CA1179957A - Procedure for the extraction of anabolic, respiration- promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue- culture purposes - Google Patents

Procedure for the extraction of anabolic, respiration- promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue- culture purposes

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Publication number
CA1179957A
CA1179957A CA000402970A CA402970A CA1179957A CA 1179957 A CA1179957 A CA 1179957A CA 000402970 A CA000402970 A CA 000402970A CA 402970 A CA402970 A CA 402970A CA 1179957 A CA1179957 A CA 1179957A
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Prior art keywords
culture
yeast
active substances
procedure
promoting
Prior art date
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Expired
Application number
CA000402970A
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French (fr)
Inventor
Karl-Heinz Jaeger
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Elkawi AG
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Elkawi AG
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06Fungi, e.g. yeasts
    • A61K36/062Ascomycota
    • A61K36/064Saccharomycetales, e.g. baker's yeast
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K10/00Animal feeding-stuffs
    • A23K10/10Animal feeding-stuffs obtained by microbiological or biochemical processes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06Lysis of microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor
    • C12N1/16Yeasts; Culture media therefor
    • C12N1/18Baker's yeast; Brewer's yeast
    • C12N1/185Saccharomyces isolates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/645Fungi ; Processes using fungi
    • C12R2001/85Saccharomyces
    • C12R2001/865Saccharomyces cerevisiae

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Polymers & Plastics (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • General Chemical & Material Sciences (AREA)
  • Botany (AREA)
  • Animal Husbandry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physiology (AREA)
  • Medical Informatics (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Food Science & Technology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Compounds Of Unknown Constitution (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

S u m m a r y The procedure is intended to make it possible to extract anabolic, respiration-promoting, low-molecular active substances. The active substances can be used for prophylactic, therapeutic and cell-culture and tissue-culture purposes. The active substances are obtained from a yeast plasmolysate, by the disintegrat-ion of yeast fungi at the moment of their maximum growth. The disintegration is achieved by the use of ultrasound under the simultaneous influence of proteo-lytic enzymes. The residue is subjected to dialysis and very carefully concentrated.

Description

Procedure for the extraction of anabolic, respiration-promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue-culture purposes ======================--===============================

This invention concerns a procedure for ~he extraction of anabolic, respiration-promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue-culture purposes.

It is known that yeast fungi of the Saccharomycetes family have a high content of active substances such as vitamins, enzymes and trace elements. To be mentioned in particular are the riboflavins and their derivatives, the vitamins of the B-complex, or the active substances of the respiratory chain, such as cytochrome C, which are extremely valuable for both humans and animals.

Apart from these known growth-promoting substances, yeast fungi also contain growth factors, some unknown, which can be described as true anabolics, such as are contained, for example, in embryonal tissue and particularly also in calf's blood, without which the growth of cells in vitro would not take place.

It was interesting to observe that such chemically still unidentified growth-promoting and respiration-promoting factors from yeast fungi possess a great simil-arity, from the functional point of view, to active sub-stances from calf 15 serum, which is positively manifested in the increased resistance of humans and animals against bacterial, viral and other lesions.

Efforts were then made to find therapeutically usable substances of this type in yeast plasmolysates, especially such substances with a regenerative action on degradation processes.

~ 3 ~ 11~9957 It was subsequently shown, however, that it was not a simple matter to identify or isolate substances with regenerative action from a yeast plasmolysate, since a yeast plasmolysate contains a large number of substances with the most widely different actions.

Regenerative processes are known to need a high level of metabolic efficiency, which is linked with an increase in the internal respiration. In order to detect the desired substances with regenerative and respiration-promoting properties from yeast plasmolysates, and also to determine the most suitable production process for the active principle, certain test procedures had to be used, by means of which the presence and the concentration of these substances could be demonstrated. Methods used for tissue cultures in vitro proved particularly suitable for this purpose.

Up till now it has not been possible to present the desired substances from yeast cells in a chemically pure form, since they are present in the yeast cell in only very small concentrations.

Surprisingly, it could be estahlished that low-molecular active substances with respiration-promoting or metabolism-activating and regenerative properties could be extracted from a yeast plasmolysate produced by the process which is the subject of this invention.

In the non-denatured yeast cell these active sub-stances can be detected either not at all or only with difficulty, since they are present in bound or inactive form and thus escape direct detection, so that they can also not react therapeutically. After the discovery of such active substances the task was to develop a process enabling these substances from yeast cells to be separ-ated from the high-molecular components of the cells, concentrated and presented in a form usable in therapy.

~ 4 ~ 1179957 Tests have shown that the usual procedures, such as freezing, salting out, heating, acid or alkaline plasmo-lysis, trituration etc., of the yeast cells, do not provide satisfactory results. The problem of how to destroy the yeast-cell membrane as thoroughly as possible, in order to obtain a high yield of active substance, has not yet been satisfactorily solved.

