CA1310927C - Process for preparing algae having improved biological effects - Google Patents
Process for preparing algae having improved biological effectsInfo
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- CA1310927C CA1310927C CA000561888A CA561888A CA1310927C CA 1310927 C CA1310927 C CA 1310927C CA 000561888 A CA000561888 A CA 000561888A CA 561888 A CA561888 A CA 561888A CA 1310927 C CA1310927 C CA 1310927C
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/60—Edible seaweed
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/12—Unicellular algae; Culture media therefor
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Abstract
PROCESS FOR PREPARING ALGAE HAVING IMPROVED BIOLOGICAL
EFFECT
A b s t r a c t The invention relates to the preparation of selenium--rich algae having an improved biological effect. Under sterile cultivating conditions, in a photosynthetic way or with the exclusion of air, an alga strain is developed which is capable to suitably incorporate selenium, and culti-vated on a culture medium containing selenium in a concentra-tion between 10-7 mole/litre and 2x10-3 mole/litre.
EFFECT
A b s t r a c t The invention relates to the preparation of selenium--rich algae having an improved biological effect. Under sterile cultivating conditions, in a photosynthetic way or with the exclusion of air, an alga strain is developed which is capable to suitably incorporate selenium, and culti-vated on a culture medium containing selenium in a concentra-tion between 10-7 mole/litre and 2x10-3 mole/litre.
Description
~ 3 1 0927 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 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.
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 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.
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 ~ - 4 ~ 1 3 1 0927 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 131~927 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 1310q27 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 , ~ !
. - ', 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 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 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 lO- I310927 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:
I ~ 1 09~7 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:
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~
- 14 - ~3tO927 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.
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.
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 ~ - 4 ~ 1 3 1 0927 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 131~927 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 1310q27 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 , ~ !
. - ', 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 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 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 lO- I310927 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:
I ~ 1 09~7 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:
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~
- 14 - ~3tO927 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.
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.
Claims (14)
1. 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 2x10-3 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 2x10-3 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.
2. A process as claimed in claim 1, wherein the inorganic selenium compound(s) in the culture medium is sodium selenite, selenium dioxide or a mixture thereof.
3. A process as claimed in claim 2, wherein the inorganic selenium compound is either sodium selenite or selenium dioxide.
4. A process as claimed in claim 1, wherein the organic selenium compound(s) in the culture medium is selenocysteine, selenomethionine or both thereof.
5. A process as claimed in claim 4, wherein the organic selenium compounds is selenocysteine or selenomethionine.
6. A process as claimed in claim 1, wherein the algae strain is Chlorella or Scenedesmus strain.
7. A process as claimed in claim 1, wherein the algae strain is Aphanocapsa thermalis.
8. A process as claimed in claim 1, wherein the algae strain is Nostoc commune.
9. A process as claimed in claim 6, wherein the algae strain is Chlorella minitissima or Chlorella fusca.
10. A process as claimed in claim 1, wherein the algae strain is a fresh-water blue algae strain.
11. A process as claimed in claim 10, wherein the algae strain is Spirulina sp.
12. A process as claimed in claim 6, wherein the algae strain is Scenedesmus obtisiusculus or Scenedesmus obliquus.
13. A process as claimed in claim 1, wherein the algae strain is inoculated into a culture medium containing inorganic but not organic nutritive materials.
14. A process as claimed in claim 1, wherein after selecting the algae capable of incorporating selenium the said algae are inoculated into a culture medium containing inorganic but not organic selenium compounds.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HU88575A HU202272B (en) | 1988-02-09 | 1988-02-09 | Process for producing algae |
| HU575/88 | 1988-02-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1310927C true CA1310927C (en) | 1992-12-01 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000561888A Expired - Fee Related CA1310927C (en) | 1988-02-09 | 1988-03-18 | Process for preparing algae having improved biological effects |
