NZ234286A - Killing bacillus spores by heating, cooling and subsequent sterilisation; culture media and culturing of fungi (especially tree mushrooms) - Google Patents

Description

E,-,.-". r, 33 4 2 8 6 ... I 1 I I --^C C\C- \ CA\ OL,-r .^-U .lO^r ' j ! tor^ ; •' , . ^s- 1 ir« *\ *.... . ti-i*1 K'• *. r.O. •« ■ • * 28 OCT 1992. v3£'. > ,•••- 0 No.: Date: NEW ZEALAND PATENTS ACT, 1953 - Z;:,;,LAND CFRCE j 28 JUN1990 RECEIVED ■W COMPLETE SPECIFICATION SUBSTRATE AMD METHOD FOR CULTURE OF FUNGI, INCLUDING SHIITAKE (LENTINUS EDODES) AV.i //We, MAUI SHIITAKE TRADING COMPANY, INC., a Hawaii corporation, USA, having a principal place of business at 295 Hiwalani Loop, Pukalani, Maui, 96768, United States of America hereby declare the invention for which t / we pray that a patent may be granted to-me-/us, and the method by which it is to be performed, to be particularly described in and by the following statement:- (followed by page la) 2 3 4 2 SUBSTRATE AND METHOD FOR CULTURE OF FUNGI, INCLUDING SHIITAKE (LENTINUS EDODES) BACKGROUND OF THE INVENTION This invention relates to the cultivation of mushrooms and other fungi, especially shiitake (Lentinus edodes).

Inventors have long sought a method for efficiently and quickly cultivating fungi, especially Shiitake, because of its great demand and relatively limited supply.

Shiitake and other mushrooms are usually cultivated on logs or in cellulose based substrates. Among the methods using a cellulose based substrate are those described in Patent No. 4,127,965 to Mee and Patent No. 4,637, 163 to Pellinen. Mee also teaches the use of a cellulose based substrate in a microorganism impermeable flexible container which is then sealed and sterilized. However, as taught by Patent No. 4,674,228 issued to Murata, removal of the mycelium from such containers often causes damage that reduces productivity. Other methods also have been tried. For example, Patent No. 4,735,014 to Weber teaches the use of hemp stalks and Patent No. 4,741,122 to Becsy teaches the use of agricultural wastes.

There are many drawbacks to the various methods for 23 4 2 I ■ ? < growing shiitake currently in use. Growing shiitake on logs in the traditional manner is slow and inefficient. Cultivation of shiitake in microorganism impermeable •flexible containers (commonly known as "space bags") offers advantages over traditional methods, but still does not provide a satisfactory production rate.

Thus, it is an object of this invention to provide an improved method of cultivating fungi, especially shiitake.

It is a further object of this invention to provide an improved culture medium for the culture of fungi, including shiitake.

It is a further object of this invention to provide a more efficient and faster method of raising fungi, including shiitake.

SUMMARY OF THE INVENTION The invention is a new substrate for the growth of fungi, especially shiitake, created using a new method of sterilizing the substrate to allow cultivation of the desired fungi without contamination by competing organisms.

The new substrate is grain that is essentially cellulose free and that has been sterilized in accordance with the process described herein. As indicated above, the prior art in the growth of mushrooms and other fungi 23 k requires growth on logs, sawdust or other substrates containing a major portion of cellulose. However, cellulose is not necessary for the cultivation of shiitake. Shiitake mushrooms have the ability to break down cellulose for essential nutrients, but can be more efficiently grown in a substrate containing these materials in an already usable form. Similarly, shiitake can break down lignin, which is a constituent of wood, but again shiitake can be cultivated more efficiently by providing the breakdown products instead of the lignin.

Prior art references have taught the use of grain as a nutritional supplement in a cellulose based substrate. See for example, Han, et. al, Physiology and Ecology of Lentinus Edodes (Berk) sing., Mushroom Science XI, Proceedings of the Eleventh International Scientific Congress on the Cultivation of Edible Fungi (1981). However, the substrate of this invention is essentially free of cellulose and the grain itself is the substrate.

The grain substrate must be sterilized for the cultivation of fungi, including shiitake. Unsterilized grain contains various bacteria and microorganisms that compete with mushrooms and other fungi and therefore reduce production efficiency. Further, conventional heat sterilization techniques, such as steam sterilization, are insufficient to sterilize the grain against all competing microorganisms. Accordingly, conventionally sterilized grain is unsuitable as a substrate. In fact, one prior art reference states that, in view of the well-established use of tree logs and the amount of energy necessary to sterilize a substrate, "widespread large scale use of any sterilized substrate to produce shiitake mushroom appears unlikely." San Antonio, "Cultivation of the Shiitake Mushroom", Hortscience, Vol. 16(2), April 1981.

The main problem with conventional heat sterilization of grain substrates is that certain bacteria, primarily of the genus Bacillus, form heat resistant spores that will survive such sterilization even though the bacteria themselves are killed. Accordingly, even though a grain substrate may be conventionally heat sterilized, it will still contain spores of Baci1lus bacteria which will contaminate the substrate and render it unsuitable for production of fungi, including shiitake. This invention solves the problem of bacterial contamination in the grain so that an appropriately sterile substrate is provided.

In the invention, the substrate is boiled to kill the bacteria that are present. The substrate is then cooled to induce any heat resistant spores to germinate. The 4 2 3 4 2 substrate then is steam sterilized after such germination, but before the bacteria have matured sufficiently to form heat-resistant spores.

Of course, non-heating methods of sterilizing the grain substrate also can be used, such as irradiation. However, irradiation of the substrate would require greater governmental regulation and may affect marketability of the resulting mushrooms.

The substrate of the invention thus provides a more efficient medium for cultivation of mushrooms, including shiitake, because the nutrients required by the mushrooms are furnished directly, rather than being furnished in the form of cellulose and lignin that must be enzymatically broken down by the mushrooms. The invention also provides a more efficient method of cultivating mushrooms because competing microorganisms, including bacteria, are eliminated from the substrate.

