U.S. patent application number 10/343604 was filed with the patent office on 2004-03-18 for isolation of glucan particles and uses thereof.
Invention is credited to Al-Ghazawi, Ahmad, Dutler, Hans, Freimund, Stefan, Kappeli, Othmar, Sauter, Martin, Schoberl, Helmut, Schwarz, Eugen, Thomas, Lutz.
Application Number | 20040054166 10/343604 |
Document ID | / |
Family ID | 8169434 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040054166 |
Kind Code |
A1 |
Sauter, Martin ; et
al. |
March 18, 2004 |
Isolation of glucan particles and uses thereof
Abstract
The present invention relates to the isolation of novel glucan
particles but also to mannoprotein from natural sources such as
yeast cell walls, novel isolation methods, and the use of products
thereof.
Inventors: |
Sauter, Martin; (Herznach,
CH) ; Freimund, Stefan; (Zurich, CH) ; Dutler,
Hans; (Zurich, CH) ; Kappeli, Othmar;
(Wurenlos, CH) ; Al-Ghazawi, Ahmad; (Waltham
Cross, GB) ; Schwarz, Eugen; (Bensheim, DE) ;
Thomas, Lutz; (Wolfenbuttel, DE) ; Schoberl,
Helmut; (Darmstadt, DE) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
8169434 |
Appl. No.: |
10/343604 |
Filed: |
August 4, 2003 |
PCT Filed: |
July 31, 2001 |
PCT NO: |
PCT/EP01/08851 |
Current U.S.
Class: |
536/123.12 ;
435/101 |
Current CPC
Class: |
A61K 8/73 20130101; B01J
20/26 20130101; C08B 37/0024 20130101; A61K 2800/412 20130101; A23L
29/271 20160801; A61Q 19/00 20130101; A23K 20/163 20160501; B01J
2220/52 20130101 |
Class at
Publication: |
536/123.12 ;
435/101 |
International
Class: |
C12P 019/04; C08B
037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 2000 |
EP |
00116764.2 |
Claims
1. Glucan particles with a molecular weight (M.sub.W or M.sub.n) of
more than 100 000 after solubilization by carboxymethylation having
preserved porous and fibrous structural properties.
2. Glucan particles according to claim 1 with a size range of 0.1
to 25 micrometers, preferably 0.5 to 15 micrometers, and most
preferably 2 to 10 micrometers.
3. Glucan particles according to claims 1 or 2, which are insoluble
in water and in most of the known organic solvents, having
activated pores and which are showing an increased stability at
high pH-values.
4. Glucan particles according to claims 1 to 3 with gel forming
properties.
5. Glucan particles according to at least one of the claims 1 to 4
forming a stable gel in organic solvents or acidified water, when
an aqueous suspension of these particles is heated to a temperature
higher than 80.degree. C.
6. Process for isolating glucan particles from unicellular
organisms or glucan-containing plants, comprising the steps a)
extracting mannoproteins with water at temperatures above the
boiling point of water and/or treatment of the raw materials or of
the product from step a) b) with proteases or by non-denaturating
chemical means for the removal of proteins and/or c) with lipases,
or by solvent extraction for the removal of lipids on condition
that steps b) and c) may be proceeded in any sequence and if
necessary in dependence on the starting material one or two steps
may be left out.
7. Process according to claim 6 comprising as step a) treatment
with water at temperatures above the boiling point of water under
elevated pressure and adjusted pH for extracting mannoprotein.
8. Process according to at least one of the claims 6 or 7 wherein
in step a) mannoproteins are extracted from an aqueous cell wall
suspension in the concentration range of 1-20% by weight,
preferably 10-20%, most preferably 13-17%, with adjusted pH in the
range of 5-9, preferably pH 6-8, most preferably pH 6.5-7.5, at a
temperature in the range of 120-130.degree. C. for a defined period
of time in the range of 0.1-24 hours, preferably 1-10 hours, most
preferably 3-7 hours under elevated pressure.
9. Process according to at least one of the claims 6-8, wherein the
mannoprotein is removed from the recovered aqueous solution (water
extracts) from step a) by adding and mixing with an alcohol and
precipitating.
10. Process according to claim 9 wherein the alcohol is selected
from the group methanol, ethanol, propanol, and butanol.
11. Process according to claim 6 comprising as step b) treatment
with proteases after pH adjustment at optimal level for the removal
of contaminating proteins.
12. Process according to claim 6 wherein in step b) protein
contaminants are extracted either with aqueous alkaline solution
selected from the group sodium carbonate and sodium hydrogen
carbonate at low concentration and low temperature or with sodium
dodecylsulfate at a concentration in the range of 0.1-5% by weight,
preferably 1-3% by weight, most preferably 1.5-2.5% by weight.
13. Process according to claim 6 comprising as step c) treatment
with lipases after pH adjustment at optimal level for removal of
contaminating lipids.
14. Process according to claim 6 wherein in step c) lipid
contaminants are extracted either with organic solvents, that are
miscible with water, selected from the group acetone ethanol,
methanol, isopropanol and butanol or mixtures thereof; or with
organic solvents that are not miscible with water, selected from
the group isobutylketone, hexane, chloroform, methylenechloride,
tetrachloroethylene and ethylacetate or mixtures thereof, or with
mixtures of organic solvents that are miscible with water with
organic solvents that are not miscible with water; or extraction
with supercritical CO.sub.2, or extraction with supercritical
CO.sub.2 and organic solvents as modifiers.
