JPH0121758B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD This invention relates to a novel yeast that has both excellent flocculating properties and excellent alcoholic fermentation ability. In recent years, fermented alcohol, which can be obtained without petrochemistry, has been attracting attention as an energy alternative to petroleum. It is manufactured from sugar cane, molasses extracted from sugar cane, cellulosic or starchy materials such as sweet potatoes, potatoes, and corn, and by fermenting them using the action of microorganisms. Generally, in alcohol fermentation, alcohol productivity is proportional to the bacterial cell concentration in the fermenter. Therefore, as a means to increase the bacterial cell concentration in the fermenter, it is possible to use yeast that has excellent flocculating properties. That is, when yeast has excellent flocculating properties, the sedimentation rate of yeast becomes faster, and therefore solid-liquid separation can be performed quickly and easily. For example, in batch fermentation, the bacterial cells can be deposited by simply allowing the fermentation liquid to stand still, and the fermentation liquid and the bacterial cells can be easily separated and the cells can be reused. In continuous fermentation, a tower-type fermenter is used, which mainly consists of a small-diameter flow section, a large-diameter sedimentation section for bacterial cell sedimentation connected above the flow section, and a bacterial cell sedimentation member built into this. Thereby, even if the amount of culture medium supplied increases, the bacterial cells can be sedimented and their outflow can be prevented. As described above, the use of yeast that has flocculating properties has many advantages over the use of yeast that does not have flocculating properties, and therefore new flocculating yeasts are in demand. Prior art and its problems In the past, attempts have been made to obtain flocculating yeast in order to meet the above-mentioned demands, but the conventional yeasts are wild strains obtained from nature (for example, fermentation research institute preserved bacteria). IFO-2018). However, although such wild strains had no particular problems in terms of flocculation, they were not satisfactory in terms of alcohol fermentation ability (see Table 3 below). The present invention was made in view of the above-mentioned circumstances, and an object of the present invention is to provide a new yeast that has excellent flocculating properties and is also excellent in alcohol fermentation ability. Means for Solving the Problems This invention has: - a flocculating property with a DF value of 5; - a mixture of 340 g of blackstrap molasses, ammonium sulfate, and potassium pyrosulfite with a pH of 4.5, which is centrifuged, By adding 7 ml of the preculture solution of the bacterial strain to 70 ml of the clear solution and performing batch culture, 55.7
g/ and 56.0 g/ of ethanol. Yeast Saccharomyces cerevisiae FR _M17 V _M2 -1 (Feikoken Bacteria No. 7792). (hereinafter referred to as "yeast according to the present invention"). In this specification, the degree of flocculation of yeast is measured using the Gilliland test (European test) shown below.
Journal of Applied Microbiology and
Biotechnology Vol. 7, pp. 227-234, 1979)
The DF value calculated by is displayed. That is, after culturing the test bacterial strain in YPG medium (Note 1) at 30°C for 16 hours, the sedimentation rate of the bacterial cells, the volume and hardness of the sedimented bacterial cells were compared with the control bacterial cells by visual observation,
The degree of aggregation is displayed in six stages from DF value 0 to 5 shown in Table 1.

[Table] The yeast according to the present invention has the following mycological properties. That is, this yeast: - Has a flocculating property with a DF value of 5, and exhibits a remarkable sedimentation property in liquid culture. - Fermenting blackstrap molasses (for example, blackstrap molasses with 15% total sugar content) to produce 7-9 vol% ethanol.・ Forms somewhat hard colonies on the agar plate.・Has spore-forming ability. The yeast culture medium according to this invention includes a carbon source,
Conventional media containing nitrogen sources, inorganic ions and, if necessary, organic micronutrients can be used. Carbohydrates such as glucose, galactose, fructose, sucrose, starch hydrolyzate, fruit juice, and cellulose decomposition products are often used as carbon sources. A particularly suitable medium is 1 g of yeast extract, 2 g of polypeptone, 2 g of glucose, 100 ml of distilled water.
