JPH0352955B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Method) The present invention relates to a yeast strain with high alcohol fermentation ability in the production of alcohol using, for example, sugar beet molasses as a raw material. (Prior Art) Cane molasses is used in many ways, such as as a raw material for alcohol fermentation or for culturing baker's yeast. On the other hand, sugar beet molasses discharged after sugar is separated from sugar beets is currently only used as a feed additive, and its uses are limited. One possible expansion of the use of sugar beet molasses is the production of spirits through alcoholic fermentation, but the present inventors have previously identified 180 flocculant strains belonging to the genus Saccharomyces cerevisiae (National Tax Agency Brewing Laboratory No. ) and the killer strain 909-1 (National Tax Agency Brewing Laboratory number) were found to exhibit excellent alcoholic fermentation ability for sugar beet molasses and cane molasses. (Problems to be solved by the invention) However, when producing alcohol by industrial alcohol fermentation using yeast strains,
Killer properties against wild yeast, flocculating properties, etc. are required, but the above-mentioned yeast strains only have either killer properties or flocculating properties. (Means for Solving the Problems) In view of the above-mentioned circumstances, the present invention provides that even when alcoholic fermentation is performed using sugar beet molasses as a raw material, the alcohol content and fermentation ratio are high, and the killer property and
As a result of intensive experiments and research to find a yeast strain that has both properties of flocculation, we found that the yeast strain represented by the strain H-1 of cerevisiae of the genus Saccharomyces and its mutants. It has been discovered that a yeast strain represented by Saccharomyces cerevisiae strain M-9 is a yeast strain suitable for the above purpose. The Saccharomyces genus cerevisiae strains H-1 and M-9 according to this invention have been submitted to the National Institute of Microbiology, Agency of Industrial Science and Technology as No. 8885 and No. 9401. It has been deposited as. The above H-1 strain has parent strains 909-1 of Saccharomyces cerevisiae and 180 of Saccharomyces cerevisiae, and the parent strain 909-1 (2x The 180 strain (diploid) of cerevisiae belonging to the genus Saccharomyces is a homothallic flocculent yeast. The H-1 strain is produced as follows. 909-1 strain (heterotallik, 2
After sporulating the spores (ploid) and exposing and dispersing the spores with cell wall lytic enzyme (Zymolyase 100T),
Grow colonies on complete medium (YPD plates). The grown colonies were screened based on their ability to ferment alcohol using sugar beet molasses, survival rate in the second half of fermentation, etc., and excellent haploids (mating type a) were isolated. This will be referred to as A-4 stock. On the other hand, 180 strains (completely homothallic, diploid) of the flocculating yeast cerevisiae of the genus Saccharomyces were sporulated, and the above A-4 strain was spore-paired to each of the four ascospore molecules formed. cells (spore to
(cell) to join directly. Mating occurs with a probability of approximately 1/2, but while confirming the formation of zygotes, these zygotes are cultured on a complete medium to obtain several colonies. These hybrid strains were screened for their ability to ferment alcohol using sugar beet molasses and their survival rate in the second half of fermentation, and among them, strains with high fermentation ability and survival rate in the second half of fermentation were selected. This is H according to this invention.
