JP2010041991A - Method for producing mannosyl erythritol lipid - Google Patents
Method for producing mannosyl erythritol lipid Download PDFInfo
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- JP2010041991A JP2010041991A JP2008322881A JP2008322881A JP2010041991A JP 2010041991 A JP2010041991 A JP 2010041991A JP 2008322881 A JP2008322881 A JP 2008322881A JP 2008322881 A JP2008322881 A JP 2008322881A JP 2010041991 A JP2010041991 A JP 2010041991A
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- -1 mannosyl erythritol lipid Chemical class 0.000 title claims abstract description 24
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、食用植物や日常的に接触する植物由来の微生物によりバイオサーファクタントの一種であるマンノシルエリスリトールリピッドを製造する方法に関するものである。より具体的には、食用植物や日常的に接触する植物由来の微生物を用いたマンノシルエリスリトールリピッドのうちの１種類を選択的に製造する方法に関するものである。 The present invention relates to a method for producing mannosyl erythritol lipid, which is a kind of biosurfactant, using microorganisms derived from edible plants and plants that come into daily contact. More specifically, the present invention relates to a method for selectively producing one type of mannosyl erythritol lipid using an edible plant or a microorganism derived from a plant that is in daily contact.
糖脂質は、脂質に１〜数十個の単糖が結合した物質であり、生体内において細胞間の情報伝達に関与し、神経系及び免疫系の機能維持にも重要な役割を果たしていることなどが明らかにされつつある。また、糖脂質は、糖の性質に由来する親水性と脂質の性質に由来する親油性の二つの性質を合わせ持つ両親媒性物質であり、このような性質を有する両親媒性物質は界面活性物質と呼ばれている。石油化学工業が隆盛となるまでは、レシチン、サポニン等の生体成分由来の界面活性剤（バイオサーファクタント）が利用されていた。 Glycolipids are substances in which one to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. Glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants (biosurfactants) derived from biological components such as lecithin and saponin were used.
近年、石油化学工業の発展により合成界面活性剤が開発され、その生産量が飛躍的に増加し、日常生活には無くてはならない物質となっている。現在、一般に用いられている合成界面活性剤の種類は、細かな構造の違いまで考慮すると、数千種類にものぼる。親水基・疎水基の種類が異なるものから、それぞれのドメインの組成が同じでも分子量や立体構造の違いによって親水性疎水性バランス（ＨＬＢ）の異なる同族体など、あらゆる化合物が開発されており、これらを単一種類で用いるだけでなく、混合して用いることで、幅広い産業分野において要求される性能に対応している。しかしながら、このような合成界面活性剤の使用量の拡大に伴って環境汚染が広がり、社会問題が生じている。従って、安全性が高く、環境に対する負荷を低減できる生分解性の高い界面活性剤の開発が望まれている。 In recent years, synthetic surfactants have been developed due to the development of the petrochemical industry, and their production has increased dramatically, making them indispensable for daily life. At present, there are several thousand types of synthetic surfactants that are generally used in consideration of fine structural differences. A variety of compounds have been developed from different types of hydrophilic groups and hydrophobic groups, including homologues with different hydrophilic and hydrophobic balance (HLB) due to differences in molecular weight and steric structure even if the composition of each domain is the same. In addition to being used in a single kind, it can be used in combination to meet the performance required in a wide range of industrial fields. However, as the amount of such synthetic surfactants used increases, environmental pollution spreads and social problems arise. Therefore, development of a highly biodegradable surfactant that has high safety and can reduce the burden on the environment is desired.
微生物などが生産する界面活性物質であるバイオサーファクントは、生分解性が高く、低毒性で環境に優しく、新規な生理機能を持つといわれている。このことから、食品、化粧品、医薬品、化学工業、環境分野等にこれらのバイオサーファクタントを幅広く応用することは、環境調和型の社会を実現し、高機能な製品を提供する上で極めて有意義である。微生物由来のバイオサーファクタントは、糖脂質系、アシルペプタイド系、リン脂質系、脂肪酸系及び高分子系の界面活性物質の５つに分類されている。なかでも、糖脂質系の界面活性物質については最もよく研究されており、酵母が生産する糖脂質系の界面活性物質としてカンジダ属酵母によるソホロースリピッド（特許文献１参照）やマンノシルエリスリトールリピッド（非特許文献１から６参照）などが知られている。 Biosurfactants, which are surface-active substances produced by microorganisms, are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. Therefore, the wide application of these biosurfactants to food, cosmetics, pharmaceuticals, chemical industry, environmental fields, etc. is extremely meaningful in realizing an environmentally conscious society and providing highly functional products. . Biosurfactants derived from microorganisms are classified into five types: surfactants of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among them, glycolipid-based surfactants have been most studied, and as glycolipid-based surfactants produced by yeast, sophorose lipids produced by Candida spp. (See Patent Document 1) and mannosyl erythritol lipids (non- Patent Documents 1 to 6) are known.
マンノシルエリスリトールリピッド（ｍａｎｎｏｓｙｌ ｅｒｙｔｈｒｉｔｏｌ ｌｉｐｉｄ、以下、「ＭＥＬ」と略記することもある）は、界面活性能以外にも抗微生物活性、白血病細胞、神経系細胞などの細胞分化誘導活性、糖タンパク質結合活性、抗炎症・抗アレルギー活性、アポトーシス誘導活性、癌細胞増殖抑制活性など多様な機能が知られている。 Mannosyl erythritol lipid (hereinafter sometimes abbreviated as “MEL”) has antimicrobial activity, cell differentiation induction activity such as leukemia cells and nervous system cells, glycoprotein binding activity, Various functions such as anti-inflammatory / anti-allergic activity, apoptosis-inducing activity, and cancer cell growth-inhibiting activity are known.
ＭＥＬはマンノースにエリスリトールと０〜２個の脂肪酸と２個のアセチル基が結合した糖脂質であり、アセチル基の結合位置と数によって、４種類のＭＥＬ（ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃ、ＭＥＬ‐Ｄ）が存在することが知られている。これまで説明したようにＭＥＬの生産については多くの微生物菌株の発見、及び培養技術の改良が成されている。 MEL is a glycolipid in which erythritol, 0 to 2 fatty acids and 2 acetyl groups are bound to mannose. Depending on the binding position and number of acetyl groups, MEL (MEL-A, MEL-B, MEL-) C, MEL-D) is known to exist. As explained so far, many microorganism strains have been discovered and culture techniques have been improved for MEL production.
ＭＥＬの生産方法については、これまで酵母を利用する方法等、多くの報告がされており、生産量向上を目的として菌株の選択、培養条件、培地組成が検討されている。酵母によるＭＥＬ生産に関する報告としては、たとえば、Ｃａｎｄｉｄａ ｓｐ． Ｂ‐７株を用いて５質量％の大豆油から５日間で３５ｇ/Ｌ（生産速度：０．３ｇ／Ｌ／ｈ、原料収率：７０質量％）のＭＥＬを生産した例（非特許文献１及び２参照）、Ｃａｎｄｉａ（Ｐｓｅｕｄｏｚｙｍａ） ａｎｔａｒｃｔｉｃａ Ｔ‐３４を用いて８質量％の大豆油から８日間で３８ｇ/Ｌ（生産速度：０．２ｇ／Ｌ／ｈ、原料収率：４８質量％）のＭＥＬを生産した例（非特許文献３及び４参照）、Ｐ． ａｎｔａｒｃｔｉｃａ Ｔ‐３４を用いて６日間隔で計３回の逐次流加により２４日後に２５質量％のピーナッツ油から１１０ｇ／Ｌ（生産速度：０．２ｇ／Ｌ／ｈ、原料収率：４４質量％）のＭＥＬを生産した例（非特許文献５参照）、Ｃａｎｄｉｄａ ｓｐ． ＳＹ‐１６株を用いて１０質量％の植物油脂から回分培養法により２００時間で５０ｇ／Ｌ（生産速度：０．２５ｇ／Ｌ／ｈ、原料収率：５０質量％）のＭＥＬを生産し、また、２０質量％の植物油から流加培養法により２００時間で１２０ｇ／Ｌ（生産速度：０．６ｇ／Ｌ／ｈ、原料収率：５０質量％）のＭＥＬを生産した例（非特許文献６参照）、Ｐｓｅｕｄｏｚｙｍａ ａｐｈｉｄｉｓを用いて８０質量％の植物油脂から流加培養法により２４時間で１３．９ｇ／Ｌ（生産速度：０．５７ｇ／Ｌ／ｈ、原料収率：９２質量％）のＭＥＬを生産した例（非特許文献７参照）などがある。また、醤油醸造工程において副産物として生産されるしょうゆ油を原料としてＰ． ａｎｔａｒｃｔｉｃａ Ｔ‐３４を用いて７日間で８質量％のしょうゆ油から１７ｇ／Ｌ（生産速度：０．１ｇ／Ｌ／ｈ、原料収率：２１質量％）のＭＥＬの生産が可能であることも報告されている（特許文献２参照）。また、Ｕｓｔｉｌａｇｏ ｍａｙｄｉｓがＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃの混合物を生産することが知られている（非特許文献１４参照）。 Many methods for producing MEL have been reported so far, such as a method using yeast, and selection of strains, culture conditions, and medium composition have been studied for the purpose of improving the production amount. As a report on MEL production by yeast, for example, Candida sp. Example of producing 35 g / L (production rate: 0.3 g / L / h, raw material yield: 70 mass%) of MEL from 5 mass% soybean oil using B-7 strain in 5 days (non-patent literature) 1 and 2), 38 g / L (production rate: 0.2 g / L / h, raw material yield: 48 mass%) in 8 days from 8 mass% soybean oil using Candia (Pseudozyma) antarctica T-34 (See Non-Patent Documents 3 and 4), p. 110 g / L (production rate: 0.2 g / L / h, raw material yield: 44 mass) from 25% by mass of peanut oil after 24 days by continuous feeding three times at an interval of 6 days using an Antarctica T-34 %) Of MEL (see Non-Patent Document 5), Candida sp. Using SY-16 strain, 50 g / L (production rate: 0.25 g / L / h, raw material yield: 50 mass%) of MEL is produced from 10 mass% of vegetable oils and fats by batch culture in 200 hours. Moreover, the example which produced 120 g / L (production rate: 0.6 g / L / h, raw material yield: 50 mass%) of MEL from 20 mass% vegetable oil by the fed-batch culture method in 200 hours (nonpatent literature 6) MEL of 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) in 24 hours by fed-batch culture method using 80% by mass of vegetable oil using Pseudozyma aphidis (See Non-Patent Document 7). In addition, soy sauce oil produced as a by-product in the soy sauce brewing process is used as a raw material. It is also possible to produce 17 g / L (production rate: 0.1 g / L / h, raw material yield: 21% by mass) of MEL from 8% by mass soy sauce oil in 7 days using antitica T-34 It has been reported (see Patent Document 2). It is also known that Ustilago maydis produces a mixture of MEL-A, MEL-B, and MEL-C (see Non-Patent Document 14).
また、シュードザイマ（Ｐｓｅｕｄｏｚｙｍａ）属の酵母を用いて、ＭＥＬ‐Ａと、ＭＥＬ‐Ｂと、ＭＥＬ‐Ｃとを選択的に量産する方法が報告されている（例えば、非特許文献８参照）。このように、マンノシルエリスリトールリピッドのバリエーションは大きく拡充され、実用化に向けた研究が展開されている。 In addition, a method for selectively mass-producing MEL-A, MEL-B, and MEL-C using yeast belonging to the genus Pseudozyma has been reported (for example, see Non-Patent Document 8). Thus, the variation of mannosyl erythritol lipid has been greatly expanded, and research for practical application is being developed.
ＭＥＬ‐Ａは、これまで説明した微生物の他にも、Ｐｓｅｕｄｏｚｙｍａ ｒｕｇｕｌｏｓａ、Ｐｓｅｕｄｏｚｙｍａ ｐａｒａｎｔａｒｃｔｉｃａ、Ｐｓｅｕｄｏｚｙｍａ ｆｕｊｉｆｏｒｍａｔａを用いて量産可能であることが報告されている（例えば、非特許文献９参照）。 In addition to the microorganisms described so far, MEL-A has been reported to be mass-produced using Pseudozyma rugulosa, Pseudozyma parantactica, and Pseudozyma fujiformata (see, for example, Non-Patent Document 9).
ＭＥＬ‐ＢについてはＫｌｕｔｚｍａｎｏｍｙｃｅｔｏｕｓ ｓｐ．Ｉ‐１１（特許文献３参照）、Ｃａｎｄｉｄａ ｓｐ．ＳＹ‐１６（非特許文献６参照）、Ｐｓｅｕｄｏｚｙｍａ ｔｓｕｋｕｂａｅｎｓｉｓを用いて量産可能であることが知られている。（非特許文献１０）。 Regarding MEL-B, Klutzmanomycetous sp. I-11 (see Patent Document 3), Candida sp. It is known that mass production is possible using SY-16 (see Non-Patent Document 6) and Pseudozyma tsukubaensis. (Non-Patent Document 10).
ＭＥＬ‐Ｃについては、Ｐｓｅｕｄｏｚｙｍａ ｈｕｂｅｉｅｎｓｉｓ（非特許文献１１参照）、Ｐｓｅｕｄｏｚｙｍａ ｇｒａｍｉｎｉｃｏｌａ（非特許文献１２参照）、Ｐｓｅｕｄｏｚｙｍａ ｓｈａｎｘｉｅｎｓｉｓ（非特許文献１３参照）を用いて量産可能であることが報告されている。 About MEL-C, it is reported that mass production is possible using Pseudozyma hubiensis (refer nonpatent literature 11), Pseudozyma gramicola (refer nonpatent literature 12), and Pseudozyma shanxiensis (refer nonpatent literature 13).
このように、多くのＭＥＬ生産微生物の発見に伴って、製造可能なＭＥＬの構造のバリエーションは拡大し、ＭＥＬの製造技術は大きく向上してきている。
ところで、これらＭＥＬ生産微生物は様々な自然環境中から単離されたものであるため、人体に対する病原性は知られていないが、その安全性に対する知見はほとんどない。特に、微生物生産物を食品用途等へ展開するためには、使用する微生物の単離源が重要視される。例えば、酒やパンの製造に関わる出芽酵母、乳酸菌や枯草菌（納豆菌）などは、食品分野で活躍する微生物として一般的によく知られている。これらの微生物は全て食品中から容易に単離可能な微生物である。 By the way, since these MEL-producing microorganisms have been isolated from various natural environments, their pathogenicity to the human body is not known, but little is known about their safety. In particular, in order to develop a microbial product for food use or the like, the isolation source of the microorganism to be used is regarded as important. For example, budding yeast, lactic acid bacteria, Bacillus subtilis (natto), and the like that are involved in the production of sake and bread are generally well known as microorganisms that play an active role in the food field. These microorganisms are all microorganisms that can be easily isolated from food.
しかしながら、上述したＰｓｅｕｄｏｚｙｍａ属の単離源は不明であるため、これらに由来するＭＥＬは食用に適しているとは言い難い。 However, since the isolation source of the above-mentioned Pseudozyma genus is unknown, it is difficult to say that MEL derived from these is suitable for food.
そこで、食用植物又は日常的に接触する植物群から単離した微生物を用いたＭＥＬ製造技術を開発することで用途範囲は拡大し、かつ安全性の面で大きな利点を得られることが期待される。 Therefore, by developing MEL production technology using microorganisms isolated from edible plants or plants that come into daily contact, it is expected that the range of applications will be expanded and great advantages will be obtained in terms of safety. .
