U.S. patent application number 10/084517 was filed with the patent office on 2002-09-26 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 | 20020137155 10/084517 |
Document ID | / |
Family ID | 23661252 |
Filed Date | 2002-09-26 |
United States Patent
Application |
20020137155 |
Kind Code |
A1 |
Wasser, Solomon P. ; et
al. |
September 26, 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, and a process for growing them in
submerged culture. Specifically, the new strains of species of the
genus Pleurotus offer superior yields of mushroom biomass and
concentrations of biologically active compounds, for example,
cholesterol-lowering compounds, lectins, proteins, essential amino
acids, vitamins or polysaccharides. The process includes use of
defined media and a simple one-step procedure of separating the
lovastatin-containing nutriceuticals from culture broth.
Inventors: |
Wasser, Solomon P.; (Haifa,
IL) ; Reshetnikov, Sergey V.; (Kiev, UA) |
Correspondence
Address: |
Rashida A. Karmali, Esq.
Suite 2525
230 Park Avenue
New York
NY
10169
US
|
Family ID: |
23661252 |
Appl. No.: |
10/084517 |
Filed: |
February 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10084517 |
Feb 26, 2002 |
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09419205 |
Oct 15, 1999 |
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6372462 |
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Current U.S.
Class: |
435/171 ;
435/255.21 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 1/14 20130101; A61K 36/07 20130101; A61P 25/32 20180101; A61P
43/00 20180101; A61P 31/00 20180101; A61P 9/10 20180101; C12P 1/02
20130101 |
Class at
Publication: |
435/171 ;
435/255.21 |
International
Class: |
C12P 001/02; C12N
001/14; C12N 001/16; C12N 001/18 |
Claims
What is claimed is:
1. A process for producing a biomass of edible Basidiomycetes
mushrooms, said process comprising: cultivating a fungi of the
Basidiomycetes in a submerged culture on nutrient media, isolating
the resulting biomass of edible Basidiomycetes from the culture
broth, and drying the biomass of edible Basidiomycetes at a
temperature range of 40.degree. C. to 45.degree. C.
2. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the fungi of the
Basidiomycetes is a species of the genus Pleurotus comprising
Pleurotus eryngii var. ferulae or Pleurotus ostreatus.
3. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the fungi of the
Basidiomycetes produces lovastatin.
4. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the nutrient media is of
the following composition (g/l):
17 Medium A Glucose (Dextrose)- 25 Peptone- 2 Yeast extract- 1
KH.sub.2PO4-- 0.5 MgSO.sub.4 .7H.sub.2O-- 0.25 CaCl.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: FeSO.sub.4.7H2O-- 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
5. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the nutrient media is of
the following composition (g/l):
18 Medium B Glucose (Dextrose)- 50 Peptone- 0.5 Yeast extract- 1
(NH.sub.4).sub.2SO.sub.4-- 2 K.sub.2HPO.sub.4.3H.sub.2O-- 2
K.sub.2HPO.sub.4 2.5 Trace Element Mixture - 10 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
6. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the fungus is Pleurotus
eryngii var. ferulae CBS 101938.
7. The process for producing a biomass of edible Basidiomycetes
mushrooms according to claim 1, wherein the fungus is Pleurotus
ostreatus CBS 101937.
8. A composition for reducing blood levels of cholesterol 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 cholesterol-lowering compound, a
lectin, polysaccharide, fiber, protein, essential amino acids,
vitamins, fatty acids, or minerals.
10. The composition according to claim 8, wherein the dried
Basidiomycetes biomass is produced from Pleurotus eryngii var.
ferulae CB 101938.
11. The composition according to claim 8, wherein the dried
Basidiomycetes biomass is produced from Pleurotus ostreatus CB
101937.
12. The composition according to claim 8 wherein the dried
Basidiomycetes biomass is in an effective amount to reduce blood
levels of cholesterol, in combination with a pharmaceutical carrier
acceptable for oral administration.
13. The composition according to claim 8 wherein the dried
Basidiomycetes biomass is in an effective amount to inhibit
atherosclerosis.
14. The composition according to claim 8 wherein the dried
Basidiomycetes biomass is in an effective amount to inhibit a
variety of diseases including cardiovascular disease, cancer,
hepatotoxicity due to chronic alcohol intake, or cell proliferative
diseases.
15. A method for treating an individual for hypercholesterolemia,
said method comprising: administering to said individual an amount
effective to reduce the level of serum cholesterol, of a composite
containing an edible biomass of Basidiomycetes, wherein the biomass
includes a cholesterol-lowering compound.
16. The method according to claim 15, wherein said amount is
effective to reduce the level of blood cholesterol 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
Pleurotus.
