LU87172A1 - PROCESS FOR THE PREPARATION OF ALGAE HAVING AN IMPROVED BIOLOGICAL EFFECT - Google Patents

Description

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The present invention relates to a process for the preparation of algae having improved biological effects.

Seaweed has been used for ten years by humans for nutritional and 5 food purposes. The algae are therefore mainly found by the peoples of the Far East; in recent times, however, they have been widely used in severe dry conditions or in the form of shared prices in developed countries. Algae are extremely interesting nutrient carriers since their strong dried content in high concentrations contains substances which are essential for a healthy life, such as vitatins, proteins, protein complexes with ilcroelients, saccharides, fatty and saturated fatty acids.

15 Pollution of the environment has become a global problem in recent years. Currently, the conta · i-nation of the ters and oceans by toxic heavy lethals (for example the plotb, the tercure, the cadtiut, copper) as well as by agents inducing tuners (for example of the condensed aromatic polycyclic compounds 20 ), is not negligible. The fact that these agents are accumulated in algae is a difficult problem. Ri nsi, "whole algae" originating from the soil or cultivated under other natural conditions, cannot be used for purposes of human or anal nutrition, for primary, cosmetic and therapeutic products; we can only use the fractions of these "complete algae" which have been previously cleared by purification of natives harmful from the biological point of view. A purification process of this type is described for example by Caranes de Guovéa [Cosnetlcs and 30 Toiletries 25, 17 (I960)]. However, a purification operation means an intervention breaking down part of the biologically active substances of the alga, so that the biological value of the alga fraction thus obtained is significantly reduced [Zajic: Properties and Products of fllgae: Edition Planut, Het-Vork, 35 (1970) 3.

2 As a result, the preparation of sterile algae which are not spoiled by the environment has become increasingly healthy.

We know a certain number of artificial cultivation process 5 of acute which can be carried out, for example, in a basin open to the solar strap or in a space bone under conditions ensuring the sterility, either under natural or artificial lighting , or sheltered from any light ..

According to the published specification of Japanese patent Ho. 10 56.96690, algae are cultivated from strains of aaritin algae in sterilized ner water, in a solution containing various nutritive salts under a source of artificial origin.

According to the published application of Japanese Patent No. 15 45,17146, the industrial cultivation of unicellular green Chlorella algae is carried out on a sterile freshwater culture site excluding carbon dioxide and exhaust.

The cultivation of green unicellular freshwater acute is also described in the French patent specification 20 Ho. 2,103,462, according to which cultivation on an industrial scale is carried out photosynthetically using nutritive solutions containing appropriate nutritive salts. .

According to published Hungarian patent applications Nos. 4613/84 and 4614/84, the culture of the algae is accosplied in sineral water of natural origin, medicinal or thermal water or in a mixture thereof enriched by a co-compound. • metallic up to a concentration of 10 “^ no per liter.

In any of the known solutions, trebles to be used for the manufacture of ally, fodder or nedicasents are produced artificially under sterile conditions. In these methods, algae are cultivated under conditions such that the properties of the acute algae obtained are analogous to those of algae 3e developing spontaneously in nature under conditions of this place without danger, or the properties of the algae thus produced differ only 3 very little, depending on the artificial culture aode.

The algae cultivated artificially according to the methods of the prior art contain only traces, if they do, of several elements such as selenium, zinc. 5 yes 1 money.

We also know that selenium has various biological functions, as it is published in a cosplet report by Thressa et al. [Nutrition Revies 35, 7 (1977)], Shasberger [J. of Enu. Path. and Tox. 1, 305 (1980)], or Nasukasa et al. 10 [Experientia 3 £, Ί05 (1983)]. The selenium in itself, has a hypotensive effect, it improves the ischemic, hypoxic and infarcted states of the heart and it inhibits cereal Iipofuscinosis of the central nervous system; it also has a beneficial effect on periodontitis and has been shown to reduce the likelihood of the development of cancerous ladies; moreover, it is considered to be an agent inhibiting sutagenesis. It has been shown that noabrous sodifications or saladles, respectively, pod a liver necrosis, an ayonecrosis, a destruction of the erythrocyte aeabran, interstitial lesions, an elevation of ST in 20 I * ECG, Kwashiorkor's syndrosis (sub- protein diet) and multiple sclerosis are induced by selenium deficiency.