また、ウスチラゴ メイディス（Ｕ． ｍａｙｄｉｓ）はトウモロコシに感染する植物病原菌であり、Ｕ． ｍａｙｄｉｓが感染したトウモロコシは、ウイトラコーチェという名で一般的に食されている。特に、メキシコでは伝統的な高級食材として食されている。しかし、Ｕ． ｍａｙｄｉｓはＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃだけでなく、セロビオースリピッドも同時に生産するため、それぞれの成分を選択的に生産することは実用上、不可能である。しなしながら、ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃはそれぞれ物性が異なるため、それぞれを選択的に製造することができれば、新たな食品用途への利用が期待される。 U. maydis is a phytopathogenic fungus that infects corn. Maize infected with maydis is commonly eaten under the name of Vitracoche. Especially in Mexico, it is eaten as a traditional luxury food. However, U. Since maydis produces not only MEL-A, MEL-B, and MEL-C, but also cellobiose lipid at the same time, it is practically impossible to selectively produce each component. However, since MEL-A, MEL-B, and MEL-C have different physical properties, if they can be selectively manufactured, they are expected to be used for new food applications.
以上の状況から、日常的に接触する安全性の高いことが判明している植物群又は食用植物群からＭＥＬ生産微生物を単離し、さらにＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのいずれかを選択的に生産できる方法が開発されれば、ＭＥＬの用途拡大に多大な影響を与え、ひいてはバイオサーファクタントの普及に繋がるものと期待される。 From the above situation, isolate the MEL-producing microorganism from the group of plants or edible plants that are known to be highly safe in daily contact, and then select one of MEL-A, MEL-B, and MEL-C. If a method capable of selective production is developed, it is expected to have a great influence on the expansion of the use of MEL and eventually lead to the spread of biosurfactants.
本発明はこのような事情に鑑みてなされたものであり、その目的は、安全性が高く、食品用途への展開が可能なマンノシルエリスリトールリピッドを製造し、かつＭＥＬ‐Ａ、ＭＥＬ‐Ｂ又はＭＥＬ‐Ｃを選択的に製造する技術を提供することにある。 The present invention has been made in view of such circumstances, and the object thereof is to manufacture mannosyl erythritol lipid which is highly safe and can be developed for food use, and MEL-A, MEL-B or MEL. -To provide a technique for selectively producing C.
本発明者は、上記課題を解決するため鋭意検討を重ねた結果、食用植物又は日常的にヒト又は動物が接触する（物理的に接触したり、動物種によっては口に含んだりする）植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属に属する微生物を培養することで、ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのいずれかを選択的に生産することができることを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that from an edible plant or a plant that comes into contact with humans or animals on a daily basis (physical contact, or depending on the animal species, it is contained in the mouth). To cultivate an isolated microorganism belonging to the genus Ustylago, one of MEL-A, MEL-B, and MEL-C can be selectively produced, and the present invention is completed. It came.
また、当該微生物が生産するＭＥＬ‐Ａ、ＭＥＬ‐Ｂ及びＭＥＬ‐Ｃの化学構造を調べると、シュードザイマ（Ｐｓｅｕｄｏｚｙｍａ）属の微生物が生産するＭＥＬと同様の構造を有しており、その物性・機能はシュードザイマ（Ｐｓｅｕｄｏｚｙｍａ）属の微生物が生産するＭＥＬと同様であった。 In addition, when the chemical structure of MEL-A, MEL-B and MEL-C produced by the microorganism is examined, it has the same structure as MEL produced by a microorganism belonging to the genus Pseudozyma. Was similar to MEL produced by microorganisms of the genus Pseudozyma.
つまり、本発明者らは、ウスチラゴ（Ｕｓｔｉｌａｇｏ）属に属するＭＥＬ生産菌の単離源として、食用植物又は日常的にヒト又は動物が接触する植物の中から特定の植物を見出し、かつこれらのＭＥＬ生産菌を用いれば、従来報告されているＭＥＬと同様の物性・機能を有するＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのいずれかを選択的に生産することができることを見出した。本発明はかかる知見により完成されたものである。 That is, the present inventors have found a specific plant from among edible plants or plants that humans or animals are in daily contact with as an isolation source of MEL-producing bacteria belonging to the genus Ustylago, and these MELs It has been found that by using a production bacterium, any of MEL-A, MEL-B, and MEL-C having the same physical properties and functions as MEL reported heretofore can be selectively produced. The present invention has been completed based on such findings.
すなわち、本発明は以下の発明を包含する。 That is, the present invention includes the following inventions.
本発明に係る下記一般式（１） The following general formula (1) according to the present invention
（上記一般式（１）においてＲ１〜Ｒ５は、それぞれ独立して、水素原子又は炭素数１〜１８の脂肪酸基を表す）
で表される構造を有するマンノシルエリスリトールリピッドの製造方法は、サトウキビ（Ｓａｃｃｈａｒｕｍ）属、リンゴ（Ｍａｌｕｓ）属、シソ属（Ｐｅｒｉｌｌａ）属、アブラナ（Ｂｒａｓｓｉｃａ）属及びギョウギシバ（Ｃｙｎｏｄｏｎ）属の植物のいずれかから単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含むことを特徴としている。
(In the general formula (1), R 1 to R 5 each independently represents a hydrogen atom or a fatty acid group having 1 to 18 carbon atoms)
A method for producing a mannosyl erythritol lipid having a structure represented by any one of the following plants: It comprises a culture step of culturing a microorganism belonging to the genus Ustilago isolated from
また、本発明に係る下記一般式（２） Further, the following general formula (2) according to the present invention:
（上記一般式（２）においてｍ、ｎは互いに独立して１〜１６の整数である）
で表される構造を有するマンノシルエリスリトールリピッド‐Ｂの製造方法は、サトウキビ（Ｓａｃｃｈａｒｕｍ）属の植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含むことを特徴としている。
(In the above general formula (2), m and n are each independently an integer of 1 to 16)
The method for producing mannosyl erythritol lipid-B having the structure represented by the formula is characterized by comprising a culture step of culturing a microorganism of the genus Ustilago isolated from a plant of the genus Saccharum.
さらに、本発明に係るＭＥＬ‐Ｂの製造方法では、上記微生物がウスチラゴ サイタミネア（Ｕｓｔｉｌａｇｏ ｓｃｉｔａｍｉｎｅａ）であることがより好ましい。 Furthermore, in the method for producing MEL-B according to the present invention, it is more preferable that the microorganism is Ustyago scitaminea.
さらに、本発明に係るＭＥＬ‐Ｂの製造方法では、上記培養工程にて用いる培地は糖を炭素源とし油脂の含有量が０〜５重量％以下であることがより好ましい。 Furthermore, in the method for producing MEL-B according to the present invention, it is more preferable that the medium used in the culturing step contains sugar as a carbon source and the fat content is 0 to 5% by weight or less.
また、本発明に係る下記一般式（３） Further, the following general formula (3) according to the present invention:
（上記一般式（３）においてｍ、ｎは互いに独立して１〜１６の整数である）
で表される構造を有するマンノシルエリスリトールリピッド‐Ｃの製造方法は、ギョウギシバ（Ｃｙｎｏｄｏｎ）属の植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含むことを特徴としている。
(In the general formula (3), m and n are each independently an integer of 1 to 16)
The method for producing mannosyl erythritol lipid-C having the structure represented by the formula is characterized by comprising a culture step of culturing a microorganism of the genus Ustilago isolated from a plant of the genus Cynodon.
さらに、本発明に係るＭＥＬ‐Ｃの製造方法では、上記微生物がウスチラゴ シノドンティス（Ｕｓｔｉｌａｇｏ ｃｙｎｏｄｏｎｔｉｓ）であることがより好ましい。 Furthermore, in the method for producing MEL-C according to the present invention, it is more preferable that the microorganism is Ustyago synonodontis (Ustilago cynodontis).
また、本発明に係る下記一般式（４） Further, the following general formula (4) according to the present invention:
（上記一般式（４）においてｍ、ｎは互いに独立して１〜１６の整数である）
で表される構造を有するマンノシルエリスリトールリピッド‐Ａの製造方法は、リンゴ（Ｍａｌｕｓ）属、シソ属（Ｐｅｒｉｌｌａ）属及びアブラナ（Ｂｒａｓｓｉｃａ）属のいずれかの植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含むことを特徴としている。
(In the above general formula (4), m and n are each independently an integer of 1 to 16)
A method for producing mannosylerythritol lipid-A having a structure represented by the formula: genus Ustylago isolated from any plant of the genus Apple (Malus), Perilla (Brassilla) and Brassica It includes a culture step of culturing the microorganisms.
また、本発明に係る製造方法は、上記培養工程にて得られた培養液から菌体を取り除き、硫酸アンモニウムを添加して沈殿物を得る濃縮工程をさらに含むことがより好ましい。 Moreover, it is more preferable that the production method according to the present invention further includes a concentration step of removing cells from the culture solution obtained in the culture step and adding ammonium sulfate to obtain a precipitate.
本発明に係る製造方法は、上述のようにサトウキビ（Ｓａｃｃｈａｒｕｍ）属、リンゴ（Ｍａｌｕｓ）属、シソ属（Ｐｅｒｉｌｌａ）属、アブラナ（Ｂｒａｓｓｉｃａ）属及びギョウギシバ（Ｃｙｎｏｄｏｎ）属の植物のいずれかから単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する工程を含んでいるので、安全性が高く、食品用途等への展開が可能なマンノシルエリスリトールリピッドを製造し、かつＭＥＬ‐Ａ、ＭＥＬ‐Ｂ又はＭＥＬ‐Ｃを選択的に製造することができるという効果を奏する。 As described above, the production method according to the present invention is isolated from any plant of the genus Saccharum, the genus Apple, the genus Perilla, the genus Brassica, and the genus Cynodon. A mannosyl erythritol lipid which is highly safe and can be developed for food use and the like, and includes MEL-A, MEL-B or MEL, because it includes a step of culturing microorganisms belonging to the genus Ustilago -There is an effect that C can be selectively produced.
また、本発明によれば、これまでに報告されたＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃの３種類のうちのいずれかを選択的に生産可能なシュードザイマ（Ｐｓｅｕｄｏｚｙｍａ）属の微生物、及び、上記３種類のうちのいずれかを選択的に生産することができないウスチラゴ メイディス（Ｕ． ｍａｙｄｉｓ）とは異なり、食用植物又は日常的に接触する植物を単離源とする微生物を使用して、かつ、３種類のＭＥＬのいずれかを選択的に生産できる。つまり、本発明により、安全性が高い上記３種類のいずれかのＭＥＬを、新たな食品用途に展開することが可能となった。 According to the present invention, a microorganism belonging to the genus Pseudozyma that can selectively produce any one of the three types of MEL-A, MEL-B, and MEL-C reported so far, and Unlike U. maydis, which cannot selectively produce any one of the above three types, using microorganisms that are isolated from edible plants or plants that come into daily contact, and Any one of the three types of MEL can be selectively produced. That is, according to the present invention, any one of the above three types of MEL having high safety can be developed for new food applications.
特に、本発明にて生産するＭＥＬ‐Ｂ及びＭＥＬ‐ＣはＭＥＬ‐Ａに比べて水溶性が高く、期待される応用範囲がより広いことから、ＭＥＬの構造・機能的バリエーションの拡充に貢献するだけでなく、用途展開の効率化にも貢献できるという更なる効果を奏する。 In particular, MEL-B and MEL-C produced in the present invention are higher in water solubility than MEL-A and expected to have a wider range of applications, contributing to the expansion of MEL structural and functional variations. In addition, it has the further effect that it can contribute to the efficiency of application development.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明に係る上記一般式（１）で表される構造を有するマンノシルエリスリトールリピッドの製造方法は、サトウキビ（Ｓａｃｃｈａｒｕｍ）属、リンゴ（Ｍａｌｕｓ）属、シソ属（Ｐｅｒｉｌｌａ）属、アブラナ（Ｂｒａｓｓｉｃａ）属及びギョウギシバ（Ｃｙｎｏｄｏｎ）属の植物のいずれかから単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する工程を含めばよい。 The method for producing a mannosyl erythritol lipid having the structure represented by the above general formula (1) according to the present invention includes the genus Saccharum, the genus Apple, the genus Perilla, the genus Brassica, and A step of culturing a microorganism of the genus Ustilago isolated from any plant of the genus Cynodon may be included.
サトウキビ属、リンゴ属、シソ属、アブラナ属及びギョウギシバ属の植物は、いずれも食用植物であるか、又は日常的に接触する植物である。よって、これらの植物から単離されたウスチラゴ属の微生物は安全性が高いといえる。なお、本明細書において「植物から単離される」とは、植物中、植物の表面、植物の根圏などの植物から単離されることを意味し、具体的には植物中等から、ここに生息する菌を取得することを意味する。 Sugarcane, apple genus, perilla genus, rapeseed genus and sorghum genus are all edible plants or plants that come into daily contact. Therefore, it can be said that the microorganisms of the genus Ustylago isolated from these plants are highly safe. In the present specification, the term “isolated from a plant” means being isolated from a plant such as a plant, the surface of the plant, or the rhizosphere of the plant. It means to get the fungus to do.
サトウキビ属の植物の具体例としては特に限定されないが、サッカリウム オフィシナルム（Ｓ． ｏｆｆｉｃｉｎａｒｕｍ）、サッカリウム スポンタネウム（Ｓ． ｓｐｏｎｔａｎｅｕｍ）、サッカリウム ロブスツム（Ｓ． ｒｏｂｕｓｔｕｍ）、サッカリウム バーバリ（Ｓ． ｂａｒｂｅｒｉ）、サッカリウム サイネンセ（Ｓ． ｓｉｎｅｎｓｅ）、サッカリウム エヂュール（Ｓ． ｅｄｕｌｅ）等が挙げられる。中でもサッカリウム オフィシナルムが好ましい。 Specific examples of sugarcane plants include, but are not limited to, S. officinarum, S. spotanem, S. robustum, S. barberi , S. potassium senense, S. potassium edule, and the like. Among them, the potassium potassium officinalum is preferable.
なお、ウスチラゴ属の微生物は感染する宿主の種類によって分類されているため、サトウキビから単離されたウスチラゴ属の微生物は全てＵｓｔｉｌａｇｏ ｓｃｉｔａｍｉｎｅａである。同様に、後述するバミューダグラスから単離されたウスチラゴ属の微生物は全てＵｓｔｉｌａｇｏ ｃｙｎｏｄｏｎｔｉｓである。 Since microorganisms belonging to the genus Ustylag are classified according to the type of host to be infected, all microorganisms belonging to the genus Ustyago isolated from sugar cane are Ustilago scitaminea. Similarly, all the microorganisms of the genus Ustylago isolated from the Bermuda glass described later are Ustilago cynodontis.
リンゴ属の植物の具体例としては特に限定されないが、リンゴ（Ｍ． ｄｏｍｅｓｔｉｃａ）、クラブアップル（Ｍ． ｐｕｍｉｌａ）、ヒメリンゴ（Ｍ． ｂａｃｃａｔａ）等が挙げられる。中でもヒメリンゴ（Ｍ． ｂａｃｃａｔａ）が好ましい。 Although it does not specifically limit as a specific example of a plant of an apple genus, an apple (M. domestica), crab apple (M. pumila), a hime apple (M. baccata) etc. are mentioned. Of these, M. baccata is preferable.
