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Production of fungal extracellular immune stimulating compoundsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, PolysaccharideProduction of fungal extracellular immune stimulating compounds description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060094689, Production of fungal extracellular immune stimulating compounds. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL AREA OF THE INVENTION [0001] The present invention is directed to a method for producing an immune stimulant in liquid culture. The invention is in one embodiment directed to culturing Lentinus edodes under particular conditions in a fermentation broth. This application is a non-provisional claiming priority from Norwegian patent application No. NO 2001 4256 filed 3 Sep. 2001, which is hereby incorporated by reference in its entirety. All patent and non-patent references cited in that application, or in the present application, are also hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION [0002] It is known that health-promoting effects are attributed to glucans from fungi and yeasts. "Shiitake" fungus (Lentinus edodes) has been attributed effects which can be exploited for many medicinal purposes such as immunestimulation, anti-virus, anti-tumour, etc. Studies of lentinan have shown that it stimulates the immune system of the host in a variety of ways, such as activation of T helper cells, increased production of Interleukin 1 and Interleukin 2, increased antibody production in various forms of cancer, and decreasing the cholesterol level in the blood. (Herbs for Health, January/February, 1997; K. Jones, "Shiitake: Medicine in a mushroom", p. 40-50, 54; Anticancer Res, Vol. 17(4A), 1997; H. Matsouka, "Lentinan potentiates immunity and prolongs the survival time of some patients", p. 2751-2755; Adv Appl Microbiol, Vol. 39, 1993; S. C. Jong, "Medicinal and therapeutic value of the shiitake mushroom", p. 153-184, Int J Immunopharmacol, Vol. 14, 1992; K. Irinoda, "Stimulation of microbiocidal host defence mechanism against aerosol influenza virus infection by lentinan, p. 971-977., Jpn J Cancer Res, Vol. 76(1), 1985; D. Herlyn, "Monoclonal antibody-dependent murine macrophage-mediated cytotoxicity against human tumors is stimulated by lentinan, p. 37-42). [0003] One active ingredient of Lentinus edodes is termed lentinan, a polysaccharide based compound described as a beta-(1,3) glucan backbone With beta-(1,6) side chains. [0004] "Solid-state" reactors are routinely used for culturing fungi such as Lentinus edodes. This is a technology which is used for many purposes such as composting, production of biological products such as enzymes, soy sauce, acetic acid, and the like. [0005] For the production of lentinan, Lentinus edodes can be cultivated on a suitable solid matrix provided by stems of tree or chips of wood to which is often added chemical compounds supporting the growth of mycelium and development of the fruiting bodies, where most of the lentinan is localised. The fruiting bodies are harvested, either by hand or mechanically, and are subsequently dried and ground to a powder which can be used as it is, or used in tablets, or sent for further processing such as extraction of lentinan. [0006] The methods used for culturing fungi such as Lentinus edodes, harvesting and subsequently drying the fruiting bodies, and optionally extraction of lentinan, are well known for the skilled person. The cultivation time can be from four to ten weeks with few possibilities for controlling the process. This results in a fungal growth, and in turn, in an amount of lentinan produced which is not the same for each batch carried out. Also, extraction of lentinan from the fungal material is time consuming and process intensive. [0007] Kim et al. (2001, Biotechnology Letters, 23, 513-517) describes extracellular polysaccharides produced by Phellinus lintus in submerged culture; Kim et al. (2002: Letters in Applied Microbiology, 34, 56-61) describes mycelial growth and exobiopolymer production by submerged culture of various edible mushrooms. There is no data available to indicate that any of these extracellular polymers are immune stimulating. [0008] It is generally known to grow mycelium of Basidiomycetes in submerged culture and to obtain active ingredients from the growth medium, see e.g. Aouadi et al 1992 (Carbohydr Polym 17:177-184) and Lee et al 1995 (Prog Plant Polym Carbohydr Res, B. Behrs Verlag, Hamburg Del.) and Hatvani 2001 (Int J Antimicrob Agents 17:71-). However, none of the references discloses the isolation of extracellular components with immune stimulating activities. Furthermore, none of the references disclose that extracellular polysaccharides have different activities from their intracellular counterparts. [0009] It is therefore an object of the present invention to provide novel methods for production of extracellular immune stimulating agents with higher level of activity than the hitherto known intracellular counterparts. SUMMARY OF THE INVENTION [0010] The invention in a first aspect, relates to a method for cultivating a fungal mycelium, preferably a fungus from the class of basidiomycetes, such as a fungus of the genus Lentinus, such as Lentinus edodes, in a liquid medium of sterile water to which nutrient compounds are added in predetermined concentrations. The liquid medium supports fungal growth and stimulates the production of extracellular compounds, such as immune stimulating agents, such as lentinan produced by fungal mycelium of the genus Lentinus, such as e.g. Lentinus edodes. [0011] By fungal mycelium is intended any fungal biomass, which can be grown in submerged culture according to the invention. The fungal biomass may be in the form of single hyphae, spores, aggregates of mycelium, and partly differentiated mycelium. [0012] Lentinan as used herein refers to the polysaccharide, which can be isolated from Lentinus edodes (Berk.) Sing. The primary structure is a .beta.1,3-D-glucan having 2 .beta.