JP5733697B2 - New ethanol-producing yeast - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Description
本発明は、エタノール生産酵母、及び、エタノール生産酵母を用いたバイオエタノール生産方法等に関する。詳細には、耐熱性、耐塩性、高凝集性などの優良形質を有する新規エタノール生産酵母、及び、当該酵母を使用したエタノール生産方法、酵母含有飼料生産方法、発酵食品製造方法に関する。 The present invention relates to an ethanol-producing yeast, a bioethanol production method using the ethanol-producing yeast, and the like. Specifically, the present invention relates to a novel ethanol-producing yeast having excellent characteristics such as heat resistance, salt resistance, and high cohesiveness, and an ethanol production method, a yeast-containing feed production method, and a fermented food production method using the yeast.
近年の環境問題、石油資源枯渇問題等を背景として、バイオエタノール生産に関する取り組みが多くなされている。バイオエタノールとは、砂糖製造の際の副産物（廃糖蜜、バガスなど）や廃木材、大麦、とうもろこしなどの植物を原料として得られる、主に燃料用として用いられるエタノールであり、上記原料に含まれるグルコース等を酵母などによって発酵させて生産される。 Many efforts have been made to produce bioethanol against the background of environmental problems and oil resource depletion problems in recent years. Bioethanol is ethanol used mainly for fuel, which is obtained from plants such as by-products (such as waste molasses and bagasse) and waste wood, barley and corn during sugar production. Produced by fermenting glucose or the like with yeast or the like.
しかし、世界的な食糧不足等の問題もあり、大麦やとうもろこしなどの食糧をバイオエタノール生産原料に使用するのはあまり好ましくなく、したがって、廃糖蜜や廃木材などの従来利用価値があまり高くなかったものを主な原料としたバイオエタノール生産方法の開発の必要性が強く主張されている。けれども、これらの使用には、酵素等でのセルロース含有材料の液化・糖化や着色物質の除去などの原料前処理が必要であるなど様々な問題点がある（特許文献１〜３）。
However, due to problems such as global food shortages, it is not preferable to use foods such as barley and corn as raw materials for bioethanol production, and therefore, the conventional utility value of waste molasses and waste wood was not so high. The necessity of developing a bioethanol production method using food as the main raw material is strongly asserted. However, these uses have various problems such as requiring raw material pretreatments such as liquefaction / saccharification of cellulose-containing materials with enzymes or the like and removal of colored substances (
特に、サトウキビ由来の製糖副産物である廃糖蜜は、近年の製糖技術向上などによりエタノール生産原料という意味での品質がより低下している。つまり、廃糖蜜中に含まれる糖類の含有量が低く、且つ、塩類、ポリフェノールなどの糖以外の成分の含有量がより高くなる傾向にある。また、その粘性もより高くなる傾向にある。また、産地毎の品質のばらつきもある。 In particular, the molasses, which is a sugar production byproduct derived from sugarcane, has a lower quality in the sense of an ethanol production raw material due to recent improvements in sugar production technology. That is, the content of saccharides contained in the molasses is low, and the content of components other than sugars such as salts and polyphenols tends to be higher. Moreover, the viscosity tends to be higher. There are also variations in quality from production area to production area.
一例としては、沖縄県の宮古島でサトウキビからの製糖時にでる宮古島産廃糖蜜は、総灰分（塩分やポリフェノールなどの糖以外の成分）が１４〜１９重量％と外国産廃糖蜜の約２〜３倍も含まれ、糖濃度は４０〜５０重量％と外国産廃糖蜜（糖濃度は６０重量％前後）よりも１割以上低い。このような廃糖蜜では、例えば、廃糖蜜を全糖濃度１５重量％に希釈して酵母によるエタノール発酵生産を行う場合でも、総灰分が高すぎる等の原因で発酵が進まず、総灰分を一般的な廃糖蜜希釈液と同程度となるまで調整（さらなる希釈）して使用した場合でも、今度は糖濃度が低すぎて発酵後のエタノール濃度が低くなりすぎ蒸留で問題が生じる。よって、従来このような廃糖蜜は好ましくない発酵基質として敬遠されてきた。 As an example, Miyakojima's waste molasses produced during sugar production from sugarcane in Miyakojima, Okinawa Prefecture has a total ash content (components other than sugar such as salt and polyphenols) of 14 to 19% by weight, which is about 2 to 3 times that of foreign molasses. The sugar concentration is 40-50% by weight, which is 10% or more lower than that of foreign waste molasses (sugar concentration is around 60% by weight). In such molasses, for example, even when the molasses is diluted to a total sugar concentration of 15% by weight and ethanol fermentation production is performed by yeast, the fermentation does not proceed due to the total ash being too high, etc. Even if it is used after adjusting (further dilution) to the same level as a typical waste molasses dilution, the sugar concentration is too low and the ethanol concentration after fermentation becomes too low, causing problems in distillation. Thus, conventionally, such molasses has been avoided as an unfavorable fermentation substrate.
