JP3289065B2 - Method for testing characteristics of enokitake mushroom cells and method for producing strains - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for assaying the characteristics of Enokitake mushroom cells, and more particularly, to a method for easily examining the characteristics of Enokitake mushroom cells using the color of a medium as an index. The present invention also relates to a method for testing the characteristics of Enokitake mushroom cells, which easily identifies and selects mutant strains of Enokitake mushrooms.

[0002] The present invention also relates to a method for producing an Enokitake mushroom strain, and more particularly to a method for easily producing a strain having stable characteristics.

[0003]

2. Description of the Related Art Enokitake is an edible mushroom that has been developed and established since the earliest in production system for institutional cultivation. In 1992, the production amount exceeded 100,000 tons, and it was the most produced mushroom.

The cultivation of edible mushrooms is carried out by using inoculum grown from the parent fungus. In recent years, mutations in inoculum have become a serious problem. Poor growth, coloring variation of fruiting bodies, etc. have become problems. Basic research is needed to elucidate these genetic causes, but if mutations in strains can be identified, the spread of damage can be suppressed.

[0005] Mutation countermeasures currently employed in Enokitake mushrooms include cryopreservation of the mother bacterium, quality test of the inoculum by enzyme activity analysis, cultivation test, and characteristics of the clonal strain obtained from mitosis, protoplasts and hyphal fragments. There is a test. However, both have problems. Hereinafter, conventional methods for countermeasures for mutation will be described individually. (1) Cryopreservation of the mother bacterium The cryopreservation of the mother bacterium is performed by freezing the mother bacterium with liquid nitrogen and periodically updating the mother bacterium, thereby limiting the number of subcultures of the inoculum and causing spontaneous mutations. This is to reduce the frequency.

[0006] However, there are many parts that have not been established in the method of freezing and thawing the mother bacterium, and the characteristics of the thawed mother bacterium may not be stable. (2) Inoculum quality assay by isozyme analysis and enzyme activity analysis Mutant strains can be detected by investigating and comparing isozyme band patterns or enzyme activities of peroxidase, laccase, cellulase and the like. It is effective for detecting some mutant strains.

[0007] However, this assay method is difficult to detect unless some of the inoculum has grown or expanded due to passage or the like. Furthermore, isozymes or enzyme activities that are considered to be effective for detecting a certain mutant strain may not be applicable to all other mutant strains having the same symptoms. In addition, the operation of crushing the bacteria to prepare a sample for measuring isozymes and enzyme activities is complicated, and is not suitable for testing a large number of strains. (3) Cultivation test The cultivation test is a test method in which a part of the inoculum is used as a sample for cultivation every time the fungus is subcultured to check whether there is any abnormality in its characteristics. By providing a large number of cultivation test monitors (the number of samples tested by cultivation), it is possible to check for signs of mutation at an early stage.

However, in many cases, preferable cultivation conditions of enokitake are different for each variety, and the conditions are delicate. In the cultivation test, the results differ depending on the cultivation conditions, and the evaluation of the quality of the sample is divided. Tends to. When the evaluation is divided, in addition to those which are clearly evaluated as defective, there may be cases where even seed lots with different evaluations of quality are discarded. Also, the same labor, labor and time as actual cultivation are required for the test. (4) Characteristic test of clonal strains obtained from mitosis, protoplasts, and hyphal fragments This characteristic test is performed by periodically cloning inoculum, protoplasts, or hyphal fragments to produce clonal strains and producing clones. Mutants are detected by investigating strains as specimens. Even if a mutation is chimerically generated inside the inoculum, if a mutant clone strain can be found among them, the bud of the mutation can be detected at an early stage.
In addition, by examining the variation among clone strains, it is possible to determine the stability of the inoculum.

[0009] The characteristic test method using the clone strain is a highly accurate mutation detection technique. However, in order to improve the detection accuracy, it is necessary to use a large number of clone strains as specimens and investigate their characteristics. There are various restrictions and labor problems.

