JPS6365310B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD This invention relates to a method for producing yeast that has both excellent flocculation properties and excellent ethanol 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 method for producing yeast that has excellent flocculation properties and is also satisfactory in alcohol fermentation ability. Means for Solving the Problems The method for producing yeast with flocculation and fermentation ability according to the present invention is based on Saccharomyces.
A yeast that does not have flocculating ability belonging to the genus Saccharomyces is sporulated, the resulting spores are subjected to mutation treatment, and a mutant yeast with excellent flocculating ability obtained from the mutant spores and other flocculants belonging to the genus Saccharomyces are produced. This method is characterized by the protoplast fusion with a yeast that has no sex. In this specification, the degree of flocculation of yeast is determined by 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 with shaking, the sedimentation rate of the bacterial cells, the volume and hardness of the sedimented bacterial cells were compared with the control strain by visual observation,
The degree of aggregation is displayed in six stages from DF value 0 to 5 shown in Table 1.

[Table] In the yeast production method according to the present invention, any yeast that does not have flocculating properties can be used as long as it has excellent alcohol fermentation ability and spore forming ability. In this invention, sporulation treatment is carried out according to conventional methods. Normally, yeast that does not have flocculating properties
The method is to culture on YPG agar medium (Note 2) and then smear it on sporulation agar medium (Note 3). To obtain cells derived from single spores, the asci are lysed using a lytic enzyme for lysing the yeast cell wall, 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, etc., can be carried out by any of the chemical methods of growth on a selective medium after contact with a mutagenic agent, but the method using ethyl methanesulfonate is particularly preferred. . Further, protoplast fusion is performed by a conventional method. 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. As the culture medium used in the production method of this invention, a conventional culture medium containing a carbon source, a nitrogen source, 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 yeast extract 1
g, polypeptone 2g, glucose 2g, distilled water
This is a medium consisting of 100 ml, and the pH of this medium is 5.5 without adjustment. 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. In a preferred embodiment of the present invention, the yeast Saccharomyces cerevisiae with a DF value of 0, which is a preserved strain of the Fermentation Research Institute, is used.
Saccharomyces cerevisiae) IFO-0224 (hereinafter simply referred to as IFO-0224) was subjected to sporulation treatment, the resulting spores were subjected to mutation treatment, and the yeast Saccharomyces cerevisiae with a DF value of 4 obtained from the mutant spores was
cerevisiae) RM-17 (hereinafter simply referred to as RM-17) and the yeast Saccharomyces cerevisiae with a DF value of 0.
cerevisiae) VM-2 (hereinafter simply referred to as VM-2) and cultured the fused yeast, the yeast Saccharomyces with a DF value of 5 was produced.
Obtain FR _M17 V _M2-1 (Feikoken Bibori No. 7792). In the above embodiment, the starting yeast IFO-0224 has 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. Furthermore, FR _M17 V _M2-1 obtained in the above embodiment is
It has the following mycological properties. In other words, this yeast has a flocculating property with a DF value of 5, and exhibits a remarkable sedimentation property in liquid culture. - Blackstrap molasses (for example, blackstrap molasses containing 15% total sugar)
is fermented to produce 7 to 9 vol% ethanol. ● Forms somewhat hard colonies on an agar plate. ●Has spore-forming ability. RM-17 also requires adenine and histidine for growth, and VM-2 requires isoleucine and valine. 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,
Published by North-Holland Publishing, 1970). In other words, yeast belonging to this genus: ●Proliferate by multipolar budding. ● Forms ascospores. ●Does not assimilate nitrates. ●Lack or form 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 alcoholic 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 ethanol production 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
Saccharomyces cerevisiae
IFO-0224 on YPG agar medium (Note 2) at 30℃
The cells were cultured 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. Then, so that the number of spores is ¹⁰⁷ /ml,
The asci were suspended in 1 ml of sterile water and washed with phosphate buffer (Note 4) after bacterial collection. 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. Add 0.1 ml of ethyl methanesulfonate as a mutagen to this suspension, and add 0.1 ml of ethyl methanesulfonate as a mutagenic agent to
It was shaken at ℃ for 2 hours. The spores were thus mutated. After harvesting, the mutant spores were suspended in 0.2 ml of phosphate buffer, 3 ml of 5% sodium thiosulfate aqueous solution was added to the suspension, and the suspension was shaken at 30°C for 10 minutes. The mutagen was thus neutralized. After harvesting, the mutant spores were diluted with 1 ml of phosphate buffer.
The mutant spores were washed twice and suspended in 5 ml of the same buffer, and the suspension was sonicated for 3 minutes under ice cooling to disperse the mutant spores in the suspension. After collecting bacteria, the suspension was diluted with sterile water to a concentration of 1/10 ⁵ to 1/10 ⁶ , and 0.1 ml of the diluted suspension was spread on YPG agar medium (Note 2) and cultured at 30°C for 48 hours. A colony derived from a single spore was obtained. Using the colony plate thus obtained as a master plate, mutant strains were detected by the replica method. That is, the colony on the master plate was replicated on a minimal medium (Note 6) using sterilized velvet cloth, cultured on the same medium at 30°C for 4 days, and strains that could not grow on the minimal medium were identified as auxotrophic mutants. Bacteria were caught from the master plate. As a result, among the 25 strains on the master plate, the strain Saccharomyces cerevisiae (RM) with excellent aggregation ability was found.
-17 (Feikoken Bibori No. 7770) was obtained. This strain was an adenine and histidine auxotrophic strain. (b) Preparation of VM-2 A non-flocculating yeast, Saccharomyces cerevisiae, EY-1 was provided by the Biochemical Engineering Laboratory, Department of Applied Technology, Institute of Microbial Technology, Agency of Industrial Science and Technology.
(Feikoken Bacteria Serial No. 7793) was mutated in the same manner as for the preparation of RM-17, and the yeast Saccharomyces cerevisiae VM-
2 (Feikoken Bibori No. 7788) was obtained. (c) Protoplast fusion of RM-17 and VM-2 RM-17 was cultured with shaking in 10 ml of YPD medium at 30°C for 16 hours, and after harvesting, it was washed with 1 ml of sterile water.
Next, use this as the protoplast preparation solution (Note 7)
The suspension was suspended in approximately 2 ml, shaken at 30°C for 1 hour, and washed twice with 1 ml of isotonic solution (Note 8) after bacterial collection. VM-2 was also processed using the same operations as above. Next, the same amount of RM-17 treated bacteria and VM-2 treated bacteria obtained in this way (cell number
After collecting bacteria, the mixture was suspended in ^0.1 ml of isotonic solution, and 2 ml of polyethylene glycol aqueous solution (Note 9) was added to the suspension. This suspension was allowed to stand at 30°C for 15 minutes to complete protoplast fusion. After collecting the bacteria, suspend the bacteria in 1 ml of isotonic solution and dilute the suspension for 20 minutes.
It was left standing at ℃ 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. In this state, the fusion strain was cultured at 30°C for 4 days to obtain a fusion strain with excellent aggregation properties.
22 strains were isolated, one of which was the yeast Saccharomyces FR _M17 V _M2 -1.
(Feikoken Bibori No. 7792). Note that the parental strains (RM-17 and VM-2) used for protoplast fusion could not grow on the above minimal medium. Measurement of flocculation and alcohol fermentation ability IFO−2018, IFO−0224, RM−17, EY−
1. Regarding VM-2 and FR _M17 V _M2-1 ,
The DF value, which indicates the degree of flocculation, and the alcohol fermentation ability were measured. 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 potassium pyrosulfite.
After mixing and dissolving 0.2g/, adjust the pH to 4.5 with sulfuric acid.
The mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant liquid obtained in this way was
ml each, add 7 ml of the preculture solution of each strain to each solution, perform batch culture by intermittent stirring at 30°C (repeated stirring for 30 seconds and standing still for 10 minutes), and 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
Since VM-2 is a mutant strain, its alcohol fermentation ability is inferior to that of the wild-type parent strain, but the fusion strain FR _M17 V _M2-1 has better flocculation than the wild strain and is capable of alcohol fermentation. In terms of performance, it is comparable to wild strains. 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 / Glucose 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 0.05% Zymolyase 20T (manufactured by Seikagaku Corporation) in 0.1M phosphate buffer (PH7.5) 2 ml of the dissolved solution and 2-mercaptoethanol
Mixed solution with 1.4μ (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- Mercaptophosphate buffer 1.4μ and Zymolyase 20T
(manufactured by Seikagaku Corporation) in 0.1M phosphate buffer (PH
A mixture of 0.2ml of a 0.25% solution in 7.5) (Note 8) Isotonic solution 0.6M potassium chloride aqueous solution (Note 9) Polyethylene glycol aqueous solution Calcium chloride 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/