また、サトウキビが栽培されている比較的温暖な地域において、サトウキビ廃糖蜜を原料としてエタノール発酵生産を行う場合、発酵生産に用いる通常酵母の耐熱性から発酵温度の上限は３０℃前後であるため、温暖な地域でこの上限温度を超えないようにするにはチラー水などによって発酵槽を冷却する必要がある。これは、エネルギー効率や作業性という点で好ましくない。 In addition, in a relatively warm area where sugarcane is cultivated, when performing ethanol fermentation production using sugarcane waste molasses as a raw material, the upper limit of the fermentation temperature is around 30 ° C. from the heat resistance of normal yeast used for fermentation production, In order to prevent the upper limit temperature from being exceeded in a warm area, it is necessary to cool the fermenter with chiller water or the like. This is not preferable in terms of energy efficiency and workability.
このような背景技術の中で、例えば高い灰分含量且つ低い糖含量の廃糖蜜などの従来敬遠されてきた原料から簡便にエタノール発酵生産をすることができ、かつ、温暖な地域においても厳密な温度管理の必要がない耐熱性を備えた、バイオエタノール発酵生産に用いる新規酵母の開発が強く望まれていた。 Among such background technologies, for example, ethanol fermentation can be easily produced from raw materials that have been avoided in the past, such as molasses having a high ash content and a low sugar content. There has been a strong demand for the development of a novel yeast used in bioethanol fermentation production that has heat resistance that does not require management.
本発明は、３５℃以上、例えば３５〜４０℃という高温条件下でも効率よくエタノールの発酵生産が可能であり、且つ、全糖濃度を１５重量％に調整した際の総灰分が３．５重量％以上の廃糖蜜などの従来発酵原料として適していないとされていた原料からでもエタノールの発酵生産が可能な新規エタノール生産酵母及びこれを用いた工業用エタノール等の生産方法を提供することを目的とする。 In the present invention, ethanol can be efficiently fermented and produced even at a high temperature of 35 ° C. or higher, for example, 35 to 40 ° C., and the total ash content is 3.5% when the total sugar concentration is adjusted to 15% by weight. The purpose of the present invention is to provide a novel ethanol-producing yeast capable of fermenting ethanol even from raw materials that have not been suitable as conventional fermentation raw materials such as molasses or more of molasses and production methods for industrial ethanol and the like using the same And
上記目的を達成するため、本発明者らは鋭意研究を行い、高温条件下における増殖能、エタノール生産能、耐塩性などを指標としたスクリーニング行程を経て選抜された、バガスより単離した３８〜４０℃でのエタノール生産能や耐塩性、凝集性などに優れた新規エタノール生産酵母、サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株（ＮＩＴＥ Ｐ−８９３）を見いだし、本発明に至った。 In order to achieve the above-mentioned object, the present inventors conducted intensive research and isolated from bagasse selected through a screening process using growth ability, ethanol production ability, salt tolerance, etc. under high temperature conditions as 38 to A novel ethanol-producing yeast, Saccharomyces cerevisiae MY17 strain (NITE P-893), which is excellent in ethanol production ability at 40 ° C., salt resistance, aggregation properties, etc., has been found, and the present invention has been achieved.
すなわち、本発明の実施形態は次のとおりである。
（１）エタノール生産酵母、サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株（ＮＩＴＥ Ｐ−８９３）。
（２）（１）に記載のエタノール生産酵母において、１又は数個の遺伝子を遺伝子工学的手法、薬剤処理や紫外線処理などの突然変異誘発法等により改変、導入若しくは欠失して得られ、耐熱性、耐塩性、全糖濃度（糖度）を１５重量％に調整した際の総灰分が３．５重量％以上（例えば４．０重量％以上）の廃糖蜜からのエタノール生産性、高凝集性のいずれの形質も有する形質転換体。
（３）（１）又は（２）に記載の酵母を発酵菌として使用して、全糖濃度を１５重量％に調整した際の総灰分が３．５重量％以上の廃糖蜜を原料として用い（酵素等による液化・糖化を行うことなく）、３５℃以上、好ましくは３８〜４０℃の温度で発酵させることを特徴とする、エタノールの生産方法。
（４）全糖濃度を１５重量％に調整した際の総灰分が４．５重量％以上の廃糖蜜（例えば宮古島産廃糖蜜）を用いることを特徴とする、（３）に記載のエタノールの生産方法。
（５）（３）又は（４）に記載の方法において得られる、発酵後に上清（エタノール含有液）を除去した発酵残渣を配合することを特徴とする、酵母含有飼料の製造方法。
That is, the embodiment of the present invention is as follows.