An advantage of the invention is the shortening of incubation times for the shiitake. The invention shortens the incubation time for forming mycelium to 21 days, as opposed to log cultivation, which requires 8 months to 1 year for incubation, and sawdust based substrates, which require approximately 80 days for incubation.

A further advantage of the invention is the 23 l\ s fi yt> i.W V increase in yield per pound of substrate. One hundred pounds of the substrate of the invention yields approximately 300 pounds of shiitake within 5 months. By comparison, 100 pounds of logs yields approximately 10 to 15 pounds of shiitake over more than 3 years, and 100 pounds of sawdust based substrate yields approximately 80 pounds of shiitake over 8 months.

A further advantage of the invention is that no special spawn material is necessary. The same material used for fruiting can be used as a spawn material to start new production units, so that production can be increased immediately instead of waiting for new spawn to be grown. Similarly, no spawn is wasted if production is decreased.

A still further advantage of the invention is that production units may be kept in incubation beyond the 21 day period for up to 6 months if, for example, market conditions ; are unfavorable. This also allows stockpiling of colonized units for large seasonal production outputs.

In the practice of the invention, various nutritional supplements (including proteins, sugars, ■■■•' starches and vitamins) are boiled in water until they are dispersed throughout the mixture. The grain for the substrate is then added and boiled for approximately one hour in order to kill the bacteria present and cause the 6 2 3 4 2 8 6 i absorption of the dispersed nutritional supplements into the grain. The grain is then allowed to cool to induce germination of any heat-resistant spores. While the grain is cooling, it is mixed with permeability enhancing powders to prevent caking and packed into microorganism impermeable sterilizable containers, such as polypropylene bags. The bags are then steam sterilized in accordance with conventional practice before the germinated bacteria have matured sufficiently to form spores.

After sterilization of the bags, colonization of the bags is accomplished by introducing either pure spawn of the desired fungi or by introducing previously colonized grain. The bags are then shaken to mix the spawn or previously colonized grain with the grain in order to decrease the incubation time. The bags are then incubated for approximately three weeks at approximately 80 degrees Fahrenheit. During this time, the spawn will digest most, if not all, of the substrate to form a mycelium.

The mycelium can then be induced to fruit by subjecting the bags to a cold shock of 40 to 65 degrees Fahrenheit for 5 to 15 days under cool white fluorescent lighting. After the cold shock, fruiting to maturation is accomplished by removing the mycelium from the containers and exposing them to an intermittent chilled water mist, or 7 234286 t ' l otherwise placing the mycelium in a high humidity environment.

Alternatively, fruiting can be induced using only a cold water spray under lighted conditions BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart of a preferred method of preparing the substrate of the invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT Figure 1 of the drawings sets forth generally a preferred method of preparing the substrate of the invention.

The ingredients in the substrate are preferably chosen to provide optimum nutrition for the fungi to be grown without requiring additional artificial supplements. This use of all-natural materials therefore makes sale and marketing of the cultivated fungi easier because fewer regulatory requirements are imposed. The preferred ingredients, their ranges and the optimum amounts are set forth below for preparing batches of the substrate.

Ingredient Range Optimum Amount Whole Sorghum grain 150-300 lbs 200 Whole Oat grain 0-50 lbs 35 Russet Potatoes 5-20 lbs 10 Rolled Barley grain 0.5-15 lbs 5 Maple pea sprouts 0-15 lbs 5 8 \ • V / # 2 3 4 9 fl 11 \j Brewer's yeast powder 2-35 lbs 6 Hulled sunflower seed 0-10 lbs 2 Soybean meal 0-2.5 lbs 1.5 Corn gluten meal 0-2.5 lbs 1.5 Whole Garlic 0.5-4 lbs 1.5 Sunflower oil 0-20 tablespoons 10 Wheat germ oil 0-20 tablespoons 10 Molasses 0-20 tablespoons 6 Water 20-35 gallons 25 Milk 0-1 gallon .25 The preferred coating ingredients, the ranges and the optimum for every two batches of the above substrate are set forth below: Limestone powder 25-75 lbs 50 Gypsum powder 100-200 lbs 160 Cottonseed meal 0-60 lbs 40 The maple pea sprouts are preferably grown for 6 to 12 days under a mist system. Commercial bean sprouts may also be used, but more roots and larger cotyledons are available with maple pea sprouts.

Sorghum provides vitamins, carbohydrates, starches, protein and minerals such as Copper, Iron, Manganese, Zinc and Selenium. Oats provide vitamins, minerals, carbohydrates, starches, proteins and salicylic acid. 9 I 2 3 4 2 8 6 ( ' Salicylic acid promotes shiitake fruiting. Rolled barley grain provides vitamins and carbohydrates and absorbs excess water. Soybean meal provides a source of minerals, proteins and vitamins. Brewer's yeast powder provides high amounts of vitamins, especially B vitamins that promote mycelial growth. Sunflower seed and sunflower oil provide vitamins, minerals, proteins and saturated and unsaturated oils. The sunflower seed and oil also promote heavier secondary mycelial growth.

The pea sprouts promote a heavier amount of fruitings to occur. This allows some control over the size of the mushrooms. More sprouts allow for more mushrooms to form but the mushrooms are smaller in size. Fewer sprouts allow for fewer mushrooms to form but the mushrooms are larger in size. With no sprouts added, mushrooms with individual weights of from 3/4 lb to 1-1/2 lbs may form on the substrate.

