WO1993012243A1 - A new fungal glukan and its preparation - Google Patents

A new fungal glukan and its preparation Download PDF

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Publication number
WO1993012243A1
WO1993012243A1 PCT/CS1991/000002 CS9100002W WO9312243A1 WO 1993012243 A1 WO1993012243 A1 WO 1993012243A1 CS 9100002 W CS9100002 W CS 9100002W WO 9312243 A1 WO9312243 A1 WO 9312243A1
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Prior art keywords
glukan
glukp
glucose
fungal
homopolymer
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PCT/CS1991/000002
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French (fr)
Inventor
L^¿udovít KUNIAK
S^¿tefan KARACSONYI
Jozef AUGUSTI^´
Anastázia GINTEROVA^´
S^¿tefan SZE^´CHE^´NYL
Dus^¿an KRAVA^´RIK
Július DUBAJ
Ján VARJU^´
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Slovenská Technická Univerzita
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Priority to AU90395/91A priority Critical patent/AU9039591A/en
Publication of WO1993012243A1 publication Critical patent/WO1993012243A1/en

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • This invention concerns fungal ⁇ (1->3) glukan from oyster fungus (Pleurotus ostreatus) and the process of its preparation.
  • ⁇ (1->3) glukans are usually differentiated by the type of microorganism which produce them.
  • the basic chain is ⁇ (1->3) glukan, which can however be branched in different ways and the difference in structure in branches may result in changes in pharmalogical properties.
  • the best known microbial ⁇ (1->3) glukans are lentilan, schyzophylan and skleroglukan (Chichara G. and all: Nature 222, 687 (1969), Chichara G. and all: Cancer Res.30, 2776 (1981), Komatsu N. and all: Gann 60,137 (1969), Singh P.P. and all: Carbohydr. Res. 37,245 (1974) ).
  • the subject matter of the invention is a new homopolymer ⁇ (1->3) glukan , which is branched in the main chain in the position -0-6 with bonding ⁇ (1->6) and ⁇ (1->4) with frequency of 1 branch to each fourth anhydroglucose unit in th main chain of the ⁇ (1->3) glukan.
  • the new polysacharide is isolated from fruit- body of the oyster fungus, preferably from stipes, by alkali hydrolysis of the protein part of fruit-body and by extraction of soluble glukans in aqueous sodium hydroxide or potassium hydroxide in concentration 0,1 to 0,2 mol/1 at a temperature of 95 to 100 °C for 2 to 3 hours.
  • the insoluble ⁇ (1->3) glukan is collected by filtration and washed, then bleached with sodium chlorite (NaCIO 2 ) at 50 to 60 °C at a pH of 3,5 to 4,5 for 6 to 12 hours.
  • the bleached product is washed with water, dehydrated with organic solvent, preferably with ethanol or acetone and finally the organic solvent is removed by vaccum drying at temperatures up to 60 °C.
  • the structure of the isolated polysacharide was analysed to show that the new fungal polysacharide is ⁇ (1->3) glukan, in which each fourth anhydroglukose unit is substituted on -0-6 with a non-reducing terminal unit.
  • the branch chains of the polysacharide there are 4,4 resp. 3,7 units bonded ⁇ (1->6) and ⁇ (1->4). It is differentiated markedly from the yeast glukan by number of branches and the type of bonding of the branches.
  • the fungal glukan using the new method can be isolated from fruit-bodies of the oyster fungus by a much simpler technique than the method of isolation from yeast.
  • concentration of sodium hydroxide resp. potassium hydroxide is 0,1 to 0,2 mol/1 which is 5to 10 times lower than isolation from yeast.
  • extraction temperature of the protein is lower, usually temperature of 90 to 100°C is sufficient, which allows extraction in unpressurized vessels.
  • the insoluble glukan does not have to be treated in disintegrators and centrifuge, which reduces the investment of the prouduction unit.
  • oyster fungus is a rich source of ⁇ (1->3) glukan, since with the new process it is possible to obtain yields of about 50 weight % of the dry raw material. This is 5 times the yield of yeast glukan based on dry yeast.
  • the main advantage of the new process is the fact that from oyster fungus it is possible to isolate a polysacharide previously unknown structure as it refers to the branching of the main chain of ⁇ (1->3) glukan and furthemore that the isolation at high yields is technologically significantly simplified enabling meeting of ecological demands.
  • the washed fungal glukan is transfered from the filter medium back in the reactor, diluted with deionized water to 50 liters and acidified with acetic acid to pH 3,5 to 4,6. Then 30 g of sodium chorite (NaClO 2 ) dissolved in 2 liters o deionized water is added to the suspension of glukan in the reactor, heated to 50°C and kept at that temperature for 6 to 12 hours.
  • sodium chorite NaClO 2
  • the unreacted chlordioxid is eliminated by addition of disulphite (Na 2 S 2 O 5 ) and the insoluble product is collected by filtration , then washed thoroughly with deionized water, which is displaced from the polysacharide by ethanol , followed by acetone and after removal of the solvent by sunction the product is vacuum dried at 60°C.
  • the product is white, fibrous polysacharide ⁇ (1->3) glukan with the yield of 505 g, nitrogen content less than 0,3%, ash content less than 0,2 and molecular weight of 240000.
  • Example 1 The same product as in Example 1, except that potassium hydroxide in concentration of 0,2 mo1/1 is used as the base.
  • the resulting product has the same properties as that in Example 1.
  • the invention has utility particulary in veterinary and human medicine as an immunostimulator, as an adjuvant invaccination, in galenic pharmacology in preparation of ointments with specific activity.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Materials Engineering (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

