JP2013169198A - Sake yeast and method for producing alcoholic drink or food using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide sake yeast retaining high fermentability while having high stress resistance, and a method for producing alcoholic drinks or foods characterized by using the sake yeast.SOLUTION: There are provided sake yeast transformed with a recombinant vector expressing RIM15 gene, and a method for producing alcoholic drinks or foods characterized by using the sake yeast.

Description

  The present invention relates to a sake yeast that has acquired excellent resistance to stress while maintaining high fermentative power by expressing a RIM15 gene that enhances stress tolerance, and a method for producing alcoholic beverages or foods using the sake yeast. is there.

  Currently, most sake breweries throughout the country produce sake using “Kyokai Yeast” sold by the Japan Brewing Association. A common feature of the currently used yeast is that the sake fermentation rate is high in sake moromi and the final ethanol yield is also high. This feature makes it possible to efficiently produce sake that is known to have a relatively high alcohol content among all brewed sake.

  On the other hand, compared to laboratory yeast, it was confirmed that the yeast was weak against environmental changes such as heat shock and ethanol stress, and that many cells were killed even at the end of the sake mash with high ethanol concentration (non-patented). Reference 1). It is known that the death of yeast and accompanying autolysis leads to an increase in the chromaticity and amino acid content of sake and significantly impairs the quality of sake. For this reason, improvement of the stress tolerance of the yeast is strongly desired.

  In recent years, the development of multi-acid sake yeast has been widely conducted with the aim of diversifying the quality of sake. However, with regard to the production of malic acid that has a relatively refreshing acidity among organic acids, It has been reported that there is a relationship (Non-Patent Document 2). Specifically, since the expression of stress response-related genes was commonly observed in several malic acid high-producing sake yeast strains, the high malic acid-producing strains may have acquired stress tolerance. It was suggested. From this, it is considered that the improvement of the stress tolerance of the yeast can contribute to the quality improvement of sake.

  Saccharomyces cerevisiae grows and vigorously consumes nutrients in the environment in an environment suitable for fermentation and increases the number of cells, but when nutrients are depleted and the environment becomes unsuitable for growth, it is detected. Signal transduction pathways for growth inhibition and stress response work quickly and shift to a state called stationary phase with low metabolic activity. The stationary phase transition regulator Rim15p plays a central role in this signal transduction pathway (see Non-Patent Document 3).

  On the other hand, the present inventors have identified a novel mutation that deletes the C-terminal of Rim15p and deletes the structure and function of the factor in the currently used yeast (see Non-Patent Document 4). . From this, it was considered that in the yeast, high metabolic activity could be maintained due to the difficulty of shifting to the stationary phase even if the ethanol concentration in sake mash is increased. Therefore, when the stationary phase transition regulator gene (RIM15) was introduced into the yeast to improve stress tolerance, it was expected that the high ethanol-producing ability of the yeast was reduced.

H. Urbanczyk, C. Noguchi, H. Wu, D. Watanabe, T. Akao, H. Takagi, and H. Shimoi; J. Biosci. Bioeng. 112, 44-48 (2011) T. Oba, H. Suenaga, S. Nakayama, S. Mitsuiki, H. Kitagaki, K. Tashiro, and S. Kuhara; Biosci. Biotechnol. Biochem. 75, 2025-2029 (2011) E. Swinnen, V. Wanke, J. Roosen, B. Smets, F. Dubouloz, I. Pedruzzi, E. Cameroni, C. De Virgilio, and J. Winderickx; Cell Dev. 1, 3 (2006) Abstracts of the 63rd Annual Meeting of the Biotechnology Society of Japan, published on August 25, 2011, published by the Japan Society for Biotechnology, page 64, 1Jp12

  Then, the subject of this invention is providing the manufacturing method of alcoholic beverage or fermented food characterized by using sake yeast which has high stress tolerance while maintaining high fermentability, and using such yeast.

  The inventors of the present invention have revealed that the ethanol fermentation rate is remarkably improved when the RIM15 gene is disrupted in laboratory yeast having a normal structure of Rim15p (Reference Examples 1 and 2 described later). Therefore, it was speculated that the loss-of-function mutations in the RIM15 gene of Japanese yeast would greatly contribute to the high fermentability of the yeast.