15. Process according to claims 6 and 14 wherein in step c) lipid
contaminants are extracted with a mixture of methanol and
chloroform in a ratio of volume of 1:1 or of hexane and isopropanol
in a ratio of volume of 3:2.
16. Mannoprotein obtained from step a) according to at least one of
the claims 6-10.
17. Glucan particles obtainable by a process according to at least
one of the claims 6-8, 11-15.
18. Glucan particles obtainable by a process according to claims
6-8 by protease treatment after extracting mannoproteins with
water.
19. Glucan particles obtainable by a process according to at least
one of the claims 6-8, 11, 13, by lipase treatment or solvent
extraction after extracting mannoprotein with water and removing
protein contaminants with protease.
20. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 as adsorbents and carriers, as
chromatographic active material or as adsorbents for toxic
environmental compounds.
21. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of pharmaceutical or cosmetic formulations
or as additive in food and feed or in products needed in
agriculture in crop protection.
22. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of pharmaceutical formulations with immune
system activating properties.
23. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of pharmaceutical formulations with
antitumor activity or for administration in combination with
chemotherapy or dialysis.
24. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of pharmaceutical formulations to improve
the host-defences to bacterial or virus infections.
25. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of pharmaceutical formulations with
prophylactic activity against diseases of age.
26. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of formulations with cholesterol reduction
activity.
27. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of formulations with an glucose regulating
effect.
28. Use of glucan particles according to at least one of the claims
1-5, 17-19 or obtainable from a process according to at least one
of the claims 6-8 or 11-15 and/or mannoprotein according to claim
16 for the preparation of formulations with improving effects on
cardiovascular diseases, on autoimmune conditions like HIV,
arthritis, lupus, allergic asthma or multipe sclerosis.
Description
FIELD OF INVENTION
[0001] The present invention relates to the isolation of novel
glucan particles from natural sources such as yeast cell walls,
novel isolation methods, and the use of products.
BACKGROUND
[0002] The cell walls of unicellular organisms and of plants mainly
consist of polysaccharides, partly associated with proteins.
Important functions are: Rigidity, physical protection of the
cells, osmotic stability, selective permeability support, cell/cell
adhesion, binding of compounds and extracellular enzyme support.
Since the polysaccharides form a three dimensional network, cell
walls may serve as a resource for particles that maintain certain
useful properties of the native cell wall such as selective
adsorption capacity. The final properties of the particles depend
on the starting material (e.g. yeast strain or other microbial or
plant cells) and on the level of preservation of the structural
integrity during isolation.
[0003] Therefore, a prerequisite for the isolation of functional
particles are non-denaturing isolation procedures.
[0004] In the following the structure and composition of the yeast
cell wall, an important potential resource of said particles, is
outlined.
[0005] The precise structure and composition of the yeast cell wall
strongly depends on the type of yeast strain and culture conditions
(R. Bonaly, H. Moulki, A. Touimi Benjellouen, M. Pierrefitte,
Biochim. Biophys. Acta 244, 484 (1971)). A shortage of amino acids,
for example, reduces the protein content in the cell wall. Yeasts
are unicellular organisms with a rigid cell wall made of
polysaccharides. The cell shape is oval to round with an average
diameter of 5-13 .mu.m. The cell walls show a thickness of about 70
nm and account for 15-25% of the yeast dry weight (J. S. D. Bacon,
V. C. Farmer, D. Jones, I. F. Taylor, Biochem. J. 114, 557 (1969)).
As mentioned, the overall composition of the cell wall varies and
depends on the special strain and on culture conditions. This forms
the basis for the isolation of a great number of cell wall
particles with different properties.
[0006] In general, the main components of the yeast cell wall are
mannan (typically about 30% by weight), glucan (also about 30% by
weight), protein (15% by weight), lipids (about 10% by weight) and
chitin (about 2% by dry weight). The latter is exclusively located
in the budding scar of the yeasts.
[0007] The Mannoprotein Component
[0008] By definition mannan, is a polymer that is exclusively
composed of mannose units. In yeasts, mannan is associated with
protein in both, the external surface of the yeast cell wall, as a
muscigenous polysaccharide and in the inner cell membrane. It
accounts for 20-50% of the dry weight (C. E. Ballou, Adv.
Microbiol. Physiol. 14, 93 (1976). Mannan is linked to a
core-peptide chain as an oligomer or polymer (R. Sentandreu, D. H.
Northcote, Biochem. J. 109, 419 (1968)). The complex consists of
5-50% proteins. Oligomeric mannan is bonded directly to serine and
threonine (R. Sentandreu, D. H. Northcote, Carbohydr. Res. 10, 584
(1969)) whereas polymeric mannan is bonded to aspargine via
N-acetylglucosamine. The many individual aspects relating to the
mannoprotein complex, including that the mannose units are linked
by .alpha.-1,6, .alpha.-1,2 and .alpha.-1,3-linkages were compiled
and reviewed by Ballou et al. (C. E. Ballou, Adv. Microbiol.
