WO2000008201A9 - Method for obtaining high-molecular biologically active immunomodulating polysaccharides from saccharomyces cerevisiae yeast - Google Patents

Method for obtaining high-molecular biologically active immunomodulating polysaccharides from saccharomyces cerevisiae yeast

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Publication number
WO2000008201A9
WO2000008201A9 PCT/EP1999/005681 EP9905681W WO0008201A9 WO 2000008201 A9 WO2000008201 A9 WO 2000008201A9 EP 9905681 W EP9905681 W EP 9905681W WO 0008201 A9 WO0008201 A9 WO 0008201A9
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water
cell wall
glucan
cell
insoluble glucan
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PCT/EP1999/005681
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German (de)
French (fr)
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WO2000008201A1 (en
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Werner-Michael Kulicke
Franziskus Karl Thomas Kath
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Kulicke Werner Michael
Franziskus Karl Thomas Kath
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Application filed by Kulicke Werner Michael, Franziskus Karl Thomas Kath filed Critical Kulicke Werner Michael
Priority to AU55106/99A priority Critical patent/AU5510699A/en
Publication of WO2000008201A1 publication Critical patent/WO2000008201A1/en
Publication of WO2000008201A9 publication Critical patent/WO2000008201A9/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast

Definitions

  • the invention relates to a method for obtaining high-molecular, biologically active immunomodulating polysaccharides from yeast Saccharomyces Cerevisiae.
  • the importance of the immune system to ward off diseases in human and animal organisms is well known. It is also generally known that there are polysaccharides in the cell wall of yeast which have an immunostimulating effect in animal and human organisms and can be used to ward off diseases. These polysaccharides are mannan and glucan.
  • the mannan consisting of 1, 2-, 1, 3- and 1, 6- ⁇ -glycosidically linked mannose units, is bound to a protein matrix and sits on the outer cell wall of the yeast cell, while the glucan 1, 3- ß-glycosidically linked with a few 1,6-ß-glycosidic side chains and is located in the inner cell wall.
  • the object of the invention is to design the method of the type mentioned at the outset in such a way that an optimal immunomodulating effect is achieved when the required process steps are reduced.
  • Extractions in the isolation of the polysaccharides avoid degradation, since extreme pH values are avoided by the use of enzymes.
  • the number of procedural steps required is significantly reduced.
  • the cell walls are first separated from the cell interior via a mechanical cell disruption and the cell wall sections obtained are cleaned and dried. Freeze drying is advantageous. However, the use of other freezing methods is also possible. Then there is an impact of technical Enzymes on the isolated cell wall sections of the yeast Saccharomyces Cerevisiae, whereby the substances can be isolated purely.
  • Both mannan and glucan are obtained, which can be used directly for immunostimulating purposes, for example in skin creams. Since the glucan is insoluble in water, it can be converted into water-soluble derivatives by chemical reactions.
  • mannan components can be isolated that have molecular weights in the range of 20,000 to 400,000 g / mol, show a specific, immunomodulating reaction and are not cytotoxic. Under certain circumstances, these Mannan components can also be used to combat asthma diseases, since they showed an immunosuppressive effect in in vitro tests.
  • Commercial enzymes can advantageously be used to isolate the polysaccharides, such as protease (SIGMA-ALDRICH), pronase (MERCK), both from Streptomyces griseus, Lyticase, also called zymoiase (SIGMA-ALDRICH), as well as enzyme cocktails from Helix pomatia and from Cytophaga ( MERCK). However, it is also possible to use other suitable enzymes or enzyme cocktails.
  • glucan derivatives and mannan are effective even without dose-effect optimization, and this even in the in vivo test to check the anti-tumor activity. A tumor does not grow again even after renewed incubation.
  • glucan derivatives and mannan there is no toxicity or only a low toxicity.
  • the agent obtained according to the invention can be used in a variety of ways. For wound healing, it is possible to use the agent physically as a moisture-storing cover and for open wounds to activate immune cells. The agent also works against pathogenic bacteria and viruses. In addition, the agent according to the invention has a regulating effect on autoimmune diseases such as e.g. Asthma and epilepsy.
