JP2013198407A - Method for breeding high-temperature-resistant ethanol-productive yeast, and method for producing fermented unrefined sake using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-temperature-resistant yeast strain capable of efficiently producing ethanol by fermentation even under temperature conditions at 35°C or higher, preferably at 38-40°C, to provide a method for breeding the same, and to provide a method for producing an ethanol-containing unrefined sake fermented product using such a strain.SOLUTION: The high-temperature-resistant variant yeast strain is produced by the following steps: a step (a) of conducting a fed-batch cultivation at 25°C or higher cultivation temperature of ethanol-fermentative yeast belonging to genus Saccharomyces to effect ethanol fermentation to produce fermented unrefined sake with ethanol concentration of 15% or higher; a step (b) of cultivating the yeast cell survived in the step (a) at 37°C or higher in an agar medium containing ≥10% ethanol and selecting the grown colony as a candidate strain; and a step (c) of cultivating the candidate strain selected in the step (b) in a liquid medium at 38-42°C followed by making a secondary selection based on the quantity of carbon dioxide production.

Description

  The present invention relates to a method for breeding a mutant strain that imparts high-temperature tolerance to yeast cells and is capable of producing ethanol even under high-temperature conditions, a method for producing fermentation mash containing ethanol using the same, and the like.

  Yeast is widely used for ethanol production including alcohol production and bioethanol production. In these, it is important to increase ethanol productivity under various conditions. Sake brewing in the liquor manufacturing field has been affected by a decline in raw material utilization due to high temperature failure of raw rice due to the impact of global warming in recent years and an increase in production costs due to cooling of strawberries because it is warm even in winter. In addition, in shochu brewing that is fermented at a relatively high temperature, measures for improving the ethanol yield are desired. Further, in the bioethanol production field, it is desired to increase the fermentation temperature to improve the raw material utilization rate and to reduce the cooling cost for cooling the saccharified liquid and removing the fermentation heat of the mash. Therefore, an ethanol-producing yeast having a strong fermenting ability under higher temperature conditions is desired.

  The method for obtaining yeast with high temperature tolerance is performed by selecting and isolating yeast with fermentability and high temperature tolerance from nature, and improving to high temperature resistant yeast by mutation treatment and gene recombination technology. A method for producing ethanol using the prepared yeast is disclosed. For example, it belongs to Saccharomyces cerevisiae, which has ferulic acid decarboxylation ability, excellent citric acid resistance, and high growth ability at high temperature (40 ° C) in an environment with the same citric acid concentration. A method for producing shochu and awamori using a new brewing yeast (Patent Document 1) and a yeast of the genus Kluyveromyces that express at least two types of cellulolytic enzymes are mixed with biomass and cultured at high temperature. Selected from a method of producing ethanol from cellulose (Patent Document 2), an ethanol-fermenting heterothalism yeast (Patent Document 3) having heat resistance and acid resistance obtained from ethanol-fermenting homothalism yeast, and a screening step. Zyomomonas mobilis that can efficiently produce ethanol under high temperature conditions of 35 ℃ or higher Ethanol production method using a field isolate of S. cerevisiae and Kluyveromyces which can efficiently produce ethanol under temperature conditions of 37 ° C or higher, preferably 40-49 ° C or higher An ethanol production method using a genus yeast (Patent Document 5) is known. However, these inventions pay attention only to high-temperature resistance, and many ethanol productivity is not high, and they did not provide an efficient method for obtaining yeast having both high-temperature resistance and high ethanol fermentation ability. Therefore, development of the efficient breeding technique of the yeast which has high temperature tolerance and fermentability which can be applied in the field of ethanol fermentation production technology and the ethanol fermentation production technique using the bred yeast has been desired.

JP 2006-67812 A JP 2011-160727 A JP 2009-171912 A JP 2009-60836 A Table 2008/062558

  Yeast is widely used in bioethanol production, alcoholic beverage production, etc., and mutants having various traits have been obtained depending on the purpose. In bioethanol production, raw materials are liquefied and saccharified at a high temperature (over 60 ° C), and then fermented mash containing ethanol is produced by ethanol-fermenting microorganisms, which are distilled to produce pure ethanol. ing. Therefore, for the purpose of reducing energy in each process, search and improvement of fermented microorganisms capable of ethanol fermentation even at high temperatures (about 40 ° C.) have been performed. In sake brewing, yeast strains with excellent ethanol resistance have been bred for the purpose of preventing the death of yeast in the late stage and suppressing the deterioration of the quality of sake. Furthermore, since shochu brewing has a relatively high fermentation temperature (about 25 to 32 ° C.), attempts have been made to acquire high-temperature resistant yeast for the purpose of improving ethanol yield.

