JP2011055714A - Method for producing valuable substance by simultaneous saccharifying fermentation - Google Patents
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- 230000004151 fermentation Effects 0.000 title claims abstract description 106
- 238000004519 manufacturing process Methods 0.000 title description 6
- 239000000126 substance Substances 0.000 title description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 57
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 13
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 13
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- 108010059892 Cellulase Proteins 0.000 description 8
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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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
Description
本発明は、セルロース系バイオマスを原料とした場合の同時糖化発酵に用いる発酵用微生物を含む醪の利用方法に関する。 The present invention relates to a method for using koji containing a fermentation microorganism used for simultaneous saccharification and fermentation using cellulosic biomass as a raw material.
化石燃料の代替燃料として、近年植物由来のアルコールが注目されている。特に食料と競合しないセルロース系バイオマス由来のエタノール生産が世界的に検討されている。セルロース系バイオマスは主にリグニン、セルロース、ヘミセルロースといった構成成分からなる。リグニンは芳香族系化合物のポリマーが主な構成成分であり、セルロースはグルコースなどのヘキソース、ヘミセルロースはヘキソースとペントースから主に構成されている。これら構成成分は互いに複雑に絡み合うことで構造を維持していることから、エタノールに変換するための発酵用微生物が資化するためには原料を単糖もしくはオリゴ糖まで分解するという前処理および糖化といった過程が必要となる。前処理および糖化の方法としては、物理的粉砕、アルカリ処理、酸処理、メカノケミカル処理、爆砕、水熱処理、水による離解、脱水による水分調整等、物理的および化学的方法や、セルラーゼ産生微生物による処理や酵素単体を作用させる生物学的処理がある。現時点では、どの前処理もコストの問題やエネルギー回収、廃棄物の問題等があり、より効果的な前処理および糖化の検討が世界中でなされている。 In recent years, plant-derived alcohol has attracted attention as an alternative fuel for fossil fuels. In particular, production of ethanol derived from cellulosic biomass that does not compete with food is being studied worldwide. Cellulosic biomass is mainly composed of components such as lignin, cellulose, and hemicellulose. Lignin is mainly composed of an aromatic polymer, cellulose is mainly composed of hexose such as glucose, and hemicellulose is mainly composed of hexose and pentose. Since these components maintain their structure by being intricately intertwined with each other, pretreatment and saccharification in which the raw material is decomposed into monosaccharides or oligosaccharides in order to assimilate the microorganism for fermentation to convert to ethanol Such a process is necessary. Pretreatment and saccharification methods include physical and chemical methods such as physical grinding, alkali treatment, acid treatment, mechanochemical treatment, explosion, hydrothermal treatment, water disaggregation, water adjustment by dehydration, and cellulase-producing microorganisms. There are biological treatments in which treatments and simple enzymes act. At present, every pretreatment has a problem of cost, energy recovery, waste, etc., and more effective pretreatment and saccharification are being studied all over the world.
セルロース系バイオマスのアルコール化として有効な方法の１つとして同時糖化発酵が挙げられる。この方法は、適切な前処理を行ったセルロース系バイオマス原料に、酵素と発酵用微生物を同時に作用させることにより、同一反応槽内で糖化と発酵を行うというものである。セルロースを加水分解する酵素であるセルラーゼによる糖化は他の方法に比べて過分解物の発生がなく糖への変換率も高いが、生成するグルコースによる競争阻害の問題もあり、反応時間が長くなる。同時糖化発酵は酵素による加水分解産物を直ちに発酵微生物が資化するため競争阻害が起こりにくく、結果的に反応時間の短縮が図れるうえ、反応槽等の施設費の軽減にも繋がると期待される。しかしセルラーゼの一般的な至適温度は５０℃付近であるのに対して発酵用微生物の成育至適温度は３０℃付近であり、一般的な成育限界温度も４０℃程度である。酵素反応において反応温度は非常に重要なファクターであり、５℃異なるだけでも活性が数倍以上異なる場合がある。 Simultaneous saccharification fermentation is mentioned as one of the effective methods for alcoholation of cellulosic biomass. In this method, saccharification and fermentation are performed in the same reaction tank by simultaneously causing an enzyme and a microorganism for fermentation to act on a cellulosic biomass material that has been appropriately pretreated. Saccharification with cellulase, an enzyme that hydrolyzes cellulose, does not generate excessively decomposed products and has a high conversion rate to sugar, but there is also a problem of competition inhibition by the generated glucose, resulting in a longer reaction time. . Simultaneous saccharification and fermentation is expected to lead to a reduction in the cost of facilities such as reaction tanks as well as to shorten the reaction time because the fermentation microorganisms immediately assimilate the hydrolyzed product of the enzyme, thereby preventing competition inhibition. . However, the general optimum temperature for cellulase is around 50 ° C, whereas the optimum growth temperature for fermentation microorganisms is around 30 ° C, and the typical growth limit temperature is about 40 ° C. In an enzymatic reaction, the reaction temperature is a very important factor, and even if the temperature is different by 5 ° C., the activity may differ by several times.