  • the application can take place via ointments, emulsions, gel solutions, by spraying or microencapsulation, orally by pills or solutions or intramuscularly, for example by spraying into the abdominal cavity or intravenously, which has the greatest potential for action.
  • the agent according to the invention can be used in HIV infections, tumors in both humans and animals, tuberculosis, acute sepsis, bacteria, fungi, increased interferon production, to increase the number of leukocytes, to heal wounds, for example to protect against radiation damage and Infections and also to increase transplant immunity, to
  • the yeast cells 1 are mechanically torn in the first process stage A by the action of shear forces, so that the cell walls are separated from the cell interior.
  • a grinding container 2 is filled with a suspension of yeast cells and glass beads and shaken at a high frequency in a vibrating mill 3, for example. Glass beads with a diameter of 0.75-1.5 mm are preferably used.
  • the frequency of the vibratory mill 3 is, for example, 1600 s ' 1 .
  • the cell wall material 4 obtained in this way is then cleaned in a further process stage B by centrifugation, or else is cleaned on microporous membranes by washing.
  • the cell wall material 4 thus prepared is then freeze-dried.
  • the cell wall material 4 is subjected to an enzymatic digestion.
  • Specific enzymes such as pronase or glucanase or specific enzyme cocktails such as zymoiase, cytophaga, helicase or cellulase destroy the protein matrix in the outer cell wall of the cell wall material 4 and release soluble mannan 5.
  • the insoluble glucan 6 is separated by centrifugation and processed separately.
  • the Mannan 5 can be purified by dialysis or by chromatography.
  • the water-insoluble glucan obtained can be chemically converted into a soluble form by carboxymethylation, phosphation, sulfation, sulfonation, glycosidation or the like.
  • the solubility and thereby the bioavailability can be increased by the action of ultrasound and undesirable side effects for a person to be treated can be prevented.
  • the water-insoluble glucan can be autoclaved briefly at temperatures up to 200 ° C.
  • the water-insoluble glucan is autoclaved for more than 5 minutes, this can take place at temperatures up to 150 ° C.
  • the water-soluble glucan can also be subjected to a stretching process in a solvent DMSO. For this purpose, it is possible for the water-soluble glucan to be passed through an aperture plate, a porous ball bed, a roller mill or a device for a capillary flow.

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Abstract

The invention relates to a method for obtaining high molecular biologically active immumodulating polysaccharide from Saccharomyces cerevisiae yeast. In a first step, the yeast cells are mechanically disintegrated under the effect of shear forces in such a way that the cell walls are separated from the inner part of the cell. In a second step, the cell wall material obtained is purified and dried. The cell wall material is then subjected to enzymatic digestion in a third step and the aqueous insoluble glucan thus formed is obtained as a solid by centrifugation and the aqueous soluble mannan is obtained in the supernatant. Said method makes it possible to obtain high yields of glucan and mannan with intact native structure.

Description

Verfahren zur Gewinnung hochmolekularer biologisch aktiver immunmodulierender Polysaccharide aus Hefe Saccharomyces CerevisiaeProcess for the production of high-molecular biologically active immunomodulating polysaccharides from yeast Saccharomyces Cerevisiae
Die Erfindung betrifft ein Verfahren zur Gewinnung hochmolekularer biologisch aktiver immunmodulierender Polysaccharide aus Hefe Saccharomyces Cerevisiae.The invention relates to a method for obtaining high-molecular, biologically active immunomodulating polysaccharides from yeast Saccharomyces Cerevisiae.