シソ属の植物の具体例としては特に限定されないが、シソ（Ｐｅｒｉｌｌａ ｆｒｕｔｅｓｃｅｎｓ）が好ましい。 Although it does not specifically limit as a specific example of the plant of Perilla genus, Perilla frucescens is preferable.
アブラナ属の植物の具体例としては特に限定されないが、ミズナ（Ｂ． ｒａｐａ）、チンゲンサイ（Ｂ． ｃｈｉｎｇｅｎｓａｉ）、ハクサイ（Ｂ． ｈａｋｕｓａｉ）、カラシナ（Ｂ． ｋａｒａｓｈｉｎａ）、コマツナ（Ｂ． ｋｏｍａｔｓｕｎａ）等が挙げられる。中でも、ミズナ（Ｂ． ｒａｐａ Ｌ． ｖａｒ． ｎｉｐｐｏｓｉｎｉｃａ）が好ましい。 Specific examples of Brassica plants are not particularly limited, but examples include Mizuna (B. rapa), B. chinensai, B. khassai, B. karasina, B. komatsusuna and the like. Can be mentioned. Among them, Mizuna (B. rapa L. var. Nipposica) is preferable.
ギョウギシバ属の植物の具体例としては特に限定されないが、バミューダグラス（ギョウギシバ、Ｃ． ｄａｃｔｙｌｏｎ）、アフリカギョウギシバ（Ｃ． ｔｒａｎｓｖａａｌｅｎｓｉｓ）、マクニスバーミューダグラス（Ｃ． ｍａｂｅｎｎｉｓｉｉ Ｈｕｒｃｏｍｂｅ）、オニギョウギシバ（Ｃ． ｐｌｅｃｔｏｓｔａｃｈｙｓ）、キノドン インコンプレツス（Ｃ． ｉｎｃｏｍｐｌｅｔｕｓ）、等が挙げられる。中でもバミューダグラスが好ましい。 Specific examples of the plant belonging to the genus Gorga are not particularly limited, but include Bermudagrass (C. dactylon), African ginkgo biloba (C. tranvaalensis), Macnis vermudgrass (C. mabennisii Hurcombe), Onigyo chy p. , Quinodon incompress (C. incompletetus), and the like. Of these, Bermuda glass is preferable.
また、これらの植物から単離されたウスチラゴ属の微生物はＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃの３種のＭＥＬのうちのいずれかを選択的に生産することができる。 In addition, microorganisms belonging to the genus Ustillago isolated from these plants can selectively produce any one of three types of MELs, MEL-A, MEL-B, and MEL-C.
本明細書において「選択的に生産する」とは、生産する対象物が複数種類想定し得る場合、そのうちの一つの対象物を他の対象物に比べて多く生産ことを意味する。具体的には、生産する上記一般式（１）で表されるＭＥＬの全量に対して、８５重量％以上を占める割合にて、３種のＭＥＬのうちの１種を製造することを意味する。 In this specification, “selectively producing” means that when a plurality of types of objects to be produced can be assumed, one of the objects is produced more than other objects. Specifically, it means that one of the three types of MEL is produced at a ratio of 85% by weight or more with respect to the total amount of MEL represented by the general formula (1) to be produced. .
〔１．植物からの単離方法〕
上述した各植物からウスチラゴ属の微生物を単離する方法としては特に限定されない。従来公知の方法を適用してもよい。例えば、植物を破砕して水又は適切な緩衝液に懸濁させ、懸濁液を栄養塩固形培地に塗布し、形成されたコロニーをＭＥＬ合成確認用培地に植えて、ＭＥＬ形成能を有する菌株を単離した上で、従来公知の同定方法によりウスチラゴ属であることを確認してもよい。また、植物を破砕して、栄養塩液体培地に混合して培養した後、ＭＥＬの生産が確認できた液体培地を栄養塩固形培地に塗布して、形成されたコロニーを合成確認用培地に植えて、ＭＥＬ形成能を確認した上で、ウスチラゴ属か否かを同定してもよい。ＭＥＬ合成確認用培地としてはＭＥＬの合成を確認できるものであれば特に限定されないが、例えば、培地に大豆油等の油脂を混合しておけばよい。当該油脂が乳化するか否かでＭＥＬの形成を確認することができる。
[1. (Isolation method from plant)
It does not specifically limit as a method of isolating the microorganism of the genus Ustyago from each plant mentioned above. A conventionally known method may be applied. For example, the plant is crushed and suspended in water or an appropriate buffer, the suspension is applied to a nutrient solid medium, the formed colonies are planted in a medium for MEL synthesis confirmation, and a strain having MEL forming ability And may be confirmed to be of the genus Ustilago by a conventionally known identification method. In addition, the plant is crushed, mixed in a nutrient liquid medium and cultured, and then a liquid medium that has been confirmed to be produced by MEL is applied to the nutrient solid medium, and the formed colonies are planted in the medium for confirmation of synthesis. Then, after confirming the ability to form MEL, it may be identified whether or not it belongs to the genus Ustilago. The medium for confirming MEL synthesis is not particularly limited as long as MEL synthesis can be confirmed. For example, oil such as soybean oil may be mixed in the medium. The formation of MEL can be confirmed by whether or not the oil or fat is emulsified.
〔２．本発明に係るＭＥＬ‐Ｂの製造方法に用いる微生物〕
本発明に係る上記一般式（２）で表される構造を有するＭＥＬ‐Ｂの製造方法は、サトウキビ（Ｓａｃｃｈａｒｕｍ）属の植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含めばよい。
[2. Microorganism used in the method for producing MEL-B according to the present invention]
The method for producing MEL-B having the structure represented by the general formula (2) according to the present invention comprises a culturing step of culturing a microorganism belonging to the genus Ustilago isolated from a plant belonging to the genus Saccharum. Include it.
つまり、本発明に係るＭＥＬ‐Ｂの製造方法では、これまでの説明で例示した植物のうち、サトウキビ属の植物から単離されたウスチラゴ属の微生物を用いるとよい。 That is, in the method for producing MEL-B according to the present invention, among the plants exemplified in the above description, a microorganism belonging to the genus Ustyago isolated from a sugar cane plant may be used.
サトウキビ族の植物から単離されたウスチラゴ属の微生物の具体例としては、特に限定されないが、Ｕ． ｓｃｉｔａｍｉｎｅａが挙げられる。この微生物はマンノシルアルジトールリピッド生産微生物として知られていなかったものであるが、本発明者等により、シュードザイマ アンタクチカ等の既知のマンノシルアルジトールリピッド生産菌と同等の生産性を有することが初めて確認されたものである。しかも、ウスチラゴ サイタミネアはサトウキビから分離された微生物であり、マンノシルアルジトールリピッドの中のＭＥＬ‐Ｂのみを選択的に生産することができる。 Specific examples of the microorganism of the genus Ustylago isolated from sugarcane plants are not particularly limited. scitaminea. Although this microorganism was not known as a mannosyl alditol lipid-producing microorganism, the present inventors have confirmed for the first time that it has the same productivity as known mannosyl alditol lipid-producing bacteria such as pseudozyma antactica. It is a thing. Moreover, Ustyago cythaminea is a microorganism isolated from sugarcane and can selectively produce only MEL-B in mannosyl alditol lipids.
Ｕ． ｓｃｉｔａｍｉｎｅａは従来公知の方法でサトウキビ属の植物から単離して得ればよいが、ＮＢＲＣ３２７３０株を用いてもよい。ＮＢＲＣ３２７３０株を用いれば極めて高効率にＭＥＬ‐Ｂを選択的に生産することができる。また、Ｕ． ｓｃｉｔａｍｉｎｅａを培養する際は、炭素源として油脂を用いずに、糖を用いればよい点で有利である。即ち、従来公知のＭＥＬ生産菌では炭素源として油脂を含む培地で培養する必要があった。これでは、培養後に油脂を除去しＭＥＬを精製する必要があったが、これには煩雑な作業を要した。しかし、糖を炭素源として、油脂を含まず（含んだとしても炭素源の５重量％以下）であれば、容易に精製できるので、高純度のＭＥＬ‐Ｂを簡便に製造することができる。 U. Although scitaminea may be obtained by isolation from a sugarcane plant by a conventionally known method, NBRC32730 strain may be used. If NBRC32730 strain is used, MEL-B can be selectively produced with extremely high efficiency. In addition, U.S. When cultivating scitamine, it is advantageous in that sugar may be used without using fats and oils as a carbon source. That is, conventionally known MEL-producing bacteria have to be cultured in a medium containing fats and oils as a carbon source. In this case, it was necessary to remove the fats and oils and purify the MEL after culturing, but this required complicated work. However, since sugar can be used as a carbon source and oil and fat are not included (even if it is included, 5% by weight or less of the carbon source), it can be easily purified, so that high-purity MEL-B can be easily produced.
〔３．本発明に係るＭＥＬ‐Ｃの製造方法に用いる微生物〕
本発明に係る上記一般式（３）で表される構造を有するＭＥＬ‐Ｃの製造方法は、ギョウギシバ（Ｃｙｎｏｄｏｎ）属の植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含めばよい。
[3. Microorganisms used in the method for producing MEL-C according to the present invention]
The method for producing MEL-C having the structure represented by the general formula (3) according to the present invention includes a cultivation step of culturing a microorganism belonging to the genus Ustilago isolated from a plant belonging to the genus Cynodon. Include it.
つまり、本発明に係るＭＥＬ‐Ｃの製造方法では、これまでの説明で例示した植物のうち、ギョウギシバ属の植物から単離されたウスチラゴ属の微生物を用いるとよい。ギョウギシバ属の植物から単離されたウスチラゴ属の微生物の具体例としては、特に限定されないが、ウスチラゴ シノドンティス（Ｕ． ｃｙｎｏｄｏｎｔｉｓ）が挙げられる。ウスチラゴ シノドンティスはバミューダグラスから分離された微生物であり、ＭＥＬのうちのＭＥＬ‐Ｃを選択的に生産することができる。なお、バミューダグラスは、グラウンドや庭に敷き詰められるシバであり、安全かつ日常的に接触頻度の高い植物である。 That is, in the method for producing MEL-C according to the present invention, among the plants exemplified in the above description, a microorganism belonging to the genus Ustyago, which is isolated from a plant belonging to the genus Gorilla, may be used. Specific examples of the microorganism belonging to the genus Ustyago isolated from a plant belonging to the genus Gyolia include, but are not limited to, U. cynodontis. Ustyago Shinodontis is a microorganism isolated from Bermuda grass and can selectively produce MEL-C of MEL. Bermuda grass is a plant that is spread on the ground and in the garden, and is a plant that is safe and has a high daily contact frequency.
ウスチラゴ シノドンティスは従来公知の方法でギョウギシバ属の植物から単離して得ればよいが、ＮＢＲＣ７５３０株を用いてもよい。ＮＢＲＣ７５３０株を用いれば極めて高効率にＭＥＬ‐Ｃを選択的に生産することができる。 The ustillago sinodontis may be obtained by isolation from a plant belonging to the genus Gorilla by a conventionally known method, but the NBRC7530 strain may be used. If NBRC7530 strain is used, MEL-C can be selectively produced with extremely high efficiency.
〔４．本発明に係るＭＥＬ‐Ａの製造方法に用いる微生物〕
本発明に係る一般式（４）で表される構造を有するＭＥＬ‐Ａの製造方法は、リンゴ（Ｍａｌｕｓ）属、シソ属（Ｐｅｒｉｌｌａ）属及びアブラナ（Ｂｒａｓｓｉｃａ）属のいずれかの植物から単離されたウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を培養する培養工程を含めばよい。
[4. Microorganisms used in MEL-A production method according to the present invention]
The method for producing MEL-A having the structure represented by the general formula (4) according to the present invention is isolated from any plant of the genus Apple (Malus), the genus Perilla and the genus Brassica. A culture step of culturing a microorganism belonging to the genus Ustilago can be included.
つまり、本発明に係るＭＥＬ‐Ａの製造方法では、これまでの説明で例示した植物のうち、リンゴ属、シソ属及びアブラナ属のいずれかの植物から単離されたウスチラゴ属の微生物を用いればよい。当該微生物は、従来公知の方法でリンゴ属、シソ属及びアブラナ属の植物から単離して得ればよい。 That is, in the method for producing MEL-A according to the present invention, among the plants exemplified in the above description, a microorganism of the genus Ustyago isolated from any one of the genus Apple, Perilla and Brassica is used. Good. The microorganism may be obtained by isolation from a plant of the genus Apple, Perilla and Brassica by a conventionally known method.
〔５．培養工程〕
上述した本発明に係るＭＥＬの製造方法、ＭＥＬ‐Ａの製造方法、ＭＥＬ‐Ｂの製造方法、ＭＥＬ‐Ｃの製造方法（なお、説明の便宜のため、これらの製造方法の総称として、単に「本発明に係る製造方法」と表記することもある）における、培養工程については、従来公知の方法でそれぞれの発明におけるウスチラゴ属の微生物を培養すればよい。
[5. (Culture process)
The above-described MEL manufacturing method, MEL-A manufacturing method, MEL-B manufacturing method, MEL-C manufacturing method according to the present invention described above (for convenience of explanation, these manufacturing methods are simply named as “generic name”. As for the culturing step in the “production method according to the present invention”), the microorganism of the genus Ustylago in each invention may be cultured by a conventionally known method.
培養に用いる培地としては、ウスチラゴ属がＭＥＬを生産する条件で培養可能なものであれば特に限定されない。ウスチラゴ属を培養すると通常ＭＥＬを生産するので、一般にウスチラゴ属等の酵母の培養に用いられる培地を用いればよい。例えばＹＰＤ培地（例えば水１Ｌにイーストエキストラクト１０ｇ、ポリペプトン２０ｇ及びグルコース２０ｇを溶解させて作製する）等の酵母に対して一般に用いられる培地を使用することができる。 The medium used for the culture is not particularly limited as long as it can be cultured under the conditions in which the genus Ustylago produces MEL. When cultivating the genus Ustylag, MEL is usually produced, and therefore, a medium generally used for culturing yeasts such as the genus Ustylag may be used. For example, a medium generally used for yeast such as YPD medium (for example, prepared by dissolving 10 g of yeast extract, 20 g of polypeptone and 20 g of glucose in 1 L of water) can be used.
また、培養に用いる培地には油脂を添加してもよい。油脂を添加する場合、培地中の油脂の含有量としては、特に限定されるものではないが、０〜８重量％以下が好ましく、０〜５重量％以下がさらに好ましい。油脂は培養開始時にその全量を添加しても、培養中に連続的または間欠的に添加してもよい。油脂の具体的な種類としては、植物油脂又は動物油脂等のいずれでもよく、目的に応じて適宜選定することができる。植物油脂としては、大豆油、菜種油、コーン油、ピーナッツ油、綿実油、ベニバナ油、ごま油、オリーブ油、バーム油等が挙げられ、これらの中でも、大豆油が好ましい。動物油脂としては、牛脂、豚脂、魚脂等が挙げられる。これらは、１種を単独で又は２種以上を適宜混合して用いてもよい。 Moreover, you may add fats and oils to the culture medium used for culture | cultivation. When fats and oils are added, the content of fats and oils in the medium is not particularly limited, but is preferably 0 to 8% by weight or less, and more preferably 0 to 5% by weight or less. The total amount of the oil / fat may be added at the start of the culture, or may be added continuously or intermittently during the culture. As a specific kind of fats and oils, any of vegetable oils and animal fats and the like may be used, and can be appropriately selected according to the purpose. Examples of vegetable oils include soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, and balm oil. Among these, soybean oil is preferable. Examples of animal fats include beef tallow, pork tallow and fish tallow. You may use these individually by 1 type or in mixture of 2 or more types as appropriate.
