Title of Invention	"A PROCESS FOR PRODUCING A MEDIUM FOR MUSHROOM BED CULTIVATION"
Abstract	A mushroom bed cultivation method for cultivating a fruit body of Agaricus and a medium used for such a method are provided. The method includes the step of trig Agaricus in a medium including a grain. The medium further includes a nutrient, a medium matrix, and the like, as necessary.

Full Text

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION: The present invention relates to a mushroom bed cultivation method for cultivating a fruit body of Agari-cus and a medium used for such a method. 2. DESCRIPTION OF THE RELATED ART: Various types of mushrooms belonging to genus Agaricus of the family Agaricaceae (hereinafter, comprehensively referred to "Agaricus") are known, including Agaricus blazei Murrill, Agaricus bisporus, Agaricus sylvaticus, and Agaricus arvensis. For example, Agaricus blazei Murrill is also referred to as "hime-matsutake" or "kawari-haratake" (Takashi IMIZUNO and Masamitsu KAWAI, ed. , "Kinoko no Kagaku/Seikagaku, published by Gakkai Shuppan Center, pp. 223-228 (1992)). Agaricus blazei Murrill grows naturally in the mountains of Piedade in the suburbs of Sao Paulo, which is located in the southeast part of Brazil, and the inhabitants there have been eating its fruit bodies for ages. Recently, it was reported that an extract from fruit bodies of Agaricus is effective in curing adult diseases and cancer, and that Agaricus is a promising medicinal mushroom. Thus, a demand for fruit bodies of Agaricus is expected to increase. Fruit bodies of Agaricus have been in demand more in Europe and the United States than in Japan so far, but is considered to be more and more demanded in Japan as a medicinal mushroom. Today, fruit bodies of Agaricus are merely obtained by picking natural Agaricus or cultivating in greenhouses such as vinyl plastic hothouses or prefabricated greenhouses, in warm regions by a "ridge culture method" described below. Natural Agaricus can only be obtained in warm regions as can be appreciated from the fact that Agaricus naturally grows in the southeast part of Brazil. The "ridge culture method" is the only method conventionally available for cultivating Agaricus. This method is carried out in the following manner. Ridges are formed of compost which is prepared by fermenting straw of rice, sugarcane baggasse or the like, and then the ridges are covered with soil to form ridge beds. Parts of the ridges where the compost exists are inoculated with a spawn of Agaricus, and then fruit bodies of Agaricus are grown.. The ridge culture method is carried out in the greenhouses, but the yield of fruit bodies of Agaricus is very low due to its roughness and is susceptible to influences of the weather since the greenhouses are built outdoors. Accordingly, stable production of fruit bodies of Agaricus throughout the year cannot be realized. SUMMARY OF THE INVENTION A mushroom bed cultivation method for cultivating a fruit body of Agaricus according to the present invention includes the step of culturing Agaricus in a medium including grain. In this specification, the term "mushroom bed" refers to both a medium used for mushroom cultivation and a medium inoculated with a spawn. According to the present invention, a medium used for such a method is also provided. In one embodiment of the invention, the grain includes a seed of at least one of foxtail millet, Deccan grass, Chinese millet, rice plant and wheat. In one embodiment of the invention, the medium includes a seed of each of foxtail millet, Deccan grass and Chinese millet. In one embodiment of the invention, the medium includes 35 to 45% of foxtail millet, 35 to 45% of Deccan grass, and 10 to 30% of Chinese millet in a dry weight ratio. In one embodiment of the invention, the medium further includes at least one of a nutrient and a medium matrix. In one embodiment of the invention, the medium includes the nutrient at a ratio of 0 to 300 weight parts and the medium matrix at a ratio of 0 to 300 weight parts with respect to 100 weight parts of the grain. Thus, the invention described herein makes possible the advantages of providing a method for cultivating a fruit body of Agaricus in a mushroom bed stably, intensively, and in a large quantity without any influence of the weather, and a medium used for such a method. These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the photograph of fruit bodies of agaricus obtained by a method in Example 1 on day 7; and Figure 2 is a photograph of fruit bodies of agaricus obtained by a method in Example 2 on day 7. In accordance with the present invention there is disclosed a process for producing a medium for mushroom bed cultivation used for cultivating a fruit body of agaricus, comprising the following steps: (a) Providing 100 weight parts of a grain mixture comprising at least one of the following i) foxtail millet ii) Deccan grass iii) Chinese millet iv) Rice plant v) Wheat; (b) adding a nutrient at a ratio of 0-300 weight parts with respect to 100 weight parts of the grain; (c) adding a medium matrix at a ratio of 0 to 300 weight parts with respect to 100 weight parts of the grain; (d) combining said ingredients with twice their weight of water; (e) boiling until most of the water is evaporated, and pressure sterilizing the resulting product DESCRIPTION OF THE PREFERRED EMBODIMENT Hereinafter the mushroom bed cultivation method and a medium used for such a method according to the present invention will be described in detail. 