A new glukan is produced by oyster fungus (Pleurotus ostreatus). Said glukan is a homopolymer β (1←3) glukan, which is substitued in the -0-6 position on each fourth anhydroglucose unit with glucose or disaccharides which contain the (1←6) or (1←4) bonding. The proportion or glucose to disaccharide in the branches is 2:1. A process for preparing said fungal glukan is described. The glukan is isolated from fruit-body of oyster fungus.

Description

A new fungal glukan and its preparation
This invention concerns fungal β(1->3) glukan from oyster fungus (Pleurotus ostreatus) and the process of its preparation.
It is known that microbial β(1->3) glukans have important pharmacological properties:
a) they stimulate natural cell killers (Lotzova E., Gutterman J.U.: J. Immunol. 123,607 (1979)),
b) they increase the immunity against certain viral infec tions (Kovalenko A.G., Votselko S.K.: Prikl. Biokhim. Mikrobiol 13, 46 (1977), Akenov O.A. and all: Deistvil Interferona. Mater. Symp. "Inhibitors of viruses" 99-106 (1970), Indulev M.K.Ed., Zinatne, Riga (1972),
c) they increase imunity against bacterial illnesses (Komarsu N. and all: US patent No. 3,943 247 (1976), Veisberg G.E., Golosova T.V. -.Zh.Mikrobiol. Epidemiol. Immunol. 41, 96 (1964) ).
d) they have an adjuvant (additive) effect with cytostats (Eliznakov E.G.: Advant. Exp. Med. Biol. 15, 315 (1971),
e) they have an antitumor effect (Sankyo Co. LZd.: Belg. 883,444 (1980), Hiyachi H. and all: Ger. Offen. 3,032,636 (1981)
Structurally the β(1->3) glukans are usually differentiated by the type of microorganism which produce them. The basic chain is β(1->3) glukan, which can however be branched in different ways and the difference in structure in branches may result in changes in pharmalogical properties. The best known microbial β(1->3) glukans are lentilan, schyzophylan and skleroglukan (Chichara G. and all: Nature 222, 687 (1969), Chichara G. and all: Cancer Res.30, 2776 (1981), Komatsu N. and all: Gann 60,137 (1969), Singh P.P. and all: Carbohydr. Res. 37,245 (1974) ).
In Czechoslovakia it is known the yeast glukan, which isolated from the cell walls of bakery yeast and which has in the branches β(1->6) bonds. Isolation of the yeast glukan is technologically difficult and the yields are low, only about -10 weight % of the dry cell walls of the yeast.
We were therefore seeking better sources of microbial glukan and a simpler method of preparation, which is the subject matter of this invention.
The subject matter of the invention is a new homopolymer β (1->3) glukan , which is branched in the main chain in the position -0-6 with bonding β (1->6) and β (1->4) with frequency of 1 branch to each fourth anhydroglucose unit in th main chain of the β (1->3) glukan. The new polysacharide is isolated from fruit- body of the oyster fungus, preferably from stipes, by alkali hydrolysis of the protein part of fruit-body and by extraction of soluble glukans in aqueous sodium hydroxide or potassium hydroxide in concentration 0,1 to 0,2 mol/1 at a temperature of 95 to 100 °C for 2 to 3 hours. After then the insoluble β (1->3) glukan is collected by filtration and washed, then bleached with sodium chlorite (NaCIO2) at 50 to 60 °C at a pH of 3,5 to 4,5 for 6 to 12 hours. The bleached product is washed with water, dehydrated with organic solvent, preferably with ethanol or acetone and finally the organic solvent is removed by vaccum drying at temperatures up to 60 °C.
