NL8800692A - METHOD FOR PREPARING ALGAE WITH IMPROVED BIOLOGICAL ACTIONS - Google Patents

Description

NL 34929-Kp / vD

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Process for the preparation of algae with improved biological effects.

The present invention relates to a process for the preparation of algae with improved biological activity.

Algae have been used by humans for food and feed purposes for centuries. For example, algae were mainly consumed by the peoples of the Far East; however recently they have also been used in dry form or in tablet form in developed countries.

Algae are the carriers of very valuable food materials, since their dry form contains high concentrations of substances essential for a healthy life, such as vitamins, proteins, complexes of protein microelements, saccharides, polyunsaturated fatty acids and the like.

In recent years, pollution of the environment 15 has become a global problem. Today, the pollution of seas and oceans by toxic heavy metals (eg lead, mercury, cadmium, copper) as well as tumor inducers (eg condensed polycyclic aromatics) is not negligible. It is a difficult problem that these agents accumulate in the algae organism. As a result, "complete algae" obtained from the sea or grown under other natural conditions cannot be used for human or animal nutrition purposes, forage purposes, cosmetics and therapy; only 25 such fractions of the "complete algae" can be used, which have been previously purified from the biologically harmful materials. A purification method of this type is described, for example, by Carames de Guovëa ^ Cosmetics and Toiletries 95 ^, 47 (1980) _ /. However, a purification method means an intervention decomposition of some of the biologically active substances of the algae, whereby the biological value of the algae fraction thus obtained decreased considerably ^ Zajic: Properties and Products of Algae: Edition Planum, New York, (1970 ) _7 ·

As a result, the preparation of sterile algae is becoming increasingly important free from environmental damage.

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A number of methods are known for the artificial cultivation of algae, which can be carried out eg in an open basin under sunlight or in a closed space under conditions, which guarantee sterility either under natural or artificial lighting, or by excluding any light.

According to the published Japanese patent application No. 56.96690, algae are cultivated using maritime algae strains in sterilized seawater, in a solution containing various nutrient salts under the influence of an artificial light source.

According to Japanese published patent application No. 45.17146, the industrial cultivation of unicellular green Chlorella algae is performed on a sterile freshwater culture medium, excluding carbon dioxide and light.

The cultivation of unicellular green freshwater algae is further described in French Patent No. 2,103,462 according to which patent industrial cultivation is photosynthetically realized using nutrient solutions containing suitable nutrient salts.

According to the published Hungarian patent applications Nos. 4613/84 and 4614/84, the cultivation of the algae is carried out in a mineral water of natural origin, medicinal or thermal water or in a mixture thereof enriched with a metal compound to a concentration of 10 mol / l.

In any of the known solutions, algae, to be used for food, feed or medicine preparation, can be artificially prepared under sterile conditions. In these processes, the algae are grown under conditions resulting in properties of the algae thus obtained, which were similar to those of algae, which grew spontaneously in nature under harmless environmental conditions; or the properties of the algae thus obtained were only slightly different depending on the way in which the artificial cultivation took place.

The algae cultured artificially using the methods of the prior art. 880 0692.

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if only, they contain traces of various elements such as selenium, zinc or silver.

It is further known that selenium has a versatile biological activity, as published in an extensive publication of Thressa et al., Nitration Review 3, 7, (1977) Shamberger [j. or Env. Path, and Tox. 4, 305 (1980) J or Masukawa et al. / Experiences 39, 405 (1983) /. Hence, selenium is known per se to have a hypotensive action, improve ischemic, hypoxy, and infarct states of the heart, and further inhibits cerodal lipofuscinosis of the central nervous system; in addition, selenium has a beneficial effect on periodonititis and has been shown to reduce the possibility of developing cancerous diseases. In addition, selenium was found to be a mutagenesis inhibitor. A number of abnormalities or diseases, such as hepatic necrosis, myonecrosis, erythrocyte membrane destruction, interstitial lesions, STG elevation in the ECG, kwashi orcor syndrome (protein malnutrition) and multiple sclerosis have been induced due to a selenium deficiency. .

The beneficial effect of selenium is mainly based on its activating action, targeting the glutathione peroxidase enzyme, which is the most important endogenous 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 therefore has to be constantly supplemented. Hitherto, selenium has been exclusively introduced into the organism through inorganic compounds (selenium dioxide, sodium selenite and the like).