[0010] As described above, there are several methods for countermeasures for the variation of Enokitake mushrooms, but none of them is sufficiently satisfactory.

[0011]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above, and aims to suppress the production loss due to the presence of a mutant in a seed bacterium and to stably produce Enokitake mushrooms. That is the task.

[0012] Specifically, an object of the present invention is to provide a simpler method for examining characteristics of various kinds of enokitake mushrooms that can be identified and selected.

[0013] Another object of the present invention is to provide a method for producing a strain of Enokitake mushroom having stable characteristics and a method for producing a strain having a stable fruiting body forming ability.

[0014]

Means for Solving the Problems The present inventors have conducted intensive studies from the above viewpoints. As a result, when enokitake mushroom cells were cultured in a medium containing bromothymol blue (hereinafter referred to as "BTB"), the color of the medium was increased. And found that the present invention was completed based on such findings.

That is, the present invention is a method for examining the characteristics of Enokitake mushroom cells, which comprises cultivating Enokitake mushroom cells in a medium containing bromothymol blue and selecting Enokitake mushroom cells using the color of the medium as an index.

The present invention is also a method for testing the characteristics of Enokitake mushroom cells, which comprises culturing Enokitake mushroom cells in a medium containing bromothymol blue and selecting mutants of Enokitake mushroom cells using the color of the medium as an index.

The present invention is also a method for testing the characteristics of Enokitake mushroom cells, which comprises cultivating Enokitake mushroom cells in a medium containing bromothymol blue, and judging the fruiting body forming ability of the Enokitake mushroom cells using the color of the medium as an index. .

Further, the present invention is characterized in that enokitake mushroom cells are cultured in a medium containing bromothymol blue, the color of the medium is detected by measuring the absorbance of the medium, and the absorbance is used as an index. This is a method for testing the characteristics of Enokitake mushroom cells.

The present invention also provides a method for culturing Enokitake mushroom cells in a medium containing bromothymol blue, collecting the Enokitake mushroom cells from a medium exhibiting a specific color using the color of the medium as an index, and purifying the obtained cells to obtain a strain. This is a method for producing an enokitake mushroom strain.

Further, the present invention is characterized in that enokitake mushroom cells are cultured in a medium containing bromothymol blue, and the cells of the medium whose medium color changes from yellow to yellowish white are collected and purified to obtain a strain. It is a method for producing an Enokitake mushroom strain having a fruiting body forming ability.

[0021] The present invention also relates to a method for culturing Enokitake mushroom cells to obtain a strain, comprising a test medium in which Enokitake mushroom cells are cultured in a medium containing bromothymol blue, and a method in which Enokitake mushroom cells are cultured in a medium containing bromothymol blue. The absorbance at a wavelength of 615 nm of the control culture medium in which the body was not cultured was measured, and the cells were collected and purified from a test medium having a degree of decolorization of 70 to 100%, which was obtained from the measured value by the formula 1, and the strain was isolated. A method for producing an Enokitake mushroom strain having a fruiting body-forming ability, characterized in that the method comprises:

[0022]

(Equation 2)

Hereinafter, unless otherwise specified, the enokitake mushrooms are simply referred to as "cells", and the enokitake mushrooms are simply referred to as "strains".

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION
The cells are cultured in a medium containing, and cells having different characteristics are selected using the color of the medium as an index. When the cells are cultured in a medium containing BTB, the color of the medium containing BTB is generally yellowish-white, yellow, yellow-green, green, blue-green, or blue. In the characteristic test method of the present invention, differences in the characteristics of the cells are identified as differences in the color of the medium in which the cells are cultured, and the cells are selected. The characteristic test method of the present invention can identify differences in cell characteristics by a simple operation of culturing cells in a medium containing BTB.