培地に添加する油脂類以外の他の成分としては、当該技術分野で通常用いられる炭素源、窒素源、無機塩類、及び必要な栄養源等が用いられる。炭素源としては、該微生物が資化し得るものであればよく、炭水化物（グルコース、マンノース、グリセロール、マンニトール等の単糖、ショ糖、麦芽糖、乳糖等のオリゴ糖、デンプン等）、有機酸（酢酸、プロピオン酸、マレイン酸、フマル酸、リンゴ酸等）、アルコール類（エタノール、プロパノール等）が用いられる。窒素源としては、塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウム、リン酸アンモニウム等の無機酸若しくは有機酸のアンモニウム塩、又はペプトン、酵母エキス、麦芽エキス、肉エキス、コーンスチープリカー等の含窒素化合物が用いられる。無機塩類としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム等が用いられる。また、上記の炭水化物、有機酸、窒素源を含む植物体の搾汁（例えば、サトウキビ、トウモロコシ、穀類、果実など）も炭素源および窒素源を含む成分として使用可能である。 As other components other than fats and oils added to the medium, carbon sources, nitrogen sources, inorganic salts, necessary nutrient sources and the like that are usually used in the technical field are used. Any carbon source may be used as long as it can be assimilated by the microorganism, and includes carbohydrates (monosaccharides such as glucose, mannose, glycerol, and mannitol, oligosaccharides such as sucrose, maltose, and lactose, starch), organic acids (acetic acid) Propionic acid, maleic acid, fumaric acid, malic acid, etc.) and alcohols (ethanol, propanol, etc.) are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium phosphate, or nitrogen-containing compounds such as peptone, yeast extract, malt extract, meat extract, corn steep liquor and the like are used. As the inorganic salts, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. Moreover, the juice (for example, sugarcane, corn, cereals, fruit, etc.) of the plant body containing said carbohydrate, organic acid, and a nitrogen source can also be used as a component containing a carbon source and a nitrogen source.
ウスチラゴ サイタミネアを培養する場合、油脂を添加しなくても好適にＭＥＬ‐Ｂを生産することができる。培地に油脂を含ませない場合、炭素源としては糖を用いることが好ましい。糖としては、グルコース、マンノース、グリセロール、マンニトール等の単糖、ショ糖、麦芽糖、乳糖等のオリゴ糖、デンプン等の多糖が単独であるいは二種以上を組み合わせて用いられる。より好ましくは、スクロース、グルコース、フルクトース、マンノースのいずれかが単独で用いられる。培地中の糖の含有量としては、５〜２０重量％が好ましい。油脂類を添加しない場合、培養後、ＭＥＬの精製が容易に行なえるという利点等がある。なお、当該利点から、ウスチラゴ サイタミネアを培養する場合、油脂を含有しない培地で培養することが好ましいが、油脂を培地に含有させる場合であっても５重量％以下とすることが、容易に精製できることから好ましい。つまり、炭素源として糖を用いる場合、油脂の含有量は０〜８重量％以下であることが精製の観点から好ましく、０〜５重量％以下がさらに好ましい。 When cultivating ustillago cythaminea, MEL-B can be suitably produced without the addition of fats and oils. When oils and fats are not included in the medium, it is preferable to use sugar as the carbon source. As the sugar, monosaccharides such as glucose, mannose, glycerol and mannitol, oligosaccharides such as sucrose, maltose and lactose, and polysaccharides such as starch may be used alone or in combination of two or more. More preferably, any of sucrose, glucose, fructose and mannose is used alone. The content of sugar in the medium is preferably 5 to 20% by weight. When oils and fats are not added, there is an advantage that MEL can be easily purified after culturing. In addition, from the said advantage, when cultivating ustillago cythaminea, it is preferable to culture in a medium that does not contain fats and oils, but even when fats and oils are contained in the medium, it can be easily purified to 5% by weight or less. To preferred. That is, when sugar is used as the carbon source, the fat content is preferably 0 to 8% by weight or less from the viewpoint of purification, and more preferably 0 to 5% by weight or less.
培養温度は、２０〜３０℃、好ましくは２２〜２８℃である。培養日数としては、ＭＥＬの生産量に応じて適宜設定すればよいが、３〜２０日間が好ましい。培養方法としては、振盪培養、通気撹拌培養等の公知の一般的な微生物の培養方法を適用することができる。良好な微生物の生育及びＭＥＬの生産のためには、培養液に酸素を供給することが好ましく、そのためには、ジャーファメンターを用いる場合では、空気を通気しながら撹拌するか、振とう培養を行なえばよい。 The culture temperature is 20-30 ° C, preferably 22-28 ° C. The culture days may be appropriately set according to the production amount of MEL, but 3 to 20 days is preferable. As a culture method, known general microorganism culture methods such as shaking culture and aeration stirring culture can be applied. For good growth of microorganisms and production of MEL, it is preferable to supply oxygen to the culture solution. For this purpose, when using a jar fermenter, agitation with air or shaking culture is recommended. Just do it.
本発明において、例えば、前記ウスチラゴ サイタミネア（Ｕｓｔｉｌａｇｏ ｓｃｉｔａｍｉｎｅａ）又はウスチラゴ シノドントィス（Ｕｓｔｉｌａｇｏ ｃｙｎｏｄｏｎｔｉａ）を用いてＭＥＬ‐Ｂ又はＭＥＬ‐Ｃを生産する場合、増殖能の高い菌株を用いた方が、単位培養液及び単位時間当たりのマンノシルエリスリトールリピッド生産量が高くなるため好ましい。そのため、まず前培養によって菌体を活性化させ、これを本培養の培地に接種して培養を行なうことが好ましい。例えば、種培養、本培養及びＭＥＬ生産培養の順にスケールアップしていくことが好ましい。これらの培養における、具体的な培地組成、培養条件を例示すると以下のとおりである。 In the present invention, for example, when producing MEL-B or MEL-C using the above-mentioned Ustyago scitamine or Ustyago cynodontia, the unit culture solution and This is preferable because the production amount of mannosyl erythritol lipid per unit time becomes high. For this reason, it is preferable that the cells are first activated by preculture and inoculated into the medium for main culture. For example, it is preferable to scale up in the order of seed culture, main culture, and MEL production culture. Examples of specific medium composition and culture conditions in these cultures are as follows.
グルコース１０〜５０ｇ／Ｌ、酵母エキス１〜２ｇ／Ｌ、硝酸ナトリウム１〜５ｇ／Ｌ、リン酸２水素カリウム０．１〜２ｇ／Ｌ、及び硫酸マグネシウム０．１〜１ｇ／Ｌの組成の液体培地２ｍＬが入った試験管に、保存菌株を１白金耳接種し、２２〜２８℃で１〜３日振とう培養を行なう（種培養）。次に、２０〜３００ｇ／Ｌ、好ましくは５０〜１００ｇ／Ｌの植物油脂等の油脂類を加えた上記と同じ組成の液体培地５０〜１００ｍＬの入った坂口フラスコに上記の種培養を行った培養液を接種して、２２〜２８℃で３〜７日間振とう培養を行なう(本培養）。さらに、上記の本培養と同じ組成の液体培地１〜２Ｌが入ったジャーファメンターに上記の本培養を行った培養液を接種して、２２〜２８℃で１〜２Ｌ／分の通気速度と６００〜１０００ｒｐｍの撹拌速度で培養する（ＭＥＬ生産培養）。このときの培養温度は２２〜２８℃、培養時間は、７〜２８日間である。この培養においては、培養途中から油脂類を培養容器中に流下させて、培地中の油脂類濃度を５０〜１００ｇ／Ｌに保持することが好ましい。 Liquid with composition of glucose 10-50 g / L, yeast extract 1-2 g / L, sodium nitrate 1-5 g / L, potassium dihydrogen phosphate 0.1-2 g / L, and magnesium sulfate 0.1-1 g / L A test tube containing 2 mL of the medium is inoculated with 1 platinum loop of the stock strain and cultured with shaking at 22 to 28 ° C. for 1 to 3 days (seed culture). Next, the culture which performed said seed culture to the Sakaguchi flask containing 50-100 mL of liquid culture medium of the same composition as the above which added oils and fats, such as vegetable oils and fats of 20-300 g / L, preferably 50-100 g / L The solution is inoculated and shake-cultured at 22-28 ° C. for 3-7 days (main culture). Further, a jar fermenter containing 1 to 2 L of liquid medium having the same composition as that of the main culture is inoculated with the culture solution obtained by performing the main culture, and an aeration rate of 1 to 2 L / min at 22 to 28 ° C. Culturing is performed at a stirring speed of 600 to 1000 rpm (MEL production culture). The culture temperature at this time is 22 to 28 ° C., and the culture time is 7 to 28 days. In this culture, it is preferable that oils and fats flow down into the culture vessel from the middle of the culture to maintain the oils and fats concentration in the medium at 50 to 100 g / L.
微生物菌株の培地への使用量は、例えば、菌体を接種する場合、培地１Ｌあたり、１０〜２００ｍＬ、好ましくは５０〜１００ｍＬの種培養液あるいは本培養液中に含まれる量であればよい。 The amount of the microorganism strain used in the medium may be, for example, an amount contained in 10 to 200 mL, preferably 50 to 100 mL of the seed culture solution or the main culture solution, per 1 L of the medium when inoculating the cells.
本発明に係る製造方法における、培養工程は、培養液中の所望のＭＥＬ生成量が最高に達した時点で終了させることができるように、培養液中のＭＥＬをガスクロマトグラフィー、高速液体クロマトグラフィー、薄層クロマトグラフィー等の周知の方法により測定しながら行なうことが好ましい。 In the production method according to the present invention, the culturing step can be terminated when the desired MEL production amount in the culture solution reaches the maximum, so that the MEL in the culture solution is gas chromatographed or high performance liquid chromatography. It is preferable to carry out the measurement while measuring by a known method such as thin layer chromatography.
培養液中に蓄積されたＭＥＬは、培養終了後、培養液を抽出することによって取得できる。 MEL accumulated in the culture solution can be obtained by extracting the culture solution after completion of the culture.
抽出は、酢酸エチル等の有機溶媒を用いて行なう。抽出は当該分野において通常行われる方法に従って行なえばよく、例えば、培養液１Ｌあたり０．５〜１．５Ｌ程度の有機溶媒を用いて１〜数回抽出操作を行ない、酢酸エチル層を合わせて溶媒を留去する。 Extraction is performed using an organic solvent such as ethyl acetate. Extraction may be carried out according to a method usually performed in the art. For example, the extraction operation is performed once or several times using about 0.5 to 1.5 L of organic solvent per 1 L of the culture solution, and the ethyl acetate layer is combined with the solvent. Is distilled off.
酢酸エチル抽出物に含まれるＭＥＬを分画するには、シリカゲルカラムクロマトグラフィーによる分離精製を行なう。酢酸エチル抽出物をシリカゲルカラムに供し、クロロホルムとアセトンを溶出液として用い、当該抽出物中の成分を分離精製する。クロロホルムのみを流した後、クロロホルムとアセトンの混合液で当該抽出物中の成分を溶出する。この時、クロロホルムとアセトンの混合液中におけるアセトンの割合は、段階的に上げていく。例えば、９：１から始め、次に、８：２あるいは７：３としていく。各溶出液は収集し、ＴＬＣにて精製度を確認する。 In order to fractionate MEL contained in the ethyl acetate extract, separation and purification by silica gel column chromatography is performed. The ethyl acetate extract is applied to a silica gel column, and components in the extract are separated and purified using chloroform and acetone as eluents. After flowing only chloroform, the components in the extract are eluted with a mixture of chloroform and acetone. At this time, the ratio of acetone in the mixed liquid of chloroform and acetone is increased stepwise. For example, start from 9: 1, then 8: 2 or 7: 3. Each eluate is collected and the degree of purification is confirmed by TLC.
また、本発明に係る製造方法においては、抽出する前に濃縮を行なってもよい。濃縮方法としては特に限定されないが、上記培養工程にて得られた培養液から菌体を取り除き、硫酸アンモニウムを添加して沈殿物を得る濃縮工程を行なうことが好ましい。この沈殿物中にＭＥＬが大量に濃縮されているので、当該沈殿物を上述の抽出に供する等、有機溶剤を用いてＭＥＬ‐Ｂを回収すれば、高純度なＭＥＬ‐Ｂを容易に回収できる。特に、工場等の大規模スケールの場合、濃縮技術は保管及び運搬のみでなく有機溶剤の使用量を削減するという観点からも重要な技術である。この濃縮工程によればＭＥＬを容易に回収することができる。 In the production method according to the present invention, concentration may be performed before extraction. Although it does not specifically limit as a concentration method, It is preferable to perform the concentration process which removes a microbial cell from the culture solution obtained at the said culture | cultivation process, and adds ammonium sulfate, and obtains a deposit. Since a large amount of MEL is concentrated in the precipitate, high-purity MEL-B can be easily recovered by recovering MEL-B using an organic solvent, such as subjecting the precipitate to the above-described extraction. . In particular, in the case of a large scale such as a factory, the concentration technique is an important technique not only from storage and transportation but also from the viewpoint of reducing the amount of organic solvent used. According to this concentration step, MEL can be easily recovered.
添加する硫酸アンモニウムの濃度としては、特に限定されないが、例えば、培養液に対して１０質量％以上、３０質量％以下にするとよい。 Although it does not specifically limit as a density | concentration of the ammonium sulfate to add, For example, it is good to set it as 10 to 30 mass% with respect to a culture solution.
上記培養において、微生物の形態は、特に限定されず、微生物の菌体、菌体処理物（例えば、菌体破砕物）などをいう。 In the above culture, the form of the microorganism is not particularly limited, and refers to a microorganism cell, a treated cell of the microorganism (for example, a disrupted cell).
微生物の菌体または菌体処理物は固定化して用いることもできる。固定化法としては、従来公知の担体結合法、架橋化法、包括法などの方法が挙げられる。担体結合法では、担体に菌体を固定化させるが、固定化は物理的吸着、イオン結合、共有結合のいずれであってもよい。担体としては多糖（アセチルセルロース、アガロース）、無機物質（多孔質ガラス、金属酸化物）、合成高分子（ポリアクリルアミド、ポリスチレン）等が用いられる。架橋化法では、グルタルアルデヒド等の二官能性試薬を用いて菌体同士を架橋、結合させることによって固定化する。また、包括法では、多糖（アルギン酸、カラギーナン）、ポリアクリルアミド、コラーゲン、ポリウレタン等の高分子ゲルの格子や半透膜カプセルに菌体を包み込むことによって固定化する。 The microbial cells or the processed microbial cells can be immobilized and used. Examples of the immobilization method include conventionally known methods such as a carrier binding method, a crosslinking method, and a comprehensive method. In the carrier binding method, the cells are immobilized on the carrier, and the immobilization may be any of physical adsorption, ionic bond, and covalent bond. As the carrier, polysaccharides (acetylcellulose, agarose), inorganic substances (porous glass, metal oxide), synthetic polymers (polyacrylamide, polystyrene) and the like are used. In the crosslinking method, the cells are immobilized by crosslinking and bonding with each other using a bifunctional reagent such as glutaraldehyde. In the inclusion method, the cells are immobilized by wrapping the cells in a lattice of a polymer gel such as polysaccharide (alginate, carrageenan), polyacrylamide, collagen, polyurethane, or a semipermeable membrane capsule.
