JP5916026B2 - Bunashimeji strain and method for producing Bunashimeji fruiting body - Google Patents

Description

  The present invention relates to a bunashimeji strain that hardly causes inoculum sclerosis and a method for producing a bunashimeji fruiting body using the strain. Furthermore, the present invention relates to a method for selecting a Bunashimeji strain that hardly causes inoculum sclerosis.

  In recent years, artificial cultivation techniques for mushrooms have been developed, and various edible mushroom fruit bodies (shiitake, enokitake, oyster mushrooms, nameko, maitake, beech shimeji, hatake shimeji, hon-shimeji, etc.) have been provided.

  Usually, mushrooms are artificially cultivated using raw wood such as Japanese oak, beech, and cucumber as a raw material, and a medium in which nutrient sources such as rice bran, wheat bran, and corn bran are mixed with sawdust and corn cob. There is a fungus bed cultivation using a medium for solid cultivation filled in a container such as a bottle, a bag, or a box. Among these cultivation methods, the fungus bed cultivation method using polypropylene bottles is relatively easy to increase in size and mechanization, and therefore has been promoted in a factory system for large facilities. However, in the factory system of large equipment, various obstacles at the time of mushroom cultivation that have not been seen so much in small-scale cultivation have come to be seen.

  For example, in Buna shimeji, there may be a phenomenon that the inoculum part on the fungus bed turns brown and hardens during culture ripening. In addition, a rotting odor is frequently generated at this time. When these phenomena occur, there are many examples in which browning occurs on the fungus floor after the fungus is scraped, and the mycelium is not regenerated or regenerated very slowly and does not emerge, that is, fruit bodies cannot be harvested. This phenomenon is called inoculum sclerosis and is regarded as an important issue because it reduces the production efficiency of fruiting bodies. However, the cause and mechanism of inoculum sclerosis have not been elucidated, and measures such as lowering the culture temperature have only been attempted as countermeasures (for example, Non-Patent Documents 1 and 2).

Fungus bed mushroom / cultivation disorder case collection, pp. 64-66, published by Nagano Prefecture Economic Business Agricultural Cooperative Federation, March 1995 Report of Nagano Vegetable Flower Experiment Station, No. 11, pp. 23-26, 2001

  In view of the above-mentioned present situation, an object of the present invention is to provide a Buna-Shimeji strain which is less likely to cause inoculum sclerosis than before in large-scale production of Buna-Shimeji and a method for producing a Buna-Shimeji fruiting body using the strain. In addition, a method for selecting Buna shimeji strains that hardly cause inoculum sclerosis is also provided.

  As a result of intensive studies on inoculum sclerosis, which is an important issue in the large-scale production of Bunashimeji fruiting bodies, the present inventors have surprisingly found that small amounts are usually used to increase the yield of Bunashimeji fruiting bodies. Inoculum caused by large-scale production by cultivating Bunashimeji fruiting bodies using solid culture medium (bacteria bed culture medium or culture medium) containing a large amount of magnesium compound such as magnesium aluminate metasilicate It was found that the same phenomenon as sclerosis can occur artificially. Moreover, it discovered that it was possible to select the Buna shimeji strain which hardly causes inoculum sclerosis using the culture medium for solid cultivation, and found the Buna shimeji strain which hardly caused inoculum sclerosis using the method. Furthermore, the present inventors have found that by using the strain, it is possible to produce a stable buna-shimeji fruiting body having a good yield in large-scale production, thereby completing the present invention.

In summary of the present invention:
[1] In cultivation using a solid cultivation medium containing softwood sawdust and rice bran as main constituents and containing about 6.4% by weight of magnesium aluminate metasilicate in dry weight ratio, an inoculum part on the surface of the solid cultivation medium Buna shimeji strain that does not cause browning of
[2] Bunashimeji strain according to [1], which does not cause browning of the inoculum part in cultivation using a solid culture medium comprising coniferous sawdust, rice bran, corn cob meal, soybean hulls, bran, and magnesium aluminate metasilicate.
[3] Hypyzygus marmoreus K-4975 (FERM BP-11321), Hypyzygus marmoreus K-4979 (FERM BP-11322), Hypyzygus marmorus K13980, FERM BP-113M Or a mutant strain derived from these strains and selected from strains having properties equivalent to those strains [1] or [2]
[4] A method for producing a fruit body of Bunashimeji using the Bunashimeji strain according to [1],
[5] Coniferous sawdust and rice bran are the main constituents, and the dry weight ratio is about 5.2 wt% or more magnesium aluminate metasilicate, the dry weight ratio is about 0.5 wt% or more magnesium oxide, and the dry weight ratio A method for selecting Buna shimeji strains that are less prone to inoculum sclerosis, comprising cultivating Buna shimeji strains using a solid culture medium comprising at least one magnesium compound selected from about 2% by weight or more of magnesium alumina hydroxide;
[6] The method for selecting a Bunashimeji strain according to [5], wherein the medium for solid cultivation comprises coniferous sawdust, rice bran, corn cob meal, soybean hulls, bran, and a magnesium compound,
[7] A selection method for the Buna shimeji strain according to [5] or [6], which is a selection method for selecting a Buna shimeji strain that does not cause browning of the inoculum portion on the surface of the solid culture medium,
About.
As one aspect of the above invention [3], cultivation using a solid cultivation medium containing softwood sawdust and rice bran as main constituents and containing about 6.4% by weight of magnesium aluminate metasilicate in a dry weight ratio , Which does not cause browning of the inoculum portion on the surface of the solid culture medium, Hypyzygus marmoreus K-4975 (FERM BP-11321), Hypsizygus marmoreus K-4979 (FERM BP-11322), Hypsizygus marMorus13M4 , And a mutant strain derived from at least one strain selected from Hypsizymus marmoreus K-4981 (FERM BP-11324), and having the same properties as these strains. It is.

