JP2005034133A - Method for producing ethanol by fermentation under aerobic-condition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing ethanol, capable of performing fermentation under an aerobic condition capable of maintaining the propagation and activity of yeast, and a method for producing ethanol or a fermented alcoholic beverage by using an NF1-1 strain. <P>SOLUTION: This method for producing ethanol by the fermentation comprises obtaining a new mutated strain of the yeast having an ethanol fermentation capability under an aerobic condition, and performing the ethanol fermentation under the aerobic condition by using the mutated strain. Thereby, it becomes possible to perform the ethanol fermentation while maintaining the propagation and activity of the yeast, and to produce ethanol in a high productivity. The method for producing ethanol can be applied to the production of ethanol in a wider range such as the production of ethanol or various fermented alcoholic beverages, etc., and especially, since the method of producing ethanol has a high productivity and a high yield based on sugar, it can effectively be used for the production of ethanol by using various biomass such as waste biomass, etc. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing ethanol by fermentation, characterized by performing fermentation under aerobic conditions using a mutant strain of yeast having ethanol fermentation ability under aerobic conditions, and production of the ethanol The present invention relates to a method for producing ethanol or a fermented alcoholic beverage using the method, and further to a novel yeast mutant strain capable of ethanol fermentation under aerobic conditions used for producing the fermented ethanol and a method for obtaining the same.

  The production technology of ethanol by fermentation of yeast has been used for the production of various fermented foods and fermented alcoholic beverages since ancient times. In recent years, the production technology of ethanol by fermentation of yeast can be broadly classified industrially, such as production technology by fermentation of industrial alcohol such as ethanol, and production technology of fermented alcoholic beverages such as alcoholic beverages. As a technology in various fields. As raw materials for production by fermentation of industrial alcohol such as ethanol, starch raw materials and saccharide raw materials are mainly used. Examples of the starchy raw material include rice, wheat, corn, sweet potato, potato, cassava, and the like. These starchy raw materials are saccharified using rice bran, enzymes, and the like and used for ethanol fermentation.

  As a sugar raw material, molasses produced as a by-product of a sugar factory is a main carbohydrate raw material, and sugar cane juice is also used in the sugar production area. In addition, recently, mandarin orange juice waste liquid, which was developed as part of the utilization of unused resources and recovered by squeezing the residue after mandarin orange production, has also been used as a raw material for producing fermented ethanol. Unlike starchy raw materials, saccharide raw materials do not require processing or saccharification of raw materials, so they are advantageous in many aspects such as facilities, manufacturing technology, cost, and energy balance, and are most frequently used as raw materials for ethanol production. Yes. In recent years, from the viewpoint of recycling of waste biomass, a method of performing ethanol fermentation using a starch or sugar-containing production residue produced during the production of various foods and drinks has also been reported.

  The use of ethanol production technology by yeast fermentation for the production of fermented alcoholic beverages such as alcoholic beverages includes fermented malt beverages such as sake and beer, fruit wines such as wine and apple wine, and distilled spirits such as whiskey and shochu. Since ancient times, it has been used as a manufacturing technology for many alcoholic beverages. The raw materials for producing these fermented alcoholic beverages include rice, wheat, malt, buckwheat, sweet potato, sugar cane, fruits such as grapes and apples, and raw materials containing starches and sugars such as honey. Is used after being saccharified with koji or enzyme, and various fermented alcoholic beverages are produced by performing ethanol fermentation with yeast using these production raw materials. Recently, from the viewpoint of effective use of food and beverage production residues, a method of producing a fermented alcoholic beverage by ethanol fermentation using a residue obtained by squeezing citrus fruits and collecting fruit juice as a raw material has been disclosed. (Japanese Patent Laid-Open No. 2002-153231).

  Production technology by fermentation of industrial alcohol such as ethanol and production technology of fermented alcoholic beverages such as alcoholic beverages are basically based on the production of ethanol by fermentation of yeast. The fermentation phenomenon is a phenomenon of ethanol fermentation in which yeast decomposes sugars anaerobically into ethanol and carbon dioxide, and these fermentations all proceed under anaerobic conditions. Therefore, ethanol production is promoted by fermentation with yeast under anaerobic conditions. On the other hand, ethanol production under the anaerobic conditions inhibits yeast growth, and the yeast activity gradually increases. To drop. In other words, it is necessary for the yeast to grow and activate under aerobic conditions, but ethanol production by yeast fermentation requires the fermentation to proceed under anaerobic conditions. Lower fermentation will lead to a decrease in yeast activity. Therefore, in either case of fermentation for the production of industrial alcohol or fermentation for the production of fermented alcoholic beverages, when performing ethanol fermentation with yeast, the yeast is cultured under aerobic conditions. Both the step of growing and activating and the step of fermenting and producing ethanol using the grown and activated yeast are employed, but both the growth and activation of yeast and ethanol fermentation are employed. A fermentation method that satisfies the conditions simultaneously has not been developed.