  On the other hand, from the conventional knowledge, it was considered necessary to restore the function of Rim15p that is deficient in the yeast to enhance the stress tolerance of the yeast. In order to prove this, when a transformed yeast was prepared by introducing the RIM15 gene derived from a laboratory yeast having a normal structure into the parent yeast strain, the transformed yeast was significantly higher than the parent strain. It was found to show stress tolerance.

  Furthermore, as a result of conducting a fermentation test of such transformed yeast, surprisingly, the transformed yeast showed a fermentation rate almost equal to that of the parent strain. As suggested in Reference Examples 1 and 2 below, the lack of Rim15p is perfectly consistent with yeast stress tolerance reduction and fermentability improvement, so the introduction of RIM15 gene can predict a reduction in fermentation rate. It was. Nevertheless, the fact that the fermentation rate does not decrease even when the RIM15 gene is restored is a phenomenon that has been found for the first time by analysis using a yeast strain. Therefore, the present inventors consider that the phenomenon can be used to improve stress tolerance while maintaining the high fermentability of the yeast, and more detailed results of the fermentation test using the transformed yeast. By analyzing the above, the present invention has been completed.

That is, the gist of the present invention is as follows.
[1] Sake yeast transformed with a recombinant vector expressing RIM15 gene;
[2] The sake yeast according to [1], wherein the sake yeast is Saccharomyces cerevisiae K701 UT-1T [pAUR112-ScRIM15] (reception number NITE AP-1219);
[3] A method for producing alcoholic beverages or foods using the sake yeast according to [1] or [2]; and [4] alcoholic beverages or foods are sake, shochu, beer, wine, breads And the production method according to [3] above, which is at least one selected from the group consisting of soy sauce and soy sauce.

  Since the transformed yeast of the present invention has a characteristic of high stress tolerance, the use of this can suppress the death of the yeast during the production of alcoholic beverages or fermented foods. As a result, it becomes possible not only to produce alcoholic beverages or fermented foods more efficiently, but also to provide a method for producing alcoholic beverages or fermented foods characterized by taste ingredients including amino acids and organic acids. Become.

FIG. 1 is a graph showing carbon dioxide generation rate (left) and total carbon dioxide generation amount (right) when sake production is performed using the parent strain laboratory yeast BY4743 strain and gene disruption strain (BY4743 Δrim15). is there. The dotted line shows the parent strain, and the solid line shows the RIM15 gene disrupted strain. FIG. 2 is a graph showing the carbon dioxide generation rate (left) and the total carbon dioxide generation amount (right) when an ethanol fermentation test was conducted using the parent strain laboratory yeast BY4743 strain and gene disruption strain (BY4743 Δrim15). It is. The dotted line shows the parent strain, and the solid line shows the RIM15 gene disrupted strain. FIG. 3 is a diagram showing the results of a stress tolerance test using sake yeast (K701 UT-1T [pAUR112]) and a transformed strain (K701 UT-1T [pAUR112-ScRIM15]) as control strains. The dotted line shows the data of the control strain and the solid line shows the data of the transformed strain. Fig. 4 shows the carbon dioxide generation rate (left) when sake was produced using the sake yeast (K701 UT-1T [pAUR112]) and the transformed strain (K701 UT-1T [pAUR112-ScRIM15]) as control strains. ) And the total amount of carbon dioxide generated (right). The dotted line shows the data of the control strain and the solid line shows the data of the transformed strain.

  Hereinafter, the present invention will be specifically described. The liquor or food of the present invention is not limited, but examples of the liquor include sake, shochu, beer, wine and the like. Examples of foods include breads and soy sauce.

  As the yeast used for transformation in the present invention, sake yeast belonging to Saccharomyces cerevisiae is desirable. Specific examples include “Kyokai Yeast” (Kyokai No. 7, Kyokai No. 701, Kyokai No. 9, etc.) distributed by the Japan Brewing Association.

  As a gene cloning method, a known gene cloning method in yeast, for example, a method of amplifying a target gene using a PCR method, a method of performing shotgun cloning, etc. may be appropriately selected and used. As an effective gene cloning method, The PCR method is preferred.

  The sequence of the RIM15 gene is described on the SGD (Saccharomyces Genome Database) homepage (http://genome-www.stanford.edu/Saccharomyces). A target gene can be obtained by PCR using yeast genomic DNA having the RIM15 gene structure as a template. Examples of yeast having a normal RIM15 gene structure include laboratory yeasts (X2180, BY4743, etc.) belonging to Saccharomyces cerevisiae. Examples of DNA primers used for amplifying the RIM15 gene include RIM15-F (SEQ ID NO: 1 in the sequence listing), RIM15-R (SEQ ID NO: 2 in the sequence listing), and the like.

