Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR PREPARING ALGAE HAVING IMPROVED BIOLOGICAL
EFFECTS
This inven-tion relates to a process for preparing
algae having improved biological effects
Algae have from time immemorial been used by the
mankind for the purpose of nutrition and feeding. Thus,
algae are mainly consumed by the peoples of the Far East;
recently, however, they are utilized in dry form or in the
form of tablets in the developed countries, too. Algae are
the carriers of highly valuable nutritive materials since
their dried form contains high concentrations of substances
which are essential for a healthy life such as vitamins,
proteins, complexes of protein microelements, saccharides,
polyunsaturated fatty acids and the like.
In recent years, the environmental pollution became
a worldwide problem. Today, the contamination of seas and
oceans by toxic heavy metals(e.g. lead, mercury, cadmium,
copper) as well as tumour-inducing agents (e.g. condensed
polycyclic aromatic compounds) is not negligible. It is a
difficult problem that these agents are accumulated in the
organism of the algae. Thus, "complete algae" obtained from
the sea or cultivated under other natural conditions cannot
be used for the purposes of human or animal nutrition, feed-
ing, cosmetics and therapy; only such fractions of the
A 4220-1402 PT
1 3 1 Oq27
"complete algae" can be used which have previously been
purified of the biologically harmful materials. A purifying
process of this type is described e.g. by Carames de Cuovéa
/ Cosmetics and Toiletries 95, 47 (1980)7. However, a purify-
ing operation means an intervention decomposing a part ofthe biologically active substances of the alga whereby the
biological value of the thus-obtained alga fraction is
significantly decreased / Zajic: Properties and Products
of Qlgae: Edition Planum, New York, (1970)7.
As a consequence, the preparation of sterile algae
being free from environmental damages is becoming more and
more important.
A number of processes are known for the artificial
cultivation of algae which may be performed e.g. in an open
basin under sunlight or in a closed space under conditions
ensuring sterility either under natural or artificial
illumination, or by exclusion of any light.
According to the published Japanese patent application
No. 56.96690 algae are cultivated by using maritime alga
strains in sterilized sea-water, in a solution containing
various nutritive salts under artificial light source.
According to the published Japanese patent application
No. 45.17146 the industrial cultivation of unicellular green
Chlorella algae is carried out on a sterile fresh-water
culture medium under the exclusion of carbon dioxide and
light.
The cultivation of unicellular fresh-water green algae
is also disclosed in the French patent specification No.
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2,103,462 according to which the industrial-scale cultivation
is photosynthetically realized by the use of nutritive solu-
tions containing appropriate nutrient salts.
According to the published Hungarian patent applications
Nos. 4613/84 and 4614/84 the cultivation of algae is accomplished
in a mineral water of natural origin, medicinal or thermal
water or in a mixture thereof enriched by a metal compound
up to a concentration of 10 2 mole/litre.
In any of the known solutions, algae to be used for
manufacturing food, fodder or medicines are artificially
produced under sterile conditions. In these processes, algae
are cultivated under conditions resulting in properties of
the thus-obtained algae similar to those of algae spontaneously
growing in the nature under harmless environmental conditions;
or the properties of the thus-produced algae are different
only to a low extent, depending on the manner of the
artificial cultivation.
The algae artificially cultivated by using the processes
of the prior art contain only traces, if any, of several
elements such as selenium, zinc or silver.
It is known further that selenium has a versatile
biological function as published in a comprehensive paper
of Thressa et al. / Nutrition Review 35, 7 (1977)7, Shamberger
L J. of Env. Path. and Tox. 4, 305 (1980)7 or Masukawa et al.
/ Experientia 39, 405 (1983)7. Thus, it is known that selenium
in itself has a hypotensive effect, improves the ischaemic,
hypoxic and infarction s-tates of the heart and inhibits the
ceroidal lipofuscinosis of the central nervous system; it
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also exerts a beneficial effect on periodontitis and proved
to diminish the probability of the development of cancer
diseases; furthermore, it is considered to be a mutagenesis-
-inhibiting agent. A number of alterations or diseases,
respectively, such as liver necrosis, myonecrosis, destruc-
tion of the erythrocyte membrane, interstitial laesions,
ST-elevation in the ECG, kwashiorcor syndrome (protein
malnutrition) and multiplex sclerosis proved to be induced
by a selenium deficiency.