Recently, ethanol fermentation by yeast is possible only under anaerobic conditions, and the desired ethanol production cannot be obtained under aerobic conditions. Discloses a method of performing ethanol fermentation under aerobic conditions (Japanese Patent Laid-Open No. 2001-286276). In this method, basidiomycetes having amylase activity and alcohol dehydrogenase activity are used to produce alcoholic beverages such as sake, beer and wine, but this technique itself does not use yeast. It is not used as a substitute for the conventional technology for producing industrial alcohol or fermented alcoholic beverages using yeast. The only known ethanol fermentation using yeast is, for example, a method of producing yeast by anaerobic culture, such as ethanol fermentation production of biomass (Takashi Saiki, Yoshinori Saijo, Junro Tochizo, etc. Supervision "Fermentation Handbook" pp43-48, Kyoritsu Publishing (2001)). Therefore, in any of the production of industrial alcohol or fermented alcoholic beverages, conventionally, examples of fermenting ethanol under aerobic conditions, such as ethanol fermentation production in aerobic shaking culture or aeration culture, I can't find it.
JP 2001-286276 A. Japanese Patent Application Laid-Open No. 2002-153231. Takashi Saiki, Yoshinori Saijo, Toshiro Kuroshiro et al. “Fermentation Handbook” pp 43-48, Kyoritsu Shuppan (2001).

  An object of the present invention is to provide a method for producing ethanol by fermentation capable of performing fermentation under aerobic conditions in which the growth and activity of yeast are maintained, and ethanol or a fermented alcoholic beverage using the alcohol production method. It is another object of the present invention to provide a novel yeast mutant strain capable of ethanol fermentation under aerobic conditions and a method for obtaining the same, which are used for producing the fermented ethanol.

  As a result of intensive studies to solve the above problems, the present inventor obtained a novel yeast mutant strain having ethanol fermentation ability under aerobic conditions, and used the yeast mutant strain under aerobic conditions. By performing ethanol fermentation, it is found that yeast can be ethanol-fermented under aerobic conditions where the growth and activity of yeast are maintained, and high-production ethanol can be produced, thereby completing the present invention. It came to. Saccharomyces cerevisiae NF1-1 strain (FERM BP-8400) can be mentioned as a yeast mutant strain having ethanol fermentation ability under aerobic conditions used in the method for producing ethanol of the present invention. Conventionally, since fermentation of ethanol by yeast had to be performed under anaerobic conditions, the production of ethanol under the anaerobic conditions would inhibit the growth of the yeast, leading to a decrease in its activity. However, since the method for producing ethanol by fermentation according to the present invention enables ethanol fermentation under aerobic conditions, it is possible to maintain the growth and activity of yeast during the fermentation, and to achieve high production. Ethanol can be produced.

  The method for producing ethanol of the present invention can be applied to the production of ethanol by fermentation in a wide range such as production of ethanol by fermentation and production of various fermented alcoholic beverages. In particular, the ethanol production method of the present invention has high productivity and high sugar yield, and therefore can be effectively used for production of ethanol using various biomass such as waste biomass. In addition, when ethanol is produced from waste biomass resources using the ethanol production method of the present invention, the productivity of ethanol increases when using tomato juice or oyster juice as waste biomass resources. This includes a method for producing ethanol using tomato juice and / or oyster juice, or a saccharide fermentation raw material or waste biomass to which the juice is added.

  Furthermore, the present invention includes a method for obtaining a mutant strain of a novel yeast having ethanol fermentation ability under aerobic conditions, which is used in the method for producing ethanol of the present invention.