〔６．ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのいずれかを主成分とするマンノシルエリスリトールリピッドの用途〕
上述した本発明に係る製造方法によれば、ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのいずれかを選択的に製造することができる。これにより、ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのいずれかを主成分とするＭＥＬを得ることができるともいえる。なお、主成分とは、ＭＥＬ‐Ａ、Ｂ、Ｃのうちの１種において、他の２種の量に比べて高い含有量で含まれているものを意味する。
[6. Uses of mannosyl erythritol lipid mainly comprising any of MEL-A, MEL-B and MEL-C)
According to the manufacturing method according to the present invention described above, any one of MEL-A, MEL-B, and MEL-C can be selectively manufactured. Thereby, it can be said that MEL which has any of MEL-A, MEL-B, and MEL-C as a main component can be obtained. The main component means that one of MEL-A, B, and C contains a higher content than the other two.
上記の方法により得られたマンノシルエリスリトールリピッドの主成分であるＭＥＬ‐Ａ、ＭＥＬ‐Ｂ又はＭＥＬ−Ｃの構造は、シュードザイマ属の酵母が生産する既知のＭＥＬと同じである。これらＭＥＬは、温度範囲が室温から７０℃まで、濃度範囲が０．０００１％から９０％までの幅広い条件下でリオトロピック液晶を形成することが可能である。リオトロピック液晶は、高濃度の水溶性成分と油溶性成分の双方を内包安定化できるという性質を有している。従って、当該マンノシルエリスリトールリピッドは、食品等の種々の組成物に含有させることにより、その界面活性作用により主成分の分散性、製品の酸化安定性向上をはかることができる。食品の形態は特に限定されないが、マンノシルエリスリトールリピッドの生理活性を活用した機能性食品・動物飼料などが挙げられる。 The structure of MEL-A, MEL-B, or MEL-C, which is the main component of mannosyl erythritol lipid obtained by the above method, is the same as the known MEL produced by Pseudozyma yeast. These MELs can form lyotropic liquid crystals under a wide range of temperature ranges from room temperature to 70 ° C. and concentration ranges from 0.0001% to 90%. The lyotropic liquid crystal has the property that it can stabilize both the high-concentration water-soluble component and the oil-soluble component. Therefore, when the mannosyl erythritol lipid is contained in various compositions such as foods, it is possible to improve the dispersibility of the main component and the oxidation stability of the product due to its surface activity. The form of the food is not particularly limited, and examples thereof include functional foods and animal feeds utilizing the physiological activity of mannosyl erythritol lipid.
また前記のとおり、マンノシルエリスリトールリピッドは界面活性能以外にも抗微生物活性、細胞分化誘導活性、糖タンパク質結合活性、抗炎症・抗アレルギー活性、アポトーシス誘導活性、癌細胞増殖抑制活性など種々の生理活性作用を有する。従って、ＭＥＬ、公知の種々の方法にて各種製剤形態に調製し、それ自体を有効成分とする医薬（例えば、抗癌剤など）として用いることができる。各種製剤は、製剤上通常用いられる賦形剤、増量剤、結合剤、湿潤剤、崩壊剤、潤滑剤、界面活性剤、分散剤、緩衝剤、保存剤、溶解補助剤、防腐剤、矯味矯臭剤、無痛化剤、安定化剤、等張化剤等などを適宜選択し、常法により製造することができる。 As described above, mannosyl erythritol lipid has a variety of physiological activities such as antimicrobial activity, cell differentiation-inducing activity, glycoprotein binding activity, anti-inflammatory / anti-allergic activity, apoptosis-inducing activity, and cancer cell growth-inhibiting activity in addition to its surface activity. Has an effect. Therefore, it can be prepared in various preparation forms by various known methods such as MEL and used as a medicament (for example, an anticancer agent) containing itself as an active ingredient. Various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffering agents, preservatives, solubilizers, preservatives, flavoring agents that are commonly used in the formulation. Agents, soothing agents, stabilizers, tonicity agents and the like can be appropriately selected and produced by conventional methods.
以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。 Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.
〔実施例１〕
（１：ＭＥＬ生産菌の探索）
ウスチラゴ属はトウモロコシ、サトウキビ、シバ、ススキといった植物体中に生息する糸状菌であり、人類が生活の中で日常的に接触してきた微生物群である。ウスチラゴ属に属する微生物は５００種以上存在するが、そのうちトウモロコシから分離されるＵｓｔｉｌａｇｏ ｍａｙｄｉｓのみがバイオサーファクタント生産菌として知られていた。Ｕ． ｍａｙｄｉｓがＭＥＬ生産能を有することは知られているが、ＭＥＬのうち、ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ及びＭＥＬ‐Ｃを混合物として生産するので、ＭＥＬ‐Ａ、ＭＥＬ‐Ｂ又はＭＥＬ‐Ｃのいずれかを選択的に製造することはできない。そこで、全くバイオサーファクタント生産能は知られていないが、食用植物あるいは日常的に接触頻度の高い植物から単離された微生物に着目し、油脂含有培地中で培養し、それらの中からバイオサーファクタント生産微生物の探索を試みた。
[Example 1]
(1: Search for MEL-producing bacteria)
The genus Ustilago is a filamentous fungus that inhabits plants such as corn, sugarcane, buckwheat, and pampas grass, and is a group of microorganisms that humans have come into contact with on a daily basis. There are more than 500 types of microorganisms belonging to the genus Ustylago, of which only Ustilago maydis isolated from corn was known as a biosurfactant producing bacterium. U. It is known that maydis has the ability to produce MEL, but among MEL, MEL-A, MEL-B and MEL-C are produced as a mixture, so any of MEL-A, MEL-B or MEL-C Cannot be selectively manufactured. Therefore, although biosurfactant production ability is not known at all, paying attention to microorganisms isolated from edible plants or plants with high daily contact frequency, cultivate in oil-containing medium and produce biosurfactant from them I tried to search for microorganisms.
本実施例では、サトウキビ属及びギョウギシバ属の植物から単離されたウスチラゴ属の微生物を用いた。具体的にはサトウキビ属由来のウスチラゴ属の微生物としてＵ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０を用いた。また、ギョウギシバ属由来のウスチラゴ属の微生物として、Ｕ． ｃｙｎｏｄｏｎｔｉｓ ＮＢＲＣ７５３０及びＮＢＲＣ９７２７を用いた。 In this example, a microorganism of the genus Ustylago isolated from a plant of the genus Sugarcane and the genus Drosophila was used. Specifically, as a microorganism of the genus Ustyago derived from the sugarcane genus, U. scitamine NBRC32730 was used. In addition, as a microorganism of the genus Ustylago derived from the genus Gorilla, U. cynodontis NBRC7530 and NBRC9727 were used.
（２：Ｕ． ｓｃｉｔａｍｉｎｅａ及びＵ． ｃｙｎｏｄｏｎｔｉｓの培養）
（２−ａ：種培養）
保存培地（麦芽エキス３ｇ／Ｌ、酵母エキス３ｇ／Ｌ、ペプトン５ｇ／Ｌ、グルコース１０ｇ／Ｌ、寒天３０ｇ／Ｌ）に保存しておいた上記のＵ． ｃｙｎｏｄｏｎｔｉｓとＵ． ｓｃｉｔａｍｉｎｅａを、グルコース２０ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地２ｍＬが入った試験管に１白金耳接種し２５℃で振とう培養を２日間行なった。
(2: Cultivation of U. spitaminea and U. cynodontis)
(2-a: seed culture)
The above-mentioned U.S. stored in a storage medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L, glucose 10 g / L, agar 30 g / L). cynodontis and U.S. A test tube containing 2 mL of liquid medium having a composition of 20 g / L glucose, 1 g / L yeast extract, 1 g / L sodium nitrate, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. 1 platinum ear was inoculated and cultured at 25 ° C. with shaking for 2 days.
（２−ｂ：本培養）
上記２−ａで得られた菌体培養液を、４０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌの組成の液体培地２０ｍＬが入った坂口フラスコに接種し、振とう培養を２５℃にて７日間行なった。
(2-b: Main culture)
The bacterial cell culture solution obtained in 2-a above was prepared by using 40 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g. A Sakaguchi flask containing 20 mL of a liquid medium having a composition of / L was inoculated, and shaking culture was performed at 25 ° C. for 7 days.
それぞれの微生物を培養して得た培養液の酢酸エチル抽出液を薄層クロマトグラフィー分析した。この結果を図１に示す。図１は、Ｕ． ｓｃｉｔａｍｉｎｅａ及びＵ． ｃｙｎｏｄｏｎｔｉｓの培養液を薄層クロマトグラフィーに供した結果を示す。薄層クロマトグラフィーについては、Ｓｉｌｉｃａ ｇｅｌ ６０Ｆ（Ｗａｋｏ社製）を用い、展開溶媒としてはクロロホルム：メタノール：７Ｎアンモニア＝６５:１５:２とした。検出は、展開後のプレートにアンスロン試薬を吹き付け、１１０℃で５分間加熱した。 The ethyl acetate extract of the culture solution obtained by culturing each microorganism was analyzed by thin layer chromatography. The result is shown in FIG. FIG. scitamine and U.S. The result of having used the culture solution of cynodontis for the thin layer chromatography is shown. For thin layer chromatography, Silica gel 60F (manufactured by Wako) was used, and the developing solvent was chloroform: methanol: 7N ammonia = 65: 15: 2. For detection, an anthrone reagent was sprayed on the developed plate and heated at 110 ° C. for 5 minutes.
なお、比較のためＭＥＬの標準サンプル及びＵ． ｍａｙｄｉｓＮＢＲＣ５３４６及びＮＢＲＣ６９０７から得たＭＥＬを薄層クロマトグラフィーに供した。標準サンプルとしてはシュードザイマ アンタクチカ（Ｐｓｅｕｄｏｚｙｍａ ａｎｔａｒｃｔｉｃａ）ＪＣＭ１０３１７株を、２−ａ及びここに記載した方法と同じ方法で種培養及び本培養して、原料油脂等の不純物を取り除いて得た。Ｕ． ｍａｙｄｉｓＮＢＲＣ５３４６及びＮＢＲＣ６９０７由来のＭＥＬについても、同様にして得た。これらの標準サンプル、ＮＢＲＣ５３４６及びＮＢＲＣ６９０７由来のＭＥＬを薄層クロマトグラフィーに供した。図１のレーンＳが標準サンプルを薄層クロマトグラフィーに供した結果を示している。 For comparison, a standard sample of MEL and U.I. MELs obtained from maydis NBRC5346 and NBRC6907 were subjected to thin layer chromatography. As a standard sample, Pseudozyma antarctica (Pseudozyma antarctica) JCM10317 strain was seed-cultured and main-cultured by the same method as 2-a and the method described here to remove impurities such as raw material fats and oils. U. The MELs derived from maydisNBRC5346 and NBRC6907 were obtained in the same manner. MELs derived from these standard samples, NBRC5346 and NBRC6907 were subjected to thin layer chromatography. Lane S in FIG. 1 shows the result of subjecting the standard sample to thin layer chromatography.
図１に示すように、両株ともＭＥＬを生産した。Ｕ． ｓｃｉｔａｍｉｎｅａはＭＥＬ‐Ｂを選択的に生産したことが確認できた（図１、レーンＡ）。また、Ｕ． ｃｙｎｏｄｏｎｔｉｓはＭＥＬ‐Ｃを選択的に生産したことが確認できた（図１、レーンＢ１及びＢ２）。以上の結果から、Ｕ． ｃｙｎｏｄｏｎｔｉｓはＭＥＬ‐Ｃのみを選択的に生産し、また、Ｕ． ｓｃｉｔａｍｉｎｅａはＭＥＬ‐Ｂのみを選択的に生産することは明らかである。 As shown in FIG. 1, both strains produced MEL. U. It was confirmed that scitamine selectively produced MEL-B (FIG. 1, lane A). In addition, U.S. It was confirmed that cynodontis selectively produced MEL-C (FIG. 1, lanes B1 and B2). From the above results, U.S. cynodontis selectively produces only MEL-C, and It is clear that scitaminea selectively produces only MEL-B.
（３：Ｕ． ｓｃｉｔａｍｉｎｅａ及びＵ． ｃｙｎｏｄｏｎｔｉｓにより生産されるＭＥＬの構造解析）
（３‐ａ：精製標品の高速液体クロマトグラフィーによる分析）
上記２−ａ及び２−ｂと同じ方法でＵ． ｓｃｉｔａｍｉｎｅａ及びＵ． ｃｙｎｏｄｏの培養を行ない、培養終了後の培養液を採取し、酢酸エチルで抽出し、酢酸エチル可溶分中のＭＥＬを高速液体クロマトグラフィー（ＨＰＬＣ）を用いて検出した。結果を図２に示す。図２はＵ． ｓｃｉｔａｍｉｎｅａ及びＵ． ｃｙｎｏｄｏｎｔｉｓが生産するＭＥＬを高速液体クロマトグラフィーで分析した結果を示す図である。なお、図２の各サンプルの結果の上に記載されている「ＭＥＬ‐Ａ：Ｂ：Ｃ」は、培養液中に含まれていたＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃの重量比である。また、比較のため、上記２−ｂで用いた標準サンプル及びＵ． ｍａｙｄｉｓ ＮＢＲＣ５３４６から得たＭＥＬを同様にＨＰＬＣに供して検出した。高速液体クロマトグラフィーによる検出には、ＤＰ‐８０２０（ＴＯＳＯＨ社製）を用いた。カラムとしては、Ｉｎｅｒｔｓｉｌ ＳＩＬ １００Ａ ５μｍ、４．６×２５０ｍｍ（ＧＬ ｓｃｉｅｎｃｅ社製）を用い、クロロホルムとメタノールを１００：０〜０：１００のグラジエントをかけて１ｍＬ／ｍｉｎの流速で流した。
(3: Structural analysis of MEL produced by U. spitaminea and U. cynodontis)
(3-a: Analysis of purified sample by high performance liquid chromatography)
In the same manner as 2-a and 2-b above, U.S. scitamine and U.S. Cynodo was cultured, the culture solution after completion of the culture was collected, extracted with ethyl acetate, and MEL in the ethyl acetate-soluble component was detected using high performance liquid chromatography (HPLC). The results are shown in FIG. FIG. scitamine and U.S. It is a figure which shows the result of having analyzed MEL which cynodontis produces by the high performance liquid chromatography. Note that “MEL-A: B: C” described above the results of each sample in FIG. 2 is the weight ratio of MEL-A, MEL-B, and MEL-C contained in the culture solution. is there. For comparison, the standard sample used in 2-b and the U.S. MEL obtained from maydis NBRC5346 was similarly subjected to HPLC for detection. DP-8020 (manufactured by TOSOH) was used for detection by high performance liquid chromatography. As the column, Inertsil SIL 100A 5 μm, 4.6 × 250 mm (manufactured by GL science) was used, and chloroform and methanol were applied at a flow rate of 1 mL / min with a gradient of 100: 0 to 0: 100.
なお、Ｕ． ｓｃｉｔａｍｉｎｅａについては、大豆油の代わりにグルコース１００ｇ／Ｌを培地に加えて培養することで得られたＭＥＬ‐ＢもＨＰＬＣによる検出に供した。 In addition, U.S. For scitamine, MEL-B obtained by adding 100 g / L of glucose to the medium instead of soybean oil and culturing was also subjected to detection by HPLC.
