JPS6344880A - Novel flocculating yeast, production thereof and alcoholic fermentation method using said yeast - Google Patents

Abstract

PURPOSE:To obtain a yeast of the genus Saccharomyces and carry out alcoholic fermentation with improved ethanol productivity using said yeast, by subjecting different auxotrophic strains of yeast of the genus Saccharomyces to protoplast fusion. CONSTITUTION:An auxotrophic stain of a yeast Saccharomyces cerevisiae IFO-1953 and auxotrophic strain of a yeast Saccharomyces S1 (FERM-P No.7794) are subjected to protoplast fusion. the resultant yeast Saccharomyces S1X1953-AA (FERM P-No.8895) has improved flocculation degree of 5 expressed in terms of DF value. The alcoholic fermentation is carried out by putting a preculture fluid of the above-mentioned yeast in a fermenter 1 containing a raw material by using an apparatus shown in the figure according to batch cultivation. When the culture medium is then continuously fed to perform alcoholic fermentation, flocks, are formed in the fermenter 1 by improved flocculation property of this yeast to afford the aimed high-concentration alcohol.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a novel yeast with excellent flocculating properties, its I! This paper relates to a production method and an alcoholic fermentation method using the same.

BACKGROUND OF THE INVENTION In recent years, fermented alcohol, which can be obtained without petrochemistry, has been attracting attention as an energy alternative to petroleum. This includes sugar cane, molasses, and even sweet potatoes5.
It is produced by fermenting starchy or cellulosic materials such as potatoes and corn 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 addition, in continuous fermentation, a basic fermenter is used, which mainly consists of a small-diameter flow section, a large-diameter sedimentation section for bacterial cell sedimentation connected above this, and a bacterial cell sedimentation member built into this. Thereby, even if the supply layer of the culture medium 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 desired.

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
The purpose of the present invention is to provide a novel flocculating yeast that has excellent flocculating properties and can be obtained in a laboratory.

The present inventors first developed a yeast (FRH) that has excellent flocculating properties and alcohol fermentation ability by mutation treatment and protoplast fusion method from yeast that can grow well in blackstrap molasses.
1□VM21'>81 was created. However, when continuous fermentation is carried out using this yeast and the dilution rate is increased, D=
At 0°5h-1, there was a tendency for the cohesiveness to become slightly weaker.

Therefore, yeast IFO-1953, a type culture with remarkable flocculation, and the above yeast (FRH1□v, 2-
1) By performing protoplast fusion with S4, a new strain with excellent flocculation and high ethanol productivity was obtained, and the invention was completed.

Means for Solving the Problems The first aspect of the present invention is a novel yeast, that is, a yeast SaCCalomyces having excellent flocculating properties.
es) S 1X 1953-AA (Feikoken Bibori No. 8895) itself, the second invention is a method for producing yeast 5IX1953 AA of the present invention, and the third invention is a method for producing yeast 5IX1953 AA of the present invention. This is an alcoholic fermentation method using yeast 5IX1953-AA.

First, the yeast St x1953-AA itself of the present invention will be explained.

In this specification, the degree of flocculation of yeast
of flocculation) is the Gilliland test shown below.
(European Journal of App
Lied Hicrobiology and Bio
Technology Vol. 7, No. 227-234, 1
979) is displayed as the DF value determined by That is, after culturing the test bacterial strain in YPD 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, and the results are shown in Table 1. The degree of aggregation is displayed in six stages from DF value O to 5.

(The following are blank spaces) Table 1 The yeast S1×1953 AA of 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 (e.g. blackstrap molasses containing 15% total sugar) to produce 7-9 vol% ethanol.

- Forms somewhat hard colonies on agar plate.

・Has spore-forming ability.

The yeast S1x1953 AA of the present invention also has various properties (presence or absence of fermentability and assimilation, physiological properties) as shown in Table 2 below.