1. Medium for mushroom bed cultivation for cultivating fruit bodies of agaricus: A medium used for cultivating fruit bodies of agaricus in a mushroom bed cultivation method includes a grain. In this specification, the grain includes all the cultivated species of Poaceae such as, for example, foxtail millet, Deccan grass, Chinese millet, rice, sugarcane, wheat, barley and rye. Foxtail millet, Deccan grass and Chinese millet are particularly preferable and the medium preferably includes at least one of these three types of grain and more preferably includes these three types of grain. According to the present invention, a seed or a plant body of grain other than seed such as, for example, straw, wheat chaff, rice chaff, or sugarcane baggasse can be used. The grain can be included in the medium in any form without any limitation. For example, the grain can be in granules such as, for example, crushed corncob, or seeds of foxtail millet and Deccan grass deprived of the seed coat; small pieces such as, for example, cut straw; or powders such as, for example, rice bran, corn bran, wheat bran, or crushed barley. In the case where a plant body other than the seed such as straw is used, the plant body is preferably used in the form of small pieces having a diameter of 15 mm or less and a length of 15 mm or less. One type of grain or a combination of arbitrary types of grain can be used in the present invention. The medium can include components other than grain such as, for example, a nutrient, a medium matrix, and an additive as necessary. Usable nutrients include any material generally used as the nutrient in the art. For example, cow manure, poultry manure, bark, bean-curd refuse, bean pod, coffee grounds, sake lees, or byproduct of food production is usable. Usable medium matrices include sawdust, wood chips, cotton seed coat bran, coconut peat, moss, and soil for horticulture. Usable additives include lime oyster shell, molasses, and liquid fertilizer for plants. The medium includes the nutrients in an amount of 0 to 300 weight parts, preferably of 10 to 100 weight; parts, and more preferably of 20 to 50 weight parts with respect to 100 weight parts of the grain. The medium matrix is included in the medium in an amount of 0 to 300 weight parts, preferably of 50 to 200 weight parts, and more preferably of 100 to 150 weight parts with respect to 100 weight parts of the grain. The additive is included in the medium in an amount of 10 weight parts or less with respect to 100 weight parts of the grain. The grain is included in the medium at a ratio of 30 weight % or more, preferably of 35 weight % or more, and more preferably of 40 weight % or more based on the total medium weight. The above-mentioned "weight" is "dry weight", which refers to a weight measured after drying each component for 5 hours at a temperature of 90 °C. In this specification, the term "weight" used regarding the medium composition refers to the dry weight. As described above, the medium can include only grain. A preferable medium including only grain includes, for example, 35 to 45 weight % of foxtail millet, 35 to 45 weight % of Deccan grass, and 10 to 30 weight % of Chinese millet. A preferable medium also including components other than grain includes, for example, 30 to 40 weight % of grain, 10 to 20 weight % of nutrient, and 40 to 50 weight % of medium matrix. The above-described components are mixed and kneaded with water in a method well known to those skilled in the art. Preferably, the mixture of the components is kneaded with water in about twice the weight amount of the mixture and then boiled until most of the water is evaporated. Then, the mixture is cooled. Thus, the medium used for mushroom bed cultivation method is prepared. The medium is accommodated in an appropriate type of culturing container as necessary. Any container which can withstand the sterilization process is usable regardless of the shape, size or material. For example, a bottle having a capacity of 850 ml and a diameter of 58 mm which is most commonly used in mushroom cultivation can be used, or other types of containers for mushroom bed cultivation, e.g., a bag for bag cultivation can be used. 2. Sterilization: The medium for mushroom bed cultivation obtained above is sterilized as it is or after being put into a culturing container. The sterilization can be performed within general sterilization standards with no specific limitation. Exemplary sterilization methods include a high pressure vapor sterilization method and a normal pressure vapor sterilization method. For example, high pressure vapor sterilization performed at 121°C for 60 minutes is preferable. For performing vapor sterilization of a large quantity of medium, the sterilization is preferably performed for a longer time period. 