Physiol. 14,93 (1976); C. E. Ballou, Adv. Enzymol. 40, 239
(1974)).
[0009] The Glucan Component
[0010] Glucan is a glucose polymer and accounts for 30-60% of the
dry weight. The majority of the polyglucoside is linked via
.beta.-1,3 glycosidic linkages and only 10-20% by .beta.-1,6
glycosidic linkages (S. Peat, J. R. Turvey, J. M. Evans, J. Chem.
Soc. 3868 (1958)). If glucan is treated with approximately 3%
caustic soda at 75.degree. C., a maximum of one-third of the glucan
is solubilized (J. S. Bacon, V. C. Farmer, D. Jones, Biochem. J.
114, 557(1969)). Consequently the glucan is divided into (1) an
alkali insoluble fraction (glucan A), and (2) an alkali soluble
fraction (glucan B) (G. H. Fleet, D. J. Manners, J. Gen. Microbiol.
94, 180 (1976)).
[0011] Glucan A accounts for 80-85% of the cell wall glucan and
consists primarily of .beta.-1,3 glycosidic linkages as well as of
about 3% .beta.-1,6 gly-cosidic linkages. 80-85% of the glycosidic
linkages of glucan B (15-20% of the total glucan) are .beta.-1,3
and 8-12% are .beta.-1,6 glycosidic linkages. 3-4% of the glucose
units are branchings. The .beta.-1,6 glycosidic linkages are
selectively hydrolysed by acetylosis. It is proposed that the
.beta.-1,3 glucan chains are linked via .beta.-1,6 intermediate
chains (J. S. D. Bacon, V. C. Farmer, D. Jones, Biochem. J. 114,
557 (1969)). Using electron microscopy it was possible to
demonstrate a fibrillar structure for the .beta.-1,3 component and
an amorphous structure of the 1,6 component (M. Kopecka, J. Basic
Microbiol. 25, 161 (1985)).
[0012] Chitin and Lipid Components
[0013] Chitin (N-acylated poly-glucosamine) is located exclusively
in the budding scars, where it forms a ring (E. Cabib, B. Browers,
J. Biol. Chem. 246, 152 (1971)). As a lipid compound dolichol
phosphate was isolated from the cell walls (P. Jung, W. Tanner,
Eur. J. Biochem. 37, 1 (1973)). The rest of the lipid component
consists of glycerol esters of various fatty acids.
[0014] The Structure of the Yeast Cell Wall
[0015] Electron microscopic investigation of the process of
biosynthesis and assembly of the glucans in Candida albicans
reveals the development of the fibrous network of the cell wall.
The triple helices which appear as microfibrils with a diameter of
approx. 2 nm are self-assembled end-to-end and side by side and are
twisted together leading to fibrils of 4-8 nm in diameter. These
fibrils finally associate to flat ribbon-shaped bundles, 8-16 nm
thick and 100-200 nm wide and thus form the basic network structure
of the cell wall. The interfibrillar spaces of the network at this
stage have dimensions of about 100-200 nm and most likely mark the
origin of the pores which are present in the cell wall at the final
stage and which constitute the structural basis for their ability
to adsorb compounds with great significance in a large number of
different areas.
[0016] They are gradually filled with the additional components and
manno-proteins which are known to form anchors to the membrane
lipids.
[0017] Isolation of Yeast Cell Wall Components
[0018] Fractionation of the cell walls, as e.g. of Saccharomyces
cerevisiae starts either from whole cells or from cell walls e.g.
obtained by autolysis; both starting materials may be used in dry
or wet form. In some cases the cells or cell walls are pre-treated
mechanically (by sonification or by treatment with glass beads).
The starting material as well as the mechanical disruption greatly
influence the purity of the resulting fraction. A large number of
different methods were reported for the isolation of cell wall
components (F. M. Klis, Yeast 10, 851 (1994)). They can be grouped
(1) in methods for the isolation of mannoprotein, and (2) in
methods for the isolation of glucan.
[0019] A common reagent of chemical methods for the isolation of
mannoprotein is sodium hydroxide of varying concentrations and
using a wide range of temperatures and treatment times (Int. Patent
WO 94/04163 (1994); D. L. Williams, R. B. McNamee, E. L. Jones, H.
A. Pretus, H. E. Ensley, I. Williams, N. R. Di Luzio, Carbohydr.
Res. 219, 203 (1991)). Depending on the reaction conditions, such
treatments also solubilize more or less glucan (see above
definition of soluble and insoluble glucan). In some cases, organic
bases like ethylene diamine and buffers like citrate salts find
application to solubilize mannoproteins (R. Sentandreu, D. H.
Northcote, Biochem. J. 109, 419 (1968); T. Nakajima, C. Ballou, J.
Biol. Chem. 249, 7679 (1974)). Extraction with a 2% boiling
sodium-dodecyl-sulfate (SDS) in the presence or absence of reducing
agents, like mercaptoethanol, represents a widely used approach to
free glucan from mannoproteins and other proteins (E. Valentin, E.
Herrero, F. I. J. Pastor, R. Sentandreu, J. General Microbiol. 130,
1419 (1984); F. I. J. Pastor, E. Valentin, E. Herrero, R.