Die Bedeutung des Immunsystems zur Abwehr von Krankheiten in menschlichen und tierischen Organismen ist bekannt. Es ist ferner allgemein bekannt, daß es in der Zellwand von Hefe Polysaccharide gibt, die in tierischen und menschlichen Organismen immunstimulierend wirken und zur Abwehr von Krankheiten eingesetzt werden können. Diese Polysaccharide sind Mannan und Glucan. Das Mannan, bestehend aus 1 ,2-, 1 ,3- und 1 ,6-α-glykosidisch verknüpften Mannose-Einheiten, ist an eine Protein-Matrix gebunden und sitzt auf der äußeren Zellwand der Hefezelle, während das Glucan 1 ,3-ß- glykosidisch verknüpft ist mit wenigen 1 ,6-ß-glykosidischen Seitenketten und sich in der inneren Zellwand befindet. Zur Isolierung dieser beiden Polysaccharide wurden zahlreiche Verfahren entwickelt, bei denen durch Einwirkung von Säuren und Laugen die beiden Zellwandkomponenten getrennt werden. Bei diesen herkömmlichen Verfahren sind jedoch viele Verfahrensschritte erforderlich und darüber hinaus wird die native Struktur der Zeliwand-Polysaccharide beschädigt, so daß die immunabwehrfördemden Effekte beeinträchtigt werden.The importance of the immune system to ward off diseases in human and animal organisms is well known. It is also generally known that there are polysaccharides in the cell wall of yeast which have an immunostimulating effect in animal and human organisms and can be used to ward off diseases. These polysaccharides are mannan and glucan. The mannan, consisting of 1, 2-, 1, 3- and 1, 6-α-glycosidically linked mannose units, is bound to a protein matrix and sits on the outer cell wall of the yeast cell, while the glucan 1, 3- ß-glycosidically linked with a few 1,6-ß-glycosidic side chains and is located in the inner cell wall. To isolate these two polysaccharides, numerous processes have been developed in which the two cell wall components are separated by the action of acids and alkalis. In these conventional methods, however, many process steps are required and, moreover, the native structure of the cell wall polysaccharides is damaged, so that the immune defense-promoting effects are impaired.
Die Aufgabe der Erfindung besteht darin, das Verfahren der eingangs genannten Art so zu gestalten, daß bei einer Verminderung der erforderlichen Verfahrensschritte ein optimaler immunmodulierender Effekt erzielt wird.The object of the invention is to design the method of the type mentioned at the outset in such a way that an optimal immunomodulating effect is achieved when the required process steps are reduced.
Erfindungsgemäß erfolgt die Lösung der Aufgabe durch die Merkmale des Anspruchs 1. Vorteilhafte Ausgestaltungen der Erfindung werden in den abhängigen Ansprüchen beschrieben.According to the invention, the object is achieved by the features of claim 1. Advantageous embodiments of the invention are described in the dependent claims.
Nach der Erfindung wird gegenüber den herkömmlichen sauren und alkalischenAccording to the invention, compared to the conventional acidic and alkaline
Extraktionen bei der Isolierung der Polysaccharide eine Degradation vermieden, da durch den Einsatz von Enzymen extreme pH-Werte vermieden werden. Außerdem wird die Anzahl der erforderlichen Verfahrensschritte deutlich reduziert. Gemäß der Erfindung werden zunächst über einen mechanischen Zellaufschluß die Zellwände vom Zellinneren abgetrennt und die erhaltenen Zellwandabschnitte gereinigt und getrocknet. Vorteilhaft ist eine Gefriertrocknung. Es ist aber auch die Anwendung anderer Gefrierverfahren möglich. Dann erfolgt eine Einwirkung von technischen Enzymen auf die isolierten Zellwandabschnitte der Hefe Saccharomyces Cerevisiae, wodurch die Substanzen rein isoliert werden können. Man erhält sowohl Mannan als auch Glucan, welche direkt für immunstimulierende Zwecke z.B. in Hautcremes eingesetzt werden können. Da das Glucan in Wasser unlöslich ist, kann es durch chemische Umsetzungen in wasserlösliche Derivate überführt werden. Hierzu sind zahlreiche Derivatisierungsmethoden bekannt. Es können spezielle Mannan- Komponenten isoliert werden, die Molmassen im Bereich von 20.000 bis 400.000 g/mol haben, eine spezifische , immunmodulierende Reaktion zeigen und dabei aber nicht cytotoxisch wirken. Diese Mannan-Komponenten können unter Umständen auch zur Bekämpfung von Asthma-Erkrankunen genutzt werden, da sie in in vitro Tests eine immunsuppressive Wirkung zeigten. Zur Isolierung der Polysaccharide können vorteilhaft kommerzielle Enzyme eingesetzt werden, wie Protease (SIGMA-ALDRICH), Pronase (MERCK), beide aus Streptomyces griseus, Lyticase, auch Zymoiyase genannt (SIGMA-ALDRICH), wie auch Enzymcocktails aus Helix pomatia und aus Cytophaga (MERCK). Es ist aber auch