JP2954694B2 - How to get higher polyploids - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method of breeding yeast, and more particularly, to a method of producing a higher polyploid with extremely high efficiency by subjecting yeast to pressure treatment. It is about a completely new way of acquiring.

(Conventional technology and problems) As a method for obtaining a higher polyploid strain of yeast, conventionally,
A cell fusion method using a protoplast generation technique, a mutation method for artificially causing mutagenesis, and a hybridization method for performing direct sexual conjugation are known.

However, the cell fusion method requires a fusion technique such as a polyethylene glycol method or an electrofusion method and a regeneration technique, and in particular, a technique that poses a problem in breeding such that the cell fusion itself or processing takes a long time is inevitable. . In addition, the mutagenesis method requires the use of chemicals such as ultraviolet rays, γ-rays, and benomyl as mutagenic agents, but all of these require careful handling, especially when the chemicals are highly toxic or carcinogenic. Not only is there a problem in terms of safety due to the nature of the strain, but also in the field of breeding, there is a problem in the selection of the target strain and techniques in screening such as concentration.
In addition, the hybridization method also includes techniques that are problematic in breeding, such as the provision of auxotrophic markers. In these methods, the frequency of induction of higher polyploid strains is
Although it is 10 ^-2 , the cell fusion method has a low value of 10 ^-4 , and the mutation method has a further lower value of ^-6 .

As described above, all of the conventional methods not only include techniques that are problematic in breeding as structural defects, but the cell fusion method has a low induction frequency of the target strain, and the mutation method has a further problem. The frequency of induction is low, and it takes a lot of time and effort to select the target strain.Also, although the hybridization method can be evaluated tentatively in terms of induction frequency, a cumbersome and delicate preparation step is indispensable for implementing the hybridization method. Therefore, as a whole, a lot of trouble is inevitable.

On the other hand, the method according to the present invention relates to a method for obtaining a higher fold aqueous strain by pressure-treating yeast, but the present method is sufficient only by pressurizing yeast,
It does not include any breeding problems and does not require any special pretreatment. In addition, the present method has a high polyploid strain induction frequency of 10 ⁻¹ , which is the highest value among the methods known hitherto. Moreover, when each method is considered as a total work load, if this method is assumed to be 1 time, the cell fusion method, the mutation method, and the hybridization method will be 10 times, 20 times, and 30 times, respectively.
These conventional methods must be said to be particularly unsuitable as industrial methods.

Pressure treatment is used for pressure sterilization or egg gelation for killing bacteria by pressure treatment (for example, at a low pressure of 1000 bar or less and for 4 hours or more). And the technology of obtaining the higher polyploid is not known at all, and the various effects achieved by it have no suggestion from the prior art, and the present invention elucidates a completely new technical idea. is there.

(Means for Solving the Problems) The present invention requires less effort than conventional techniques, does not require delicate operations, and has a high frequency, that is, extremely high reproducibility, in a short time. The purpose of this study was to develop a method for obtaining higher polyploids.

Therefore, as a result of examination from various directions to achieve the above purpose,
In view of the fact that yeast is used for food, we focused on physical treatment rather than chemical treatment in terms of safety. And when the pressure treatment used to kill bacteria was dared to be applied to yeast, contrary to common technical knowledge, rather than unexpectedly, the yeast did not die but higher order multiples I have found a very useful finding that the body is produced and that it is produced at a very high frequency. We also discovered that the use of a dye-containing medium makes it very easy to sort out higher polyploids, thus solving the problem of screening for the target strain, which was one of the structural disadvantages of the conventional method. Successful. Furthermore, it was also confirmed that the higher-order polyploid yeast thus obtained, for example, baker's yeast, did not have any color different from diploid baker's yeast conventionally used in baking properties and the like.

