U.S. patent application number 10/084544 was filed with the patent office on 2002-11-07 for process for producing, methods and compositions of glucuronoxylomannan as nutriceutical agent from higher basidiomycetes mushroom.
Invention is credited to Reshetnikov, Sergey V., Wasser, Solomon P..
Application Number | 20020164773 10/084544 |
Document ID | / |
Family ID | 23661260 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164773 |
Kind Code |
A1 |
Wasser, Solomon P. ; et
al. |
November 7, 2002 |
Process for producing, methods and compositions of
glucuronoxylomannan as nutriceutical agent from higher
basidiomycetes mushroom
Abstract
The present invention describes new and distinct strains of
higher Basidiomycetes mushrooms grown in submerged cultures.
Specifically, the new strain of species of the genus Tremella offer
superior yields of one-cell biomass and exocellular
heteropolysaccharide glucuronoxylomannan, niacin and essential
amino acids.
Inventors: |
Wasser, Solomon P.; (Haifa,
IL) ; Reshetnikov, Sergey V.; (Kiev, UA) |
Correspondence
Address: |
Rashida A. Karmali, Esq.
Attorney for Applicants
230 Park Avenue, Suite 2525
New York
NY
10169
US
|
Family ID: |
23661260 |
Appl. No.: |
10/084544 |
Filed: |
February 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10084544 |
Feb 26, 2002 |
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09419207 |
Oct 15, 1999 |
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6383799 |
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Current U.S.
Class: |
435/254.1 ;
424/195.15 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
37/04 20180101; A61K 36/07 20130101; C12N 1/14 20130101; C12P 19/04
20130101 |
Class at
Publication: |
435/254.1 ;
424/195.15 |
International
Class: |
C12N 001/16; A61K
035/84; A01N 065/00; C12N 001/14; C12N 001/18 |
Claims
What is claimed is:
1. A process for producing a one cell biomass and exocellular
polysaccharides of edible Basidiomycetes mushrooms, said process
comprising: cultivating a fungi of the Basidiomycetes in submerged
culture on nutrient media, and isolating the resulting biomass of
edible Basidiomycetes and exocellular polysaccharides from the
culture broth by alcohol precipitation.
2. The process for producing a biomass and exocellular
polysaccharides of edible Basidiomycetes mushrooms according to
claim 1, wherein the fungi of the Basidiomycetes is a species of
the genus Tremella is selected from the group consisting of
Tremella mesenterica.
3. The process for producing a biomass of edible Basidiomycetes
mushrooms and exocellular polysaccharide according to claim 1,
wherein the fungi of the Basidiomycetes produces nutrients
comprising a hypoglycemic compound, essential amino acids or
niacin.
4. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the nutrient media is of
the following composition:
8 Medium A Sucrose 25 Peptone 2.0 Yeast extract 2.25
KH.sub.2PO.sub.4 1.0 MgSO.sub.4.7H.sub.2O 0.5
5. The process for producing a biomass and exeocellular
polysaccharides of edible Basidiomycetes mushrooms according to
claim 1, wherein the nutrient media is of the following
composition:
9 Medium B Sucrose 40 Peptone 1.0 Yeast extract 1.1
NaHPO.sub.47H.sub.2O 1.0 NaH.sub.2PO.sub.47H.sub.2O 0.5
KH.sub.2PO.sub.4 1.0 Mg acetate 1.0 KC1 1.0
6. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the fungi is Tremella
mesenterica CBS 101939.
7. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the fungi is selected from
the group consisting of Tremella luciformis, T aurantia, T.
encephala, and T. mesenterica.
8. A composition for reducing blood levels of glucose in a mammal,
said composition comprising a nutriceutical formulation of dried
Basidiomycetes biomass.
9. The composition according to claim 8 wherein the dried
Basidiomycetes biomass comprises a glucose-lowering compound,
glucuronoxylomannan, polysaccharide, fiber, protein, essential
amino acids, vitamins or niacin.
10. The composition according to claim 8, wherein the dried
Basidiomycetes biomass and exocellular polysaccharides is produced
from Tremella mesenterica CB 101939.