このような問題はあるが、同時糖化発酵は効率的なアルコール生産にとっては大きな可能性を持った方法であり、耐熱性微生物の適用、セルラーゼ等酵素機能を付加させた微生物の育種、低温でも高活性なセルラーゼの開発等が行われている。 Despite these problems, simultaneous saccharification and fermentation is a method with great potential for efficient alcohol production. Application of thermostable microorganisms, breeding of microorganisms with enzyme functions such as cellulase, and high temperature even at low temperatures. Active cellulases have been developed.
現状、発酵用微生物の成育限界温度付近では、発酵用微生物の継続的な活性の維持が困難なため、成育限界温度よりも３〜８℃程度低い温度での同時糖化発酵が検討されており、その場合、使用微生物にもよるが、増殖に最も適した成育最適温度よりも５〜１０℃程度高い温度で反応が行われることになる（非特許文献１）。 Currently, in the vicinity of the growth limit temperature of the fermentation microorganism, since it is difficult to maintain the continuous activity of the fermentation microorganism, simultaneous saccharification and fermentation at a temperature about 3 to 8 ° C. lower than the growth limit temperature has been studied. In this case, although depending on the microorganism used, the reaction is carried out at a temperature about 5 to 10 ° C. higher than the optimum growth temperature most suitable for growth (Non-patent Document 1).
一方で、同時糖化発酵を行う際の発酵用微生物にかかる培地成分や施設費といったコストは決して低くなく（非特許文献２）、生産物に対するコストとして非常に重要である。 On the other hand, costs such as medium components and facility costs for fermentation microorganisms during simultaneous saccharification and fermentation are not low (Non-Patent Document 2), and are very important as costs for products.
以上を踏まえて、セルロース系バイオマスの同時糖化発酵を考えると、同時糖化発酵後の醪には主に未発酵および難発酵残渣、酵素、発酵用微生物が含まれている。醪または醪を含む反応液を次に行う同時糖化反応に利用することで発酵用微生物の再利用ができ、発酵用微生物の新規投入を減量するもしくは削減することができる。すなわち前培養に要するコストが軽減され、さらに再利用した醪中に含まれる酵素も再利用され、効率的かつ安価に同時糖化発酵を行うことができる方法を提供することを目的とする。 Based on the above, considering the simultaneous saccharification and fermentation of cellulosic biomass, the cocoons after the simultaneous saccharification and fermentation mainly contain unfermented and difficult-to-ferment residues, enzymes, and fermentation microorganisms. By using soot or a reaction solution containing soot for the subsequent simultaneous saccharification reaction, the fermentation microorganisms can be reused, and the amount of new input of fermentation microorganisms can be reduced or reduced. That is, an object of the present invention is to provide a method in which the cost required for the pre-culture is reduced, and the enzyme contained in the reused koji is reused, so that simultaneous saccharification and fermentation can be performed efficiently and inexpensively.
上記課題を解決するための本発明は、以下の技術的手段より構成される。 The present invention for solving the above-described problems comprises the following technical means.
（１）セルロース系バイオマスを原料とする同時糖化発酵方法において、同時糖化発酵反応を行った後に生じた醪の一部を次に行う同時糖化発酵反応に加えることを特徴とする同時糖化発酵方法。 (1) In the simultaneous saccharification and fermentation method using cellulosic biomass as a raw material, a simultaneous saccharification and fermentation method, wherein a part of the koji produced after the simultaneous saccharification and fermentation reaction is added to the subsequent simultaneous saccharification and fermentation reaction.
（２）同時糖化発酵反応を行った後に生じた醪を固液分離した後に生じた固形分を、次に行う同時糖化発酵反応に加える、前記（１）の同時糖化発酵方法。 (2) The simultaneous saccharification and fermentation method according to (1), wherein the solid content generated after solid-liquid separation of the koji produced after the simultaneous saccharification and fermentation reaction is added to the subsequent simultaneous saccharification and fermentation reaction.