die Verwendung anderer geeigneter Enzyme oder Enzymcocktails möglich.Extractions in the isolation of the polysaccharides avoid degradation, since extreme pH values are avoided by the use of enzymes. In addition, the number of procedural steps required is significantly reduced. According to the invention, the cell walls are first separated from the cell interior via a mechanical cell disruption and the cell wall sections obtained are cleaned and dried. Freeze drying is advantageous. However, the use of other freezing methods is also possible. Then there is an impact of technical Enzymes on the isolated cell wall sections of the yeast Saccharomyces Cerevisiae, whereby the substances can be isolated purely. Both mannan and glucan are obtained, which can be used directly for immunostimulating purposes, for example in skin creams. Since the glucan is insoluble in water, it can be converted into water-soluble derivatives by chemical reactions. Numerous derivatization methods are known for this. Special mannan components can be isolated that have molecular weights in the range of 20,000 to 400,000 g / mol, show a specific, immunomodulating reaction and are not cytotoxic. Under certain circumstances, these Mannan components can also be used to combat asthma diseases, since they showed an immunosuppressive effect in in vitro tests. Commercial enzymes can advantageously be used to isolate the polysaccharides, such as protease (SIGMA-ALDRICH), pronase (MERCK), both from Streptomyces griseus, Lyticase, also called zymoiase (SIGMA-ALDRICH), as well as enzyme cocktails from Helix pomatia and from Cytophaga ( MERCK). However, it is also possible to use other suitable enzymes or enzyme cocktails.
Der Vorteil der enzymatischen Isolierung von Hefeinhaltsstoffen besteht darin, daß Glucanderivate und Mannan bereits ohne Dosis-Wirkungsoptimierung wirksam sind und dies sogar im in vivo Test zur Überprüfung der Anti-Tumoraktivität. Ein Tumor wächst auch nach erneuter Inkubation nicht wieder an. Durch Anwendung der Glucanderivate und Mannan besteht auch keine Toxizität, bzw. nur eine geringe Toxizität.The advantage of the enzymatic isolation of yeast ingredients is that glucan derivatives and mannan are effective even without dose-effect optimization, and this even in the in vivo test to check the anti-tumor activity. A tumor does not grow again even after renewed incubation. By using the glucan derivatives and mannan there is no toxicity or only a low toxicity.
Das nach der Erfindung gewonnene Mittel kann vielfältig angewandt werden. Für die Wundheilung ist es möglich, das Mittel rein physikalisch als feuchtigkeitsspeichemde Abdeckung und bei offenen Wunden zur Aktivierung von Immunzellen zu verwenden. Ferner wirkt das Mittel gegen pathogene Bakterien und Viren. Außerdem hat das erfindungsgemäße Mittel eine regulierende Wirkung auf Autoimmunkrankheiten wie z.B. Asthma und Epilepsie.The agent obtained according to the invention can be used in a variety of ways. For wound healing, it is possible to use the agent physically as a moisture-storing cover and for open wounds to activate immune cells. The agent also works against pathogenic bacteria and viruses. In addition, the agent according to the invention has a regulating effect on autoimmune diseases such as e.g. Asthma and epilepsy.
Die Anwendung kann über Salben, Emulsionen, Gellösungen, durch Spritzen oder Mikroverkapselung, oral durch Pillen oder Lösungen oder intramuskulär z.B. durch Spritzen in die Bauchhöhle oder intravenös erfolgen, was ein höchstes Wirkungspotenzial hat. Der Einsatz des erfindungsgemäßen Mittels ist möglich bei HIV- Infektionen, Tumoren sowohl bei Menschen wie auch Tieren, Tuberkulose, akuter Sepsis, Bakterien, Pilze, gesteigerter Interferonproduktion, zur Steigerung der Leukozytenzahl, zur Wundheilung wie z.B. zum Schutz vor Strahlenschäden und Infektionen und darüber hinaus zur Erhöhung der Transplantationsimmunität, zurThe application can take place via ointments, emulsions, gel solutions, by spraying or microencapsulation, orally by pills or solutions or intramuscularly, for example by spraying into the abdominal cavity or intravenously, which has the greatest potential for action. The agent according to the invention can be used in HIV infections, tumors in both humans and animals, tuberculosis, acute sepsis, bacteria, fungi, increased interferon production, to increase the number of leukocytes, to heal wounds, for example to protect against radiation damage and Infections and also to increase transplant immunity, to
Bekämpfung von Trauma, zur Erhöhung von Blutbildung und zur Impfunterstützung.Combating trauma, increasing blood formation and vaccination support.