The object of the present invention is to provide algae with special biological properties and, due to their increased selenium content, algae capable of maintaining selenium's biological activity.

The invention is based on the recognition that under certain conditions the algae can not only be maintained as living in selenium-containing solutions, i.e. they do not decay, but in addition they grow and thus 8800692. - 4 - ** are grown in a nutrient solution containing selenium in a high concentration. This recognition is surprising since it has been anticipated in the prior art that the algae will be killed by the toxic selenium.

Furthermore, the invention is based on the recognition that selenium is incorporated under certain conditions in the organism of an algae grown on a selenium-containing kwsek medium.

The invention therefore relates to a process for the preparation of algae with improved biological activity, wherein the selected algae strain is cultivated under sterile conditions on a culture medium consisting of fresh water and nutrient salts in the presence of carbon dioxide and light by a photosynthetic route, or with the Exclusion of light on a culture medium which additionally contains a source of carbon, hydrogen, oxygen and nitrogen, after which the algae thus obtained are separated. According to the present method, inorganic and / or organic selenium compound (s) are added to the culture medium in -7 -3 at a concentration of 10 to 2x10 mol / l, after which the culture medium is inoculated with a pure algae culture, which is capable of rapidly selenium to record. The pure culture is obtained by inoculating the selected strain on a liquid culture medium containing inorganic or optionally organic nutrient materials, followed by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. From the individual constituents of the strain thus obtained by mutation, those which are capable of rapidly absorbing selenium are grown after mutation on a culture medium containing selenium and at a growth rate at least equal to that of the wild strain . After inoculation of the culture medium with the pure culture thus obtained and after the cultivation period, the alga is separated and optionally dried to a water content of choice and finally decomposed by means of sound waves or by grinding in a manner known per se.

According to the invention, pure algae is cultivated, which is capable of incorporating selenium, preferably prepared from unicellular green or blue algae, such as Chlorella sp., Scenedesmus sp. or Spirulina sp. : such that after the treatment with N-methyl-N1-nitro-N-nitrosoguanidine .8800692 i-5 the cells are carefully washed and dispersed on a series of solid culture media prepared by half dilution and supplemented with -7 a selenium compound to a concentration between 10 and -3 2x10 mol / l. The most preferred strains of those developed here with rapid growth and ability to rapidly build in selenium are selected and grown further in a liquid medium, thereby exposing the pure alga culture thus obtained to industrial production.

Thus, selenium is incorporated during the growth of the organism of the algae treated in the manner described above, at a concentration which is on average 10 times higher than that in the original algae strain.

Distilled water is used as liquid in the culture medium used for the industrial cultivation of the algae strain capable of rapidly building in selenium. In addition to the commonly used known food materials, these culture media are also supplemented with inorganic and / or organic selenium compounds. The culture medium thus prepared and sterilized is diluted with the algae strain obtained in the manner described above, which is capable of rapidly absorbing selenium. In the course of its growth, the algae absorb selenium in their own organism without the unwanted toxic impurities described above.

The algae thus prepared were separated from the culture medium and carefully dried in a manner known per se under mild conditions at an optimum temperature of 65 ° C and not higher than 80 ° C. The dried algae are suitably decomposed by grinding to a particle size of about 1 yam. Alternatively, the wet algae concentrate can be decomposed using sound waves and then dried under the above conditions.

The algae thus obtained are in the form of powder containing 250 to 4000 yig / g selenium, which can be consumed directly or used as such or in food, feed, cosmetics or together with therapeutically useful and biologically active substances and / or their additive, in the form of any commercially available preparation of these products, .0800692-6- * preferably in the form of tablets and capsules or in other form.

The main advantages of the method according to the invention can be summarized as follows: a) The algae can be grown in a simple device using an easy to carry out inexpensive method.

b) No purification or multistage work-up is necessary after cultivation.

C) The algae are grown under sterile conditions, excluding environmental contamination, the algae obtained being particularly suitable for human consumption.

d) The present process allows the preparation of algae with a high selenium content and with improved biological and physiological action compared to that of the known algae.

e) The algae obtained according to the present method can be preferably and extensively used, eg for food, in the cosmetic industry, for therapeutic purposes and other fields.

The use of algae grown according to the present method is especially recommended in therapy, since selenium is an activator with respect to the prosthetic group of the glutathione peroxidase enzyme. Selenium is not accumulated in the organism, so that its supplementation can be made possible with the aid of the algae obtained by the present method, whereby all health conditions, which are due to selenium deficiency, can be medicated.