  In addition, Saccharomyces yeasts have been conventionally used for ethanol fermentation production, and improvements have also been made. Recent improved technologies in the field of bioethanol production have been improved using genetic engineering techniques to enable simultaneous saccharification and fermentation using only yeast strains and to assimilate sugars that cannot be assimilated by Saccharomyces yeasts. . However, even if such improvements are made, the temperature range in which Saccharomyces yeast can efficiently undergo ethanol fermentation is around 30 ° C., and the saccharification reaction is often insufficient in this temperature range. In addition, in the fermentation production of ethanol in this temperature range, in order to keep the fermenter at an appropriate temperature, a cooling facility for removing fermentation heat is often required.

  An object of the present invention is to provide a high temperature resistant yeast strain capable of efficiently producing ethanol under a temperature condition of 35 ° C. or higher, preferably 38 ° C. to 40 ° C., a breeding method thereof, and such a high temperature resistant yeast strain. An object of the present invention is to provide a method for producing a fermented mash containing ethanol.

  The conventional method for obtaining a yeast strain having ethanol resistance is that the parent yeast strain is shake-cultured in a liquid medium at 25-30 ° C. for about 48 hours, and the obtained yeast cells are subjected to a mutation treatment or as it is. The yeast strain obtained by applying to an agar medium supplemented with ethanol and selecting from the grown colonies, or by culturing the parent yeast strain in a liquid medium at 25-30 ° C. for about 48 hours with shaking. A method has been used in which the body is immersed in a high-concentration (about 20%) ethanol treatment solution and allowed to stand at 4 ° C. for about one month to obtain surviving cells as ethanol-resistant yeast. In these methods, since the yeast cells to be treated are only subjected to simple fermentation, the ethanol concentration is a cell that has produced no more than about 10%, so that even though it has ethanol tolerance, it exceeds 15%. Concentration ethanol productivity could only be confirmed by a small preparation test using parallel double fermentation. In addition, since the fermentation temperature cannot be set extremely high in the small preparation test, it was very difficult to study ethanol productivity under high temperature conditions.

  In addition, the method of screening and isolating high-temperature resistant strains from the natural world requires examination of microorganisms of many genera and species, and for effective strains, it is necessary to clarify the genera and species, which is very complicated. In addition, it is necessary to study a method for efficiently producing ethanol, and it takes time to obtain useful ethanol-producing microorganisms.

  On the other hand, the present inventors constructed a fed-batch culture system for the purpose of developing a technology for producing high-concentration ethanol, and applied this fed-batch culture system to produce high-concentration (18% or more) ethanol in a short period (within 4 days). (See “Study on Development of High Concentration Continuous Production System for Ethanol” in 2007 Saga Industrial Technology Center Research Report). However, we hypothesized that it is effective to impart high-temperature tolerance to the yeast used for fermentation in order to efficiently produce higher concentrations of ethanol. In addition, the developed fed-batch culture system can be ethanol-fermented under various conditions, so it was considered that high-temperature resistant yeast can be efficiently bred using this system. Therefore, sake yeast (Association No. 901) was subjected to mutation treatment as a parent strain, and the treated cells were subjected to ethanol fermentation at 30 ° C. using a fed-batch culture system. The microbial cells that survived in the fermented mash were spread on an agar medium containing 15% ethanol, and colonies grown by culturing at 38, 39, 40, and 42 ° C. were obtained as high-temperature resistant strains. When the obtained high-temperature-resistant strain was subjected to a fermentation test at 30 ° C. using a fed-batch culture system, it was confirmed that the ethanol concentration at which mortality starts and the ultimate ethanol concentration were improved as compared with the parent strain. The same experiment was conducted at 39 ° C., and fermentation mash containing about 12% high concentration of ethanol could be obtained. Furthermore, the pre-culture conditions for enhancing the high-temperature resistance of yeast strains (sake mother's production conditions) were also examined, and when using a liquor obtained under specific pre-culture conditions, 10.7% ethanol even at a fermentation temperature of 40 ° C. From the fact that it can be produced, it has been found that it has excellent ethanol productivity and high temperature resistance at the same time. The present invention has been completed based on the above findings.

  As described above, in the present invention, the yeast cells that have been fermented to an ethanol concentration of 17% or more by using a fed-batch culture system (conditions for almost 100% killing) are subjected to high-temperature treatment and processed. Since yeast cells have experience in producing high-concentration ethanol, it is relatively easy to obtain mutant strains that have become resistant to high temperatures. In addition, since the fed-batch culture system can freely set the fermentation temperature, it is possible to easily study ethanol productivity under high temperature conditions.