The structure of the isolated polysacharide was analysed to show that the new fungal polysacharide is β(1->3) glukan, in which each fourth anhydroglukose unit is substituted on -0-6 with a non-reducing terminal unit. In the branch chains of the polysacharide there are 4,4 resp. 3,7 units bonded β (1->6) and β (1->4). It is differentiated markedly from the yeast glukan by number of branches and the type of bonding of the branches.
We further found that the fungal glukan using the new method can be isolated from fruit-bodies of the oyster fungus by a much simpler technique than the method of isolation from yeast. For the hydrolysis of the protein component the concentration of sodium hydroxide resp. potassium hydroxide is 0,1 to 0,2 mol/1 which is 5to 10 times lower than isolation from yeast. Also the extraction temperature of the protein is lower, usually temperature of 90 to 100°C is sufficient, which allows extraction in unpressurized vessels. After hydrolysis of the proteins, the insoluble glukan does not have to be treated in disintegrators and centrifuge, which reduces the investment of the prouduction unit. We determined that oyster fungus is a rich source of β (1->3) glukan, since with the new process it is possible to obtain yields of about 50 weight % of the dry raw material. This is 5 times the yield of yeast glukan based on dry yeast.
The main advantage of the new process is the fact that from oyster fungus it is possible to isolate a polysacharide previously unknown structure as it refers to the branching of the main chain of β (1->3) glukan and furthemore that the isolation at high yields is technologically significantly simplified enabling meeting of ecological demands.
Example 1
10 kg stipes (in dry 10 to 11%) of freshly cut oyster fungus (Pleurotus ostreatus) is defibred in kitchen mixer with 5 times the amount of sodium hydroxide water solution in concentration of 0,15 mol/1 and the defibred suspension is heated to 95 to 100°C and allowed to react under constant mixing for 2 hours at the stated temperature. It is advantegeous to use as the reactor an enameled 100 liter Lampart heated with steam externally. At the end of the reaction the mixture is filtered with a cloth filter medium on an appropriete filter and the insoluble product on the filter medium is washed with deionized water with total volume of 50 liters. The washed fungal glukan is transfered from the filter medium back in the reactor, diluted with deionized water to 50 liters and acidified with acetic acid to pH 3,5 to 4,6. Then 30 g of sodium chorite (NaClO2) dissolved in 2 liters o deionized water is added to the suspension of glukan in the reactor, heated to 50°C and kept at that temperature for 6 to 12 hours. The unreacted chlordioxid is eliminated by addition of disulphite (Na2S2O5) and the insoluble product is collected by filtration , then washed thoroughly with deionized water, which is displaced from the polysacharide by ethanol , followed by acetone and after removal of the solvent by sunction the product is vacuum dried at 60°C. The product is white, fibrous polysacharide β (1->3) glukan with the yield of 505 g, nitrogen content less than 0,3%, ash content less than 0,2 and molecular weight of 240000.
Example 2
The same product as in Example 1, except that potassium hydroxide in concentration of 0,2 mo1/1 is used as the base. The resulting product has the same properties as that in Example 1.
The invention has utility particulary in veterinary and human medicine as an immunostimulator, as an adjuvant invaccination, in galenic pharmacology in preparation of ointments with specific activity.