3. Inoculation of Agaricus: A spawn of Agaricus is prepared by picking a hypha or a spore from natural or cultivated fruit bodies of Agaricus and proliferating the hypha or germinating the spore in the medium, or alternatively by proliferating a hypha of the strains which are stored in an institution for microorganism deposit or a research institute. formation of the fruit bodies. The maturing is completed in about 5 to about 20 days, preferably in about 10 tc about 15 days, although the maturing is not necessary. 4.2 Induction of formation of the fruit bodies: After the spawn is cultured, the induction of formation of the bruit bodies is performed; i.e., the culture obtained in section 4,1, supra, is cultured under induction conditions. Before the induction, the surface of the culture can be optioncilly covered with soil, for example, peat moss which is generally used in horticulture. The thickness of the soil is preferably between about 1.0 cm to about 3.5 cm although not specifically limited. Induction can be performed under any conditions which allow Agaricus to grow with no specific limitation. Preferably, the temperature is between about 20 °C and about 30°C, the humidity is between about 80% and about 98%, and the CO2 concentration is about 2500 ppm or less. More preferably, thei temperature is between about 22°C and about 28°C, the humidity is between about 85% and about 95%, and the C02 concentration is between about 300 ppm and about 2200 ppm. Still more preferably, the temperature is about 25°C, the humidity is about 95%RH, and the CO2 concentration is between about 600 ppm and cibout 2000 ppm. The culture can be illuminated with J.ight, but is preferably kept in the darkness. The humidity and the C02 concentration for induction are preferably higher than those for culturing. The number of days required for the induction is not specifically limited, but the induction is completed when formation of young fruit bodies is recognizable with human eye. 4.3 Growth of the fruit bodies: The young fruit bodies obtained in section 4.2, supra, can be grown under any conditions which allow Agaricus to grow with no specific limitation. Preferably, the temperature is between about 20 °C and about 30°C, the humidity is between about 75% and about 95%, and the CO2 concentration is about 2500 ppm or less. More preferably, the temperature is between about 22 °C and about 28°C, the humidity is between about 80% and about; 90%, and the C02 concentration is between about 300 pprn and about 2000 ppm. Still more preferably, the temperature is about 25°C, the humidity is about 90%RH, and the CO2 concentration is between about 600 ppm and about 1500 ppm. The fruit bodies need to be illuminated with light. The fruit bodies are preferably illuminated at; about 50 Lux to about 500 Lux for about 1 to about 8 hours per day, more preferably at about 100 Lux to about 500 Lux for about 2 to about 6 hours per day, and still more preferably at about 200 Lux to about 500 Lux for about 3 to about 5 hours per day. The illuminance and the CO2 concentration are appropriately controlled in order to obtain fruit bodies of a desirable form. The number of days required for growth can vary in accordance with the growth conditions of Agaricus. The fruit bodies of Agaricus are harvested when grown to a desirable size. Examples Hereinafter, illustrative and non-limiting examples of the present invention will be described with reference to figures. Example 1: Cultivation of Agaricus blazei Murrill in a bottle 1. Preparation of a medium:: Seeds of Deccan grass, foxtail millet and Chinese millet were mixed at a dry weight ratio of 2:2:1, and then water of twice the weight amount of the resultant mixture was added cind boiled. When most of the water was evaporated, boiling was stopped and the resultant mixture was cooled. Thus, a medium was prepared. When the medium was cooled to a temperature of 20°C or lower, 600 g of the medium was put into each of a plurality of polypropylene bottles having an opening of a diameter of 58 mm and a capacity of 850 ml, and the bottles were capped. Next, high pressure vapor sterilization was performed at a pressure of 1 atm (gauge pressure) at a temperature of 121°C for 60 minutes. Then, the bottles were cooled in a clean room until the temperature of the medium decreased to about 18°C. 2. Inoculation of Agaricus blazei Murrill: Spores were harvested from fruit bodies of commercially available Agaricus blazei Murrill, and germination and propagation of hypha were performed on a potato dextrose agar medium (PDA medium) under the conditions of a temperature of 25°C, a humidity of 65%RH and in the darkness for 25 days. A PDA medium was newly inoculated with hypha pieces having a size of 5 mm x 5 mm obtained by culturing and proliferating the hypha, and the hypha pieces were cultured under the same conditions. This process was repeated three times to purify the hypha of Agaricus blazei Murrill, A mixture of the hypha pieces obtained in this manner and the medium having the same composition as that of the medium described in section 1, supra, was used cis a spawn. Inoculation was performed by mixing 15 g of the spawn and the sterilized medium contained in each of 16 bottles obtained in section 1, supra. (The volumetric ratio of the spawn and the medium was 1:40.) 