18. The method according to claim 17 wherein the species of the
fungi is selected from the group consisting of Pleurotus ostreatus,
Pleurotus eryngii var. ferulae, Hypsizygus marmoreus, Lepista nuda,
Pleurotus cystidiosus, P. dijamor, P. pulmonarius, P. salignus,
Grifola frondosa, and Hericium erinaeus
19. The method according to claim 14 wherein the Basidiomycetes
biomass is produced from Pleurotus eryngii var. ferulae CB
101938.
20. The method according to claim 14 wherein the Basidiomycetes
biomass is produced from Pleurotus ostreatus CB 101937.
21. A nutrient medium suitable for growth of the Pleurotus biomass,
the medium comprising of the following ingredients (g/l):
19 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
CaCl.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
22. A nutrient medium suitable for growth of the Pleurotus biomass,
the medium comprising of the following ingredients (g/l):
20 Medium B Glucose (Dextrose)- 50 Peptone- 0.5 Yeast extract- 1
(NH.sub.4).sub.2SO.sub.4-- 2 K.sub.2HPO.sub.4 3H.sub.2O-- 2
K.sub.2HPO.sub.4 2.5 Trace Element Mixture - 10 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
Description
FIELD OF THE INVENTION
[0001] 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.
[0002] 1. Background of the Invention
[0003] 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 U.S. dollars. The
most popular species of cultivated edible mushrooms include
Agaricus bisporus (J. Ige) 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 (Bull.: Fr.) Cooke, Agrocybe aegerita
(Brig.) Sing.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 2. Summary of the Invention
[0009] 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.
[0010] 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.
[0011] 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.degree. C.
[0012] 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.
[0013] 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.
[0014] 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
[0015] FIG. 1 shows a top view of the cultivated mushroom Pleurotus
ostreatus CBS 101937 fruiting bodies.
[0016] FIG. 2 shows a view of Pleurotus ostreatus CBS 101937
fruiting in the petri dish with malt agar medium.
[0017] FIG. 3 shows a surface of submerged culture pellet of
Pleurotus ostreatus CBS 101937. Scanning electron microscopy (SEM),
.times.1500.
[0018] FIG. 4 shows a part of a surface of submerged culture pellet
of Pleurotus ostreatus CBS 101937. SEM, .times.3000.
[0019] FIG. 5 shows a single clamp connection on a hypha of
Pleurotus ostreatus CBS 101937, typical for Basidiomycetes. SEM,
.times.4000.
[0020] FIG. 6 shows a clamp connection on a Pleurotus ostreatus CBS
101937 hypha proliferating by new hyphae with clamps. SEM,
.times.4800.
[0021] FIG. 7 shows a conidium-like structure on a hypha of
Pleurotus ostreatus CBS 101937, a typical for the genus Pleurotus.
SEM, .times.6000.
[0022] FIG. 8 shows a top view of the Pleurotus eryngii var.
ferulae fruiting bodies.
[0023] FIG. 9 shows a view of Pleurotus eryngii var. ferulae CBS
101938 submerged culture biomass in the form of pellets.
Magnification .times.10.
[0024] FIG. 10 shows a view of Pleurotus eryngii var. ferulae BS
101938 submerged culture biomass in the form of pellets.
Magnification .times.40.
[0025] FIG. 11 shows a single clamp connection on a hypha of
Pleurotus eryngii var. ferulae CBS 101938, typical for
Basidiomycetes. SEM, .times.7800.
[0026] FIG. 12 shows a conidium-like structure on a hypha of
Pleurotus eryngii var. ferulae BS 101938, typical for the genus
Pleurotus. SEM, .times.6000.
DETAILED DESCRIPTION OF THE INVENTION
[0027] 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 cholesterol-lowering compounds in an efficient and
economic way, and ii) in a nutriceutical composition that is edible
and useful as a dietary supplement.
[0028] A process according to the invention comprises the submerged
cultivation of edible higher Basidiomycetes mushrooms, producers of
lovastatin, belonging to the genus Pleurotus on a nutrient media
including nitrogen, mineral salts and a source of carbon - mono- or
polysaccharides including glucose; one-step isolation procedure
from a fermentation broth obtained from the submerged culture and a
method of preparing a hypocholesterolemic, hypolipidemic, protein,
or mineral functional food.
[0029] The field of this invention relates to edible higher
Basidiomycetes mushrooms from the genus Pleurotus and a process for
culturing them for the production of a biomass with a high
concentration of lovastatin, as a dietary supplement with
hypocholesterolemic activity, which is suitable for human
consumption.