  That is, the present invention provides [1] (a) ethanol-fermenting yeast belonging to the genus Saccharomyces by fed-batch culture at a culture temperature of 25 ° C. or higher, thereby fermenting ethanol to a fermented mash having an ethanol concentration of 15% or higher. (B) a step of culturing the yeast cells surviving in step (a) on an agar medium containing 10% or more of ethanol at 37 ° C. or higher and selecting the grown colonies as candidate strains; A method for preparing a high-temperature-resistant mutant yeast strain characterized by sequentially comprising a) and (b), and the candidate strain selected in (c) step (b) following step [b] A high-temperature-resistant mutant yeast strain according to [1], further comprising a step (c) of culturing in a medium at 38 to 42 ° C. and secondarily selecting based on a large amount of carbon dioxide generation. The production method of [ [3] The method for producing a high-temperature resistant mutant yeast strain according to [1] or [2], wherein the fed-batch culture is performed at a culture temperature of 30 ° C. or higher as ethanol fermentation in the step (a), [4] In the step (a), a fermentation mash of 17% or more is produced, and the method for producing a high-temperature resistant mutant yeast strain according to any one of the above [1] to [3], or [5] step (b) The method for producing a high-temperature tolerant mutant yeast strain according to any one of [1] to [4], wherein the culture is performed at 39 to 42 ° C. in an agar medium.

  Furthermore, the present invention provides [6] a high-temperature resistant mutant yeast strain obtained by the production method according to any one of [1] to [5], and [7] a Saccharomyces cerevisiae which is a high-temperature resistant mutant yeast strain. H2023-4 (NITE AP-1261) or [8] Manufacture of moromi fermented product characterized by performing ethanol fermentation by fed-batch culture using the high-temperature resistant mutant yeast strain described in [6] or [7] above [9] A method for producing a moromi fermented product according to [8] above, wherein ethanol fermentation is performed under a temperature condition of 38 ° C. to 40 ° C., and [10] a logarithmic growth phase at 15 to 25 ° C. [8] or [8], wherein a culture which is statically cultured from the first phase to the middle of the logarithmic growth phase and then shake-cultured at 28 to 32 ° C. from the late logarithmic phase to the early stationary phase is used as the preculture. 9] The method for producing the moromi fermented product described above, or [11] a culture cultured at 20-30 ° C. from the late logarithmic growth phase to the early stationary phase and then allowed to stand at 0-10 ° C. for 3-60 days The present invention relates to a method for producing a moromi fermented product according to [8] or [9], which is used as a product.

  The present invention relates to a method for obtaining a yeast strain that is excellent in ethanol productivity and at the same time has a high temperature resistance, and to the obtained high temperature resistant yeast, and a method for producing ethanol using the resistant yeast, and uses the high temperature resistant yeast of the present invention. As a result, the fermentation mash of each ethanol concentration of 18.8% in culture at 30 ° C for 84 hours, 12.0% in culture at 39 ° C for 47 hours, and 10.7% in culture at 40 ° C for 45 hours is obtained. Can be manufactured. Moreover, fermentation mash containing a higher concentration of ethanol can be obtained by pre-culturing under specific conditions to enhance the tolerance of yeast. As described above, the high-temperature resistant yeast of the present invention can efficiently produce ethanol under high temperature conditions such as 38 ° C. to 40 ° C. In the field of liquor production, the fermentation temperature is set high to improve the raw material utilization rate. In addition, in the field of ethanol fermentation production such as bioethanol production, the equipment for removing fermentation heat is downsized or unnecessary, so that the production capacity of fermentation production equipment is expected to be reduced. Simplification and the like are possible, and the equipment cost can be reduced and the efficiency of ethanol fermentation production can be improved.

It is the schematic of a fed-batch culture system. It is a figure which shows the fermentation course (30 degreeC) by the parent strain (association 901) and the high temperature tolerance yeast (H2023-4 strain) of this invention. It is a figure which shows the fermentation progress (39 degreeC) by the high temperature tolerance yeast (H2023-4 strain) of this invention. It is a figure which shows the fermentation progress (40 degreeC) by the difference in the preculture conditions using the high temperature tolerance yeast (H2023-4 strain) of this invention. It is a figure which shows the fermentation progress (40 degreeC) by the difference in the preculture conditions using a parent strain (association 901). It is a figure which shows the fermentation progress (40 degreeC) by the high temperature tolerance yeast (H2023-4 strain | stump | stock) of this invention using the liquor culture | cultivated by shaking at 30 degreeC for 24 hours. It is a figure which shows the fermentation process (40 degreeC) by the high temperature tolerance yeast (H2023-4 strain | stump | stock) of this invention using the liquor which carried out stationary culture at 15 degreeC for 60 hours, and was shake-cultured at 30 degreeC for 12 hours, and strengthened tolerance after that. is there.