(Margin below) Table 2 In addition, Saccharomyces (Saccharomyces)
) yeasts belonging to the genus are known to have the following mycological properties (J. Lodder [Theve
asts, A TaXOnOmiC5tudy” 2nd edition, N0rth-HOIland Publisher
ng Publishing, 1970), the yeast of the present invention also has these properties.

In other words, yeast belonging to this genus are - Propagates by multipolar budding.

- Forms ascospores.

・Does not utilize nitrates.

・Lack of or only a few mycelial threads are formed.

-Mature ascus does not cleave easily.

・The shape of the spores is spherical or oval.

・Ferment glucose well.

・Does not form a film on the wort medium.

As a medium for the yeast of the present invention, 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. As a preculture medium, yeast extract 1q1
Polypeptone 20. glucose 2q1 distilled water 1007f
A medium consisting of t/ is often used.

The pH of this medium is 5.5 without adjustment.

Cultivation is carried out at a temperature of 25 to 40°C, preferably 30 to 37°C, and p
) 13.0 to 7.0, preferably pH 3.5 to 6.0.

Next, the method for producing yeast of the present invention will be explained.

The yeast of the present invention is the yeast Satucharomyces cerevisiae (
Saccharomyces cerevisiae)
The auxotrophic strain of I F 0-1953 (hereinafter referred to as yeast H2-1953) and the yeast Satucharomyces (
Saccharomyces ) (F RH17VM
2-1) Manufactured by protoplast fusion with an auxotrophic strain (hereinafter referred to as yeast m S + ) of S. be done.

Yeast)-12-1953 and yeast St are both D
It has a cohesive property with an F value of 5.

Protoplast fusion is performed by conventional methods. Usually, each bacterial cell suspension is prepared at a concentration of 107 to 108 cells/rIJI, and after mixing preferably equal amounts of these suspensions, the mixture is treated with a protoplast preparation solution containing yeast cell wall lytic enzyme. Alternatively, each bacterial cell suspension is treated with the same preparation solution and then mixed.

Yeast 1-12-1953 and yeast S1 were yeast IFO-195, respectively, as demonstrated in the Examples below.
3 and yeast (FRH1□VM2-1) Sl are subjected to sporulation treatment, the resulting spores are subjected to mutation treatment, and the resulting mutant spores are cultured, thereby obtaining the mutant spores with good reproducibility.

Sporulation treatment is carried out according to conventional methods. Usually yeast is Y
After culturing on a PD agar medium (Note 2), a method is used in which the cells are smeared onto a sporulation agar medium (Note 3). To obtain cells derived from single spores, you can lyse the asci using a lytic enzyme for lysing yeast cell walls and then separate the spores using a micromanipulator, or use the same lytic enzyme to separate the spores. After dissolving the spores, the spores are dispersed by ultrasonication, and the spores are then cultured on a nutrient agar medium.

The mutation treatment is performed by subjecting the spores or asci obtained by the sporulation treatment to known mutation treatments, such as ultraviolet rays, X-rays, γ
Physical method of irradiating rays, ethyl methanesulfonate,
N-methyl-N'-nitro N-nitrosoguanidine,
Although this can be done by any chemical method involving contact with a mutagenic agent such as 4-nitroquinoline-N-oxide followed by growth on a selective medium, the method using ethyl methanesulfonate is particularly preferred.

In the above series of manufacturing processes, the culture medium and culture conditions are as follows:
The medium and culture conditions for the yeast itself are the same as described above.

Next, a method for producing yeast as a raw material for the yeast of the present invention will be explained.

(F RHI3 V 82 1 ) S + is the yeast Saccharomyces F
It is produced by subjecting R-Old 7VM2-1 (Feikoken Kyoiku No. 7792) to sporulation treatment and culturing the obtained spores.

This sporulation treatment is also performed in the same manner as described above.

Yeast FRH1□■H2-1 is a yeast strain of Saccharomyces cerevisiae that has flocculating properties.
S cerevisiae) RM-17 (Feikoken Bibori No. 7770) and the non-flocculating yeast Saccharomyces c.
erevisiae) VM-2 (Microtechnical Laboratory No. 77
No. 88) and protoplast fusion treatment and culturing the resulting fused bacterial cells. This protoplast fusion is also performed in the same manner as described above.