[0030] Hypercholesterolemia is one of the main risk factors of
cardiovascular disease, causing high blood pressure and
atherosclerosis. So many efforts have been made to find methods or
substances which would reduce cholesterol uptake from the digestive
tract, or destroy cholesterol by enzyme reactions. The most
practical way to protect the human body from high cholesterol
levels is to inhibit the cholesterol-synthesizing ability of an
organism.
[0031] One of the best known hypocholesterolemic pharmacological
agents of natural origin, which is the competitive inhibitor of
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG CoA
reductase)--the key enzyme in cholesterol metabolism--is lovastatin
(mevinolin, MSD 803), described by Monaghan et al. (U.S. Pat. No.
4,231,938).
[0032] Mevinolin contains a naphthalene ring system, a 0-hydroxy
lactone and a methylbutyric acid. It belongs to the series of
similar compounds such as ML-236, compactin, monacolin K. These
compounds are metabolites of mold fungi from the genera
Aspergillus, Penicillium, Eupenicillium, Paecilomyces, Trichoderma,
Phoma, Hypomyces, Pythium, Doratomyces and Gymnoascus. Later, the
presence of lovastatin was detected in a submerged culture of three
higher Basidiomycetes species: Pleurotus ostreatus, P. saca and P.
sapidus. The discovery of lovastatin in fruit bodies of P.
ostreatus helps to interpret the hypocholesterolemic activity of
Pleurotus fruit body preparations previously described in a series
of experiments. It was found that addition of 2 to 4% of P.
ostreatus fruit bodies to the hyperlipidemic diet efficiently
prevented accumulation of cholesterol and triacylglycerols in both
sera and liver of rats with experimentally or genetically induced
hyperlipidemia. This effect was attributed to the fiber pulp
complex of the P. ostreatus mushroom, which limits the absorption
of cholesterol in the gastrointestinal tract. Later it was found
that a 30% ethanol extract of P. ostreatus is equivalent in
activity to a whole oyster mushroom and suggested that this effect
may be related to the lovastatin content in the fruit bodies. Bobek
et al., 1996, Nhrung: 222-224.
[0033] When fruiting bodies of the edible and commercially
available mushroom Pleurotus ostreatus were investigated, it was
found that they contained significant amounts of lovastatin, which
varies in quantity when obtained from submerged or surface
fermented different Pleurotus strains. Moreover, the lovastatin
content in fruit bodies of P. ostreatus is not constant even in the
same strain and depends mainly on substrate variability, on fruit
body size and age, and is concentrated presumably in the lamellae
and basidiospores but not in stipe or cap tissue.
[0034] The best producers of lovastatin are different strains of
Aspergillus terreus, a common contaminant mold on food, possessing
such toxic metabolites as terrein, patulin, citrinin, and
citreoviridin. This leads to the necessary purification of
lovastatin from other Aspergillus secondary metabolites. As
described in U.S. Pat. No. 4,231,938, the purification process
includes extraction of lovastatin from the culture broth with ethyl
acetate or XAD-2 resin and the subsequent procedure of
concentration, washing, reconcentration and crystallization. Some
other proposed processes for isolation of lovastatin differ by the
use of a more selective resin (EP 877089 A1) or of less toxic
solvent butyl acetate in the first step of extraction (U.S. Pat.
No. 5,712,130), but all these modified processes deal with the
extraction procedure of the whole culture broth including both
culture medium and cell biomass.
[0035] A process for extraction lovastatin from the submerged
culture broth of Pleurotus ostreatus, P. sapidus and P. saca was
proposed by adding an equal volume of methanol. The final product
is extracted by shaking the culture broth with methanol, separating
the mycelium by filtering, and obtaining the lovastatin in the
lactone or acid form depending from the previous adjustment pH of
the culture broth to 3,0 or 7,7 (DE Pat. 440259).
[0036] The general efficiency of processes for lovastatin
production is determined by the productivity of fungi strains and
the number of steps in the extraction procedure. Aspergillus
strains are more productive than those of Pleurotus, but they
produce a wide range of toxic substances, beside lovastatin, and
involve complex extraction methods for lovastatin purification.
[0037] Although such methods are certainly appraised as rational
and practical, they still involve extraction processes of large
volumes of culture broth by solvents of different toxicity.
[0038] The strains of Pleurotus used in the present invention were
obtained from the growing fruit bodies collected in Israel by a
tissue-culture method. Pieces of cap tissue were put onto malt
extract agar, pH-6.3 and incubated at 27.degree. C. The
morphological characteristics of obtained cultures were typical of
Basidiomycetes by the presence of clamp connections at each
septa.