The beneficial action of selenium is mainly based on
its activating effect directed to the glutathione-peroxidase
enzyme which is the most important endogenic inhibitor of
the harmful peroxidation processes. Being an indispensable
constituent of the prosthetic group of the glutathione-
-peroxidase enzyme, selenium is one of the most important
and indispensable substances of life which is not accumulated
in the organism and thus it has to be continuously supplied.
Until now selenium has exclusively been introduced to the
organism by inorganic compounds (selenium dioxide, sodium
selenite and the like).
The aim of the present invention is to provide algae
having particular biological properties and, owing to their
increased selenium content, capable to assert the biological
effect of selenium.
The invention is based on the recognition that, under
certain conditions, the alga can not only be maintained as
alive in selenium-containing solutions, i.e. it does not
perish, but it also grows and thus it can be cultivated in a
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nutritive solution containing selenium in a high
concentration. This recognition is surprising since,
according to the prior art, the algae was expected to be
killed by the poisonous selenium.
Further on, the invention is based on the
recognition that selenium is incorporated in the organism
of an algae cultivated on a selenium-containing culture
medium under certain conditions.
Thus, the present invention provides a process
for the preparation of selenium-rich algae which comprises
inoculating an algae strain into an liquid culture medium
containing inorganic and organic nutritive materials,
treating the medium with N-methyl-N'- nitro-N-
nitrosoguanidine, cultivating the algae strain in the
culture medium also containing selenium in a concentration
of between 10 7 and 2x103 mole/litre and then selecting the
algae capable of incorporating selenium and having a growth
rate which is at least identical to that of the original
strain, thereafter cultivating the selected algae capable
of incorporating selenium under sterile conditions in an
aqueous culture medium containing inorganic or organic
selenium compound(s) in a concentration of between 10 7 and
2x103 mole/litre, fresh water and nutritive salts either
photosynthetically in the presence of carbon dioxide and
light or, in the absence of light, in such a medium
additionally containing a source of carbon, hydrogen,
oxygen and nitrogen, and thereafter separating the
selenium-rich algae thus obtained.
According to the invention, the pure algae culture
which is capable to incorporate selenium is preferably
prepared from unicellular green or blue algae such as
Chlorella sp., Scenedesmus sp. or Spirulina sp. in such a
way that, after the treatment with N-methyl-N'-nitro-N-
nitrosoguanidine which results in mutation of the algae,
the cells are carefully washed out and dispersed onto a
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series of solid culture media prepared by halving dilution
and supplemented with a selenium compound to a
concentration between 10-7 and 2~10-3 mole/litre. The most
preferred strains of the here developing ones having a
rapid growth and capable of readily incorporating selenium
are selected and further cultivated in a liquid medium,
then the thus-maintained pure algae culture is subjected to
industrial production.
Thus, selenium is incorporated during the growth
to the organism of the algae treated as described
hereinabove, in a concentration which is in average 104
times higher than that in the original algae strain.
Distilled water is used as liquid in the culture
medium employed for the industrial cultivation of the algae
strain which is capable of readily incorporating selenium.
In addition to the commonly used known nutritive materials,
these culture media are supplemented also with inorganic
,
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and/or organic selenium compounds. The thus-prepared and
sterilized culture medium is inoculated with the alga strain,
obtained as described above,which is capable of readily
incorporating selenium. In the course of its growth, the
alga incorporates selenium to its own organism without the
undesired toxic contaminations discussed above.
The thus-prepared alya is separated from the culture
medium and carefully dried in a known manner under mild
conditions at an optimum temperature of 65 C and not higher
than 80 C. The dried alga is suitably decomposed by grinding
to a particle size of about 1 /um. Alternatively, the wet
alga concentrate is decomposed by supersound and then dried
under the conditions defined above.