(1) Ethanol-producing yeast, Saccharomyces cerevisiae MY17 strain (NITE P-893).
(2) In the ethanol-producing yeast described in (1), one or several genes are obtained by modification, introduction or deletion by a genetic engineering method, a mutagenesis method such as drug treatment or ultraviolet treatment, Heat resistance, salt resistance, ethanol productivity from molasses with high total ash content of 3.5% by weight (for example, 4.0% by weight or more), high aggregation when total sugar concentration (sugar content) is adjusted to 15% by weight A transformant having any sex trait.
(3) Using the yeast described in (1) or (2) as a fermenting bacterium and using waste molasses with a total ash content of 3.5% by weight or more as a raw material when the total sugar concentration is adjusted to 15% by weight A method for producing ethanol, wherein fermentation is performed at a temperature of 35 ° C. or higher, preferably 38 to 40 ° C. (without liquefaction or saccharification by an enzyme or the like).
(4) Ethanol production according to (3), characterized in that it uses waste molasses (for example, Miyakojima molasses) with a total ash content of 4.5 wt% or more when the total sugar concentration is adjusted to 15 wt%. Method.
(5) A method for producing a yeast-containing feed, characterized in that a fermentation residue obtained by removing the supernatant (ethanol-containing liquid) after fermentation obtained in the method according to (3) or (4) is blended.
（６）（１）又は（２）に記載の酵母を発酵菌として使用し、３５℃以上、好ましくは３８〜４０℃の温度で発酵させることを特徴とする、エタノール含有発酵食品（泡盛、焼酎など）の製造方法。 (6) Ethanol-containing fermented food (Awamori, shochu) characterized in that the yeast described in (1) or (2) is used as a fermenter and fermented at a temperature of 35 ° C or higher, preferably 38-40 ° C. Etc.) manufacturing method.
本発明の酵母をエタノール生産に用いることで、３５℃以上、例えば３８℃という高温条件下でも（つまり温暖な地域においても冷却操作を行うことなく）効率よくエタノールの発酵生産が可能であり、且つ、全糖濃度を１５重量％に調整した際の総灰分が３．５重量％以上、更には４．５重量％以上の廃糖蜜からでもエタノールの発酵生産を行うことができる。また、当該酵母は十分な凝集性を有するため、エタノール発酵後の分離操作が非常に簡便である。そして、この分離後のエタノール発酵残渣は、再度エタノール発酵菌として利用することもできるし、これを配合材料として用いることで、新規な酵母含有飼料を製造することもできる。 By using the yeast of the present invention for ethanol production, ethanol can be efficiently fermented and produced even under high temperature conditions of 35 ° C. or higher, for example, 38 ° C. (that is, without cooling operation even in a warm region) Furthermore, ethanol can be produced by fermentation from molasses having a total ash content of 3.5% by weight or more, and even 4.5% by weight or more when the total sugar concentration is adjusted to 15% by weight. Moreover, since the said yeast has sufficient cohesiveness, the separation operation after ethanol fermentation is very simple. And the ethanol fermentation residue after this isolation | separation can also be utilized as an ethanol fermentation bacterium again, and a novel yeast containing feed can also be manufactured by using this as a compounding material.
まず、本発明においては、新規エタノール生産酵母サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株（ＮＩＴＥ Ｐ−８９３）を使用する。このＭＹ１７株は、宮古島のバガスから単離されたものであり、耐熱性、耐塩性、高凝集性などの有用形質を有する。そして、全糖濃度を１５重量％に調整した際の総灰分が３．５重量％以上の廃糖蜜からのエタノール生産性が非常に高いという特徴を有する株である。 First, in the present invention, a novel ethanol-producing yeast Saccharomyces cerevisiae MY17 strain (NITE P-893) is used. This MY17 strain is isolated from bagasse on Miyakojima and has useful traits such as heat resistance, salt resistance, and high aggregability. And it is a strain | stump | stock which has the characteristics that ethanol productivity from waste molasses whose total ash content is 3.5 weight% or more when adjusting total sugar concentration to 15 weight% is very high.