Yeast RM-17 is a strain of the yeast Saccharomyces cerevisiae, which is kept by the Fermentation Research Institute.
myces cerevisiae) I F O-0
224 was subjected to sporulation treatment, the resulting spores were subjected to mutation treatment,
produced by culturing mutant spores, and yeast VM
-2 is yeast Satsuriro' that does not have flocculating properties? Saccharomyces cerevis
iae) It is produced by subjecting EY-1 (Feikoken Kyoiku No. 7793) to sporulation treatment, subjecting the obtained spores to mutation treatment, and culturing the mutant spores. This sporulation treatment and mutation treatment are also performed in the same manner as described above.

Effects of the Invention Since the present invention is configured as described above, by carrying out alcohol fermentation using the flocculating yeast of the present invention, the bacterial cell concentration in the alcohol fermenter can be maintained at a high level in both batch fermentation and continuous fermentation. As a result, ethanol productivity can be significantly improved.

EXAMPLES Next, the present invention will be explained so that it can be reproduced by a person skilled in the art.

■ Production example (a) Preparation of yeast S, which has flocculating properties (F RH17VM21) S+ to YP
The cells were cultured on a D agar medium (Note 2) at a temperature of 30°C for 24 hours, then spread on a sporulation agar medium (Note 3), and cultured at a temperature of 30°C for 3 to 5 days. In this way, spores were formed.

Then the number of spores is 10'/ro11. Suspend the ascus in 1 ml of sterile water, and after collecting the bacteria, add phosphate buffer (
Washed with Note 4). Next, the asci were treated with a lytic enzyme solution (Note 5).
) 211II at 30° C. for 1 hour to lyse the asci. Then, after collecting the bacteria, the released spores were added to sterile water (1 w/w).
and suspended in phosphate buffer solution.

Add 0.1w of ethyl methanesulfonate to this suspension as a mutagenic agent! and the suspension was shaken for 2 hours at 30°C. The spores were thus mutated. Then, after collecting the bacteria, the mutant spores were added to 0.2w of phosphate buffer! and add 5% to the suspension.
% aqueous sodium thiosulfate solution 3III 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 volume of phosphate buffer, suspended in 5N of the same buffer, and the suspension is sonicated for 3 minutes on ice to make the mutant spores into a suspension. dispersed inside.

After collecting the bacteria, the suspension is diluted with sterile water to a concentration of 1/105 to 1/1/2.
The suspension was diluted to 10'', and 0.1 ml of the diluted suspension was spread on a YPD agar medium (Note 2) and cultured at 30°C for 48 hours to obtain a colony derived from a single spore.

Using the plate of the colony thus obtained as a master plate, mutant strains were detected by the replica method. That is, using sterilized velvet cloth, the colonies on the master plate were replicated on a minimal medium (Note 6), 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 collected from the master plate.

As a result, an auxotrophic strain S1 was obtained. Obtained yeast S1
The cohesiveness of (FR, 1□VM,, -1)S was not decreased compared to that of (FR, 1□VM,, -1)S.

(b) Preparation of yeast 1-IZ-1953 IFO-1953, which has flocculating properties, was subjected to sporulation and modification using the same method as in the preparation of yeast S, and auxotrophic strain H2-1953 was produced. Obtained.

(c) Protoplast fusion enzyme ff1s+ of yeast S and yeast H2-1953 was cultured with shaking at 30°C for 16 hours in YPD medium 10, and washed with mixture 1 after harvesting.

Next, this was mixed into a protoplast preparation solution (Note 7) of approximately 211/
The suspension was shaken at 30° C. for 1 hour, and after harvesting the bacteria, the mixture (Note 8) was washed twice per day.

Yeast H2-1953 was also treated in the same manner as above.