  As a method for producing the high-temperature-resistant mutant yeast strain of the present invention, ethanol fermentation yeast belonging to the genus Saccharomyces is fed-batch cultured at a culture temperature of 25 ° C. or higher, and ethanol-fermented to give an ethanol concentration of 15% or higher. The step (a) for producing fermentation moromi and the step of culturing the yeast cells surviving in the step (a) at 37 ° C. or higher in an agar medium containing 10% or more of ethanol, and selecting the grown colonies as candidate strains (B) is not particularly limited as long as it is a method sequentially comprising, and the high-temperature resistant mutant yeast strain of the present invention is a high-temperature resistant mutant yeast strain obtained by the above-described method for producing a high-temperature resistant mutant yeast strain of the present invention. If it is, it will not be restrict | limited in particular, Specifically, high temperature tolerance mutant yeast strain H2023-4 (reception number: NITE AP-1261, receiving organization: independent administrative agency product evaluation) Surgery NITE Patent Microorganisms Depositary Center, receipt date: February 2012 27, 2007) can be mentioned. Furthermore, the method for producing the moromi fermented product of the present invention is not particularly limited as long as it is a method of performing ethanol fermentation by fed-batch culture using the high-temperature resistant mutant yeast strain of the present invention. Saccharomyces cerevisiae can be exemplified, and in particular, sake yeast used for sake brewing and shochu yeast used for shochu brewing can be preferably exemplified, and these yeasts have been subjected to mutation treatment in advance. It may be a stock.

  The step (a) in the method for producing a high-temperature resistant mutant yeast strain of the present invention is performed by subjecting an ethanol-fermenting yeast strain (parent strain) belonging to the genus Saccharomyces to which high-temperature resistance has been imparted (chemical, physical treatment), or Used as it is and fed-batch culture at a culture temperature of 25 ° C. or higher, preferably 30 ° C. to 32 ° C., to produce a fermented mash having an ethanol concentration of 15% or higher, preferably 17% to 18%. This is a process (most of the yeast grown in this process is killed), and the process (b) is the yeast that survived in the process (a), for example, the fermented moromi obtained, The body is sedimented, and the yeast cells are concentrated about 20 times by decantation, and 0.1 mL of the concentrated cells is 37 ° C. or higher, preferably 39 ° C. to 42 ° C. in an agar medium containing 10% or more of ethanol. In this step, the colonies grown and grown are selected as candidate strains. Further, the candidate strains selected in step (b) are cultured in a liquid medium at 38 to 42 ° C. It is preferable to include a step (c) for subsequent selection.

  Manufacture of fermented moromi and fed-batch culture in fermented moromi started with saccharides that can be assimilated and fermented by ethanol-fermenting yeast, or containing saccharides as raw materials, and adjusted to a sugar concentration that does not inhibit fermentation of ethanol-fermenting yeast A culture solution and a fed sugar solution having a sugar concentration higher than the sugar concentration of the starting culture solution are prepared, ethanol fermentation is performed by inoculating the starting culture solution with ethanol fermentation yeast, and the sugar concentration of the fermentation solution is directly or Add the fed sugar solution to the fermentation solution while monitoring indirectly, continue the ethanol fermentation while controlling the sugar concentration of the fermentation solution, and add the fed sugar solution within 30-80% of the total ethanol fermentation period It is done by ending.

  The sugar raw material used for ethanol fermentation is not particularly limited as long as it is a saccharide that can be assimilated and fermented by ethanol-fermenting yeast or a substance containing the saccharide. Specifically, glucose, fructose, maltose, sucrose, xylose , Galactose, cellobiose or starch saccharified solution, molasses, sugar cane, sugar beet juice, etc., particularly glucose and molasses can be preferably exemplified, and two or more of these can be used in combination. Moreover, various nutrient sources, such as a nitrogen source added to normal ethanol fermentation, can be appropriately added to the fermentation medium. The aeration and stirring conditions of the medium are preferably a slight oxygen condition in which ethanol fermentation proceeds more than respiration, for example, 0.02 to 0.1 vvm, and the stirring speed is high in a high-concentration sugar fed-batch medium. A speed that diffuses and does not damage the yeast cells, for example, 150 to 500 rpm is preferable.

  In order to prepare a starting culture solution using these sugar raw materials, it is important that the sugar concentration does not inhibit fermentation, although it varies depending on the yeast strain. Specifically, the sugar concentration is 50 to 300 g / L, Preferably, it is adjusted to 100 to 250 g / L, more preferably 120 to 220 g / L, and still more preferably 150 to 200 g / L.