[0039] Using the criteria specified for fruit bodies in the
standard authority "The genus Pleurotus (Fr.) Kummer (2)", Oswald
Hilber, published in 1997, and by comparison with known species,
they were determined as Pleurotus ostreatus (Jacq. Fr.) Kumm and
Pleurotus eryngii (DC.: Fr.) Qul. var. ferulae Lanzi respectively.
According to the Budapest Treaty on the international recognition
of the deposit of microorganisms for purposes of patent procedure
two strains were deposited in the Centraalbureau voor
Schimmelcultures, Netherlands:
1 Species Dep. No Dep. Date Pleurotus ostreatus (Jacq. Fr.) CBS
101937 June 14, 1999 Kumm. Pleurotus eryngii (DC.: Fr.) Qul. CBS
101938 June 14, 1999 Var. ferulae Lanzi
[0040] All species of the genus Pleurotus are edible, and some of
them, especially P. ostreatus, P. pulmonarius (.about.P.
sajor-caju) and related species are commercially cultivated.
[0041] The culture of these strains to produce lovastatin is
carried out in aqueous media such as those employed for good
mycelium growth and biomass accumulation. Such media contain
sources of carbon, nitrogen and inorganic salts assimilated by the
growing culture.
[0042] All species of genus Pleurotus are capable to utilize
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.
[0043] The best sources of nitrogen possessing it 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 I to 1.5 g
of pure N per liter of culture medium.
[0044] 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.
[0045] 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. Pleurotus mycelium of the
foregoing species stopped its growth.
[0046] Lovastatin is produced by both surface and submerged
culture. However, in surface culture at stationary conditions of
growth on a liquid medium the lovastatin content did not reach a
maximum value that is expected for this type of a medium. Hence,
the surface method of cultivation can be used only for screening
for lovastatin producing strains.
[0047] The fermentation in submerged culture includes one or more
stages of seed development under controlled conditions. 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 inoculum
flask inoculated from surface agar culture (tube or Petri dish) was
kept at stationary conditions of growth in a constant temperature
chamber at about 27.degree. C. until the mycelium mat covered all
the surface of the medium. Then the mycelium mat was transferred to
the sterile vessel of a waring commercial laboratory blender with
water and homogenized at a low speed setting. Ten milliliters of
mycelium homogenate was transferred to 100 ml of sterile medium and
the seed flask was shaken 6-7 days at 100-120 rpm until growth is
satisfactory in the form of pulp. The seed flask content was
transferred to 1 l of sterile medium for the fermentation process,
so the two-step seed development included the scaling process of
transferring inoculum to pure medium in the proportion 1:10.
[0048] The following examples are provided to illustrate and in no
way limit the scope of the process of the present invention and
should not be construed as being limiting.
EXAMPLE 1
A. Fermentation
[0049] A tube with a 8-10 days old pure culture of Pleurotus
eryngii var. ferulae CBS 101938 on malt agar pH 6.2 was used for
inoculation into 100 ml of medium A in a 250 ml Erlemneyer flask
(inoculum flask). 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
CaCl.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.
[0050] The inoculated flasks were incubated at stationary
conditions of growth in a temperature chamber at 27.degree. C.
After 6-8 days of growth a mycelium mat which covered all the
surface of the culture medium was placed into the sterile vessel of
a waring commercial laboratory blender, 100 ml of sterile water
added and the mycelium homogenized at a low speed setting during 30
seconds.
[0051] New 250 ml Erlenmeyer flasks with 100 ml of sterile medium A
were each inoculated with 10 ml of mycelium homogenate (seed
flasks) and incubated in the refrigerated orbital incubator at 120
rpm, 27.degree. C. for 6 days.
[0052] The seed flask content (100 ml of pulp biomass) was used to
inoculate 1000 ml of medium B in two-liter Erlemneyer flasks and
the fermentation process was carried out at 27.degree. C. on a 120
rpm shaker. Medium B has the following composition (g/l):
3 Medium B Glucose (Dextrose)- Peptone- 0.5 Yeast extract- 1
(NH.sub.4).sub.2SO.sub.4- 2 K.sub.2HPO.sub.4.3H.sub.2O- 2
K.sub.2HPO.sub.4 2.5 Trace Element Mixture- 10 ml Trace Element
Mixture (g/l): FeSO.sub.4.7H.sub.2O- 0.5 g 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 medium B is between
6.2 to 6.5.
B. Testing of Lovastatin
[0053] The incubated broth was filtered through a filter cloth to
remove the mycelium and washed twice with distilled water. Fresh
mycelium (10 g) was disintegrated in 40 ml of water on a Virtis
Gardiner laboratory homogenizer and 30 ml of homogenate taken for
preparation of samples, while 10 ml was used to estimate the dry
weight of the biomass.