-1 stock. This H-1 strain “The Yeast”a
taxonomic study 3rd ed (NJWKreger−van
The results of the TAXONOMIC TESTS (edited by Rij) are shown below. Spore Formation: Positive Fermentation of sugars: Glucose, galactose, sucrose, maltose, raffinose All positive Carbon source assimilation: Glucose, galactose, sucrose, maltose, trehalose, raffinose, α-methyl-D-gluco Both sides are positive, both meletitose and DL-lactic acid are weakly positive Nitrate assimilation: None Vitamin requirement: None Growth at 37℃: Yes Growth in 10% saline medium: Yes As a result, H-1 strain is Saccharomyces ) was identified as belonging to the genus cerevisiae. Furthermore, the present inventors conducted research with the aim of obtaining a yeast with strong fermentation power superior to the above-mentioned strain H-1. That is, according to the research conducted by the inventors of the present application, a phenomenon was observed in which when yeast is cultured in distilled waste liquid (hereinafter abbreviated as waste) after alcoholic fermentation of sugar beet molasses, it becomes extinct. It is thought that some kind of growth-inhibiting substance exists in the waist, but by obtaining a mutant strain of the H-1 strain that is resistant to that inhibitor,
The conclusion was reached that yeast with stronger alcoholic fermentation power could be obtained. Therefore, the method for producing M-9 strain, which is a mutant strain of H-1 strain, is carried out as follows. That is, strain H-1 is repeatedly cultured in waste after alcohol fermentation of sugar beet molasses, and the cultured cells are inoculated onto a waste agar medium and cultured to grow a colony. The grown colonies were screened for their ability to ferment alcohol using sugar beet molasses and their survival rate in the second half of fermentation, and excellent strains were isolated. This is W
-9 stocks. Furthermore, the W-9 strain is repeatedly cultured in sugar beet molasses containing 2-deoxyglucose (2DOG), and the cultured strain is inoculated onto a sugar beet molasses agar medium containing 2DOG and cultured to grow colonies. As a result, a colony in which catabolite repression has been released is obtained. This colony was screened again for its ability to ferment alcohol using sugar beet molasses based on its survival rate in the second half of fermentation, and strains with excellent fermentation ability were isolated. This is the M-9 strain according to this invention. When the M-9 strain was subjected to the same taxonomic test as the H-1 strain, similar results were obtained. As a result, the M-9 strain was identified as a genus Saccharomyces cerevisiae and a mutant strain of the H-1 strain. (Effect of the invention) Furthermore, according to research by the present inventors, the H-1 strain and the M-9 strain exhibit excellent alcoholic fermentation ability for sugar beet molasses, etc., as well as flocculating properties, killer properties, continuous usability, It also has excellent properties such as reproducibility of fermentation power. In particular, strain M-9 has superior properties to strain H-1, which is the parent strain, in terms of flocculation, continuous utilization, and reproducibility of fermentation power. In addition to sugar beet molasses, raw materials for alcohol production using the above bacterial strains include cane molasses, sweet potato, potato, corn, mackerel, rice, agricultural waste, and their hydrolysates. can do. Furthermore, since the strain according to the present invention has killer properties, the sterilization process can be simplified and, for example, non-cooked products can be used as raw materials.
The amount of the above bacterial strain used for alcohol fermentation varies depending on the type of raw material, but in the case of batch fermentation using sugar beet molasses as the raw material, an inoculum amount of 10 ⁵ to 10 ⁸ cells/ml is sufficient, and in the case of continuous use. The cell concentration in the tank may be 10 ⁵ to 10 ⁹ cells/ml. Furthermore, alcohol fermentation conditions generally range from PH4 to
6. The temperature is 25 to 37°C (preferably around 30°C). The alcohol fermentation method may be a batch fermentation method using a stirring tank or a continuous fermentation method. The batch method is mainly suitable for the production of spirits, and the continuous method is mainly suitable for the production of fuel alcohol. In addition, since the H-1 strain and M-9 strain according to this invention have good flocculation and sedimentation properties, when using a continuous utilization method, high concentration fermentation can be carried out, and the sedimentation tank also requires special separation means. It has the advantage that it can be made compact without the need for a (Example) Examples of the present invention will be shown below. Example 1 (Breeding method for strain H-1) A killer yeast, Saccharomyces genus Cerevisiae strain 909-1 (heterotrich diploid) was grown in a sporulation medium (potassium acetate 1%, yeast extract 0.2%). , glucose 0.2%, agar 2%). After exposing and dispersing the 4 molecules of ascospores formed, the spores were dissected using a micromanipulator and placed in a complete medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar). Fermentation tests were conducted on the inoculated, cultured, and grown colonies under the following conditions. [Fermentation test conditions] Pre-culture: Sugar beet molasses with a total sugar content of 10%, pH 5.0,
The culture medium was adjusted to contain KH ₂ PO ₄ at 700 ppm, and after autoclaving, one platinum loopful was inoculated from the storage slant and cultured with shaking at 30°C for 2 days. Fermentation test: Adjust the culture medium so that the total sugar content of sugar beet molasses is 24% and pH 5.0, autoclave it, and add the pre-cultured yeast suspension to 250ml at 4.0x.