本発明においては、ＭＹ１７株を遺伝子工学的手法（ベクター等の利用による人為的改変）や突然変異処理（化学薬剤処理、紫外線照射処理など）により、１又は数個の遺伝子を改変、導入若しくは欠失して得られ、ＭＹ１７株が有する上記有用形質をいずれも有する形質転換体を使用しても良い。また、ＭＹ１７株と別の株を細胞融合することによって得られる、上記有用形質をいずれも有する細胞融合株を使用しても良い。 In the present invention, the MY17 strain is altered, introduced, or deleted by genetic engineering techniques (artificial modification using vectors etc.) or mutation treatment (chemical drug treatment, ultraviolet irradiation treatment, etc.). A transformant obtained by losing all of the above-mentioned useful traits possessed by the MY17 strain may be used. Moreover, you may use the cell fusion strain which has the said useful character obtained by carrying out cell fusion of MY17 strain | stump | stock and another strain | stump | stock.
なお、サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株は、独立行政法人製品評価技術基盤機構・特許微生物寄託センター（〒２９２−０８１８ 日本国千葉県木更津市かずさ鎌足２−５−８）に、２０１０年（平成２２年）２月１日付けで寄託されており、その受託番号はＮＩＴＥ Ｐ−８９３である。 The Saccharomyces cerevisiae MY17 strain was established in 2010 by the National Institute for Product Evaluation Technology and Patent Microorganism Depositary Center (Kazusa Kamashichi 2-5-8, Kisarazu City, Chiba Prefecture, Japan 292-0818). Deposited on February 1, 2010, and the deposit number is NITE P-893.
サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株の主な菌学的性質を示すと、以下の通りである。
（ａ）ＹＭ液体培地で生育させたときの菌の形態
（１）栄養細胞の大きさ：５〜１０μｍ程度
（２）栄養細胞の形状：楕円形
（３）増殖の形式：出芽
（ｂ）胞子形成の有無
胞子形成する。
The main bacteriological properties of Saccharomyces cerevisiae MY17 strain are as follows.
(A) Bacteria morphology when grown in YM liquid medium (1) Vegetative cell size: about 5-10 μm (2) Vegetative cell shape: oval (3) Growth type: budding (b) Spore Presence or absence of sporulation.
本発明においては、ＭＹ１７株又はその形質転換体を用いてエタノール発酵を行う。エタノール発酵の発酵原料としては、砂糖製造の際の副産物（廃糖蜜、バガスなど）や廃木材、大麦、とうもろこしなどの植物原料等が幅広く使用でき、特に限定はされない。なお、上記菌株は、上述の通り、全糖濃度を１５重量％に調整した際の総灰分が３．５重量％以上の廃糖蜜からでもエタノール生産性が非常に高いという特徴を有しているため、従来は敬遠されてきた発酵基質である上記のような廃糖蜜でも原料として使用できる。 In the present invention, ethanol fermentation is performed using the MY17 strain or a transformant thereof. As fermentation raw materials for ethanol fermentation, by-products (such as waste molasses and bagasse) during sugar production and plant raw materials such as waste wood, barley and corn can be widely used, and are not particularly limited. In addition, as mentioned above, the strain has a feature that ethanol productivity is very high even from molasses having a total ash content of 3.5% by weight or more when the total sugar concentration is adjusted to 15% by weight. Therefore, the above-mentioned waste molasses, which is a fermentation substrate that has been avoided in the past, can also be used as a raw material.
例えば、全糖濃度を１５重量％に調整した際の総灰分が３．５重量％以上の廃糖蜜だけでなく、全糖濃度を１５重量％に調整した際の総灰分が４．０重量％以上の廃糖蜜、あるいは宮古島産廃糖蜜のような全糖濃度を１５重量％に調整した際の総灰分が４．５重量％以上の廃糖蜜でも使用することができる。範囲としては、全糖濃度を１５重量％に調整した際の総灰分が３．５〜８．０重量％、更には４．５〜８．０重量％が例示される。糖度調整前の廃糖蜜では、例えば、全糖濃度３５〜５０重量％、総灰分１０〜１９重量％のものが使用できる。そして本発明において、これらの廃糖蜜は、従来のように吸着カラム等での着色物質の除去などの原料前処理は全く必要なく、そのまま、あるいは単に希釈するだけで使用できる。 For example, not only waste molasses having a total ash content of 3.5% by weight or more when the total sugar concentration is adjusted to 15% by weight, but also a total ash content of 4.0% by weight when the total sugar concentration is adjusted to 15% by weight. Even the above molasses or the molasses with a total ash content of 4.5 wt% or more when the total sugar concentration is adjusted to 15 wt%, such as Miyakojima molasses, can be used. Examples of the range include a total ash content of 3.5 to 8.0% by weight, and further 4.5 to 8.0% by weight when the total sugar concentration is adjusted to 15% by weight. As the molasses before adjusting the sugar content, for example, one having a total sugar concentration of 35 to 50% by weight and a total ash content of 10 to 19% by weight can be used. In the present invention, these molasses can be used as they are or simply by diluting them without the need for raw material pretreatment such as removal of colored substances in an adsorption column or the like as in the prior art.