  The sugar solution fed to the starting culture solution must have a sugar concentration and a fed-batch amount that can achieve the target ethanol concentration, and the sugar concentration must be adjusted to a sugar concentration higher than the sugar concentration of the starting culture solution. There is. Specifically, the sugar concentration can be adjusted to 300 to 800 g / L, preferably 400 to 700 g / L, more preferably 400 to 600 g / L. On the other hand, about the amount of the fed-batch liquid, if the amount of the fed-batch medium is too large, the fermentation period becomes longer because it is diluted, and if the amount is too small, the concentration of the fed-batch medium increases too much and the diffusion becomes worse. 0.2 to 1.2 times, preferably 0.4 to 0.9 times, more preferably 0.5 to 0.7 times the amount of the starting culture solution, and flow from the sugar concentration and the amount of the starting culture solution. It is necessary to determine the sugar concentration and amount of the sugar solution to be added. At this time, the average sugar concentration needs to be 300 to 320 g / L. For example, when the starting culture solution is 1 L of 200 g / L sugar solution, the sugar concentration when fed sugar concentration is 500 g / L. The liquid is about 530 to 660 mL.

  Producing high-concentration ethanol when the addition of the fed sugar solution is completed within 30 to 80% of the total period of ethanol fermentation, preferably within 35 to 70%, and more preferably within 40 to 60% Can do. If the addition of the fed sugar solution is less than 30% of the total period of ethanol fermentation, the yeast cells are inhibited from fermentation by high sugar pressure, so that ethanol fermentation is not sufficiently performed and high concentration ethanol can be obtained. In addition, if the ethanol fermentation rate is reduced to 70% of the total period of ethanol fermentation, the fed-batch medium is supplied when the ethanol fermentation rate is reduced, and the produced ethanol is diluted by the fed-batch medium. The ethanol concentration is lowered, and it takes a long time to obtain a predetermined high concentration ethanol. Therefore, up to 80% of the total period of ethanol fermentation is the upper limit of the addition end time of the fed sugar solution.

  As a method for monitoring the sugar concentration of the fermentation broth, a direct or indirect method may be used, and a direct method may include a method using a biosensor, and an indirect method includes fermentation. For the sugar concentration of the liquid, a method using a refractometer such as a refractive Brix meter, a vibrating densimeter, or a viscometer can be employed. These devices can adopt a flow cell system or a system installed directly in the fermenter so that the sugar concentration can be continuously monitored during the fermentation period. Various control methods such as on / off control, PID control, and PI control can be used as a control method for keeping the sugar concentration constant by feeding the sugar solution. In the present invention, it is preferable to use a refractive Brix meter to easily control the sugar concentration of the fermentation broth. The refraction type Brix meter is calibrated with a sucrose solution whose concentration is known in advance, and the Brix value indicates the percent concentration of soluble solids contained in the aqueous solution. Soluble solids are all substances that are soluble in water, such as sugar, salts, proteins, acids, etc., and the measured value is the sum of those values. Therefore, the method using this refractive Brix meter is consistent with the glucose concentration at the start of ethanol fermentation, but is detected as a value higher than the glucose concentration as the ethanol fermentation proceeds and the ethanol concentration rises. It can be advantageously used because it is easy to use and maintain.

  The high-concentration ethanol produced by the method for producing a moromi fermented product according to the present invention can be used as an ethanol beverage, through distillation treatment, as an alternative energy to replace car gasoline or as industrial ethanol such as petrochemical raw materials.

  As a fed-batch culture system used for fed-batch culture, a fermenter, a fed-batch sugar solution storage tank, an aeration unit that ventilates the fermented liquid in the fermenter, an agitation unit that stirs the fermented liquid in the fermenter, If it is a device equipped with a control means for feeding the fed sugar solution from the fed sugar solution storage tank to the fermentation tank based on a signal from the sensing means for directly or indirectly monitoring the sugar concentration of the fermentation liquid, a signal from the sensing means For example, the apparatus shown in FIG. 1 can be specifically mentioned. In FIG. 1, 1 is a fed sugar solution storage tank, 2 is a flow meter, 3 is a fermenter, 4 is a relay unit, 5 is A flow cell type refractometer, 6 is a control computer, 7 and 8 are liquid feeding pumps, 9 is an air pump, and 10 is a stirring means in the fermenter.

  The fermenter 3 is fermented to a flow unit of a flow means of a refraction type saccharimeter 5 of a flow cell type, an aeration means for inflowing a predetermined amount of oxygen measured by an air pump 9 with a flow meter 2, an agitation means 10 for agitating the fermentation liquid It is equipped with a detecting means for detecting the sugar content by inflow and outflow of liquid, and a feeding means for feeding a sugar solution having a higher concentration than the fed sugar solution storage tank to the fermentation tank, both via a pump The high-concentration sugar solution and the sugar solution used for detection means are flowed in and out. The amount of oxygen aerated in the fermenter is such an amount that it is in a slightly oxygen state although it varies depending on the type of yeast, the state of the yeast, the type of medium, the fermentation temperature, and the like. Stirring is carried out by rotating a stirrer having a stirring blade by a drive device, but it varies depending on the type of yeast, the state of the yeast, the sugar concentration (viscosity) of the fed sugar solution, etc., as well as the amount of oxygen, but usually around 250 rpm is preferred.