Next, the treated cells of yeast s1 and yeast H2 obtained in this way
-1953 treated cells in the same amount (108 cells/1 ml)
After bacterial collection, the mixture was suspended to an isotonic ratio of 0.1%, and a 2N 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, the cells were suspended in an isotonic solution of 1 wl, and the suspension was incubated at 20°C for 15 minutes.
It was left standing for a minute. Then, the suspension is isotonicly transferred to the bottom.
- The diluted suspension was diluted to 1/10 to 1/102, and the diluted suspension was smeared on a minimal medium (Note 6), and overlaid with a medium for overlay (Note 10). In this state, culture was carried out at 30°C for 4 days, resulting in aggregation (
A fusion strain with a DF value of 5) was isolated, and this strain was transformed into yeast S
It was set as 1x1953-A.

In addition, the parent strains S and H2- used for protoplast fusion
1953 could not grow on the above minimal medium.

(d) S, x1953-A strain, which is a fused strain of yeast S1 x 1953'A strain acclimated to molasses medium, was incubated in YPD medium 1.
A preculture solution was obtained by culturing with shaking at 30°C for 16 hours. Next, ammonium sulfate 4.18g/l and potassium pyrosulfite 0.2g were added to Philippine blackstrap molasses 4189/I.
The preculture solution was inoculated into a medium 11 obtained by mixing and dissolving /l and adjusting the pl to 14.5 with sulfuric acid. After culturing this with stirring for 24 hours, let it stand for 5 minutes, remove the culture medium leaving 300 d, and add new medium to make 11.
Culture was restarted. After continuing the culture for 10 times in this way,
The obtained yeast was designated as S1×1953 AA (Feikokenbokuki No. 8895).

■ Measurement of flocculation and alcohol fermentation ability yeast IFO-
1953, FRM1□VM2-1) S+, S+ and S'+

The DF value was determined by the method described above.

In addition, alcohol fermentation ability was determined by the following method.

That is, Philippine blackstrap molasses 475q/l, ammonium sulfate 4.75a/1 and potassium pyrosulfite o, 2a.
/li was mixed and dissolved, the pH was adjusted to 4.5 with sulfuric acid, the preculture solution was added to the obtained medium 11, and this was mixed in a mini jar fermenter (Model M-100 manufactured by Tokyo Rikakikai Co., Ltd.). Batch culture was carried out at a temperature of 35°C, and the amount of ethanol produced after 144 hours was measured by gas chromatography.

= 20- at! The I determination results are shown in Table 3 below.

-21-,.

Table 3 ■ Usage Example (Continuous Alcohol Fermentation) Continuous alcohol fermentation was carried out using yeast S and x1953-AA 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 main body is a glass fluidized bed fermenter (1) with an actual volume of 700 m, and is configured to be able to control temperature and I)H.

Then, the fermentation raw material is supplied to the bottom of the tank (1) by the pump (2), and the reaction liquid is returned from the top to the bottom of the tank by the pump (3), and the bacterial cells at the top of the tank are transferred to the ridge (4). It is supposed to flow out from.

YPD medium (Note 1) I in a 5'OOd Sakaro flask
After adjusting OC)d and sterilizing it at a temperature of 121°C for 10 minutes, yeast S was stored in a YPD agar slant medium (Note 2).
One white fungus was inoculated with the Sho X1953-AA strain and cultured at 30°C overnight.

In this way, a preculture of active yeast S1x1953AA was obtained.

100 d of the above preculture solution was put into a fermenter (1) containing 600 d of Philippine blackstrap molasses medium (attention), and batch culture was carried out at a fermentation temperature of 30° C. for 8 hours. Next, the continuous fermentation molasses medium (Note 12) was diluted with tap water, and the diluted solution was fed into the fermenter (1) at a flow rate of 35 d/hour (dilution rate -0,05 hours -1).
Continuous fermentation was carried out by gradually increasing the amount of culture medium supplied.

