量分析計を用いて網羅的に測定・定量した。
結果を以下の表１に示す。
The results are shown in Table 1 below.
例２：KA社の味噌の熟成前後の菌叢解析
次に、オルニチンが高含量で含まれているKA社の味噌について、菌叢解析を行った。検体より抽出したDNAを鋳型として、細菌の16S rRNA遺伝子中のV3-V4領域をPCRにて増幅し、次世代シーケンサーを用いて増幅物の配列を網羅的に解読した。得られた配列データは、微生物群集解析用プログラム（QIIME, Caporaso JG. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods. 2010; 7(5): 335-336.）を用いて解析し、検体に含まれる細菌の種類や存在割合を見積もった。
使用したサンプルは、以下のとおりであった。
従って、テトラゲノコッカス・ハロフィルスが味噌の熟成において、重要な役割を果たしていると考え、これを単離することとした。
Example 2: Bacterial flora analysis before and after aging of KA company's miso Next, KA company's miso containing a high content of ornithine was subjected to a bacterial flora analysis. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using the DNA extracted from the sample as a template, and the sequence of the amplified product was comprehensively sequenced using a next-generation sequencer. The obtained sequence data were analyzed using a microbial community analysis program (QIIME, Caporaso JG. et al. QIIME allows analysis of high-throughput community sequencing data. Nature methods. 2010; 7(5): 335-336.). We analyzed and estimated the types and proportions of bacteria contained in the samples.
The samples used were as follows.
Therefore, Tetragenococcus halophilus was considered to play an important role in maturation of miso, and was isolated.
例３：オルニチン高含有味噌からの微生物の単離
テトラゲノコッカス・ハロフィルスの単離は、塩濃度を変化させた（０％、５％、１０％ＮａＣｌ）ＭＲＳ寒天培地（乳酸菌用培地）上で、３０℃で３～４日間培養して行った。１９２株を単離・保存した。
その後、Int J Food Microbiol, 153:269-274, 2012に記載されるように、アルギニン代謝スクリーニング培地に菌株を接種し、アルギニンを代謝する菌株を選抜した（図３）。培養７日後に、アルギニン代謝率が最も高い菌株として、KA03033株を見出した（図４）。
Example 3: Isolation of microorganisms from ornithine-rich miso Tetragenococcus halophilus was isolated on MRS agar medium (lactic acid bacteria medium) with varying salt concentrations (0%, 5%, 10% NaCl). , and cultured at 30° C. for 3 to 4 days. 192 strains were isolated and preserved.
The strains were then inoculated into an arginine metabolism screening medium to select strains that metabolize arginine, as described in Int J Food Microbiol, 153:269-274, 2012 (Figure 3). After 7 days of culture, KA03033 strain was found as a strain with the highest arginine metabolic rate (Fig. 4).
例４：KA03033株の増殖速度の解析
KA03033株の増殖速度と、乳酸菌基準株NBRC12172の増殖速度を、所定の時間における吸光度として比較した。KA03033株は、４８時間で静止期へ移行したのに対し、NBR12172株は、７２時間で静止期へ移行した。結果を図６に示す。
したがって、KA03033株の増殖速度は、NBR12172株よりも速く、アルギニン代謝能力が増殖速度に起因することが示唆された。
Example 4: Analysis of growth rate of strain KA03033
The growth rate of the KA03033 strain and the growth rate of the lactic acid bacteria reference strain NBRC12172 were compared as absorbance at a given time. The KA03033 strain entered stationary phase at 48 hours, whereas the NBR12172 strain entered stationary phase at 72 hours. The results are shown in FIG.
Therefore, the growth rate of the KA03033 strain was faster than that of the NBR12172 strain, suggesting that the growth rate was attributed to the ability to metabolize arginine.
例５：KA03033株の菌種同定
KA03033株について、16S rRNA遺伝子の増幅とシーケンス解析を行った。KA03033株の16S rRNA遺伝子配列の部分配列を配列番号１に示す。
Nucleotide BLASTのデータベースと照合したところ、テトラゲノコッカス・ハロフィルスである可能性が極めて高いことが示された。
Example 5: Identification of strain KA03033
The KA03033 strain was subjected to 16S rRNA gene amplification and sequence analysis. A partial sequence of the 16S rRNA gene sequence of the KA03033 strain is shown in SEQ ID NO:1.
Collation with the Nucleotide BLAST database indicated that it was highly likely to be Tetragenococcus halophilus.
例６：微生物メタボローム解析
次いで、KA03033株、Tetragenococcus
halophilus基準株NBRC12172、およびプロバイオティック乳酸菌（Lactobacillus
rhamnosus GG, LGG株）について、微生物メタボローム解析を行った。
結果を以下の表３に示す。
図６のシトルリンの生産量が高いという結果は、KA社の味噌ではオルニチンが高含量で含まれているという表１の結果とは、相反するものであり、驚くべきことであった。
Example 6 Microbial Metabolome Analysis Next, microbial metabolome analysis was performed on strain KA03033, Tetragenococcus halophilus type strain NBRC12172, and probiotic lactic acid bacteria ( Lactobacillus rhamnosus GG, LGG strains).
The results are shown in Table 3 below.
The result that the amount of citrulline produced in FIG. 6 is high contradicts the result in Table 1 that the KA miso contains a high amount of ornithine, which is surprising.
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