Ferner ist ebenso eine umfangreiche Anwendung bei Tieren zur Immunförderung möglich.Furthermore, extensive use in animals for immune promotion is also possible.
Im folgenden wird das erfindungsgemäße Verfahren anhand der in der Zeichnung schematisch dargestellten Verfahrensabläufe näher erläutert.The method according to the invention is explained in more detail below with reference to the method sequences shown schematically in the drawing.
Die Hefezellen 1 werden in der ersten Verfahrensstufe A durch Einwirkung von Scherkräften mechanisch zerrissen, so daß die Zellwände vom Zellinneren getrennt werden. Hierzu wird ein Mahlbehälter 2 mit einer Suspension aus Hefezellen und Glasperlen gefüllt und in z.B. einer Schwingmühle 3 mit hoher Frequenz geschüttelt. Als Glasperlen werden vorzugsweise solche mit einem Durchmesser von 0,75 - 1 ,5 mm verwendet. Die Frequenz der Schwingmühle 3 beträgt z.B. 1600 s'1. Das so gewonnene Zellwandmaterial 4 wird dann in einer weiteren Verfahrensstufe B durch Zentrifugation gereinigt oder aber an mikroporösen Membranen durch Waschen gereinigt. Das so aufbereitete Zellwandmaterial 4 wird dann gefriergetrocknet. In der dann folgenden Verfahrensstufe C wird das Zellwandmaterial 4 einem enzymatischen Aufschluß unterzogen. Spezifische Enzyme wie Pronase oder Glucanase oder spezifische Enzymcocktails wie Zymoiyase, Cytophaga, Helicase oder Cellulase zerstören in der äußeren Zellwand des Zellwandmaterials 4 die Proteinmatrix und setzen lösliches Mannan 5 frei. Das unlösliche Glucan 6 wird durch Zentrifugation abgetrennt und gesondert aufbereitet. Das Mannan 5 kann durch Dialyse oder chromatographisch gereinigt werden.The yeast cells 1 are mechanically torn in the first process stage A by the action of shear forces, so that the cell walls are separated from the cell interior. For this purpose, a grinding container 2 is filled with a suspension of yeast cells and glass beads and shaken at a high frequency in a vibrating mill 3, for example. Glass beads with a diameter of 0.75-1.5 mm are preferably used. The frequency of the vibratory mill 3 is, for example, 1600 s ' 1 . The cell wall material 4 obtained in this way is then cleaned in a further process stage B by centrifugation, or else is cleaned on microporous membranes by washing. The cell wall material 4 thus prepared is then freeze-dried. In the subsequent process step C, the cell wall material 4 is subjected to an enzymatic digestion. Specific enzymes such as pronase or glucanase or specific enzyme cocktails such as zymoiase, cytophaga, helicase or cellulase destroy the protein matrix in the outer cell wall of the cell wall material 4 and release soluble mannan 5. The insoluble glucan 6 is separated by centrifugation and processed separately. The Mannan 5 can be purified by dialysis or by chromatography.
