NL9700003A - Method of inoculating Fistulina hepatica - Google Patents

Abstract

The invention relates to a method for inoculating Fistulina hepatica, wherein inocula (inoculums) of Fistulina hepatica are mixed with a sterilized solid culture medium for Fistulina hepatica to disperse the inocula in all of the medium. Preferably, a solid culture medium is used which comprises (a) a wood material (ligneous material) and (b) a nutrient comprising a saccharide and an organic nitrogen compound and which has a pH in the range of from 3.5 to 5.5.

Description

Method for inoculating Fistulina hepatica

The invention relates to a method for inoculating Fistulina hepatica. which makes it possible to spread the fungus threads in the medium during the artificial cultivation of Fistulina hepatica.

Fistulina hepatica is also called "steak mushroom" and grows naturally on the chinquapin tree in May and June or October. The fruit body (hat) of Fistulina hepatica has a liver-like or bovine-like shape and a red to dark reddish brown surface. The fruit body has a diameter of about 10 to 20 cm.

Slices of the fresh fruit body of Fistulina hepatica can be eaten as such or after frying with butter and it is known to be a very tasty mushroom. Thus, if Fistulina hepatica can be grown in large quantities all year round, it can enrich meals and also provide excellent processed foods.

The usual method of inoculating mushrooms involves inoculating mushroom inocula on the surface of a solid culture medium. This method has the drawback that it takes a long time to spread the inocula over the solid culture medium.

The object of the invention is to overcome the above drawback. The invention therefore relates to a method for inoculating Fistulina heoatica. characterized in that Fistulina heoatica inocula is mixed to disperse the inocula in the entire medium.

Figure 1 is a rough sketch showing fruiting bodies of Fistulina heoatica growing from a vessel through perforations in the vessel. In Figure 1, 1 indicates the container, 2 indicates the perforations, and 3 indicates the mature fruit body.

In the present invention, the mushroom inocula are inoculated on or in the culture medium. The advantage of this method is that the fungal threads of Fistulina hepatica can be spread in a short period of time if the inocula have been inoculated by dispersion over the entire solid medium and cultivation is then carried out.

Although the culture medium for Fistulina hepatica can be either liquid or solid, the solid medium is preferred. Any suitable solid medium can be used. In addition, a solid medium consisting of (a) a wood material and (b) a food consisting of a saccharide and organic nitrogen and having a pH in the range of 3.5 to 5.5. suitable for practical artificial cultivation, because the fungus threads of Fistulina hepatica can be spread over the culture medium in a short period of time. The preferred wood material (a) is, for example, a wood powder such as sawdust. The wood materials also include other powders obtained by pulverizing wood with a pulverizer or by chopping and wood shavings or shavings obtained with a planer or cutter.

Examples of the wood materials include deciduous trees such as Fagus, Castanopsis, Pasania, Quercus, Betula, Alnus, Zelkova, Chamaecyparis, Cryptomeria, Abies, Pinus and Larix. In particular, the beech, chinquapin, Quercus mongolica var. grosseserrata, zelkova tree, evergreen oak, alder and white birch; and coniferous trees such as the Japanese cedar, fir, Japanese larch, Japanese red pine and hinoki cypress. The birch, pasania, Castanopsis, Quercus serrata and Quercus mongolica var. gross serrata are particularly preferred.

For spreading the Fistulina hepatica hyphae over the culture medium in a short period of time, at least 50 wt.%, Preferably at least 70 wt.%, More preferably at least 80 wt.%. of the wood material a sieve with an aperture of 11.10 mm but does not pass that of 850 µm, in particular it passes a sieve with an aperture of 6.00 mm but does not pass that of 1.00 mm.

The food (b) comprises a saccharide and an organic nitrogen. The food (b) is a mixture of one or more saccharides selected from glucose, saccharides, starches, α-starches and malt extract and one or more organic nitrogen compounds selected from amino acids, peptons and yeast extracts. Examples of foods that substantially include both (a) saccharide (s) and (a) organic nitrogen compound (s) are corn food additives such as granulated or pulverized corn on the cob and corn bran; soybean food additives such as bean curd waste, defatted soybeans and soybean powder; food additives selected from wheat germ and the untreated rye and oat grain powders; rice bran; wheat bran; and koji.