図２に示すように、Ｕ． ｓｃｉｔａｍｉｎｅａから得られたＭＥＬのピークは、ＪＣＭ１０３１７株とは異なった。また、Ｕ． ｓｃｉｔａｍｉｎｅａから得られたＭＥＬのメインピークは、ＪＣＭ１０３１７株から得たＭＥＬ中のＭＥＬ‐Ｂとのピークと一致した。Ｕ． ｓｃｉｔａｍｉｎｅａは、高いＭＥＬ‐Ｂ生産能を有し、一週間で約９．６ｇ／ＬのＭＥＬ‐Ｂを生産した。なお、ＭＥＬ−Ｂおよび後述のＭＥＬ−Ｃの生産量は、精製したＭＥＬ−ＢおよびＭＥＬ−Ｃを用いて作成した検量線を用い、ＨＰＬＣの分析結果から定量した。 As shown in FIG. The peak of MEL obtained from scitamine was different from that of JCM10317 strain. In addition, U.S. The main peak of MEL obtained from scitaminea coincided with the peak of MEL-B in MEL obtained from JCM10317 strain. U. scitaminea had high MEL-B production capacity and produced about 9.6 g / L MEL-B in one week. The production amounts of MEL-B and MEL-C described below were quantified from the HPLC analysis results using a calibration curve prepared using purified MEL-B and MEL-C.
また、図２に示すように、Ｕ． ｃｙｎｏｄｏｎｔｉｓから得られたＭＥＬのピークはＪＣＭ１０３１７株から得たＭＥＬのピークとは異なった。また、Ｕ． ｃｕｎｏｄｏｎｔｉｓから得たＭＥＬのメインピークは、ＪＣＭ１０３１７株から得たＭＥＬ中のＭＥＬ‐Ｃのピークと一致した。Ｕ． ｃｙｎｏｄｏｎｔｉｓは、高いＭＥＬ‐Ｃ生産能を有し、一週間で約１．４ｇ／ＬのＭＥＬ‐Ｃを生産した。 As shown in FIG. The MEL peak obtained from cynodontis was different from the MEL peak obtained from the JCM10317 strain. In addition, U.S. The main peak of MEL obtained from Cunodontis coincided with the peak of MEL-C in MEL obtained from JCM10317 strain. U. cynodontis had high MEL-C production capacity and produced about 1.4 g / L MEL-C in one week.
なお、Ｕ． ｍａｙｄｉｓ ＮＢＲＣ５３４６については、ＭＥＬ‐Ａ：ＭＥＬ‐Ｂ：ＭＥＬ‐Ｃ：ＣＬｓの重量比は２１：０：９：２３であり、ＭＥＬ中の７割程度しかＭＥＬ‐Ａを生産せず、ＭＥＬの３種類のうちの１種類を選択的に製造するものではないことが分かった。また、ＭＥＬの生産の際、セロビオースリピッド（ＣＬｓ）を大量に混合することが示された。 In addition, U.S. For Maydis NBRC5346, the weight ratio of MEL-A: MEL-B: MEL-C: CLs is 21: 0: 9: 23, and only about 70% of MEL is produced. It was found that one of the types was not selectively produced. It was also shown that cellobiose lipids (CLs) were mixed in large quantities during MEL production.
（３‐ｂ：ＮＭＲによる構造解析）
Ｕ． ｃｉｔａｍｉｎｅａの培養液の酢酸エチル可溶分から、主成分であるＭＥＬ‐Ｂをシリカゲルカラムクロマトグラフィーによって精製分離した後、１Ｈと１３ＣＮＭＲによる構造解析を行なった。結果を表１に示す。また、標準的なＭＥＬの構造解析結果の例としてＰ． ａｎｔａｒｃｔｉｃａ ＪＣＭ １０３１７株が生産するＭＥＬの分析結果も表１に示す。表１によれば、Ｕ． ｓｃｉｔａｍｉｎｅａが生産する物質は典型的なＭＥＬ‐Ｂの構造を有することが確認できた。なお、ＮＭＲによる測定ついては、ＭＥＬ精製標品重クロロホルム中に溶解し、３０℃で、Ｖａｒｉａｎ ＩＮＯＶＡ（４００ＭＨｚ）を用いて行なった。
(3-b: Structural analysis by NMR)
U. The main component MEL-B was purified and separated by silica gel column chromatography from the ethyl acetate-soluble content of the culture solution of citamine, and then structural analysis was performed by 1 H and 13 C NMR. The results are shown in Table 1. Further, as an example of a standard MEL structural analysis result, P.I. Table 1 also shows the results of analysis of MEL produced by the Antarctica JCM 10317 strain. According to Table 1, U.S. It was confirmed that the substance produced by scitamine has a typical MEL-B structure. In addition, about the measurement by NMR, it melt | dissolved in the MEL refinement | purification sample heavy chloroform, and performed it at 30 degreeC using Varian INOVA (400MHz).
また、Ｕ． ｃｙｎｏｄｏｎｔｉｓの培養液の酢酸エチル可溶分から、主成分であるＭＥＬ‐Ｃをシリカゲルカラムクロマトグラフィーによって精製分離した後、１Ｈ及び１３Ｃ ＮＭＲによる構造解析を行った。結果を下記表２に示す。また、標準的なＭＥＬの構造解析結果の例としてＰ． ａｎｔａｒｃｔｉｃａ ＪＣＭ １０３１７株が生産するＭＥＬの分析結果も表２に示す。表２によれば、Ｕ． ｓｃｉｔａｍｉｎｅａが生産する物質は典型的なＭＥＬ‐Ｂの構造を有することが確認できた。 In addition, U.S. The main component MEL-C was purified and separated by silica gel column chromatography from the ethyl acetate-soluble content of the culture solution of cynodontis, and then structural analysis was performed by 1 H and 13 C NMR. The results are shown in Table 2 below. Further, as an example of a standard MEL structural analysis result, P.I. Table 2 also shows the results of analysis of MEL produced by the Antarctica JCM 10317 strain. According to Table 2, U.I. It was confirmed that the substance produced by scitamine has a typical MEL-B structure.
〔実施例２〕
（１：微生物の単離）
本実施例では、アブラナ（Ｂｒａｓｓｉｃａ）属（ミズナ）、リンゴ（Ｍａｌｕｓ）属及びシソ属（Ｐｅｒｉｌｌａ）属の植物からそれぞれウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物を単離した。
[Example 2]
(1: Isolation of microorganisms)
In this example, microorganisms belonging to the genus Ustilago were isolated from plants belonging to the genus Brassica (Mizuna), the genus Apple (Malus) and the genus Perilla.
具体的にはヒメリンゴ（Ｍ． ｂａｃｃａｔａ）、シソ（Ｐｅｒｉｌｌａ ｆｒｕｔｅｓｃｅｎｓ）、ミズナ（Ｂｒａｓｓｉｃａ ｒａｐａ Ｌ． ｖａｒ． ｎｉｐｐｏｓｉｎｉｃａ）の切片を培養液（４０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌ）中に浸し、２５℃で３〜７日間振とう培養し、培地中の大豆油の乳化を指標として、バイオサーファクタント生産性を判定した。生育してきた微生物を、寒天培地（４０ｇ/Ｌのグルコースと酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、硫酸マグネシウム０．５ｇ/Ｌ、及び寒天３０ｇ/Ｌ）上で単離した後、さらに、得られた微生物を２５℃で３〜７日間振とう培養し（培養液：４０ｇ/Ｌの大豆油と酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌ）、培養液中に分泌生産されたＭＥＬを薄層クロマトグラフィーにて検出することで、ＭＥＬ生産微生物を単離した。 Specifically, slices of Japanese apple (M. baccata), perilla (Frulacescens) and Mizuna (Brassica rapa L. var. Nipposinica) were cultured in a culture solution (40 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L). L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L), and cultured with shaking at 25 ° C. for 3 to 7 days. Surfactant productivity was determined. Growing microorganisms were added to agar medium (40 g / L glucose and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, and agar 30 g / L. L) After the above isolation, the obtained microorganism was further cultured with shaking at 25 ° C. for 3 to 7 days (culture solution: 40 g / L soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, The MEL-producing microorganism was isolated by detecting MEL secreted and produced in the culture broth by 0.5 g / L potassium dihydrogen phosphate and 0.5 g / L magnesium sulfate) by thin layer chromatography.
なお、得られた微生物のＩＴＳ１領域における約２６０ｂｐの配列をＢＬＡＳＴ解析により比較した結果、より具体的には、ウスチラゴ ツラガナ（Ｕｓｔｉｌａｇｏ ｔｒａｇａｎａ）の近縁種であることが分かった。ＩＴＳ１領域のＰＣＲによる増幅は、プライマーＡ（配列番号１）及びプライマーＢ（配列番号２）を用いて行なった。ＰＣＲのテンプレートとして用いた上記微生物のゲノムＤＮＡは、ＧｅｎＴＬＥ(ＴａＫａＲａ社製)を用いて抽出した。その後、増幅した遺伝子断片の塩基配列を解析した。その結果を配列番号３（ミズナ由来の微生物Ａ１）、配列番号４（ヒメリンゴ由来の微生物Ｂ１）、配列番号５（シソ由来の微生物Ｃ１）、配列番号６（シソ由来の微生物Ｃ２）、配列番号７（シソ由来の微生物Ｃ３）に示す。得られた遺伝子配列について、ＤＮＡデータベース（ＤＤＢＪ）にアクセスし、ＢＬＡＳＴ解析によって相同性検索を行ったところ、ウスチラゴ ツラガナ（Ｕｓｔｉｌａｇｏ ｔｒａｇａｎａ）の対応するＩＴＳ１領域の塩基配列と９４％一致した。このことから、本実施例で同定を行なった微生物はウスチラゴ属の微生物（Ｕｓｔｉｌａｇｏ ｓｐ．）であることが分かった。 In addition, as a result of comparing the sequence of about 260 bp in the ITS1 region of the obtained microorganism by BLAST analysis, more specifically, it was found to be a related species of Ustilago tragana (Ustilago tragana). Amplification of the ITS1 region by PCR was performed using primer A (SEQ ID NO: 1) and primer B (SEQ ID NO: 2). Genomic DNA of the microorganism used as a PCR template was extracted using GenTLE (TaKaRa). Thereafter, the base sequence of the amplified gene fragment was analyzed. As a result, SEQ ID NO: 3 (microorganism A1 derived from Mizuna), SEQ ID NO: 4 (microorganism B1 derived from Japanese apple), SEQ ID NO: 5 (microorganism C1 derived from perilla), SEQ ID NO: 6 (microorganism C2 derived from perilla), SEQ ID NO: 7 This is shown in (Perilla-derived microorganism C3). When the obtained gene sequence was accessed to the DNA database (DDBJ) and homology search was performed by BLAST analysis, it was 94% identical with the base sequence of the corresponding ITS1 region of Ustyago tragana. From this, it was found that the microorganism identified in this example was a microorganism belonging to the genus Ustilago (Ustilago sp.).
（２：培養及び生産されたＭＥＬの確認）
本実施例２で得た微生物を使用し、また、大豆油の代わりにオリーブ油を用いた以外は、実施例１の２−ａ及び２−ｂに記載の方法で微生物の培養を行なった。
(2: Confirmation of cultured and produced MEL)
The microorganisms were cultured by the method described in 2-a and 2-b of Example 1 except that the microorganisms obtained in Example 2 were used and olive oil was used instead of soybean oil.
次に、得られたＭＥＬを実施例１に記載の方法と同じ方法で薄層クロマトグラフィーに供した。結果を図３に示す。図３は本実施例で得たＭＥＬを薄層クロマトグラフィーに供した結果を示す図である。図３に示すように、本実施例で得た微生物はいずれもＭＥＬ‐Ａを選択的に生産することが確認された。 Next, the obtained MEL was subjected to thin layer chromatography by the same method as described in Example 1. The results are shown in FIG. FIG. 3 is a diagram showing the results of subjecting the MEL obtained in this example to thin layer chromatography. As shown in FIG. 3, it was confirmed that all the microorganisms obtained in this example selectively produced MEL-A.
また、上記微生物Ｃ１から実施例１と同じ方法で得た酢酸エチルの抽出液をＨＰＬＣ解析に供した。結果を図４に示す。なお、図４に記載されている「ＭＥＬ‐Ａ：Ｂ：Ｃ」は、培養液中に含まれていたＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃの重量比である。図４に示すように、上記のウスチラゴ ツラガナ近縁種から得られたＭＥＬのピークはＪＣＭ１０３１７株から得たＭＥＬ−Ａのピークと一致した。上記のウスチラゴ ツラガナの近縁種は、高いＭＥＬ‐Ａ生産能を有し、一週間で約１１．６ｇ／ＬのＭＥＬ‐Ａを生産した。なお、ＭＥＬ−Ａの生産量は、ＭＥＬ−Ａの精製標品を用いて作成した検量線を用い、ＨＰＬＣの分析結果から定量した。また、ＭＥＬ‐Ａの化学構造は、実施例１の３−ａ及び３−ｂに記載の方法に従って行ない、その結果を表３に記載した。表３によれば、上記のウスチラゴ ツラガナ近縁種が生産する物質は典型的なＭＥＬ‐Ａの構造を有することが確認できた。 In addition, an ethyl acetate extract obtained from the microorganism C1 in the same manner as in Example 1 was subjected to HPLC analysis. The results are shown in FIG. In addition, “MEL-A: B: C” described in FIG. 4 is a weight ratio of MEL-A, MEL-B, and MEL-C contained in the culture solution. As shown in FIG. 4, the MEL peak obtained from the above-mentioned species related to Ustillago tsuragana coincided with the MEL-A peak obtained from the JCM10317 strain. The above-mentioned species of Ustirago tsuragana has a high ability to produce MEL-A and produced about 11.6 g / L of MEL-A in one week. In addition, the production amount of MEL-A was quantified from the analysis result of HPLC using the analytical curve created using the refinement | purification sample of MEL-A. Further, the chemical structure of MEL-A was determined according to the method described in 3-a and 3-b of Example 1, and the results are shown in Table 3. According to Table 3, it was confirmed that the substance produced by the above-mentioned species related to Ustirago tsuragana has a typical MEL-A structure.
〔実施例３〕
本実施例では、様々な炭素源を用いたＵ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０の培養を行ないＭＥＬの生産を試みた。
Example 3
In this example, U.S. using various carbon sources. Citaminea NBRC32730 was cultured and MEL production was attempted.
培地としては、炭素源１０重量％、硝酸ナトリウム０．３重量％、リン酸２水素カリウム０．０３重量％、硫酸マグネシウム０．０３重量％、酵母エキス０．１重量％を含み、残部を水とし、全量を１０ｍｌとした液体培地を用いた。また、培養期間を７日間、培養温度を２５℃として、２００ｒｐｍにて振とう培養した。 The medium includes 10% by weight of carbon source, 0.3% by weight of sodium nitrate, 0.03% by weight of potassium dihydrogen phosphate, 0.03% by weight of magnesium sulfate, and 0.1% by weight of yeast extract, with the balance being water. And a liquid medium with a total volume of 10 ml was used. Further, the culture was carried out at 200 rpm with a culture period of 7 days and a culture temperature of 25 ° C.
炭素源としては、スクロース、グルコース、フルクトース、マンノース、ガラクトース、ソルビトール、マンニトール、リビトール、アラビトール、イノシトール、エリトリトール、メチルオレイン酸、オリーブ油をそれぞれ用いた。 As the carbon source, sucrose, glucose, fructose, mannose, galactose, sorbitol, mannitol, ribitol, arabitol, inositol, erythritol, methyl oleic acid, and olive oil were used.