  INDUSTRIAL APPLICABILITY According to the present invention, a bunashimeji strain that is less prone to inoculum sclerosis is provided, and a large-scale commercial production method of a stable bunashimeji fruiting body by using the strain is provided. In addition, a simple screening method for Bunashimeji strains that hardly cause inoculum sclerosis is provided.

  In the specification of the present application, “inoculum sclerosis” refers to the inoculation of the solid culture medium in the production of bunashimeji fruiting bodies, and then the inoculum on the surface of the solid culture medium turns brown and hardens during culture ripening. This refers to a phenomenon in which a spoiled odor is generated and the hyphae do not regenerate on the surface of the fungus after the fungus is scraped or the regeneration is extremely delayed. The solid culture medium in which this phenomenon has occurred does not lead to germination, that is, fruit bodies cannot be harvested.

  The fungus bed cultivation method of beech shimeji mushroom in the present specification is not particularly limited. For example, solid culture medium in which nutrient sources such as rice bran, wheat bran, corn bran and the like are mixed in sawdust, corn cob, etc., containers such as bottles, bags, boxes, etc. The cultivation method using the culture medium for solid cultivation filled in is mentioned. As the medium for solid cultivation, a known medium for solid cultivation used at the time of cultivation of Bunashimeji fruiting body can be used. For example, a medium for solid cultivation containing coniferous sawdust and rice bran as main constituents is preferable, and a medium for solid cultivation containing coniferous sawdust, rice bran, corn cob meal, soybean hulls, bran, and a magnesium compound is preferable.

  In the present specification, “coniferous sawdust and rice bran as main constituents” means that in solid culture medium, coniferous sawdust is a main component as a medium substrate, and rice bran is a main component as a nutrient. . That is, it shows that only coniferous sawdust is used as a medium base material or more coniferous sawdust than other medium base materials, and only rice bran or rice bran is used as a nutrient material. For example, when 850 mL bottles are used in bottle cultivation, softwood sawdust is 50 to 95 g, preferably 60 to 75 g in dry weight, and rice bran is 45 to 110 g, preferably 45 to 90 g, in dry weight.

In the present specification, the “magnesium compound” is not particularly limited as long as it is a compound containing magnesium, and magnesium oxo acid salt, halide, oxide, hydroxide, inorganic salt, organic acid salt, or the like is used. Can do. For example, magnesium aluminate metasilicate, magnesium carbonate, magnesium nitrate, magnesium sulfate, magnesium hydrogen carbonate, magnesium percarbonate as the oxo acid salt, magnesium fluoride, magnesium hydrogen fluoride, magnesium chloride, magnesium bromide as the halide, Magnesium iodide, magnesium oxide as the oxide, magnesium hydroxide as the hydroxide, magnesium hydroxide alumina (coprecipitate of aluminum hydroxide and magnesium hydroxide), inorganic salts as magnesium aluminate, magnesium hydride, Examples thereof include magnesium sulfide, magnesium hydrogen sulfide (magnesium hydrosulfide), magnesium permanganate, magnesium chromate, and magnesium chlorate. These magnesium compounds may be used alone or in combination. In the selection method of Bunashimeji strains that hardly cause inoculum sclerosis according to the present invention, magnesium aluminate, magnesium oxide, or magnesium aluminate hydroxide is particularly preferably used. In addition, the content of the magnesium compound in the solid cultivation medium in the selection method varies depending on the magnesium compound to be used, and may be appropriately selected. For example, when using magnesium aluminate metasilicate, the total dry weight of the solid cultivation medium About 5.2% by weight (for example, when the loss on drying of a commercial product is 18%, about 12 g / 850 mL bottle in wet weight) or more, preferably about 5.2 to 8.4% by weight (for example, When the loss on drying of a commercial product is 18%, the wet weight is about 12 to 20 g / 850 mL bottle), more preferably about 6.4% by weight (for example, when the loss on drying of a commercial product is 18%, the wet weight is about 15 g / 850 mL bottle), in the case of magnesium oxide, about 0.5 wt% (for example, about 1 g / 850 mL bottle of a commercial product) in a dry weight ratio to the total dry weight of the medium for solid cultivation More preferably, about 0.5 to 3% by weight (for example, about 1 to 6 g / 850 mL bottle of commercial product), more preferably about 0.8% by weight (for example, about 1.5 g / 850 mL of commercial product) Bottles), in the case of an alumina magnesium hydroxide compound, the dry weight ratio to the total dry weight of the medium for solid cultivation is about 2% by weight (for example, when the loss on drying of a commercial product is 10%, the wet weight is about 4 g / 850 mL bottle). ) Or more, preferably about 2 to 4% by weight (for example, about 4 to 8 g / 850 mL bottle when the loss on drying of a commercial product is 10%), more preferably about 2.5% by weight (for example, commercially available product When the loss on drying is 10%, the wet weight is about 5 g / 850 mL bottle).
In addition,% and a ratio in this-application specification show the value of a weight reference | standard unless there is particular notice.