  Specifically, the present invention relates to a method for producing ethanol by fermentation, characterized in that fermentation is carried out under aerobic conditions using a mutant strain of yeast having alcohol-fermenting ability under aerobic conditions (claims) The method for producing ethanol by fermentation according to claim 1, wherein the yeast mutant having ethanol fermentation ability under aerobic conditions is a mutant belonging to the genus Saccharomyces (claim 2). 3) or a yeast mutant strain capable of ethanol fermentation under aerobic conditions is Saccharomyces cerevisiae NF1-1 strain (FERM BP-8400). The fermentation according to any one of claims 1 to 3, wherein the production method (Claim 3) and fermentation under aerobic conditions are performed while supplying oxygen to the liquid fermentation raw material. The method for producing tanol (Claim 4) and the supply of oxygen to the liquid fermentation raw material are performed by aeration of the liquid fermentation raw material and / or stirring of the liquid fermentation raw material. Using the ethanol production method (Claim 5) or the ethanol production method by fermentation according to any one of Claims 1 to 5, the ethanol is produced and accumulated by aerobic fermentation with a liquid fermentation raw material. The method for producing ethanol according to claim 6, wherein a starch or a sugar fermentation raw material is used as the liquid fermentation raw material (claim 6). 7) The method for producing ethanol (Claim 8) or the waste biomass resource according to claim 6, wherein the waste biomass resource is used as a liquid fermentation raw material in the medium. 9. A method for producing ethanol according to claim 8, wherein tomato juice and / or oyster juice, or a saccharide fermentation raw material or waste biomass to which said fruit juice has been added is used as a fermentation raw material. The method for producing a fermented alcoholic beverage (Claim 10), wherein the brewing raw material is aerobically fermented using the method for producing fermented ethanol according to any one of Items 1 to 5, It consists of a fermented malt beverage, a fruit liquor, or a distilled liquor, comprising the method for producing a fermented alcoholic beverage according to claim 10 (claim 11).

  The present invention also provides a method for isolating a yeast mutant having an ethanol fermentation ability under aerobic conditions, wherein the yeast is subjected to a mutation treatment, and the yeast subjected to the mutation treatment is purely separated using a separation medium. 13. An ethanol fermentation under aerobic conditions according to claim 12, characterized in that an acquisition method (Claim 12), ethyl methanesulfonate treatment is used as a yeast mutation treatment, and a medium containing cycloheximide is used as a separation medium. The method for separating and obtaining yeast mutants having the ability (Claim 13), and the selection of yeast having ethanol fermentation ability under aerobic conditions, the separated yeast, and further stationary or shaking culture using a liquid medium It isolate | separates and acquires the yeast mutant which has the ethanol fermentation ability of Claim 12 or 13 characterized by performing by measuring the generation amount of the carbon dioxide in Or Saccharomyces cerevisiae NF1-1 strain (FERM BP-8400) having high ethanol fermentation ability under aerobic conditions and having the properties of foam-free yeast (Claim 15) .

  The method for producing ethanol by fermentation according to the present invention enables ethanol fermentation by yeast under aerobic conditions in which the growth and activity of yeast are maintained, so that high-production ethanol can be produced. It can be applied as a highly productive ethanol production method to the production of alcohol by fermentation in a wide range, such as the production of alcohol and various fermented alcoholic beverages. In particular, the ethanol production method of the present invention makes it possible to perform fermentation while maintaining both yeast activity maintenance and fermentation, and has high production efficiency and high sugar yield. It can be effectively used for the production of ethanol using various biomass such as waste biomass. In addition, since the ethanol production method of the present invention enables production of ethanol with high productivity, as an effective reuse technology in the field of recycling waste biomass, such as effective utilization of unused agricultural products. Can be expected. Furthermore, a mutant strain of yeast having the ability to ferment alcohol under aerobic conditions, such as the Saccharomyces cerevisiae NF1-1 strain in the present invention, can be obtained by using the strain for the production of a fermented alcoholic beverage. It becomes possible to produce fermented liquor efficiently in a short time.

  The present invention comprises producing ethanol by performing fermentation under aerobic conditions using a mutant strain of yeast having ethanol fermentation ability under aerobic conditions. In the present invention, first, an industrially usable yeast mutant strain capable of producing ethanol by fermentation under an aerobic condition is obtained. To obtain a mutant strain, the yeast is subjected to a mutation treatment, and the yeast subjected to the mutation treatment is purely isolated using a separation medium, and a mutant strain having an ethanol fermentation ability under aerobic conditions is isolated and obtained. To do. As the yeast mutation treatment, known mutation treatment means used for microorganism mutation treatment can be used as appropriate. Examples include γ-rays, X-rays and β-rays, chemical mutating agents such as ethyl methanesurufonate (EMS) and sodium azide. It can be mentioned as a suitable mutation treatment.

  As yeast used for the mutation treatment, yeast conventionally used for ethanol fermentation can be selected according to each purpose, but yeast of the genus Saccharomyces that is widely used in the production of ethanol fermentation and various fermented alcoholic beverages For example, Saccharomyces cerevisiae can be mentioned as a representative yeast. The yeast subjected to the mutation treatment is purely separated using a separation medium such as a medium containing cycloheximide. Purely isolated yeast mutants are subjected to static or shaking culture using a liquid medium in order to select yeast mutants with high ethanol fermentation ability under aerobic conditions. Measure and isolate yeast mutants with high ethanol fermentation ability.