Sentandreu, Biophys. Acta 802, 292 (1984)). Treatment of whole
cells with pure water at temperatures of up to 135.degree. C. was
also applied, yielding a highly contaminated mannoprotein fraction
(S. Peat, W. J. Whelan, T. E. Edwards, J. Chem. Soc. 29 (1961); N.
Shibata, K. Mizugami, S. Susuki, Microbiol. Immunol. 28, 1283
(1984); Y. Okubo, T. Ichikawa, S. Susuki, J. Bact. 136, 63
(1978)).
[0020] Enzymatic methods were alternatively used for releasing the
manno-proteins. For this purpose, proteases and glucanases are
used, acting on the protein part of the mannan or the glucan fixing
the mannoprotein (.beta.-1,6 glucan).
[0021] The mannan-free glucan is further purified by procedures
that include acid treatment such as acetic acid or HCl.
[0022] The summarised chemical procedures for isolation and
purification of cell wall components will more or less affect the
nativity of the polymers, which is primarily reflected in the
occurrence of increased amounts of soluble glucan and in a
disturbance of the structure of the insoluble glucan fraction. It
is especially the lafter negative impact of existing glucan
isolation procedures that make the insoluble glucan less suitable
for adsorbent applications. When such chemical treatments are used
under milder conditions, the pores of the glucan skeleton are not
properly activated, i.e. freed from physically or chemically bound
pore filling material. This also yields insoluble glucan not
optimal for adsorption.
[0023] Therefore, it is the objective of this invention to provide
simple and effective methods for the isolation of glucan particles,
which are characterised by a native structure and active pores.
[0024] The respective process for isolating glucan particles with
such features from cells, cell walls, or cell wall fragments of
unicellular organisms like yeasts or fungi or of cell wall residues
of glucan-containing plants, comprises the steps a)-c) which may be
proceeded in any sequence (FIG. 1). These steps are characterised
as follows:
[0025] a) extracting mannoproteins with water at temperatures above
the boiling point of water from suspensions,
[0026] b) removal of contaminating proteins with protease or
non-denaturing chemical means,
[0027] c) removal of contaminating lipids with lipases or by
solvent extraction.
[0028] The extraction of step is preferably proceeded with adjusted
pH under elevated pressure.
[0029] Depending on the starting material one or more steps may be
deleted. Cells, cell walls or cell wall fragments of e.g. yeast or
fungi or of uni-cellular organisms other than yeast or cell wall
residues of glucan-containing plant tissues are used as starting
material.
[0030] According to the invention these new glucan particles are
obtainable from these starting materials by combining steps c) and
b) or steps b) and c) respectively.
[0031] As far as appropriate, residual non-glucan components may be
removed by non-denaturing chemical means, such as extraction of
non-glucan residues with NaOH at low concentration and temperature,
with 2% sodium dodecylsulfate solution at elevated temperature, and
with organic solvents, such as acetone, at room temperature or at
elevated temperatures.
[0032] If useful starting materials are treated according to the
invention and if the steps of the inventive process are carried out
in a suitable sequence the basic structure of the isolated glucan
particles remains intact and shows properly active pores. Naturally
this means that each single step of the process has to be adapted
to the treated material.
[0033] It has been found that glucan particles can be isolated with
preserved porous and fibrous structural properties. These particles
are having a molecular weight (M.sub.W or M.sub.n) of more than 100
000, especially more than 400 000, having essentially retained its
native structure after solubilization by carboxymethylation. Their
particle sizes are in a range of 0.1 to 25 micrometers, preferably
0.5 to 15 micrometers, and most preferably 2 to 10 micrometers. In
contrast to known glucan these glucan particles are insoluble in
water and in most of the known organic solvents, while they are
having activated pores and are showing an increased stability at
high pH-values. Additionally, these glucan particles are able to
form stable gels; for example they are forming a stable gel in
organic solvents or acidified water, when an aqueous suspension of
these particles is heated to a temperature higher than 80.degree.
C.
[0034] Glucan particles according to the present invention may be
obtained by extracting mannoproteins from an aqueous cell wall
suspension in the concentration range of 1-20% by weight,
preferably 10-20%, most preferably 13-17% with adjusted pH in the
range of 5-9, preferably pH 6-8, most preferably pH 6.5 to 7.5, at
a temperature in the range of 100-150.degree. C., preferably
110-140.degree. C., most preferably 120-130.degree. C., for a
defined period of time, e.g. 3-7 hours, under elevated
pressure.
[0035] According to the described treatment lipid and protein
containing glucan particles are obtained as solid fraction and
mannoprotein as the soluble fraction, which may be isolated.
[0036] The extracted mannoprotein can be isolated from the aqueous
solution by precipitation. It is found, that this precipitation can
be induced by mixing the solution with an alcohol. Suitable
alcohols are short chained alcohols. Preferably an alcohol selected
from the group methanol, ethanol, propanol or butanol is used. Most
preferably the precipitation is carried out with ethanol.
Alternatively the mannoprotein may be concentrated by
ultra-filtration before precipitation with alcohols.
[0037] Lipid and protein containing glucan particles, removed by
centrifugation or filtration, are subsequently treated with a
protease at pH values and temperatures required for optimum
protease activity for 1-12 hours, preferably 3-8 hours and most
preferably 4-6 hours. Lipid containing glucan particles result from
this treatment and can be isolated.