The pH of this medium is not adjusted.
It is 5.5. Culture at a temperature of 25-40℃, preferably 30-37℃, and a pH of
It is carried out at a pH of 3.0 to 7.0, preferably 3.5 to 6.0. Next, the method for producing yeast according to the invention will be explained. The yeast according to the present invention is a yeast called Saccharomyces cerevisiae, which has flocculating properties.
cerevisiae) RM-17 (hereinafter referred to simply as RM-17), and the non-flocculating yeast Saccharomyces cerevisiae VM-2 (February 2011). No. 7788) (hereinafter simply referred to as VM-2) by protoplus fusion treatment and culturing the fused bacterial cells. Protoplast fusion is performed by conventional methods. Usually, each cell suspension with a concentration of 10 ⁷ to ^{10 9} cells/ml is prepared, these suspensions are preferably mixed in equal amounts, and then the mixture is mixed with a protoplast preparation solution containing yeast cell wall lytic enzyme. or, each bacterial cell suspension is treated with the same preparation solution and then mixed. RM-17 is a preserved bacteria of the Fermentation Research Institute.
Yeast Saccharomyces cerevisiae IFO-0224 (hereinafter referred to as
(simply referred to as IFO-0224) is subjected to sporulation treatment, the resulting spores are subjected to mutation treatment, the mutant spores are cultured, the mutant strain is detected from the obtained colony by the replica method, and the mutant strain is isolated. Manufactured. Sporulation treatment is carried out according to conventional methods. Normally, yeast that does not have flocculating properties are grown on YPG agar medium (Note 2).
After culturing on a spore-forming agar medium (Note 3), a method is used. To obtain cells derived from single spores, the asci are lysed using a lytic enzyme for lysing yeast cell walls, and then the spores are separated using a micromanipulator, or the asci are separated using a lytic enzyme. After dissolving the spores, the spores are dispersed by ultrasonication, and the spores are then cultured on a nutrient agar medium. The mutation treatment may also include physical methods of irradiating the spores or asci obtained by sporulation treatment with ultraviolet rays, X-rays, gamma rays, ethyl methanesulfonate, N-methyl-N'-nitro -N-nitrosoguanidine, 4-nitroquinoline-N-oxide, or any other chemical method that involves contact with a mutagenic agent followed by growth on a selective medium, but a method using ethyl methanesulfonate is particularly preferred. . Detection of mutant strains is performed by the replica method. That is, a plate of colonies obtained by culturing mutant spores on a complete medium such as YPG agar medium is used as a master plate, and the colonies on the same plate are treated with predetermined nutritional elements using sterilized cloth. Transfer it to a plate of minimal medium without containing it, and culture it in the same medium. Colonies that require the above nutritional elements do not grow on minimal medium plates, so auxotrophic strains can be easily detected by comparing the colonies on this plate with those on the master plate. Can be done. The detected auxotrophic strain is then picked from the master plate and separated from other bacterial cells. In this way, yeast having the desired flocculating properties is obtained. In the series of production steps described above, the medium and culture conditions are the same as those of the yeast itself described above. IFO-0224 has a DF value of 0 and shows no cohesiveness. Further, yeast belonging to Satucharomyces cerevisiae has various properties (presence or absence of fermentability and assimilation, physiological properties) as shown in Table 2 below.