  The method for obtaining the yeast mutant of the present invention will be described with an example. In the present invention, in order to obtain a microorganism capable of producing ethanol by fermentation under aerobic conditions, which can be used industrially, Sake yeast Kyokai No. 7 yeast was selected as a yeast strain to be mutated.

  The yeast strain was subjected to mutation treatment by treatment with ethyl methanesulfonate. The yeast strain subjected to the mutation treatment was purely isolated by plate culture containing cycloheximide. The isolated mutant strains were further collected using a froth flotation method by accumulating foam-free yeast and isolated by plating. The isolated mutant strain is subjected to stationary or shaking culture with the medium A having the composition shown in Table 1, and the amount of carbon dioxide generated is measured. In this example, mutant strains with excellent carbon dioxide content in shaking culture were selected. The yeast mutant strain obtained in this example and having the ability to produce ethanol highly by fermentation under aerobic conditions was transferred to NF1-1 strain (FERM BP-8400) at the National Institute of Advanced Industrial Science and Technology (AIST). ). The yeast mutant is a novel yeast mutant belonging to the cerevisiae species of the genus Saccharomyces that has an aerobic ability to produce ethanol and has an optimum pH for growth on the acidic side.

本発明のエタノールの製造方法は、液状発酵原料を用いた工業用アルコール等のエタノールの製造や各種発酵アルコール飲料の製造に用いることができる。

The ethanol production method of the present invention can be used for the production of ethanol such as industrial alcohol using liquid fermentation raw materials and the production of various fermented alcoholic beverages.

  In order to produce ethanol by fermentation using the method for producing ethanol of the present invention, the yeast mutant yeast strain obtained in the present invention is used, fermentation is performed under aerobic conditions, and ethanol is added to the medium. Generate and accumulate. As the medium, a liquid medium is usually used. Fermentation under aerobic conditions is performed while supplying oxygen to the liquid fermentation raw material by an appropriate means. Usually, oxygen is supplied to the liquid fermentation raw material by aeration of the liquid fermentation raw material and / or stirring of the liquid fermentation raw material. In the present invention, by supplying oxygen to the liquid fermentation raw material and performing aerobic fermentation, the growth and activity of yeast can be maintained, and ethanol can be produced and accumulated with high efficiency.

  As a fermentation raw material at the time of producing ethanol using the method of the present invention, a fermentation raw material usually used when producing ethanol using yeast can be used. As the fermentation raw material, starch or saccharide fermentation raw material is used. Examples of the starchy raw material include rice, wheat, corn, sweet potato, potato, cassava, and the like. These starchy raw materials are saccharified using rice bran, enzymes, and the like and used for ethanol fermentation. As the sugar raw material, molasses produced as a by-product of the sugar factory is the main carbohydrate raw material, and sugar cane juice produced in the sugar production area is also used. In addition, fruit juices such as mandarin orange juice waste liquid collected by pressure squeezing the residue after producing mandarin orange juice, which was developed as part of the utilization of unused resources, are also used, including sucrose, glucose and Fructose is used for fermentation. Unlike starchy raw materials, saccharide raw materials do not require raw material processing or saccharification, so they are advantageous raw materials in many aspects such as facilities, manufacturing technology, cost, energy balance, etc. it can.

  As a fermentation raw material in the production of ethanol using the method for producing ethanol of the present invention, starch and carbohydrates produced in the production of various foods and beverages that have recently been attracting attention from the viewpoint of recycling waste biomass, The manufacturing residue etc. which contain can be used. In the production of ethanol by fermentation according to the present invention, it should be particularly noted that ethanol productivity is particularly high when tomato juice or oyster (pepper) juice is used as waste biomass, and the juice or molasses added with the juice It is that ethanol can be manufactured with high production using sugar fermentation raw materials, such as these, and waste biomass as a liquid fermentation culture medium.

  By way of example, the production of ethanol of the present invention will be described. In the present invention, the mutant yeast strain of the present invention is used, and the fermentation raw material as described above is used for fermentation under aerobic conditions. To produce ethanol in the medium. During fermentation, the concentrations of sucrose, glucose and fructose contained in the medium are preferably adjusted to 10% W / V or more. For example, when using fruit juice, molasses, etc., the addition method is a method of adding the whole amount at the same time at the start of culture, a method of adding sequentially or continuously and making the total amount become the above amount, etc. Either method is acceptable. As the nitrogen source, ammonium sulfate is used, and the amount added to the medium is preferably 0.5% W / V. There is no need to add minerals or inorganic salts. The pH at the start of fermentation of the microorganism is optimum at 6.0, so it must be adjusted with about 2N sodium hydroxide.