The thus-obtained alga is a powder containing 250 to
4000 /ug/g of selenium which may directly be consumed or
used in itself or in foods, fodders, cosmetics or together
with therapeutically useful and biologically active substances
and/or as their additive, in the form of any commercially
available formulation of these products, preferably in the
form of tablets and capsules or in other forms.
The main advantages of the process according to the
invention can be summarized as follows:
a) The alga can be cultivated in a simple equipment by using
an easily practicable cheap process.
b) After cultivation no purification or multistep working-up
is needed.
c) The alga is cultivated under sterile conditions, with
exclusion of environmental contamina~ions whereby the
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obtained alga is unambiguously usable for human consump-
tion.
d) It renders possible the preparation of algae with a high
selenium content and possessing improved biological
and physiological effects as compared to those of known
algae.
e) The algae obtained by the process of the invention can
preferably and widely be used, e.g. for alimentation,
in the cosmetic industry, for therapeutical purposes and
in other areas.
The use of the algae cultivated by the process of the
invention is most preferred in the therapy since selenium
is an activating agent of the prosthetic group of the
glutathione-peroxidase enzyme. Selenium is not accumulated
in the organism, so its supplementation is rendered possible
with the algae obtained by the process of the invention where-
by all health damages can be medicated which are the
consequences of selenium deficiency.
The process of the invention is illustrated in detail
by the following non-limiting Examples.
Example 1
A Scenedesmus obtisiusculus culture is maintained in
a 250 ml flask in a shaken culture on Bold~s liquid culture
medium containing 100 to 500 /ug/ml of N-methyl-N'-nitro-N-
-nitrosoguanidine at 25 to 27 C for 30 minutes. After this
treatment, the cells are carefully washed with water and
dispersed onto a Bold's culture medium series solidified
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by agar. This culture medium series contains selenium in a
concentration regularly increasing to a double value each
from 3.125 /ug/ml to 400 /ug/ml. The colonies formed from
the cells growing and surviving on this cul-ture medium series
are isolated and the selected cell lines are propagated on
a laboratory scale in a Bold's nutritive solution containing
at least 20 /ug/ml of selenium. Those colonies are propagated
which most preferably incorporate selenium and have a growth
rate nearly indentical to that of the wild (control) strain
in a culture medium containing as least 20 /ug/ml of selenium.
The selenium content of the alga is determined by
drying at 65 C the cells previously isolated and carefully
washed with water, decomposing them by supersound and then
measuring the selenium content by using atomabsorption method.
The selenium content of the thus-obtained alga powder
is as follows:
Strain Selenium content
(/ug/g of alga powder)
Untreated wild I strain 50
Untreated wild II strain 30
____________________________________________________________
Code No. FM-I-120 strain 1300
Code No. FM-1-1871 strain 2400
Code No. FM-441/87 strain 1600
Code No. FM-449/87-strain 1800
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Example 2
40 mg of sodium selenite are added to 8 litres of
Knop-Pringsheim's culture mediurn filled in an alga-cultivat-
ing glass bottle of 10 litres volume. The thus-obtained
nutritive solution is sterilized at 121 C under an over-
pressure of 1 bar for 30 minutes. Then, the sterile solution
is cooled and inoculated with a pure culture of Scenedesmus
obtisiusculus which is capable to readily incorporate selenium.
Sterile air containing 5% by volume of carbon dioxide is
bubbled through the culture medium at 25 C while the system
is illuminated by an electric discharge tube working with
4000 lux at a wavelength of 440 to 520 and 640 to 700 /um.
After a cultivation period of 14 days the alga is
separated from the culture medium, then washed with water.
The thus-obtained alga mass is decomposed by supersound and
carefully dried at a temperature below 65 C. The selenium
content of the thus-obtained alga powder is 1200 /ug/g.
Example 3
8.0 9 of sodium nitrite, 0.8 9 of magnesium sulfate
heptahydrate, 0.8 9 of dipotassium hydrogen phosphate, 2.5 ml
of Arnon's trace element solution as well as 5 9 of glucose,
0.1 9 of cysteine and 0.1 g of metnionine are dissolved in
8 litres of distilled water filled in an alga-cultivating
fermentor of 10 litres volume. The thus-obtained nutritive
solution is supplemented with 50 mg of sodium selenite, then
the solution is flown through a sterilizing filter and
inoculated under carefully maintained sterile conditions
.. . .
with a pure alga culture of Scenedesmus obtisiusculus
capable t,o readily incorporate selenium. After a cultivat-
ing perio~ of 4 days at 25 to 28 C in the dark, the algais separated from the culture medium and washed with water.