Garlic provides natural antibacterial action in order to resist bacterial growth after boiling and sterilization of the substrate. Molasses provides sugars and wheat germ oil provides saturated and unsaturated oils as well as vitamin D. Corn gluten meal provides vitamins, minerals, protein and selenium. Potatoes provide starch. Milk provides cassein and cheese can be substituted instead of milk. 23 4 2 The coating ingredients serve additional functions besides increasing permeability of the substrate. Limestone powder adjusts the pH of the substrate to neutral (approximately 7 to 8). The gypsum powder also provides long term pH maintenance and makes the grain substrate loose and powdery. The cottonseed meal provides protein and oil.

It should be noted that the prior art teaches that, under certain conditions, calcium inhibits fruiting of mycelium. However, the substrate of this invention contains substantial amounts of calcium from the limestone and gypsum powder.

The size and number of mushrooms can be controlled prior to colonization by the amount of substrate that is packed in the bags, with larger bags that contain more substrate producing larger and more mushrooms. For example, eight pound bags will produce 3/4 pound mushrooms for approximately 6 months.

Mushroom size and number also can be controlled after colonization by allowing individual colonized units to come into contact with each other. The individual units will form one large continuous unit forming larger and more numerous mushrooms than an individual unit.

Fully colonized units can be placed on shelving or strung on rods to maximize production per unit area. 11 23 4 2 The following example illustrates the use of this invention using the optimum amounts set forth above.

EXAMPLE The water is boiled in a 60 gallon capacity steam kettle with a bottom spigot. The potatoes are sliced and then added to the boiling water together with the milk, garlic, corn gluten meal, wheat germ oil, sunflower oil, molasses, hulled sunflower seed, brewer's yeast powder and soybean meal. The mixture is then boiled until all components break into small pieces. The mixture is preferably mixed with a portable paint mixer to help break clumps into small pieces. Maple pea sprouts are then added to the boiling mixture, which is stirred with a large paddle until the sprouts are soft. The oat grain, barley grain and sorghum grain are then added, together with sufficient water only to cover the grain. The mixture is then boiled and stirred until the water level falls below the grain level by 3 to 4 inches and the heat source is then turned off. . After approximately one hour, any remaining liquid is drawn off from the bottom of the pot. At this point, the grain should be half-cooked and semi-hard. The grain is then allowed to cool for approximately 24 hours, at which time it is removed from the pot.

Two batches of grain are then placed in a large 2 C ( flat bin and the limestone powder, gypsum powder and cottonseed meal are mixed with the grain until all the grain is coated with powder. The grain should appear coated and should not stick in clumps. Two batches will yield approximately 1,200 pounds of prepared substrate.

The prepared substrate is then packed into double polypropylene plastic bags (1.5 mil.). Each of these double bag units has a polypropylene collar, a cotton plug and an aluminium foil cover over the plug. The bags from 4 batches of the grain (approximately 2,400 pounds) are then loaded in a steam retort (5 foot diameter, 13 feet long) and steam-sterilized at 250° F, 15 pounds per square inches steam pressure for 7 hours. Each load is then cooled for 24 hours before seeding.

After the bags of substrate have been sterilized, they are preferably seeded under sterile conditions in laminar airflow hoods. Seeding is accomplished by introducing pure spawn or, preferably, colonized grain from previous production runs. Approximately 5 to 10 tablespoons of colonized grain is added into each 2-pound bag. Each of the bags is then shaken to mix the colonized grain throughout the new unit. This thorough mixing of the previously colonized grain with the substrate reduces the normal incubation time considerably. Thus, a 2-pound bag 13 23 4 2 will usually be fully colonized after approximately 3 weeks of incubation at 80° F. Usually 15 new 2-pound units may be started from each colonized 2-pound unit. The preferred size of bag is 8 pounds because of the disproportionately greater number of buds per 8 pound bag when compared with 2 pound and 4 pound bags.

After approximately 3 weeks, the grain substrate will be mostly or completely digested, leaving only the mycelium in the bag. The bag can be retained in the mycelial stage for approximately 3 to 4 months for shipment or storage. When mushroom production is desired, the bags containing the mycelium are subjected to a cold shock by chilling them at 40 to 65° F for 5 to 15 days under cool white fluorescent lighting of 25 to 100 lux. The preferred cold shock is at a temperature of 45° F for 7 to 9 days, although a cold water bath for 24 to 48 hours also may be used.

The bags can be shipped in a refrigerated container during this cold shock stage.

As an alternative to the cold shock method of inducing fruiting, the mycelium may be removed from the bags and exposed to an intermittent cold water mist. It is preferred that the misting take place during daylight hours and also during a 2 hour period during the night. The water 14 2^4? 4 Skw C ? ' used for misting is chilled to 50 to 75° F and misting occurs for 2 to 120 seconds at 2 to 10 minute intervals for 6 to 15 hours during the daylight period. Approximately 10 to 20 days after the mycelium is exposed to mist, shiitake mushrooms may be harvested. Subsequent crops from the bags may occur 20 to 30 days apart. The relative humidity in the misting environment must be at least 80%.

As an alternative to the intermittent chilled water mist, the mycelium may be removed from the bags and allowed to fruit using previously known methods.

After the substrate has been spent, it may be used for other purposes, such as compost, animal feed, mushroom compost for other mushrooms or insect feed.