  Culturing is performed under aerobic conditions with aeration and agitation. The culture temperature is 25-30 ° C, preferably about 30 ° C. The pH during the culture may not be adjusted. The culture time may be cultured until the production amount of ethanol reaches the maximum. Preferably, it is 32 hours. The culture solution thus obtained may be purified according to a general fermented ethanol production process (by Takashi Saiki, edited by the Japan Institute of Energy: Biomass Handbook, pp 157-165, Co-Ohmu Publishing Office (2002)).

  Ethanol obtained by the fermentation of the present invention can be used in various fields such as fuel, industrial, and beverage (by Takashi Saiki, edited by the Japan Institute of Energy: Biomass Handbook, pp157-165, Co-Ohmu Publishing Office) (2002)).

  The method for producing ethanol of the present invention can be applied to the production of various fermented alcoholic beverages. Traditionally, fermentation for the production of fermented alcoholic beverages using yeast had to be carried out under anaerobic conditions, and ethanol production under this anaerobic condition inhibited the growth of yeast and reduced its activity. Although it was supposed to be invited, since it became possible to perform ethanol fermentation under aerobic conditions by the method for producing ethanol by fermentation of the present invention, while maintaining the growth and activity of yeast during the fermentation The fermentation of fermented alcoholic beverages can proceed.

  The method for producing a fermented alcoholic beverage of the present invention includes various types of alcoholic beverages using yeasts such as sake, fermented malt beverages such as beer, fruit liquors such as wine and apple liquor, and distilled spirits such as whiskey and shochu. It can be applied to the production of fermented alcoholic beverages.

  In order to produce a fermented alcoholic beverage using the method for producing a fermented alcoholic beverage of the present invention, the yeast of the present invention having ethanol fermentability under aerobic conditions is used as the yeast, and fermentation is performed under aerobic conditions. Other than the above, in the other fermentation conditions, the production method of the fermentation raw material, etc., there is basically no difference from the conventional production method of fermented alcoholic beverages. For example, as a raw material for producing fermented alcoholic beverages, corresponding to each fermented alcoholic beverage, fruits such as rice, wheat, malt, buckwheat, sweet potato, sugar cane, grape and apple, and starch and sugar such as honey When a starchy fermentation raw material is used, as is conventionally done in the production of fermented alcoholic beverages, starch is saccharified with koji or an enzyme and used. Recently, a fermented alcoholic beverage is produced by fermenting ethanol by the method of the present invention using a residue obtained by collecting fruit juices such as citrus fruits and apples as a fermentation raw material, which has attracted attention from the viewpoint of effective use of food and beverage production residues. This can also be done as appropriate.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Productivity of ethanol using aerobically yeast mutants with ethanol fermentation ability)
5 ml of the liquid medium A shown in Table 1 was dispensed into a 15 ml test tube and steam sterilized at 121 ° C. for 15 minutes. This seed medium was inoculated with 1 ase of a slant culture of the yeast mutant NF1-1 strain (FERM BP-8400) which has an aerobic ethanol fermentation ability, and left to stand at 30 ° C. for 24 hours. It was. As the main medium, 100 ml of the molasses liquid medium shown in Table 2 was dispensed into a 500 ml Erlenmeyer flask, the above seed culture was added at a concentration of 5%, stationary culture (anaerobic conditions) and rotary shaking (180 rpm) ( Aerobic conditions) and cultured at 30 ° C. The culture was terminated when carbon dioxide generation was not observed. For comparison, Kyoto No. 7 yeast was also cultured under the same conditions. Fermentation was completed in 7 days in static culture, and fermentation was completed in 2 days in shake culture. After completion of the culture, the cells were removed by centrifugation (10,000 rpm, 15 minutes). The obtained supernatant was quantified by gas chromatography to determine the production amount, and the productivity was compared. The ethanol production amount and productivity results are shown in Table 3. As shown in Table 3, the NF1-1 strain, which was lower in productivity than static No. 7 yeast in static culture, was higher than the No. 7 yeast in shake culture, and had the highest productivity. It was.

(Productivity of ethanol using tomato medium and oyster medium)
5 ml of each liquid medium whose sugar content shown in Table 4 was adjusted to 20% was dispensed into a 15 ml test tube and steam sterilized at 121 ° C. for 15 minutes. This seed medium was inoculated with 1 ase of a slope culture of the yeast mutant NF1-1 strain (FERM BP-8400) and allowed to stand at 30 ° C. for 24 hours to form a seed culture. As the main medium, 100 ml of each liquid medium shown in Table 4 was dispensed into a 500 ml Erlenmeyer flask and steam sterilized at 121 ° C. for 15 minutes. The seed culture was transplanted to a main medium having the same composition so as to have a concentration of 5%, and cultured under conditions of 180 rpm and 30 ° C. until no carbon dioxide generation was observed. For comparison, the medium A shown in Table 1 as a synthetic medium was also used at the same time. After completion of the culture, the cells were removed by centrifugation (10,000 rpm, 15 minutes). The obtained supernatant was quantified by gas chromatography to determine the production amount, and the productivity was compared.