The cells were inoculated at 10 ⁷ cells/ml. We obtained strain A-4, which has the best fermentation ability and has a high survival rate even in the second half of fermentation. On the other hand, Saccharomyces
180 strains (completely homothallic, diploid) of the genus Cerevisiae were cultured on a sporulation medium, and each of the four ascospore molecules formed and the above A-
The four strains are directly mated by spore to cell. This is cultured on a complete medium at a temperature of about 30°C, and the formation of zygotes is confirmed over time under a microscope. Those that have formed zygotes are sexually crossed (mating).
The above-mentioned fermentation test was carried out again, and among them, a strain with high fermentation power and high survival rate in the second half of fermentation was selected. In this way, strain H-1 was obtained. The appearance rate of strains having the same fermentation power as the H-1 strain produced by the above production method is 70% or more. Example 2 (Ethanol fermentation test using strain H-1 at 30°C using beet molasses as raw material) Preculture: Same as Example 1 Fermentation test: Sugar beet molasses with total sugar content of 24%, pH 5.0,
The culture medium was adjusted to contain 700 ppm of KH ₂ PO ₄ . Using this culture medium, use a 1000ml fermentation bottle (actual amount of preparation: 500ml).
Strain H-1 was cultured under anaerobic conditions at a culture temperature of 30° C., pH 5.0, slow stirring (100 to 200 rpm), and ethanol fermentation was performed. For comparison, 909-1 strain, which is the parent strain of H-1 strain,
A-4 strain and 180 strain were subjected to ethanol fermentation under the same conditions. The results are shown in FIGS. 1 and 2. From the results shown in Figures 1 and 2, the ethanol production rate of the parent strain 909-1 is relatively fast, but it reaches a plateau at 12V/V% after 3 days, and the cell survival rate also rapidly declines. On the other hand, the ethanol production rate of the A-4 strain isolated from the 909-1 strain did not decrease even after 3 days, but the ethanol production remained at about 12.5 V/V%. On the other hand, 180 stocks have been a little slow to rise, but
Even after 5 days, ethanol is being produced gradually, and the survival rate is extremely high, but ethanol production remains at 12V/V%. On the other hand, the initial ethanol production rate of the H-1 strain exceeds that of the 909-1 strain, the A-4 strain, and the 180 strain, and the ethanol production reaches 13 V/V% after 5 days. Therefore, the final ethanol concentration is also 909-1 strain,
The result will be 1V/V% higher than that of 180 stocks in a short time. Example 3 (Ethanol fermentation test using H-1 strain at 30°C using cane molasses as raw material) Preculture: 2% YM medium (commercial product manufactured by Difco,
After autoclaving YM broth, inoculate one platinum loopful from the storage slant and culture with shaking at 30°C for 2 days. Fermentation test: Adjust the culture medium so that cane molasses has a total sugar content of 24% and a pH of 5.0, add (NH ₄ ) ₂ SO ₄ 0.1% and KH ₂ PO ₄ 0.1% as aids, and after autoclaving, 4.0 precultured yeast suspension to 250ml
H-1 strain was inoculated at ×10 ⁷ cells/ml and ethanol fermentation was performed. For comparison, 909-1 strain, 180 strain, and Taiken 396 strain, which has been used as a practical strain for cane molasses alcohol fermentation, were tested under the same conditions. The results are shown in FIG. As is clear from Figure 3, when comparing cane molasses with sugar beet molasses of the same sugar concentration, fermentation started extremely quickly in the ethanol fermentation test using any of the yeast strains mentioned above, and after 3 days it reached 13V/V.