After formation of the mycelium, but before fruiting, the mycelium also may be used as animal feed or for human food. Useful biochemicals also may be extracted from the mycelium.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as described in the claims. For example, and not by way of limitation, the substrate described herein is suitable for growing many ( 23 4 2 species of mushrooms, including those listed in Mushroom List 1, which is attached hereto and incorporated herein by reference, and many genera of fungi, including those listed in Fungal List 2, which is attached hereto and incorporated herein by reference. Many of these fungi are useful for their biochemical or other properties. Thus, the substrate can be used for growing penicillin mold, weed molds, yeasts and medicinal mushrooms. Accordingly, no limitation is to be inferred except as set forth in the claims. 16 * ft > mushroom Scientific Name Agaricus arvensis Agaricus augustus Agaricus bernardii Agaricus bisporus Agaricus bitorquis Agaricus campestris Agaricus excellans Agaricus langei Agaricus macrosporus Agaricus silvaticus Agaricus silvicola Agaricus vaporarius Agrocybe aegerita Armillaria Caligata Armillaria ponderosa Armillariella mellea Armillariella tabescens Auricularia polytricha Auricularia auricula Calvatia craniiformis Calvatia gigantea Clitocybe geotrapa Coorinus comatus Dictyphora duplicata Flammulina velutipes Galerina mutabilis Ganoderma lucidum Grifola frondosa Grifola umbellata Hericium coralloides Hericium erinaceus Laetiporus sulphureus 23 4 2 8 6 list l Common Name Horse Mushroom The Prince Common Field Mushroom Wood Mushroom Brown Swordbelt Wood Ear Wood Ear Skull-shaped Puffball Giant Puffball Shaggy Inky Cap Netted Stinkhorn Enoki Reishi Hen of the Woods Zhu Ling Pom Pom Sulfur Polypore 17 Lentinus edodes Shi itake Lepiota naucina Lepiota procera Lepiota rachodes Leoista nuda Leucopaxillus giganteus Lycoperdon gemmatus Lycoperdon pyriforme Lyophyllum cecastes Lyophyllum ulmarium Macrolepiota procera Marasmius oreades Morchella angusticeos Morchella deliciosa Morchella esculenta Morchella conica Morchella crassipes Morchella elata Morchella semilibera Morchella vulgaris Panellus serotinus Panus sp.

Pholiota adiposa Pholiota nameko Pleurotus columbinus Pleurotus cornucopiae Pleurotus cystidiosus Pleurotus eryngii Pleurotus flabellatus Pleurotus florida Pleurotus ostreatus Pleurotus pulmonarius Pleurotus sajor-caju Pleurotus salmoned stramineus Sparassis crispa Stropharia rugosoannulata Smooth Lepiota Parasol Mushroom Scaly Lepiota Wood Blewit Gem-Studded Puffball Pear-Shaped Puffball Honshimeji Parasol Fairy Ring Black Morel White Morel Conical Morel Thick-Footed Morel Common Morel Fat Pholiota Nameko Blue Oyster Canary Abalone Pink Oyster Florida Oyster Oyster Phoenix Cauliflower Wine Red Stropharia 18 Tremella fusciformis Trichblomopsis rutilans Volvariella bakeril Volvariella bombycina Volvariella volvacea White Jelly Paddy Straw FUNGAL LIST 2 Lish of Funaal Genera That Mav be Grown on the Substrate Abortiporus Absidia Achlya Acremonium Acrophialophora Acrospeira Actinomucor Agaricus Agrocybe Aleurodiscus Allescheria Alternaria Alysidium Amanita Amauroascus Amylomyces Backusella Beauveria Bispora Bjerkandera Blakeslea Blastomyces Boletopsis Cadophora Calbovista Calcarisporium Caldariomyces Calocera Calocybe Calonectria Calvatia Camarops Candida Cantharellus Celphalosporium Cephaliophora Cephaloascus Ceratocystis Cercospora Cerinomyces Ceriosporopsis Cerrena Chaetomella Chaetomium Amylostereum Anomoporia Antrodia Apiotrichum Arachnomyces Armillariella Arthrinium Arthrobotrys Arthrographis Ascotricha Ashbya Aspergillus Athelia Aureobasidium Auricularia Boletus Bondarzewia Botryodiplodia Botryotrichum Botrytis Bovista Byssochlamys Coccospora Cochliobolus Colletotrichum Collybia Columnocystis Conidiobolus Coniella Coniophora Coniothyrium Conoplea Coprinus Cordyceps Coridus Coriolus Corticium Cortinarius Coryne Corynespora Coryneum Craterellus Craterellus 23 4 2 8 6 Chalara Chalaropsis Choariephora Chondrostereum Chroogomphus Chrysosporium Circinella Cladosportium Clavariadelphus Claviceps Clavicorona Clavispora Clavulina Clitocybe Clitopilus Dacrymyces Dacryopinax Dactylium Daedalea Debaryomyces Dekkera Dendryphion Dentinum Dermaloma Dichomitus Echinodontium Elsinoe Emericella Emericellopsis Entoloma Favolus Fernsj onia Filobasidium Fistulina Flammula Ganoderma Geotrichum Gerlachia Gibberella Gilmaniella Gliocladium Gliomastrix Gloeophyllum Crebrothecium Cryphonectria Cryptococcus Cryptoporus Cryptosporiopsis Cunninghamella Curvularia Custingophora Cyanthus Cylindrocarpon Cylindrocephalum Cylindrocladium Cystostereum Cytospora Cytospora Dictyostelium Diheterospora Diplocarpon Diplodia Discina Discula Ditiola Doratomyces Dothistroma Drechslera Epicoccum Eupenicillium Eutypa Exophiala Flammulina Fomes Fomitopsis Fusarium Fuscoboletinus Gnomonia Gomphidius Gomphus Grandinia Graphium Grifola Guepiniopsis Gymnopilus 21 © 23 4 2 8 6 Gloeoporus Gyrodon Gloeosporium Gyromitra Glomerrella Gyroporus Hanseniaspora Humicola Hansenula Humicolopsis Haploporous Hyalodendron Helicostylum Hydnum Helminthosporium Hygrophoropsis Helvella Hygrophorus Hendersonula Hymenochaete Hericium Hyphopichia Heterobasidion Hypornyces Hirschioporus Hypomyces Hormodendrum Hypoxylon Incrustoporia Irpex Inocybe Isaria Inonotus Ishnoderma Kloeckera Kluyveromyces Laccaria Lenzites Lactarius Leptosphaerulina Laetisaria Leucopaxillus Laurilia Libertella Leccinum Linderina Lentinellus Lipomyces Lentinula Lycoperdon Lentinus Lyophyllum Lentodium Macrophomina Monascus Mammaria Monilinia Marasmiellus Monochaetia Marasmius Monodictus Melanconium Monosporium Melanoleuca Mortierella Memnoniella Mucor Meruliopsis Myceliophythora Merulius Mycena Merulius Mycocentrospora Metarrhizium Mycosphaerella Metschnikowia Myriococcum Micronectrie11a Myrothecium Mollisia 22 r> 234 2 8 Naematoloma Nectri a Neocosmospora Odontia Oedocephalum Oidiodendron Omphalotus Onnia Pachybasium Pachysolen Paecilomyces Panellus Panus Papularia Papulaspora Pellicularia PeniciIlium Peniophora Perenniporia Periconia Pestalotia Pestalotiopsis Peziza Phaeocoriolellus Phaeolus Phanerochaete Phellinus Phialomyces Phialophora Phlebia Phlogiotis Pholiota Phoma Phoma Phomopsis Phycomyces Radulodon Ramaria Ramaricium Resinicium Retinocyclus Rhinocladiella Rhizoctonia Rhizomucor Neurospora Nodulisporium Nomuraea Oosporidium Ophiostoma Osmoporus Ostenia Oudemansiella Phylloporus Physarum Phytophthora Pichia Piptoporus Piricularia Pithomyces Pleurocybella Pleurotus Plicatura Pluteus Podospora Polyozellus Polyporus Poria Potebniamyces Preussia Psathyrella Pseudeurotium Pseudofusarium Pseudohydnum Pseudospiropes Ptychogaster Pulcherricium Pycnoporus Pyrenochaeta Pyrenophora Pythium Rhizopus Rhodosporidium Rhodotorula Rigdoporus Robi1larda Rosellinia Russula 23 C 0 £s. y 8 f) Saccharomyces Saccharomycopsis Sacodon Saprolengnia Sarcosphaera Schizophyllum Schizosaccharomyces Schwanniomyces Sclerotinia Sclerotium Scolecobasidium Scopulariopsis Scytalidium Scytinostroma Sebacina Sepedonium Septomyxa Septoria Seroula Sirodesmium Sistotrema Sordaria Talaromyces Taphrina Termitomyces Tetracladium Thamnidium Thamnostylum Thanatephorus :Thermoascus Thermomyces Thielavia Thielaviopsis Torulaspora Torulopsis Trametes Tremella Ulocladium Valsa Valsaria Vararia Verpa Wallemia Wardomyces Sphaceloma Spicari a Spiroidium Spondylocladium Spongipellus Sporidesmium Sporidiobolus Sporobolomyces Sporothrix Sporotrichum Stachybotrys Staurophoma Steccherinum Stemphylium Stereum Stibella Strobilomyces Stromatinia Suillus Syncephalastrum Syringospora Tricellua Trichocladium Trichoderma Tricholoma Trichophyton Trichosporon Trichothecium Trichurus Tridentaria Trigonopsis Truncatella Tuber Tympanis Tyromyces Utilago Verticicladiella Verticillium Volucrispora Volutella Whetzelinia 24 Xeromphalina Xylaria Ya rrowia Zalerion Zygodesmus Zygorhynchus Xylobolus Xylogone Yeasts Zygosaccharomyc Zygosporium Zythia D