表４に示したトマト抽出液は、廃棄される完熟トマトから加熱抽出後、遠心分離（５，０００ｒｐｍ、１５分）して調製し、カキ抽出液は、廃棄される庄内柿から発明者の一人が開発した方法（河東田茂義・末永秀清、軟化したカキ‘平核無’果実の微生物による利用、山形農林学会報、第４４号、ｐｐ．２５−２７、１９８７）により調製した。エタノール生成量及び生産性は表５に示した。比較のためにきょうかい７号酵母の結果を加えた。エタノールの収量が最高値に達するまでの発酵時間は、トマト培地が１日、カキ培地が２日及び合成培地である培地Ａが４日であった。表５の結果は、廃棄バイオマスとして用いたトマト果汁及びカキ果汁のエタノール生産性が高く、しかも本発明で得られた菌株ＮＦ１−１はきょうかい７号と比較してすぐれていることを示していた。

The tomato extract shown in Table 4 is prepared by heating and extracting from discarded ripe tomatoes and then centrifuging (5,000 rpm, 15 minutes). (By Shigeyoshi Kawatoda and Hideki Suenaga, Utilization of softened oysters 'no plain' fruit by microorganisms, Yamagata Agricultural and Forestry Research Institute, No. 44, pp. 25-27, 1987). The amount of ethanol produced and productivity are shown in Table 5. For comparison, results of Kyoto No. 7 yeast were added. The fermentation time until the yield of ethanol reached the maximum value was 1 day for tomato medium, 2 days for oyster medium, and 4 days for medium A, which is a synthetic medium. The results in Table 5 indicate that the ethanol productivity of tomato juice and oyster juice used as waste biomass is high, and that the strain NF1-1 obtained in the present invention is superior to Kyoto No. 7. It was.

(Productivity of ethanol by aeration stirring conditions)
5 ml of molasses medium shown in Table 2 was dispensed into a 15 ml test tube and steam sterilized at 121 ° C. for 15 minutes. A slant culture of NF1-1 strain (FERM BP-8400) was inoculated into this preculture medium for 1 ase and statically cultured at 30 ° C. for 24 hours to obtain a preculture. As a seed medium, 100 mL of the molasses medium shown in Table 2 was dispensed into a 500 ml Erlenmeyer flask, the above preculture was added at a concentration of 5%, and the mixture was subjected to rotary shaking (180 rpm) (aerobic condition). Culturing for 24 hours was used as a seed culture. On the other hand, as the main medium, 7 L of molasses medium shown in Table 2 was charged into a 10 L jar fermenter without sterilization. The seed culture was transplanted to the main medium so as to have a concentration of 5%, and cultured under the conditions of a stirring rate of 200 rpm, aeration of 0.5 vvm, and 30 ° C. Samples were taken at regular intervals, and the cells were removed by centrifugation (10,000 rpm, 15 minutes). The resulting supernatant was quantified for ethanol by gas chromatography to determine the amount produced.

  The culture was terminated when no increase in the amount of ethanol produced was observed. For comparison, Kyoto No. 7 yeast was also cultured under the same conditions. The ethanol production and productivity results were 93.9 g / L (productivity of about 2.9 g / (L · h)) at 32 hours for the NF1-1 strain. The maximum value in 32 hours was 79.8 g / L (productivity: about 2.5 g / (L · h)). The amount of these products produced is 1.3-1.5 g / (L · h) productivity per culture volume of batch method using molasses as a fermentation raw material that has been reported so far. Supervision: Alcohol Handbook, 9th edition, Tech Press Publishing (1997)) is twice as high. In particular, both the amount of ethanol produced and the productivity of the NF1-1 strain were excellent. Furthermore, in the case of Kyokai No. 7 yeast, foam is remarkably formed under these culture conditions, and an antifoaming agent must be added up to 12 hours after the start of the culture when ethanol production is started. For one strain, a slight amount of antifoaming agent may be added at the start of culture.

  Table 6 summarizes the conventional ethanol fermentation conditions with the NF1-1 strain. The effectiveness of aeration culture with the NF1-1 strain is shown.