11. The composition according to claim 8, wherein the dried
Basidiomycetes biomass and is produced from a fungi selected from
the group consisting of Tremella luciformis, T. aurantia, T.
encephala, and T. mesenterica.
12. The composition according to claim 8 wherein the dried
Basidiomycetes biomass and exocellular polysaccharides is used in
an effective amount to reduce blood levels of glucose, in
combination with a pharmaceutical carrier acceptable for oral
administration.
13. The composition according to claim 8 wherein the dried
Basidiomycetes biomass and exocellular polysaccharides is used in
an effective amount to inhibit diabetes.
14. The composition according to claim 8 wherein the dried
Basidiomycetes biomass and exocellular polysaccharides is used in
an effective amount to inhibit a variety of diseases including
human immunodeficiency.
15. A method for treating an individual for hyperglycemia, said
method comprising: administering to said individual an amount
effective to reduce the level of serum glucose, of a composition
containing an edible biomass of Basidiomycetes and exocellular
polysaccharides, wherein said product includes a glucose-lowering
compound.
16. The method according to claim 15, wherein said amount is
effective to reduce the level of blood glucose from about 20% to
50%.
17. The method according to claim 15, wherein the biomass of
Basidiomycetes is produced from fungi of a species of the genus
Tremella.
18. The method according to claim 17 wherein the species of the
fungi is selected from the group consisting of Tremella luciformis,
T. aurantia, T. encephala, and T. mesenterica.
19. The method according to claim 14 wherein the Basidiomycetes
biomass is produced from Tremella mesenterica CB 101939.
Description
FIELD OF THE INVENTION
[0001] 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,
BACKGROUND OF THE INVENTION
[0002] 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 (Qul.) 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 (Bul.: Fr) Cooke, Agrocybe aegerita (Brig)
Sing.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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 .beta.-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.
[0007] 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 C E 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.
[0008] 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 4 wk 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.
[0009] 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 L H et al., 1989, Drug Dev. Res. 17:
109-117. In addition, Tremella polysaccharides may have
hepatoprotective functions in cases of chronic hepatitis Xiong H Z
et al., 1985, Chin. J. Antibiot. 10: 363-365.
[0010] 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.
[0011] 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 monocytes to secrete interleukin-6 with
high potency, indicating that the activity may be caused by a
common structure, (1.fwdarw.3)-mannan in the four heteroglycans and
their fragments. So, the change of molecular weight had no obvious
influence on the activity of the heteroglycans, Gao Q. et al.,
1996, Carbon Res. 288:135-142.
[0012] Naturally growing or artificially cultivated fruit bodies of
Tremella mushrooms have been extensively used during the last
decade for development of different types of Tremella health-care
nutritive or medicinal food, drink or beverage (CN Patt. 1125065;
1109300; 1102305; 1099946; 1091263; 1082362; 1072829; 1066964;
1044036; 1204474; 1178122; 114871; JP Pat. 6153879; 6339354;
60075279).
[0013] These Tremella-by products can be classified as "mushroom
nutriceuticals", which are refined/partially defined extracts from
either the mycelium or the fruiting body of the mushroom, which are
consumed in the form of capsules or tablets as a dietary supplement
or functional foods, and which have potential therapeutic
applications. Chang ST, 1999, Int. J. of Medicinal Mushrooms 1:
1-7. In this way nutriceuticals differ from "nutraceuticals" which
are consumed as part of the normal diet and may have been
modified/enriched in some way to provide health-giving benefits.
Enriched compositions of nutriceuticals may thus be prepared by
selecting the appropriate strains of mushroom and/or by optimizing
the culture cultivation conditions. Hence the regulatory
requirements for approval of nutriceuticals for human consumption
may be more stringent than those for nutraceuticals.
[0014] However, it is found that nutraceutical products from
fruiting bodies of medicinal mushrooms are very diverse in quality
and quantity of different nutrients, and there are heretofore, no
standard protocols for guaranteeing reproducible high product
quality. Chang S T et al, 1999, Int. J. of Medicinal Mushrooms 1:
139-146. So, it is generally desirable to have nutriceutical
compositions that are relatively uniform as to the type and levels
of nutrients present in them. Generally, it has been found in
cultivating different mushroom strains, that the polysaccharides
extracted from the fruit bodies and from mycelia in pure cultures
are not essentially the same, although both may be
pharmacologically active. A slight difference was observed in
xylose : glucuronic acid : mannose proportions in Tremella
fuciformis polysaccharide from fruit bodies (1.0:2.77:4.9) and
those obtained from pure cultures of different haploid yeast-like
budding strains -1:0.8-1.3:2.1-3.5, Kakuta M. et al., 1979, Agric.