  The RIM15 gene amplified by the method described above can be connected to an expression vector and introduced into yeast. As the expression vector, any of a single copy type, a multi copy type, and a chromosomal integration type can be used. In addition, as a selection marker used at the time of transformation, an auxotrophic marker such as an amino acid or a resistance marker for a drug can be used. A specific example of an effective expression vector is pAUR112 (manufactured by Takara Bio Inc.). A known method can be adopted as a method for preparing a recombinant vector having the RIM15 gene using such an expression vector. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include a lithium acetate method, an electroporation method, and a spheroplast method.

  An example of sake yeast transformed with a recombinant vector that expresses the RIM15 gene is the Saccharomyces cerevisiae K701 UT-1T [pAUR112-ScRIM15] strain. This strain is named K701 UT-1T [pAUR112-ScRIM15], and received the NITE AP-1219 (Receipt date: February 2, 2012) at the National Institute of Technology and Evaluation for Microorganisms. It is deposited as (currently in the process of obtaining the deposit number).

  The method for producing an alcoholic beverage or food of the present invention can be carried out by employing the sake yeast of the present invention as yeast in the production process using yeast.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited at all by these Examples.

Reference example 1
Sake production test using Saccharomyces cerevisiae BY4743 strain and RIM15 gene-disrupted strain which was the parent strain was conducted. The BY4743 strain and BY4743 Δrim15 strain are both available from EUROSCARF as Y20000 and Y37281, respectively.

  Sake was produced using the parent strains BY4743 and BY4743 Δrim15 in the following manner.

One-stage charging was performed by mixing 40 g of rice, 10 g of glutinous rice, 80 mL of water, and 17.8 μL of 90% lactic acid. Alpha rice with a 70% polishing rate was used as the hanging rice, and dried rice with a 70% polishing rate was used as the polished rice. Each yeast is cultured overnight in a YPD medium (contains 1% yeast extract, 2% peptone, 2% glucose) and then washed with sterilized distilled water so that the yeast count becomes 1 × 10 ⁷ cells / mL. Was added at the time of charging. The fermentation temperature was 15 ° C. The preparation test was repeated four times for each strain. After the preparation, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The results are shown in FIG.

  For the peak value of the carbon dioxide generation rate and the total amount of carbon dioxide generation, a significant difference test was also performed between the parent strain and the gene-disrupted strain. Furthermore, on the 20th day after preparation, the ethanol concentration in sake recovered by centrifugation was measured, and the average ethanol concentration was calculated for each strain. In addition, the obtained average ethanol concentration was also subjected to a significant difference test between the parent strain and the gene-disrupted strain. The ethanol concentration was measured using a gas chromatograph GC-17A manufactured by Shimadzu Corporation.

  RIM15 gene-disrupted strain has a higher peak carbon dioxide generation rate during fermentation than the parent strain (parent strain: 14.7 ± 0.8mg / 30min, RIM15 gene-disrupted strain: 20.5 ± 0.7mg / 30min, risk rate less than 0.1% And significantly larger than the parent strain), and the total amount of carbon dioxide generation was also large (parent strain: 5.60 ± 0.00 g, RIM15 gene disruption strain: 10.07 ± 0.24 g, significantly higher than the parent strain with a risk rate of less than 0.1%). Furthermore, the final ethanol concentration is 11.2 ± 0.3% by volume for the parent strain, whereas the RIM15 gene disruption strain is significantly higher at 17.0 ± 0.2% by volume (significantly higher than the parent strain at a risk rate of less than 0.1%). From the results, it was found that when the RIM15 gene disrupted strain was used, the ethanol fermentation rate was fast and the ethanol productivity was excellent.

Reference example 2
An ethanol fermentation test was performed using the above Saccharomyces cerevisiae BY4743 strain and BY4743 Δrim15 strain.

An ethanol fermentation test using 50 mL of a high-concentration glucose-containing YPD medium (containing 1% yeast extract, 2% peptone, and 20% glucose) was performed. Each yeast was cultured in a YPD medium with shaking overnight, and then added to a high-concentration glucose-containing YPD medium so that the yeast density was OD ₆₆₀ = 0.1 / mL, followed by stationary culture for 5 days. The fermentation temperature was 30 ° C. The fermentation test was repeated four times for each strain. After the start of culture, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The results are shown in FIG. The peak value of carbon dioxide generation rate and the total amount of carbon dioxide generation were also tested for significant differences between the parent strain and the gene-disrupted strain.