BTB used in the present invention is a dye generally used as a pH reagent. As a medium to which BTB is added, a medium usually used for cultivation of Enokitake can be used, and a medium containing lactose as a sugar content is preferable. The content of lactose in the medium is preferably 2 to 5% (W / V). The BTB content in the medium is preferably 0.0001 to 0.03 g / L,
Particularly preferably, it is 0.001 to 0.003 g / L. In addition, the initial pH of the medium containing BTB is preferably adjusted to 6.0 to 7.5 so that the color of the medium becomes blue-green. The medium may be liquid or solid, but preferably a liquid medium is used. The culture medium is used in test tubes or microplates, but the use of microplates tends to produce results in a shorter time than the use of test tubes. In consideration of this, a 24-well microplate is particularly preferable. With a 24-well microplate, results can be obtained in about 4 days. Although it is not necessarily certain, it is inferred that the period during which the test results are obtained tends to be influenced by the volume of the medium.

The cells to be subjected to the assay may be live cells, and for example, those that are generally used as inoculum or those that are obtained by thawing stored cells can be used. More specifically, one example of the experimental operation is to inoculate a seed bacterium on an agar medium or the like, form a colony, take out a colony fragment with a cork borer or the like, and use this colony fragment as a cell of one specimen to be subjected to a test. And the like. In addition, as a culture medium for forming a colony, for example, a 1.8% agar Petri dish medium containing 2% malt extract and the like can be mentioned.

Each sample is inoculated into a culture medium containing BTB in a test tube or a microplate and cultured. Culture is
Static culture or shaking culture may be used, but shaking culture is preferably performed. In the case of stationary culture, a color difference may occur in the medium of one specimen, and it is considered that shaking culture often makes it easier to identify the color of the medium.

When the culture of the cells is performed for about 4 to 10 days, the color of the medium may change, and the cells can be distinguished from each other with respect to the characteristic of changing the color of the medium containing BTB.
Also, when comparisons between strains are not so strict,
As a simpler assay method, there is a method of inoculating a mycelium liquid-cultured or a mycelium suspension obtained by aseptically cutting a mycelium liquid-cultured with a homogenizer into a BTB medium as a specimen. In some cases.

If the color of the culture medium of each specimen is the same, it can be determined that the cells have the same characteristics. Also,
If the color of the culture medium of each sample is different, it can be determined that the cells have different characteristics. Therefore, it is possible to select desired Enokitake mushroom cells using the color of the medium as an index. In addition, a medium in which the cells are not cultured is provided as a control,
By comparing this with the medium in which the specimen is cultured, differences in color and changes can be more easily distinguished.

The color of the medium can be identified by visual observation.
Also, by measuring the absorbance of the medium and comparing or examining the absorbances, it is possible to determine the difference in the color of the medium. According to the absorbance, the color of the culture medium is converted into numerical data, and a more objective test can be performed. When measuring the absorbance, the measurement wavelength is preferably 550 to 700 nm, more preferably 600 to 650 nm, and particularly preferably about 615 nm. The measurement of the absorbance of the medium can be performed according to a usual method.

Further, a medium in which the cells were cultured (test medium)
If the degree of decolorization is determined based on the absorbance of the medium and the absorbance of the medium in the control where no cells are cultured, numerical data that can be more easily compared and selected can be obtained. For example, 615 nm
The degree of decolorization when the absorbance is measured by the formula (1) is a numerical value obtained by Expression 1. By the degree of decolorization, it is possible to identify as a numerical value how much the color of the control medium has changed in the test medium.

[0032]

(Equation 3)

Culture conditions other than those described above when culturing using a medium containing BTB can be adjusted as appropriate, and can be generally determined according to the conditions for growing and growing Enokitake mushrooms. Generally, the preferred temperature is between 15 and 2
The temperature is about 5 ° C., and the light and dark conditions are preferably dark conditions.

By applying the above-described characteristic test method, there are provided, as more specific embodiments, a characteristic test method for selecting mutants and a characteristic test method for judging the ability of cells to form fruiting bodies.