  The selection method of Bunashimeji strain that is less likely to cause inoculum sclerosis according to the present invention can be carried out by bottle cultivation, bag cultivation, box cultivation, and the like, which are fungus bed cultivation methods. As an example, the sorting method of the present invention by bottle cultivation will be described. Buna shimeji mushroom bed cultivation method consists of each process such as preparation of fungus bed medium, bottling, sterilization, inoculation of inoculum, fungus bed culture, fungi-squirrel (shark shaving), budding, growth, harvesting, etc. (Kaihei No. 05-268742). Among these steps, in the bacterial bed medium preparation step, the magnesium compound such as magnesium aluminate metasilicate is added to the solid culture medium so that the dry weight ratio to the total dry weight of the solid culture medium is about 5.2% by weight. Added. In the selection method of Buna shimeji strain of the present invention that hardly causes inoculum sclerosis, the medium for solid cultivation containing a large amount of magnesium compound is not particularly limited, and a known solid cultivation medium used for cultivation of Buna shimeji fruiting body is used. Can be used. In particular, in the present invention, a medium mainly composed of coniferous sawdust (for example, cedar wood) and rice bran is preferably used. For example, for solid cultivation comprising coniferous sawdust, rice bran, corn cob meal, soybean hulls, bran, and a magnesium compound. A culture medium may be used.

  Next, the steps after bottling are performed. As the inoculum to be inoculated, a liquid inoculum or solid inoculum of Bunashimeji strain to be screened for whether or not inoculum sclerosis is unlikely to be used can be used.

  As a strain of Buna shimeji which can perform the selection method of Buna shimeji which is difficult to cause inoculum sclerosis of the present invention, a strain indicated as Lyophyllum ulmarium, a strain indicated as Hypsyzymus marmoreus and suitable for cultivation Examples of these mutant strains, and crossbreds obtained by known crossbreeding using these strains as parent strains.

  To determine whether or not inoculum sclerosis is likely to occur, observe the state of the surface of the fungus bed before and after fungi, the state of membrane regeneration after fungi, and any or all states selected from the growth state of fruiting bodies Can be selected. For example, in the case of Buna shimeji spp. Which are prone to inoculum sclerosis, brown and / or off-flavor occurs in the state of the fungus bed surface before the fungi. In such a case, the surface of the fungus after the fungus is often brown and / or has a strange odor. Furthermore, in the membrane regeneration after the fungi, the membrane is not regenerated at all or only a part of the membrane is regenerated. In the growth state of the fruiting body, the yield of the fruiting body is drastically reduced and the quality (shape) of the fruiting body is decreased as compared with the case where a solid culture medium containing no magnesium compound or a small amount is used. As shown in the examples below, when the fungus bed surface after browning is browned, even if fruiting bodies are normally generated, the yield is reduced. Therefore, in a solid culture medium containing a large amount of magnesium compound, a strain that does not cause browning of the inoculum portion on the surface of the solid culture medium can be said to be an excellent strain that hardly causes inoculum sclerosis.

  By selecting a Buna shimeji strain that does not enter the above state, it is possible to select a Buna shimeji strain that hardly or does not cause inoculum sclerosis. In addition, as described in Examples below, a Buna shimeji strain that hardly or hardly causes inoculum sclerosis may be selected by comparison with a conventionally known Buna shimeji strain. In the screening method of the present invention, 5 strains or more, preferably 8 or more crops are cultivated per strain in the same period, in the same medium and under the same conditions, and whether or not inoculum sclerosis is unlikely or does not occur is selected. Is preferred.

Examples of Bunashimeji strains selected using the method of the present invention include the following strains.
・ Hypzygus marmoreus K-4975 (FERM BP-11321)
・ Hypzygus marmoreus K-4799 (FERM BP-11322)
・ Hypzygus marmoreus K-4980 (FERM BP-11323)
・ Hypzygus marmoreus K-4981 (FERM BP-11324)

These strains are 62g sawdust (cedar wood) by dry weight, 60g rice bran by wet weight (water content about 10%), 30g corn cob meal (water content about 10%), 20g soybean hull (water content about 10%) ), 20 g of bran (water content of about 10%) and 15 g of magnesium aluminate metasilicate (in the case of 18% loss on drying), then grown at 25 ° C. using a medium for solid cultivation prepared with a water content of 65% No browning occurs in the inoculum part.
Further, these strains are known as parent strains, for example, mutant strains obtained by mutating spores of the strains or by mating the mycelium of the strains and the hyphae of other strains, which are equivalent to the strains Mutants having the property of i.e., the property that inoculum sclerosis hardly occurs are also encompassed in the present invention. Such mutant strains can be easily obtained by those skilled in the art by conventional methods. Further, whether or not the mutant strain has the same properties as the strain can be selected by the method described in the present specification. Furthermore, in cultivation using a solid cultivation medium comprising coniferous sawdust and rice bran as main constituents and comprising about 6.4% by weight magnesium aluminate metasilicate in a dry weight ratio, browning of inoculum on the surface of the solid cultivation medium A Bunashimeji strain having properties equivalent to at least one strain selected from the aforementioned strains that do not cause the symptom is also encompassed in the present invention.