それぞれの炭素源を用いることで得られた培養液の酢酸エチル抽出液１μｌを、実施例１に記載の方法と同じ方法で薄層クロマトグラフィーに供した。結果を図５に示す。図５は本実施例にて得た培養液の酢酸エチル抽出液を薄層クロマトグラフィーに供した結果を示す図である。図５に示すように糖を炭素源としてＵ． ｓｃｉｔａｍｉｎｅａを培養することでＭＥＬ‐Ｂを選択的に生産できることが示された。一方、糖アルコールを炭素源とした場合、ＭＥＬ‐Ｂを生産しなかった。 1 μl of the ethyl acetate extract of the culture solution obtained by using each carbon source was subjected to thin layer chromatography in the same manner as described in Example 1. The results are shown in FIG. FIG. 5 shows the results of subjecting the ethyl acetate extract of the culture solution obtained in this example to thin-layer chromatography. As shown in FIG. It was shown that MEL-B can be selectively produced by culturing scitamine. On the other hand, when sugar alcohol was used as the carbon source, MEL-B was not produced.
次に、上記酢酸エチル抽出液を実施例１の記載と同じ方法でＨＰＬＣ解析に供した。結果を図６に示す。なお、図６に記載されている「ＭＥＬ‐Ａ：Ｂ：Ｃ」は、培養液中に含まれていたＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃの重量比である。また、図６に示すように、ＭＥＬのピークはＪＣＭ１０３１７株から得たＭＥＬ−Ｂのピークと一致した。そのＭＥＬ−Ｂ生産量は、一週間で約９．８ｇ／Ｌに達した。なお、ＭＥＬ−Ｂの生産量は、ＭＥＬ−Ｂの精製標品を用いて作成した検量線を用い、ＨＰＬＣの分析結果から定量した。図６から明らかなように、炭素源として、スクロース等の糖質を用いた場合、メチルオレイン酸やオリーブ油を用いた場合と比較して、培地中に油脂等の混在はなく、少なくとも９５％以上の純度である。この結果は、スクロース等の糖質を炭素源することでＭＥＬの精製プロセスを大幅に簡略化できることを示している。また、スクロース等の糖質を多く含む溶液（例えば、サトウキビ、トウモロコシ、穀類、果実などの搾汁）であればMEL生産のための培地と成り得ることも、この結果から容易に類推できる。このように、スクロース等の糖質を原料として用いた場合、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０のＭＥＬ生産量は、従来のＭＥＬ生産微生物を凌駕するものである。 Next, the ethyl acetate extract was subjected to HPLC analysis in the same manner as described in Example 1. The results are shown in FIG. Note that “MEL-A: B: C” shown in FIG. 6 is a weight ratio of MEL-A, MEL-B, and MEL-C contained in the culture solution. Further, as shown in FIG. 6, the MEL peak coincided with the MEL-B peak obtained from the JCM10317 strain. The MEL-B production amount reached about 9.8 g / L in one week. In addition, the production amount of MEL-B was quantified from the analysis result of HPLC using the analytical curve created using the refinement | purification sample of MEL-B. As is clear from FIG. 6, when a saccharide such as sucrose is used as the carbon source, there is no mixture of fats and oils in the medium as compared with the case where methyl oleic acid or olive oil is used, and at least 95% or more. Of purity. This result shows that the purification process of MEL can be greatly simplified by using a saccharide such as sucrose as a carbon source. In addition, it can be easily inferred from this result that a solution containing a large amount of sugar such as sucrose (for example, juice from sugarcane, corn, cereals, fruits, etc.) can be a medium for MEL production. Thus, when a saccharide such as sucrose is used as a raw material, U.S. The amount of MEL produced by scitamine NBRC32730 exceeds that of conventional MEL-producing microorganisms.
〔比較例〕
本比較例では、オオムギ（Ｈｏｒｄｅｕｍ）属、マダケ（Ｐｈｙｌｌｏｓｔａｃｈｙｓ）属、ススキ（Ｍｉｓｃａｎｔｈｕｓ）属、メヒシバ（Ｄｉｇｉｔａｒｉａ）属（アキメヒシバ）、及びヒエ（Ｅｃｈｉｎｏｃｈｌｏａ）属の植物からそれぞれウスチラゴ（Ｕｓｔｉｌａｇｏ）属の微生物が、ＭＥＬを生産するか否か確認した。
[Comparative Example]
In this comparative example, the genus Ustilago from plants of the genus Hordeum, the genus Phyllostachys, the genus Miscanthus, the genus Digitaria (Akihishiba), and the genus Echinochlo, respectively. Whether or not to produce MEL was confirmed.
具体的にはオオムギ属の植物から単離されたＵ． ｎｕｄａ ＮＢＲＣ６９０６、マダケ属の植物から単離されたＵ． ｓｈｉｒａｉａｎａ ＮＢＲＣ ３０１４９、Ｕ． ｓｈｉｒａｉａｎａ ＮＢＲＣ ８８０９、ススキ属の植物から単離されたＵ． ｋｕｓａｎｏｉ ＮＢＲＣ９０７０、アキメヒシバから単離されたＵ． ｒａｂｅｎｈｏｒｓｔｉａｎａ ＮＢＲＣ８９９５、ヒエ属の植物から単離されたＵ． ｔｒｉｃｈｏｐｈｏｒａ ＮＢＲＣ１００１５５、Ｕ． ｔｒｉｃｈｏｐｈｏｒａ ＮＢＲＣ１００１５６、Ｕ． ｔｒｉｃｈｏｐｈｏｒａ ＮＢＲＣ１００１５７、Ｕ． ｔｒｉｃｈｏｐｈｏｒａ ＮＢＲＣ１００１５８、Ｕ． ｔｒｉｃｈｏｐｈｏｒａ ＮＢＲＣ１００１５９、Ｕ． ｔｒｉｃｈｏｐｈｏｒａ ＮＢＲＣ１００１６０を用いた。 Specifically, it has been isolated from a plant of the genus Barley. nuda NBRC 6906, a U.S. shiraiana NBRC 30149, U.S.A. shiraiana NBRC 8809, a U.S. strain isolated from a plant of the genus Susuki. Kusanoi NBRC 9070, a U.S. rabenhorstiana NBRC8995, a U. cerevisiae isolated from a plant of the genus Panax. trichophora NBRC100155, U.S.A. trichophora NBRC100156, U.S.A. trichophora NBRC100157, U.I. trichophora NBRC100158, U.S.A. trichophora NBRC100159, U.S.A. Trichophora NBRC100160 was used.
また、比較のため実施例１にて用いたＵ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０、ＭＥＬ‐Ａ生産菌として公知のＵ． ｍａｙｄｉｓ（ＮＢＲＣ５３４６及びＮＢＲＣ６９０７）も用いた。 For comparison, the U.I. scitamine NBRC32730, a U.K. known as MEL-A producing bacterium. Maydis (NBRC5346 and NBRC6907) were also used.
微生物の培養、培養液からＭＥＬの抽出、薄層クロマトグラフィーについては実施例２に記載の方法と同じ方法で行なった。結果を図７に示す。図７は本比較例における薄層クロマトグラフィーの結果を示す図である。 Microbial culture, MEL extraction from the culture, and thin-layer chromatography were performed in the same manner as described in Example 2. The results are shown in FIG. FIG. 7 shows the results of thin layer chromatography in this comparative example.
図７に示すように、本発明に係る製造方法で用いるＵ． ｓｃｉｔａｍｉｎｅａ以外はＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃのうちの一つを選択的に生産することは確認できなかった。 As shown in FIG. 7, the U.S. used in the manufacturing method according to the present invention. It was not possible to confirm that one of MEL-A, MEL-B, and MEL-C was selectively produced except for scitamine.
〔実施例４〕
本実施例では、Ｕ． ｓｃｉｔａｍｉｎｅａ に分類される様々な菌株について、実施例３と同様にして培養し、ＭＥＬ生産能力があるかどうか調べた。結果を図８に示す。図８は実施例４にて得た培養液の酢酸エチル抽出液を薄層クロマトグラフィーに供した結果を示す図である。図８に示すように、本実施例にて用いたＵ． ｓｃｉｔａｍｉｎｅａ は全てＭＥＬ‐Ｂ生産能力を有することが明らかとなった。
Example 4
In this embodiment, U.S. Various strains classified as scitamine were cultured in the same manner as in Example 3 and examined for their ability to produce MEL. The results are shown in FIG. FIG. 8 shows the results of subjecting the ethyl acetate extract of the culture solution obtained in Example 4 to thin layer chromatography. As shown in FIG. All of the scitamines were found to have MEL-B production capacity.
〔実施例５〕
本実施例では、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０によって生産されたＭＥＬ‐Ｂの、回収・分離方法について検討した。
Example 5
In this embodiment, U.S. A method for recovering and separating MEL-B produced by scitamine NBRC32730 was examined.
まず、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０を培養液（スクロース１００ｇ/Ｌ、酵母エキス１ｇ/Ｌ、硝酸ナトリウム１ｇ/Ｌ、リン酸２水素カリウム０．５ｇ/Ｌ、及び硫酸マグネシウム０．５ｇ/Ｌ）中に浸し、２５℃で７日間振とう培養し、得られた培養液を実験に用いた。図９に本実施例におけるＭＥＬ‐Ｂの回収方法及び結果を示す。 First, U.I. dipping scitamine NBRC32730 in a culture solution (sucrose 100 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L and magnesium sulfate 0.5 g / L) at 25 ° C. The culture was shaken for 7 days, and the resulting culture was used for the experiment. FIG. 9 shows the recovery method and results of MEL-B in this example.
図９に示すように、得られた培養液に酢酸エチルを等量添加して５８００ｇで５分間遠心分離した。遠心分離後の酢酸エチル抽出画分を画分１とした。また、培養液を５８００ｇで５分間遠心分離すると、上層、沈殿層、これらの中間層が生じるため、当該上層、当該中間層、当該沈殿層のそれぞれを分取して、それぞれに酢酸エチルを等量（培養液と等量）添加し、５８００ｇで５分間遠心分離した。当該上層、当該中間層、当該沈殿層のそれぞれから得られた酢酸エチル抽出画分を、画分２、３、４とした。 As shown in FIG. 9, an equal amount of ethyl acetate was added to the obtained culture broth and centrifuged at 5800 g for 5 minutes. The fraction extracted with ethyl acetate after centrifugation was designated as fraction 1. In addition, when the culture solution is centrifuged at 5800 g for 5 minutes, an upper layer, a precipitate layer, and an intermediate layer thereof are generated. Therefore, the upper layer, the intermediate layer, and the precipitate layer are separated, and ethyl acetate is added to each. An amount (equal to the culture solution) was added and centrifuged at 5800 g for 5 minutes. The ethyl acetate extract fractions obtained from each of the upper layer, the intermediate layer, and the precipitation layer were designated as fractions 2, 3, and 4.
また、上記培養液を４００ｇで５分間遠心分離した。得られた上層および沈殿層を分取して、それぞれに酢酸エチルを等量（培養液と等量）添加し、５８００ｇで５分間遠心分離した。当該上層、当該沈殿層のそれぞれから得られた酢酸エチル抽出画分を画分５、６とした。画分１〜６を薄層クロマトグラフィーに供してＭＥＬ‐Ｂの存在を確認した。 The culture broth was centrifuged at 400 g for 5 minutes. The obtained upper layer and precipitated layer were separated, and an equal amount of ethyl acetate (equivalent to the culture solution) was added to each and centrifuged at 5800 g for 5 minutes. The ethyl acetate extract fractions obtained from the upper layer and the precipitated layer were designated as fractions 5 and 6, respectively. Fractions 1-6 were subjected to thin layer chromatography to confirm the presence of MEL-B.
図９に示すように、培養液に等量の酢酸エチルを添加することで、ＭＥＬ‐Ｂを回収することができることが示された。また、培養液を５８００ｇで遠心分離すると、上層、中間層、沈殿層が得られ、それぞれに培養液と等量の酢酸エチルを添加することで、ＭＥＬ‐Ｂを回収することができるが、この場合、ＭＥＬ‐Ｂは遠心分離によって一部分が沈殿することが示された。一方、培養液を４００ｇで遠心分離すると、上層及び沈殿層が得られ、それぞれに培養液と等量の酢酸エチルを添加してＭＥＬ‐Ｂを回収した結果、沈殿にはＭＥＬ‐Ｂが検出されず、全て上層から検出された。すなわち、培養液を４００ｇで遠心分離することで、ＭＥＬ‐Ｂを沈殿させることなく、菌体と完全に分離し、ＭＥＬ‐Ｂ溶液を回収することが可能であることが示された。 As shown in FIG. 9, it was shown that MEL-B can be recovered by adding an equal amount of ethyl acetate to the culture solution. Moreover, when the culture solution is centrifuged at 5800 g, an upper layer, an intermediate layer, and a precipitation layer are obtained, and MEL-B can be recovered by adding the same amount of ethyl acetate as the culture solution. In some cases, MEL-B was shown to partially precipitate by centrifugation. On the other hand, when the culture broth was centrifuged at 400 g, an upper layer and a precipitate layer were obtained. MEL-B was recovered by adding the same amount of ethyl acetate as the culture broth to each, and MEL-B was detected in the precipitate. All were detected from the upper layer. That is, by centrifuging the culture solution at 400 g, it was shown that the MEL-B solution can be recovered by completely separating from the bacterial cells without precipitating MEL-B.
また、画分１〜６におけるＭＥＬ‐Ｂの含有量は、それぞれ、２．３ｇ／ｌ、０．７ｇ／ｌ、０．５ｇ／ｌ、１．８ｇ／ｌ、０．４ｇ／ｌ、１．５ｇ／ｌであった。 The contents of MEL-B in fractions 1 to 6 are 2.3 g / l, 0.7 g / l, 0.5 g / l, 1.8 g / l, 0.4 g / l, and 1. It was 5 g / l.
〔実施例６〕
本実施例では実施例５において得た画分５（本実施例において以下「培養液」いう。）を用いて、ＭＥＬ‐Ｂの濃縮方法について検討した。
Example 6
In this example, a method for concentrating MEL-B was examined using fraction 5 obtained in Example 5 (hereinafter referred to as “culture medium” in this example).
検討した条件は（ａ）培養液を６０℃の環境下に３時間静置、（ｂ）培養液を４℃の環境下に３時間静置、（ｃ）培養液のｐＨを２に調整して、４℃の環境下に３時間静置、（ｄ）培養液に硫酸アンモニウムを２０質量％添加して、４℃の環境下に３時間静置、（ｅ）培養液に塩化ナトリウムを１０質量％添加して、４℃の環境下に３時間静置の５通りである。 The conditions examined were: (a) the culture broth was left in an environment of 60 ° C. for 3 hours, (b) the broth was left in a 4 ° C. environment for 3 hours, (c) the pH of the broth was adjusted to 2. (D) 20% by mass of ammonium sulfate was added to the culture solution and left at 4 ° C for 3 hours. (E) 10% sodium chloride was added to the culture solution. %, And left in a 4 ° C. environment for 3 hours.