[0054] Dry submerged mycelium samples were ground into a powder of
which 0.5 g was presoaked in 30 ml of distilled water using a
magnetic stirrer for 30 min. The sample pH was adjusted to 7.7
using 4% NaOH, then 5 ml of methanol and 10 ml of ethyl acetate for
UPLC (Carlo Erba) were added. The extraction process in the
two-phase solvent system H20:MeOH:ethyl acetate--3:0.5:1 was
performed on a magnetic stirrer for 30 min. This was followed by
centrifugation at 5000 rpm and 4.degree. C. for 10 min and a probe
from the upper ethyl acetate layer was taken for HPLC analysis
using a Meta Chem nylon syringe filter of 0.45u pore size (Part No.
4104).
[0055] The same extraction process was done with culture broth
filtrate after separation from the mycelium. The samples were
analyzed on an HPLC Meta Chem Inertsil 5.mu. ODS-2 250.times.4.6
min column using 0.05 M sodium phosphate pH 3.0:acetonitrile
(45:55, v/v) (Monaghan et al., 1980) at a flow rate of I ml/min. at
25.degree. C. as the eluent. A 655A Hitachi UV detector set at 237
nm was used for lovastatin detection. Standard samples were
prepared by dissolving Mevacor tablets (Merk) in water:MeOH:ethyl
acetate (3:05:1, v/v) at pH 7.7.
[0056] Lovastatin content in the submerged myceliurn was determined
at different stages of culture growth (Table 1).
4TABLE 1 Time of growth, Biomass, Lovastatin, mg/g, days g/l dry
weight dry biomass 6 3.95 0.46 8 7.76 0.41 10 8.56 0.52 12 12.20
0.41
[0057] It was found that lovastatin content in P. eryngii var.
ferulae CBS 101938 submerged mycelium is stable during all phases
of growth. No lovastatin was detected in the culture broth filtrate
samples after separation from the mycelium. biomass.
EXAMPLE 2
A. Fermentation
[0058] A tube with a 8-10 days old pure culture of Pleurotus
eryngii var. ferulae CBS 101938 on malt agar pH 6.2 was used for
inoculating of 100 ml of medium A in a 250 ml Erlenmeyer flask
(inoculum flask). Medium A has the following composition (g/l):
5 Medium A Glucose (Dextrose)- Peptone- 0.5 Yeast extract- 1
KH.sub.2HPO.sub.4 0.5 MgSO.sub.4.7H.sub.2O 0.25
CaCl.sub.2.2H.sub.2O 0.1 Corn Steep liquor 10 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.
[0059] The inoculated flasks were incubated at stationary
conditions of growth in a temperature chamber at 27C. After 6-8
days of growth a mycelium mat which covered all the surface of the
culture medium was placed into a sterile vessel of waring
commercial laboratory blender, 100 ml of sterile water was added
and the mycelium homogenized at low speed setting during 30
seconds.
[0060] New 250 ml Erlenmeyer flasks with 100 ml of sterile medium A
were each inoculated with 10 ml of mycelium homogenate (seed
flasks) and incubated in the refrigerated orbital incubator at 120
rpm, 27.degree. C. during 6 days.
[0061] The seed flask content (100 ml of pulp biomass) was used to
inoculate 1000 ml of medium A in two-liter Erlemneyer flasks and
the fermentation process was carried out at 27.degree. C. on a 120
rpm shaker.
B. Testing of Lovastatin
[0062] Lovastatin content in the submerged mycelium was determined
according to the method described in Example 1.
[0063] Lovastatin content in the submerged mycelium was determined
at different stages of culture growth (Table 2).
6TABLE 2 Time of growth, Biomass, Lovastatin, mg/g, days g/l dry
weight dry biomass 6 10.11 0.1 8 11.23 0.24
[0064] Lovastatin synthesis by P. eryngii var. ferulae CBS 101938
on the fermentation medium A is lower than the same strain on the
fermentation medium B (Example 1). However, it is a characteristic
feature of this strain on a culture medium, which can provide a
good yield of biomass, about 10 g/l dry weight.
[0065] No lovastatin was detected in the culture broth filtrate
samples after separation from the mycelium biomass.
EXAMPLE 3
A. Fermentation
[0066] A tube with a 8-10 days old pure culture of Pleurotus
ostreatus CBS 101937 on malt agar pH 6.2 was used for inoculating
into 100 ml of medium A in a 250 ml Erlenmeyer flask (inoculum
flask). Medium A has the following composition (g/l):
7 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
CaCl.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.4.7H.sub.2O 0.5
ZnSO.sub.4.7H.sub.2O 0.05 CuSO.sub.4.7H.sub.2O 0.02 After 30 min.
sterilization at 120.degree. C., pH of the medium A is between 6.2
to 6.5.