The various properties of Hypsizygus marmoreus K-4975 (FERM BP-11321) are shown.
(1) Growth state on malt extract agar medium (25 ° C.):
Use malt extract agar medium (malt extract 5%, agar 3%). Vigorous growth on day 7, colony diameter is 52 mm. It produces white and dense mycelia, aerial hyphae. Mycelia grow throughout the petri dish on the 12th day. The mycelium is white, dense and straight. The back side is uniform and there is no discoloration.
(2) Growth state on potato / glucose agar medium (25 ° C.):
PDA agar medium (Difco Potato Dextrose Agar, manufactured by Becton, Dickinson and Company USA) was used. Vigorous growth on the 7th day, colony diameter is 55 mm. A large amount of white and dense hyphae and aerial hyphae are produced. Mycelia grow throughout the petri dish on the 12th day. On the 20th day, the entire surface is covered with dense mycelia and swells in a mat shape. There are many aerial hyphae. The back side is uniform and there is no discoloration.
(3) Growth state on the Tuapec-Docx agar medium (25 ° C.):
Tourpec Dox Agar (NaNO ₂ 0.2%, K ₂ HPO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.05%, KCl 0.05%, FeSO ₄ · 7H ₂ O 0.001%, Sucrose 3%, agar 1.5%). Medium growth on day 7, colony diameter 45 mm. Mycelium grows in a dendritic form and is extremely dilute and has few aerial hyphae. On the 10th day, the colony diameter was 65 mm, and on the 15th day, hyphae grew on the entire petri dish. The mycelium is dendritic, white and dilute. The back side is uniform and there is no discoloration.
(4) Growth condition on Sabouraud agar medium (25 ° C):
Sabouraud agar medium (glucose 4%, peptone 0.1%, agar 1.5%) is used. Small growth on day 10, colony diameter 31 mm. White, thin cotton-like mycelium, colony diameter 55 mm on the 20th day. Mycelium is white and dense. The back side is uniform and there is no discoloration.
(5) Growth state on oatmeal agar medium (25 ° C.):
Oatmeal agar medium (1000 g or less of water is added to 30 g of oatmeal and boiled for 1 hour in a hot water bath. After filtering with 2-3 layers of gauze, water is added to the resulting filtrate to make 1000 mL. %) Is used. Vigorous growth on the 7th day, colony diameter is 54 mm. The hyphae are well branched and stretched, and there are many aerial hyphae. On the 10th day, hyphae grow on the entire petri dish, and a large amount of cotton-like aerial hyphae is produced. Mycelium is white. The back side is uniform and there is no discoloration.
(6) Growth state on synthetic mucor agar medium (25 ° C.):
Synthetic mucor agar (glucose 4%, asparagine 0.2%, KH ₂ PO ₄ 0.05%, MgSO ₄ .7H ₂ O 0.025 g / L, thiamine 0.5 mg / L, agar 1.5%) use. Medium growth on day 10, colony diameter 50 mm. The mycelium is white and extends linearly. On the 15th day, the mycelium grows throughout the petri dish and produces aerial hyphae. Mycelium is white. The back side is uniform and there is no discoloration.
(7) Growth state on YpSs agar medium (25 ° C.):
YpSs agar medium (soluble starch 1.5%, yeast extract 0.4%, K ₂ HPO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.05%, agar 2%) vigorous growth on the 7th day, The colony diameter is 64 mm. The mycelium is white dense and produces a large amount of aerial hyphae. Grows on a mat. On the 10th day, the mycelium grows throughout the petri dish and produces a large amount of aerial hyphae. The back side is uniform and there is no discoloration.
(8) Growth state in phenol oxidase assay medium (25 ° C.):
On PDA agar medium supplemented with 0.5% gallic acid, small growth on day 7, colony diameter was 27 mm. The mycelium is white, fluffy and produces aerial hyphae. The medium is browned and the browning radius is 26 mm. On the 15th day, the mycelium grows in the entire petri dish, and the mycelial surface shows the concentration of mycelia, and produces aerial mycelia. The whole medium is browned. The back side is evenly browned.
(9) Optimal growth temperature:
A precultured 5 mm diameter agar inoculum was inoculated on a PDA agar medium and precultured at 25 ° C. to regenerate mycelia. Then, it culture | cultivated for 7 days at each temperature, and measured the extending | stretching amount of the unidirectional mycelium. As a result, when measuring mycelial elongation for 7 days, the optimum growth temperature was 24-26 ° C. Moreover, although some growth was recognized at 5 degreeC, it hardly grew at 35 degreeC.
(10) Optimal growth pH:
60 mL each of PGY liquid medium (glucose 2%, polypeptone 0.2%, yeast extract 0.2%, KH ₂ PO ₄ 0.5%, MgSO ₄ · 7H ₂ O 0.5%) in a 100 mL Erlenmeyer flask Dispensed and sterilized, adjusted to pH with acid or alkali, inoculated with an agar inoculum having a diameter of 5 mm, and after standing at 25 ° C. for 15 days, the dry weight of the cells was measured. As a result, the optimum growth pH was 6-8. Moreover, the pH range in which this strain can grow was 3.5-10.

Hereinafter, various properties of the strain were examined using the same medium and conditions as described above.
The various properties of Hypsizygus marmoreus K-4799 (FERM BP-11322) are shown.
(1) Growth state on malt extract agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 46 mm. It produces white and dense mycelia, aerial hyphae. On the 13th day, hyphae grew on the entire petri dish. The mycelium is white, dense and straight. The back side is uniform and there is no discoloration.
(2) Growth state on potato / glucose agar medium (25 ° C.):
Vigorous growth on day 7, colony diameter is 53 mm. A large amount of white and dense hyphae and aerial hyphae are produced. Mycelia grow throughout the petri dish on the 12th day. On the 20th day, the entire surface is covered with dense mycelia and swells in a mat shape. There are many aerial hyphae. The back side is uniform and there is no discoloration.
(3) Growth state on the Tuapec-Docx agar medium (25 ° C.):
Small growth on day 7, colony diameter 33 mm. Mycelium grows in a dendritic form and is extremely dilute and has few aerial hyphae. On the 10th day, the colony diameter was 50 mm, and on the 17th day, hyphae grew on the entire petri dish. The mycelium is dendritic, white and dilute. The back side is uniform and there is no discoloration.
(4) Growth condition on Sabouraud agar medium (25 ° C):
Small growth on day 10, colony diameter is 34 mm. White, thin cotton-like mycelium, colony diameter 65 mm on the 20th day. Mycelium is white and dense. The back side is uniform and there is no discoloration.
(5) Growth state on oatmeal agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 51 mm. The hyphae are well branched and stretched, and there are many aerial hyphae. On the 12th day, hyphae grow on the entire petri dish, and a large amount of cotton-like aerial hyphae is produced. Mycelium is white. The back side is uniform and there is no discoloration.
(6) Growth state on synthetic mucor agar medium (25 ° C.):
Medium growth on day 10, colony diameter 55 mm. The mycelium is white and extends linearly. On the 15th day, the mycelium grows throughout the petri dish and produces aerial hyphae. Mycelium is white. The back side is uniform and there is no discoloration.
(7) Growth state on YpSs agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 62 mm. The mycelium is white dense and produces a large amount of aerial hyphae. Grows on a mat. On the 10th day, the mycelium grows throughout the petri dish and produces a large amount of aerial hyphae. The back side is uniform and there is no discoloration.
(8) Growth state in phenol oxidase assay medium (25 ° C.):
Small growth on day 7, colony diameter 21 mm. The mycelium is white, fluffy and produces aerial hyphae. The medium is browned and the browning radius is 26 mm. On the 15th day, the mycelium grows in the entire petri dish, and the mycelial surface shows the concentration of mycelia, and produces aerial mycelia. The whole medium is browned. The back side is evenly browned.
(9) Optimal growth temperature:
When the mycelial elongation for 7 days was measured, the optimum growth temperature was 24 to 26 ° C. Moreover, although some growth was recognized at 5 degreeC, it hardly grew at 35 degreeC.
(10) Optimal growth pH:
The optimum growth pH was 6-8. Moreover, the pH range in which this strain can grow was 3.5 to 10.