Claims (91)

? 3 4 2 8 6 WHAT/f/WE CLAIM lo. 0LAIMS What is claimed is-:

1. A method for killing Baci1lus bacteria spores in a culture medium, comprising: heating said culture medium for approximately 1 hour to kill Bacillus bacteria cells; cooling said culture medium for substantially 8 to 24 hours to induce germination of spores; and killing said germinated spores by sterilizing said culture medium.

2. A method for preparing a substrate for culture of fungi, comprising: preparing a grain mixture by mixing water in substantially one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain and minor portions of starch, protein and nutrient sources; boiling said grain mixture for a sufficient time to allow dispersal of said starch, protein and nutrient sources into said grain mixture; cooling said grain mixture for a sufficient time to allow spores of any heat resistant bacteria to germinate; and sterilizing said grain mixture before said germinated spores mature sufficiently to produce more spores.

3. A method as described in claim 2, further comprising: 26 M.Z. 32.r>iV " i:A 23 4 2 draining said grain and water after said boiling step.

4. A method as described in claim 3, further comprising: mixing a permeability improving additive into said grain mixture.

5. A method as described in claim 4, wherein said starch, protein and nutrient supplements are preselected to meet the nutritional requirements of said fungi.

6. A method for preparing a substrate for culturing of fungi, comprising: boiling approximately 25 gallons of water in a 60 gallon capacity steam kettle; adding approximately 10 pounds of sliced Russet potatoes, 6 pounds of brewer's yeast powder, one quart of milk, 2 pounds of hulled sunflower seed, 1.5 pounds of soybean meal, 1.5 pounds of whole garlic, 1.5 pounds of corn gluten meal, 10 tablespoons of wheat germ oil, 10 tablespoons of sunflower oil, 6 tablespoons of molasses to form an intermediate mixture; boiling said intermediate mixture using a heat source; mixing said intermediate mixture to break clumps into small pieces; adding approximately 5 pounds of maple pea sprouts; 27 stirring said intermediate mixture until said sprouts are soft; adding approximately 200 pounds of whole sorghum grain, 35 pounds of whole oat grain and 5 pounds of rolled barley grain to form a grain mixture; adding a sufficient amount of water to immerse said grain mixture; boiling and stirring said grain mixture only until the water level falls below said grain mixture level by substantially 3 to 4 inches; removing said heat source; approximately one hour after removal of said heat source, draining said grain mixture; step, mixing said grain mixture with approximately 25 pounds of limestone powder, 80 pounds of gypsum powder and 20 pounds of cottonseed meal until said grain mixture is completely coated; introducing measured portions of said grain mixture into sterilizable microorganism impermeable containers; and steam sterilizing said sterilizable microorganism impermeable containers.

