Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1179957
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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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.
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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.
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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.
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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
.
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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
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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.