The various properties of Hypsizygus marmoreus K-4980 (FERM BP-11323) are shown.
(1) Growth state on malt extract agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 45 mm. It produces white and dense mycelia, aerial hyphae. On the 13th day, hyphae grew on the entire petri dish. The mycelium is white, dense and straight. The back side is uniform and there is no discoloration.
(2) Growth state on potato / glucose agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 47 mm. A large amount of white and dense hyphae and aerial hyphae are produced. Mycelia grow throughout the petri dish on the 12th day. On the 20th day, the entire surface is covered with dense mycelia and swells in a mat shape. There are many aerial hyphae. The back side is uniform and there is no discoloration.
(3) Growth state on the Tuapec-Docx agar medium (25 ° C.):
Small growth on day 7, colony diameter is 38 mm. Mycelium grows in a dendritic form and is extremely dilute and has few aerial hyphae. On the 10th day, the colony diameter was 55 mm, and on the 15th day, hyphae grew on the entire petri dish. The mycelium is dendritic, white and dilute. The back side is uniform and there is no discoloration.
(4) Growth condition on Sabouraud agar medium (25 ° C):
Small growth on day 10, colony diameter is 41 mm. White, thin cotton-like mycelium, colony diameter is 61 mm on the 20th day. Mycelium is white and dense. The back side is uniform and there is no discoloration.
(5) Growth state on oatmeal agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 46 mm. The hyphae are well branched and stretched, and there are many aerial hyphae. On the 13th day, hyphae grow on the entire petri dish, and a large amount of cotton-like aerial hyphae is produced. Mycelium is white. The back side is uniform and there is no discoloration.
(6) Growth state on synthetic mucor agar medium (25 ° C.):
Medium growth on day 10, colony diameter 47 mm. The mycelium is white and extends linearly. On the 15th day, the mycelium grows throughout the petri dish and produces aerial hyphae. Mycelium is white. The back side is uniform and there is no discoloration.
(7) Growth state on YpSs agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 59 mm. The mycelium is white dense and produces a large amount of aerial hyphae. Grows on a mat. On the 10th day, the mycelium grows throughout the petri dish and produces a large amount of aerial hyphae. The back side is uniform and there is no discoloration.
(8) Growth state in phenol oxidase assay medium (25 ° C.):
Small growth on day 7, colony diameter 29 mm. The mycelium is white, fluffy and produces aerial hyphae. The medium is browned and the browning radius is 27 mm. On the 15th day, the mycelium grows in the entire petri dish, and the mycelial surface shows the concentration of mycelia, and produces aerial mycelia. The whole medium is browned. The back side is evenly browned.
(9) Optimal growth temperature:
When the mycelial elongation for 7 days was measured, the optimum growth temperature was 24 to 26 ° C. Moreover, although some growth was recognized at 5 degreeC, it hardly grew at 35 degreeC.
(10) Optimal growth pH:
The optimum growth pH was 6-8. Moreover, the pH range in which this strain can grow was 3.5-10.