Biol. Chem. 43: 1659-1668. The Tremella fuciforms polysaccharide
had a hypocholesterolemic effect, which is characteristic for fruit
bodies polysaccharide, when tested in rats with the addition of
submerged culture-derived polysaccharide to a high-cholesterol
diet.
[0015] Alternatively, acidic technology has been proposed to
produce Tremella proteoglycan from artificially growing mycelium on
a semisolid medium after fruit bodies have been removed from this
substrate (CN Pat. 1071060). A pure culture of Tremella was used
for production of a fermented beverage, when Tremella strains were
inoculated in a liquid medium made with potato as the main raw
material. The fermented liquor was directly made into a Tremella
polysaccharide beverage after filtration and also could be
concentrated and dried in order to obtain crude Tremella
polysaccharide powder (CN Pat. 1069866; 1057954). This high-level
nutritive beverage which fully embodies the nutritive and medicinal
value of Tremella is proposed for enhancing the effect on human
immunity and exerting antiageing, anti-tumor, or anti-hyperlipemia
effects.
[0016] A special method for increasing the growth rate of
Basidiomycetes species Coriolus versicolor pure culture was
developed (U.S. Pat. No. 4,159,225). It was found that when
dycariotic mycelium of this species was subjected to submerged
culture while undergoing a mechanical treatment such as grinding or
shearing in a liquid medium, the mycelium lost clamp connection,
which is an intrinsic morphological characteristic, and changed
into a monocariotic mycelium, and that the thus-formed monocariotic
mycelium was stable and also had a unique characteristic in its
extremely high propagation rate as compared with the known
dycariotic mycelium.
[0017] A unique feature of some Heterobasidiomycetes species, to
which the Tremella species belongs, is that their monocariotic
(haploid) strains are able to grow in the form of yeast-like
budding cells, and these monocariotic strains can be obtained not
by grinding dycariotic mycelium but by development of
monobasidiosporous cultures from the basidiospore print. This
biological phenomenon was used for the production of Tremella
fuciformis food and beverage, when yeast-like cells obtained by
cultivating T-9 haploid strain (FERM No. 9419) in a submerged
culture were blended with other components to form a food and
beverage having an inhibitory action on a rise in cholesterol (JP
Pat. 1020070). Heretofore, one cell cultures of Tremella have not
been used to produce compounds having a hypoglycemic activity, and
in particular, one cell cultures of Tremella mesenterica have not
been used for any purpose.
SUMMARY OF THE INVENTION
[0018] The present invention relates to cultivation in submerged
culture containing defined nutrient medium, of a one cell culture
of the edible Basidiomycetes mushrooms comprising the genus
Tremella including, but not limited to Tremella mesenterica, T.
fuciformis, T. aurantia, and T. encepuala.
[0019] 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
hypoglycemic 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
glucuronoxylomannan, having a hypoglycemic activity, by submerged
culturing of haploid yeast-like budding cultures of an edible
mushroom selected from Tremella mesenterica.
[0020] In the second aspect, the invention provides a method to
concentrate the hypoglycemic compound together with mushroom cells
thus enabling the simple separation of the edible one-cell biomass
and exocellular polysaccharides from the fermentation broth by
alcohol precipitation, 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 drying after
extraction with alcohol.
[0021] In accordance with the invention, compositions including
glucuronoxylomannan, a glucose lowering compound is described,
which, when orally consumed or ingested, lowers the blood levels of
glucose. The preventive and/or treatment method of the invention
therefore involves reduction of risk posed by elevated glucose in
subjects at high risk of having diabetes.
[0022] The present invention can provide methods and compositions
including nutriceutical components generally beneficial for
promoting health, for example, glucuronoxylomannan, vitamins,
protein rich in the essential amino acids and free amino acids.