エタノール発酵条件は、バイオエタノール生産を行う際の定法で行うことができ、特に限定はされない。例えば、上記菌株を前培養した後に基質に加え、３３〜３８℃で２０〜４８時間発酵させる方法が例示される。なお、ＭＹ１７株又はその形質転換体は、上述の通り３５℃以上、例えば３８〜４０℃でも高い発酵性、増殖性を有しているため、発酵温度を３５℃以上（例えば３５〜４０℃）で設定しても問題はない。そのため、宮古島のようなサトウキビが栽培されている温暖な地域でもエタノール生産が可能である。 Ethanol fermentation conditions can be performed by a conventional method for producing bioethanol, and are not particularly limited. For example, after pre-culturing the above strain, it is added to the substrate and fermented at 33-38 ° C. for 20-48 hours. In addition, since MY17 strain or its transformant has high fermentability and proliferation even at 35 ° C. or higher, for example, 38 to 40 ° C. as described above, the fermentation temperature is 35 ° C. or higher (for example, 35 to 40 ° C.). There is no problem even if you set with. Therefore, ethanol production is possible even in a warm area where sugarcane is cultivated, such as Miyakojima.
そして、上述のとおり、ＭＹ１７株又はその形質転換体は例えば廃糖蜜培地における凝集性（菌体の沈降性）が十分に高いため、エタノール発酵終了後に上清と菌体を含む発酵残渣の分離が非常に容易である。つまり、エタノール発酵終了後に遠心分離などの分離工程を設ける必要がなく、単に一定時間静置するのみで分離が可能であるため、非常に簡便かつ効率的である。 And as above-mentioned, since MY17 stock | strain or its transformant has sufficient aggregation property (sedimentation property of a microbial cell) in a waste molasses medium, for example, separation of the fermentation residue containing a supernatant and a microbial cell is completed after ethanol fermentation. It is very easy. That is, it is not necessary to provide a separation step such as centrifugation after the end of ethanol fermentation, and separation is possible simply by leaving for a certain period of time, so that it is very simple and efficient.
なお、発酵後に上清を除去した発酵残渣は、エタノール発酵生産の種菌として再度用いることもできるし、これを乾燥等させて飼料に配合することで新規な酵母を含む配合飼料（酵母含有飼料）を製造することもできる。 In addition, the fermentation residue from which the supernatant was removed after fermentation can be used again as an inoculum for ethanol fermentation production, or it can be dried, etc., and mixed with feed to contain new yeast (yeast-containing feed). Can also be manufactured.
さらには、本発明のＭＹ１７株又はその形質転換体を用いて、３５℃以上の温度で発酵させることで、泡盛や焼酎などに代表されるエタノールを含む発酵食品を製造することもできる。この発酵食品の製造においても、宮古島のような温暖な地域でも生産が可能であることが特徴である。 Furthermore, the fermented food containing ethanol represented by Awamori, shochu, etc. can also be manufactured by fermenting at the temperature of 35 degreeC or more using MY17 stock | strain of this invention, or its transformant. A feature of this fermented food production is that it can be produced even in a warm area such as Miyakojima.
このように、本発明の新規な酵母（ＭＹ１７株又はその形質転換体）は、温暖な地域での発酵困難であった廃糖蜜からのバイオエタノール生産、当該発酵残渣を用いた酵母含有飼料生産、発酵食品生産において特に有用である。 Thus, the novel yeast of the present invention (MY17 strain or transformant thereof) is a bioethanol production from molasses that has been difficult to ferment in a warm region, a yeast-containing feed production using the fermentation residue, Particularly useful in fermented food production.
以下、本発明の実施例について述べるが、本発明はこれらのみに限定されるものではない。 Examples of the present invention will be described below, but the present invention is not limited to these examples.
（新規エタノール生産酵母のスクリーニング）
宮古島のサトウキビ、バガス、土壌、草花や廃糖蜜等を中心に計１５５点の試料を採取し、これらから合計１，１２２株の酵母を分離した。酵母の分離は次のように行った。採取試料を、３倍希釈した廃糖蜜を用いて、３３℃での気泡発生を観察し、廃糖蜜液中のエタノール濃度測定を行った。試料５点について、特にエタノール生成能が高かったことから、３倍希釈廃糖蜜寒天培地（塩化カリウムにより塩濃度を高めたものも併用）に塗布し、３５℃、４８時間後にできたコロニーのうち、塩濃度の高い廃糖蜜培地で増殖がよく、大きなコロニーとなった株を、試料ごとに４個ずつ選択した。
(Screening of new ethanol-producing yeast)
A total of 155 samples were collected mainly from sugarcane, bagasse, soil, flowers, and molasses on Miyakojima, and a total of 1,122 strains were isolated from these samples. Yeast separation was performed as follows. Using the molasses diluted 3 times for the collected sample, the generation of bubbles at 33 ° C. was observed, and the ethanol concentration in the molasses liquid was measured. Of the five colonies, the ethanol-producing ability was particularly high, so it was applied to a 3-fold diluted molasses agar medium (also used in combination with a salt concentration increased by potassium chloride). Four strains were selected for each sample, which grew well on a molasses medium with a high salt concentration and became large colonies.
