JPH0824487B2 - Method for producing liquid inoculum of basidiomycete - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a liquid inoculum of basidiomycetes, and more particularly to a method for producing a liquid inoculum of basidiomycetes suitable for flooring using a film bag method.

[Conventional technology]

Conventionally, the basidiomycete inoculum has been a solid inoculum having a wood-processed seed piece or sawdust as a main culture medium.

[Problems to be Solved by the Invention]

The present inventor has considered a cultivation method using a culture bottle as an improvement plan of the conventional technique, but it is not practical because a healthy fruiting body cannot be obtained (the fruiting body generated is small when the floor is small, which is not practical). ). Therefore, it was studied to make a film bag bed which is 4 to 5 times as large as an ordinary culture bottle for mass production of mushrooms.

Flooring by the film bag method using solid inoculum is performed by attaching the inoculation port, inoculation port and polyurethane stopper,
In addition, it requires a great deal of labor such as the preparation work of these, such as a rubber cord for attaching the inoculation port to the film bag, and the recovery work of these, which is unsuitable for mass production. In addition, these equipments are also quite expensive and not suitable for practical use.

In the case of a film bag floor, even if you try to inoculate a hole in the center of the floor and inoculate the inoculum into the center of the floor through the hole, the hole will collapse during the installation of the inoculation port, so you can inoculate only on the floor surface Inevitably, it suffers from the drawback that the number of culture days becomes long.

In the automation of floor preparation by the film bag method, the medium is compression-molded, wrapped in a film bag, etc., directly sealed and then sterilized and cooled, and liquid inoculum is injected from the top of the film bag using an injection needle method. The present inventor has determined that inoculation in the center is the best method. Therefore, we have conducted research for the development of liquid inoculum.

Conventionally, considerable research has been conducted on a method of culturing basidiomycetes in a liquid culture and proliferating the cells, but there were many problems and it was difficult to put them into practical use. The reason is,
When growing basidiomycetes in liquid culture, take out the inoculum from the slant culture medium and inoculate it into the liquid culture medium, shake it and grow it, and then use it as a seed in a large tank such as a jar amentor. Although the method of culturing was adopted,
This equipment requires enormous cost, and there is a problem in that labor cost, maintenance cost, management cost, etc. are too large. In terms of technical aspects, the growth of basidiomycetes is extremely slow compared to general filamentous fungi such as Aspergillus niger, Kumonosubi, and hairy mildew, and the number of days of culture is long, and further,
In the liquid culture, the bacterial cells adhere to the wall surface of the culture vessel and propagate, or because the hyphae grow in the liquid by spreading the hyphae in a cloud shape or in a radial shape, this is entangled, and it is easy for pellets to be easily inconvenient, In addition, even if the culture was successful, there were many problems such as the fact that when the culture medium was stopped, the bacterial cells precipitated, and it was not possible to inoculate with a dispenser, and it was not practically applicable.

Therefore, an object of the present invention is to provide a liquid inoculum that can be produced extremely advantageously so that floor formation by the film bag method or the like can be reasonably automated.

[Means for solving the problem]

In order to achieve the above object, in the present invention, in shredding the microbial mass obtained by stationary culture of basidiomycetes in a liquid medium, the shredded pieces are injected. It is shredded into fine pieces that are small enough to pass through the needle, and sterilized to increase the survival rate of the cells when the shredded cells are inoculated to the floor. When mixed with water, suppress evaporation of water, swell when water is added, dissolve in water, swell in water and swell in water, as a substance that exhibits a gel form, sodium polyacrylate is added to the sterilized water. I did.

[Action]

The present inventors confirmed the following facts. That is, when the cells that have been statically cultivated are finely shredded with each culture and the solution containing the shredded cells is directly or diluted in sterile water and inoculated into the floor, the survival rate is poor. (At 15-20% or less)
Although it is poor in practicality, the chopped basidiomycete or a liquid containing the fungus can be mixed with sterilized water containing sodium polyacrylate to significantly increase the survival rate (mostly 100%). It was confirmed as a result of the experiment. That is, when it is diluted with mere sterile water, it almost dead and does not survive, but when diluted with sterilized water to which sodium polyacrylate is added, even if it is diluted at a high magnification, it is also shredded at a high magnification. Even if you inoculate a very small amount of inoculum,
We found a surprising fact that the hyphae grow vigorously with 100% survival.

In the case of mere sterilized water, the more chopped the basidiomycetes, the lower the survival rate.However, when sterilized water containing sodium polyacrylate as described above is used, basidiomycetes are used. The survival rate is extremely high and the rate at which hyphae spread on the floor is fast.

Furthermore, the above-mentioned sodium polyacrylate not only suppresses the drying of shredded cells and protects the cells, but also prevents the precipitation of shredded cells and keeps the cells in the liquid evenly. It also has the effect of suspending and dispersing in the water, which is very useful for mass production.

The liquid inoculum of basidiomycetes obtained by the method of the present invention was developed for the purpose of making a film bag floor, but in addition to this, a floor using a culture container such as a bottle, or spreading on the floor like a Matsuroom room, etc. Needless to say, the present invention can be applied to all beds such as a bed to be built up, or a bed made by aseptically mixing a liquid inoculum with a medium to form a block.