[0038] For obtaining intact mannoprotein the protease treatment on
the glucan particles has to be done after the mannoprotein fraction
has been separated.
[0039] Lipid containing glucan particles removed by centrifugation
or filtration, are subsequently treated with a lipase at pH values
and temperatures required for optimum protease activity for 1-12
hours, preferably 1-5 hours and most preferably 2-4 hours. From the
treatment glucan particles result and may be isolated.
[0040] Alternatively lipid containing glucan particles may be
obtained from lipid and protein containing glucan particles by
extraction of protein containing contaminants either with aqueous
alkaline solutions such as earth alkali hydroxide, like NaOH,
sodium carbonate and sodium hydrogen carbonate solutions at low
concentration and low temperature or with sodium dodecylsulfate at
a concentration in the range of 0.1-5% by weight, preferably 1-3%
by weight, most preferably 1.5 to 2.5% by weight.
[0041] Glucan particles may alternatively be obtained from lipid
containing glucan particles by cold or hot organic solvent
extraction with solvents that are miscible with water, e.g.
selected from the group acetone, ethanol, methanol, isopropanol and
butanol or mixtures thereof,
[0042] or with solvents that are not miscible with water, e.g.
selected from the group dialkylketones, e.g. isobutylmethylketone,
hydrocarbons, e.g., hexane, chlorinated hydrocarbons, e.g.
chloroform, methylenchloride, tetrachloroethylene and ester
solvents, e.g. ethylacetate, or mixtures thereof,
[0043] or with mixtures of organic solvents that are miscible with
water with organic solvents that are not miscible with water, e.g.
methanol/chloroform in a ration of volume of 1:1 or
hexane/isopropanol in a ration of volume of 3:2;
[0044] or with supercritical fluids, e.g. supercritical
CO.sub.2;
[0045] or with supercritical CO.sub.2 and organic solvents as
modifiers.
[0046] Therefore, the process for isolating insoluble native glucan
particles with properly activated pores from cells, cell walls, or
cell wall fragments of unicellular organisms like yeast or fungi or
of cell wall residues of glucan-containing plants, comprises the
steps a)-c) mentioned above which may be proceeded in any sequence.
These steps are characterised as follows:
[0047] a) extracting mannoproteins with water at temperatures above
the boiling point of water from suspensions with adjusted pH under
elevated pressure,
[0048] b) treatment with proteases after pH adjustment at high
level and removal of proteins,
[0049] c) treatment with lipases after pH adjustment at high level
and removal of contaminating lipids,
[0050] or if appropriate by solvent extraction.
[0051] Optionally non-glucan residues can be removed by
non-denaturing chemical means and as already said above depending
on the starting material one or more steps may be deleted.
[0052] Thus glucan particles are prepared which are insoluble in
water and most of the common solvents, especially most of the
common organic solvents. A unique advantage is their stable
three-dimensional structure, which is nearly unchanged in the
presence of adsorbed substances or if their surface reacts with
active groups. The particles according to the invention possess
activated open pores.
[0053] A valuable by-product of the present process is a
mannoprotein, which may be recovered from step a) of the process.
For this purpose the recovered aqueous fraction of step a) is added
to and mixed with an alcohol. This alcohol may be a short-chained
alcohol, especially one of the group methanol, ethanol, propanol
and butanol. The extracted mannoprotein may be recovered after
precipitation by cooling for several hours.
[0054] Distinguishing properties of glucan particles isolated
according to the present invention are also:
[0055] 1. Solubility in DMSO
[0056] Glucan particles according to the invention swell markedly
in DMSO but can easily be centrifuged which means that they are not
truly dissolved. For comparisons, glucan particles isolated by
harsh conditions (conventional glucans) dissolve in DMSO (e.g. D.
L. Williams, H. A. Pretus, H. E. Ensley, I. W. Browder, Carbohydr.
Res. 253, 293 (1994)) which allows the characterisation in solution
like the chromatographic determination of the molecular weight.
[0057] 2. Swelling/Gel Formation
[0058] After heating to a temperature higher than 80.degree. C. and
subsequent cooling of an aqueous suspension, glucan particles
prepared as described above swell and yield a voluminous gel. This
gel is stable for several years when it is stored in organic
solvents like methanol or acidified water.
[0059] 3. Stability at High pH-Values
[0060] Glucan particles as described are much more stable at high
pH values (>pH 10) as compared to conventional glucans, which
are solubilized at high pH values.
[0061] 4. Molecular Weight
[0062] Glucan particles as described are insoluble in water and in
common organic solvents. Therefore, for the determination of the
molecular weight it is necessary to solubilize the glucan particles
by an as much as possible mild derivatization method. For example,
carboxymethylation under common, only slightly degrading conditions
(alkaline isopropanol, chloroacetic acid) yields a water soluble
product. Analysis of the product by FFFF (flow
field-flow-fractionation) resulted in M.sub.W=880000 and
M.sub.n=581000. (Comparison: M.sub.W=35300 and M.sub.n=35000 for
underivatzed glucan (D. L. Williams, H. A. Pretus, H. E. Ensley, I.