The method according to the invention is further elucidated by means of the following non-limiting Examples.

EXAMPLE I

A scenedesmus obtisiusculus culture was maintained in a 250 ml shake culture flask on Bold's liquid culture medium containing 100 to 500 yag / ml N-methyl-N'-nitro-N-nitrosoguanidine at 25-27 ° C for 30 min. After this treatment, the cells were carefully washed with. 8800692.

Water and then dispersed on a Bold's culture medium series, which was solidified with agar. This culture medium series contained selenium at a concentration, which usually increased to a double value each from 3.125 yig / ml to 400 yig / ml. The colonies formed from the cells, grown and survived on this culture medium series, were isolated and the selected cell lines were multiplied on a laboratory scale in a Bold's nutrient solution containing at least 20 yag / ml selenium. Those colonies were multiplied, which preferably take up the selenium and have a growth rate practically identical to that of the wild (control) strain in a culture medium containing at least 20 yag / ml selenium.

The selenium content of the algae was determined by drying at 65 ° C, whereby the cells were first isolated and carefully washed with water, followed by decomposition thereof by sound waves, the selenium content being measured using atomic absorption method.

The selenium content of the algae-20 powder thus obtained was as follows:

Strain selenium content (yug / g algae powder) untreated "wild" I strain 50 untreated. "Wild" II strain 30 25 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

EXAMPLE II

40 mg of sodium selenite was added to 8 L of Bud-Pringsheim's culture medium, which was transferred to a 10 L algae culture glass bottle. The culture solution thus obtained was sterilized at 121 ° C under an overpressure of 12 bar for 30 minutes. the sterile solution cooled and inoculated with a pure culture of Scenedesmus obtisiusculus capable of rapidly incorporating selenium. Sterile air with 5% by volume of carbon dioxide was bubbled through the culture medium at 25 ° C while the system was exposed. a 8 0 0 6 9 2.

- 8 -f using an electric discharge tube operating at 4000 lux and wavelengths from 440 to 520 and 640 to 700 µm.

After a 14-day culture period, the algae were separated from the culture medium and then washed with water. The algae mass thus obtained was decomposed by means of sound waves and then carefully dried at a temperature below 65 ° C. The selenium content of the algae powder thus obtained was 1200 µg / g.

EXAMPLE III

8.0 sodium nitrite, 0.8 g magnesium sulfate heptahydrate, 0.8 g dipotassium hydrogen phosphate, 2.5 ml Arnon's trace element solution as well as 5 g glucose, 0.1 g cysteine and 0.1 g methionine were dissolved in 8 L distilled water which was transferred to a 10 L algae culture fermentor. The nutrient solution thus obtained was supplemented with 50 mg of sodium selenite, the solution was passed through a sterilizing filter and then inoculated under pure sterile conditions with pure algae culture of Scenedesmus obtisiusculus, who was able to absorb selenium quickly. After a culture period of 4 days at 25 to 28 ° C in the dark, the algae were separated from the culture medium and washed with water. The algae mass thus obtained was decomposed by means of sound waves and then dried at a temperature below 65 ° C. The selenium content of the algae powder thus obtained was 1380 µg / g.

EXAMPLE IV

The procedure described in Example II was followed at 30 provided that instead of the heat treatment the culture medium was sterilized by passing it through a G-5 type sterilizing bacterium filter. The selenium content of the algae powder thus obtained was 1300 µg / g.

EXAMPLE V

The procedure described in Example II or III was followed with the proviso that instead of Scenedesmus obtisiusculus strain, a Chlorella vulgaris algae strain was used, which was capable of rapidly building in selenium, which strain was subjected to the one described in Example I. 6800692 - 9 - mutation treatment.

The selenium content of the algae powder thus obtained was as follows: strain selenium content 5 (ig / g algae powder) "Wild" I strain 140 "Wild" II strain 140 DV-35-42 strain 3200 10 DV-78-20 strain 2300 DV- 104-21 strain 1500

EXAMPLE VI

The method described in Example II or III was followed, it being understood that instead of the Scenedes-mus obtisiusculus strain, a Chlorella minitissima algae strain was used, which was able to rapidly build in selenium. The selenium content of the algae powder thus obtained was 1400 µg / g.