7. A substrate for culturing of fungi prepared according to the method of any one of the preceding claims. substantially 8 to 24 hours after said draining 28

8. A method for culturing tree mushrooms, comprising: preparing a grain mixture by mixing water in substantially one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain and minor portions of starch, protein and nutrient sources; boiling said grain mixture for approximately 1 hour; cooling said grain mixture for substantially 8 to 24 hours; introducing said grain mixture into a microorganism impermeable sterilizable container; sterilizing said container and said grain mixture; introducing tree mushroom spawn into said grain mixture; shaking said container to mix said tree mushroom spawn throughout said grain mixture; incubating said tree mushroom spawn in said container to allow said tree mushroom spawn to consume said grain mixture and to form mycelium; chilling said container and said mycelium for 7 to 9 days at a temperature of approximately 45 degrees Fahrenheit; removing said mycelium from said containers; and misting said mycelium with chilled water until tree 29 mushrooms of the desired size are grown.

9. A substrate for culture of fungi, comprising by weight: approximately 50% sorghum grain; approximately 26.6% water; approximately 1% yeast powder; approximately 1.67% potatoes; approximately .3% garlic; approximately .3% barley grain; approximately 4.2% limestone powder; and approximately 16% gypsum powder; wherein said sorghum grain, water, yeast powder, potatoes, garlic and barley grain is prepared by: boiling for a sufficient time to kill any microorganisms to form an intermediate mixture; cooling said intermediate mixture for a sufficient time to allow any spores to germinate; and heat sterilizing said intermediate mixture before said germinated spores have matured sufficiently to form new spores.

10. A substrate according to claim 9, wherein said limestone powder and said gypsum powder is mixed into said intermediate mixture after said cooling step.

11. A method for sterilizing a culture medium containing sporulation capable microorganisms, comprising: first, sterilizing said culture medium to kill said microorganisms; r i r. — r ^ (

■ ^ L C second, inducing germination of any spores in said sterilized culture medium to form germinated spores; and third, sterilizing said culture medium to kill said germinated spores before said germinated spores have matured sufficiently to be capable of forming further spores. 12. A method according to claim 11, wherein said first sterilizing step is performed by boiling said culture medium in water for approximately one hour, wherein said inducing step is performed by cooling said culture medium for a period from 8 to 24 hours, and wherein said third sterilizing step is performed by steam sterilizing said culture medium to approximately 250° Fahrenheit for approximately seven hours.

13. A method for culturing tree mushrooms, comprising: preparing a grain mixture by mixing water in substantially one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain and minor portions of starch, protein and nutrient sources; boiling said grain mixture for approximately 1 hour; cooling said grain mixture for substantially 8 to 24 hours; introducing said grain mixture into a microorganism impermeable 'sterilizable container; sterilizing said container and said grain mixture; introducing tree mushroom spawn into said grain mixture; shaking said container to mix said tree mushroom spawn throughout said grain mixture; incubating said tree mushroom spawn in said container to allow said tree mushroom spawn to consume said grain mixture and to form mycelium; and inducing said mycelium to fruit.

14. A method for culturing tree mushrooms, according to claim 13, further comprising: mixing a permeability improving additive into said grain mixture during said cooling step.

15. A method for culturing tree mushrooms, according to claim 13, wherein: said grain comprises: sorghum grain.

16. A method for culturing tree mushrooms, according to claim 13, wherein: said protein comprises maple pea sprouts.

17. A method for culturing tree mushrooms, according to claim 13, wherein: said sterilizing step is accomplished by steam sterilizing said container and said grain mixture at a 32 temperature of approximately 250°F and a pressure of approximately 15 pounds per square inch, for approximately 7 hours.

18. A method for culturing tree mushrooms, according to claim 13, wherein said tree mushroom spawn comprises: grain that has been previously colonized with tree mushroom spawn.

19. A method for culturing tree mushrooms, according to claim 13, wherein: said inducing step is accomplished by removing said mycelium from said containers and exposing said mycelium to an intermittent cold water mist.

20. A method for culturing tree mushrooms, according to claim 19, wherein said intermittent cold water mist is accomplished by using water chilled to 50 to 75° F for 2 to 120 seconds at 2 to 10 minute intervals for 6 to 15 hours during daylight hours and for 2 hours at night.

21. A method for culturing tree mushrooms according to claim 13, wherein: said starch, protein and nutrient sources are preselected to meet the nutritional requirements of said mushrooms.

22. A method for culturing tree mushrooms 33 2 3 4 2 8 8 according to claim 13, further comprising adding whole garlic to said grain mixture before said boiling step.

23. A method for culturing tree mushrooms according to claim 13, wherein: said tree mushroom spawn is shiitake mushroom spawn.

24. A method for culturing tree mushrooms according to claim 13, wherein: said tree mushroom spawn is oyster mushroom spawn.

25. A method for culturing tree mushrooms according to claim 13, wherein: said tree mushroom spawn is morel mushroom spawn.

26. A method for culturing mushrooms, comprising: boiling between 25 and 35 gallons of water; adding between 5 and 20 pounds of Russet potatoes, between 2 and 35 pounds of brewer's yeast powder and between 1/2 and 4 pounds of garlic to form an intermediate mixture; boiling said intermediate mixture using a heat source; mixing said intermediate mixture to break clumps into small pieces; adding between 150 and 300 pounds of whole sorghum grain and between 1/2 and 15 pounds of rolled barley grain to form a grain mixture; adding a sufficient amount of water to immerse said 34 grain mixture; boiling and stirring said grain mixture only until the water level falls below said grain mixture level; removing said heat source; approximately one hour after reraoval of said heat source, draining said grain mixture; substantially 8 to 24 hours after said draining step, mixing said grain mixture with between 12 and 37 pounds - of limestone powder and 50 to 100 pounds of gypsum powder until said grain mixture is completely coated; introducing measured portions of said grain mixture into sterilizable microorganism impermeable containers; sterilizing said sterilizable microorganism impermeable containers and said grain mixture; introducing mushroom spawn into said grain mixture; mixing said mushroom spawn throughout said grain mixture; incubating said mushroom spawn in said container to allow said mushroom spawn to consume said grain mixture and to form mycelium; and inducing said mycelium to fruit.