  First, a fermentable sugar solution (starting culture solution) having a predetermined concentration is charged into the fermenter 3, and after further yeast is charged, fermentation is started while gently stirring with the stirring means 10 while maintaining the temperature at a predetermined temperature. The relay unit 4 is operated by the control computer 6 while the sugar concentration measured by the flow cell type refractometer 5 is monitored over the entire fermentation period, and the power supply of the liquid feed pump 8 is turned on. By turning OFF, the sugar solution is appropriately fed through the pump 8 from the fed sugar solution storage tank 1 in which a high concentration sugar solution higher than the sugar concentration of the starting culture solution is accommodated. For example, in the flow cell type refractometer 5, the measurement is performed at a predetermined interval with time, but the measurement can be performed more accurately when the interval is, for example, 10 seconds or even shorter. Thus, it is possible to always measure an accurate sugar concentration, and based on the measured value, it is possible to finish feeding a desired sugar solution, maintaining the sugar concentration, or feeding the sugar solution. After the fermentation for a predetermined time, the feeding of the high-concentration sugar solution is terminated, and the fermentation is continued thereafter, and the fermentation is terminated by checking the production of ethanol.

  The method for producing a moromi fermented product of the present invention is a method of performing ethanol fermentation by fed-batch culture using the high-temperature-resistant mutant yeast strain of the present invention, and the culture temperature is not particularly limited, but is 30 ° C or higher, preferably 35 ° C. As described above, it is particularly preferable to perform ethanol fermentation by fed-batch culture under a temperature condition of 38 ° C to 40 ° C. For fed-batch culture, it is preferable to use a liquor mother (preculture) that has been subjected to a resistance enhancement treatment under preculture conditions. In the production of such a liquor, the culture temperature is set at 15 to 25 ° C., the medium is cultured until the middle of the logarithmic growth phase, preferably the first phase of the logarithmic growth phase, and then at 30 ° C. for 6 to 12 hours in the early stationary phase, preferably the logarithmic growth phase. Culturing until late, or after culturing until 20-30 ° C. at the initial stationary phase, preferably late in the logarithmic growth phase, preferably at 0-10 ° C., preferably 0-6 ° C. for 3-60 days, preferably Can enhance the tolerance of yeast by treating for 5 to 30 days. The preculture obtained in this manner can also be subjected to ethanol fermentation in step (a) of the method for producing a high-temperature resistant mutant yeast strain of the present invention. Such enhanced resistance improves ethanol productivity during high-temperature fermentation, and can enhance high-temperature resistance and ethanol resistance.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations. In the following examples, a Brix meter modified to a flow cell type (Atago Co., RX-5000) was used for measuring Brix, and a gas chromatograph (manufactured by Shimadzu Corporation) was used for measuring the ethanol concentration. Using GC9A, FID detector), the absolute density curve method by the total volume injection method was used, and the bacterial cell density was collected in a micro test tube in which 1 mL of the culture solution was previously weighed, and centrifuged at 15000 rpm for 3 minutes. After removing the supernatant, the wet weight was measured, and the previous empty weight was subtracted.

[Acquisition of yeast high temperature resistant mutants]
Association 901 yeast which is sake yeast is slurried 1 platinum from slant, glucose concentration is changed to 15% MY15 medium (yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, glucose 15% ) Inoculate 7.5 mL, shake culture at 30 ° C. for 18 to 36 hours, inoculate 0.1 mL each of 3 7.5 mL of new MY15 medium, and culture at 28 ° C. for 18 hours with shaking . Three of these cultures are added to 900 mL of 2MY15 medium (0.6% yeast extract, 0.6% malt extract, 1.0% peptone, 15% glucose), and the culture temperature is 28 using a fed-batch culture system. Fermented at 5 ° C. From the stage when Brix decreased by 1.0 after the start of culture, 600 mL of 1 / 2MY55 medium (yeast extract 0.15%, malt extract 0.15%, peptone 0.25%, glucose 55%) was kept at a constant Brix value. Fermentation was continued until the Brix value of the medium did not decrease. After completion of fermentation, the ethanol concentration of the fermentation mash is measured to confirm that the ethanol concentration has reached 17% or more (17.8%), and the produced fermentation mash is transferred to another container at 4 ° C for 1 week. The floating cells were allowed to settle and settle. After the bacterial cells settled, the supernatant was transferred to another container by decantation, and the bacterial cell suspension was concentrated. MY plate containing 10% ethanol (0.1% concentrated cell suspension, yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, glucose 1.0%, agar 2.0) %) And cultured at 39 ° C., 40 ° C., and 42 ° C. to grow colonies, which were obtained as candidate strains for high-temperature resistant mutants. The obtained candidate strain was inoculated into 7.5 mL of 2MY20 medium (0.6% yeast extract, 0.6% malt extract, 1.0% peptone, 20% glucose) at 38 ° C, 39 ° C, 40 ° C, The amount of carbon dioxide generation at 41 ° C. and 42 ° C. was measured, and a strain having a large amount of carbon dioxide generation at the highest temperature was obtained as a high-temperature resistant mutant (H2023-4 strain).