（３）前記醪の一部もしくは前記固形分は、次に行う同時糖化発酵反応に使用される前に、微生物が成育できる限界温度以下まで昇温させられる、前記（１）または（２）に記載の同時糖化発酵方法。 (3) A part of the koji or the solid content is heated to a temperature lower than a limit temperature at which microorganisms can grow before being used in a simultaneous saccharification and fermentation reaction to be performed next. (1) or (2) The simultaneous saccharification and fermentation method described.
（４）前記昇温度は、同時糖化発酵反応の温度よりも２℃以上高い温度である、前記（３）の同時糖化発酵方法。
(4) The simultaneous saccharification and fermentation method according to (3), wherein the elevated temperature is a
（５）前記醪の一部もしくは前記固形分は、同時糖化発酵反応終了時の温度よりも低い温度に降温させた後に、次の同時糖化発酵反応に使用される、前記（１）または（２）の同時糖化発酵方法。 (5) A part of the koji or the solid content is used for the next simultaneous saccharification and fermentation reaction after the temperature is lowered to a temperature lower than the temperature at the end of the simultaneous saccharification and fermentation reaction. ) Simultaneous saccharification and fermentation method.
（６）前記降温は、同時糖化発酵反応終了時の温度よりも２℃以上低い温度である、前記（５）の同時糖化発酵方法。
(6) The simultaneous saccharification and fermentation method according to (5), wherein the temperature drop is a
（７）前記降温の際に発酵用微生物が要求する栄養塩を加える、前記（５）の同時糖化発酵方法。 (7) The simultaneous saccharification and fermentation method according to (5), wherein a nutrient salt required by the fermentation microorganism is added during the temperature drop.
次に、本発明についてさらに詳細に説明する。セルロース系バイオマスを原料として同時糖化発酵を行った際の反応液は、反応生成物だけでなく、使用発酵微生物、使用酵素の残留分、未反応残渣、難反応残渣、反応不可物質で構成されており、反応条件や原材料の種類、原材料の前処理方法によってこれらの比率が変化する。このような反応生成物以外の成分をも含んだ残渣もしくは残渣を含む同時糖化発酵反応液の一部、好ましくは１０〜５０％を次に行う同時糖化発酵反応に利用することで、使用酵母の軽減もしくは削減が期待され、さらに残留した使用酵素や未反応残渣の反応継続による反応収率の向上といった効果も期待できる。微生物を含む反応残渣もしくは反応液を再利用する方法は、下水処理や、生ゴミを用いたアルコール生産（特開２００８−１０４４５２号公報、特に、請求項１５、段落［００１０］〜［００１１］）において報告されているが、セルロース系バイオマスを用いたアルコール製造に関しての報告はない。さらに、セルロース系バイオマスを用いた同時糖化発酵は、他の原料、例えばデンプン系原料を用いた場合と比べて反応時間が長いため、雑菌汚染の可能性が高く、さらに使用発酵微生物の活性が低下しやすくなる。また、セルロース系バイオマス中に含まれるセルロースは高度に結晶化しているものが残存していることが多く、同時糖化発酵後の残渣中に含まれる未反応残渣の相対量比率が高く、その結果発酵用微生物の相対量比率が低くなるため、残渣の返送の検討はされていないのが現状である。 Next, the present invention will be described in more detail. The reaction solution when performing simultaneous saccharification and fermentation using cellulosic biomass as raw material is composed of not only reaction products but also fermentation microorganisms used, residues of enzymes used, unreacted residues, difficult-to-react residues, and unreacted substances. These ratios vary depending on reaction conditions, types of raw materials, and raw material pretreatment methods. By using a residue containing such components other than the reaction product or a part of the simultaneous saccharification and fermentation reaction solution containing the residue, preferably 10 to 50% in the subsequent simultaneous saccharification and fermentation reaction, Reduction or reduction is expected, and the effect of improving the reaction yield by continuing the reaction of the remaining used enzyme and unreacted residue can also be expected. Methods for reusing reaction residues or reaction liquids containing microorganisms include sewage treatment and alcohol production using garbage (Japanese Patent Application Laid-Open No. 2008-104452, particularly claim 15, paragraphs [0010] to [0011]). However, there is no report on alcohol production using cellulosic biomass. Furthermore, simultaneous saccharification and fermentation using cellulosic biomass has a longer reaction time compared to the case of using other raw materials, such as starch-based raw materials. It becomes easy to do. In addition, the cellulose contained in cellulosic biomass often remains highly crystallized, and the ratio of unreacted residues contained in the residue after simultaneous saccharification and fermentation is high, resulting in fermentation. Since the relative amount ratio of microorganisms for use becomes low, the return of the residue has not been studied at present.