-1,6-glucopyranoside branchings for every 5 .beta.-1,3 linear linkages. The molecular weight may vary from 400,000 to 800,000 (Merck Index 12.sup.th Edition, 1996, Monograph No 5462). [0013] The cultivation of the fungal mycelium, such as fungi of the genus Lentinus, such as Lentinus edodes, results in the production of one or more extracellular agents, such as e.g. immune stimulating agents, in the form of e.g. a polysaccharide, such as e.g. a beta-glucan, including lentinan isolatable from Lentinus edodes, a polypeptide, a glycosylated polypeptide or a proteinacious polysaccharide compound, a proteoglucan, such as e.g. a polypeptide associated alpha-glucan or a polypeptide associated alpha-mannan, including KS-2 isolatable from Lentinus edodes, a lipid, or a secondary metabolite, which can be isolated and/or purified, and optionally fractionated, from the extracellular fraction of the fermentation broth of a bioreactor following cultivation of the basidiomycete fungal mycelium in question. [0014] The extracellular fraction of the liquid fermentation medium is also termed the supernatant and this fraction can be separated from the fungal mycelium by e.g. centrifugation or filtration, or indeed by any other means available for obtaining a liquid fraction essentially without any fungal mycelium present therein. The term "essentially without any fungal mycelium present therein" shall denote that the concentration of fungal mycelium, including fractions thereof, has been reduced at least by a factor of 10.sup.3, such as reduced by a factor of at least 10.sup.4, for example a factor of at least 10.sup.5, such as reduced by a factor of at least 10.sup.6. [0015] Besides being easier to isolate and process, fungal extracellular agents such as immune stimulating agents according to the present invention are surprisingly more potent than fungal associated agents, i.e. agents being either fungal intracellular agents, or agents which are removed from a liquid fermentation broth along with the removal of the fungal mycelium and fractions thereof by filtration, precipitation, or otherwise. [0016] In a further aspect the invention relates to an immune stimulating agent obtainable from the extracellular part of the liquid growth medium according to the method of producing said immune stimulating agent. [0017] This immune stimulating agent has surprisingly turned out to have a higher immunostimulating activity than the corresponding extract obtainable from the mycelium of the fungus, although the mycelium is known as the primary source of e.g. lentinan. In a particularly preferred embodiment, this extract is obtainable by removal of mycelium, removal of water from the medium, one or more rounds of precipitation with alcohol and washing with acid and/or base. [0018] According to the invention there is also provided a composition comprising the immune stimulating agent obtainable as described above and a physiologically acceptable carrier. Furthermore, there is provided a pharmaceutical composition comprising the immune stimulating agent according to the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition may in particular be used for treatment or prophylaxis in connection with an immune compromised condition. [0019] Fungi belonging to the genus of Lentinus, such as Lentinus edodes, represent one example of fungi according to the present invention. In other preferred aspects of the invention a method is provided wherein the fungus is selected from the group of fungi consisting of Auricularia auricula-judae, Coriolus versicolor, Grifola frondosa, Flammulina velutipes, Schizophyllum commune, Sclerotinia scleroticum, Trametes versicolor, Tremella fuciformis, Agaricus blazei, Cordyceps sinensis, Ganoderma lucidum, Hericium erinaceus, lonotus obliquus, Pleurotus ostreatus, and Polyperus umbellatus. DETAILED DESCRIPTION OF THE INVENTION [0020] For the method of the present invention, "liquid-state" cultivation of Lentinus mycelium, or fractions thereof, is used. In short, this involves dissolving in water the nutrient compounds a microbial organism such as fungal mycelium, or fractions thereof, require for growth, transferring the solution to a bioreactor and inoculating the bioreactor with cells or spores of the microbial organism such as a fungal mycelium, or fractions thereof, to be cultivated. This is done under sterile conditions and with control of the environment in order to give the microbial organism a suitable chemical and physical environment. The technology related to "liquid-state" cultivation of microbial organisms is well known for the skilled person. Continue reading about Production of fungal extracellular immune stimulating compounds... Full patent description for Production of fungal extracellular immune stimulating compounds Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Production of fungal extracellular immune stimulating compounds patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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