JPS60149379A - Production of basidiomycetous fruiting body - Google Patents

Abstract

PURPOSE:To obtain a basidiomycetous fruiting body, e.g. an edible mushroom, at a low cost without a change from a dark to a lighted places nor temperature change, by cultivating a basidiomycetous mold starter in a nutrient culture medium containing anthranilic acid, p-aminobenzoic acid, etc. to induce the basidiomycetous fruiting body. CONSTITUTION:A basidiomycetous mold starter is inoculated into a nutrient culture medium containing at least one of anthranilic acid, p-aminobenzoic acid or a metal salt or a mixture thereof and further maltose, polypeptone, a salt such as KH2PO4 or thiamin hydrochloride, etc. and cultivated therein to induce a basidiomycetous fruiting body and give the aimed mushroom. Examples of the basidiomycete to be used include edible mushrooms such as Lentinus edodes (SHIITAKE mushroom), Flammulina velutipes, Panus ostreatus, Pholiota nameko, Auricularia auricula, Lyophyllum aggregatum, etc. and Polyporus orcularius, Polyporaceae versicolor, Elfvinga applanata, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing basidiomycete fruiting bodies. More specifically, the basidiomycete inoculum is cultured in a nutrient medium supplemented with at least one of anthranilic acid (also known as 〇-aminobenzoic acid), p-aminobenzoic acid, or their potassium, sodium, or calcium salts, or a mixture thereof. , relates to a method for producing basidiomycetes, which is characterized by inducing basidiomycete fruiting bodies.

Basidiomycete fruiting bodies have not only been enjoyed as edible mushrooms for a long time, such as shiitake mushrooms, enoki mushrooms, hiratake mushrooms, nameko mushrooms, wood ear fungi, and matsutake mushrooms, but also in recent years have been used as sources of various useful physiologically active substances and medicines. It has started to attract particular attention.

For the formation of basidiomycete fruiting bodies, not only the supply of sufficient nutrients but also environmental stimuli such as sufficient lighting and appropriate temperature changes are considered essential. However, in the production of basidiomycete fruiting bodies, temperature treatments such as switching from dark to light irradiation or lowering the culture temperature midway through cultivation are time-consuming and are factors that increase costs. It became.

Therefore, the present inventors cultivated the basidiomycete inoculum only in the dark and at a constant temperature without changing from dark to light or changing the temperature when producing basidiomycete fruiting bodies. A natural microbial product was used as a basidiomycete fruiting body inducer, which makes it possible to form basidiomycete fruiting bodies on top of the basidiomycete.

By chance, we were able to find the basidiomycete fruiting body inducer in the broth of TA-7, a strain belonging to the actinobacteria group. Upon identification, it was determined that the basidiomycete fruiting body inducing substance was anthranilic acid. Furthermore, the present invention was completed after discovering that p-aminobenzoic acid, a related substance, has a similar effect on inducing fruiting bodies of basidiomycetes. That is, the present invention cultivates a basidiomycete inoculum in a nutrient medium supplemented with anthranilic acid, p-aminobenzoic acid, a metal salt thereof, or a mixture thereof,
This is a method for producing a basidiomycete fruiting body, which is characterized by inducing a basidiomycete fruiting body.

In the present invention, the term "basidiomycetes" is not limited to edible mushrooms such as shiitake, enokitake, oyster mushroom, nameko, wood ear fungus, and cymen, but broadly refers to basidiomycetes in general, such as amis gitake, versicolor, and sarunokoshika.

The medium for producing basidiomycete fruiting bodies in the present invention includes carbohydrates such as starch, maltose, glucose, and sucrose, peptone, amino acids, soybean protein, fish meal, organic and inorganic nitrogen sources such as ammonium sulfate, thiamine, yeast extract, Appropriate combinations of micronutrients such as C3L, nucleic acid-related substances, etc., or appropriate combinations of these with wood chips, oak waste, rice bran, paper waste, etc., or basidiomycete fruiting body inducers such as anthranilic acid in potato broth medium, etc. Add appropriate amount.

The inoculum can be inoculated by the usual method, and 20 to 30 minutes after inoculation.
C., preferably around 25.degree. C., for 10 to 40 days in the dark. In this way, the basidiomycete fruiting body is induced or
Further, by continuing the culture under light irradiation, mature basidiomycete fruiting bodies can be obtained.

Anthranilic acid, p-aminobenzoic acid, or their metal salts added to the culture medium in the present invention are chemically synthesized and easily available. Therefore, the TA-7 strain can be obtained without going to the trouble of culturing and producing it. And anthranilic acid with a purity of not only special reagent grade but also first grade reagent and second grade reagent,
Any of p-aminobenzoic acid or their metal salts or mixtures thereof may be suitably used in the present invention.

Next, a test example regarding the optimal amount of anthranilic acid, which is a basidiomycete-inducing substance, to be added to a medium in the present invention and the ability of anthranilic acid-related compounds to induce basidiomycete fruiting bodies will be described.