Nachstehend werden zwei Ausführungsbeispiele der Erfindung beschrieben:Two embodiments of the invention are described below:
1. Ausführungsbeispiel1st embodiment
5,0 g Hefezellwände aus dem mechanischen Aufschluß wurden in 115 ml Trispuffer Lösung (Tri(hydroxymethyl)aminoethan, mit Salzsäure auf pH = 7,5 eingestellt) suspendiert und auf 37,0°C temperiert. 10,6 mg Pronase E (Streptomyces griseus, Fa. MERCK) wurden in 5 ml Puffer-Lösung gelöst und zum Reaktionsgemisch hinzugegeben. Nach sechs Stunden wurde der Feststoff abzentrifugiert und der Überstand dialysiert. Nach Gefriertrocknung erhielt man Mannan aus dem Überstand, während im Feststoff 2,9 g Glucan zu finden sind. 2. Ausführungsbeispiel5.0 g of yeast cell walls from the mechanical digestion were suspended in 115 ml of Tris buffer solution (tri (hydroxymethyl) aminoethane, adjusted to pH = 7.5 with hydrochloric acid) and temperature-controlled at 37.0 ° C. 10.6 mg of Pronase E (Streptomyces griseus, MERCK) were dissolved in 5 ml of buffer solution and added to the reaction mixture. After six hours the solid was centrifuged off and the supernatant dialyzed. After freeze-drying, mannan was obtained from the supernatant, while 2.9 g of glucan can be found in the solid. 2nd embodiment
1 ,55 g Hefezellwände aus dem mechanischen Aufschluß wurden in 60 ml Trispuffer-1.55 g of yeast cell walls from the mechanical digestion were placed in 60 ml of Tris buffer
Lösung (Tri(hydroxymethyl)aminoethan, mit Salzsäure auf pH = 7,5 eingestellt) suspendiert und auf 37,0°C temperiert. 462 mg Protease (Streptomyces griseus, Fa. SIGMA) wurden in 5 ml Puffer-Lösung gelöst und zum Reaktionsgemisch hinzugegeben. Nach 6,5 Stunden wurde der Feststoff abzentrifugiert und der Überstand dialysiert. Nach Gefriertrocknung erhielt man 352 mg Mannan aus dem Überstand, während im Feststoff 522 mg Glucan zu finden sind.Solution (tri (hydroxymethyl) aminoethane, adjusted to pH = 7.5 with hydrochloric acid) and heated to 37.0 ° C. 462 mg of protease (Streptomyces griseus, SIGMA) were dissolved in 5 ml of buffer solution and added to the reaction mixture. After 6.5 hours the solid was centrifuged off and the supernatant dialyzed. After freeze-drying, 352 mg of mannan were obtained from the supernatant, while 522 mg of glucan can be found in the solid.
Das gewonnene wasserunlösliche Glucan kann chemisch durch Carboxymethylirung, Phosphatierung, Sulfatierung, Sulfonierung, Glycosidierung oder dergleichen in eine lösliche Form gebracht werden. Bei dem gewonnenen wasserunlöslichen Glucan kann durch Einwirkung von Ultraschall die Löslichkeit und dadurch die Bioverfügbarkeit erhöht und für eine zu behandelnde Person unerwünschte Nebenwirkungen verhindert werden. Es ist auch möglich, das gewonnene wasserunlösliche Glucan sowohl chemisch, wie auch durch Ultraschall zu behandeln. Ferner ist es möglich, das gewonnene wasserunlösliche Glucan bei unterschiedlichen Temperaturen zu behandeln. Kurzzeitig kann das Autoklavieren des wasserunlöslichen Glucans bei Temperaturen bis 200 C erfolgen. Bei einer Zeit des Autoklavierens des wasserunlöslichen Glucans länger als 5 min kann dies bei Temperaturen bis 150 C erfolgen. Das wasserlösliche Glucan kann auch in einem Lösungsmittel DMSO einem Dehnströmvorgang unterworfen werden. Hierzu ist es möglich, daß das wasserlösliche Glucan durch eine Lochblende, eine poröse Kugelschüttung, eine Rollenmühle oder eine Einrichtung für eine Kapillarströmung geführt wird. The water-insoluble glucan obtained can be chemically converted into a soluble form by carboxymethylation, phosphation, sulfation, sulfonation, glycosidation or the like. In the water-insoluble glucan obtained, the solubility and thereby the bioavailability can be increased by the action of ultrasound and undesirable side effects for a person to be treated can be prevented. It is also possible to treat the water-insoluble glucan obtained both chemically and by ultrasound. It is also possible to treat the water-insoluble glucan obtained at different temperatures. The water-insoluble glucan can be autoclaved briefly at temperatures up to 200 ° C. If the water-insoluble glucan is autoclaved for more than 5 minutes, this can take place at temperatures up to 150 ° C. The water-soluble glucan can also be subjected to a stretching process in a solvent DMSO. For this purpose, it is possible for the water-soluble glucan to be passed through an aperture plate, a porous ball bed, a roller mill or a device for a capillary flow.