Of these, koji is particularly preferred. It is also possible to add a saccharide and / or an organic nitrogen compound to the foodstuff which mainly comprises the saccharide (s) and the organic nitrogen compound (s). Although the amount of saccharide and organic nitrogen compound present in the food is not particularly limited, the saccharide content is preferably 0.1 to 90 wt.% Based on the food, and the organic nitrogen compound content is preferably 0.02 to 50 wt%. The ratio of the wood material to the food, which is not particularly limited, is usually 1: 1 to 9: 1. preferably 3: 1 to 9: 1 (on a dry basis). If desired, inorganic compounds and metal salts, such as phosphates, magnesium salts and calcium salts, can be added to the solid medium.

The solid medium containing the wood material and foodstuff has a water content of 50 to 70 wt. %, preferably 50 to 60% by weight, and particularly preferably 56 to 58% by weight, based on the entire medium.

The pH of the solid medium after heating sterilization is 3.5 to 5.5. preferably 4 to 5.2 and more preferably 4.2 to 5.0.

The pH of the medium varies depending on the type and amount of food added. Thus, the pH of the medium is adjusted by adding, for example, a heating inorganic or organic acid, such as a hydrochloric acid solution, a lactic acid solution, an acetic acid solution or a succinic acid solution, to the culture medium before or after heating sterilization. The pH of the medium is determined by suspending 5 van of the medium in 50 ml of distilled water, keeping the suspension for 10 minutes and determining the pH of the suspension.

After sterilizing the solid medium according to a conventional method, the inocula in solid or liquid form are mixed homogeneously in the solid medium described above under sterile conditions to disperse the microbes in the solid medium and then cultivate them. The sterilized solid medium is mixed with the inocula under sterile conditions, which is done with a clean room work table or the like, to form the solid medium which comprises a substantially homogeneous mixture with the inocula, after which cultivation begins. In addition, if the inocula which can be poured into openings of the sterilized medium, such as liquid inocula, are used, the solid medium containing the inocula dispersed homogeneously therein is used to start the cultivation. Although the amount of inocula to be dispersed in the solid medium is not particularly limited, it is preferably at least 0.4 parts by weight, more preferably 0.4 to 20 parts by weight, and most preferably 1 to 10 parts by weight per 100 parts by weight of solid medium.

In particular, the solid medium is added to a vessel and then sterilized. Preferably, this vessel is the same vessel used for growing the fruit body of Fistulina hepatica towards the outside of the vessel after inoculating the inocula. The vessel must have at least a bottom and side walls. Preferred is a sealed vessel with a filter that is permeable to gases such as oxygen and carbon dioxide, but impermeable to spores and the like, and particularly preferred is a plastic bag in which holes can easily be cut in the walls. Of these, a translucent or semi-translucent vessel is preferred, because the primordia of the fruit body of Fistulina hepatica growing in the vessel can be observed from the outside.

Particular preference is given to flexible plastic bags made of a polyolefin film, such as polyethylene or polypropylene, a polyester film, such as polyethylene terephthalate, or a nylon film or a multilayer film thereof. The thickness of the film is preferably about 10 to 80 µm. Although a filter is not necessary if the plastic film as such is permeable to gases such as oxygen and carbon dioxide, it is usually preferred to provide a filter made of polytetrafluoroethylene, polyvinylidene fluoride, a cellulose mixed ester or polycarbonate, which vent pores with a size from 0.05 to 2 µm. Such a filter is commercially available. An example of this is Durapore Membrane Filter (Nippon Millipore Limited).

In the present invention, a vessel, other than the vessel described above, can be used to sterilize the solid medium, after which the contents of the vessel are transferred to the vessel described above at the time of inoculation. After adding the solid medium to the vessel, the vessel is preferably sterilized by heating at, for example, 100 to 120 ° C

for 30 to 300 minutes.

While the amount of solid medium added to the vessel prior to inoculation and cultivation is not particularly limited, it is preferably 50 to 83% based on the capacity of the vessel.

In the present invention, the inocula inoculated in the medium in the vessel are grown by any suitable method for spreading the Fistulina hepatica hyphae into the medium, obtaining a medium completely filled with mycelium, and mycelium is fully grown to bear fruit. In particular, the inocula are mixed in the sterilized solid medium under sterile conditions to homogeneously disperse them throughout the medium and then the solid medium is stored at 15 to 32 ° C for 15 to 60 days. More preferably, the cultivation is carried out in a dark place at a temperature around 25 ° C and at a relative humidity of 50 to 83%, so that the fungus threads spread throughout the solid medium in a time shorter than in conventional methods.