W. Browder, Carbohydr. Res. 253, 293 (1994)); M.sub.W=110000 and
M.sub.n=25000 for glucan phosphate (D. L. Williams, R. B. McNamee,
E. L. Jones, H. A. Pretus, H. E. Ensley, I. W. Browder, N. R. Di
Luzio, Carbohydr. Res. 219, 203 (1991)).
[0063] 5. Microscopy
[0064] Microscopy techniques were used to show the structural
features of the glucan isolated according to the invention.
Electronmicrography shows the porous surface (FIG. 2) and confocal
fluorescence microscopy demonstrates the shape and size of the
glucan particles (FIGS. 3 and 4).
[0065] 6. Particle Size Distribution
[0066] By means of light scattering a particle size distribution
has been determined (FIG. 5).
[0067] Determined particle sizes of the prepared new glucan are in
a size range of 0.1 to 25 micrometers. Most of the particles show
particle sizes in the range of 0.5 to 15 micrometers, especially in
the range of 2 to 10 micrometers.
[0068] 7. Determination of Purity
[0069] For the determination of the purity of distinct fractions of
glucan particles, the elemental composition of the main possible
components are used:
[0070] Pure glucan: C 44.45H 6.22
[0071] Pure mannan: C 44.45H 6.22
[0072] Triglycerides: C .about.72H .about.14
[0073] Protein: C .about.53H .about.6.5 N .about.17
[0074] In summary the preparation of glucan particles is
characterised by the following steps:
[0075] a) Preparation of protein- and lipid-containing glucan
particles from glucan containing starting material, in particular
from yeast cell walls by heating an aqueous suspension of yeast
cell walls for several hours at elevated temperature above the
boiling point of water under elevated pressure.
[0076] b) Preparation of lipid-containing glucan particles by
treating particles from step a with proteases
[0077] c) Preparation of glucan particles by treating
lipid-containing glucan particles from step b) with lipases or
[0078] preparation of glucan particles by combining steps b and c
or steps c and b respectively. or
[0079] in variation of the lipase treatment: solvent extraction,
e.g. with acetone.
[0080] Therefore, the described products are useful in a wide range
of applications: as carrier in cosmetic or pharmaceutical
formulations, as additive for feed and food, as adsorbent for toxic
environmental compounds, as active material in chromatography or
for immobilisation of substances in different fields of
application, such as biotechnology as well as in chemical
processing. Glucan particles according to the invention may be used
for the formulation of products needed in agriculture in particular
in crop protection, since these products have a potential as health
promoting agent for animals and humans. Glucan particles according
to the invention are also useful for the preparation of
pharmaceutical formulations with immune system activating
properties as well as for formulations with anti tumour activity or
for administration in combination with chemotherapy or dialysis.
Since materials prepared according to the present invention are
able to stimulate the activity of the immune system, these glucan
particles can be used to prepare pharmaceutical formulations to
improve the host-defences to bacterial or virus infections as well
as such with glucose regulating effect or with improving influence
on cardiovascular diseases, in treatment of HIV, and other auto
immune conditions like arthritis, lupus, allergic asthma, multiple
sclerosis and so on. They are also useful for the preparation of
pharmaceutical formulations with prophylactic activity against
diseases of age and such with cholesterol reduction activity.
[0081] As health promoting agent for animals and humans glucan
particles of the present invention may be contained in food
supplement or dietary compositions.
[0082] They can be taken or administered to warm blooded mammals in
need thereof in various forms such as dried powder mixed with
liquid, as a pill, tablet or capsule as part of other formulations
for a regulated diet. In addition to the inventive compounds, a
variety of fillers, flavouring agents, binders, minerals and
vitamins as well as typical adjuvants used in the art can be used
for the preparation of the administration forms. Sorbitol as a
sweetener can be mentioned as well as dicalcium phosphate and
magnesium stearate as mineral agents are also suitable.
[0083] Glucan particles according to the invention, that can be
isolated as a powder, may be used as food or dietary supplement,
which can be used in conjunction with a dietary plan.
[0084] In preparing the dietary products of the invention, a dry
granulation technique may be used that is well understood in the
art. Typical equipment used is a roll compactor known as a
"Chilsonator" made by the Fitzpatrick Company. The Chilsonator
densifies the blended powder by passing the powder between high
pressure rollers, which compresses the powder and removes the air.
The densified material is reduced to a uniform granule size and can
be compressed into tablets after addition of a lubricant in
accordance with conventional practice. The blending of the
dehydrated powdered glucans and other ingredients and conventional
excipients can be carried out with a powder blending machine. This
equipment is well known in the art.
[0085] The food supplement, dietary and pharmaceutical compositions
of this invention will contain glucan particles, which can be
isolated according to the described process, together with a solid
or liquid pharmaceutically acceptable non-toxic carrier. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk,
silica gel, magnesium carbonate, magnesium stearate, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene glycol, water, ethanol and the like.
These compositions can take the form of tablets, pills, capsules,
powders, sustained-release formulations and the like. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain an effective therapeutic amount of the active ingredients
together with a suitable amount of carrier so as to provide the
form for proper administration to the host. These formulations may
also contain mannoprotein as such or in combination with glucan
particles according to this invention.
