Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using fungi
Reexamination Certificate
1999-10-15
2002-04-16
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using fungi
C435S256800, C435S254100, C424S195150
Reexamination Certificate
active
06372462
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a process for culturing a variety of higher Basidiomycetes mushrooms to produce superior yields of biologically active nutriceuticals. The nutriceutical agents are isolated by a simple one-step process, and are formulated for use as dietary supplements to achieve normal human bodily functions in general, and to control particular abnormal factors, for example, hypercholesterolemia, in particular.
BACKGROUND OF THE INVENTION
Mushrooms or macrofungi with distinctive fruiting bodies of sufficient size to be seen with the naked eye, include about 10,000 species of varying degrees of edibility. Approximately 100 species have been tested for cultivation and only seven to eight have been cultivated on an industrial scale. The world production of cultivated edible mushrooms in 1994 was estimated to be about five million tons and was valued at about ten billion US dollars. The most popular species of cultivated edible mushrooms include
Agaricus bisporus
(J. Lge) Imbach,
A. bitorquis
(Quél.) Sacc.,
Lentinus edodes
(Berk.) Sing, Pleurotus spp., Auricularia spp.,
Volvariella volvacea
(Fr.) Sing.,
Flammulina velutipes
(Fr.) Sing,
Tremella fuciformis
Berk.,
Hypsizygus marmoreus
(Peck) Bigel.,
Pholita nameko
(T. Ito) S. Ito et Imai,
Grifola frondosa
(Dicks.: Fr.) S. F. Gray,
Hericium erinaceus
(Bull.: Fr.) Pers.,
Dictyophora indusiata
(Vent.: Pers.) Fischer,
Stropharia rugosoannulata
Farl. apud Murr.,
Lepista nuda
(Bull.: Fr.) Cooke,
Agrocybe aegerita
(Brig.) Sing.
The cultivation of fruiting bodies of mushrooms deals with living organisms, for example, the mushroom itself and other microorganisms which may either be harmful or beneficial. Therefore, the methods employed in mushroom cultivation require modifications depending upon the region being cultivated, substrates available, environmental conditions and species of microorganisms encountered.
The cultivation of mushrooms for fruiting bodies production is a long-term process needed from one to several months for the first fruiting bodies to appear. Moreover, in the case with
Pleurotus ostreatus
it is known that lovastatin is concentrated presumably in the lamella and basidiospores but not in the stipe or cap tissue, and its amount depends on fruiting body size, age, and substrate composition. Therefore, the submerged culturing of lovastatin producers allows production of the end product which has a constant composition, in a short period, by using controlled conditions such as ecologically pure culture medium of defined composition.
Lovastatin (=mevinolin) (=MSD 803), is a useful hypocholesterolemic pharmacological agent of natural origin. It is a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMG CoA reductase), the key enzyme in cholesterol metabolism. Monaghan et al, U.S. Pat. No. 4,231,938. The best producers of lovastatin are different strains of
Aspergillus terreus
a common contaminant mold on food, which contains several toxic substances including terrein, patulin, citrinin, and citreoviridin. As a result, lovastatin isolated from Aspergillus requires an extensive purification process involving extraction of lovastatin from the culture broth with ethyl acetate or XAD-2 resin and subsequent steps of concentration, washing, reconcentration, and recrystallization. U.S. Pat. No. 4,231,938. Alternate processes for isolation of lovastatin include use of different resins or less toxic solvent, such as butyl acetate in the first step of extraction of the culture broth. The culture broth which is extracted includes both the culture medium and the cell mass. For example, even for strains of Pleurotus grown in submerged cultures, lovastatin was extracted from the culture broth. DE 4,402,259 and Gunde-Cimerman et al, 1993, Microbiology Letters III: 203-206.
In general, the efficiency of lovastatin production is determined by the amount of lovastatin produced by the various fungi strains together with the efficiency of the extraction procedure employed. The Aspergillus strains are more productive than the Pleurotus strains in producing greater amounts of lovastatin. U.S. Pat. No. 4,231,938. However, the Aspergillus strains produce a wide range of toxic substances besides the cholesterol lowering lovastatin, and this requires complex and additional extraction and purification procedures to obtain lovastatin. Not only are these procedures more expensive but they require use of large numbers of solvents, which in turn are toxic e.g. benzene, toluene, acetonitrile, or ethyl acetate. Hence, working with these solvents endangers the health of persons involved, and requires multi-step purification procedures. Accordingly, there is need for methods to produce cholesterol-lowering compounds with a high activity, preferably from sources that are not toxic, and by using simple, rapid and inexpensive manufacturing processes. Moreover, different cholesterol-lowering compounds have varying degrees of activity. The process of the present invention involves production of a cholesterol lowering compounds from edible Basidiomycetes mushrooms grown in submerged cultures. The mycelium is grown on nutrient media especially formulated to produce high yields of the cholesterol-lowering compound and other nutrients.
SUMMARY OF THE INVENTION
The present invention relates to cultivation in submerged culture containing defined nutrient media of a mycelium of the edible Basidiomycetes mushrooms comprising
Pleurotus ostreatus, Pleurotus eryngii
var.
ferulae, Hypsizygus marmoreus, Lepista nuda, Pleurotus cystidiosus, P. dijamor, P. pulmonarius, P. salignus, Grifola frondosa,
and
Hericium erinaeus.
In a first aspect, the invention provides a method of cultivating submerged cultures of one or more Basidiomycetes mushrooms having the trait to produce one or more substances having hypocholesterolemic activity. The use of the nutrient media of the invention, comprising a saccharide containing glucose in the molecule, an organic or mineral source of nitrogen and a variety of salts, is especially suited to enhance the production of the cholesterol- lowering nutrients and other essential nutrients.
In the second aspect, the invention provides a method to concentrate the hypocholesterolemic compound mainly in the mushroom cells thus enabling the simple separation of the edible biomass from the fermentation broth, thereby requiring no further extraction, concentration, purification or complex separation procedures. The simple separation of the edible Basidiomycetes from the culture broth of the present invention is followed by the drying of the final nutriceutical product at 40-45° C.
In accordance with the invention, compositions including a cholesterol-lowering compound are described, which, when orally consumed or ingested, inhibit the biosynthesis of mevalonic acid by inhibition of 3-hydroxy-3-methylglutaryl A reductase coenzyme (HMG-COA reductase, E.C.1.1.1.34) and thus reducing cholesterol levels in blood of a mammal. The preventive and/or treatment method of the invention therefore involves reduction of risk posed by elevated cholesterol in subjects at high risk of having cardiovascular disease.
The present invention can provide methods and compositions including nutriceutical components generally beneficial for promoting health, for example, lovastatin, polysaccharides, proteins and essential amino acids, vitamins, fiber, fatty acids, or minerals.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.
REFERENCES:
patent: 3716579 (1973-02-01), Knauseder et al.
patent: 4231938 (1980-11-01), Monaghan et al.
patent: 4369253 (1983-01-01), Takita et al.
patent: 5712130 (1998-01-01), Hajko et al.
patent: 877089 (1998-11-01), None
Hadar et al. Applied and Environmental Microbiology (1986), vol. 51, No. 6, p. 1352.*
ATCC catalog; Filamentous Fungi, 19th edition, Jong et al., eds. p. 424, entry #90212, 1996.*
Microbiology Letters III: 333-338, 1993, vol. 113 Bobek et
Reshetnikov Sergey V.
Wasser Solomon P.
Coe Susan D.
Karmali Rashida A.
MedMyco Ltd.
Prats Francisco
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