% or more. Among these, the H-1 strain, 909-1 strain, and 180 strain were found to rise faster than the conventional practical strain, Taiken396 strain, and the H-1 strain related to this invention was particularly quick to rise. . Example 4 (Coagulation sedimentation test of H-1 strain) H-1 strain and 909-1 strain as a control were
When cultured at 30°C, anaerobic conditions, and stirring, the turbidity (OD: absorbance at a wavelength of 660 nm and a 10 mm cell) and the absorption rate of the culture solution during stirring and after standing for 1 minute after stirring were determined. The volume percentage (V/V%) of bacterial cells is shown below. Turbidity (0.D) During stirring (0.D) After 1 minute of standing (0.D) H-1 strain 8.56 0.12 909-1 strain 8.44 7.20 Volume percentage of bacterial cells in culture solution (%) Stirring Time (%) After 1 minute of standing (%) H-1 strain 100 5 (95% is supernatant) 909-1 strain 100 98 (2% is supernatant) As is clear from the above results, 909-1 strain When used, it remained in a suspended state even if it was left to stand for 1 minute after stirring, but in the case of strain H-1, when stirring was stopped, it immediately flocculated and sedimented, and was completely sedimented and separated within 1 minute. In this case, the bacterial cells are sedimented and compacted to less than 1/10 of the total volume of the culture solution, and the supernatant does not contain bacterial cells and is transparent, exhibiting strong cohesive force. Due to the properties of this H-1 strain, the bacterial cells can be isolated from the culture solution within a few minutes by simply leaving it standing, without using equipment such as centrifuges or separators in the bacterial cell isolation process after culturing. This makes it possible to immediately send the supernatant liquid to a distillation process, etc., and to recycle and reuse the precipitated bacterial cells. Example 5 (Continuous utilization of H-1 strain) Pre-culture: Sugar beet molasses with a total sugar content of 10%, pH 5.0,
After adjusting the culture medium to contain 700 ppm of KH ₂ PO ₄ , one ear of white fungus was inoculated from the slant and cultured with shaking at 30°C for 2 days. Fermentation test: 20ml of preculture solution was inoculated into 250ml of sugar beet molasses adjusted to 28% total sugar and pH 5.0 so that the initial number of bacteria was 5 x 10 ⁷ cells/ml, stirred at 30℃, and fermented for 4 days. After fermentation, the supernatant was discarded, the sugar beet molasses was added again, fermentation was carried out, and this operation was repeated four times. All fermentation is carried out under anaerobic conditions with a fermentation plug attached, and stirring is done using a magnetic stirrer at approximately 200 rpm.
I did it at

[Table] Example 6 (Killer properties of strain H-1) Using sugar beet molasses with a total sugar content of 24% and a pH of 5.0 as a culture medium, Kyokai No. 701 (K-701) was cultured with 5 times the number of yeast.
10 ⁸ cells/ml and H-1 strain at yeast number 5×10 ⁷ cells/ml.
ml, mixed culture was carried out with stirring at 30°C, and one day after culturing, the number of viable bacteria of the K-701 strain was counted using a β-alanine plate. In addition, as a control, only the K-701 strain was inoculated and cultured under the same conditions, and the number of viable bacteria was counted.

[Table] As is clear from the above results, H
It can be seen that the K-701 strain, which existed 10 times as much as the -1 strain, becomes about 1/1000 times as large and dies after one day. On the other hand, the number of bacteria of the H-1 strain remained unchanged, and as a result, it became clear that the H-1 strain had killer properties against other yeasts. Example 7 (Breeding method of M-9 strain) The killer flocculant yeast obtained in Example 1, Saccharomyces cerevisiae strain H-1, was grown in West medium (TOC 50000 PPM, total sugar content 2.5%). , PH5.0), and after culturing at 30°C, the viable bacterial cells were again inoculated into the West medium and cultured, and this process was repeated for 10 days.
I did it repeatedly. The cultured bacterial cells were inoculated onto West's agar medium (agar 2%), cultured at 30°C, and a fermentation test was conducted on the grown colonies under the following conditions. [Fermentation test conditions] Pre-culture: Sugar beet molasses with a total sugar content of 10%, pH 5.0,
The culture medium was adjusted to contain KH ₂ PO ₄ at 700 ppm, and after autoclaving, one platinum loopful was inoculated from the storage slant and cultured with shaking at 30°C for 2 days. Fermentation test: Adjust the culture medium so that the total sugar content of sugar beet molasses is 24% and pH 5.0, autoclave it, and add the pre-cultured yeast suspension to 250ml at 4.0x.
