JPH04183386A - Collection of high-order ploid - Google Patents

Abstract

PURPOSE:To collect a high-order ploid in a short time and in extremely high frequency without requiring delicate operation by subjecting yeast to high- pressure treating under a specific hydrostatic pressure, culturing the yeast in a dyestuff-containing agar medium after the treatment and extinguishing change of color tone. CONSTITUTION:A suspension of yeast such as yeast belonging the genus Saccharomyces is packed into a container such as plastic bag having gas barrier properties, sealed by using a pressure solvent such as hexane and subjected to high-pressure treatment under hydrostatic pressure of 1,500-3,000bar. Then the yeast after the pressure treatment is applied to malt juice agar medium containing a dyestuff such as Aniline blue and cultured to form a colony. A variant can be simply and accurately screened by distinction according to change of color tone. Consequently, a high-order ploid of the yeast is collected simply and in high frequency and the variant is extremely simply and accurately screened to greatly contribute to thremmatology.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for breeding yeast, and more specifically, it relates to a method for breeding yeast, and more specifically, it is possible to produce higher-order polyploids with extremely high efficiency by pressurizing yeast. It concerns an entirely new method of acquisition.

(Conventional technology and problems) How to obtain higher polyploid strains of yeast.

Conventionally, cell fusion methods using protoplast generation technology,
A mutation method that artificially induces mutagenesis.

Crossing methods that involve direct sexual conjugation are known.

However, the cell fusion method requires fusion and regeneration technologies such as the polyethylene glycol method and electrofusion method, and in particular, techniques that pose problems in breeding, such as requiring a long time for cell fusion itself and processing, cannot be avoided. In addition, the mutation method requires the use of ultraviolet rays, gamma rays, or chemicals such as benomyl as mutagenic agents, but all of these require careful handling, and especially in the case of chemicals, they are highly toxic. Not only is there a problem in terms of safety due to carcinogenicity, but also problems in screening techniques such as selection and enrichment of target strains have become a problem in terms of breeding. Crossing methods also include techniques that pose problems in breeding, such as the provision of auxotrophic markers. And in these ways.

The frequency of high-order polyploid strain induction is lo-2 in the hybridization method, 10-4 in the cell fusion method, which is low, and even lower at 104 in the mutation method.

In this way, all conventional methods not only include techniques that pose breeding problems due to their structural defects, but also the cell fusion method has a low frequency of inducing the desired strain, and the mutation method has even more problems. In addition, the induction frequency is low, and it takes a lot of effort and time to select the desired strain, and although the hybridization method can be evaluated in terms of the induction frequency, a troublesome and delicate preparatory stage is essential for implementing the hybridization method. In the end, a great deal of effort is unavoidable.

On the other hand, the method according to the present invention is related to a method for obtaining a high-order polyploid strain by pressure-treating yeast, but in this method, pressure-treating yeast alone is sufficient, and it is not suitable for breeding. It does not involve any problematic technology and does not require any special pre-processing. Moreover, this method has a high-ploidy strain induction frequency of 10-1, which is the highest value among conventionally known methods. Moreover, when looking at the overall workload of each method, if this method is 1 times as much, the cell fusion method,
The mutation method and hybridization method are 10 times and 20 times, respectively.

30 times, and these conventional methods are particularly unsuitable as industrial methods.

Although pressure treatment is used for pressure sterilization to kill bacteria by pressure treatment (for example, at a low pressure of 1000 bar or less and for 4 hours or more) and for gelling eggs, it is not possible to pressure treat yeast as in the present invention. There is no known technology for obtaining such higher-order multiples, and there is no suggestion from the prior art regarding the various effects achieved by it, and the present invention has elucidated a completely new technical idea. be.

(Means for Solving the Problems) The present invention requires less time and effort than conventional techniques, does not require delicate operations, and can be performed in a short time and with extremely high frequency, that is, with extremely high reproducibility.

This was done for the purpose of developing a method for obtaining higher polyploid yeast.

Therefore, as a result of consideration from various aspects in order to achieve the above objectives, #
Considering that the meat would be used for food, my mother decided to use physical treatment instead of chemical treatment for safety reasons. When we dared to apply pressure treatment, which was used to kill bacteria, to yeast, we found that, completely unexpectedly, or rather contrary to common technical knowledge, the yeast did not die, but instead increased in high-order multiples. We discovered the extremely useful knowledge that the body is generated, and that it is generated at a very high frequency. They also discovered that the selection of higher-order polyploids can be carried out extremely easily by using a single-dye-containing medium, which has helped solve the problem of screening for target strains, which was one of the structural shortcomings of conventional methods. Successful. And furthermore,
It was also confirmed that the higher-order polyploid yeast, such as baker's yeast, obtained in this manner is not similar in bread-making properties to the conventionally used diploid baker's yeast.

The present invention was completed as a result of further studies based on these useful new findings, and will be described in detail below.