前述の２０株について、３倍希釈糖蜜を用いて、３５℃、静置条件での７２時間後のエタノール生成能から１０株を選択した。選択した１０株について、同様の条件で、温度を３８℃に上げたときのエタノール生成能を確認するとともに、３８℃での撹拌培養時、２４時間でのエタノール生成能を複数回にわたり確認し、最も発酵能の高い株１株（ＭＹ１７株と命名）を選択した。 For the 20 strains described above, 10 strains were selected from the ethanol-producing ability after 72 hours at 35 ° C. under static conditions using 3-fold diluted molasses. For the selected 10 strains, under the same conditions, the ethanol production ability when the temperature was raised to 38 ° C. was confirmed, and the ethanol production ability in 24 hours was confirmed multiple times during the stirring culture at 38 ° C., One strain having the highest fermentability (named MY17 strain) was selected.
前述の選択株（ＭＹ１７株）については、ゲノムＤＮＡを抽出し、ＩＴＳ（ｉｎｔｅｒｎａｌ ｔｒａｎｓｃｒｉｂｅｄ ｓｐａｃｅｒ）領域の塩基配列を確認することで菌株の同定を行ったところ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅと同定された。なお、ＭＹ１７株のＩＴＳ領域の塩基配列は配列表（ＩＴＳ１：配列番号１、ＩＴＳ２：配列番号２）及び図１に示した。これらの配列は、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅの標準株であるＳ２８８ｃ株と３塩基の違いが見られ、過去に解析した産業酵母（Ｂｉｏｓｃｉｅｎｃｅ， Ｂｉｏｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｂｉｏｃｈｅｍｉｓｔｒｙ，７１，１６１６−１６２０（２００７））と比較すると清酒酵母や焼酎酵母等と完全に一致する配列であったが、ワイン酵母やウイスキー酵母とは一致しなかった。 The above-mentioned selected strain (MY17 strain) was identified as Saccharomyces cerevisiae when genomic DNA was extracted and the strain was identified by confirming the base sequence of the ITS (internal transcribed spacer) region. The nucleotide sequence of the ITS region of the MY17 strain is shown in the Sequence Listing (ITS1: SEQ ID NO: 1, ITS2: SEQ ID NO: 2) and FIG. These sequences differed from the S288c strain, which is a standard strain of Saccharomyces cerevisiae, by 3 bases, and compared with industrial yeasts analyzed in the past (Bioscience, Biotechnology and Biochemistry, 71, 1616-1620 (2007)). The sequence was completely identical to that of shochu yeast or shochu yeast, but was not identical to wine yeast or whiskey yeast.
（ＭＹ１７株の発酵温度特性評価）
ＹＰＤ培地５０ｍｌ、３０℃、４８時間の条件で前培養したＭＹ１７株を、糖度１５％に調整した宮古島産廃糖蜜４５０ｍｌに添加し、撹拌子（５００ｒｐｍ）により撹拌し、１２時間後、１８時間後、２４時間後のエタノール生成能を測定した。温度は、３０℃、３３℃、３５℃、３８℃、４０℃とした。
(Fermentation temperature characteristic evaluation of MY17 strain)
MY17 strain pre-cultured under the conditions of 50 ml of YPD medium at 30 ° C. for 48 hours was added to 450 ml of Miyakojima waste molasses adjusted to a sugar content of 15%, stirred with a stir bar (500 rpm), 12 hours later, 18 hours later, The ethanol production ability after 24 hours was measured. The temperature was 30 ° C, 33 ° C, 35 ° C, 38 ° C, 40 ° C.