First, a description will be given of a characteristic test method for selecting mutants. When cells are cultured in a medium containing BTB,
As described above, the color of the medium may change. When a plurality of samples are tested, the difference in the color of the culture medium for each sample can be inferred that there is a high probability that there is a difference between the samples. In addition, if the color of the culture medium when normal cells are cultured is tested in advance, a mutant different from the normal strain can be easily identified and selected. In addition, when a certain strain is assayed by the method of the present invention, it can be inferred that the greater the difference in the color of the medium, the more various variants are contained in this strain. As described above, mutants can be selected by using the color of the medium as an index by the characteristic test method of the present invention.

Next, a characteristic test method for judging the fruiting body forming ability will be described. When the Enokitake mushroom cells are cultured in a medium containing BTB, a difference in the color of the medium occurs due to a difference in fruiting body forming ability. Specifically, when a strain having a high fruiting body forming ability is cultured, the color of the medium containing BTB tends to take on a yellowish-yellowish white color. And the cells having almost no fruiting body-forming ability tend to exhibit a deeper blue color than the blue-green color of the medium in the control group. By using the color of such a medium as an index, it is possible to determine the fruiting body-forming ability of the cells.

It should be noted that the ability to form fruiting bodies is high because
This means that when enokitake is cultivated using a certain bacterial body (strain) as a seed fungus, there is little or no occurrence of enokitake which does not form fruiting bodies or does not sufficiently form fruiting bodies.

An example of an index for specifying the color of a medium by the degree of decolorization is shown below. For example, when the absorbance at a wavelength of 615 nm is measured, the range of the degree of bleaching that can be evaluated as having a high fruiting body forming ability is preferably 70 to 100%, and particularly preferably 80 to 100%.

If the mutants are contained in the parent fungi and inoculum of the enokitake, the growth of the enokitake will vary, and the production will not be stable. However, according to the characteristic test method of the present invention, it is possible to easily know whether or not the mutant is contained in the inoculum or the like, and furthermore, the ability to form fruiting bodies, which is an important trait for production of Enokitake, is obtained. Since it can be easily discriminated, the property test method of the present invention is expected to greatly contribute to the production of enokitake.

Furthermore, the present invention provides a method for producing an Enokitake mushroom strain having stable properties. The bacterial strain production method of the present invention is an application of the above-described method of the present invention for determining the characteristics of bacterial cells. That is, after culturing the cells in a medium containing BTB, performing the characteristic test method of the present invention in which the cells are selected using the color of the medium as an index, and then collecting the cells from the medium exhibiting a specific color. And purifying the cells to obtain a strain.
If a medium of a specific color, for example, yellow is defined as yellow, and cells are collected from this medium alone, a strain with stable characteristics can be obtained, and the relationship between the color of the medium and a specific trait is examined in advance. By doing so, cells having the specific trait can be collected based on the results thereafter, and a strain having the specific trait stabilized can be obtained.

The cells collected from the medium exhibiting a specific color are as follows:
If necessary, the strain can be grown under ordinary culture conditions to obtain a required amount of strain. In addition, by repeating the selection of cells using a medium containing BTB and the collection of cells from a medium having a specific color, a strain with extremely high purity and stable characteristics can be obtained. Further, even if the strain is once established as a strain, the mutant may be generated by long-term storage and passage, but the characteristics of the strain can be maintained by the same method as the strain production method of the present invention. it can.

Next, a method for producing a strain having a high fruiting body-forming ability will be described as a specific embodiment of the method for producing a strain of the present invention.

Bacteria having the fruiting body-forming ability can be selected by the above-described characteristic assay method. That is, B
The cells may be cultured in a medium containing TB, and a medium in which the color of the medium is yellow to yellowish-white may be selected. Purification is performed by collecting cells from the yellow-yellowish white medium. The collected cells may be used as a strain as they are, or may be further cultured and grown to a required amount. Furthermore, by repeating the procedure of performing a characteristic test and collecting and culturing the cells from a yellow-yellowish white medium, a strain having extremely excellent fruiting body-forming ability can be obtained.