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

In order to carry out the present invention, the yeast may be subjected to pressure treatment,
Known means can be used extensively as appropriate,
Various yeast strains with altered genetic properties are induced, for example, tetraploids and other higher polyploids are induced at a high rate. For this purpose, for example, a method of applying a hydrostatic pressure to the yeast using a pressure treatment device can be used, and a commercially available pressure treatment device can be used as appropriate. The conditions for the pressure treatment are, for example, hydrostatic pressure, pressure 1500 to 3000 bar, preferably 2
It is preferred to add 000 to 2500 bar. That is, 3000bar
If higher pressure is applied, the yeast is killed, and if the pressure is lower, it is not efficient. As a pressure solvent,
Organic solvents such as hexane, water and other aqueous solvents, mixed solvents thereof, and other commonly used pressure solvents can be appropriately used.

At that time, as the yeast, a yeast suspension, a wet cake, a yeast block, the yeast cells themselves, etc. can be used widely, and commercially available live yeast, semi-live yeast, etc. can also be used freely. it can. These may be housed in a plastic bag or other container having gas barrier properties in a conventional manner, sealed, and subjected to a pressure treatment. The temperature may be maintained in the range of 0 to 35 ° C, preferably 20 to 30 ° C. If necessary, the temperature may be freely selected without being limited to the above range. Processing time is also 3-20
Minutes, preferably 5 to 15 minutes, but it is of course possible to employ other processing times if necessary.

High-pressure treatment induces strains with altered genetic properties.However, this method not only has the remarkable effect of greatly increasing the frequency of mutagenesis in yeast, which had been low in the past, but also has a significant effect. The present invention has a remarkable effect that the identification or screening of a mutant strain, which has been extremely difficult, can be performed quickly and accurately by the present invention for the first time, and the present invention is particularly remarkable in breeding science.

Therefore, in the present invention, a dye-containing medium is used. That is, the yeast after the pressure treatment, after appropriate dilution if necessary, applied to a dye-containing agar medium, for example, 20 ~ 30 ° C.,
Culture for about 1 to 5 days to form a colony. Since the color tone is different between the mutated colony and the non-mutated colony, identification may be performed according to the difference in color tone. Therefore, according to the present invention, the screening of mutant strains can be performed industrially simply and accurately without skill by the present invention. It became possible. As a medium, a medium in which yeast can grow is appropriately used, and a wort agar medium, a YM agar medium, a YCB (DCB
ifco) agar medium and the like are used without limitation. As the dye, aniline blue, Ponceau 3R and other various dyes can be used.

For example, culturing yeast after pressurizing a medium containing aniline blue and Ponceau 3R results in a change in color tone of the mutated colonies and a ploidy of these colonies, as is clear from the examples described later. It contained a great number of changed ones, and it was confirmed that the mutant strain could be identified and screened very easily by the change in the color tone of the colony on the dye medium. Also, since this phenomenon is also observed in mutant strains by the hybridization method,
The method of the present invention for identifying mutant strains by color change is extremely versatile and can be widely used as a general breeding method for strains.

As described above, the present invention is not only excellent as a method for breeding yeast, but also effective in producing a large amount of the contents because large cells can be obtained. For example, DN
A, mass production of nucleic acid related substances such as RNA, proteins such as SCP, other yeasts, antibiotics, sorting active substances becomes possible,
The present invention makes a very important contribution not only in the field of breeding but also in the field of biotechnology such as the fermentation industry and the gene-related industry.

For example, in the case of baker's yeast, the obtained higher polyploid is a diploid baker's yeast conventionally used in various bread-making properties and safety, such as fermentation power in bread, flavor, color and structure of bread. There was no disadvantage in terms of application to bread making due to the mutation treatment.

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

Example 1 Saccharomyces cerevisiae
visiae), yeast suspensions of the yeast strains FA-1 and P-554 were placed in a plastic container and sealed.

The sealed yeast was subjected to a hydrostatic pressure of 2500 bar using a pressure treatment device (KP5B manufactured by Hikari Kogaku Kiki Co., Ltd.). At that time, the temperature was 25 ° C, the pressure holding time was 10 minutes,
Hexane was used as the pressure solvent. In addition, P-55 which is a hybrid strain of sake yeast x baker's yeast and baker's yeast x baker's yeast.
7 and P-559 were treated in the same manner.