3. Cultivation of the fruit: bodies: 3.1 Culturing: The spawn in the medium contained in the bottles was cultured under the conditions of a temperature of 25°C, a humidity of 60 to 70%RH, a CO2 concentration of 1500 ppm or less and in the darkness. Hypha spreading was completed in 20 days, but the hypha was still cultured for another 20 days to be matured. The total culturing period was 40 days. In this way, a matured mushroom bed was obtained. 3.2 Induction of formation of the fruit bodies: After the culturing was over, induction was performed in two methods of (1) removing the caps of the bottles without scraping off the surface of the mushroom bed, and (2) covering a surface of the mushroom bed with peat moss to a thickness of 1 cm without scraping off the surface of the mushroom bed. In either case, induction was performed under the conditions of a temperature of 25°C, a humidity of 95%RH a C02 concentration of 2000 ppm or less and in the darkness. The number of days required to form the fruit bodies was not substantially influenced by whether the surface of the mushroom bed was covered with peat moss or not. In either case, formation of young fruit bodies was observed in 14 to 17 days. From these results, it was found that fruit bodies of Agaricus blazei Murrill are formed whether or not the surface of the mushroom bed is covered with peat moss. 3.3 Growth of the fruit bodies: The young fruit bodies obtained in section 3.2, supra, were grown under the conditions of a temperature of 25°C, a humidity of 90%RH, and a CO2 concentration of 1500 ppm or less. During this stage, the fruit bodies were illuminated at 200 to 500 Lux for 3 to 5 hours per day. The fruit bodies grew to have a stipe length of 5 cm on day 7 as shown in Figure 1. The yield of the fruit bodies per bottle was about 50 g on average. The composition of the. medium used in this example is particulate, and thus it was difficult to uniformly distribute the moisture in the medium. However, it was found that inoculation performed by mixing the medium and the spawn has an advantage of progressing the culturing more smoothly and thus forming the fruit bodies of Agaricus more stably. Example 2: Cultivation of Agaricus blazei Murrill in a bag 1. Preparation of a medium: Rice bran and wheat chaff as grain, cow manure as a nutrient, and sawdust as a medium matrix were mixed at a weight ratio shown in Table 1 below, and then kneaded with water. Different types of sawdust have different water contents. In this example,, sawdust having a water contents of 63% was used. Heat-resistive polypropylene bags were each filled with 1500 g of the resultant mixture. Next, high pressure vapor sterilization was performed at a pressure of 1 atm (gauge pressure) at a temperature of 121°C for 60 minutes. Then, the bags were cooled in a clean room until the temperature of the medium decreased to about 18°C. Table 1 Composition of the medium (per bag) (Table Removed) 2. Inoculation of Agaricus blazei Murrill: A spawn was obtained in the same manner as described in section 2 of Example 1. Inoculation was performed by mixing 80 g of the spawn and the sterilized medium contained in each of 20 bags obtained in section 1, Example 2, supra. (The volumetric ratio of the spawn and the medium was 1:25.) After the inoculation, the bags were closed. 3. Cultivation of the fruit bodies: 3.1 Culturing: The spawn in the medium in the bags was cultured under the conditions of a temperature of 23 to 25°C, a humidity of 60 to 70%RH, a CO2 concentration of 1500 ppm or less and in the darkness. Hypha spreading was completed in about 40 days, but the hypha was still cultured for another 10 days to be matured. The total culturing period was 50 days.. In this way, a matured mushroom bed was obtained. 3.2 Induction of formation of the fruit bodies: After the culturing was over, induction was performed as follows. On about day 50, top parts of the bags were cut off at about 5 cm from the surface of the mushroom bed, and the surface was covered with peat moss or mountain soil for horticulture. The surface of the mushroom bed was not scraped off. Induction was performed under the conditions of a temperature of 18 to 22°C (3 to 5°C lower than the temperature in section 3.1 in Example 2, supra), a humidity of 90 to 95%RH, a C02 concentration of 800 ppm or less and in the darkness. During the induction, water was sprayed to prevent the peat moss or the mountain soil covering the surface of the mushroom bed from becoming dried. From around day 20 after the start of induction of young fruit bodies was observed on the surface of the mushroom bed. This point was set as the start of "growth". 