To carry out the present invention, yeast may be pressure-treated, and a wide variety of known means can be appropriately used for this purpose, and various yeast strains with altered genetic properties are induced, for example, 4
Polyploids and other higher-order polyploids are induced at a high rate. 1 for that
For example, a method such as applying hydrostatic pressure to yeast using a pressure treatment device can be used, and as the pressure treatment device, commercially available devices can be used as appropriate. The conditions for the pressure treatment are, for example, hydrostatic pressure, preferably 1500 to 3000 bar, preferably 2000 to 2500 bar. In other words.

This is because applying a pressure higher than 3000 bar will kill the yeast, and lower pressure is not efficient.

As the pressure solvent, organic solvents such as hexane, water and other aqueous solvents, mixed solvents thereof, and other commonly used pressure solvents can be used as appropriate.

At that time, yeast suspensions, wet cakes, yeast blocks, yeast cells themselves, etc. can be used in a wide range of ways, and commercially available live yeast, semi-live yeast, etc. can also be used freely. can. These may be placed in a plastic bag or other container having gas barrier properties, sealed, and subjected to pressure treatment according to a conventional method. The temperature may also be maintained within the range of 0 to 35°C, preferably 20 to 30°C, but if necessary, it may be freely selected without being limited to the above range. The processing time is also 3
The treatment time may be 20 minutes, preferably 5 to 15 minutes, but it is of course possible to adopt a treatment time other than the above if necessary.

High-pressure treatment induces strains with altered genetic properties, but water droplets not only have the remarkable effect of greatly increasing the frequency of mutation induction in yeast, which had traditionally been low in induction frequency, but also The present invention is particularly noteworthy in the field of breeding, as the present invention has succeeded in quickly and accurately carrying out the extremely difficult identification or screening of mutant strains for the first time.

For this purpose, a single dye-containing medium is used in the present invention. That is, the yeast after the pressure treatment is diluted as appropriate if necessary, and then applied to an agar medium containing one dye.

For example, the cells are cultured at 20 to 30°C for about 1 to 5 days to form colonies. Colonies that have mutated and colonies that have not mutated have different colors, so they can be identified based on the difference in color. Therefore, with the present invention, for the first time,
It has become possible to screen for mutant strains easily and accurately on an industrial scale without requiring any skill. As the medium, a medium in which yeast can grow is appropriately used, including but not limited to wort agar medium, YM agar medium, YCB (Difco) agar medium, and others. As the dye, aniline blue, Ponceau 3R, and various other dyes can be used.

For example, when pressurized yeast is cultured in a medium containing aniline blue and Ponceau 3R, as is clear from the examples below, mutated colonies change in color, and these colonies have no ploidy. It was confirmed that there were a large number of altered strains, and that mutant strains could be identified and screened very easily by the change in color tone of colonies on the dye medium. In addition, this phenomenon is also observed in mutant strains produced by hybridization.
The method of identifying mutant bacterial strains based on changes in color tone according to the present invention is extremely versatile and can be widely used as a general breeding method for bacterial strains.

As described above, the present invention is not only excellent as a yeast breeding method, but also enables large cells to be obtained, so it is also effective in producing a significant amount of the contents.
It has become possible to mass-produce nucleic acid-related substances such as NA and RNA, proteins such as SEP, and other enzymes, antibiotics, and physiologically active substances, and is useful not only in the field of breeding but also in the technical field of biotechnology such as fermentation industry and gene-related industry. The present invention also makes an extremely important contribution.

In addition, for example, in the case of baker's yeast, the obtained higher polyploid is
It is completely similar to the conventionally used diploid baker's yeast in terms of bread-making properties such as fermentation power, bread flavor, color, structure, etc., and safety, and it is suitable for use in bread-making through mutation processing. No shortcomings were observed at all.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Saccharomyces cerevisiae
Yeast strain FA- belonging to
The yeast suspensions of strains 1 and P-554 were placed in a plastic container and sealed.

Hydrostatic pressure of 2500 bar was applied to the sealed yeast using a pressure treatment device (manufactured by Hikari Kokatsu Kiki Co., Ltd.: KP5B type). At that time, the temperature was 25℃, and the pressure holding time was 10
Hexane was used as the pressure solvent. In addition, strains P-557 and P-559, which are crossbreeding methods of sake yeast x baker's yeast and baker's yeast and span yeast, were also treated in the same manner.

After the pressure treatment, each yeast suspension was diluted and added to Anili.
The cells were plated on a YCB (Difco) agar medium containing ne blue and Ponceau 3R, and incubated at 25°C for 3
Colonies were allowed to form for several days, and changes in colony color were observed to obtain the results shown in Table 1.

Table 1 P-554 diploid 114150 P-557 tetraploid 014010 P-559 tetraploid 017018 As is clear from the above colony strain count results, high-order polyploidy occurs extremely frequently by performing pressure treatment. It has been found that higher-order polyploids can be identified and screened at an extremely high rate and accurately by observing changes in the color tone of colonies formed using a dye-containing medium.