Culturing can thus continue under suitable conditions. The fungus threads of Fistulina hepatica disperse in the medium to form mature mycelium, which is then preferably grown at 10 to 30 ° C for 1 to 30 days to form the primordia of the fruit body. More preferably, the cultivation is carried out at 15 to 20 ° C and at a relative humidity of at least 70% (especially 75 to 90%), the illuminance being controlled in the range of 50 to 1000 lx. It is particularly preferred that the mature mycelium is obtained by growing in a dark place and then growing in a light place at a temperature that is 2 to 15 ° C, preferably 5 to 12 * C, lower than the culture temperature.

After thus forming the primordia of the fruit body, pieces are cut from the vessel around the primordia of the fruit body, so that each fruit body of Fistulina hepatica can grow out of the vessel through the perforation. In particular, the vessel is partially cut open in such a way that the wall of the vessel is cut open to form a perforation near the primordium of the fruiting body or else part of the wall or top surface of the vessel cut open to form a round perforation with a diameter of about 2 cm around the primordium of the fruit body. The size and shape of the perforation are such that the growing fruiting body is not physically contacted with the culture vessel because its growth is inhibited by the wall of the vessel. However, it is desirable that the surface area of the perforation be as small as possible. The culture vessel must be handled with care, because if the medium or the primordium of the fruit body is damaged or strongly stimulated, the fruit body does not grow or the primordium of the fruit body or small fruit body tends to dissolve.

According to the above-described treatment, the fruit body of Fistulina hepatica grows out of the vessel through the perforation formed by the cut-out piece, and as a result, in the present invention, a large mature fruit body is obtained. Because the majority of the medium is thus covered by the culture vessel, it is prevented from drying out as such and the temperature and high moisture content in the medium are kept constant, so that the fruit body of Fistu-linfl hepatica can grow extremely efficiently. is going to be.

It is preferred that the mature fruit body is obtained by keeping the temperature at 10 to 30 ° C for 10 to 40 days. More preferred conditions (include a temperature of 15 to 20 ° C, a relative humidity of at least 90 ° (especially 95 to 98 °) and an illuminance of 50 to 1000 lx. It is particularly preferred that the cultivation , after forming the primordia of the fruit body, it is carried out under conditions of high humidity, which is achieved by increasing the moisture content by at least 5%.

The fruit bodies of Fistulina hepatica that grow out of the vessel through the perforations in the vessel are shown in Figure 1. In Figure 1, 1 shows the vessel, 2 shows the perforations in the vessel, 3 shows the fruit bodies of Fistulina hepatica, and 4 a filter again.

The present invention is further illustrated by the following examples.

Example I

To a mixture of 534 g (100 wt% based on the whole) of birch chips of such size that they did not pass a sieve with a 2 mm aperture but a sieve with a 6 mm aperture and 178 g of dry koji became water added to obtain a solid medium with a water content of 60% by weight. Then the veal thus obtained was added to medium as a 2.5 kg mushroom cultivation bag and provided with a filter (trade name: Kinopack; a product of Nissho Kabushiki Kaisha) to obtain a density of 0.5 g. / cm3 and then sterilized by heating at 121 ° C for 60 minutes. The solid medium thus sterilized by heating had a pH ve 4.9. Subsequently, under sterile conditions, 17 g inocula of Fistulina heoatica (obtained by cultivation in a medium of the same composition as the medium described above) was added to the solid medium. is sterilized by heating to disperse the inocula homogeneously in the solid medium.

The solid medium was stored in a dark place for 25 days at a temperature of 25 ° C and at a humidity of 85% in order to spread the fungus threads of Fistulina hepatica in the medium.

Comparative example 1

A solid medium with a water content of 60% was prepared in the same manner as in Example I, except that 775 g of the same birch chips and 178 g of the same dry koji as in Example I were used.

Then, the solid medium thus obtained was added to a mushroom cultivation bag with a capacity of 2.5 kg and equipped with a filter (trade name: Kinopack; a product of Nissho Kabushiki Kaisha) to obtain a density of 0.5 g / cm3 and then sterilized by heating at 121 ° C for 60 minutes. The solid medium which was sterilized by heating Eddus had a pH of 4.9. Then, under sterile conditions, 17 g inocula of Fistulina hepatica (obtained by cultivation in a medium with the same composition as the above described medium) was inoculated on the surface of the solid medium sterilized by heating.

The solid medium was stored in a dark place at a temperature of 25 ° C and at a humidity of 85%. It took 55 days to spread the Fistulina hepatica hyphae in the medium.

Thus, this method took a total of 55 days to spread the Fistulina hepatica hyphae in the medium, while only 25 days were required to spread them in the medium of Example I.