本発明はセルロース系原料を用いた同時糖化発酵における醪もしくは固液分離後の固形物の再利用に関するものであり、その適応はセルロース系原料に対してセルラーゼをはじめとする原料を分解する酵素を用いて反応させる際に、同時に発酵用微生物を作用させる同時糖化発酵を行っているが、ここで、「同時」というのは、厳密に同時である必要はなく、糖化と発酵の反応が重複して行われてさえいれば、多少の時間的なずれがあっても本発明に言う「同時」に含まれるものとする。また、セルロース系原料は、糖化によりグルコース等を生じさせるようなものを含んでさえいれば良く、その種類および含有量の多少についてなんら制限はない。さらに、使用酵素および発酵用微生物の種類および使用量についても、本発明の目的とする糖化および発酵を生じさせることができるのであれば、なんらの制限も課されない。さらに、同時糖化発酵時の温度、ｐＨ、溶存酵素量、攪拌速度、攪拌方法、栄養添加物といった条件についても何らの制限はない。 The present invention relates to the reuse of koji or solid after solid-liquid separation in simultaneous saccharification and fermentation using cellulosic materials, and its adaptation is to apply enzymes that decompose cellulase and other materials to cellulosic materials. When saccharification and fermentation are performed, simultaneous saccharification and fermentation using a microorganism for fermentation is performed at the same time. However, the term “simultaneous” does not need to be strictly the same, and the saccharification and fermentation reactions overlap. As long as it is performed, even if there is a slight time lag, it is included in the “simultaneous” referred to in the present invention. In addition, the cellulosic raw material only needs to contain a substance that generates glucose or the like by saccharification, and there is no limitation on the kind and the amount of the content. Furthermore, no restrictions are imposed on the types and amounts of the enzymes used and the microorganisms used for fermentation as long as the saccharification and fermentation targeted by the present invention can be caused. Furthermore, there are no restrictions on conditions such as temperature, pH, amount of dissolved enzyme, stirring speed, stirring method, and nutritional additive during simultaneous saccharification and fermentation.
本発明は、同時糖化発酵の醪もしくは醪の固液分離後の固形物を次に行う同時糖化発酵に用いるものであり、これにより以下のような効果を得ることができる：
（１）発酵用微生物の新規投入を減量もしくは削減することができる。
The present invention is used for simultaneous saccharification and fermentation by performing the simultaneous saccharification and fermentation of the koji or the solid after the solid-liquid separation of the koji, thereby obtaining the following effects:
(1) A new input of fermentation microorganisms can be reduced or reduced.
（２）発酵用微生物の減量もしくは削減により前培養に要するコストが軽減される。 (2) The cost required for pre-culture is reduced by reducing or reducing the microorganisms for fermentation.
（３）同時糖化発酵の醪もしくは醪の固液分離後の固形物を次に行う同時糖化発酵に用いる際に、発酵用微生物の成育限界温度付近まで反応液の温度を上昇させることで、雑菌汚染の危険性が減少する。 (3) When using the saccharification fermentation coagulation or the solid material after solid-liquid separation of the coffin for the subsequent simultaneous saccharification fermentation, the temperature of the reaction solution is increased to near the growth limit temperature of the microorganism for fermentation. The risk of contamination is reduced.
（４）同時糖化発酵の醪もしくは醪の固液分離後の固形物を次に行う同時糖化発酵に用いる際に、発酵用微生物の成育最適温度付近まで反応液の温度を低下させることで、発酵用微生物の活性を上昇させることができる。 (4) When using the saccharification fermentation coagulation or the solid material after solid-liquid separation of the coffin for the subsequent simultaneous saccharification fermentation, the temperature of the reaction solution is lowered to near the optimum growth temperature of the fermentation microorganism, The activity of microorganisms for use can be increased.
次に実施例に基づいて本発明を具体的に説明するが、本発明は以下の実施例に何ら限定されるものではない。 EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited to a following example at all.