[0023] Still other objects and advantages of the invention will in
part be obvious and will in part be apparent from the
specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1. Shows a view of germination of basidiospores, both
by hyphae or budding cells. Objective.times.100, phase
contrast.
[0025] FIG. 2. Shows a view of cells of haploid strain; one cell is
proliferating by haploid hypha. Objective.times.100, phase
contrast.
[0026] FIG. 3. Shows a view of preparation of Tremella mesenterica
in Indian ink. White area around cells indicates a polysaccharide
slime envelope. It is well evident, that the polysaccharide matrix
around yeast-like budding cells is much more voluminous that of the
hypha. Objective.times.100.
[0027] FIG. 4. Shows a view of dycariotic myceliurn with clamps
originating from crossed compatible haploid strains. Haploid cells
are visible in the field of view. Objective.times.40, phase
contrast.
[0028] FIG. 5 shows a view of Tremella mesenterica CBS 101939
submerged culture. Objective.times.100, phase.
DETAILED DESCRIPTION OF THE INVENTION
[0029] An object of the present invention was to overcome the
deficiencies of the state of the art and to provide: i) a process
for producing glucose-lowering compounds in an efficient and
economic way, and ii) a nutriceutical composition that is edible
and useful as a dietary supplement.
[0030] The strain of Tremella used in the present invention was
obtained from the naturally growing fruit body collected in Israel
on deciduous wood.
[0031] A basidiospore print was obtained from a fresh fruit body
situated under a sterile Petri dish in a moist chamber with slowly
decreasing humidity. The basidiospores were transferred into
sterile water and spore suspension was spread on the surface of
malt agar (Pronadisa pH 6.5) in Petri dishes. The primary colonies
appearing from individual basidiospores were transferred
aseptically onto the slants of malt agar. Using the criteria
specified for fruit bodies in the standard authority "Morphological
and molecular studies in the genus Tremella", Chee-Jen Ch.,
published in 1998, Bibl. Mycologica, Band 174, and by comparison
with known species, collected fruit body was determined as Tremella
mesenterica Retz.: Fr.
[0032] In accordance with the Budapest Treaty on the international
recognition of the deposit of microorganisms for purposes of patent
procedure, one strain, showing the best yield of polysaccharide in
preliminary tests, was deposited in the Centraalbureau voor
Schimmelcultures, Netherlands and was given Acc. No CBS 101939.
1 Species Dep. No Dep. Date Tremella mesenterica Retz.: Fr. CBS
101939 June 14, 1999
[0033] Tremella mesenterica has a very complex life cycle. In
contrast to other Basidiomycetes mushrooms a single basidiospore
germinates on a nutrient medium broth by hypha and by yeast-like
budding cells (FIG. 1). Monobasidiosporous culture is haploid, i.e.
contains only one nucleus in each cell (FIG. 5). When two
compatible haploid cells, originating from different basidiospores,
come into contact, a plasmogamy and caryogamy occurs and dycariotic
mycelium develops (FIG. 4). The dycariotic mycelium cannot grow in
the form of budding cells, under any conditions of cultivation, so
a yeast-like type of growth is genetically determined by a haploid
status of mushroom culture. The haploid culture is more plastic,
because on poor media or under conditions of exhaustion yeast-like
cells can form haploid hypha (FIG. 2). One-cell fungi cultures,
like other microorganisms, are more acceptable for biotechnological
processes, than mycelial ones. This is especially important for
Basidiomycetes dyeariotic cultures, which grow in the form of
sterile mycelium, and a special procedure for preparing
Basidiomycetes inoculum is needed, that includes dycariotic
mycelium homogenization. The haploid yeast-like budding culture of
the present invention, is the most optimal form of growth not only
from biotechnological considerations, but as defined by its
physiological attribute of producing a larger amount of
polysaccharide than mycelium form (FIG. 3).
[0034] In order to produce polysaccharide the culture of T.
mesenterica CBS 101939 strain was carried out in aqueous media such
as those employed for good growth and biomass accumulation. Such
media contain sources of carbon, nitrogen and inorganic salts
assimilated by the growing culture. T. mesenterica is capable of
utilizing lignocellulose materials, so a wide range of
carbohydrates including pentoses, hexoses and polysaccharides are
good sources of carbon for their growth. Glucose, sucrose and
starches such as grain, corn meal and the like are the main
ingredients that can be used either alone or in combination as
sources of carbon. The amount of carbohydrate usually varies
between about 3% and 5% by weight of the medium to provide a high
yield of biomass.