The various properties of Hypyzygus marmoreus K-4981 (FERM BP-11324) are shown.
(1) Growth state on malt extract agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 48 mm. It produces white and dense mycelia, aerial hyphae. Mycelia grow throughout the petri dish on the 12th day. The mycelium is white, dense and straight. The back side is uniform and there is no discoloration.
(2) Growth state on potato / glucose agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 48 mm. A large amount of white and dense hyphae and aerial hyphae are produced. On the 13th day, hyphae grew on the entire petri dish. On the 20th day, the entire surface is covered with dense mycelia and swells in a mat shape. There are many aerial hyphae. The back side is uniform and there is no discoloration.
(3) Growth state on the Tuapec-Docx agar medium (25 ° C.):
Small growth on day 7, colony diameter is 30 mm. Mycelium grows in a dendritic form and is extremely dilute and has few aerial hyphae. On the 10th day, the colony diameter was 45 mm, and on the 17th day, hyphae grew on the entire petri dish. The mycelium is dendritic, white and dilute. The back side is uniform and there is no discoloration.
(4) Growth condition on Sabouraud agar medium (25 ° C):
Small growth on day 10, colony diameter 36 mm. White, thin cotton-like mycelium, colony diameter is 68 mm on the 20th day. Mycelium is white and dense. The back side is uniform and there is no discoloration.
(5) Growth state on oatmeal agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 48 mm. The hyphae are well branched and stretched, and there are many aerial hyphae. On the 12th day, hyphae grow on the entire petri dish, and a large amount of cotton-like aerial hyphae is produced. Mycelium is white. The back side is uniform and there is no discoloration.
(6) Growth state on synthetic mucor agar medium (25 ° C.):
Medium growth on day 10, colony diameter 50 mm. The mycelium is white and extends linearly. On the 15th day, the mycelium grows throughout the petri dish and produces aerial hyphae. Mycelium is white. The back side is uniform and there is no discoloration.
(7) Growth state on YpSs agar medium (25 ° C.):
Vigorous growth on the 7th day, colony diameter is 55 mm. The mycelium is white dense and produces a large amount of aerial hyphae. Grows on a mat. On the 10th day, the mycelium grows throughout the petri dish and produces a large amount of aerial hyphae. The back side is uniform and there is no discoloration.
(8) Growth state in phenol oxidase assay medium (25 ° C.):
Small growth on day 7, colony diameter is 30 mm. The mycelium is white, fluffy and produces aerial hyphae. The medium is browned and the browning radius is 27 mm. On the 15th day, the mycelium grows in the entire petri dish, and the mycelial surface shows the concentration of mycelia, and produces aerial mycelia. The whole medium is browned. The back side is evenly browned.
(9) Optimal growth temperature:
When the mycelial elongation for 7 days was measured, the optimum growth temperature was 24 to 26 ° C. Moreover, although some growth was recognized at 5 degreeC, it hardly grew at 35 degreeC.
(10) Optimal growth pH:
The optimum growth pH was 6-8. Moreover, the pH range in which this strain can grow was 3.5-10.

  All of these 4 strains formed a band in the opposite culture with the other 3 strains. Furthermore, a band was formed even in the counter-culture with lyophilum urmarium M-8171 (FERM BP-1415) and various known strains, and it was revealed that the strain was a new strain. Moreover, it is a solid consisting of magnesium aluminate metasilicate having coniferous sawdust and rice bran as main constituents and a dry weight ratio of about 6.4% by weight (for example, about 15 g / 850 mL bottle in the case of 18% loss on drying of a commercial product). It is a new strain that does not cause inoculum sclerosis in cultivation using a cultivation medium. In addition, coniferous sawdust and rice bran are the main constituents, and the dry weight ratio is about 6.4% by weight (for example, when the loss on drying of a commercial product is 18%, the wet weight is about 15 g / 850 mL bottle) magnesium aluminate metasilicate. In the cultivation using the medium for solid cultivation consisting of the above, all the strains have a high yield as compared with Riophylamumumarium M-8171 (FERM BP-1415). Furthermore, even when cultivated in a normal medium, that is, a medium that does not contain a large amount of magnesium compound, the above-mentioned strains have a lower incidence of inoculum sclerosis than conventional strains.

As mentioned above, although bottle cultivation was mentioned as an example, this invention is not limited to the selection method by the said bottle cultivation.
By using the strain obtained by the above-described screening method, it is possible to produce bunashimeji fruit bodies by bottle cultivation, bag cultivation, box cultivation or the like. As an example, the production method of Bunashimeji fruiting body by bottle cultivation is described. This method includes preparation of bacterial bed medium, bottling, sterilization, inoculation of inoculum, fungus bed culture, fungi (sardine oysters), sprout, growth, harvest, etc. It consists of each process. For example, the strain obtained by the above-mentioned selection method can be used as an inoculum when performing fungus bed cultivation described in Japanese Patent Application Laid-Open No. 05-268742.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited only to the scope of the following examples.

Example 1
Sawdust (cedar wood) 62g in dry weight, wet weight rice bran 60g (water content 10%), corn cob meal 30g (water content 10%), soybean hull 20g (water content 10%), bran 20g (water content 10%) ) Well mixed with magnesium aluminate metasilicate (weight loss on drying 18%, manufactured by Fuji Chemical Industry Co., Ltd., trade name: Neusilin) 2.5 g or 15 g / bottle (each dried relative to the total dry weight of the medium for solid cultivation) After adding the water content to 65%, the polypropylene 850 mL wide-mouth culture bottle (Blowbin S-850, manufactured by Shin-Etsu Agricultural Materials Co., Ltd.) was added at a weight ratio of about 1.1% by weight and about 6.4% by weight. ). An inoculation hole having a diameter of about 1 cm was formed in the center of the surface of the filling, and after plugging, pasteurized at 120 ° C. for 60 minutes and cooled to room temperature to prepare a medium for solid cultivation.

  A total of 434 crosses were obtained by carrying out cross-bred for 11 strains of the Hypzygus marmoreus strain possessed by the present inventors. Cultivating test for inoculating each of the mating strains into the above medium, and a new mating strain in which the surface of the fungus bed before the fungi was not browned in a solid cultivation medium containing 15 g / 850 mL bottle of magnesium aluminate metasilicate 4 strains of Hypzygus marmoreus K-4975 (FERM BP-11321), K-4799 (FERM BP-11322), K-4980 (FERM BP-11323), K-4981 (FERM BP-11324) Selected.

  A solid culture medium containing 2.5 g or 15 g / 850 mL bottle of magnesium aluminate metasilicate was prepared. K-4975, K-4979, K-4980, K-4981, K-4529 (strains selected from the above-mentioned mating strains without going through the selection process), known from Buna-Shimeji, which is commercially available from JA Zenno Nagano as a comparison. 8 solid inoculums separately prepared by the above method (hereinafter referred to as a commercially available strain) and lyophilum ulmarium M-8171 (FERM BP-1415) were separately inoculated into a solid culture medium at 25 ° C. and humidity The mycelium was cultured for 30 days under the condition of 55%, and further cultured for 45 days to obtain a fruiting body generating group. Thereafter, the cap was removed, and the mycelium layer 1 cm above the fungus bed surface was left in the center, and the fungus was scrubbed, and the condition of the fungus bed surface during fungi scavenging was investigated. The results are shown in Table 1.