EXAMPLE VII

The procedure described in Example II or III was followed, except that the nutrient solution was allowed to warm to a temperature of not more than 50 ° C using an Aphanocapsa thermalis algae strain, which was thermoduric and capable of rapid selenium. The selenium content of the algae powder thus obtained was as follows: selenium content (yWg / g algae powder) 30 "Wild" I strain 130 DV-12-220 strain 1100 DV-12-340 strain 1550

EXAMPLE VIII

The method described in Example II was followed, except that instead of the Scenedesraus obtisiusculus strain, a Spirulina sp. algae strain, belonging to the blue algae, was used, which was able to quickly build in selenium. The selenium content of the algae-40 powder thus obtained was as follows:. 8800692.

τ - 10 -,) strain of selenium algae powder) "Wild" I strain 50 "Wild" II strain 30 5 HE-87-104 strain 1100 HE-89-241 strain 1200 HE-89-302 strain 1500

EXAMPLE IX

The procedure described in Example II was followed except that the temperature of the nutrient solution was lowered to 8 ° C using a filamentous blue Nostoc commune algae strain which was cold resistant and capable of build in selenium quickly. The selenium content of the algae powder thus obtained was as follows: selenium content (yag / g algae powder) "Wild" strain 140 BK-1218-2 strain 1140 20 ---

EXAMPLE X

3 5x10 M sodium selenite was added to 8 L of Bold's culture medium which was sterilized by filtration and transferred to a 10 L algae culture bottle.

The culture medium thus obtained was inoculated with a pure culture of Chlorella fusca, prepared in the manner described in Example I, which was able to rapidly build in selenium. The algae were grown for 14 days in the manner described in Example II. Then the algae mass was filtered off, carefully washed with water and dried below 80 ° C. The selenium content of the algae powder thus obtained was 2700 µg / g.

EXAMPLE XI

A Scenedesmus obliquus strain, cultured in the manner described in Example I and capable of incorporating selenium, was inoculated into a culture bottle containing 8 1 7

Bold's nutrient solution and 10 M sodium selenite. The method of Example I was then followed to yield an algae powder with a selenium content of 300 µg / g.

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EXAMPLE XII

The procedure of Example II was followed, except that the isolated algae were dried at 65 ° C and then decomposed by grinding to a particle size of 1 µm. The selenium content of the algae powder thus obtained was 1200 yag / g.

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Claims (10)

1. Process for the preparation of algae with improved biological activity, wherein the selected algal strain is grown under sterile conditions on a culture medium consisting of fresh water and nutrient salts in the presence of carbon dioxide and light by a photosynthetic route, or excluding light on a culture medium additionally containing a source of carbon, hydrogen, oxygen and nitrogen, after which the algae thus obtained are separated, characterized in that inorganic and / or organic selenium compound (s) are added to the culture medium or -7 -3 are added in a concentration between 10 and 2x10 mol / l, followed by inoculation of the culture medium with a pure algae culture, which is capable of rapidly building in selenium, after which the resulting algae are optionally dried after cultivation and decomposed by sound waves or by grinding to obtain the pure algae culture by inoculating the selected algae strain on a liquid culture breeding medium containing inorganic or optionally organic feed materials, followed by treatment with N-20 methyl-N'-nitro-N-nitrosoguanidine, after which the strain is grown on a culture medium, which additionally contains selenium, and finally the individual, which is rapidly selenium which have a growth rate at least equal to that of the original strain are selected. 2. Process according to claim 1, characterized in that sodium selenite and / or selenium dioxide are used as inorganic selenium compound (s) in the culture medium. 3. Process according to claim 1, characterized in that selenocysteine and / or selenomethionine are used as organic selenium compound (s) in the culture medium. 4. Process according to claim 1, characterized in that as selected algae strain Chlorella or 35 Scenesmus algae strain is used. 5. Process according to claim 1, characterized in that as the algae strain Aphanocapsa becomes thermalis. 8fc006U t - 13 - »ί used. 6. Method according to claim 1, characterized in that Nostoc commune is used as selected algae strain. Method according to claim 1, characterized in that the selected algae strain is Chlorella minitissima or Chlorella fusca. 8. A method according to claim 1, characterized in that a fresh water blue algae strain is used as the selected algae strain. 9. A method according to claim 8, characterized in that a spirulina sp. is used. 10. Method according to claim 1, characterized in that the selected algae strain is Scenedesmus obtisiusculus or Scenedesmus obliquus. 8800692