[0035] The best sources of nitrogen, usually in organic form,
include yeast hydrolysates or extract, bacteriological peptone,
corn steep liquor and the like. The sources of nitrogen either
alone or in combination are used in the range of 0.5% to 4% by
weight depending on N content in the source, but about 1 to 1.5 g
of pure N per liter of culture medium.
[0036] Among inorganic salts, which can be incorporated in the
culture media, are salts possessing cations of potassium, ammonium
and magnesium. Sodium is not needed for growth at all. Useful
cations can be obtained in the form of phosphate, or sulfate and
chloride. The main microelements Fe, Mn, Zn and Cu are available
from any type of inorganic salts.
[0037] The fermentation is carried out at temperatures ranging from
20.degree. C. to 28.degree. C. The optimal temperature for growth
in a refrigerated orbital incubator is 27.degree. C., and
28.degree. C. is maximal; further increase of temperature is
detrimental and at 30.degree. C. Tremella mesenterica cells stopped
their growth. The fermentation in submerged culture includes one
stage of seed development. The liquid nutrient medium for the first
step of inoculum preparation may be any suitable combination of
carbon and nitrogen sources, preferably glucose or sucrose, and
peptone or yeast extract. The seed flask inoculated from surface
agar culture (tube or Petri dish) is filled with 100 ml of sterile
medium and the seed flask is shaken 4-6 days at 100-120 rpm until
growth is satisfactory. The seed flask is transferred to I liter of
sterile medium, which may itself serve as inoculum in proportion 1
to 10 volumes.
[0038] The invention is not to be limited in scope by the
embodiment disclosed in the examples which is intended as an
illustration of one aspect of the invention and it is contemplated
that the scope of the invention encompasses any number of species
and genera of the Basidiomycetes mushrooms grown in submerged
cultures in the form of yeast-like budding cells. It should include
those that may be adjusted or modified, within the range of this
invention, depending on its objective or usage.
EXAMPLE I
[0039] A tube with a 6-8 days old pure culture of Tremella
mesenterica CBS 101939 on malt agar pH 6.5 was used for inoculation
into 100 ml of medium A in a 250 ml Erlenmeyer flask. The culture
from the agar surface was washed off with sterile water. Medium A
has the following composition (g/l):
2 Medium A Glucose (Dextrose) 25 Peptone 2 Yeast extract 1
KH.sub.2PO.sub.4 0.5 MgSO.sub.4.7H.sub.2O 0.25 CaC1.sub.2.2H.sub.2O
0.1 Corn Steep liquor 2.5 ml Trace Element Mixture 10 ml 10% KOH
2.5 ml Trace Element Mixture (g/l): FeSO.sub.4.7H.sub.2O 0.5
MnSO.sub.4.H.sub.2O 0.1 ZnSO.sub.4.7H.sub.2O 0.05
CuSO.sub.4.5H.sub.2O 0.02 After 30 min. of sterilization at
120.degree. C., pH of the medium A is between 6.2 to 6.5.
[0040] The inoculated flasks were incubated in the refrigerated
orbital incubator at 200 rpm, 27.degree. C. After six days of
growth the whole content of the seed flasks was transferred to
inoculate 1000 ml of medium B in two-liter Erlenmeyer flasks and
the fermentation process was carried out at 27.degree. C. in the
refrigerated orbital incubator at 200 rpm. Medium B has the
following composition (g/l):
3 Medium B Sucrose 30 NaNO.sub.3 0.75 NaH.sub.2PO.sub.4 H.sub.2O
1.0 KC1 1.0 MgSO.sub.4.7H.sub.2O 0.3 Before sterilization pH of the
medium was adjusted to 6.5.
[0041] After 90 h of cultivation the culture broth was centrifuged
at 4.degree. C., 5000 rpm for 10 min. The biomass was determined in
grams of dry weight after desiccation at 105.degree. C. until
constant weight was attained. Polysaccharide prediction was
determined by the weight method from supernatant of centrifuged
culture broth using ethanol for precipitation, 2-3 volumes to
culture supernatant. After 24 h at 4.degree. C. the precipitated
crude biopolymer was separated by filtration and dried to constant
weight.