  As is apparent from Table 1, when 2.5 g of magnesium aluminate metasilicate, which is usually used for increasing the yield in the manufacture of bunashimeji fruit bodies, was added, all the strains grew normally, whereas bunashimejiko When 15 g of magnesium aluminate metasilicate, which is not normally used in the production of the entity, is added, 4 out of 5 newly obtained crosses (K-4975, K-41979, K-4980, And K-4981) were normal. However, one strain (K-4529) was accompanied by browning of the fungus bed surface and generation of off-flavors in all test sections. Furthermore, browning and off-flavor generation were also observed in all test sections for the commercially available strain and M-8171.

  Next, after adding 20 mL of tap water to all these media and absorbing them sufficiently, excess water was removed by inversion dehydration, and then the environment was illuminance 20 lux, temperature 15 ° C., humidification was Humiai 100 (manufactured by Kakimiya Seisakusho). ) Was controlled to a humidity of 90% or more and the carbon dioxide concentration was controlled to be in the range of 1000 to 3000 ppm. After budding and growing for 14 days, the membrane regeneration state was confirmed. The results are shown in Table 2.

  As apparent from Table 2, when 2.5 g of magnesium aluminate metasilicate was added, the membrane regeneration state was normal in all strains. On the other hand, when 15 g was added, the membrane regeneration state of K-4975, K-4799, K-4980, K-4981, and commercially available strains was normal, but for K-4529 and M-8171 Some of the membranes were partially regenerated and some were not regenerated at all.

  Furthermore, the growth was continued under the same conditions, and a bunashimeji fruiting body (mature fruiting body) was obtained. Table 3 shows the results of the average yield and the number of days of growth (the number of days until the mature fruit body is harvested after wiping the fungus). In the table, the average yield is “average yield of fruiting body = total weight of fruiting body / number of generated test bottles (including non-occurring bottles)”, and the number of growing days is “number of growing days = total number of growing days required for mature fruiting body / number of harvested bins” (Non-occurrence bins are not included) ”.

  As can be seen from Table 3, when 15 g of magnesium aluminate metasilicate was added, K-4975, K-4799, K-4980 and K-4981 had good yields, no special days of growth, and fruiting bodies. The quality of the was also good. On the other hand, with regard to K-4529, a commercially available strain, and M-8171, a significant decrease in yield and deterioration in quality were observed.

  From the results of Tables 1 to 3, K-4975, K-41979, K-4980 and K-4981 are inoculum sclerosis by using a medium for solid cultivation containing a large amount of magnesium aluminate metasilicate which is a magnesium compound. It was clarified that it is a strain that hardly occurs. Thus, these strains do not cause inoculum sclerosis or occur more frequently than conventional strains even in large-scale commercial production using a normal medium, that is, a medium that does not contain a large amount of magnesium compound. It became clear that it was extremely low. In addition, it was revealed that by using a solid culture medium containing a large amount of magnesium aluminate metasilicate, which is a magnesium compound, it is possible to select a strain that hardly or hardly causes inoculum sclerosis.

Example 2
Whether 15 g of magnesium aluminate metasilicate was added or not was affected by the temperature at which the mycelium was cultured had an influence on the selection of strains that hardly or did not cause inoculum sclerosis.

  In the same manner as described in Example 1 except that a medium for solid cultivation to which 15 g of magnesium aluminate metasilicate was added and the temperature at the time of culturing the mycelium was 25 ° C. and 27 ° C., beech shimeji (Hypzygyus marmoreus) ) K-4975 (FERM BP-11321), K-4799 (FERM BP-11322), K-4980 (FERM BP-11323), K-4981 (FERM BP-11324), K-4529, commercially available strains for comparison and The lyophilum ulmarium M-8171 (FERM BP-1415) was examined. First, the condition of the fungus bed surface during the fungi was investigated. The results are shown in Table 4.

  As is clear from Table 4, the same results as in Example 1 were obtained when 15 g of magnesium aluminate metasilicate was added and the mycelium culture temperature was 25 ° C. Even when the mycelium culture temperature is 27 ° C. in the same medium, as in the case of 25 ° C., 4 strains (K-4975, K-41979, K-4980) among the 5 newly obtained cross strains. , And K-4981) were normal. However, one strain (K-4529) was accompanied by browning and off-flavor in all test sections. Moreover, also about the commercially available strain and M-8171, it was accompanied by browning and off-flavor in all test sections.

  Next, Table 5 shows the results of confirming the membrane regeneration state after water absorption, inversion dehydration, budding and growth in the same manner as in Example 1.

  As is clear from Table 5, the same results as in Example 1 were obtained when 15 g of magnesium aluminate metasilicate was added and the mycelium culture temperature was 25 ° C. Even when the mycelium culture temperature was 27 ° C. in the same medium, the membrane regeneration state of K-4975, K-4799, K-4980, K-4981, and the commercial strain was normal, but K-4529 and As for M-8171, only a part of the membrane was regenerated and a membrane was not regenerated at all.

  Next, Table 6 shows the results of the average yield and the number of days of growth (number of days until the mature fruit body is harvested after wiping the fungus).

  As is apparent from Table 6, the same results as in Example 1 were obtained when 15 g of magnesium aluminate metasilicate was added and the mycelium culture temperature was 25 ° C. Even when the mycelium culture temperature is 27 ° C. in the same medium, K-4975, K-4799, K-4980 and K-4981 have good yield, no special delay in growth, and the quality of fruiting bodies is also low. It was good. On the other hand, with regard to K-4529, a commercially available strain, and M-8171, a significant decrease in yield and deterioration in quality were observed.