Claims

P atent a sprü che 1. Verfahren zur Gewinnung hochmolekularer biologisch aktiver immunmodulierenderP atent a sprü che 1. Process for obtaining high-molecular biologically active immunomodulating
Polysaccharide aus Hefe Saccharomyces Cerevisiae, dadurch gekennzeichnet, daß in einer ersten Verfahrensstufe A die Hefezellen durch Einwirkung von Scherkräften mechanisch soweit zerrissen werden, daß die Zellwände vom Zellinneren getrennt sind und daß dann die Zellwandbestandteile von den Bestandteilen des Zellinneren getrennt werden, daß dann in einer zweiten Verfahrensstufe B das in der ersten Verfahrensstufe A gewonnene Zellwandmaterial gereinigt und getrocknet wird und daß dann das Zellwandmaterial in einer dritten Verfahrensstufe C einem enzymatischen Aufschluß unterzogen und das hierbei entstehende wasserunlösliche Glucan durch Zentrifugation als Feststoff und wasserlösliche Mannan im Überstand gewonnen wird.Polysaccharides from yeast Saccharomyces Cerevisiae, characterized in that in a first process step A, the yeast cells are mechanically torn apart by the action of shear forces to such an extent that the cell walls are separated from the cell interior and that the cell wall components are then separated from the components of the cell interior, that then in one second process stage B, the cell wall material obtained in the first process stage A is cleaned and dried, and then the cell wall material is subjected to an enzymatic digestion in a third process stage C and the water-insoluble glucan formed in this process is obtained by centrifugation as a solid and water-soluble mannan in the supernatant.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß das in der ersten Verfahrensstufe A gewonnene Zellwandmaterial gefriergetrocknet wird.2. The method according to claim 1, characterized in that the cell wall material obtained in the first process stage A is freeze-dried.
3. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die in der ersten3. The method according to claim 1, characterized in that in the first
Verfahrensstufe A gewonnenen Zellwandbestandteile durch Zentrifugation von den Bestandteilen des Zellinneren getrennt und vor der Gefriertrocknung gereinigt werden.Process stage A obtained cell wall components separated from the components of the cell interior by centrifugation and cleaned before freeze-drying.
4. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die in der ersten Verfahrensstufe A gewonnenen Zellwandbestandteile an mikroporösen Membranen von den Bestandteilen des Zellinneren getrennt und durch Waschen vor der Gefriertrocknung gereinigt werden.4. The method according to claim 1, characterized in that the cell wall components obtained in the first process stage A on microporous membranes are separated from the components of the cell interior and are cleaned by washing before freeze-drying.
5. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die gefriergetrockneten Zellwandbestandteile in einem Puffersystems bei einem pH-Wert von 3,0 bis 11 bei5. The method according to claim 1, characterized in that the freeze-dried cell wall components in a buffer system at a pH of 3.0 to 11 at
Temperaturen zwischen 20°C und 50°C über einen Zeitraum von vier bis 30 Stunden mit enzymatischen Aufschlußmitteln zur Reaktion gebracht werden.Temperatures between 20 ° C and 50 ° C are reacted with enzymatic disintegrants over a period of four to 30 hours.
6. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß als enzymatisches Aufschlußmittel Proteasen aus Streptomyces griseus und/oder Lyticase (Zymoiyase) verwendet werden. 6. The method according to claim 1, characterized in that proteases from Streptomyces griseus and / or lyticase (zymoiase) are used as the enzymatic disintegrant.
1 7. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß als enzymatiscnes1 7. The method according to claim 1, characterized in that as enzymatiscnes
Aufschlußmittel Enzymcocktails aus Helix pomatia und/oder aus Cytophaga verwendet werden.Disintegrant enzyme cocktails from Helix pomatia and / or from Cytophaga can be used.