[0067] The inoculated flasks were incubated at stationary
conditions of growth in a temperature chamber at 27.degree. C.
After 6-8 days of growth a mycelium mat which covered all the
surface of the culture medium was placed into the sterile vessel of
a waring commercial laboratory blender, 100 ml of sterile water was
added and the mycelium homogenized at a low speed setting during 30
seconds.
[0068] New 250 ml Erlenmeyer flasks with 100 ml of sterile medium A
were each inoculated with 10 ml of mycelium homogenate (seed
flasks) and incubated in the refrigerated orbital incubator at 120
rpm, 27.degree. C. during 6 days.
[0069] The seed flask content (100 ml of pulp biomass) was used to
inoculate 1000 ml of medium B in two-liter Erlenmeyer flasks and
the fermentation process was carried out at 27.degree. C. on a 120
rpm shaker. Medium B has the following composition (g/l):
8 Medium B Glucose (Dextrose)- 50 Peptone- 0.5 Yeast extract- 1
(NH.sub.4)SO.sub.4- 2 K.sub.2HP.sub.4.3H.sub.2O- 2 KH.sub.2P.sub.4-
2.5 Trace Element Mixture- 10 ml Trace Element Mixture (g/l):
FeSO.sub.4.7H.sub.2O 0.5 MnSO.sub.4.7H.sub.2O 0.1
ZnSO.sub.4.7H.sub.2O 0.05 CuSO.sub.4.7H.sub.2O 0.02 After 30 min.
of sterilization at 120.degree. C., pH of the medium B is between
6.2 to 6.5.
B. Testing of Lovastatin
[0070] Lovastatin content in the submerged myceliurn was determined
according to the method described in Example 1.
[0071] Lovastatin content in the submerged mycelium was determined
at different stages of culture growth (Table 3).
9TABLE 3 Time of growth, Biomass, Lovastatin, mg/g, days g/l dry
weight dry biomass 6 3.65 0.44 8 9.30 0.23 10 9.30 0.20 12 5.50
0.15
[0072] It was found that the maximum accumulation of lovastatin in
Pleurotus ostreatus CBS 101937 mycelium is related to the
exponential stage of growth, with accumulation being somewhat less
at the beginning and during the stationary stage.
[0073] No lovastatin was detected in the culture broth filtrate
samples after separation from the myceliurn biomass.
EXAMPLE 4
[0074] Lovastatin is known to be stable in pure solutions, for
example its concentrate in toluene complete lactonisation at
106.degree. C. for 2 hours (U.S. Pat. No. 5,712,130). However, the
stability of lovastatin found in the fresh biomass of mushroom, and
the effect of the drying procedure used were not known. By carrying
out a series of tests under different conditions, it was found that
the stability of lovastatin in the biomass was temperature
sensitive. Therefore, low temperatures are recommended in general
for drying processes of composite biological material in order not
to destroy many biologically active compounds.
[0075] Samples of Pleurotus eryngii var. ferulae CBS 101938
submerged mycelium obtained according to the process described in
example 1 were pretreated before the extraction procedure for
lovastatin determination at different temperatures (Table 4).
10 TABLE 4 Lovastatin, mg/g, dry Sample Treatment biomass Pleurotus
eryngli var. 40.degree. C. 0.46 feruale CBS 101938 45.degree. C.
0.45 6 days on medium B, 90.degree. C. 0.18 example 1
[0076] Therefore, the temperature for the drying process of
submerged Pleurotus mycelium should not be higher than 45.degree.
C. Low extraction of lovastatin after high-temperature treatment
may be due not only to destruction of its molecule but to the
attachment to denatured biopolymers. This process can lead to a
weak absorption of lovastatin in the digestive tract from the
denatured mycelium biomass.
Nutriceutical Formulations and Biochemical Composition
[0077] Nutriceutical compositions containing cholesterol-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 ethoxyquin, which are
listed as generally safe for use by the Food and Drug
Administration.
[0078] The cholesterol-lowering compound can be used to reduce the
blood levels of cholesterol and for the treatment of
hyperlipidemias including hypercholesterolemia and associated
disease states such atherosclerosis, cardiovascular disease and
pancreatitis. The cholesterol-lowering compound can also be used in
normal subjects as a preventative measure to prevent the occurrence
of these disorders. It is desirable that human serum cholesterol
levels be maintained below 180 mg/dl, with values of 240 mg being
considered clinically high and values of 160 mg being considered
too low.