図２に、これらの温度条件でのエタノール生成能を示す。グラフは、それぞれ、３０℃、３３℃、３５℃、３８℃、４０℃における、それぞれ、１２時間後、１８時間後、２４時間後の希釈廃糖蜜中でのエタノール濃度（縦軸：重量％）の温度依存性（横軸：発酵温度）として表した。１２時間後のエタノール濃度から、至適発酵温度は３３℃から３８℃であった。一方、２４時間後では、４０℃であっても、至適温度の時と変わらぬ、ほぼ理論収率どおりのエタノールを生産することができた。 FIG. 2 shows the ethanol production ability under these temperature conditions. The graphs show ethanol concentrations in diluted molasses after 12 hours, 18 hours, and 24 hours at 30 ° C, 33 ° C, 35 ° C, 38 ° C, and 40 ° C, respectively (vertical axis: wt%). It was expressed as temperature dependence (horizontal axis: fermentation temperature). From the ethanol concentration after 12 hours, the optimum fermentation temperature was 33 ° C to 38 ° C. On the other hand, after 24 hours, even when the temperature was 40 ° C., it was possible to produce ethanol that was almost the same as the theoretical yield, the same as at the optimum temperature.
（３８℃での半回分連続発酵試験による発酵性能の安定性の確認）
糖度１５％に調整した糖蜜を用いて、２１時間の撹拌と３時間の静置凝集による酵母回収、新規糖蜜添加を繰り返す、３８℃での連続した回分発酵試験を行った。
ＭＹ１７株は、ＹＰＤ培地６ｍｌ、３０℃、２４時間、振とう培養したのち、糖度１０％に調整した廃糖蜜５４ｍｌに添加、３０℃、２４時間、振とう培養したものを用いた。発酵試験は、前述の前培養液約６０ｍｌを、糖度１５％に調整した廃糖蜜４５０ｍｌに添加し、撹拌子（５００ｒｐｍ）により撹拌した。
(Confirmation of stability of fermentation performance by semi-batch continuous fermentation test at 38 ° C)
Using molasses adjusted to a sugar content of 15%, a continuous batch fermentation test at 38 ° C. was repeated, in which yeast was collected by 21 hours of stirring and static aggregation for 3 hours, and new molasses was added.
The MY17 strain used was 6 ml of YPD medium, cultured at 30 ° C. for 24 hours with shaking, added to 54 ml of molasses adjusted to 10% sugar content, and cultured at 30 ° C. for 24 hours with shaking. In the fermentation test, about 60 ml of the aforementioned preculture was added to 450 ml of molasses adjusted to a sugar content of 15% and stirred with a stirrer (500 rpm).
図３に、半回分発酵試験におけるＭＹ１７株のエタノール生成能を示す。グラフは、ＭＹ１７株のエタノール生成能を、希釈廃糖蜜中のエタノール濃度（縦軸：重量％）の経時変化として表した。２４時間を１サイクルとして、２１時間から３時間静置し、菌体を凝集させた。静置後、上清４８０ｍｌを新しい廃糖蜜と交換したため、静置凝集後のエタノール濃度は減少している。ＭＹ１７株は、３８℃という高温においても、少なくとも５回以上安定して発酵することができた。 FIG. 3 shows the ethanol production ability of the MY17 strain in the semi-batch fermentation test. The graph represents the ethanol production ability of the MY17 strain as a change with time in the ethanol concentration (vertical axis: wt%) in the diluted molasses. The cells were allowed to stand for 21 hours to 3 hours, with 24 hours as one cycle, to aggregate the cells. Since 480 ml of the supernatant was exchanged with fresh molasses after standing, the ethanol concentration after standing aggregation decreased. The MY17 strain could be stably fermented at least 5 times even at a high temperature of 38 ° C.
（ＭＹ１７株の耐塩性及び凝集性評価）
実施例２において３８℃でエタノール発酵生産したＭＹ１７株について、発酵終了後の凝集性を比較確認した。
ＭＹ１７株は、単にＹＰＤ培地で培養を行った場合、培養後に凝集性は示さなかった。しかし、３８℃での廃糖蜜発酵終了後には、極めて短時間で凝集沈殿すること（つまり十分な凝集性を示すこと）が明らかとなった。
(Evaluation of salt tolerance and aggregation of MY17 strain)
In Example 2, the MY17 strain produced by ethanol fermentation at 38 ° C. was compared and confirmed for cohesiveness after completion of fermentation.
The MY17 strain did not show aggregating properties after culturing when it was simply cultured in a YPD medium. However, after the end of the molasses fermentation at 38 ° C., it was revealed that the precipitate settles in a very short time (that is, exhibits sufficient cohesiveness).
さらに、総灰分１９％の宮古島産廃糖蜜を２．５倍希釈した培地（総灰分７．６重量％、塩分約５重量％）において、３７℃の温度条件でＭＹ１７株及び協会酵母Ｋ７号の増殖性（耐塩性）を確認した。この結果、ＭＹ１７株は高い増殖性（耐塩性）を示したが、協会酵母Ｋ７号はほとんど増殖することができなかった。 Furthermore, the growth of MY17 strain and association yeast No. K7 was carried out at a temperature of 37 ° C. in a medium (total ash content: 7.6 wt%, salinity: about 5 wt%) diluted 2.5 times with Miyakojima waste molasses with a total ash content of 19% (Salt resistance) was confirmed. As a result, the MY17 strain showed high growth (salt tolerance), but the association yeast K7 could hardly grow.