In the selection of cells having the fruiting body-forming ability, the color of the culture medium may be identified by the naked eye, or the culture medium may be selected by determining the absorbance and decolorization degree of the culture medium. For example, 6
When measuring the absorbance of the medium using a wavelength of 15 nm,
A preferred range of the degree of decolorization is 70 to 100%, particularly preferably 80 to 100%.

The above-mentioned method for assaying characteristics and production of the bacterial strain according to the present invention can be applied to pure white enokitake (Flammu) among enokitake mushrooms.
lina velutipes). Also,
Even when white or pure white bacterial cells are generated from the originally colored bacterial strain, it can be suitably used.

[0046]

EXAMPLES <Example 1> Normal strains, strains lacking fruiting body (strains incompletely forming fruiting bodies), and strains lacking development (strains in which fruiting bodies do not occur at all) were used as test strains. Was inoculated into a 2% malt extract, 1.8% agar Petri dish, and cultured.

The colony has a plurality of specimens (colony fragments)
When it became large enough to take out, a portion about 5 mm inside from the edge of the colony was cut out with a cork borer to obtain a colony fragment, and the colony fragment was
A test tube containing 3 to 4 mm of a YBLB medium having the composition shown in Table 1 was inoculated and cultured at 24 ° C. with shaking. In addition, a medium without inoculating the strain was also prepared as a control, and kept under the same conditions.

[0048]

[Table 1]

About one week later, the color of the culture medium began to change, and on the tenth day, the difference between the strains became apparent to the naked eye.

At this time, the culture was stopped, and the colors of the medium in the control section and the medium in which each strain was cultured were compared and observed with the naked eye. Also,
The absorbance at 615 nm was measured to quantify the difference in the color of each medium. Table 2 shows the results such as the color of the medium, the absorbance, and the degree of decolorization (%). The degree of bleaching was determined by Equation 1 (the same applies hereinafter). Note that a normal strain is a strain that normally forms fruiting bodies, in other words, a strain that produces good mushrooms.

[0051]

[Table 2]

As a result of macroscopic observation, in the medium in which the normal strain was cultured, BTB was decolorized and turned from yellow to yellowish white, and the medium of the fruiting body-deficient strain showed yellowish green to pale green to green to blue-green. It was observed that the medium of the underdeveloped strain was much darker than the control medium. Also, the absorbance,
Comparing the values for the degree of decolorization showed completely different values for each strain. <Example 2> From wild enokitake mushroom strains refrigerated at the Forestry Research Institute, three strains that form well fruiting bodies and two strains that do not form fruiting bodies were grown at 24 ° C. The test was performed. Table 3 shows the results.

[0053]

[Table 3]

Example 3 Two strains A and B were cultured to form colonies, and colony fragments were obtained from each colony. 1 ml of YBLB medium was placed in each well of a 24-well microplate, and each colony fragment was inoculated into YBLB and cultured with shaking.

As a result, the change in the color of the medium became clear four days later. All the culture media of the colony fragments obtained from the strain A exhibited yellow (FIG. 1), which revealed that the strain had no mutation. On the other hand, the culture medium of the colony fragments obtained from the B strain showed a color difference in each hole, and it was revealed that mosaic mutation occurred in the strain treated as one strain ( (Fig. 2).

From the microplate on which the strain B was cultured, the mycelium was picked up from a hole in which the color of the medium was yellow, selected, and inoculated on a 2% malt agar medium. After the colonies were formed on the agar medium, colony fragments were cut out again from 10 locations. Using 10 wells of a 24-well microplate, colony fragments obtained from 10 locations were each subjected to YB
LB medium was inoculated and cultured. The operation of collecting and culturing the mycelium from the medium exhibiting yellow was repeated, and at the stage where the mycelium was selected and cultured four times in total from the medium exhibiting yellow, the medium in all 10 wells exhibited yellow, A strain with stable properties was successfully obtained.

Table 4 shows the progress of the degree of purification.
The numerical values in Table 4 are obtained by culturing each of the colony fragments collected from 10 of the colonies in the YBLB medium.
The number of culture media (holes in the microplate) showing each color is shown.

