JPH0121754B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD This invention relates to a method for producing yeast having excellent flocculating properties. 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 demands, but the conventional yeast is a wild strain obtained from the natural world. It was isolated from a specific soil. However, the task of finding and isolating a desired bacterial strain from the natural world is extremely troublesome, and there is little certainty that the desired bacterial strain can be collected. This invention was made in view of the above-mentioned circumstances, and it is an object of the present invention to provide a method for producing flocculating yeast that allows yeast with excellent flocculating properties to be obtained in a laboratory. Means for Solving the Problems The method for producing flocculating yeast according to the present invention involves sporulation treatment of non-flocculating yeast belonging to the genus Saccharomyces, mutation treatment of the obtained spores, and mutant spores. The mutant strain was detected from the resulting colony by the replica method, and DF
This method is characterized by separating yeast having a flocculating property of value 4 or higher. 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 has excellent alcohol fermentation ability and spore forming ability can be used as the parent yeast that does not have flocculating properties. 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 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, 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 strains. In this way, yeast having the desired flocculating properties is obtained. As a yeast medium, a conventional 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 1g, polypeptone 2g
g, glucose 2 g, and distilled water 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 IFO-, which has a DF value of 0 and is kept at the Fermentation Research Institute, is used as a non-flocculating yeast.
0224 (hereinafter simply referred to as IFO-0224) was used to test the yeast Saccharomyces cerevisiae, which has a DF value of 4 as a yeast with flocculating properties.
cerevisiae) RM-17 (hereinafter simply referred to as RM-17). In another preferred embodiment, the yeast Saccharomyces having a DF value of 0 is used as the non-flocculating yeast.
Saccharomyces uvarum DF-0
(Feikoken Bacteria No. 7791) (hereinafter simply referred to as DF-0), the DF value was 4 as a yeast with flocculating properties.
The yeast Saccharomyces uvarum AM-7 (Feikoken Bibori No. 7790) (hereinafter simply referred to as AM-7) is obtained. The parent yeast DF-0 is a yeast that has flocculating properties, Saccharomyces ubarum (Saccharomyces ubarum).
uvarum) ATCC-26602 (DF value = 3), which has lost its aggregation property as a result of the mutation (DF value = 0). Each parent yeast IFO-0224 and
DF-0 has various properties (fermentability and assimilation, physiological properties) as shown in Table 2 below.

[Table] In Table 2, the fermentability of raffinose is indicated by whether it can ferment some of the constituent monosaccharides fructose, glucose, and galactose produced by cleavage of the bond. That is, fermentability 1/
3 means that only fructose is fermented, 2/3 fermentability means that fructose and glucose are fermented, and 3/3 fermentability means that all constituent monosaccharides are fermented. Also, RM-17 and RM-17 obtained in these embodiments
AM-7 has the following mycological properties. That is, these yeasts have a flocculating property with a DF value of 4, and exhibit a remarkable sedimentation property in liquid culture. - Fermenting waste molasses (e.g. waste molasses containing 15% total sugar) to produce 7-9 vol% ethanol.・ Forms somewhat hard colonies on the agar plate.・Has spore-forming ability.・RM-17 requires adenine and histidine for growth. 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,
Yeast with excellent flocculating properties can be created in the laboratory. Therefore, there is no need for the conventional troublesome work of finding a desired strain of bacteria from nature and separating it from soil, and the desired flocculating yeast can be reliably produced. 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, increasing ethanol productivity. can be significantly improved. Examples Next, examples of the present invention will be shown to demonstrate the above effects. Example 1 (Production of RM-17) Non-flocculating yeast Saccharomyces cerevisiae 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, 0.1 ml of ethyl methanesulfonate was added 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. It was dispersed into After collecting bacteria, dilute the suspension with sterile 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, the strain Saccharomyces cerevisiae RM-17 (Feikoken Bacteria Collection No. 7770) with excellent aggregation properties was obtained. This strain was an adenine and histidine auxotrophic strain. Example 2 (Production method of AM-7) Saccharomyces uvarum DF- as a yeast without flocculating property
The same operation as in Example 1 was repeated using 0 (Feikoken Bibori No. 7791). As a result, the strain Saccharomyces uvarum AM-7 (Feikoken Bacteria Collection No. 7790), which had excellent aggregation properties, was obtained among the 40 strains on the master plate. Measurement of cohesiveness Regarding RM-17 and AM-7 obtained in Example 1 and Example 2, the degree of cohesiveness, i.e.
When the DF value was measured by the method described above, the DF value was 4 in all cases. 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 0.1M 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/

Claims (1)

[Claims] 1. A yeast that does not have flocculating properties and belongs to the genus Saccharomyces is subjected to sporulation treatment,
The obtained spores are subjected to mutation treatment, the mutant spores are cultured,
1. A method for producing yeast with excellent flocculating properties, which comprises detecting mutant strains from the obtained colonies by a replica method and isolating yeast with flocculating properties with a DF value of 4 or more. 2. Saccharomyces cerevisiae as a non-flocculating yeast
The manufacturing method according to claim 1, which uses IFO-0224 or Saccharomyces uvarum DF-O (Feikokenbokuyori No. 7791). 3 The yeast Saccharomyces cerevisiae IFO-0224 with a DF value of 0 was used as the yeast without flocculation, and the yeast Saccharomyces cerevisiae with a DF value of 4 was used as the yeast with flocculation.
cerevisiae) RM-17 (Feikoken Bibori No. 7770). 4 The yeast Saccharomyces ubarum has a DF value of 0 as a yeast that does not have flocculating properties.
uvarum) DF-0 (Feikoken Bibori No. 7791),
The yeast Saccharomyces ubarum has a DF value of 4 as a yeast with flocculating properties.
uvarum) AM-7 (Feikoken Bibori No. 7790).