〔Example〕

 Examples of the present invention will be described below.

Example 1 Each of Pleurotus communis, Pleurotus cornucopiae and Ganoderma lucidum cultured in a PDA slant medium (medium consisting of potato, dextrose and agar) was taken out by a platinum loop method, and put in a 300 ml Erlenmeyer flask with 100 ml of an onion soy sauce medium. Each of the cells was inoculated, and the cells were statically cultivated in a dark room at 25 ° C for 25 days.
Shred by rotation, about 100 m of liquid containing the shredded fine cells
l diluted in 500ml, 1000ml, 1500ml, 2000ml, 2500ml simple sterilized water (see Table-1) and 500ml, 1000m
l, 1500ml, 2000ml, 2500ml diluted with sterilized water with 0.5% sodium polyacrylate added (see Table-2), and take out 10ml to each with a dispenser, length 20cm x
Width 20 cm x height 10 cm, weight 1.5 kg bed (bagasse 6, cake 3, rice bran 1-anhydrous solids weight mixing ratio, pH 6.8, moisture 65
%) Each of the 10 species was inoculated into 30 beds, and a comparison test was conducted on the survival rate of the inoculum and the number of days when the hyphae spread to the bed. However,
When sterilized water was simply used, inoculation was performed with a dispenser while shaking to prevent the shredded cells from precipitating.

Culture is 25 ℃, humidity is 60-70%, CO ₂ concentration is 700-800ppm,
I went under an illuminance of 50 lux or less.

 The results of this test are shown in Table 1 and Table 2 as an average value.

When diluted with 500 ml of simple sterile water in the case of Table-1,
Survival rate is 7 to 13%, 7 to 10% for 1000ml, 0 for 1500ml
It is about 7%, and with 2000 ml and 2500 ml, it almost die and does not survive.

As shown in Table-1, the survival rate decreases as the dilution ratio increases. As a result of careful observation of the floor, the mycelium will die and a considerable proportion of the mycelium will die, and it will take time for the mycelium to grow, and the mycelium will spread to the floor. The days are getting late.

In the case of Table-2, that is, sterilized water obtained by adding finely chopped bacterial cell liquid to 0.5% of sodium polyacrylate.
Mix to ~ 2500 ml, take this out as 10 ml inoculum,
When the above-mentioned floor was inoculated and the survival rate and the number of days of hyphae spread were observed, 100% of all of 500 to 2500 ml was lived, and the number of days when hyphae spread vigorously and hyphae spread to the floor. Is also greatly shortened.

It should be noted that comparison will be made in the case of solid seed bacteria (sawdust bacteria) by the conventional method. Since the surface of the floor is inoculated, the number of days of culturing is about 32 to 35 days with the above-mentioned fungus of Example 1, about 50 days with Abalone and 50 days with Ganoderma lucidum. From now on,
It can be seen that in the case of the liquid inoculum of the example of the present invention, the hyphae spread very quickly.

Example 2 The statically cultured cells were shredded together with the medium (under the same conditions as in Example 1), and well mixed with 1000 ml of sterilized water to which the drying-suppressing substance shown in Table 2 was added, and 0.5 ml, 1 ml, 5 ml , 10 ml each were inoculated into each of 30 beds in the same manner as in Example 1, and the survival rate and the number of days of infestation of hyphae were checked. The culture conditions were the same as in Example 1. The results are shown in Table-3. The survival rate and the number of hyphae infestation days were expressed as the average value of 30 beds.

As shown in Table-3, 0.5m of shredded liquid diluted with high magnification is used.
l, 1 ml, 2 ml, very small amount Even if inoculated on the floor 1
It can be seen that the hyphae proliferate vigorously despite the fact that they have a survival rate of 00% and are extremely small in number.

In addition, a bed in which hyphae were spread by these liquid inoculum was placed in each cultivation house and tested for the occurrence of mushrooms, but all normal mushrooms were obtained.

[Effects of the Invention] As described above, in the present invention, a liquid inoculum of basidiomycetes that is most suitable for floor construction by the film bag method can be produced extremely inexpensively and efficiently. When inoculated on the floor, the number of culturing days can be drastically shortened, and the inoculum of the inoculum can be reasonably inoculated, which is very economical and has many excellent effects such as being suitable for mass production.

 ─────────────────────────────────────────────────── ─── Continuation of front page (56) Reference JP-A-51-141259 (JP, A) JP-A-55-61730 (JP, A) JP-A-51-27561 (JP, A) JP-A-52- 640 (JP, A) JP 58-8251 (JP, B2)

Claims (1)

[Claims] 1. When shredding a microbial mass obtained by statically culturing basidiomycetes in a liquid medium together with the medium, the shredded pieces must be sufficiently passed through the injection needle. In addition to shredding into fine pieces, in order to increase the survival rate of the shredded cells when inoculated on the floor, the water content evaporates when mixed with sterilized water. The method is characterized in that sodium polyacrylate is added to the sterilized water as a substance that swells when water is added, swells when dissolved in water, dissolves in water, swells in water, and presents a gel form. A method for producing a liquid inoculum of basidiomycetes, which is suitable for floor construction using the method.