Up till now no suitable method was known for the purification of the extracted growth-promoting substances;
in particular, the procedures for deproteinization by precipitation of the non-denatured proteins of the yeast hyaloplasm by the usual methods, such as, for example, by heating or by the use of trichloroacetic acid, were not satisfactory, because with such precipitation of the protein the substances one is trying to extract are also precipitated out at the same time.

It has now been discovered that the most effective disintegration of the yeast cells is obtained by adding a sugar-containing solution, such as molasses, fruit-juice waste and similar material, to a yeast mass, then leaving this mass to ferment under adequate ventilation at 25C + 3 C for 1 to 3 days, and after this adding a proteolytic enzyme, such as papain for example, to the resultant yeast-mass suspension. Then, at the moment of optimum fermentation, the mass is spun by means of an ultrasound integrator of 50 Watt~cm2 output until the temperature of the yeast suspension has risen to about 40C, which occurs with a residue of 70-75~.
The solution is then cooled, buffered to a pH value of 6.2 and centrifuged in portions. The residues of the centrifugates are combined and then subjected to an end-dialysis, which is carried out against neutral ~uffered water. The whole is then carefully concentrated. In order to preserve the producl obtained by this procedure a well-known preservative agent is added.

~ 5 - 1179957 Surprisingly, it was observed that with the procedure which is the subject of this invention the yield of respiration-promoting active substances is three times as great as with a normally applied papain proteolysis, without the action of simultaneous ultrasound. This is all the more surprising, since it could not be foreseen.

For better understanding of the procedure, the in-vention is illustrated by the following practical example.

Practical examPle 100 litres of water containing about 5 kg of sugar-containing raw materials are added to 1 kg of yeast fungus, preferably a top-fermented or bottom-fermented yeast strain of the species, Saccharomyces cerevisiae.
The whole mixture is left to stand for 1 to 3 days at a temperature of about 25C, with good ventilation.
A proteolytic enzyme, preferably papain, is then added in the proportion of 2-5~ of the total biomass. The whole is then spun, by means of an ultrasound integrator of 50 Watt~cm2 output until the temperature of the bio-mass rises to 40C; the residue is then 70-75~. The whole solution is then brought to a pH value of 6.2 and centrifuged in portions. The centrifugates are combined and subjected to an end-dialysis, which is carried out at about 25-30C against neutral buffered water. Before the start of the end-dialysis a preservatiYe agent is added to the centrifugates, in a quantity sufficient to ensure that at the end of the final dialysis the con-centration of this agent in the dialysate is between 0.001 and 0.008%. As preservative agents, p-amino-benzoic acid methylester and/or p-aminobenzoic acid ethylester can be used to advantage.

It has been shown that this preservative lowers the oxygen uptake of, for example, a liver homogenate or a .

- 6 ~ 1~79957 yeast culture, by the active substance, by a maximum of about 1~%, compared with the active substance without any preservative agent. The end-dialysate is then care-fully concentrated to 50 mg per ml of dialysate.

The end-product is standardized and tested by being added, in a dilution of 1:100 to 1:10 000, to a normal tissue culture, in vitro, with a cell-count being carried out at the appropriate time. It has been shown that compared with a control tissue culture without the addition of active substance the cell-count increases by at least 150%.

It is also possible to carry out standardization by direct measurement of the acceleration of cell-division of cells cultured in vitro, such as fibroblasts of human or animal origin. The increased resistance through administration of the active substances obtained ~y the procedure which is the subject of this invention is determined by adding toxic metallic salts, such as lead or mercury compounds, to human or animal cells in such a way that their mitotic capacity is impaired or blocked.
The degree of deblocking of this effect which is ob-tained through the action of the growth-factors then gives the degree of resistance.

Further investigations have shown that the end-product, through stimulation of the metabolism, promotes oxygen uptake and, at the same time, the increased production of adenosine triphosphate. The end-product catalyses and activates the respiratory chain at the leYel of the cytochrome oxidase. For these reasons, in the case of a wound for example, the active substance leads to in-creased cell-division and thus to rapid epithelializ-ation and healing. From calculations of feed utiliz-ation in domestic animals it has been shown that the addition of the active substance leads to better ~ 7 ~ 1179957 utilization of the basic feed so that these animals also reach their desired degree of maturity much faster than animals whose feed contains no active substance.

It has also been shown that a fermentation process, for example, can be considerably activated by addition of this active substance. It could thus be established that the active substance can be used successfully in the brewing of beer, in that a normal fermentation process, which otherwise takes 8~2 days, for ~xample, can be reduced to 7 days. The time-saving of 1~2 days is of considerable importance fr~m the economic point of view; in both cases the quality of the beer is the same.

The growth factors produced by the procedure which is the subject of this invention are not only suitable for therapeutic use in human and veterinary medicine but can also be used in the technical field of ferment-ation processes, in the preparation of cell and tissue cultures in vitro and for the improved utilization of basic feed in animals.