  RIM15 gene-disrupted strain has a higher peak carbon dioxide generation rate during fermentation than the parent strain (parent strain: 11.3 ± 1.5mg / 15min, RIM15 gene-disrupted strain: 16.3 ± 2.7mg / 15min, less than 5% risk rate Because the total amount of carbon dioxide generated is significantly larger than the parent strain (parent strain: 2.74 ± 0.13g, RIM15 gene disruption strain: 3.23 ± 0.21g, significantly larger than the parent strain with a risk rate of less than 0.1%), When the RIM15 gene disrupted strain was used, it was found that the ethanol fermentation rate was fast and the ethanol productivity was excellent.

Example 1
[Create transformants]
In order to obtain a PCR product containing 811 bp upstream of the RIM15 gene, 5313 bp coding region of the RIM15 gene and 500 bp downstream of the RIM15 gene, primers RIM15-F (SEQ ID NO: 1 in the sequence listing), RIM15-R (SEQ ID NO: 2 in the sequence listing) ) Designed. The software for primer design used was the one attached to the SGD (Saccharomyces Genome Database) website (http://genome-www.stanford.edu/Saccharomyces).

  Genomic DNA was extracted from laboratory yeast X2180, and DNA amplification was performed by PCR using primers RIM15-F (SEQ ID NO: 1 in the sequence listing) and RIM15-R (SEQ ID NO: 2 in the sequence listing). It was cloned into the SmaI site of the pAUR112 vector. The obtained recombinant vector was introduced into a uracil-requiring strain (K701 UT-1T strain) derived from sake yeast Kyokai No. 701 by the lithium acetate method. Culturing on SC-Ura agar medium (0.67% yeast nitrogen base without amino acids, 0.77 g / L complete supplement mixture minus uracil, containing 2% glucose) at 30 ° C for 3 days. The cells were re-cultured on SC-Ura agar and grown to obtain transformants. This strain is named K701 UT-1T [pAUR112-ScRIM15], and received the NITE AP-1219 (Receipt date: February 2, 2012) at the National Institute of Technology and Evaluation for Microorganisms. Deposited as (currently in the process of obtaining the deposit number). In addition, a control strain into which an empty vector was introduced was also prepared by the same method. This strain was named K701 UT-1T [pAUR112] and received the NITE AP-1218 (Receipt date: February 2, 2012) at the National Institute of Technology and Evaluation for Microorganisms (Receipt date: February 2, 2012) (currently Deposited as deposit number).

[Stress tolerance test]
A stress tolerance test was performed using the K701 UT-1T [pAUR112] and K701 UT-1T [pAUR112-ScRIM15] strains described above. Each yeast is cultured overnight in SC-Ura medium and then added to YPD medium (yeast extract 1%, peptone 2%, glucose 2%) so that the yeast density is OD ₆₆₀ = 0.1 / mL. Then, stationary phase cells were obtained by shaking culture for 1 week. The culture temperature was 25 ° C. The cells were transferred to a 54 ° C. water bath and subjected to heat shock, and sampling was performed immediately (0 minutes), 15 minutes, and 30 minutes later. Each sample was applied on a YPD agar medium, cultured at 30 ° C. for 3 days, and the number of single colonies obtained was counted.

  The test was repeated 3 times or more for each strain. In each strain, the number of colonies when the number of colonies immediately after application of heat shock was taken as 100% was determined, and the average survival rate was calculated. The results are shown in FIG. For the mean survival rate, a significant difference test of values in both strains was also performed.

  As is clear from FIG. 3, it was shown that the transformant had a significantly higher average survival rate after heat shock treatment than the control strain at a risk rate of less than 1% (control strain (after 15 minutes)). : 0.018%, transformed strain (after 15 minutes): 10.4%, control strain (after 30 minutes): 0.0016%, transformed strain (after 30 minutes): 4.4%). From this result, it was shown that the stress tolerance of sake yeast No. 701, the parent strain, was significantly recovered by the introduction of RIM15 gene derived from laboratory yeast.