In this example, cell ploidy was determined based on cell size and DNA content as shown in Table 2. Cell size is 80X in an Elzone particle counter with a 48μ orifice tube and a 100μg volume tube.
Y (Particle Data, Inc.), and the DNA content of the cell is N, ^1g1e
et al.

Measured according to the method.

Table 2 P-554 No diploid 4.18 27
．． 5P-specific tetraploid 5.51
63.6to559 None Tetraploid
5.69 55.9 P-559 Tetraploid 5.72 63.7 Example 2 (1) P-554 strain of S. cerevisiae was subjected to pressure treatment in the same manner as in the previous example and cultured in a single dye medium. Mutant strain P-554CP) ((P) indicates pressure-treated strain) was selected and obtained. Then, P-554 and P
-554(P) was cultured in a 3012 jar apparatus under the following conditions, and general analysis was performed for each item in Table 3 to obtain the results shown in the table.

Fake J 1st grade 400g Preparation 1.4kg C/N/P 100/410.4 Ventilation
16L/win Stirring 600rpm Table 3 Umbrella The dough fermentation power was shown by the amount of gas generated in the dough containing 30% sugar and high fructose sugar.

(40g dough, 30℃, 3 hour value) (2) For these two strains, the fermentation power (3 hours) in 30% sugar dough after storing each yeast block at 30℃ for 3 days was measured. Preservability was evaluated. The results of the shelf life test are shown in Table 4.

Table 4 (3) Cell size and cell color were measured for these two strains, and the results shown in Table 5 were obtained. Note that the cell size was measured using an Elzone particle counter, and the cell color was measured using a color difference meter.

Table 5 As is clear from these results, it was found that pressure treatment did not cause any particularly large changes in the color of the cells.

(4) Using baker's yeast P-554 and P-554(P), sweet breads were produced by the sweetened dough method according to the following formulations and steps, and the bread making results shown in Table 6 were obtained.

Distribution 1 million 70- & 130 Salt 1 Shortening 5 NFDM 2 Whole eggs
5 Yeast 3 C Aunty SO・1 Knee”i Kneading time LIM2 LIM3 Kneading temperature
25.5 28.0 Fermentation time 2.5
hr Floor - 40, 55 Bench 15 As is clear from the results of the above bread making test.

It has been confirmed that the tetraploid yeast obtained by pressure treatment is completely similar to conventional diploid strains, including the dough processing properties during the process and the quality of baked goods, and is suitable for use as yeast for bread making. It turned out to be excellent.

Example 3 Kluyveromyces thermotole
A 10% suspension of R. rans) was placed in a plastic container in the same manner as in Example 1, sealed, and heated to 2500 b
A hydrostatic pressure of ar was applied. This resulted in a large strain of cells.

Example 4 Torulaspora delbreckii (Torulaspora)
Example 1 A 10% suspension of
Similarly, it was placed in a plastic container and sealed, and a hydrostatic pressure of 3000 bar was applied for 10 minutes at 25°C. This resulted in a large strain of cells.

(Effects of the Invention) According to the present invention, not only can high-order polyploids be obtained with an extremely high frequency through the simple process of pressurizing yeast, but also mutant strains can be identified by changing the color tone of colonies in a pigment-containing medium. Screening can be performed easily and accurately, and the present invention therefore makes an extremely significant contribution to breeding science.

In addition, regarding the higher order polyploid produced according to the present invention, the obtained cells were embedded in an agarose block, treated with zymolyase and protease to extract DNA, and subjected to electrophoresis using the CHEP method to extract yeast chromosomal DNA.
After examining the pattern, it was confirmed that the higher-order polyploid according to the present invention was the desired one.

Furthermore, the high-order polyploid according to the present invention not only has the breeding advantage that a diploid has become, for example, a tetraploid, but also has an actual bread-making property that is completely similar to conventional yeast. It also makes an extremely important contribution to the food industry, fermentation industry, biotechnology, and other fields by increasing the yield of useful substances secreted and produced by yeast, such as DNA, enzymes, proteins, antibiotics, and physiological substances. be.

Claims (5)

[Claims] (1) A method for obtaining higher-order polyploids, which is characterized by subjecting yeast to high-pressure treatment. (2) The method according to claim 1, wherein the high pressure treatment is a hydrostatic high pressure treatment of 1500 bar to 3000 bar. (3) The method according to any one of claims 1 to 3, wherein the yeast is a yeast of the genus Saccharomyces. (4) Claim 4 characterized in that the yeast is baker's yeast.
The method described in. (5) A method for obtaining higher-order polyploids, which comprises culturing yeast after high-pressure treatment on a dye-containing agar medium, and selecting higher-order polyploids based on a change in color tone.