[0086] The compositions of this invention can further include a
filler, flavouring agent, binder, mineral, vitamin as mixtures
thereof. Tablets can be coated with a film and/or colour layer for
protection and colour as is known in the art. These additives are
conventional components of dietary products.
[0087] It has also been found, that the isolated mannoprotein from
the hot water treatment can be used in the same manner as the
inventive glucan particles for food or ppharmaceutical
applications. Most advantageously such formulations are prepared
using the inventive glucan particles in combination with this
mannoprotein.
[0088] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilise the
present invention to its fullest extent. The preferred specific
embodiments and examples of the following are, therefore, to be
construed as merely illustrative, and not limitative of the
disclosure in any way whatsoever.
[0089] The entire disclosures of all applications, patents, and
publications cited above and below are hereby incorporated by
reference.
EXAMPLE 1
[0090] Preparation of Glucan Particles
[0091] a) Hot Water Treatment
[0092] Dry yeast cell walls (1.3 kg) were suspended in water (8.7
l) and the suspension was adjusted to pH 7 by adding an aqueous
sodium hydroxide solution (30% w/w). The suspension was heated to
120.degree. C. and stirred for 5 h. After cooling, the suspension
was diluted with water (7.3 l) and centrifuged (20 min, 4000 rpm).
The supernatant was removed and the sediment resuspended in water
(total weight: 17 kg). After centrifugation and removal of the
supernatant, the sediment was ready for the next step.
[0093] For analytical purposes, an aliquot of the sediment was
several times washed with water and centrifuged until the
supernatant was colourless and clear and then freeze-dried yielding
a pale yellow powder.
1 Yield: 63.6% Protein content: 7.9% Lipid content: 16.3% Elemental
analysis: C 50.5%, H 7.5%, N 3.3%
[0094] b) Protease Treatment
[0095] The washed sediment from step a) was resuspended in water to
a final volume of 10 l. Then the suspension was heated to
45.degree. C. and adjusted to pH 10.5 by adding an aqueous sodium
hydroxide solution (30% w/w). Savinase (7.5 ml) was added at t=0,
1.5 and 3 h. After an overall duration of 5 h, the suspension was
neutralised with acetic acid (100%) and centrifuged (30 min, 4000
rpm). After removal of the supernatant, the sediment was ready for
the next step.
[0096] For analytical purposes, an aliquot of the sediment was
several times washed with water and centrifuged until the
supernatant was colourless and clear and then freeze-dried yielding
a pale yellow powder.
2 Yield (referring to starting material): 31.6% Protein content:
3.5% Lipid content: 17.3% Elemental analysis: C 49.7%, H 7.6%, N
1.2%
[0097] c) Solvent Treatment
[0098] The moist sediment from step b) was treated with a large
excess of acetone and filtered. The residue was washed several
times with acetone until the filtrate was colourless and clear and
then dried yielding a pale yellow powder.
3 Yield (referring to starting material): 25.7% Protein content:
4.2% Elemental analysis: C 46.2%, H 6.7%, N 1.6%
[0099] Pr Paration of Mannoprotein
[0100] The first supernatant from step a) was added to ethanol
(95%) under stirring until the water content reached 30%. The
mixture was stored over night at 5.degree. C. leading to a
precipitate. The precipitate was filtered, washed several times
with ethanol and then dried yielding a white powder.
4 Yield (referring to starting material): 14.0% Protein content:
15.4% Elemental analysis: C 43.1%, H 6.2%, N 3.7%
[0101] .sup.1H and .sup.13C NMR spectra are shown in FIGS. 6 and
7.
EXAMPLE 2
[0102] Preparation of Glucan Particles on Pilot Scale
[0103] a) Hot Water Treatment
[0104] 150 kg yeast cell walls, washed free of water-soluble
components, were suspended in 850 l of tap water, and the pH was
adjusted to 7. The suspension was heated to 125.degree. C. under
stirring adapting the stirrer speed accordingly in order to prevent
heat gradients and especially local overheating, which leads to
gelation. The overpressure amounted to approximately 1.3 bar. After
5 h the suspension was cooled to 45.degree. C. Vacuum formation was
prevented during cooling by opening an air inlet valve equipped
with a sterile filter. The lipid and protein containing glucan
particles were separated by centrifugation (Westfalia SB 07
centrifuge) and washed twice with water. The washed sediment was
used in the next step.
[0105] b) Protease Treatment
[0106] Lipid and protein containing glucan particles were
resuspended in a total volume of 470 l of tap water with a
temperature of 45.degree. C. The pH was adjusted to 10.5 with a
30%-NaOH solution. Then 3.5 l of proteolytic enzyme solution
SAVINASE 16.0 L EX (Novo) containing 0.4 l of SAVINASE adapted
detergent solution (according to manufaturer specifications) were
added with stirring. After 3 h the pH dropped to 9.5 indicating
protein hydrolysis. Therefore, the pH was readjusted step-wise to
10.5 and incubation was carried on until pH remained constant
(.about.2 h). After neutralisation lipid containing (protein free)
glucan particles were harvested by centrifugation and washed twice
with water. The washed sediment was used in the next step.