  From the results of Tables 4 to 6, K-4975, K-4799, K-4980 and K-4981 are inoculum sclerosis by using a medium for solid cultivation containing a large amount of magnesium aluminate metasilicate which is a magnesium compound. It became clear that this is a strain that is unlikely to occur. Moreover, when using the culture medium for solid cultivation containing a large amount of magnesium aluminate metasilicate, it became clear that the change in the culture temperature of the mycelium does not affect the selection of the strain that hardly or hardly causes inoculum sclerosis.

Example 3
Alumina magnesium hydroxide (coprecipitate of aluminum hydroxide and magnesium hydroxide, 10% loss on drying, manufactured by Kyowa Chemical Industry Co., Ltd., trade name: KYOWARD 300) instead of magnesium aluminate metasilicate 5 g / bottle (for solid cultivation) In the same manner as in Example 2 except that about 2.5% by weight as a dry weight ratio with respect to the total dry weight of the medium was added, Hypsizygus marmoreus K-4975 (FERM BP-11321), K-4799 (FERM BP-11322), K-4980 (FERM BP-11323), K-4981 (FERM BP-11324), K-4529, as a comparison, a commercial strain and lyophilum ulmarium M-8171 (FERM BP-1415) went. First, the condition of the fungus bed surface during the fungi was investigated. The results are shown in Table 7.

  As is apparent from Table 7, the results showed the same tendency as in Example 2 under the condition where 5 g of alumina magnesium hydroxide was added and the mycelium culture temperature was 25 ° C. or 27 ° C. .

  Next, after adding 20 mL of tap water to all these media and absorbing them sufficiently, excess water was removed by inversion dehydration, and then the environment was illuminance 20 lux, temperature 15 ° C., humidification was Humiai 100 (manufactured by Kakimiya Seisakusho). ) Was controlled to a humidity of 90% or more and the carbon dioxide concentration was controlled to be in the range of 1000 to 3000 ppm, and budding and growth were continued for 12 days. Furthermore, K-4980 and K-4981 were grown for 9 days, K-4975 was grown for 10 days, K-4799, K-4529 and commercial strains were grown for 14 days, and M-8171 was grown for 15 days. It was.

  The membrane regeneration state on the 14th day at the time of budding and growth was confirmed. The results are shown in Table 8.

  As is apparent from Table 8, when the mycelium culture temperature is 25 ° C. under conditions where 5 g of alumina magnesium hydroxide is added, only some of the commercially available strains are regenerated and some are not regenerated at all. It was. For other strains, the same results as in Example 2 were obtained. Moreover, when the mycelium culture temperature was 27 ° C. in the same medium, the same results as in Example 2 were obtained.

  Next, Table 9 shows the results of the average yield and the number of growth days (the number of days until the mature fruit body is harvested after the fungi are scraped).

  As apparent from Table 9, even in the condition where 5 g of alumina magnesium hydroxide was added, K-4975, K-4799, K-4980 and K-4981 had good yields, and there was no special delay in growth days, and the fruiting bodies The quality of the was also good. On the other hand, regarding K-4529, a commercially available strain, and M-8171, fruit bodies were not generated, or a significant decrease in yield and deterioration in quality were observed.

  From the results of Tables 7 to 9, even when a solid culture medium containing a large amount of magnesium hydroxide, which is a magnesium compound, is used, K-4975, K-41979, K-4980, and K-4981 are inoculum sclerosis. It was clarified that it is a strain that hardly occurs. In addition, even when using a solid culture medium containing a large amount of magnesium aluminate instead of magnesium aluminate metasilicate, it has become clear that it is possible to select a strain that hardly or does not cause inoculum sclerosis. It was revealed that the same effect can be obtained with other magnesium compounds.

  According to the present invention, a Buna shimeji strain that does not cause browning of the inoculum portion on the surface of the medium is provided in cultivation on a solid medium for commercial production. By using this strain, stable and large-scale commercial production of Bunashimeji fruiting bodies becomes possible. In addition, the present invention provides a simple screening method for Bunashimeji strains that are less prone to inoculum sclerosis. By using this method, it is possible to select in advance whether or not it is a Buna shimeji strain that is prone to inoculum sclerosis, preventing the yield of large-scale commercially produced Buna shimeji fruit bodies, and efficiently Manufacture is possible.

Claims (4)

In cultivation using a solid culture medium containing coniferous sawdust and rice bran as main constituents and containing 6.4% by weight of magnesium aluminate metasilicate as a dry weight ratio, browning of the inoculum on the surface of the solid culture medium was caused. Not the following Bunashimeji strains.
Hypyzygus marmoreus K-4975 (FERM BP-11321),
Hypyzygus marmoreus K-4799 (FERM BP-11322),
Hypyzygus marmoreus K-4980 (FERM BP-11323),
Hypsizygus marmoreus K-4981 (FERM BP-11324 )   Coniferous sawdust and rice bran are the main constituents, 5.2 to 8.4 wt% magnesium aluminate metasilicate, 0.5 to 3 wt% magnesium oxide and 2 wt% dry weight ratio. A method for selecting a Bunashimeji strain that does not cause inoculum sclerosis, comprising culturing the Bunashimeji strain using a medium for solid cultivation comprising at least one magnesium compound selected from -4 wt% magnesium hydroxide alumina. The method for selecting a Bunashimeji strain according to claim 2 , wherein the medium for solid cultivation comprises coniferous sawdust, rice bran, corn cob meal, soybean hulls, bran, and a magnesium compound. Screening method Bunashimeji strains according to claim 2 or 3 Bunashimeji strain does not cause browning of inoculum portion of the solid cultivation medium surface is a screening method for selecting.