（ａ）〜（ｅ）の処理を行なった後、８０００ｒｐｍで５分間遠心分離を行なった。得られた上清と沈殿物とをわけて、それぞれに４ｍｌの酢酸エチルを添加して混合した。酢酸エチル中のＭＥＬ‐Ｂを薄層クロマトグラフィー及びＨＰＬＣによって検出した。また、上清中のＭＥＬ‐Ｂと沈殿物中のＭＥＬ‐Ｂとの質量の比を算出した。この結果を、ＭＥＬ‐Ｂを検出するまでのプロセスと共に図１０に示す。図１０は本実施例においてＭＥＬ‐Ｂを濃縮して得た結果を示す図である。なお、図１０において「ＴＬＣ検出結果」と「ＨＰＬＣ定量結果」との間に記載の「上清」は上清からＭＥＬ‐Ｂを検出した結果を示し、「沈殿」は沈殿物からＭＥＬ‐Ｂを検出した結果を示す。図１０に示すように、温度、ｐＨの調整においてＭＥＬ‐Ｂは沈殿せず、遠心分離後の上清及び沈殿物の両方に分布していた。一方、硫酸アンモニウムを添加することによって、ＭＥＬ‐Ｂは効率よく沈殿し、７９％のＭＥＬ‐Ｂを沈殿物として回収することができることが示された。また、ＮａＣｌを添加した場合、ＭＥＬ‐Ｂのほとんどは沈殿せず、上清画分として回収することができることが示された。以上の結果によれば、培養液から、低速遠心分離によって菌体の大部分を除いた後、硫酸アンモニウムを添加し、再度遠心分離することで、ＭＥＬ‐Ｂを濃縮できることが示された。 After performing the treatments (a) to (e), centrifugation was performed at 8000 rpm for 5 minutes. The obtained supernatant was separated from the precipitate, and 4 ml of ethyl acetate was added to each and mixed. MEL-B in ethyl acetate was detected by thin layer chromatography and HPLC. Moreover, the mass ratio of MEL-B in the supernatant and MEL-B in the precipitate was calculated. This result is shown in FIG. 10 together with the process up to detection of MEL-B. FIG. 10 is a graph showing the results obtained by concentrating MEL-B in this example. In FIG. 10, “supernatant” described between “TLC detection result” and “HPLC quantification result” indicates the result of detecting MEL-B from the supernatant, and “precipitation” indicates MEL-B from the precipitate. The result of detecting is shown. As shown in FIG. 10, MEL-B did not precipitate in adjusting the temperature and pH, and was distributed in both the supernatant and the precipitate after centrifugation. On the other hand, it was shown that by adding ammonium sulfate, MEL-B precipitates efficiently and 79% of MEL-B can be recovered as a precipitate. In addition, when NaCl was added, most of MEL-B did not precipitate, indicating that it can be recovered as a supernatant fraction. According to the above results, it was shown that MEL-B can be concentrated by removing most of the cells from the culture solution by low-speed centrifugation, adding ammonium sulfate, and centrifuging again.
〔実施例７〕
本実施例では、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０を、炭素源としてスクロース、グルコース又は大豆油のみを炭素源として添加した培地で培養し、ＭＥＬを生産するか否か確認した。まず、それぞれの菌株を培養液（炭素源（スクロース、グルコース又は大豆油）１００ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌ）中に浸し、２５℃で７日間振とう培養し、得られた培養液中のＭＥＬを薄層クロマトグラフィーにて検出した。図１１に結果を示す。図１１は本実施例で得られたＭＥＬを薄層クロマトグラフィーによって検出した結果を示す図である。
Example 7
In this embodiment, U.S. Citaminea NBRC32730 was cultured in a medium supplemented with only sucrose, glucose or soybean oil as a carbon source as a carbon source, and it was confirmed whether or not MEL was produced. First, each strain was cultured in a culture solution (carbon source (sucrose, glucose or soybean oil) 100 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0 0.5 g / L) and cultured with shaking at 25 ° C. for 7 days, and MEL in the obtained culture broth was detected by thin layer chromatography. The results are shown in FIG. FIG. 11 is a diagram showing the results of detecting the MEL obtained in this example by thin layer chromatography.
図１１に示すように、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０は、スクロース又はグルコースのみを炭素源として得られた培養液中にＭＥＬを蓄積した。また、図１１に示すように、スクロース又はグルコースを炭素源として培養したＵ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０により生産された主なＭＥＬは、薄層クロマトグラフィーの結果から、大豆油のみを炭素源として培養した場合と同様、ＭＥＬ−Ｂであった。この結果から、この菌株が、スクロース等の糖類のみを炭素源としてＭＥＬを生産できる菌株であることが示された。 As shown in FIG. scitamine NBRC32730 accumulated MEL in the culture medium obtained using only sucrose or glucose as a carbon source. Moreover, as shown in FIG. 11, U. coli cultured with sucrose or glucose as a carbon source. The main MEL produced by scitamine NBRC32730 was MEL-B, as in the case of culturing using only soybean oil as a carbon source, from the results of thin layer chromatography. From this result, it was shown that this strain is a strain capable of producing MEL using only saccharides such as sucrose as a carbon source.
さらに、本実施例では、スクロース培養で得られたＵ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０の主なＭＥＬをカラムクロマトグラフィーにて均一精製した後、ＭＥＬの脂肪酸組成および界面物性について検討した。ＭＥＬの脂肪酸組成は、塩酸メタノール処理後のヘキサン抽出液を用い、ＧＣ−ＭＳによって解析した。その結果を表４に示す。 Further, in this example, U.S. obtained by sucrose culture was used. The main MEL of scitamine NBRC32730 was uniformly purified by column chromatography, and then the fatty acid composition and interface physical properties of MEL were examined. The fatty acid composition of MEL was analyzed by GC-MS using a hexane extract after treatment with hydrochloric acid and methanol. The results are shown in Table 4.
表４に示されるように、スクロース由来のＭＥＬ−Ｂの脂肪酸組成は、Ｃ８、Ｃ１０及びＣ１２であった。一方、植物油由来のＭＥＬの脂肪酸組成は、Ｃ８、Ｃ１０、Ｃ１２及びＣ１４であった。従って、炭素源が異なると、生産されるＭＥＬの脂肪酸組成が変化することが示された。 As shown in Table 4, the fatty acid composition of sucrose-derived MEL-B was C8, C10, and C12. On the other hand, the fatty acid composition of MEL derived from vegetable oil was C8, C10, C12 and C14. Therefore, it was shown that the fatty acid composition of the produced MEL changes with different carbon sources.
また、界面物性の指標である、臨界ミセル濃度（ＣＭＣ）については、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０のＭＥＬ−Ｂは２．８×１０−６Ｍであった。このＣＭＣ測定の結果から、Ｕ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０由来のＭＥＬは、優れた界面活性を有する界面活性剤であることが示された。さらに、ＭＥＬの液晶形成能を、顕微鏡用スライドガラスにＭＥＬ標品を塗布してカバーガラスをかぶせた後、カバーガラスの横から水を浸入させて、ＭＥＬと水との接触面を光学顕微鏡下で偏光観察することにより判定した。結果を図１２に示している。図１２は本実施例において得られたＭＥＬの液晶形成能を観察した結果を示す図である。図１２に示されるように、液晶形成能が認められた。すなわち、スクロースで生産したＭＥＬは、大豆油で生産したものと脂肪酸組成が多少異なるが、優れた界面活性を有しており、かつ液晶形成能（高度な自己集合特性）をも有する、高機能な界面活性剤として使用可能であることが確認できた。なお、脂肪酸組成の違いは微生物のＭＥＬ合成経路に依存しており、大豆油等の油脂類の場合は分解反応の中間体がＭＥＬ生合成に用いられていると考えられ、一方、スクロースの場合、脂肪酸は細胞内で合成されるため、比較的短い脂肪酸が構成成分となっていると考えられる。 Regarding the critical micelle concentration (CMC), which is an index of interface physical properties, see U.S. Pat. The MEL-B of scitamine NBRC32730 was 2.8 × 10 −6 M. From the results of this CMC measurement, U. MEL derived from scitamine NBRC32730 has been shown to be a surfactant with excellent surface activity. Furthermore, the liquid crystal forming ability of MEL was applied to the slide glass for the microscope and covered with the cover glass. Then, water was infiltrated from the side of the cover glass, and the contact surface between the MEL and water was observed under the optical microscope. This was determined by observing the polarized light. The results are shown in FIG. FIG. 12 is a diagram showing the result of observing the liquid crystal forming ability of the MEL obtained in this example. As shown in FIG. 12, liquid crystal forming ability was recognized. In other words, MEL produced with sucrose has a slightly different fatty acid composition from that produced with soybean oil, but has excellent surface activity and liquid crystal forming ability (advanced self-assembling properties). It was confirmed that it can be used as a novel surfactant. The difference in fatty acid composition depends on the MEL synthesis pathway of microorganisms, and in the case of fats and oils such as soybean oil, it is considered that the intermediate of decomposition reaction is used for MEL biosynthesis, whereas in the case of sucrose Since fatty acids are synthesized in cells, it is considered that relatively short fatty acids are constituents.
〔実施例８〕
本実施例では、スクロース又はオリーブ油を炭素源として用い、炭素源の流下培養法によってＵ． ｓｃｉｔａｍｉｎｅａ ＮＢＲＣ３２７３０の培養を行ない、ＭＥＬの生産を試みた。
Example 8
In this example, sucrose or olive oil was used as a carbon source, and U.V. Citaminea NBRC32730 was cultured and MEL production was attempted.
培地としては、炭素源（スクロース及びオリーブ油）１５〜２３重量％、硝酸ナトリウム０．３重量％、リン酸２水素カリウム０．０３重量％、硫酸マグネシウム０．０３重量％、酵母エキス０．１重量％を含み、残部を水とし、全量を２０ｍｌとした液体培地を用いた。また、培養期間を７日間、培養温度を２５℃として、２００ｒｐｍにて振とう培養した。その後、炭素源のみを培養液中に添加して、７日間の培養を２回繰り返した。それぞれの培養で得られた培養液の酢酸エチル抽出液を用いて、実施例１（３−ａ）に記載のＨＰＬＣ法によってＭＥＬ生産量を定量した。結果を図１３に示す。図１３は炭素源の量とＭＥＬ生産量との関係を示す図である。図１３に示すように１５重量％のスクロースのみを炭素源としてＵ． ｓｃｉｔａｍｉｎｅａを培養することでＭＥＬ‐Ｂを生産した場合、その生産量は２１日間で１２ｇ／Ｌに達した。１５重量％のスクロール及び１重量％のオリーブ油を含む培地で培養した場合、ＭＥＬ−Ｂの生産量は２５ｇ／Ｌに達した。さらに、１５重量％のスクロール及び２重量％のオリーブ油を含む培地で培養した場合、ＭＥＬ−Ｂの生産量は３１ｇ／Ｌに達した。１５重量％のスクロール及び４重量％のオリーブ油を含む培地で培養した場合、ＭＥＬ−Ｂの生産量は３７ｇ／Ｌに達した。１５重量％のスクロール及び８重量％のオリーブ油を含む培地で培養した場合、ＭＥＬ−Ｂの生産量は３７ｇ／Ｌに達した。以上の結果から、Ｕ． ｓｃｉｔａｍｉｎｅａを用いたＭＥＬ−Ｂの培養において、スクロースのみからＭＥＬ−Ｂを生産可能であり、さらに油脂を添加することで生産量を向上させることができることが示された。 As a medium, carbon source (sucrose and olive oil) 15 to 23% by weight, sodium nitrate 0.3% by weight, potassium dihydrogen phosphate 0.03% by weight, magnesium sulfate 0.03% by weight, yeast extract 0.1% by weight %, And the balance was water, and the total volume was 20 ml. Further, the culture was carried out at 200 rpm with a culture period of 7 days and a culture temperature of 25 ° C. Thereafter, only the carbon source was added to the culture solution, and the 7-day culture was repeated twice. MEL production was quantified by the HPLC method described in Example 1 (3-a) using the ethyl acetate extract of the culture obtained in each culture. The results are shown in FIG. FIG. 13 is a diagram showing the relationship between the amount of carbon source and the amount of MEL production. As shown in FIG. When MEL-B was produced by culturing scitamine, the production amount reached 12 g / L in 21 days. When cultured in a medium containing 15% by weight scroll and 1% by weight olive oil, the production of MEL-B reached 25 g / L. Furthermore, when cultured in a medium containing 15% by weight scroll and 2% by weight olive oil, the production amount of MEL-B reached 31 g / L. When cultured in a medium containing 15 wt% scroll and 4 wt% olive oil, MEL-B production reached 37 g / L. When cultured in a medium containing 15% by weight scroll and 8% by weight olive oil, the production of MEL-B reached 37 g / L. From the above results, U.S. In the culture of MEL-B using scitamine, it was shown that MEL-B can be produced only from sucrose and that the production amount can be improved by adding oils and fats.
本発明に係る製造方法により得られたＭＥＬは、食用植物や日常的に接触する植物から単離された微生物を培養して得られたものであり、安全性がより高く、食品用途への展開等が期待される。また、培養に用いる微生物を適宜選択することにより、用途に応じて、親水性が異なるＭＥＬ‐Ａ、ＭＥＬ‐Ｂ、ＭＥＬ‐Ｃをそれぞれ選択的に得ることができるので、新たな食品用途に適用することが期待される。 The MEL obtained by the production method according to the present invention is obtained by culturing microorganisms isolated from edible plants and plants that come into daily contact, and has higher safety and development for food applications. Etc. are expected. In addition, MEL-A, MEL-B, and MEL-C with different hydrophilicity can be selectively obtained depending on the application by appropriately selecting the microorganism to be used for culture, so it can be applied to new food applications. Is expected to do.
Claims (8)
で表される構造を有するマンノシルエリスリトールリピッドの製造方法。 Cultivate a microorganism of the genus Ustilago isolated from any plant of the genus Saccharum, Malus, Perilla, Brassica and Cynodon The following general formula (1) characterized by including a culture step
The manufacturing method of the mannosyl erythritol lipid which has a structure represented by these.
で表される構造を有するマンノシルエリスリトールリピッド‐Ｂの製造方法。 The following general formula (2), which comprises a culture step of culturing a microorganism of the genus Ustilago isolated from a plant of the genus Saccharum
The manufacturing method of the mannosyl erythritol lipid-B which has a structure represented by these.
で表される構造を有するマンノシルエリスリトールリピッド‐Ｃの製造方法。 The following general formula (3), comprising a culturing step of culturing a microorganism belonging to the genus Ustylago isolated from a plant belonging to the genus Cynodon
The manufacturing method of the mannosyl erythritol lipid-C which has a structure represented by these.
で表される構造を有するマンノシルエリスリトールリピッド‐Ａの製造方法。 The following general formula, which comprises a culture step of culturing a microorganism of the genus Ustilago isolated from any plant of the genus Apple (Malus), the genus Perilla and the genus Brassica (4)
The manufacturing method of mannosyl erythritol lipid-A which has a structure represented by these.
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| CN112280806A (en) * | 2019-07-23 | 2021-01-29 | 广东现代汉方科技有限公司 | Method for preparing ceramide by emulsification fermentation and application thereof |
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| JPN6012044861; Ｂｉｏｓｃｉ． Ｂｉｏｔｅｃｈｎｏｌ． Ｂｉｏｃｈｅｍ． ｖｏｌ．７３, 2009, ｐ．７８８-９２ * |
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| JP2011182740A (en) * | 2010-03-10 | 2011-09-22 | National Institute Of Advanced Industrial Science & Technology | New microorganism and method for producing sugar type biosurfactant therewith |
| CN112280806A (en) * | 2019-07-23 | 2021-01-29 | 广东现代汉方科技有限公司 | Method for preparing ceramide by emulsification fermentation and application thereof |
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