[0042] The resulting crop of biomass on this fermentation medium B
was 3.4 g/l and polysaccharide production after 90 h was 5.8
g/l.
EXAMPLE 2
[0043] A tube with a 6-8 days old pure culture of Tremella
mesenterica CBS 101939 on malt agar pH 6.5 was used for inoculation
of 100 ml of medium A in a 250 ml Erlenmeyer flask. The culture
from the agar surface was washed off with sterile water. Before
sterilization, pH of the medium was adjusted to 6.5. Medium A has
the following composition (g/l):
4 Medium A Sucrose 25 Peptone 2.0 Yeast extract 2.25
KH.sub.2PO.sub.4 1.0 MgSO.sub.4 .cndot. 7H.sub.2O 0.5
[0044] The inoculated flasks were incubated in the refrigerated
orbital incubator at 200 rpm, 27.degree. C. After six days of
growth the content of the seed flasks was transferred to inoculate
1000 ml of medium B in two-liter Erlenmeyer flasks and the
fermentation process was carried out at 27.degree. C. in the
refrigerated orbital incubator at 200 rpm. Before sterilization, pH
of the medium was adjusted to 6.5. Medium B has the following
composition (g/l):
5 Medium B Sucrose 40 Peptone 1.0 Yeast extract 1.1 NaHPO.sub.4
.cndot. 7H.sub.2O 1.0 NaH.sub.2PO.sub.4 .cndot. 7H.sub.2O 0.5
KH.sub.2PO.sub.4 1.0 Mg acetate 1.0 KC1 1.0
[0045] After 5 days of cultivation 2 or 3 volumes of ethyl alcohol
were added to culture broth. After 24 h at 4.degree. C. the
precipitated crude product was separated by filtration and dried to
constant weight. The resulting product which consisted of both
polysaccharide and one-cell biomass was 18.4 g/l. When measured
independently according to the method described in example 1,
polysaccharide yield was 13.9 g/l and biomass--7.circle-solid.7 g/l
(some amount of polysaccharide is firmly attached with cells).
[0046] Amino acids analysis of obtained biomass showed that
essential amino acids composed 31% of total amino acids content
(Table 1). Thus, the nutriceutical composition of the present
invention is a usefull source to supply high quality protein rich
in essential amino acids.
[0047] Table 1. The amino acid composition of the Tremella
mesenterica C13S 101939 biomass.
6TABLE 1 Free Protein .mu.g/100 mg %, dry .mu.g/100 mg %, dry Amino
acids dry weight weight dry weight weight Aspartic acid 42.73 0.043
2252.27 2.652 Threonine 138.80 0.139 1700.96 1.700 Serine 204.87
0.205 2281.97 2.281 Glutamatic acid 557.08 0.557 2020.39 2.020
Proline -- 1379.40 1.379 Glycine 162.11 0.162 1929.04 1.929 Alanine
604.10 0.604 2402.32 2.402 Cysteine -- -- 294.71 0.294 Valine
102.40 0.102 802.71 0.802 Methionine 210.40 0.210 -- -- Isoleucine
68.46 0.068 230.23 0.230 Leucine 153.80 0.0153 415.59 0.415
Tyrosine 83.58 0.083 -- -- Phenylalanine 71.35 0.071 -- --
Histidine 87.14 0.087 290.92 0.290 Ornitine 41.80 0.042 187.17
0.187 Lysine 47.87 0.047 1876.85 1.576 Arginine 88.04 0.088 528.82
0.528
[0048] Among vitamins of B group, determined by microbiological
method, based on the estimation of growth characteristics of
sensitive auxotroph microorganisms, T. mesenterica biomass is
especially rich in niacin (Table 2). Thus, the nutraceutical
composition of the present invention may be used as a vitamin
supplement, in particular to supply a natural source of niacin.
[0049] Table 2 describes the group B vitamin content in Tremella
mesenterica CBS 101939 biomass.