3.3 Growth of the fruit bodies: The young fruit bodies obtained in section 3.2, supra, were grown under the conditions of a temperature of 18 to 22°C, a humidity of 90 to 95%RH, and a CO2 concentration of 800 ppm or less. During this stage, the fruit bodies were illuminated at 100 Lux for 2 to 3 hours per day. The fruit bodies grew to have a stipe length of 8 to 12 cm on day 6 or 7 as shown in Figure 2. The bodies were harvested before the pilei opened. The yield of the fruit bodies per bag was about 200 g on average. It was found that the method according to the present invention realizes more stable cultivation of the fruit bodies of Agaricus. According to the present invention, cultivation of Agaricus in a mushroom bed which has been considered to be impossible is realized. The method according to the present invention, which realizes cultivation of fruit bodies of Ageiricus in facilities regardless of the location, season or weather, opens the way for mass production. Cultivation within about 60 to 65 days is possible, which is not very different from the conven - tional mushroom cultivation.. According to the conventional "ridge culture method", cultivation is performed outdoors. Therefore, the danger of the ridge beds being contaminated by saprophyte and the danger of hypha of Agaricus being attacked by flies and pest insects in the soil are high and thus stable production of fruit bodies of Agaricus is difficult. In the method according to the present invention, the medium for mushroom bed cultivation is inoculated with a spawn after the medium is sterilized and thus the saprophyte in the medium are killed, and the inoculation is performed in a substantially sterile state. Therefore, the problems of the conventional method are overcome. Moreover, according to the present invention, the components included in the medium and the mixture ratios thereof can be relatively freely selected. Consequently, the amount of medicinal component can be increased by adding to the medium a substance which is a starting material from which the medical component is formed through the biosynthetic route of a medicinal component of Agaricus. The most promising antitumor active materials contained in Agaricus and known to those skilled in the art so far are polysaccharides including p-D-glucan. Nucleic acid, lectin, steroid, lipid and the like are also known to those skilled in the art to have an antitumor activity. Therefore, addition of glucose, sucrose, or starch to the medium is considered to be effective in increasing the antitumor active material (i.e., medicinal component contained in Agaricus). The conventional method would not work well even if the starting material of the biosynthetic route of a medicinal component of Agaricus is added when the biosynthetic route is clearly found in the future. The reason is that the starting material is degraded by other microorganisms existing in the medium, resulting in reduction in the effect of the starting material or in promotion of proliferation of the other microorganisms to inhibit proliferation of the hypha of Agaricus. Furthermore, according to the present invention, the culturing container is not limited in the size or shape and a plurality of small culturing containers can be used. Thus, the three-dimensional space in the cultivating facilities can be effectively used to improve cost effectiveness. Additionally, the fruit bodies obtained by the method according to the present invention does not have a smell of mud and thus is more acceptable as a medicine taken after decocted. Various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the scope and spirit of this invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the description as set forth herein, but rather that the claims be broadly construed. We Claim : 1. A process for producing a medium for mushroom bed cultivation used for cultivating a fruit body of agaricus, comprising the following steps: (a) Providing 100 weight parts of a grain mixture comprising at least one of the following i) foxtail millet ii) Deccan grass iii) Chinese millet iv) Rice plant v) Wheat; (b) adding a nutrient at a ratio of 0-300 weight parts with respect to 100 weight parts of the grain; (c) adding a medium matrix at a ratio of 0 to 300 weight parts with respect to 100 weight parts of the grain; (d) combining said ingredients with twice their weight of water; (e) boiling until most of the water is evaporated, and (f) pressure sterilizing the resulting product. 2. The process A$ claimed in claim 1, wherein the grain mixture of step (a) includes i) 35% to 45% of foxtail mille ii) 35% to 45% of Deccan grass iii) 10% to 30% of Chinese millet in a dry weight ratio the nutrient of step (b) is at a ratio of 0 to 300 weight parts with respect to 100 weight parts of the grain and the medium matrix of step (c) is at a ratio of 0 to 300 weight parts with respect to 100 weight parts of the grain. 3. The process as claimed in claim 2, wherein The nutrient of step (b) is at ratio 0 weight parts with respect of 100 weight parts of the grain and the medium matrix of step (c) is at ratio of 0 weight parts with respect to 100 weight parts of the grain. 4. The process as claimed in claim 1, wherein the nutrient is a manure. 5. A process for proncing a medium for mushroom bed cultivation used for cultivating a fruit body of agaricus substantially as herein described with reference to the foregoing description, examples and the accompanying drawings.

Documents:

596-del-1998-abstract.pdf

596-del-1998-claims.pdf

596-del-1998-complete specification (granted).pdf

596-DEL-1998-Correspondence-Others.pdf

596-del-1998-correspondence-po.pdf

596-del-1998-description (complete).pdf

596-del-1998-drawings.pdf

596-del-1998-form-1.pdf

596-del-1998-form-2.pdf

596-del-1998-form-3.pdf

596-del-1998-form-4.pdf

596-del-1998-pa.pdf

596-del-1998-petition-others.pdf