[0058]

[Table 4]

[0059]

According to the characteristic assay method of the present invention, the characteristics of Enokitake mushroom cells can be easily assayed by a simple operation of culturing the cells using a medium having a simple composition. In addition, the characteristic assay method of the present invention can easily evaluate the ability to form fruiting bodies, and easily determine the presence or absence of a mutant in a strain. In addition, it is possible to know the result of the test in a very short time as compared with a cultivation test or the like.

In addition, since the method for testing the characteristics of the present invention is simple in operation, even if a large number of new strains are generated by cross breeding, etc., it is easy to apply each strain to the characteristic test, and This is useful for primary selection.

Further, according to the characteristic test method of the present invention,
The cells can be cultured and used after the test without destroying the cells (eg mycelia) subjected to the test.

Therefore, the method for assaying characteristics of the present invention can be applied to the production of strains.
A strain with stable characteristics can be easily obtained. Also,
The characteristics of the strain can be maintained by the same method as the method for producing the strain of the present invention.

According to the method for testing the characteristics and the method for producing the strains of the present invention, the quality of the parent fungus and inoculum for the production of Enokitake can be stabilized, and thus the production of Enokitake with little variation in quality and little production loss can be performed. Will be able to do it.

[Brief description of the drawings]

FIG. 1 is a photograph showing a normal strain in which no mutation has occurred as a result of an assay. The leftmost column is a control group. In addition, the portion that appears dark in the approximate center of each hole (excluding the control plot) is a colony fragment cut out and inoculated with a cork borer. In the original photograph shown in FIG. 1, the color of the culture medium can be determined from the color that appears in the culture medium around the colony fragment.

FIG. 2 is a view showing a photograph indicating that the strain has a mutation as a result of the test. The leftmost column is a control group. In addition, the portion that appears dark in the approximate center of each hole (excluding the control plot) is a colony fragment cut out and inoculated with a cork borer. In the original photograph shown in FIG. 2, the color of the culture medium can be determined from the color that appears in the culture medium around the colony fragment.

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int. Cl. ⁷ , DB name) A01H 4/00 JICST file (JOIS)

Claims (7)

(57) [Claims] 1. A method for testing characteristics of Enokitake mushrooms, comprising culturing Enokitake mushrooms in a medium containing bromothymol blue and selecting Enokitake mushrooms using the color of the medium as an index. 2. A method for testing characteristics of Enokitake mushroom cells, comprising culturing Enokitake mushroom cells in a medium containing bromothymol blue and selecting mutants of Enokitake mushroom cells using the color of the medium as an index. 3. A method for examining the characteristics of Enokitake mushroom cells, comprising culturing Enokitake mushroom cells in a medium containing bromothymol blue, and judging the fruiting body forming ability of Enokitake mushroom cells using the color of the medium as an index. 4. The method according to claim 1, wherein the color of the culture medium is detected by measuring the absorbance of the culture medium. 5. An Enokitake mushroom characterized by culturing Enokitake mushroom cells in a medium containing bromothymol blue, collecting and purifying the Enokitake mushroom cells from a medium exhibiting a specific color using the color of the medium as an index, and obtaining a strain. How to produce the strain. 6. A fruiting body obtained by culturing Enokitake mushroom cells in a medium containing bromothymol blue, collecting and purifying the cells from a medium having a yellow to yellowish white color, and obtaining a strain. A method for producing an Enokitake mushroom strain having a forming ability. 7. A method for culturing Enokitake mushrooms to obtain a strain, wherein the test medium in which Enokitake mushrooms are cultured in a medium containing bromothymol blue and the culture medium containing Enokitake mushrooms are not cultured in a medium containing bromothymol blue The absorbance at a wavelength of 615 nm of the control medium was measured, and the degree of decolorization determined by the formula 1 from the measured value was 70 to 100.
% Of an Enokitake mushroom strain having a fruiting body-forming ability, wherein a strain is obtained by collecting and purifying the cells from a test medium having a percentage of test medium. (Equation 1)