27. A method for culturing mushrooms, according to claim 26, wherein said mushroom spawn is shiitake mushroom spawn. •'34 286

.28. A method for culturing mushrooms, according to claim 26, wherein said mushroom spawn is oyster mushroom spawn.

29. A method for culturing mushrooms, according to •~\ claim 26, wherein said mushroom spawn is button mushroom spawn.

30. A method for culturing mushrooms, according to claim 26, wherein said mushroom spawn is paddy straw mushroom spawn.

31. A method for culturing mushrooms, according to claim 26, wherein said mushroom spawn is enoki mushroom « spawn.

32. A mushroom cultivated according to the method of any one of claims 8 or 13 to 31.

33. A method as defined in claim 1 for killing Bacillus bacteria spores in a culture medium substantially as herein described with reference to any example thereof or to the accompanying drawing.

34. A method as defined in claim 2 or claim 6 for preparing a substrate for culturing of fungi substantially as herein described with reference to any example thereof or to the accompanying drawing. t ""v

35. A substrate for culturing of fungi as defined in claim 7 or claim 9 substantially as herein described with reference to any example thereof or to the accompanying drawing.

36. A method as defined in claim 8 or claim 13 for culturing tree mushrooms substantially as herein described with reference to any example thereof or to the accompanying drawing.

37. A method as defined in claim 11 for sterilizing a culture medium substantially as herein described with reference to any example thereof or to the accompanying drawing.

38. A method as defined in claim 26 for culturing mushrooms substanti: as herein described with reference to any example thereof or to the accompanyinc drawings. 36 /

39. A method for culturing mushrooms according to claim 13, further comprising: retaining said mycelium for substantially 3 to 4 months after said incubating step.

40. A method for culturing tree mushrooms, according to claim 13, wherein: the said inducing step is accomplished by cold shocking said containers.

41. A method for culturing tree mushrooms, according to claim 40, wherein: said cold shock is accomplished by chilling said containers at substantially 40 to 65° Fahrenheit for 5 to 15 days.

42. A method for culturing tree mushrooms, according to claim 40, wherein: said cold shock is accomplished by a cold water bath for 24 to 48 hours.

43. A method for culturing mushrooms on a substantially cellulose free medium, comprising: preparing a substantially cellulose free grain mixture by mixing water in approximately one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain; sterilizing said grain mixture to kill any microorganisms that may be present and to kill any spores that may be formed by said microorganisms; introducing mushroom spawn into said grain mixture; incubating said mushroom spawn to allow said mushroom spawn to consume said grain mixture and to form mycelium; inducing said mycelium to fruit, whereby mushroom fruiting bodies are formed; and harvesting said fruiting bodies.

44, A method according to claim 43, wherein said incubating step is carried out for between substantially 3 weeks and six months. V 'J r)-> ' r /

45. A method according to claim 43, wherein said incubating step is carried out for approximately 3 weeks.

46. A method according to claim 43, wherein said mushroom spawn comprises tree mushroom spawn.

47. A method according to claim 46, wherein said tree mushroom spawn comprises shiitake mushroom spawn.

48. A method according to claim 46, wherein said tree mushroom spawn is selected from the group consisting of oyster mushroom spawn, enoki mushroom spawn, wood ear mushroom spawn, white jelly mushroom spawn and pom pom mushroom spawn.

49. A method according to claim 43, wherein said sterilizing step comprises: boiling said grain mixture to kill any microorganisms that may be present and to induce creation of spores; inducing said spores to germinate; and sterilizing said grain mixture to kill said germinated spores.

50. A method according to claim 43, further comprising: mixing said mushroom spawn throughout said grain mixture.

51. A method according to claim 43, further comprising: adding minor portions of starch, protein and nutrient sources to said grain mixture.

52. A method for using a sterilized grain mixture, comprising: culturing tree mushroom fruiting bodies in said grain mixture, said sterilized grain mixture being free of spores.

53. A method according to claim 52, wherein said culturing step comprises: inoculating tree mushroom spawn into said grain mixture; incubating said tree mushroom spawn to allow said tree mushroom spawn to consume said grain mixture and to form mycelium; and inducing said mycelium to form fruiting bodies. - 38 -

54. A method according to claim 52, wherein said tree mushroom spawn comprises shiitake mushroom spawn.

55. A method according to claim 52, wherein said tree mushroom spawn is selected from the group consisting of oyster mushroom spawn, enoki mushroom spawn, wood ear mushroom spawn, white jelly mushroom spawn and pom pom mushroom spawn.

56. A method according to claim 52, wherein said culturing step comprises: introducing said grain mixture into a microorganism impermeable container; sterilizing said container and said grain mixture; inoculating tree mushroom spawn into said container; incubating said tree mushroom spawn in said grain mixture and said container to form mycelium; and inducing said mycelium to form tree mushroom fruiting bodies.

57. A method according to claim 52, wherein said grain mixture is prepared from an initial mixture comprising water in approximately one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain; and wherein said initial mixture has been boiled for a sufficient time to kill any spore forming microorganisms, cooled for a sufficient time to allow any spores to germinate, and sterilized before said germinated spores have matured sufficiently to form new spores.