[Table] In Table 2, the fermentability of raffinose is expressed by how many sugars it can ferment out of the constituent monosaccharides fructose, glucose, and galactose produced by cleavage of the bond. That is, fermentability 1/3
"fermentability" means the case where only fructose is fermented, "2/3 fermentability" means the case where fructose and glucose are fermented, and "fermentability 3/3" means the case where all the constituent monosaccharides are fermented. In addition, Saccharomyces
Yeast belonging to the genus are known to have the following mycological properties (J. Lodder, “The
Yeasts, A Taxonomic Study” 2nd edition,
North-Holland Publishing, 1970). That is, yeast belonging to this genus: • Propagate by multipolar budding. - Forms ascospores.・Do not assimilate nitrates. - Lacking or forming only a few fungal threads.・Mature asci do not cleave easily.・The shape of the spores is spherical or oval. - Ferment glucose well. - Does not form a film on the wort medium. Effects of the invention Since this invention is configured as described above,
It is possible to obtain a new yeast that has better flocculation than the wild strain and also has excellent alcohol fermentation ability. Therefore, by carrying out alcohol fermentation using the flocculating yeast obtained in this way, the bacterial cell concentration in the alcohol fermenter can be maintained at a high level in both batch fermentation and continuous fermentation, as explained at the beginning, and the production of ethanol can be improved. Productivity can be significantly improved. Examples Next, examples of the present invention will be shown to demonstrate the above effects. Production example (a) Preparation of RM-17 Non-flocculating yeast Saccharomyces cerevisias IFO-
0224 was cultured on a YPG agar medium (Note 2) at 30°C for 24 hours, then spread on a sporulation agar medium (Note 3), and cultured at 30°C for 3 to 5 days. In this way, spores were formed. Next, the asci were suspended in 1 ml of sterile water so that the number of spores was 10 ⁷ /ml, and after collection, the cells were washed with phosphate buffer (Note 4). The ascus was then shaken in 2 ml of lytic enzyme solution (note 5) at 30°C for 1 hour to dissolve the ascus. After bacterial collection, the released spores were washed with 1 ml of sterile water and suspended in 3 ml of phosphate buffer. To this suspension was added 0.1 ml of ethyl methanesulfonate as a mutagenic agent, and the suspension was shaken at 30°C for 2 hours. The spores were thus mutated. After collecting the bacteria, the mutant spores were suspended in 0.2 ml of phosphate buffer, and the suspension was added with 3 ml of 5% sodium thiosulfate aqueous solution.
ml was added and the suspension was shaken for 10 minutes at 30°C.
The mutagen was thus neutralized. After collection, the mutant spores are washed twice with 1 ml of phosphate buffer, suspended in 5 ml of the same buffer, and the suspension is sonicated for 3 minutes on ice to remove the mutant spores in the suspension. dispersed into. After collecting bacteria, dilute the suspension with collection water to a concentration of 1/10 ⁵ to 1/10 ⁶ to obtain a diluted suspension.
Spread 0.1ml onto YPG agar medium (Note 2) and incubate at 30℃.
After culturing for 48 hours, colonies derived from single spores were obtained. Using the colony plate thus obtained as a master plate, mutant strains were detected by the replica method. That is, using sterilized velvet cloth, replicate the colony on the master plate onto a minimal medium (Note 6), culture it on the same medium at 30°C for 4 days, and detect strains that cannot grow on the minimal medium on the master plate, This was harvested from the master plate as an auxotrophic mutant strain. As a result, out of the 25 strains on the master plate, a strain of Saccharomyces cerevisiae RM-17 (Feikoken Bacterial Collection No. 7770) with excellent aggregation properties was obtained. This strain was an adenine and histidine auxotrophic strain. (b) Preparation of VM-2 The non-agglutinating yeast Saccharomyces cerevisiae EY-1 (Saccharomyces cerevisiae), which was provided by the Biochemical Engineering Laboratory, Department of Applied Technology, Institute of Microbial Technology, Agency of Industrial Science and Technology, was used. No. 7793) was mutated in the same manner as for the preparation of RM-17, and the yeast Saccharomyces cerevisiae was produced as an auxotrophic mutant strain requiring isoleucine and valine by the replica method.