Test Example 1 Amysgitake (Favolus arcularius ATCC) was placed in each of 10 Ersinmeyer flasks containing an agar medium with the same composition as that shown in Example 1.
24461) and then 0.01-100 ng
/ Discs impregnated with anthranilic acid at various concentrations were placed around each flask and incubated in the dark for 2 hours.
Cultured at 5°C for 2 weeks. Table 1 shows the relationship between the ability to induce fruit bodies of Amis Gitake and the amount of anI-ranilic acid added.

(Left below) Table-1 As is clear from Table-1, the amount of anthranilic acid added is 0.
．． It can be seen that the ability to induce fruiting bodies is shown in the range of 1 to 3 ng/disc (equivalent to 3 to long/- as the amount added in the medium).

Test Example 2 A test was conducted under the same conditions as Test Example 1, except that an anthranilic acid-related compound was used instead of the anthranilic acid of various concentrations impregnated into the disk in Test Example 1. Table 2 shows the relationship between anthranilic acid-related compounds and the ability to induce fruit bodies of Mysgitake.

However, cases with the ability to induce fruiting bodies are indicated as (+), and cases without are indicated as (-).

(Space below) Table 2 As is clear from Table 2, in addition to anthranilic acid, the sodium salt of anthranilic acid, or its potassium salt, p-aminobenzoic acid or the sodium salt of p-aminobenzoic acid of related compounds, or its potassium salt (dose of 1 to 3 ng/disc) was found to have the ability to induce fruiting bodies of Mysgitake, but neither m-aminobenzoic acid, methyl anthranilate, nor N-methylanthranilate had the ability to induce fruiting bodies. was absent (doses of 0.01-100 ng/disc).

Tryptophan and kynurenine, which are related to anthranilic acid in the metabolic pathway, as well as folic acid, which contains a p-aminobenzoic acid group, have a concentration of 0.01 to 100 n.
It is shown that it does not have the ability to induce Amisukitake fruiting bodies when administered at 100 g/disk.

In Test Examples 1 and 2, Amis Gitake was used as the basidiomycete, but similar results were obtained with mushrooms such as Enoki mushroom, Oyster mushroom, and Shiitake.

Is the method for producing basidiomycete fruiting bodies according to the present invention extremely? The inoculum was inoculated into a culture medium to which anthranilic acid, its sodium salt, potassium salt, or calcium salt, p-aminobenzoic acid, its sodium salt, potassium salt, or calcium salt was added, and the culture medium was kept at a constant temperature in the dark. Fruiting bodies can be induced simply by culturing the mushrooms, which is an epoch-making phenomenon that could not even be imagined using conventional mushroom cultivation techniques.

The present invention will be specifically explained below with reference to Examples.

Example 1 Maltose 20g 1 Volibep I・N 1g, KH2P
O40.3g, MgSO4・71120'0.3g,
CaCl2.2H200, 1g.

Zn5O+ ・7 H2O0,3n+g, FeSO4−
7H2O0,15mg, CuSO4・5 H2O0,1
nv, MnSO4・5H200,1nw, (N114
)6 Mo7024・t1200.02mg, thiamine hydrochloride 0.1■, anthranilic acid (Wako Pure Chemical Industries, Ltd., reagent special grade) 3/'g dissolved in 1000m water (pH
5,2,)' 5 (1ml!) was dispensed into a 200me Erlenmeyer flask, and after heat sterilization, Amis Gitake (
Favolus arcularius AT, CC24
461) and cultured in the dark at 25°C for 2 weeks, several fruiting bodies were formed in the flask. On the other hand, in the case where anthranilic acid is not added, fruiting bodies are not formed in the dark, but primordia are formed on the second day after being transferred to the light, and then 3
Fruiting bodies were obtained on day one.

Example 2 10 pr of p-aminobenzoic acid instead of anthranilic acid
50me of medium having the same composition as in Example 1 except that g was added.
was dispensed into Erlenmeyer flasks with a capacity of 200 + + + (!), and after heat sterilization, the Favolus
cula-rius ATCC24461) and cultured in the dark at 25°C for 15 days, numerous fruiting bodies were formed in the flask. On the other hand, when p-aminobenzoic acid was not added, fruiting bodies were not induced in the dark, primordia were formed on the second day after transfer to the light, and fruiting bodies were formed after a further 3 days.

Example 3 3 pg of anthranilic acid was added to a potato broth medium (300 g of fresh potatoes were heat-extracted for 30 minutes to obtain a broth of looomE, and 10 g of maltose was added).
After that, it was dispensed into a 100-capacity Erlenmeyer flask, sterilized at 120°C for 15 minutes, and after cooling, it was mixed with enokitake (Flammulina velutipes IFO).
7045) and cultured in the dark at 25°C for 38 days, a large number of fruiting bodies were induced in the flask. On the other hand, no fruiting bodies were formed even after 38 days of culture in a medium without anthranilic acid. To form fruiting bodies 1
After culturing at 25°C in the dark for 4 days, the fruiting body primordium was formed by culturing in the light and lowering the temperature to 17°C for 11 days, and after 13 days of continued culture. Fruiting bodies were formed.