[Fermentation at 30 ° C]
Take H2023-4 strain from slant with 1 platinum ear and inoculate it into 10 mL of MY20 medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 20% glucose) at 25 ° C. The shaking culture was performed for about 60 hours. In addition, 0.1 mL of this culture solution was inoculated on 3 10 mL of 2MY20 medium, shaken at 30 ° C. for 12 hours, added to 900 mL of 2MY15 medium, and the culture temperature was adjusted to 30 ° C. using a fed-batch culture system. Fermented. From the stage when the Brix decreased by 1.0 after the start of the culture, 600 mL of 1 / 2MY55 medium was fed while maintaining the Brix value constant, and the fermentation was continued until the Brix value of the medium did not decrease. The result is shown in FIG.

  As shown in FIG. 2, it can be seen that the fermentation mash containing about 19% ethanol can be produced in 84 hours when the H2023-4 strain is used. In addition, when the ethanol concentration reaches about 16% in the parent strain, the cell density begins to decrease, whereas in the H2023-4 strain, there is almost no decrease in cell density up to about 18%. It can be seen that the high temperature resistance is improved.

[Fermentation at 39 ° C]
Ethanol productivity at 39 ° C. of strain H2023-4 was confirmed by the same method as in Example 2. As in the case of 30 ° C., 900 mL of 2MY15 medium was used as the starting medium, and 150 mL of 1 / 2MY50 medium (yeast extract 0.15%, malt extract 0.15%, peptone 0.25%, glucose 50 %) Was used. The result is shown in FIG.

  As shown in FIG. 3, it was possible to produce a fermented mash with an ethanol concentration of 12% in 47 hours even in a high temperature environment such as 39 ° C.

[Influence of pre-culture conditions (high temperature resistant mutant)]
When the fermentation test was performed in a high-temperature environment, ethanol productivity sometimes decreased. Considering the cause, it was considered that preculture conditions had an effect. Therefore, pre-culture conditions in 2MY20 medium were examined using the high-temperature resistant H2023-4 strain. As pre-culture, (1) static culture at 30 ° C. for 72 hours, (2) 0.1 mL of culture solution static culture at 30 ° C. for 60 hours, inoculated into 7.5 mL, and shaking culture at 30 ° C. for 12 hours (3) a stationary culture at 30 ° C. for 60 hours, and then a static culture at 4 ° C. for 12 hours, (4) a shaking culture at 30 ° C. for 24 hours, and a culture solution that is allowed to stand at 4 ° C. for 72 hours, 0.1 mL 7.5 mL, inoculated with shaking at 30 ° C. for 12 hours, (5) incubated with shaking at 30 ° C. for 24 hours, and then allowed to stand at 4 ° C. for 72 hours, (6) shaking cultured at 30 ° C. for 14 hours Then, 7.5 mL of 0.1 mL of the culture solution that was allowed to stand at 4 ° C. for 82 hours was inoculated and shake-cultured at 30 ° C. for 12 hours. (7) After shaking culture at 30 ° C. for 14 hours, 82 hours at 4 ° C. (8) 0.1 mL of the culture solution that was allowed to stand for 96 hours at 15 ° C in contact with 7.5 mL And (9) a stationary culture at 15 ° C. for 60 hours, followed by a shaking culture at 30 ° C. for 12 hours, (10) a stationary culture at 15 ° C. for 108 hours, 11) A static culture at 15 ° C. for 60 hours, followed by a shaking culture at 30 ° C. for 12 hours, and then a static culture at 4 ° C. for 45 days. (12) A static culture at 15 ° C. for 60 hours and then 12 hours at 30 ° C. After shaking culture and then inoculating 7.5 mL of 0.1 mL of the culture that was allowed to stand at 4 ° C. for 45 days and then incubating for 12 hours at 30 ° C. The amount of carbon dioxide generated at 40 ° C. was measured over time. The result is shown in FIG.

  As shown in FIG. 4, it can be seen that the amount of carbon dioxide gas generated (ethanol productivity) changes by changing the pre-culture conditions. Therefore, in order to efficiently produce ethanol by fermentation under high temperature conditions, a resistance enhancement process according to the pre-culture conditions (5), (7) and (9) is required.