[0079] The formulation is taken as a single daily dose or divided
daily doses, most preferably three doses given before, during or
after meals. Patients can be maintained on cholesterol-lowering
compounds indefinitely to regulate the synthesis of cholesterol by
the liver. Conditions to be considered in selecting dosage level,
frequency, and duration primarily include the severity of the
patient's disorder, the patient's serum cholesterol 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's 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.
[0080] Table 5 describes the general composition of the P.
ostreatus CBS 101937 submerged mycelium (% dry weight),
specifically for carbohydrates, crude fiber, crude protein, lipids
and ash.
11 TABLE 5 General nutrients Mycelium Total carbohydrate 41.0-58.0
Crude fiber 5.0-5.8 Crude protein 21.0-48.0 Total lipids 2.0-7.2
Ash 4.2-7.9
[0081] Table 6 describes the protein and amino acid composition of
the P. ostreatus CBS 101937 submerged mycelium (g/100 g crude
protein)
12 TABLE 6 Amino Acids Mycelium Crude Protein (% dry 35.3-42.0
weight) Tryptophan 0.7-0.8 Lysine 4.6-7.0 Threonine 3.1-3.5 Valine
3.0-4.5 Isoleucine 2.1-3.7 Leucine 2.9-6.3 Methionine 2.9-6.3
Cysteine 0.6-0.8 Tyrosine 1.3-2.5 Phenylalanine 2.1-2.8 Total
essential Amino acids 21.0-44.0 Histidine 2.0-3.3 Arginine 3.8-6.4
Aspartic acid 6.1-7.1 Serine 2.8-4.3 Glutamatic acid 10.0-15.7
Proline 2.6-4.6 Glycine 3.4-3.8 Alanine 4.0-5.4 Total amino acid
69.0-72.0
[0082] Table 7 describes the Estimated Nutritive Value of P.
ostreatus CBS 101937 submerged mycelium (M).
13 TABLE 7 Protein was Protein EAA index, calculated content, %
(FAO) NI M M M N total .times. 6.25 42.0 70.0 29.0 N total .times.
4.38 29.0 99.0 29.0 Protein 23.0 125.0 29.0
[0083] Table 8 describes the fatty acid composition of the P.
ostreatus CBS 101937 submerged mycelium (% of total methyl
esters).
14 TABLE 8 Submerged Fatty acid mycelium Myristic (14.0) 1.5 + 0.4
Palmitic (16.0) 18.4 + 1.9 Palmitoleic (16.1) 1.5 + 0.4 Oleic
(18.1) 18.4 + 1.6 Linoleic (18.2) 43.2 +1.5
[0084] Table 9 describes the content of minerals in Pleurotus
ostreatus CBS 101937 submerged mycelium (mg/100 g dry weight).
15 TABLE 9 Minerals Mycelium Potassium 735-1800 Phosphorus 985-1980
Sodium 80-316 Calcium 40-94 Magnesium 148-359 Sulfur 2.1 Iron
39.1-60.0 Copper 0.5-2.44 Zinc 1.85-5.70 Manganese 2.0-9.0 Cobalt
0.01-0.36 Molybdenum 0.01-0.36 Selenium (.mu.g) 0.02-0.36 Chronium
0.05-3.4 Nickel 0.05-2.9 Stannous 7.4-31.4 Vanadium 0.01-0.02
Barium 0.03-0.15 Bor Titanium 0.15-1.3 Plumbum 0.1-0.54 Silver
0.01-0.09
[0085] Table 10 describes the vitamin content in Pleurotus
ostreatus CBS 101937 submerged mycelium (mg/100 g dry weight).
16 TABLE 10 Vitamins Mycelium Symbol Compound B1 Thiamine 0.6-6.0
B2 Riboflavin 2.6-5.7 B5/PP/ Niacin 64.0-122.0 B6 Piridoxin
0.06-0.47 B7 Biotin (.mu.g) 14.0-63.0 B12 Cyanobalamin C Ascorbic
acid 70.0-108.0 D2 Calciferol (.mu.g) 0.10-0.14 Pro-D Ergosterol --
E Tocopherol 6.6--10.6 Pro-A Beta-carotene Trace
[0086] The natural cholesterol-lowering nutriceuticals are
generally ingested orally. However, the nutriceuticals of the
present invention may be extracted further from the biomass and
concentrated. These products may be administered intravenously,
intraperitoneally, subcutaneously, intramuscularly, intrathecally,
orally, rectally, topically or by aerosol. The present invention
will further be understood with reference to the following
non-limiting examples. All literature citations are expressly
incorporated herein by reference.
Health Effects of Nutriceutical Compositions
[0087] Hyperlipidemias, particularly hypercholesterolemia and
hyperlipoproteinemias, are risk factors for atherosclerosis. The
higher the circulating levels of cholesterol, usually in the form
of low density lipoproteins (LDL) containing cholesterol, the
greater the risk for atherosclerosis.