Example 4 Sucrose Log, polypeptone 2g, KH2PO
40.5g, MgSO4・711200.5g, Ca
Cl2-211200. '1g.

Thiamine hydrochloride 0.1■, ZnSO4.5) 120
0.3■, FeSO4・7 N200.15mg, Cu
SO4・51120 0.1mg, MnSO4・5N
20 0.1m1r, (N114)6 Mo702
4 ・H2!00.02mg and p-aminobenzoic acid N
Dissolve 10 pg of a-salt in 1,000 ml of water, dispense 20 ml into a 50-mn Erlenmeyer flask, and heat-sterilize it.
'a IFO7051) and incubated in the dark at 27℃ for 3
After culturing for 5 days, many fruiting bodies were induced in the flask. On the other hand, when p-aminobenzoic acid is not added, fruiting bodies are not induced, and in order to form fruiting bodies, after culturing in the dark for 14 days, transfer to a light place at 17°C and continue culturing for 11 days. was necessary.

Example 5 Of the medium composition in Example 1, 3 pg of anthranilic acid
A medium with the same composition was used, except that a mixture of anthranilic acid and p-aminobenzoic acid (ratio 1:2) was used instead, and 50-mL of the medium was dispensed into a 200-volume Erlenmeyer flask. After heat sterilization, Amis gitake (Fa
volus arcularius ATCC2446
1) and cultured in the dark at 25°C for 15 days, many fruiting bodies were induced in the flask. On the other hand, in the culture without the addition of the mixture of anthranilic acid and p-aminobenzoic acid, fruiting bodies were not induced even after 20 days of culture.

Example 6 Among the medium compositions in Example 1, anthranilic acid 3p
A medium with the same composition was used, except that 3 pg of potassium anthranilate was used instead of 3 pg of potassium anthranilate, and 50 g of the culture medium was dispensed into a 200-capacity Erlenmeyer flask, and after heat sterilization, the culture medium was incubated with Favolus arcularius AT.
CC24461) and cultured in the dark at 25°C for 14 days, a large number of Mysgitake fruiting bodies were induced in the flask. On the other hand, when potassium anthranilate is not added, Amis Gitake fruiting bodies are not induced even after 20 days of culture; in order to induce fruiting bodies, it is necessary to culture in the dark for 6 days, then transfer to a light place and culture for 5 days. there were.

Example 7 Among the medium compositions used in Example 1, anthranilic acid 3p
Calcium anthranilate 3) sg was used instead of sg, but a medium with the same composition was used, and the 50-sg was replaced with 20-sg.
0m1! Pour into Erlenmeyer flasks, heat sterilize, and add Favolus arcula.
rius ATCC24461) and inoculated in the dark for 25 minutes.
When cultured at °C for 14 days, many fruiting bodies of Mysgitake were induced in the flask. On the other hand, in the medium containing no calcium anthranilate, fruiting bodies of Mysgitake were not induced even after 20 days of culture.

Example 8 Potato broth of Example 3 10100O! 10 J of a mixture of anthranilic acid and p-aminobenzoic acid (mixing ratio 1:1) in place of 3 Jjg of anthranilic acid added in the mixture.
20me was added to a 100-capacity Erlenmeyer flask, and after heat sterilization, enokitake (Fl.
ammulina velutipes IFO704
5) and cultured in the dark at 27°C for 38 days, a large number of Enokitake fruiting bodies were induced in the flask.

On the other hand, when the mixture of anthranilic acid and p-aminobenzoic acid was not added, enokitake fruiting bodies were not induced even when cultured for 50 days.

[Brief explanation of drawings]

FIG. 1'a) shows an outline of the conventional method for producing the fruiting body of Enokitake mushroom, and FIG. 1(b) shows an outline of the conventional method for producing the fruiting body of Enokitake mushroom. Figure 2(a)
FIG. 2(b) schematically shows the method for producing the fruiting body of Enokitake mushroom of the present invention, and FIG. Patent applicant Sawao Murao C9 No. 1 (a) (b) Inoculation Light-irradiated fruiting body primordium Mature fruiting body 3 Noto fruiting body primordium Mature fruiting body 9 (- Fig. 2 (a) Inoculation Mature fruiting body (b) ) Inoculation Mature fruiting body 25°C, lh'f Office procedure amendment (method) % formula % 1. Indication of the case 1982 Patent Application No. 4496 2. Name of the invention Process for producing Basidiomycete fruiting body 3. Amendment Relation to the case of a person who does

Claims (1)

[Claims] ■ A basidiomycete inoculum is cultured in a nutrient medium supplemented with at least one of anthranilic acid, p-aminobenzoic acid, or a metal salt thereof, or a mixture thereof, to induce basidiomycete fruiting bodies. A method for producing basidiomycete fruiting bodies. 2. The method for producing a basidiomycete fruiting body according to claim 1, wherein the basidiomycete inoculum is cultured only at 11η sites. 3. The method for producing a basidiomycete fruiting body according to claim 1 or 2, wherein the basidiomycete inoculum is cultured at a constant temperature.