cerevisiae) VM-2 (Feikoken Bibori No. 7788) was obtained. (c) Protoplast fusion of RM-17 and VM-2 RM-17 was cultured in 10 ml of YPD medium at 30°C for 16 hours with shaking, and after harvesting, it was washed with 1 ml of sterile water. This was then suspended in approximately 2 ml of protoplast preparation solution (Note 7), the suspension was shaken at 30°C for 1 hour, and after bacterial collection, the cells were washed twice with 1 ml of isotonic solution (Note 8). VM-2 was also processed using the same operations as above. Next, the treated bacterial cells of RM-17 obtained in this way and
Same amount of VM-2 treated bacteria ( ¹⁰ cells/ml)
After collecting the bacteria, dilute the mixture into an isotonic solution of 0.1
ml, and 2 ml of polyethylene glycol aqueous solution (Note 9) was added to the suspension. 30% of this suspension
Protoplast fusion was completed by standing at ℃ for 15 minutes. Then, after collecting bacteria, suspend the bacterial cells in 1 ml of isotonic solution,
The suspension was allowed to stand at 20°C for 15 minutes. Next, dilute the suspension with an isotonic solution to a concentration of 1/10 to 1/10 ² , smear the diluted suspension on a minimal medium (Note 6), and add it to the overlay medium (Note 6).
10) were layered. Cultivation was _carried out at 30°C for 4 days under this _condition , and 22 fusion strains with excellent flocculation properties were isolated.
No. 7792). In addition, the parent strains (RM-17 and VM-25) used for protoplast fusion could not grow in the above minimal medium. Measurement of flocculation and alcohol fermentation ability IFO-2018, IFO-0224, RM-17, EY-1 ，
For VM-2 and FR _M17 V _M2-1 , the DF value indicating the degree of flocculation and the alcohol fermentation ability were measured, respectively. The DF value was determined by the method described above. In addition, alcohol fermentation ability was determined by the following method.
In other words, 340 g of blackstrap molasses from Okinawa, 3.4 g of ammonium sulfate, and 0.2 g of potassium pyrosulfite.
After mixing and dissolving, adjust the pH to 4.5 with sulfuric acid,
The mixture was centrifuged at 3000 rpm for 10 minutes. Take 70 ml of the supernatant liquid obtained in this way, add 7 ml of the preculture solution of each strain to each liquid, and perform batch culture by intermittently stirring (stirring for 30 seconds and standing still for 10 minutes) at 30°C. After 24 hours and 48 hours, the amount of ethanol produced in each culture solution was measured by gas chromatography. The measurement results are shown in Table 3 below.

[Table] As is clear from Table 3, RM-17 and VM
-2 is a mutant strain, so its alcohol fermentation ability is inferior to that of the wild type parent strain, but it is a fusion strain.
FR _M17 V _M2-1 has better flocculation than the wild strain, and is comparable to the wild strain in terms of alcohol fermentation ability. Example of Use (Continuous Alcohol Fermentation) Using FR _M17 V _M2-1 , continuous alcohol fermentation was carried out by the following procedure, and its alcohol fermentation ability was investigated. As the fermentation apparatus, an alcohol fermentation apparatus shown in FIG. 1 was used. This mainly consists of a glass fluidized bed fermenter 1 with an actual volume of 700 ml, and is configured to be able to control temperature and pH. Then, the fermentation raw material is supplied to the bottom of the tank 1 by pump 2, and the reaction liquid is returned from the top to the bottom of the tank by pump 3.
It is designed to flow out from the bacterial cell sedimentation section 4 at the top of the tank. YPG medium (Note 1) in a 500ml Sakaguchi flask
After adjusting 100 ml and sterilizing it at a temperature of 121°C for 10 minutes, it was stored in a YPG agar slant medium (Note 2).