Country Status (26)
| Country | Link |
|---|---|
| JP (1) | JPH01215278A (en) |
| KR (1) | KR890014024A (en) |
| CN (1) | CN1034842A (en) |
| AT (1) | AT389888B (en) |
| AU (1) | AU606138B2 (en) |
| BE (1) | BE1001675A3 (en) |
| CA (1) | CA1310927C (en) |
| CH (1) | CH676605A5 (en) |
| DD (1) | DD267994A5 (en) |
| DK (1) | DK148888A (en) |
| ES (1) | ES2006114A6 (en) |
| FI (1) | FI881310A (en) |
| FR (1) | FR2626890B1 (en) |
| GB (1) | GB2214928B (en) |
| GR (1) | GR1000446B (en) |
| HU (1) | HU202272B (en) |
| IL (1) | IL85777A (en) |
| IT (1) | IT1216140B (en) |
| LU (1) | LU87172A1 (en) |
| NL (1) | NL8800692A (en) |
| NO (1) | NO881215L (en) |
| PL (1) | PL271284A1 (en) |
| PT (1) | PT87016B (en) |
| SE (1) | SE468815B (en) |
| YU (1) | YU55488A (en) |
| ZA (1) | ZA881956B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5211003A (en) * | 1992-02-05 | 1993-05-18 | General Electric Company | Diffuser clean air bleed assembly |
| EP1731597A1 (en) * | 2005-06-06 | 2006-12-13 | Johannes Gutenberg-Universität Mainz | Selenium-enriched liquid media for the cultivation of siliceous sponges and for biogenic silica production |
| CZ300861B6 (en) * | 2007-11-02 | 2009-08-26 | Mikrobiologický ústav AV CR, v.v.i. | Industrial strain SeIV of green chlorococcale alga Scenedesmus quadricauda (Turpin) Brebisson |
| CZ300809B6 (en) * | 2007-11-02 | 2009-08-12 | Mikrobiologický ústav AV CR, v.v.i. | Industrial strain Scenedesmus quadricauda SeVI of green chlorococcale alga Scenedesmus quadricauda (Turpin) Brebisson |
| CZ300808B6 (en) * | 2007-11-02 | 2009-08-12 | Mikrobiologický ústav AV CR, v.v.i. | Industrial strain Scenedesmus quadricauda SeIV+VI of green chlorococcale alga Scenedesmus quadricauda (Turpin) Brebisson |
| FR2935394B1 (en) * | 2008-08-29 | 2013-03-08 | Eco Solution | ORGANIC SELENIUM-ENRICHED PHOTOSYNTHETIC MICROORGANISMS FROM SELENO-HYDROXYACID COMPOUNDS AND THEIR USE IN NUTRITION, COSMETICS AND PHARMACY |
| KR101413841B1 (en) * | 2009-02-04 | 2014-07-02 | 클로렐라고교 가부시끼가이샤 | Selenium-containing unicellular microalgae for animal plankton feeds and method of culturing selenium-containing animal planktons using the same |
| TWI495725B (en) * | 2009-08-28 | 2015-08-11 | Metabolium | Photosynthetic microorganisms enriched in selenium using selenohydroxy acid compounds, uses thereof in nutrition, cosmetics and pharmacy |
| MD4395C1 (en) * | 2014-11-28 | 2016-08-31 | Институт Зоологии Академии Наук Молдовы | Strain of green microalga Scenedesmus quadricauda var. quadricauda - source of proteins, glucides and lipids |
| WO2018199723A2 (en) * | 2017-04-28 | 2018-11-01 | 한남바이오 주식회사 | Selenium resistive novel microalgae |
| CN110317769B (en) * | 2019-07-18 | 2021-04-20 | 恩施硒圣植物科技有限公司 | Culture method of selenium-rich microalgae |
| GB2603709B (en) * | 2020-07-08 | 2024-08-07 | Council Scient Ind Res | Organic selenium enriched edible marine microalgal biomass |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD246789A (en) * | ||||
| FR2426404A1 (en) * | 1978-05-26 | 1979-12-21 | Brevier Jean | Culture of phytoplankton in sea water - enriched with minerals, vitamin(s) and aminoacid(s), used in cosmetics and in human and veterinary medicine |
| DE3421644A1 (en) * | 1984-06-09 | 1985-12-12 | Richard 7880 Bad Säckingen Hau | DIET DIAGRAM |
| JPH062047B2 (en) * | 1985-10-11 | 1994-01-12 | 有限会社サン・ファーム | Spirulina containing high concentration of trace elements in human body and production method thereof |
| DE3784359T2 (en) * | 1986-03-19 | 1993-09-30 | Biotechna Ltd | Biomass production. |
-
1988
- 1988-02-09 HU HU88575A patent/HU202272B/en not_active IP Right Cessation
- 1988-03-17 CH CH1010/88A patent/CH676605A5/de not_active IP Right Cessation
- 1988-03-18 SE SE8800991A patent/SE468815B/en not_active IP Right Cessation
- 1988-03-18 NO NO88881215A patent/NO881215L/en unknown
- 1988-03-18 ES ES8800828A patent/ES2006114A6/en not_active Expired
- 1988-03-18 IT IT8819836A patent/IT1216140B/en active
- 1988-03-18 PL PL27128488A patent/PL271284A1/en unknown
- 1988-03-18 GB GB8806532A patent/GB2214928B/en not_active Expired - Fee Related
- 1988-03-18 JP JP63063751A patent/JPH01215278A/en active Pending
- 1988-03-18 LU LU87172A patent/LU87172A1/en unknown
- 1988-03-18 DD DD31381788A patent/DD267994A5/en unknown
- 1988-03-18 GR GR880100171A patent/GR1000446B/en unknown
- 1988-03-18 AT AT0073588A patent/AT389888B/en not_active IP Right Cessation
- 1988-03-18 PT PT87016A patent/PT87016B/en not_active IP Right Cessation
- 1988-03-18 KR KR1019880002909A patent/KR890014024A/en not_active Application Discontinuation
- 1988-03-18 ZA ZA881956A patent/ZA881956B/en unknown
- 1988-03-18 IL IL85777A patent/IL85777A/en unknown
- 1988-03-18 NL NL8800692A patent/NL8800692A/en not_active Application Discontinuation
- 1988-03-18 DK DK148888A patent/DK148888A/en not_active Application Discontinuation
- 1988-03-18 CA CA000561888A patent/CA1310927C/en not_active Expired - Fee Related
- 1988-03-18 FI FI881310A patent/FI881310A/en not_active Application Discontinuation
- 1988-03-18 YU YU00554/88A patent/YU55488A/en unknown
- 1988-03-18 FR FR888803516A patent/FR2626890B1/en not_active Expired - Fee Related
- 1988-03-18 CN CN88101399A patent/CN1034842A/en active Pending
- 1988-03-18 BE BE8800310A patent/BE1001675A3/en not_active IP Right Cessation
- 1988-03-18 AU AU13262/88A patent/AU606138B2/en not_active Ceased
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