[Influence of pre-culture conditions (parent strain)]
In order to confirm whether the effect of Example 4 is a phenomenon characteristic only to resistant strains, the parent strain association 901 yeast was also confirmed. The preculture conditions and the like were the same as in Example 4 (9). The result is shown in FIG.

  As shown in FIG. 5, it was found that the parent strain has the same effect. Moreover, the effect was higher than that of the resistant mutant. Therefore, it can be said that the resistance-enhancement treatment under pre-culture conditions is effective for a less resistant strain.

[Confirmation of effect of resistance enhancement treatment (control)]
In order to confirm the effect of the resistance enhancement treatment, a fermentation test using a fed-batch culture system was performed. The test conditions were the same as in Example 3 except that the fermentation temperature was changed to 40 ° C. First, as a control, preculture conditions were set to a constant temperature of 30 ° C. for 24 hours. That is, using a high temperature resistant strain H2023-4, 30 mL of a culture solution obtained by shaking culture in 2MY20 medium at 30 ° C. for 24 hours was added to 900 mL of 2MY15 medium as a starting medium, and 150 mL of 1 / 2MY50 medium was used as a fed-batch medium. It was. The result is shown in FIG.

  Under these pre-culture conditions, the cell density did not increase so much, and it was considered that the cells were damaged by the high temperature. Therefore, even after 48 hours, the ethanol concentration of the fermentation mash was as low as about 9%.

[Confirmation of resistance enhancement treatment effect]
Next, a fermentation test was performed under the same conditions as in Example 6 using a culture solution prepared according to pre-cultivation conditions according to (9) of Example 4. That is, using a high-temperature resistant strain H2023-4, 30 mL of a culture solution that was statically cultured at 15 ° C. for 60 hours in 2MY20 medium and then shake-cultured at 30 ° C. for 12 hours was added to 900 mL of 2MY15 medium as a starting medium. 150 mL of 1 / 2MY50 medium was used as the supplement medium. The result is shown in FIG.

  Unlike Example 6, the cell density increased and it was found that there was little damage to the cells. Therefore, fermentation mash with an ethanol concentration exceeding 10% was obtained in 45 hours.

DESCRIPTION OF SYMBOLS 1 Feeding sugar liquid storage tank 2 Flowmeter 3 Fermenter 4 Relay unit 5 Flow cell type refractive sugar meter 6 Control computer 7 Liquid feeding pump 8 Liquid feeding pump 9 Air pump 10 Stirring means

Claims (11)

A method for producing a high-temperature resistant mutant yeast strain comprising the following steps (a) and (b) in sequence.
(A) a step of subjecting an ethanol-fermenting yeast belonging to the genus Saccharomyces to fed-batch culture at a culture temperature of 25 ° C. or higher to produce ethanol fermented moromi having an ethanol concentration of 15% or higher;
(B) a step of culturing the yeast cells surviving in step (a) on an agar medium containing 10% or more of ethanol at 37 ° C. or higher and selecting the grown colonies as candidate strains; The method for producing a high-temperature resistant mutant yeast strain according to claim 1, further comprising the following step (c) following step (b).
(C) a step of culturing the candidate strain selected in the step (b) at 38 to 42 ° C. in a liquid medium and performing a secondary selection based on the amount of carbon dioxide generation;   The method for producing a high-temperature tolerant mutant yeast strain according to claim 1 or 2, wherein fed-batch culture is performed at a culture temperature of 30 ° C or higher as ethanol fermentation in the step (a).   The method for producing a high-temperature resistant mutant yeast strain according to any one of claims 1 to 3, wherein in the step (a), 17% or more of the fermented mash is produced.   The method for producing a high-temperature resistant mutant yeast strain according to any one of claims 1 to 4, wherein in the step (b), the culture is performed at 39 to 42 ° C on an agar medium.   A high-temperature resistant mutant yeast strain obtained by the production method according to claim 1.   Saccharomyces cerevisiae H2023-4 (NITE AP-1261), a high-temperature resistant mutant yeast strain.   A method for producing a moromi fermented product, wherein ethanol fermentation is performed by fed-batch culture using the high-temperature resistant mutant yeast strain according to claim 6 or 7.   The method for producing a moromi fermented product according to claim 8, wherein ethanol fermentation is performed under a temperature condition of 38 to 40 ° C.   It is characterized by using as a pre-culture a culture which is statically cultured at 15 to 25 ° C. from the early log growth phase to the middle log growth phase and then shaken at 28 to 32 ° C. from the late log growth phase to the early stationary phase. A method for producing a moromi fermented product according to claim 8 or 9.   10. A culture obtained by shaking culture at 20-30 ° C. from the late logarithmic growth phase to the early stationary phase and then standing at 0-10 ° C. for 3-60 days is used as a preculture. The manufacturing method of the moromi fermented product of description.