以下、実施例は、原料としてリグニン含量が少なく、品質が一定していると考えられる市販ＯＡ紙を用いた。市販ＯＡ紙はコクヨ工業滋賀株式会社、ロットＮｏ．７１１０６６７８を用いた。このＯＡ紙は事前に大量の酵素を作用させて生成したグルコース量を計測した結果、１ｇのＯＡ紙より約０．８ｇのグルコースが生成した。なお、グルコースおよびエタノール濃度は王子計測株式会社製グルコースセンサー（ＢＦ−５）にて計測を行った。 In the following examples, commercially available OA paper that has a low lignin content and is considered to have a constant quality was used as a raw material. Commercially available OA paper is KOKUYO INDUSTRIAL SHIGA CORPORATION, lot no. 71106678 was used. As a result of measuring the amount of glucose produced in advance by applying a large amount of enzyme to this OA paper, about 0.8 g of glucose was produced from 1 g of OA paper. The glucose and ethanol concentrations were measured with a glucose sensor (BF-5) manufactured by Oji Scientific Co., Ltd.
（実施例１：固液分離後の醪の利用）
総反応量を１００ｇとし、反応槽中に原料であるＯＡ紙が１５重量％になるように水を用いて調整し、セルラーゼ酵素（ジェネンコア社製アクセララーゼ）をＯＡ紙１ｇ当たり１５ＦＰＵ加えた。以下の実施例における同時糖化発酵についても同様にして原料を調整した。さらにそれぞれに同時糖化発酵用微生物として酵母（サッカロマイセスセルビジエ）を反応槽当たり湿重量で１ｇ加え、３８℃で３日間反応させることにより同時糖化発酵後の醪を得た（表１）。
(Example 1: Utilization of soot after solid-liquid separation)
The total reaction amount was set to 100 g, and water was used to adjust the OA paper as a raw material to 15% by weight in the reaction tank, and cellulase enzyme (Genencore Accelerase) was added at 15 FPU per 1 g of OA paper. The raw materials were similarly prepared for the simultaneous saccharification and fermentation in the following examples. Furthermore, 1 g of yeast (Saccharomyces cerevisiae) as a microorganism for simultaneous saccharification and fermentation was added to each by wet weight per reaction tank, and reacted at 38 ° C. for 3 days to obtain strawberries after simultaneous saccharification and fermentation (Table 1).
醪作製用の反応液を３５００ｒｐｍで５分間遠心後、醪中の固形分の湿重量を計測し、その１／５量と、酵母０．５ｇを加え、それ以外は同条件で再び次の同時糖化発酵反応（実施区Ａ）を行った（表２）。 After centrifuging the reaction solution for preparing cocoons at 3500 rpm for 5 minutes, measure the wet weight of the solid content in the cocoons, add 1/5 of that amount, and 0.5 g of yeast, and then repeat the following simultaneously under the same conditions. Saccharification and fermentation reaction (Implementation Zone A) was performed (Table 2).
さらに、対照区として酵素量を１５ＦＰＵ／ｇ−紙、使用酵母量を１ｇ用いた対照区Ａおよび使用酵母量を０．５ｇ用いた対象区Ｂを行った（表３）。 Further, as a control group, an enzyme amount of 15 FPU / g-paper, a control group A using 1 g of yeast used, and a target group B using 0.5 g of yeast used were performed (Table 3).
本発明の実施条件である実施区ＡのＥｔＯＨ生成量は、図２に示されるように、対照区Ｂより多く、対照区Ａとほぼ同量であった。したがって、酵母の使用量を半減させることができた。 As shown in FIG. 2, the amount of EtOH produced in the working group A, which is an implementation condition of the present invention, was larger than that in the control group B and was almost the same as that in the control group A. Therefore, the amount of yeast used could be halved.
（実施例２：醪の利用）
実施例１と同様の原料を用い、残渣を含む同時糖化発酵反応液の調整を行った。反応容量を１００ｇで行い、反応槽中の原料であるＯＡ紙が１５重量％になるように水で調整し、セルラーゼ酵素（ジェネンコア社製アクセララーゼ）を紙１ｇ当たり１０ＦＰＵ加え、同時糖化発酵用微生物として酵母（サッカロマイセスセルビジエ）を湿重量で１ｇ加え３８℃で３日間反応させることにより同時糖化発酵後の醪を得た（表４）。
(Example 2: Use of rice cake)
Using the same raw materials as in Example 1, the simultaneous saccharification and fermentation reaction solution containing the residue was adjusted. The reaction volume is 100 g, the water in the reaction vessel is adjusted to 15% by weight with OA paper, and cellulase enzyme (Genencore Accelerase) is added at 10 FPU per gram of paper. As a result, 1 g of yeast (Saccharomyces cerevisiae) was added in a wet weight and reacted at 38 ° C. for 3 days to obtain koji after simultaneous saccharification and fermentation (Table 4).