[0107] For analytical purposes, a small amount of the sediment was
treated with an excess of acetone and filtered. The residue was
washed three times with acetone and subsequently dried.
5 Elemental analysis: C 45.1%, H 6.3%, N 1.2%
[0108] .sup.1H and .sup.13C NMR spectra including the assignment of
the signals are shown in FIGS. 8 and 9.
[0109] c) Lipase Treatment
[0110] 10 l of the 470 l hot water extracted, protease treated cell
wall suspension were further treated with 100 g of LIPOLASE 100 L
EX (Novo) containing 4 ml of LIPOLASE adapted detergent solution
(according to manufacturer specifications) at 45.degree. C. and pH
10.5 with stirring for three hours. The (protein and lipid free)
glucan particles were harvested by centrifugation, washed twice and
lyophilised.
EXAMPLE 3
[0111] Variation of Protease Treatment
[0112] Yeast cell walls were suspended in water to a final
concentration of 11%. The pH was adjusted to 10.5 by adding an
aqueous sodium hydroxide solution (30% w/w). The suspension was
heated to 50.degree. C. under stirring and the reaction was started
by the addition of Savinase (3 ml/l suspension). As a standard
procedure the addition of equal amounts of Savinase was repeated
after 1 and 3 h, respectively. Total incubation time was 4 h. The
pH was kept at 10.5 throughout the incubation by addition of
adequate amounts of sodium hydroxide whenever necessary. Finally
the suspension was neutralised by adding acetic acid and the
product was isolated by centrifugation (20 min, 5000 rpm). The
sediment was washed twice with water and fats were removed by
acetone treatment of the moist sediment as described in Example 1.
After drying the protein content was analysed and the results are
summarised in table 1.
6TABLE 1 Lipid extraction of dry cell walls. Variation of protease
treatment Protein content [%] Standard procedure 4.5 Additional
washing after every protease step Overnight incubation after third
addition 2.4 of Savinase Additional treatment with 2% SDS 1.2
overnight after third Savinase step
EXAMPLE 4
[0113] Lipid Extraction of Dry Cell Walls with Organic Solvents
[0114] 10 g of dry cell walls (named A containing 16.1% of lipids
and B containing 13.4% of lipids) were suspended in 200 ml of an
organic solvent. The suspension was heated at reflux for 2 h. After
cooling to 40.degree. C., the mixture was filtered. The residue was
washed two times with 30 ml of warmed solvent and subsequently
dried yielding a colourless or slightly yellowish powder. The
combined filtrates were evaporated yielding brownish oil. Table 2
summarises the results of the extractions.
7TABLE 2 Lipid extraction of dry cell walls. extracted lipids lipid
content cell content total Solvent walls (%) (%) n-Hexane/methanol
4:1 (v/v) A 16.1 15.5 96% Ethanol (techn.) " " 14.3 89% Methanol
(abs.) " " 14.7 91% Ethanol (techn.) B 13.4 10.9 81% Methanol
(abs.) " " 10.9 81%
EXAMPLE 5
[0115] Lipid Extraction of Spray-Dried Lipid Containing Glucan
Particles with Organic Solvents
[0116] 10 g of spray-dried lipid containing glucan particles (from
Example 1: lipid content: 28%) were suspended in 200 ml of an
organic solvent. The suspension was-heated at reflux for 2 h. After
cooling to 40.degree. C., the mixture was filtered. The residue was
washed two times with 30 ml of warmed solvent and subsequently
dried yielding a colourless or slightly yellowish powder. The
combined filtrates were evaporated yielding dark brown oil. Table 3
summarises the results of the extractions.
8TABLE 3 Lipid extraction of spray-dried lipid containing glucan
particles. extracted lipids Solvent content (%) total (%)
n-Hexane/methanol 4:1 (v/v) 28 100 n-Hexane 2 7
n-Hexane/isopropanol 4:1 (v/v) 5 17 Isopropanol 12 43 Acetone
(tech.) 10 37 Acetone/H.sub.2O 4:1 (v/v) 27 96 Ethanol (abs.) 27 96
Ethanol (tech.) 27.5 98 Methanol (abs.) 28 100
EXAMPLE 6
[0117] Adsorption by Glucan Particles
[0118] 30-70 mg of glucan particles were homogeneously suspended in
100 ml of distilled water. The compound of interest was added as
stock solution to the glucan suspension. After stirring this
mixture for some time the glucan particles were removed from the
suspension by centrifugation. The concentration of the compound in
the supernatant was determined and the amount of the compound
adsorbed by the glucan particles was calculated from the difference
between the starting and the end concentration of the compound.
[0119] Different classes of compounds, like proteins (e.g. 17 mg of
lysozyme bound per g of glucan particles, 43 mg of myoglobine bound
per g of glucan particles), flavours (e.g. 4 mg of eugenol bound
per g of glucan particles) or toxins (e.g. 2.5 mg zearalenone bound
per g of glucan particles), were adsorbed by the glucan
particles.
EXAMPLE 7
[0120] Glucan Particles as Carrier
[0121] Retinol (370 mg) was melted and glucan particles (3.0 g)
were added gradually within 10 minutes under stirring at 75.degree.
C. Stirring was continued for 10 minutes. After cooling a yellow,
free flowing powder was obtained.
* * * * *