（比較例：各種酵母での宮古島産廃糖蜜からのエタノール発酵性確認）
比較例として、清酒酵母（Ｋ７株、Ｋ９株）及び焼酎酵母（ＡＷ１０１株、ＳＨ４株、ＳＨ５株、ＫＦ−１株、ＫＦ−３株、ＣＡＮ１株、ＭＫＯ２１株、Ｉ３３株、Ｃ１４株、Ｃ４株、Ｈ５株、Ｋ２株）を用いて、ＹＭ培地の糖をスクロース１５％に変えたＹＭ改変１５％スクロース培地と、総灰分１９％の宮古島産廃糖蜜を２．５倍希釈して全糖濃度を１５％とした培地でエタノール生産試験を行った。いずれの培地も、ＯＤ６６０を０．５とした菌含有液を加え、３７℃で４８時間静置培養を行った。
(Comparative example: Confirmation of ethanol fermentability from waste molasses from Miyakojima in various yeasts)
As comparative examples, sake yeast (K7, K9) and shochu yeast (AW101, SH4, SH5, KF-1, KF-3, CAN1, MKO21, I33, C14, C4) , H5 strain, K2 strain), a YM modified 15% sucrose medium in which the sugar in the YM medium is changed to 15%, and Miyakojima waste molasses with a total ash content of 19% are diluted 2.5 times to obtain a total sugar concentration. An ethanol production test was conducted on a medium with 15%. For each medium, a bacterium-containing solution with an OD 660 of 0.5 was added, and static culture was performed at 37 ° C. for 48 hours.
この結果、いずれの菌株も、ＹＭ改変１５％スクロース培地では４８時間後に６〜７重量％のエタノールが培地中に含まれていたが、宮古島産廃糖蜜培地ではほとんどエタノール生産ができなかった（図４）。よって、宮古島産廃糖蜜が、通常の酵母ではほとんどエタノール発酵できない原料であることが示された。 As a result, all the strains contained 6 to 7% by weight of ethanol in the YM modified 15% sucrose medium after 48 hours, but almost no ethanol was produced in the Miyakojima waste molasses medium (FIG. 4). ). Therefore, it was shown that the waste molasses from Miyakojima is a raw material that can hardly be subjected to ethanol fermentation by ordinary yeast.
このように本発明は、有用形質を有する新規エタノール生産酵母に関するものであり、特にＭＹ１７株は、遺伝子組み換え、紫外線照射、薬品処理など全く人為的な変異を加えていない野生からの分離株であるため極めて安全性が高く、食品を含む様々な分野の要望に応えるものである。 As described above, the present invention relates to a novel ethanol-producing yeast having useful traits, and in particular, the MY17 strain is an isolated strain from the wild that has not been subjected to any artificial mutation such as genetic recombination, ultraviolet irradiation, and chemical treatment. Therefore, it is extremely safe and meets the demands of various fields including food.
本発明を要約すれば、以下の通りである。 The present invention is summarized as follows.
本発明は、３５℃以上の温度条件下でも効率よくエタノールの発酵生産が可能であり、且つ、従来発酵原料として適していないとされていた原料からでもエタノールの発酵生産が可能な新規エタノール生産酵母及びこれを用いた工業用エタノール等の生産方法を提供することを目的とする。 The present invention is a novel ethanol-producing yeast capable of efficiently producing ethanol by fermentation under temperature conditions of 35 ° C. or higher and capable of producing ethanol by fermentation even from a raw material that has not been conventionally suitable as a fermentation raw material. And it aims at providing the production methods of industrial ethanol etc. using this.
そして、新規エタノール生産酵母であるサッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株（ＮＩＴＥ Ｐ−８９３）又はこの株を親株とした形質転換体を用いて、廃糖蜜などを原料として３５℃以上の温度で発酵させてエタノール生産を行う。 Then, using a novel ethanol-producing yeast, Saccharomyces cerevisiae MY17 strain (NITE P-893) or a transformant having this strain as a parent strain, fermentation is performed at a temperature of 35 ° C. or more using waste molasses as a raw material. To produce ethanol.
本発明において寄託されている微生物の受託番号を下記に示す。
（１）サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）ＭＹ１７株（ＮＩＴＥ Ｐ−８９３）。
The accession numbers of the microorganisms deposited in the present invention are shown below.
(1) Saccharomyces cerevisiae MY17 strain (NITE P-893).
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