Claims (1)

[Scope of Claims] 1. Treating non-agglomerating yeast belonging to the genus Saccharomyces to form sporulation,
The method is characterized in that the obtained spores are subjected to mutation treatment, and the mutant yeast obtained from the mutant spores, which has excellent flocculation ability, is fused into protoplasts with another yeast that does not have flocculation ability and belongs to the genus Saccharomyces. , a method for producing yeast that has both excellent flocculation and alcohol fermentation ability. 2 The yeast Saccharomyces cerevisiae IFO-0224 with a DF value of 0 was subjected to sporulation treatment, the obtained spores were subjected to mutation treatment, and the mutant yeast with excellent flocculating properties obtained from the mutant spores and Saccharomyces ) Protoplast fusion with other non-flocculating yeast belonging to the genus A. . 3 The yeast Saccharomyces cerevisiae IFO-0224 with a DF value of 0 was subjected to sporulation treatment, the obtained spores were subjected to mutation treatment, and the yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) with a DF value of 4 was obtained from the mutant spores.
RM-17 (FEI No. 7770) and the yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae) VM-2 (FEI No. 7788) with a DF value of 0 are fused into protoplasts to achieve excellent flocculation. 2. The production method according to claim 1, wherein yeast is obtained which has both alcohol fermentation ability and excellent alcoholic fermentation ability. 4 The yeast Saccharomyces cerevisiae IFO-0224 with a DF value of 0 was subjected to sporulation treatment, the resulting spores were subjected to mutation treatment, and the yeast Saccharomyces cerevisiae with a DF value of 4 was obtained from the mutant spores.
Protoplast fusion of RM-17 (FEI No. 7770) and the yeast Saccharomyces cerevisiae VM-2 (FEI No. 7788) with a DF value of 0 was carried out.
Yeast Saccharomyces FR _M17 V _M2-1 with DF value of 5
7792) according to claim 1.