Claims (3)

THE EMBODIMENTS OE` THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Procedure for the extraction of anabolic, respiration-promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue-culture purposes, characterized by the disintegration of yeast fungi of the Saccharomycetes family at the moment or their maximum growth by means of ultrasound, in the presence of proteolytically active enzymes, the residue then being subjected to dialysis and the dialysate concentrated under extremely careful treatment.
2. Procedure according to claim 1, characterized by the use of papain as a proteolytic enzyme.
3. Procedure according to claim 1 wherein the yeast fungi are fermented at 25°C ? 3°C for 1 to 3 days, after which a proteolytic enzyme is added, the mass of yeast fungi is spun by means of an ultrasound integrator of about 50 Watts/cm2 until the temperature of the yeast mass has risen to about 40°C, the mass is then cooled, buffered to a pH value of 6.2 and centrifuged.
CA000402970A 1981-05-14 1982-05-14 Procedure for the extraction of anabolic, respiration- promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue- culture purposes Expired CA1179957A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH3131/81-9 1981-05-14
CH3131/81A CH651063A5 (en) 1981-05-14 1981-05-14 METHOD FOR OBTAINING ANABOLIC, BREATHABILIZING, LOW-MOLECULAR ACTIVE SUBSTANCES FOR PROPHYLACTIC, THERAPEUTIC, CELL AND TISSUE CULTURAL TECHNICAL PURPOSES.

Publications (1)

Publication Number Publication Date
CA1179957A true CA1179957A (en) 1984-12-27

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ID=4249283

Family Applications (1)

Application Number Title Priority Date Filing Date
CA000402970A Expired CA1179957A (en) 1981-05-14 1982-05-14 Procedure for the extraction of anabolic, respiration- promoting, low-molecular active substances for prophylactic, therapeutic, cell-culture and tissue- culture purposes

Country Status (6)

Country Link
EP (1) EP0065246B1 (en)
JP (1) JPS57192316A (en)
AT (1) ATE15072T1 (en)
CA (1) CA1179957A (en)
CH (1) CH651063A5 (en)
DE (1) DE3265583D1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013485A (en) * 1996-05-07 2000-01-11 Thymopharma, Ag Low-molecular weight active ingredient extract from yeasts and method for producing it
US6143295A (en) * 1995-05-10 2000-11-07 Thymopharma Ag Low- molecular active ingredient extract from yeasts and method for producing it

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH654210A5 (en) * 1983-05-20 1986-02-14 Hasunor Ag PROCEDURE TO GET PREPARED metabolically INCOME OBTAINED FROM YEAST OF ANY KIND.
DE3711054A1 (en) * 1987-04-02 1988-10-13 Socop Nahrungsmittel BIOPHYSICALLY DERIVATIZED YEAST PREPARATION, METHOD FOR THE PRODUCTION THEREOF AND FEED CONTAINING THIS Yeast PREPARATION AND PLANT GROWTH SUBSTANCES
DD284131A7 (en) * 1987-07-10 1990-11-07 �����@�������`����k�� METHOD AND DEVICE FOR PRODUCING BIOACTIVE SUSPENSIONS
AT389889B (en) * 1988-05-03 1990-02-12 Langenecker Bertwin Dr METHOD AND DEVICE FOR INACTIVATING VIRUSES CONTAINED IN A CARRIER MEDIUM
DK166459B1 (en) * 1991-05-07 1993-05-24 Agro Ferm As PROCEDURE FOR THE CONVERSION OF PLANT SAFETY TO A MEDIUM SELECTED AS A NUTRITIONAL SUBSTANCE FOR VITAMIN AND AMINO ACID REQUIRING BACTERIES CONCERNING ORGANIC ACIDS OR AMINOS ACIDS
CN101748076B (en) * 2008-12-16 2013-09-04 湖北杰康诺生物科技有限公司 Method for preparing personalized biological culture medium raw material from beer waste yeast

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR994079A (en) * 1944-12-16 1951-11-12 Lesaffre & Cie Process for the manufacture of yeast extracts by means of ultrasonic vibrations and products obtained by this process
BE712331A (en) * 1968-03-18 1968-07-31
FR2186247A1 (en) * 1972-05-31 1974-01-11 Liberas Rene Revivable lyophilised yeasts contg growth factor - for treatment of antibiotic side effects intestinal disorders, dermatoses and vitamin B deficiency
CH643296A5 (en) * 1980-05-02 1984-05-30 Nestle Sa PROCESS FOR PRODUCING A YEAST EXTRACT.

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6143295A (en) * 1995-05-10 2000-11-07 Thymopharma Ag Low- molecular active ingredient extract from yeasts and method for producing it
US6348335B1 (en) * 1995-05-10 2002-02-19 Thymopharma, Ag Low-molecular active weight ingredient extract from yeasts and method for producing it
US6013485A (en) * 1996-05-07 2000-01-11 Thymopharma, Ag Low-molecular weight active ingredient extract from yeasts and method for producing it

Also Published As

Publication number Publication date
DE3265583D1 (en) 1985-09-26
CH651063A5 (en) 1985-08-30
EP0065246A2 (en) 1982-11-24
EP0065246B1 (en) 1985-08-21
EP0065246A3 (en) 1983-07-06
JPS57192316A (en) 1982-11-26
ATE15072T1 (en) 1985-09-15

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