Example II

979 g (100 wt% based on the whole) of birch chips sized not to pass a 2 mm aperture screen but a 6 mm aperture screen was mixed with 178 g of dry koji. Water was added to the resulting mixture to obtain a solid medium with a water content of 60% by weight. Subsequently, the solid medium thus obtained was added to a mushroom cultivation bag with a capacity of 2.5 kg and fitted with a filter (trade name: Kinopack; a product of Nissho

Kabushiki Kaisha) to obtain a density of 0.5 g / cm3 and then sterilized by heating at 121 ° C for 60 minutes. The solid medium thus sterilized by heating had a pH of J9.9. Then, under sterile conditions, 17 g of Fistulina hepatica inocula (obtained by cultivation in a medium of the same composition as the solid medium described above) was added to the solid medium sterilized by heating to disperse the inocula homogeneously in the solid medium.

The solid medium was stored in a dark place for 25 days at a temperature of 25 ° C and a humidity of 852 to form the mature mycelium of Fistulina hepatica. Cultivation was then continued for 5 days at a temperature of 20 ° C, a humidity of 90 µt and an illuminance of 200 lx to form the primordia of Fistulina hepatica. Cultivation was further continued for 19 days at a temperature of 13 to 23 ° C, a humidity of 90 * or higher and an illuminance of 200 lx to obtain fruiting bodies.

Example III

781 g of birch chips sized not to pass a 2 mm aperture sieve but a 6 mm aperture sieve were mixed with 178 g of dry koji. Water was added to the resulting mixture to obtain a solid medium with a water content of 58 wt. %. Subsequently, the solid aedium thus obtained was added to a mushroom cultivation bag with a capacity of 2.5 kg and provided with a filter (trade name: Kino-pack; a product of Nissho Kabushlkl Kaisha, which is made of a polyethylene film with a high density). a thickness of 40 pa) to obtain a density of 0.5 g / cm 3 and then sterilized by heating at 121 ° C for 60 minutes. The solid aedium thus sterilized by heating had a pH of 4.9. Subsequently, under sterile conditions, 17 g inocula of Fistulina hepatica (obtained by cultivation in a medium with the same composition as the solid medium described above) was added to the solid medium. that has been sterilized by heating to homogeneously disperse the inocula in the solid medium.

The solid medium was stored in a dark place at a temperature of 25 ° C and a humidity of 85% for 25 days to obtain a medium completely filled with mycelium of Fistulina hepatica and where the mycelium is mature enough to bear fruit. to wear. Cultivation was continued for 5 days at a temperature of 20 ° C, a humidity of SO0% and an illuminance of 200 lx to form primistia of Fistulina hepatica. Parts of the walls of the bag were cut to form round perforations about 2 cm in diameter around the primordia by observing them from the outside through the bag. Then the cultivation was done for 19 days at a temperature of 13 to 23 * C, a humidity of 90? » or higher and an illuminance of 200 lx continued to grow the fruit bodies through the perforations from the bag, thereby harvesting large mature fruit bodies of Fistulina heoati. The total cultivation period was 49 days, the yield of fruit bodies per solid aedium was 65 g, and the average weight of the fruit body was 21 g.

The primordia of the Fistulina hepatica fruit body were formed in the same manner as in Example III, and cultivation was then carried out under the same conditions as in Example III, except that the bag was completely removed. The yield of fruit bodies per solid medium was 44 g and the average weight of the fruit body was 4 g.

Claims (7)

1. Method for inoculating Fistulina heoatica. characterized in that inocula of Fistulina heoatica is mixed with a sterilized solid culture medium for Fistulina hepatica to disperse the inocula in the whole medium. A method according to claim 1, characterized in that a solid culture medium is used which (a) comprises a wood material and (b) a foodstuff comprising a saccharide and an organic nitrogen compound and which has a pH in the range from 3.5 to 5.5 possession. A method according to claim 1 or 2, characterized in that the solid culture medium has a water content of 50 to 70% by weight. 4. Process according to claim 2, characterized in that at least 50 wt.%! of the wood material passes through a sieve with an aperture of 11.10 mm but does not pass through a sieve with an aperture of 850 µm. A method according to claim 2, characterized in that the ratio of the wood material to the foodstuff is in the range of 3: 1 to 9 1. Method according to any one of the preceding claims, characterized in. that at least 0.4 parts by weight of inocula per 100 parts by weight of solid culture medium is dispersed in the solid culture medium. A method according to claim 2, characterized in that the foodstuff is koji.