(Effects of tomato juice addition on ethanol productivity)
5 ml of each medium (molasses: tomato medium) in which tomato juice was added to the diluted molasses stock solution at a ratio shown in Table 7 was dispensed in a 15 ml test tube and steam sterilized at 121 ° C. for 15 minutes. A slant culture of NF1-1 strain (FERM BP-8400) was inoculated into this preculture medium for 1 ase and statically cultured at 30 ° C. for 24 hours to obtain a seed culture. Molasses of the same ratio: 100 ml of tomato medium is dispensed into a 500 ml Erlenmeyer flask, and the seed culture of the same ratio is added at a concentration of 5%, and is shaken (180 rpm) (aerobic condition) and cultured at 30 ° C. did. Samples were taken at regular intervals, and the cells were removed by centrifugation (10,000 rpm, 15 minutes). The resulting supernatant was quantified for ethanol by gas chromatography to determine the amount produced.

  The culture was terminated when no increase in the amount of ethanol produced was observed. For comparison, Kyoto No. 7 yeast was also cultured under the same conditions. The results are shown in Table 7. The fermentation time was completed in a short time according to the increase in the ratio of adding tomato juice, and in particular in the case of 5: 5, the fermentation completion time of the tomato juice medium was the same. The results of ethanol production and productivity at that time were 61.5 g / L for NF1-1 strain (productivity of about 2.6 g / (L · h)), and 61.0 g / L for Kyoto 7 The productivity was about 2.5 g / (L · h). In this case, both the amount of ethanol produced by the NF1-1 strain and the productivity were superior to those of Kyokai No. 7.

(Manufacture of fermented alcoholic beverages with tomato juice)
The ripe tomato fruit was put on a juicer, 5 ml of tomato juice filtered with a bleaching cloth was dispensed into a 15 ml test tube, and steam-sterilized at 121 ° C. for 15 minutes to obtain a preculture medium. A slant culture of Saccharomyces cerevisiae NF1-1 strain (FERM BP-8400) was inoculated into this preculture medium for 1 ase and statically cultured at 30 ° C. for 24 hours to obtain a preculture. As a seed medium, 100 ml of tomato juice prepared in the same manner as the preculture medium was dispensed into a 500 ml Erlenmeyer flask, the above preculture was added at a concentration of 5%, and the mixture was shaken (180 rpm) (aerobic condition). Culturing at 30 ° C. for 24 hours was used as a seed culture. On the other hand, sucrose was added as a main medium to 10% of tomato juice prepared in the same manner as the preculture medium. 4 liters of the main medium was charged into a 10 liter jar fermenter without sterilization. The seed culture was transplanted to the main medium so as to have a concentration of 5%, and cultured under the conditions of a stirring rate of 200 rpm, aeration of 0.1 vvm, and 30 ° C.

  Samples were taken at regular intervals, and the cells were removed by centrifugation (10,000 rpm, 15 minutes). The resulting supernatant was quantified for ethanol by gas chromatography to determine the amount produced. Fermentation was terminated when no increase in ethanol production was observed. For comparison, Kyoto No. 7 yeast was also cultured under the same conditions. The ethanol production results are shown in Table 8. Neither NF1-1 nor Kyokai No. 7 produced any more ethanol after 20 hours. In the NF1-1 strain, the maximum value is 7.6% (60.0 g / L, productivity about 3.0 g / (L · h)) in 20 hours of culture, and in the case of Kyoto 7 the maximum value is 6 in 20 hours of culture. 0.7% (52.9 g / L, productivity about 2.6 g / (L / h)). In this case, both the amount of ethanol produced and the productivity of the NF1-1 strain were excellent.

これらの発酵酒を比較するために、１０人のパネラーによって３点評価法により官能試験をおこなった。その結果、最も評価が高かったのはＮＦ１−１株による培養１２時間目が１．７５で、最も低かったのがきょうかい７号株の培養２０時間目で２．８５であった。１６時間目以降では酸味がきついため、酸味の好き嫌いで評価が分かれる結果となっていた。官能試験とトマト酒に含まれる有機酸と還元糖との関連性をみるために有機酸および還元糖の分析をおこなった。有機酸は液体クロマトグラフィー法、還元糖はジニトロフタル酸による比色定量法により定量した。その結果（トマト酒の有機酸及び還元糖量）を表９にまとめた。

In order to compare these fermented liquors, a sensory test was conducted by 10 panelists using a three-point evaluation method. As a result, the highest evaluation was 1.75 at the 12th hour of cultivation with the NF1-1 strain, and the lowest was 2.85 at the 20th hour of cultivation of the Kyoto 7 strain. Since the sourness was strong after the 16th hour, the results were divided according to likes and dislikes of the sourness. The organic acid and reducing sugar were analyzed in order to examine the relationship between the organoleptic test and the organic acid contained in tomato liquor and the reducing sugar. Organic acids were quantified by liquid chromatography, and reducing sugars were quantified by colorimetric quantification with dinitrophthalic acid. The results (organic acid and reducing sugar amount of tomato liquor) are summarized in Table 9.