The cells were inoculated at 10 ⁷ cells/ml. We obtained the W-9 strain, which has the best fermentation ability and has a high survival rate even in the second half of fermentation. Furthermore, sugar beet molasses medium (total sugar content 10% PH
2-deoxyglucose was added to 150 ppm (5.0 KH ₂ PO ₄ 700 ppm), and the W-9 strain was inoculated.
Culture was carried out. After culturing, a sugar beet molasses agar medium containing 2-deoxyglycose (agar 2%) with the above composition was used.
Pick out the colony growing on top and repeat the same operation three times. The cultured bacterial cells were inoculated onto the above agar medium and cultured, and the grown colonies were subjected to a fermentation test under the conditions described above. As a result, M-9 has the best fermentation power and has a high survival rate even in the second half of fermentation.
Acquired stock. Example 8 (Ethanol fermentation test using strain M-9 at 30°C using beet molasses as raw material) Preculture: Same as Example 7 Fermentation test: Sugar beet molasses with total sugar content of 24%, pH 5.0,
The culture medium was adjusted to contain 700 ppm of KH ₂ PO ₄ . Using this culture medium, use a 1000ml fermentation bottle (actual amount of preparation: 500ml).
strain M-9 was inoculated at a concentration of 1 x 10 ⁸ ceus/ml in a culture medium at a culture temperature of 30°C with slow stirring (100 -
200 rpm) under anaerobic conditions, and ethanol fermentation was performed. For comparison, the H-1 strain, which is the parent strain of the M-9 strain, was subjected to ethanol fermentation under the same conditions. The results are shown in FIG. From this result, the ethanol production rate of the parent strain was relatively fast, but reached a plateau at 12.8 V/V% after 5 days. On the other hand, the initial ethanol production rate of the M-9 strain exceeds that of the H-1 strain, and the ethanol production reaches 13.5 V/V% after 5 days. Therefore, the final ethanol concentration also exceeded that of the H-1 strain by 1 V/V% in a short period of time. In addition, the number of viable bacteria after 5 days of fermentation is shown in the table below.

[Table] As is clear from the results, the M-9 strain had 7 times more bacterial cells than the H-1 strain even in the latter half of fermentation, maintaining the number of bacteria at the time of inoculation. Example 9 (Continuous utilization of M-9 strain) Using the M-9 strain, experiments were conducted by repeating batch fermentation. Preculture: Same as Example 7 Fermentation test: The preculture yeast suspension was inoculated into 250ml of sugar beet molasses adjusted to 15% total sugar and pH 5.0 so that the initial bacterial count was 1 x 10 ⁸ cells/ml. , stir at 30 °C,
The mixture was fermented for 2 days, left to stand for 10 minutes after fermentation, the supernatant was discarded, sugar beet molasses with the above sugar concentration was added again, batch fermentation was carried out, and this operation was repeated 5 times. All fermentation was carried out under anaerobic conditions with a fermentation stopper attached, and stirring was done using a magnetic stirrer.
This was done by rotating at 100 rpm. The results are shown in FIG. 5 and the table below.

[Table] As is clear from Figure 5, alcohol produced through continuous use is stable at a sugar concentration of 15%, and from the table above, the number of viable yeast bacteria remains the same as the number at the time of inoculation. It became clear that Further, the present yeast easily precipitates and forms a highly dense slurry within a short period of time after the completion of fermentation and by simply standing still, so that a clear fermented liquid can be obtained in a short period of time.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the daily change in ethanol production in Example 2, FIG. 2 is a diagram showing the daily change in the number of viable bacteria in Example 2, and FIG. 3 is a diagram showing the ethanol production in Example 3. FIG. 4 is a diagram showing daily changes in production. FIG. 4 is a diagram showing daily changes in ethanol production in Example 8. FIG. 5 is a diagram showing changes in ethanol production for each alcohol fermentation in Example 9.

Claims (1)

[Scope of Claims] 1. A yeast strain with high alcohol fermentation ability represented by Saccharomyces genus Cerevisiae strain H-1 (Feikoken Bokuyori No. 8885). 2. A yeast strain with high alcohol fermentation ability represented by Saccharomyces genus cerevisiae strain M-9 (Feikoken Bibori No. 9401).