[0088] Cardiovascular disease is the leading cause of death in
women and middle-aged American men. Atherosclerosis, however, which
is known to contribute to cardiovascular disease and stroke, begins
at a much earlier age. Children and adolescents with elevated serum
cholesterol levels are more likely than their counterparts with
normal cholesterol levels to have parents with coronary heart
disease.
[0089] Reduction of hypercholesterolemia results in a delayed onset
of atherosclerosis and a decrease in progression of
atherosclerosis, thus reducing the risk of coronary heart disease
in humans. Specifically, relatively complicated plaques induced by
hyperlipidation will regress, and progression of atherosclerosis
will cease when hyper lipidemia is removed. Some forms of
hyperlipidemia, including hypercholesterolemia, are potentially
partially reversible with current techniques of preventive
management. However, none of the current techniques is completely
successful and many are associated with unwanted side effects and
complications. Taking cholesterol-lowering drugs can result in a
twenty percent reduction in serum cholesterol. However, drugs are
not always warranted for hypercholesterolemia, and some of the
hypolipemic drugs such as lovastatin, mevastatin, cholestyrmaine,
clofibrate, probucol and nicotinic acid, may have serious side
effects, including an increase in mortality through liver
complications, or less severe side effects such as constipation,
skin flushes and muscle dysfunction. Dietary therapy is usually
recommended for all patients with hypercholesterolemia but is not
always effective.
[0090] Accordingly, there is need for methods and compositions
which are effective in lowering blood lipid levels, specifically
cholesterol levels. These compositions should not in themselves
have significant side effects, and would therefore be useful in
treating disease states associated with high levels of blood
lipids. It is therefore an object of the present inventor to
provide compositions and methods of use in lowering serum
cholesterol in a subject in need thereof.
[0091] Cancer is the second highest largest single cause of death
in children and adults, claiming more than 6 million lives each
year worldwide. Chemoprevention with agents including nonsteroidal
anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen,
piroxican, indomethacin, naproxene and sulindac may be useful in
reducing morbidity and mortality form cancer. Conventional NSAIDs
inhibit both forms of the cyclooxygenase (COX) enzymes COX-1 and
COX-2, of which COX-2 which plays a key role in cancer. Thus, in
searching for new cancer chemopreventive agents, hundreds of plant
extracts have been evaluated for their COX inhibitory potential.
The nutriceutical compositions of the present invention, derived
from edible higher Basidiomycetes mushrooms inhibit COX activity
and thus have chemopreventive activity against cancer.
[0092] A large number of cellular components and secondary
metabolites derived from mushrooms, have been shown to affect the
immune system and are 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->3)-.beta.-D-glucans.
.beta.-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-.gamma.) which are
released upon activation of effector cells. The carcinostatic
effect of antitumor polysaccharides thus results from the
activation of the host's immune system.
[0093] 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
compositions grown in submerged cultures in the present invention
comprised of cellular and secondary metabolites, polysaccharides
and specifically .beta.-D-glucan, and exhibit immunomodulatory and
carcinostatic properties.
[0094] 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.
[0095] The effect of administering Pleurotus ostreatus CBS 101937
submerged mycelium to Wistar rats was studied in a 12-month series
of experiments. Various lipid parameters and survival time were
recorded.
[0096] Experimental Protocol:
[0097] The experimental group consists of 500 growing Wistar rats,
randomly selected, representing both male and female rats with
initial body weight of about 90-110 g. Animals are kept in standard
temperature conditions without regulation of light regime. A
semisynthetic diet with casein albumin balanced by main food
components, mineral elements and vitamins was used in the control
rats.
[0098] The animals are divided into two groups: the first one
received an unmodified diet (control group). The second group
received food in which 0.5; 5; 10; 25 and 50% of casein were
replaced by equal levels of protein from P. ostreatus submerged
mycelium.
[0099] During the experimental period all animals were active,
healthy and with normal appetite. The overall morphological
characteristics did not differ between the two groups. It was found
that in rats supplemented with P. ostreatus mycelium there was a
decrease in cholesterol, triglycerides and oxidized peroxidase
by-products in the blood serum and liver compared with the control
group. In addition, the percentage survival in supplemented group
was higher. These data indicate a hypocholoesterolemic,
hypolipidemic and non-toxic effect of P. ostreatus. In addition,
the data obtained suggest that the P. ostreatus submerged mycelium
can be used as food additive in amount approximately equal to 5% of
daily diet.
[0100] 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.
[0101] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
[0102] 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.
[0103] 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.
* * * * *