JPH0121755B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD This invention relates to a novel 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. The present invention was made in view of the above-mentioned circumstances, and an object of the present invention is to provide a novel flocculating yeast that has excellent flocculating properties and can be obtained in a laboratory. Means for Solving the Problems This invention is directed to the cultivation of the yeast Satucharomyces ubarum (which: - has a flocculating property with a DF value of 4, - has a sausage-shaped morphology, and - forms pseudohyphae depending on the culture conditions). Saccharomyces uvarum) AM-7 (Feikoken Bibori No. 7790) (hereinafter referred to as AM-7). 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】

[Table] AM-7 has the following mycological properties. That is, AM-7: - Has a flocculating property with a DF value of 4, and exhibits significant 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. As a medium for AM-7, 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. AM-7 is cultured at a temperature of 25-40℃, preferably 30-40℃.
It is carried out at 37° C. and at a pH of 3.0 to 7.0, preferably 3.5 to 6.0. Next, the manufacturing method of AM-7 will be explained. AM-7 is a non-flocculating yeast, Saccharomyces uvarum.
DF-0 (Feikoken Bibori No. 7791) (hereinafter simply DF-
0) 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. . The parent strain DF-0 is derived from the flocculating yeast Saccharomyces ubarum (Saccharomyces ubarum).
uvarum) ATCC-26602 (DF value = 3), which has lost its aggregation properties as a result of mutation. Furthermore, yeast belonging to Satucharomyces ubarum, such as AM-7 and DF-0, have 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. 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. The medium and culture conditions in the manufacturing process of AM-7 are the same as those for AM-7 itself described above. 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: • Proliferate by multipolar budding. - Forms ascospores.・Do not assimilate nitrates. - Lack of or only a few mycelial threads are formed.・Mature asci do not cleave easily. - Spores are spherical or oval in shape. - Ferment glucose well. - Does not form a film on the wort medium. Effect of the invention Since this invention is configured as described above,
A new yeast with excellent flocculation properties can be created in the laboratory. Therefore, there is no need for the conventional troublesome work of finding the desired bacterial strain from nature and isolating it from the soil.
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. a Production example Saccharomyces uvarum DF-0 (Feikoken Bacteria No. 7791), which does not have flocculating properties, was grown on YPG agar medium (Note 2) for 30 minutes.
The cells were cultured 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 harvesting, 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 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, among the 40 strains on the master plate, a strain of Saccharomyces uvarum AM-7 (Feikoken Bacteria No. 7790) with excellent aggregation properties was obtained. b Measurement of cohesiveness The degree of cohesiveness, ie, DF value, of the thus obtained AM-7 was measured by the method described above, and the DF value was 4. c Usage Example (Continuous Alcohol Fermentation) Continuous alcohol fermentation was carried out using AM-7 according to 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).
One strain of AM-7 was inoculated into the ear and cultured at 30°C overnight. In this way, an active AM-7 preculture was obtained. 100 ml of the above preculture solution was placed in fermenter 1 containing 700 ml of Philippine molasses medium (Note 7), 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 175 ml/hour (dilution rate =
0.25 hours ^-1 ), AM-7's excellent flocculation ability forms flocs with a diameter of 1 to 4 mm in the tank, and the bacterial cell concentration (Note 8) in the tank is as high as 47 g/1. The value was maintained. Also, the alcohol produced is 61
The alcohol productivity (Note 9) reached a high value of 16 g/hr, as shown in Figure 2. d Comparative example Using DF-0 instead of AM-7 as yeast,
The above operation was repeated with other matters being the same as in the above usage example. As a result, the medium supply amount was 70ml/hour (dilution rate =
If it exceeds 0.1 h ^-1 ), alcohol productivity (Note 9)
As shown in FIG. 2, the amount rapidly decreased from 4 g/hour. e 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) was dissolved in 0.1M phosphate buffer (PH 7.5). Mixture of 2ml of 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) Philippine molasses medium Philippine molasses 280g / Ammonium sulfate 2.8g / Potassium pyrosulfite 0.5g / Antifoaming agent 0.2g / A mixed solution consisting of: adjusted to PH4.5 with sulfuric acid (Note 8) 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 9) 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. The yeast Saccharomyces uvarum AM, which has a flocculating property with a DF value of 4, - has a sausage-shaped morphology, and - forms pseudohyphae depending on culture conditions. 7 (Microtechnology Research Institute No. 7790).