As a result, the amount of culture medium supplied was 350ml! /hour (dilution rate -0
, 25 o'clock-1), flocs with a diameter of 1 to 4 mm were formed within the premises due to the excellent flocculating property of this yeast.

In addition, the produced alcohol was obtained at a high concentration of 649/I, and the alcohol productivity (Note 13) reached a high value of 32 g/l·hr at a dilution rate of 0° and 5 layers, as shown in Figure 2.

■ As a comparative yeast, the yeast E was used instead of S1x1953-AA.
The above operation was repeated using Y=1 and making other matters the same as in the above usage example.

As a result, when the medium supply rate exceeds 70 d/hour (dilution rate = 0.1 hour - 1), alcohol productivity (Note 13) decreases to the second level.
As shown in the figure, it suddenly decreased from 4 o/l.

■Medium and reagents The media and reagents are as follows.

(Note 1) YPD medium yeast extract 10g/l Polypeptone 20g// Glucose 20g// (Note 2) YPD agar medium Yeast extract 10g// Polypeptone 20g// Glucose 20w/1 Agar 20g// (Note 3) For spore formation Medium Sodium acetate 5g/l Agar 20ci/1 (Note 4) Phosphate buffer 0.1M phosphate buffer pI-1 = 7.5 (Note 5) Lytic enzyme solution 0.1M phosphate buffer (pH 7,5 Solution 2 of 0.05% Zymolyase 20T (manufactured by Seikagaku Corporation) dissolved in )
Mixed solution of Gato and 1.4 μl of 2-mercaptoethanol (Note 6) Minimal medium Difco-Yeast Nitrogen Ba5e
WloAmino acid (manufactured by Difco)6.
7g//glucose 20ci/1 agar
20 g/l (Note 7) Protoplast preparation solution 1.5 M potassium chloride 0.8 layer, 2/15 M phosphate buffer (pH 7.5>1.ON), 2-mercaptoethanol 1.4 μl, zymolyase 20T ( (manufactured by Seikagaku Corporation) in 0.1M phosphorus buffer (pH 7.5).
% solution (Note 8) Isotonic solution 0.6M potassium chloride aqueous solution (Note 9) Polyethylene glycol aqueous solution Calcium chloride 5.69/1 Polyethylene glycol (PEG-5olo) 300 g/ 1
(Note 10) Overlay medium glucose 209/I D1fco-Yeast Nitrogen Ba5e
WloAmino acid (manufactured by Difco), 6
．． 79/1 Difco-Bact Agar (Difc
o company) (attention) Philippine molasses culture Philippine molasses 190o# Ammonium sulfate 1.9 (+// Potassium pyrosulfite 0.2 (+/l Antifoaming agent)
0.5a// adjusted to pH 4.5 with sulfuric acid (Note 12) Molasses medium for continuous fermentation Philippine molasses 700o//Ammonium sulfate 3.
! M/1 Potassium pyrosulfite 1.20/l Antifoaming agent 1.0 (+// A mixed solution adjusted to pH 4.5 with sulfuric acid (Note 13) Produced in 1 hour per 11 alcohol-producing culture solutions Weight of alcohol (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. that's all

Claims (4)

[Claims] (1) The yeast Saccharomyces (S
accharomyces)S_1×1953-AA(
Microtechnology Research Institute No. 8895). (2) The yeast according to claim 1, which has a flocculating property of 5 in terms of DF value. (3) Yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae)
aromyces cerevisiae) IFO-1
953 auxotrophic strains and the yeast Saccharomyces (Sac
charomyces) (FR_M_1_7V_M_2
-1) Protoplast fusion with an auxotrophic strain of S_1 (Feikoken Bacterial Serial No. 7794) was carried out, and the resulting fused bacterial cells were cultured.
accharomyces)S_1×1953-AA(
1. A method for producing flocculating yeast, characterized in that a method for producing flocculating yeast is obtained. (4) The yeast Saccharomyces (S
accharomyces)S_1×1953-AA(
An alcoholic fermentation method characterized in that it uses A.