5 8. Verfahren nach Anspruch 1 und 2, 6 oder 7, dadurch gekennzeichnet, daß als enzymatisches Aufschlußmittel ein Gemisch aus Enzymen und Enzymcocktails verwendet wird.5 8. The method according to claim 1 and 2, 6 or 7, characterized in that a mixture of enzymes and enzyme cocktails is used as the enzymatic disintegrant.
9. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß das gewonnene9. The method according to claim 1, characterized in that the obtained
10. wasserunlösliche Glucan chemisch durch Carboxymethylirung, Phosphatierung, Sulfatierung, Sulfonierung, Glycosidierung oder dergleichen in eine lösliche Form gebracht wird.10. Water-insoluble glucan is brought into a soluble form chemically by carboxymethylation, phosphating, sulfation, sulfonation, glycosidation or the like.
10. Verfahren nach Anspruch 1 dadurch gekennzeichnet, daß bei dem gewonnenen10. The method according to claim 1, characterized in that in the obtained
15 wasserunlöslichen Glucan durch Einwirkung von Ultraschall die Löslichkeit erhöht und für eine zu behandelnde Person unerwünschte Nebenwirkungen vermindert werden.15 water-insoluble glucan increases the solubility by the action of ultrasound and undesirable side effects are reduced for a person to be treated.
11. Verfahren nach Anspruch 9 und 10, dadurch gekennzeichnet, daß das gewonnene wasserunlösliche Glucan sowohl chemisch wie auch durch Ultraschall behandelt wird.11. The method according to claim 9 and 10, characterized in that the water-insoluble glucan obtained is treated both chemically and by ultrasound.
2020th
12. Verfahren nach Anspruch 9 bis 11 , dadurch gekennzeichnet, daß das gewonnene wasserunlösliche Glucan bei unterschiedlichen Temperaturen behandelt wird.12. The method according to claim 9 to 11, characterized in that the water-insoluble glucan obtained is treated at different temperatures.
13. Verfahren nach Anspruch 12, dadurch gekennzeichnet, daß das Autoklavieren des 25 wasserunlöslichen Glucans kurzzeitig bei Temperaturen bis 200 C erfolgt.13. The method according to claim 12, characterized in that the autoclaving of the 25 water-insoluble glucan is carried out briefly at temperatures up to 200 ° C.
13. Verfahren nach Anspruch 12 dadurch gekennzeichnet, daß das Autoklavieren des wasserunlöslichen Glucans länger als 5 min bei Temperaturen bis 150 C erfolgt.13. The method according to claim 12, characterized in that the autoclaving of the water-insoluble glucan takes place at temperatures up to 150 C for more than 5 min.
30 15. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß das wasserlösliche Glucan in einem Lösungsmittel DMSO einem Dehnströmungsvorgang unterworfen wird.15. The method according to claim 1, characterized in that the water-soluble glucan is subjected to an expansion flow process in a solvent DMSO.
16. Verfahren nach Anspruch 15, dadurch gekennzeichnet, daß das wasserlösliche Glucan durch eine Lochblende, eine poröse Kugelschüttung, eine Rollenmühle oder eine 35 Einrichtung für eine Kapillarströmung geführt wird. 16. The method according to claim 15, characterized in that the water-soluble glucan is passed through a pinhole, a porous ball bed, a roller mill or a device for a capillary flow.
PCT/EP1999/005681 1998-08-07 1999-08-05 Method for obtaining high-molecular biologically active immunomodulating polysaccharides from saccharomyces cerevisiae yeast WO2000008201A1 (en)

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KR101076209B1 (en) 2003-08-11 2011-10-24 르사프르 에 꽁빠니 Yeast cell walls for the treatment or prevention of hyperglycemia or for the stabilization of glycemia
CA2607004C (en) 2005-05-05 2016-01-26 Sensient Flavors Inc. Production of beta-glucans and mannans
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DE102016107140A1 (en) 2016-04-18 2017-10-19 Gea Mechanical Equipment Gmbh A process for recovering at least one or more beta-glucan compounds or a beta-glucan-containing solid suspension from yeast cells

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