Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1999-10-15
2002-05-07
Campell, Bruce R. (Department: 1661)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C047S001100
Reexamination Certificate
active
06383799
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a process for culturing a variety of higher Basidiomycetes mushrooms using defined media, 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 hyperglycemia in particular. Specifically, this invention relates to a method of producing a polysaccharide glucuronoxylomannan, which has medicinal properties, and a biomass rich in the essential amino acids and vitamins, from the culture broth obtained from a submerged culture of an edible higher Basidiomycetes mushroom belonging to the genus Tremella.
1. 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
Far. 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 fruit bodies production is a long-term process needing from one to several months for the first fruiting bodies to appear. Moreover, it was found that processes for extraction of polysaccharides from fruit bodies are not considered commercially feasible, since the physicochemical properties of the products resulting from these processes were not known or regulated, Otsuka, U.S. Pat. No. 4,051,314. Submerged culturing of polysaccharide producers allows obtaining the end product of constant composition in a short period under controlled conditions using ecologically pure culture medium of defined composition.
Several species of the genus Tremella have been used in folk medicine since ancient times. These are
T. fuciformis, T. mesenterica, T. aurantia
, and
T. cinnabarina
, and all of them are characterized as “strengthening health, resisting disease,” Yang Q. et al, 1989, Mushroom Science 12: 631-643. The last three species, with yellow-gold fruit bodies, are known under the common name “Kinji” in Oriental medicine and are considered to possess equal medicinal value, Ukai S. et al., 1995, JP 7, 238, 031 A.
Tremella mushrooms belong to the so-called jelly mushrooms, which form gelatinous fruit bodies. The jelly mushrooms are a set of species from different taxonomical groups of Phragmobasidiomycetes, which are able to survive long periods of drought by drying to a horny texture. When moisture is again available, they absorb water and become gelatinous. This characteristic of jelly mushrooms is due to the presence of specific water absorbing polysaccharides that compose 60-70% of the dry fruiting body. Unlike the &bgr;-1-3-glucans polysaccharides from other medicinal mushrooms, jelly mushroom polysaccharides consist of other sugars as well as glucose, and therefore belong to the class of heteropolysaccharides. A unique feature of Tremella mushrooms is that their pharmacologically active polysaccharides make up most of the structural fruit body polysaccharides while in other medicinal mushrooms pharmacologically active polysaccharides make up only a small part of the biomass. For example, in shiitake mushrooms only 31 g of lentinan was extracted from 200 kg of fresh mushrooms, Mizuno, 1999, Int. J. Medicinal Mushrooms, 1:7-27.
The main pharmacologically active substance from Tremella is the polysaccharide glucuronoxylomannan, consisting of a linear backbone of 1,3-linked alpha-D-mannose with mainly xylose and glucuronic acid in side chains. The chemical structure of Tremella glucuronoxylomannan differs among various samples of even one species, and may be in some way connected with a type of polysaccharide-based method of identification. The general proportions of xylose:glucuronic acid:mannose are given in
Tremella fuciformis
as 1.0:2.77:4.9; 2:1:4 in
T. aurantia
, and 7:1:5 in
T. mesenterica
, Fraser CE et al., 1973, Can J. Biochem. 51: 219-224. Some additional saccharides can be identified in different samples of
T. fuciformis
, such as glucose and fucose , xylobiose and fructose.
Several species of Tremella produce glucuronoxylomannan which has been shown to have hypocholesterolemic activity. Hypercholesterolemia and dyslipoproteinemia, causing high blood pressure and diabetes, are the main risk factors determining ischemic cardiopathologies and cerebral vascular accidents. Hypercholesterolemia is defined as the increase of the blood cholesterol ratio above 2 g/l. Dyslipoproteinemia coincides with the disturbance of diverse lipoprotein levels, resulting in lipid sedimentation on the arterial walls.
Tremella fuciformis
fruiting bodies, taken as 5% dried powder in a hypercholesterolemic diet (1.5% cholesterol, 5% fat), decreased the serum total cholesterol concentration in rats 19% after 4wk of mushroom diet consumption. Similar significant decreases in serum low-density lipoprotein (LDL) and triacylglycerol levels were observed, Cheung, PCK, 1996, Nutrition Res. 16: 1721-1725.
The antilipemic effect of polysaccharides from both
T. fuciformis
fruit body and pure culture has been described earlier, Janhe S. et al., 1989, J. China Pharm. Univ., 20:344-347, and it has been proposed that Tremella polysaccharides may be useful as dietary supplement in the prevention and therapy of atherosclerosis, Ryong LH et al., 1989, Drug Dev. Res. 17: 109-117. In addition, Tremella polysaccharides may have hepatoprotective functions in cases of chronic hepatitis Xiong HZ et al., 1985, Chin. J. Antibiot. 10: 363-365.
Tremella aurantia
was found to have hypoglycemic activity in normal mice and in two diabetic mouse models, streptozotocin-induced diabetes and genetic diabetes. Diabetes is defined by a fasting glycemia above 1.4 g/l evaluated on two different blood samples. Insulin-dependent diabetes (IDD) corresponds to a fault in insulin secretion; cardiovascular complications are due to the important and lasting hyperglycemia causing the persistence of proteins in the urine.
Tremella aurantia
fruit body polysaccharide (TAP) was found to be effective in reducing hyperglycemia following not only intraperitoneal, but also oral administration (0.5 g/l TAP). Similar effects in prevention and treatment of alloxan- or streptozotocin-induced diabetic mice were shown for
T. fuciformis
polysaccharides, Xue W. et al, 1989, J. China Pharm. Univ. 20: 181-183; It was proposed that the mechanism of hypoglycemic activity in normal mice was at least related the increase of insulin secretion and for the acceleration of glucose metabolism Kiho, T. et al., 1994, Yakugaku-Zasshi,114:308-315.
Acid heteroglycans from
T. fuciformis
was found to have cytokine-stimulating activity. Four kinds of acidic heteroglycans with molecular weights from 550 to 48 KDa were isolated from the fruit bodies. These and additional fragments of their acidic hydrolysate also induced
Reshetnikov Sergey V.
Wasser Solomon P.
Bell Kent L.
Campell Bruce R.
Karmali Rashida A.
MedMyco Ltd.
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