58. A method according to claim 57, wherein said boiling step is carried out for approximately 1 hour.

59. A method according to claim 57, wherein said cooling step is carried out for substantially 8 to 24 hours.

60. A method according to claim 57, wherein said sterilizing step is carried out by steam sterilizing said mixture.

61 . A method according to claim 52, wherein said grain mixture comprises a major portion of grain and minor portions of starch, protein, and nutrient sources.

62. A method according to claim 61, wherein said starch, protein and nutrient sources are preselected to meet nutritional requirements of tree mushrooms.

63. A method according to claim 53, further comprising mixing a permeability improving additive into said grain mixture.

64. A method according to claim 52, wherein said grain mixture is essentially free of cellulose.

65. A method for culturing fungi, comprising: preparing a substantially cellulose free grain mixture comprising water in approximately one to one-fourth parts by weight per part of a dry mixture comprising a major portion of grain; effecting removal of microorganisms and spores from said grain mixture; introducing said fungi into said grain mixture; incubating said fungi in said grain mixture to allow said fungi to consume said grain mixture; and harvesting said fungi.

66. A method according to claim 65, wherein said effecting step comprises: boiling said grain mixture to kill microorganisms and induce formation of spores; cooling said grain mixture to induce germination of said spores; sterilizing said grain mixture to kill said germinated spores.

67. A method for obtaining useful biochemicals, comprising: preparing a grain mixture by mixing water in approximately one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain; sterilizing said grain mixture to kill any microorganisms that may be present and to kill any spores that may be formed by said microorganisms; introducing mushroom spawn into said grain mixture; incubating said mushroom spawn to allow said mushroom spawn to consume said grain mixture and to form mycelium; and extracting useful biochemicals from said mycelium.

68. A method according to claim 67, wherein said mushroom spawn comprises tree mushroom spawn.

69. A method according to claim 68, wherein said tree mushroom spawn comprises shiitake mushroom spawn.

70. A method according to claim 68, wherein said tree mushroom spawn is selected from the group consisting of oyster mushroom spawn, enoki mushroom spawn, wood ear mushroom spawn, white jelly mushroom spawn and pom pom mushroom spawn.

71. A method according to claim 67, wherein said sterilizing step comprises: boiling said grain mixture to kill any microorganisms that may be present and to induce creation of spores; inducing said spores to germinate; and sterilizing said grain mixture to kill said germinated spores.

72. A method for using a sterilized substantially cellulose free grain mixture, comprising: culturing tree mushroom mycelia in said substantially cellulose free grain mixture without the use of a casing layer, said sterilized grain mixture being free of spores; and extracting useful biochemicals from said mycelia.

73. A method according to claim 72, wherein said culturing step comprises: inoculating tree mushroom spawn into said grain mixture; incubating said tree mushroom spawn to allow said tree mushroom spawn to consume said grain mixture and to form mycelium.

74. A method according to claim 72, wherein said tree mushroom spawn comprises shiitake mushroom spawn.

75. A method according to claim 72, wherein said tree mushroom spawn is selected from the group consisting of oyster mushroom spawn, enoki mushroom spawn, wood ear mushroom spawn, white jelly mushroom spawn and pom pom mushroom spawn.

76. A method according to claim 72, wherein said culturing step comprises: introducing said grain mixture into a microorganism impermeable container; sterilizing said container and said grain mixture; inoculating tree mushroom spawn into said container; incubating said tree mushroom spawn in said grain mixture and said container to form mycelium; and extracting useful biochemicals from said mycelium.

77. A method according to claim 72, wherein said grain mixture is prepared from an initial mixture comprising water in approximately one to one-fourth parts by weight per part of a dry mixture containing a major portion of grain; and wherein said initial mixture has been boiled for a sufficient time to kill any spore forming microorganisms, cooled for a sufficient time to allow any spores to germinate, and sterilized before said germinated spores have matured sufficiently to form new spores.

78. A method according to claim 77, wherein said boiling step is carried out for approximately 1 hour.

79. A method according to claim 77, wherein said cooling step is carried out for substantially 8 to 24 hours.

80. A method according to claim 77, wherein said sterilizing step is carried out by steam sterilizing said mixture.

81. A method according to claim 72, wherein said grain mixture comprises a major portion of grain and minor portions of starch, protein and nutrient sources.

82. A method according to claim 81, wherein said starch, protein and nutrient sources are preselected to meet nutritional requirements of tree mushrooms.

83. A method according to claim 72, further comprising mixing a permeability improving additive into said grain mixture. .A

84. A method for obtaining useful biochemicals, comprising: preparing a substantially cellulose free grain mixture comprising water in approximately one to one-fourth parts by weight per part of a dry mixture comprising a major portion of grain; effecting removal of microorganisms and spores from said grain mixture; introducing fungi into said grain mixture; incubating said fungi in said grain mixture to allow said fungi to consume said grain mixture and to form mycelium; and extracting useful biochemicals from said mycelium.

85. A method according to claim 84, wherein said effecting step comprises: boiling said grain mixture to kill microorganisms and induce formation of spores; cooling said grain mixture to induce germination of said spores; and sterilizing said grain mixture to kill said germinated spores.

86. A method as defined in claim 43 for culturing mushrooms on a substantially cellulose free medium substantially as herein described with reference to any example thereof.

87. A method as defined in claim 52 for using a sterilized grain mixture substantially as herein described with reference to any example thereof.

88. A method as defined in claim 65 for culturing fungi substantially as herein described with reference to any example thereof.

89. A method as defined in claim 67 for obtaining useful biochemicals substantially as herein described with reference to any example thereof. / I

90. A method as defined in claim 72 for using a sterilized Substantially cellulose free grain mixture substantially as herein escribed with reference to any example thereof.

91. A method as defined in claim 84 for obtaining useful biochemicals substantially as herein described with reference to any example thereof. moui -inn-r.^tce -t£/ve; c r, ,y~> p/vf-o -/ , (1. , >f5/Their authorised Agent 43 -