7 TABLE 2 Vitamins Content, .mu.g/g dry weight Thiamine, B1 1.58
+/- 0.05 Niacin, B5 500.0 +/- 24 Piridoxin, B6 1.0 +/- 0.01
Biotine, B7 0.1 0.003
Nutriceutical Formulations and Method of Use
[0050] Nutriceutical compositions containing glucose-lowering
compounds must be stable under the conditions of manufacture and
storage and may be protected from contamination by microorganisms,
such as fungi and bacteria, through the use of bacteriostatic
agents, antioxidants such as vitamin E and ethoxyqiun, which are
listed as generally safe for use by the Food and Drug
Administration.
[0051] The formulation is taken as a single daily dose or divided
daily doses, most preferably three doses given before, during or
after meals. The formulation may be used in food or for garnishing
and packaged accordingly. Patients can be maintained on
glucose-lowering compounds indefinitely to regulate the blood
glucose levels. Conditions to be considered in selecting dosage
level, frequency, and duration, primarily include the severity of
the patient's disorder, the patient's blood glucose level, adverse
side effects and the patient's need for preventive intervention as
well as the therapeutic efficacy. It is to be understood that for
any particular subject, specific dosage regimens should be adjusted
over time according to the individual patient need and the
professional judgment of the person administering or supervising
the administration of the nutriceutical compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
composition. Other concentration ranges and dosage durations can be
determined by routine experimentation.
[0052] Diabetes mellitus is a disease of metabolic dysregulation,
notably of glucose metabolism, and long-term vascular and
neurologic complications. Diabetes has several clinical forms, the
two major forms being insulin-dependent diabetes mellitus I (IDDM)
and the non-insulin-dependent diabetes mellitus II (NIDDM). IDDM is
rare, affecting one in 250 persons in the United States, where
approximately 10,000 to 15,000 new cases are reported each year.
Data suggest that the incidence of IDDM is increasing in Europe,
where the highest prevalence is found in northern Europe, for
example, more than one in every 150 Finns develop IDDM by 15 years
of age. LaPorte, R. et al., in Diabetes in America, 2.sup.nd ed. Ed
M. Harris, National institutes of Health, Bethesda, Md. NIH
Publication No 95-1498, 1995.
[0053] NIDDM is common, with an overall prevalence of 6.6 percent
in the United States. NIDDM has become one of the most frequent
chronic diseases in most industrialized nations and the projected
prevalence for the next decade is 10 percent. 600,000 new cases are
reported each year and one half of the NIDDM population are unaware
of their disorder. The increase in the prevalence of NIDDM in the
United States is commonly attributed to an aging population that is
also increasingly obese and sedentary. The prevalence of NIDDM
among persons older than 65 years exceeds 18 percent, and compared
with normal-weight individuals, obese people with a body mass index
greater than 30, are at 10 to 20 times greater risk for NIDDM.
Although genetic and immunologic markers for IDDM have been
identified, they are not enough nor sensitive enough to be used to
define IDDM or distinguish IDDM and NIDDM. Harris, M. I., et al.,
Diabetes 36: 523 (1987); Bennett, P. H., et al., in International
Textbook of Diabetes, ed Alberti KGMM, et al., John Wiley &
Sons Ltd UK 1992, p148.
[0054] The natural hypoglycemic mushroom nutriceuticals are
generally ingested orally. However, if individual agents are
further extracted from the biomass, then the purified hypoglycemic
agents may be administered intravenously, intraperitoneally,
subcutaneously, intramuscularly, intrathecally, orally, rectally,
topically, or by aerosol.
[0055] Formulations suitable for oral administration include liquid
solutions of the active compound dissolved in diluents such as
saline, water or PEG 400; capsules or tablets, each containing a
predetermined amount of the active agent as solid, granules or
gelatin; suspensions in an approximate medium; and emulsions.
[0056] Formulations suitable for parenteral administration include
aqueous and non-aqueous isotonic sterile solutions, which contain
buffers, antioxidants and preservatives. The formulations maybe in
unit dose or multi-dose sealed contains.
[0057] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the antidiabetic and other favorable metabolic
effects.
[0058] Alternatively, one may administer the compound in a local,
rather than oral manner, for example, via injection of the compound
directly into the target site, often in a depot or sustained
release formulation.