その結果、最も評価の高かったＮＦ１−１株の発酵１２時間の酒は、強い刺激的な酸味を示すピルビン酸と酢酸含量が低く、還元糖量が高かった。そのため甘いトマトの風味が生き、丸味ある飲みやすい酸味の生きたすっきりした味が保たれていた。最も評価の低かったきょうかい７号株の発酵２０時間の酒は酢酸含量が最も高かく、還元糖は１％以下であった。そのため酸味と渋みが引き立ち、くどさが感じられる結果となっていた。以上の結果から、ＮＦ１−１株を用いることで、発酵時間が１２時間以内の短時間で、新鮮で飲みやすい低アルコールの発酵トマト酒の醸造が可能である。他の果汁や野菜ジュースを単独であるいは混合しても同様に低アルコールの発酵酒の醸造が可能である。

As a result, the most evaluated 12-hour fermentation of NF1-1 strain had a low pyruvic acid and acetic acid content showing a strong stimulating acidity and a high amount of reducing sugar. Therefore, the flavor of sweet tomatoes was alive, and the lively and refreshing taste of a round and sour taste that was easy to drink was maintained. The sake No. 7 which had the lowest evaluation, the 20-hour fermentation of sake had the highest acetic acid content and less than 1% reducing sugar. As a result, the acidity and astringency were enhanced and the result was a bit of a bit of a feeling. From the above results, by using the NF1-1 strain, it is possible to brew a fresh, easy-to-drink low-alcohol fermented tomato liquor in a short time of 12 hours or less. It is possible to brew a low-alcohol fermented liquor by mixing other fruit juices and vegetable juices alone or in combination.

Claims (15)

A method for producing ethanol by fermentation, which comprises performing fermentation under an aerobic condition using a mutant strain of yeast having an alcohol fermentation ability under an aerobic condition. The method for producing ethanol by fermentation according to claim 1, wherein the mutant strain of yeast having ethanol fermentation ability under aerobic conditions is a mutant strain belonging to the genus Saccharomyces. The method for producing ethanol by fermentation according to claim 1 or 2, wherein the mutant strain of yeast having ethanol fermentation ability under aerobic conditions is Saccharomyces cerevisiae NF1-1 strain (FERM BP-8400). The method for producing ethanol by fermentation according to any one of claims 1 to 3, wherein fermentation under aerobic conditions is performed while supplying oxygen to the liquid fermentation raw material. The method for producing ethanol by fermentation according to claim 4, wherein oxygen is supplied to the liquid fermentation raw material by aeration to the liquid fermentation raw material and / or stirring of the liquid fermentation raw material. A method for producing ethanol by using the method for producing ethanol by fermentation according to any one of claims 1 to 5 to produce and accumulate ethanol by performing aerobic fermentation with a liquid fermentation raw material, and collecting the ethanol. Method. The method for producing ethanol according to claim 6, wherein starch or a saccharide fermentation raw material is used as the liquid fermentation raw material. The method for producing ethanol according to claim 6, wherein a waste biomass resource is used in the medium as a liquid fermentation raw material. 9. The method for producing ethanol according to claim 8, wherein tomato juice and / or oyster juice, or a saccharide fermentation raw material or waste biomass to which the fruit juice is added is used as a waste biomass resource. A method for producing a fermented alcoholic beverage, wherein the brewing raw material is aerobically fermented using the method for producing fermented ethanol according to any one of claims 1 to 5. The method for producing a fermented alcoholic beverage according to claim 10, wherein the fermented alcoholic beverage is a sake, a fermented malt beverage, a fruit liquor, or a distilled liquor. A method for separating and obtaining a yeast mutant having an ethanol fermentation ability under aerobic conditions, wherein the yeast is subjected to mutation treatment, and the mutation-treated yeast is purely separated using a separation medium. 13. Isolation and acquisition of a yeast mutant having ethanol fermentation ability under aerobic conditions according to claim 12, wherein ethyl methanesulfonate treatment is used as a mutation treatment for yeast, and a medium containing cycloheximide is used as a separation medium. Method. Selection of yeast having ethanol fermentation ability under aerobic conditions is carried out by further measuring the amount of carbon dioxide generated in the isolated yeast or in a stationary or shaking culture using a liquid medium. The method for separating and obtaining a yeast mutant having ethanol fermentation ability according to claim 12 or 13. Saccharomyces cerevisiae NF1-1 strain (FERM BP-8400) which has high ethanol fermentation ability under aerobic conditions and has the properties of foam-free yeast.