FR _M17 V _M2-1 strain was inoculated into one white fungus ear and cultured overnight at 30°C. In this way, a preculture of active FR _M17 V _M2-1 was obtained. 100 ml of the above preculture solution was placed in fermenter 1 containing 700 ml of Philippine molasses medium (Note 11), and batch culture was carried out at a fermentation temperature of 30° C. for 8 hours. Next, the molasses medium was continuously supplied to the fermenter 1 at a flow rate of 35 ml/hour (dilution rate = 0.05 hour ^-1 ), and the supply amount of the medium was gradually increased to carry out continuous fermentation. As a result, the medium supply amount was 175ml/hour (dilution rate =
0.25 hours ^-1 ), the excellent flocculation properties of this yeast form flocs with a diameter of 1 to 4 mm in the tank, and the bacterial cell concentration (Note 12) in the tank remains at a high value of 47 g/1. was maintained. Also, the alcohol produced is 61
The alcohol productivity (Note 13) reached a high value of 16 g/hr as shown in Figure 2. Comparative example: Using EY-1 instead of FR _M17 V _M2-1 as the yeast, and keeping the other details the same as in the above usage example,
The above operation was repeated. As a result, the medium supply amount was 70ml/hour (dilution rate =
If it exceeds 0.1 h ^-1 ), alcohol productivity (Note 13)
As shown in FIG. 2, the amount decreased rapidly from 4 g/h. Medium and reagents The medium and reagents are as follows. (Note 1) YPG medium yeast extract 10g / polypeptone 20g / glucose 20g / (note 2) YPG agar medium yeast extract 10g / polypeptone 20g / glycose 20g / agar 20g / (note 3) Sporulation medium sodium acetate 5g / agar 20g/ (Note 4) Phosphate buffer 0.1M phosphate buffer PH = 7.5 (Note 5) Lytic enzyme solution 0.1M phosphate buffer (PH 7.5) with 0.05% Zymolyase 20T (Seikagaku Corporation) A mixture of 2ml of the dissolved solution and 1.4μ of 2-mercaptoethanol (Note 6) Minimal medium Difco-Yeast Nitrogen Base W/O
Amino acid (manufactured by Difco) 6.7g / Glucose 20g / Agar 20g / (Note 7) Protoplast preparation solution 1.5M potassium chloride 0.8ml, 2/15M phosphate buffer (PH7.5) 1.0ml, 2- Mercaptoethanol 1.4μ and zymolyase
Mixture with 0.2ml of 0.25% solution of 20T (manufactured by Seikagaku Corporation) in 0.1M phosphorus buffer (PH7.5) (Note 8) Isotonic solution 0.6M potassium chloride aqueous solution (Note 9) Polyethylene glycol aqueous solution chloride Calcium 5.6g/Polyethylene glycol (PEG-6000)
300g/ (Note 10) Overlay medium glucose 20g/ Difco-Yeast Nitrogen Base W/O
Amino acid (manufactured by Difco) 6.7g / Difco-Bact Agar (manufactured by Difco) 30g / (Note 11) Philippine molasses medium Philippine molasses 280g / Ammonium sulfate 2.8g / Potassium pyrosulfite 0.5g / Antifoaming agent 0.2g / (Note 12) Bacterial cell concentration Take a certain amount of the culture solution in the fermenter, collect the bacterial cells with a centrifuge, wash them, and heat them at a temperature of 800 ml.
Calculated as the amount of bacterial cells burned at ℃ (Note 13) Weight of alcohol produced in 1 hour per 1 alcohol-producing culture solution (g)

[Brief explanation of drawings]

FIG. 1 is a flow sheet for continuous fermentation, and FIG. 2 is a graph showing the relationship between dilution rate and alcohol productivity.

Claims (1)

[Claims] 1. Has a flocculating property with a DF value of 5. - A mixture of 340 g of blackstrap molasses and ammonium sulfate and potassium pyrosulfite at pH 4.5 is centrifuged, and 70 ml of the resulting supernatant is added to the By adding 7 ml of the preculture solution of the bacterial strain and performing batch culture, 55.7
The yeast Saccharomyces cerevisiae FR _M17 V _M2-1 (Feikoken Bacterial Serial No. 7792) produces ethanol of 56.0 g/g/ and 56.0 g/g/.