Claims

Claims
1. A new fungal glukan of the formula
β β β
-3/-Glukp-/1 ->3/-Glukp-/1 ->3/-Glukp-/1 ->3/-Glukp-/1-
Figure imgf000007_0001
R,R'
wherein R represents glucose
R' represents disacharides
Glukp represents -D-glukopyranose ,
said glukan as a homopolymer β (1->3) glukan, which is substituted in the -0-6 positionon each third to fifth anhydroglucose unit with glucose or disacharides.
2. A glukan according to claim 1 of formula
β β β
-3/-Glukp-/1 ->3/-Glukp-/1 ->3/-Glukp-/1 ->3/-Glukp-/1-
Figure imgf000007_0002
R,R'
wherein R represents Glukp -/1-
R' represents Glukp -/1->6/-Glukp-/1- or
Glukp -/1->4/-Glukp-/1-
Glukp represents -D-glukapyranose
ration R:R' is 2:1,
said glukan is a homopolymer β (1->3)glukan, which is substituted in the -0-6 position on each fourth
anhydroglucose unit with glucose or sacharides which contain the (1->6) or (1->4) bonding, the proportion of glucose to disacharide in the branches is 2:1, said glukan is produced by oyster fungus (Pleurotus ostreatus).
3. A process for preparing a fungal glukan according to Claims 1 or 2, which comprises alkali hydrolysis of protein part of the fruit-body of oyster fungus, preferably of its stipes and extraction of the soluble glukans with aqueous alkali hydroxide at a concentration of 0,1 to 0,2 mol/1 at temperature 95 to 100 °C for 2 to 3 hours, after which the insoluble β (1->3) glukan is collected by filtration and after washing is bleached with sodium chlorite NaCIO2 at 50 to 60 °C at pH 3,5 to 4,5 for 6 to 12 hours, after which the bleached product is washed and dehydrated with an organic solvent, preferably ethanol or acetone, and finally the organic solvent is removed by vacuum drying at temperature up to 60 °C.
PCT/CS1991/000002 1991-12-16 1991-12-16 A new fungal glukan and its preparation WO1993012243A1 (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6284885B1 (en) 1997-09-01 2001-09-04 Seikagaku Corporation Process for preparing (1→3)-β-D-glucan from fungi
WO2002006295A1 (en) * 2000-07-18 2002-01-24 Proteome Systems Ltd The recovery of oxygen linked oligosaccharides from mammal glycoproteins
WO2002085950A1 (en) * 2001-04-23 2002-10-31 Gabrizova Leona A method of isolation of immunostimulating glucan from oyster mushroom
WO2006119783A1 (en) * 2005-05-10 2006-11-16 Mubarak City For Scientific Research And Technology Applications Production of anticancer compound from oyster mushroom fruit bodies
AT513524A1 (en) * 2012-10-26 2014-05-15 Matilda Matzner Obtaining highly clean beta-1,3 / 1, 6-D-glucan

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ305050B6 (en) * 2009-07-22 2015-04-15 Jihočeská univerzita v Českých Budějovicích, Zemědělská fakulta Process for preparing raw {beta}glucan from sporocarps of oyster cap mushroom (Pleurotus ostreatus) or from oyster cap mushroom culturing substrate

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2138329A1 (en) * 1970-07-31 1972-02-10 Ajinomoto Co, Ine , Tokio Polysaccharides and processes for their preparation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2138329A1 (en) * 1970-07-31 1972-02-10 Ajinomoto Co, Ine , Tokio Polysaccharides and processes for their preparation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CARBOHYDRATE RESEARCH. vol. 140, 1985, AMSTERDAM NL pages 93 - 100; YUKO YOSHIOKA ET AL.: 'ANTITUMOR POLYSACCHARIDES FROM P. ostreatus (FR) QUEL.: ISOLATION AND STRUCTURE OF A BETA-GLUCAN' *
CARBOHYDRATE RESEARCH. vol. 141, no. 1, 1 August 1985, AMSTERDAM NL pages 111 - 119; KAZUYOSHI IINO ET AL.: 'STRUCTURAL CHARACTERISATION OF A NEUTRAL ANTITUMOUR BETA-D-GLUCAN EXTRACTED WITH HOTH SODIUM HYDROXIDE FROM CULTURED FRUIT BODIES OF GRIFOLA FRONDOSA' *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6284885B1 (en) 1997-09-01 2001-09-04 Seikagaku Corporation Process for preparing (1→3)-β-D-glucan from fungi
WO2002006295A1 (en) * 2000-07-18 2002-01-24 Proteome Systems Ltd The recovery of oxygen linked oligosaccharides from mammal glycoproteins
WO2002085950A1 (en) * 2001-04-23 2002-10-31 Gabrizova Leona A method of isolation of immunostimulating glucan from oyster mushroom
WO2006119783A1 (en) * 2005-05-10 2006-11-16 Mubarak City For Scientific Research And Technology Applications Production of anticancer compound from oyster mushroom fruit bodies
AT513524A1 (en) * 2012-10-26 2014-05-15 Matilda Matzner Obtaining highly clean beta-1,3 / 1, 6-D-glucan
AT14719U1 (en) * 2012-10-26 2016-04-15 Matilda Matzner Obtaining highly clean beta-1,3 / 1,6-D glucan

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CS63090A2 (en) 1991-12-17
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