同時糖化発酵後の醪のうち、１／２、１／５、１／１０にあたる反応液を使用酵母の代わりに用い、水で１５重量％に調整したＯＡ紙１００ｇを加えた。ＯＡ紙１ｇに対して１０ＦＰＵのセルラーゼ酵素（ジェネンコア社製アクセララーゼ）を加えて３８℃で次の同時糖化発酵反応を行った（表５）。 Of the koji after simultaneous saccharification and fermentation, reaction solutions corresponding to 1/2, 1/5, and 1/10 were used instead of the yeast used, and 100 g of OA paper adjusted to 15% by weight with water was added. To 1 g of OA paper, 10 FPU cellulase enzyme (Genencore Accelerase) was added and the following simultaneous saccharification and fermentation reaction was performed at 38 ° C. (Table 5).
同時糖化発酵後の反応液を利用したことで、利用した反応液中にＥｔＯＨが含まれている。したがって、使用酵母の代わりに同時糖化発酵後の反応液を使用した場合に観測されるＥｔＯＨ量を算出し、同時糖化発酵後の反応液に含まれていたＥｔＯＨ量を減算した正味のＥｔＯＨ生成量（ｍＬ）を産出した。図３にこの結果を示す。発酵用微生物である酵母を新たに使用せずに、同時糖化発酵後の反応液中に含まれている酵母のみで次の同時糖化発酵が進行し、その返送割合の中で５０％の返送が最もエタノールの生成量が多く、この場合は対照区よりも多かった。 By using the reaction solution after simultaneous saccharification and fermentation, EtOH is contained in the used reaction solution. Therefore, the amount of EtOH observed when the reaction solution after simultaneous saccharification and fermentation is used instead of the yeast used, and the amount of EtOH produced is obtained by subtracting the amount of EtOH contained in the reaction solution after simultaneous saccharification and fermentation. (ML) was produced. FIG. 3 shows the result. The next simultaneous saccharification and fermentation progresses only with the yeast contained in the reaction solution after the simultaneous saccharification and fermentation without newly using the yeast for fermentation, and 50% of the return rate is returned. The amount of ethanol produced was the largest, and in this case it was more than the control.
したがって、醪の再利用により酵母の使用を削減することができることが示された。 Therefore, it has been shown that the use of yeast can be reduced by recycling the straw.
（実施例３）
実施例１の対照区Ａの同時糖化発酵反応液を、反応終了後温度を４１℃に上昇させ、６時間インキュベートした場合としない場合で雑菌汚染の度合いを調べた（表６）。
(Example 3)
The temperature of the simultaneous saccharification and fermentation reaction solution in the control group A of Example 1 was increased to 41 ° C. after the completion of the reaction, and the degree of contamination was examined with and without incubation for 6 hours (Table 6).
反応液を１０００倍に希釈し、そのうち５０μＬをニュートリエント培地に播き、３０℃で２日間インキュベートした後のコロニー数を計数した。温度を上昇させることで、雑菌の成育を約３分の２に抑えることができたことから、反応の安定性が増した。 The reaction solution was diluted 1000 times, 50 μL of the solution was seeded on a nutrient medium, and the number of colonies after incubation at 30 ° C. for 2 days was counted. By increasing the temperature, the growth of miscellaneous bacteria could be suppressed to about two thirds, and the stability of the reaction increased.
（実施例４）
実施例２の実施区Ｄを対照区Ｄとし、表４で示した醪の残りを、増殖に適した温度である３０℃で６時間インキュベートした場合（実施区Ｅ）において、同時糖化発酵反応によるエタノール生成能を比較した。醪の返送量は１／１０量とした。この結果を図４に示す。温度低下処理をすることで反応液中に残存する発酵用微生物の活性が向上したと考えられる。
Example 4
In the case where the control group D of Example 2 was designated as the control group D and the remainder of the koji shown in Table 4 was incubated at 30 ° C., which is a temperature suitable for growth, for 6 hours (execution group E), The ethanol production ability was compared. The amount of returned rice cake was 1/10. The result is shown in FIG. It is thought that the activity of the microorganisms for fermentation remaining in the reaction solution was improved by performing the temperature reduction treatment.
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