Dilute each yeast suspension after the pressure treatment,
It was spread on a YCB (Difco) agar medium containing blue and Ponceae 3R, and allowed to form colonies at 25 ° C. for 3 days. Changes in the color tone of the colonies were observed, and the results shown in Table 1 were obtained.

As is clear from the count results of the colony strains described above, by performing the pressure treatment, a higher polyploid was obtained at a very high frequency, and the change in the color tone of the colony formed using the dye-containing medium was observed. Observation revealed that higher order polyploids could be identified and screened at a very high rate and accuracy.

In this example, the ploidy of the cells was determined in terms of cell size and DNA content as shown in Table 2. The size of the cells was determined using an Elzone particle counter 80XY (Particle
Date. Inc.) and the DNA content of the cells was determined according to the method of M. Aigle et al.

Example 2 (1) S. cerevisiae strain P-554 was subjected to pressure treatment in the same manner as in the previous example, and the mutant strain P-554 was treated with a dye medium.
554 (P) [(P) indicates a pressurized strain] was selected and obtained. Next, P-554 and P-554 (P) were cultured in a 30-jar apparatus under the following conditions, and a general analysis was performed for each item in Table 3 to obtain the results in the same table.

(2) For each of these two strains, the fermentation ability of each yeast block in a 30% sugar dough after storage at 30 ° C. for 3 days (3.
H) was measured and thereby the shelf life was evaluated. Table 4 shows the results of the storage stability test.

(3) For these two strains, cell size and cell color were measured, and the results in Table 5 were obtained. The size of the cells was measured with an Elzone particle counter, and the color of the cells was measured with a color difference meter.

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

(4) Using the baker's yeasts P-554 and P-554 (P), confectionery buns were manufactured by the saccharification seeding method according to the following formulation and process, and the bread making results shown in Table 6 were obtained.

As is evident from the results of the above bread making test, the tetraploid yeast obtained by the pressure treatment was completely tinged with the conventional diploid strain, including the processability of the dough during the process and the quality of the baked product. Was confirmed to be excellent as a yeast for baking.

Example 3 Kluyveromyces thermotolerance (Kluyveromy
ces thermotolerans) in a plastic container as in Example 1 and sealed, 2,500 at 25 ° C for 10 minutes.
Bar hydrostatic pressure was applied. As a result, a large cell strain was obtained.

Example 4 Torulaspora delbrucky
A 10% suspension of the eckii) strain was placed in a plastic container, sealed as in Example 1, and subjected to a hydrostatic pressure of 3000 bar at 25 ° C. for 10 minutes. As a result, a large cell strain was obtained.

(Effect of the Invention) According to the present invention, not only a polyploid can be obtained at a very high frequency by a simple step of pressurizing yeast, but also a mutant can be extremely isolated by a change in the color tone of a colony in a dye-containing medium. It can be screened easily and accurately and therefore the present invention makes a very great contribution in breeding science.

Further, for the higher polyploid generated by the present invention,
The obtained cells are embedded in an agarose block, DNA is extracted by performing zymolyase and protease treatment, the electrophoresis is performed by the CHEP method, the yeast chromosome DNA pattern is also examined, and the higher order multiple according to the present invention is used. It was also confirmed that the body was the target.

Furthermore, the higher polyploid according to the present invention has not only a breeding advantage that a diploid has become, for example, a tetraploid,
In the actual bread-making properties, the food and fermentation industries do not have the same color as conventional yeast at all, and increase the yield of useful substances secreted and produced by yeast, such as DNA, enzymes, proteins, antibiotics, and physiological substances. It also makes a very important contribution in fields such as biotechnology.

Claims (5)

(57) [Claims] 1. A method for obtaining a higher polyploid, comprising subjecting yeast to high pressure treatment. 2. The method according to claim 1, wherein the high pressure treatment is a hydrostatic high pressure treatment at 1500 bar to 3000 bar. 3. The method according to claim 1, wherein the yeast is Saccharomyces yeast. 4. The method according to claim 4, wherein the yeast is baker's yeast. 5. A method for obtaining a higher polyploid, comprising culturing yeast after high-pressure treatment on a dye-containing agar medium and selecting a higher polyploid based on a change in color tone.