The thus-obtained alga mass is decomposed by supersound and
finally dried at a temperature below 65 C. The selenium
content of the thus-obtained alga powder amounts to 138û /ug/g.
Example 4
The process described in Example 2 is followed, except
that instead of treatment by heat, the culture medium is
sterilized by flowing through a G-5 type sterilizing
bacterium filter. The selenium content of the thus-obtained
alga powder is 1300 /ug/g.
Example 5
The process described in Example 2 or 3 is followed,
except that instead of the Scenedesmus obtisiusculus strain,
a Chlorella vulgaris alga strain is used which is capable
to readily incorporate selenium and which has been subjected
to the mutation treatment described in Example 1.
The selenium content of the thus-obtained alga powder
is as follows:
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Strain Selenium content
(/ug/g of alga powder)
"Wild" I strain 140
"WiId" II strain 140
DV-35-42 strain 3200
DV-78-20 strain 2300
DV-104-21 strain 1500
Example 6
The process described in Example 2 or 3 is followed,
except that instead of the Scenedesmus obtisiusculus strain,
a Chlorella minitissima alga strain is used which is capable
to readily incorporate selenium. The selenium content of
the thus-obtained alga powder is 1400 /ug/g.
Example 7
2D The process described in Example 2 or 3 is followed,
except that the nutritive solution is let to warm to a
temperature of at most 50 C and an Aphanocapsa thermalis
alga strain is used which is thermoduric and capable to
readily incorporate selenium. The selenium content of the
thus-obtained alga powder is as follows:
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Strain Selenium content
(/ug/g of alga powder)
"Wild" strain 130
__ _________________
DV-12-220 strain 1100
OV-12-340 strain 1550
Example 8
The process described in Example 2 is 40110w~d, except
that instead of the Scenedesmus obtisiusculus strain, a
Spirulina sp. alga strain belonging to the blue algae is
used which is capable to readily incorporate selenium. The
selenium content of the thus-obtained alga powder is as
follows:
Strain Selenium content
(/ug/g of alga powder)
"Wild" I strain 50
"Wild" II strain 30
___________________________________________________________
HE-87-104 strain 1100
HE-S9-241 strain 1200
HE-S9-302 strain 1500
r~
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Exa~ple 9
The process described in Example 2 is followed,
except that the temperature of the nutritive solution is
descreased to 8 C and a filiform blue Nostoc commune alga
strain is used which is cold-resistant and capable to readily
incorporate selenium. The selenium content of the thus-obtained
alga powder is as follows:
Strain Selenium con-tent
(/ug/g of alga powder)
"Wild" strain 140
BK-1218-2 strain 1140
_ _
ExamPle 10
5 x 10 3M of sodium selenite are added to 8 litres of
8O1d's culture medium sterilized by filtration and filled in
an alga-cultivating bottle of 10 litres volume. The thus-
-obtained culture medium is inoculated with a pure culture
2û of Chlorella fusca prepared as described in Example 1 and
capable to readily incorporate selenium. The alga is cultivated
for 14 days as described in Example 2. Then, the alga mass
is filtered, carefully washed with water and dried below
8û C. The selenium content of the thus-obtained alga powder
is 2700 /ug/g.
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Exam~]e 11
A Scenedesmus obliquus strain cultivated in the way
as described in Example 1 and capable to readily incorporate
selenium is inoculated into a cultivating bottle containing
8 litres of Bold's nutritive solution and 10 7M of sodium
selenite. Thereafter, the process of Example 10 is followed
to obtain an alga powder with a selenium content of 300 /ug/g.
Example 12
The process of Example 2 is followed, except that the
separated alga is dried at 65 C and then decomposed by
grinding to a particle size of 1 /um. The selenium content
of the thus-obtained alga powoer is 1200 /ug/g.