[Sake production test]
Using the K701 UT-1T [pAUR112] and K701 UT-1T [pAUR112-ScRIM15] strains described above, sake was produced by the method described below. One-stage charging was performed by mixing 40 g of rice, 10 g of glutinous rice, 80 mL of water, and 17.8 μL of 90% lactic acid. Alpha rice with a 70% polishing rate was used as the hanging rice, and dried rice with a 70% polishing rate was used as the polished rice. Each yeast was cultured overnight in SC-Ura medium after shaking, washed with sterilized distilled water, and added at the time of preparation so that the number of yeast was 1 × 10 ⁷ cells / mL. The fermentation temperature was 15 ° C. The preparation test was repeated three times or more for each strain.

  After the preparation, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The results are shown in FIG. For the peak value of the carbon dioxide generation rate and the total amount of carbon dioxide generation, a significant difference test was also conducted between the control strain and the transformed strain. Furthermore, at the time when 20 days have passed since the start of fermentation, the death rate of yeast in sake mash and the ethanol concentration, sake degree, acidity, amino acid degree and organic acid composition in sake recovered by centrifugation were measured, and the average for each strain The value was calculated. The results are shown in Table 1. Moreover, about the obtained average value, the significant difference test of the value in a control strain and a transformed strain was also performed.

  The yeast death rate was measured by the methylene blue staining method. The ethanol concentration was measured using a gas chromatograph GC-17A manufactured by Shimadzu Corporation. About sake degree, acidity, and amino acid degree, it analyzed according to the National Tax Agency predetermined analysis method. The organic acid composition was analyzed using a high performance liquid chromatograph LC-10AD manufactured by Shimadzu Corporation.

  As is apparent from FIG. 4, the transformed strain did not show a significant difference from the control strain in terms of the peak value of the carbon dioxide generation rate or the total amount of carbon dioxide generated during fermentation (risk rate 5%). Specifically, the peak value of the carbon dioxide generation rate is 15.5 ± 0.3 mL / 30 min for the control strain, 15.3 ± 0.2 mL / 30 min for the transformed strain, and the total carbon dioxide generation rate is 7.02 ± 0.20 for the control strain. The L and transformed strains were 7.01 ± 0.14 L, both of which were almost the same. Regarding the carbon dioxide generation rate, the control strain showed a significantly larger value from 4.5 to 8.7 days after the start of fermentation (risk rate 5%), but the transformed strain was significantly larger after 15.7 days. (Risk rate 5%), it is considered that the fermentation progressed to almost the same degree in both strains over the entire 20-day fermentation period. Furthermore, the final ethanol concentration was 19.55 ± 0.25% by volume in the control strain and 19.20 ± 0.28% by volume in the transformed strain, and no significant difference was observed (risk rate 5%). From the above results, it was confirmed that the introduction of RIM15 gene derived from laboratory yeast had very little effect on ethanol fermentation using sake yeast No.701.

  On the other hand, the death rate of yeast in sake moromi was significantly reduced in the transformed strains, and the stress tolerance recovery effect by introduction of RIM15 gene derived from laboratory yeast was observed. In this connection, it was shown that in the transformed strains the amino acid content was significantly lower and the acidity was significantly higher, in particular the malic acid content was increased by 48% compared to the control strain.

  Yeast is subjected to stress such as high-concentration ethanol and low temperature in the sake production environment. When subjected to these stresses for a long time, not only does the yeast die and the production efficiency decreases, but it also has an adverse effect on the quality of sake with the self-digestion of the yeast cells. However, in the production method of the present invention, by using sake yeast that has recovered stress resistance while maintaining high fermentability, sake having good characteristics of low amino acid content and high malic acid concentration can be efficiently obtained. It can be manufactured. In addition, the yeast of the present invention having high stress resistance is not limited to sake, and is also suitably used for the production of alcoholic beverages such as shochu, beer and wine, and foods such as breads and soy sauce.

Sequence number 1 of a sequence table is a primer for RIM15 amplification.
Sequence number 2 of a sequence table is a primer for RIM15 amplification.

Claims (4)

  Sake yeast transformed with a recombinant vector expressing the RIM15 gene.   The sake yeast according to claim 1, wherein the sake yeast is Saccharomyces cerevisiae K701 UT-1T [pAUR112-ScRIM15] (reception number NITE AP-1219).   A method for producing alcoholic beverages or foods, wherein the sake yeast according to claim 1 or 2 is used.   The production method according to claim 3, wherein the liquor or food is at least one selected from the group consisting of sake, shochu, beer, wine, breads and soy sauce.

Images