[0059] A variety of delivery systems for the pharmacological
compounds may be employed, including, but not limited to, liposomes
and emulsions. The pharmaceutical compositions also may comprise
suitable solid or gel phase carriers or excipients. Example of such
carriers or excipients include, but are not limited to, calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0060] In cases of local administration or selective uptake, the
effective local concentration of the nutraceutical compound may be
related to plasma concentration.
[0061] The invention accordingly comprises the several steps and
the relation of one or more of such steps with respect to each of
the others, and the product embodying properties, which are adapted
to effect such steps and methods, all as exemplified in the
following detailed disclosure, and the scope of the invention will
be indicated in the claims.
[0062] A large number of cellular components and secondary
metabolites derived from mushrooms, have been shown to affect the
immune system and used in a variety of disease states. Mushrooms
have been used as adaptogens and immunostimulants. An adaptogen as
defined herein, is any substance that meets specific criteria for
the category of plant derived biological response modifier, that
may modify the host's biological response by a stimulation of the
immune system. The principal component of these biological response
modifiers is (1.fwdarw.3)-.beta.-D-glucans. .beta.-D-glucan, a
polysaccharide isolated from mushrooms binds to lymphocyte surfaces
or serum specific proteins, which activate macrophage, T-helper,
natural killer cells and other effector cells. These increase the
production of antibodies as well as interleukins (IL-1, IL-2) and
interferon (IFN-r) which are released upon activation of effector
cells. The carcinostatic effect of antitumor polysaccharides thus
results from the activation of the hosfs immune system.
[0063] In addition to water-soluble .beta.-D-glucans, mushrooms
also contain .beta.-D-glucans with heterosaccharide chains of
xylose, mannose, galactose, and uronic acid, and
.beta.-D-glucan-protein complexes. The higher Basidiomycetes edible
composition obtained grown in submerged culture in the present
invention comprised of cellular and secondary metabolites,
polysaccharides and specifically 1,3-linked glucuronoxylomannan,
and exhibit immunomodulatory and carcinostatic properties.
[0064] The higher Basidiomycetes mushrooms contain dietary fibers
belonging to glucans, chitin, and heteropolysaccharides including,
but not limited to, pectinous substances, hemi-celluloses or
polyuronides. The .beta.-glucans and chitinous substances are
present primarily in the dietary fiber of mushrooms. Their
carcinostatic activity has been attributed to their physicochemical
interactions with hazardous materials such as carcinogenic
substances, thereby preventing their absorption into the intestine
and hastening their excretion. The higher Basidiomycetes edible
compositions of the present invention comprise of dietary fibers
belonging to .beta.-glucans, chitin and heteropolysaccharides,
having carcinostatic activity.
[0065] The effect of T. mesenterica submerged mycelium can be
studied in the streptozotocin-induced type I diabetes model in
Sprague Dawley male rats as described by G. S. Mahdi et al. Ann.
Nutr. Metab 35: 6.5 (1991). Plasma glucose concentrations are
measured seven days after a single does of streptozotocin in rats
maintained on a casein based diet or a diet in which T. mesenterica
replaces the protein supplied diet in which by casein in the
control group. The rats maintained of the T. mesenterica diet
generally regulate the glucose levels better than the casein fed
group suggesting a hypoglycemic role for the nutriceuticals present
in T. mesenterica.
[0066] The present invention is not to be limited in scope by the
embodiments disclosed in the examples which are intended as an
illustration of one aspect of the invention and any methods which
are functionally equivalent are within the scope of the invention.
Indeed, various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in
the art from the foregoing description. Such modifications are
intended to fall within the scope of the appended claims.
[0067] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
[0068] It will thus be seen that the objects set forth above, among
those made apparent from the preceding description, are efficiently
attained and, since certain changes may be made in carrying out the
above method and in the compositions set forth without departing
from the spirit and scope of the invention, it is intended that all
matter contained in the above description and shown in the
accompanying figures shall be interpreted as illustrative and not
in a limiting sense.
[0069] It is also to be understood that the following claims are
intended to cover all of the generic and specific features of the
invention herein described and all statements of the scope of the
invention which, as a matter of language, might be said to fall
there between.
* * * * *