The beneficial action of selenium is mainly based on its activation effect directed against the enzyme glutathione peroxidase, which is the most important endogenous inhibitor of harmful peroxidation processes. In the measurement where the selenium is an essential constituent of the prosthetic group of the enzyme. glutathione peroxidase, it is one of the most ("load-bearing and most essential for life which is not accumulated in the organized and must therefore be supplied continuously. Until now, selenium has been exclusively introduced in the organization under the forae of inorganic coaposes (seléniua dioxide, sodiua selenite or sial taire).

The object of the present invention is to provide algae having particular biological properties and which, thanks to their increased selenium content, are capable of ensuring the biological effect of selenium.

The invention is based on the observation that, under certain conditions, the acute can not only be its living content in solutions containing selenium, that is to say not to perish, but also develop and it can therefore to be cultivated in a nutritive solution containing selenium * in high concentration, This observation is surprising since, according to the prior art, it is expected that the alga will be OJ killed by the toxic selenium.

In addition, the invention is based on the observation that the selenium is incorporated into the organism of an alga cultivated on a culture site containing selenium under certain conditions.

The invention therefore relates to a process for the preparation of acute with improved biological effects, according to which the selected acute strain is cultivated under sterile conditions on a culture site consisting of fresh water and nutritive salts in the presence carbon dioxide and light photosynthetically, or excluding light on a culture site also containing a source of carbon, hydrogen, oxygen and nitrogen, after which the algae thus obtained and separated. According to the process of the present invention, one or more inorganic and / or organic coaposes of selenium are added to the culture medium at a concentration of tO "7 to 2x10" 3 aole / liter, then the culture medium is inoculated with a pure seaweed culture capable of incorporating selenium. The pure culture is obtained by inoculation of the selected algae strain on a liquid culture site containing inorganic nutritive materials and optionally organic and then treating it with H-aethyl-H'-nitro-H -nitrosoguanidine. We choose the individuals of the strain thus obtained by autation, those who are capable of easily incorporating eelenlua when they are cultivated after autation on a place of culture containing selenium and which have a growth rate identical to the size. to 5 that of the wild strain, Rprè3 the inoculation of the culture culture with the pure culture thus obtained and the culture period) the alga is separated, possibly dried to a given water content and decomposed by ultrasound or grinding of way known in 5 itself.

According to the present invention, the culture of pure algae which is capable of incorporating selenium is preferably prepared from unicellu algae blue or green ires, such as Chlorella sp., Scenedesmus sp. or Spirulina sp., so that, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, the cells are thoroughly washed and dispersed on a series of solid culture media, successively diluted half and supplemented with a selenium compound at a concentration between 10 "^ and 2x1Q" ^ mole / liter. The most advantageous strains which grow rapidly and are capable of easily incorporating selenium are selected from the strains which develop here and are again cultivated in a liquid medium, then the pure algae culture is subjected. thus maintained for industrial production.

Selenium is therefore incorporated during the growth in the organism of the alga treated as described above, at a concentration which is on average 10 ^ times higher than that of the original algal strain.

Distilled water is used as liquid in the culture medium used for the industrial culture of the acute strain which is capable of easily incorporating selenium. In addition to the commonly known known nutrients, these. . culture media are also supplemented with inorganic and / or organic selenium compounds. The culture medium 00 thus prepared and sterilized is inoculated with the algae strain, obtained as described above, which is capable of easily incorporating selenium. While developing, the alga incorporates selenium into its own organism without the unwanted toxic contaminations discussed above.

05 The Iga thus prepared 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 exceeding 80 ° C. The dried treble is suitably decoaposed by grinding to a particle size of approximately 1 μ ". In a variant, the acute oil concentrate is decoaposed by ultrasound and then dried under the conditions defined above.

The acute thus obtained is a powder containing 250 to 4000 pg / g of oil, which can be directly consumed or used in itself or in ingredients, fodder, cosmetics or with bioactive and useful active ingredients. therapeutically and / or as additives thereof, in the form of any commercially available form of these products, preferably in the form of coapriae and capsules or other forms.

The main advantages of the process according to the invention can be summarized as follows: a) The alga can be grown in simple equipment with a cheap and easy-to-use process.

b) no multi-stage purification or treatment is required after culture.

c) The alga is cultivated under sterile conditions, excluding environmental contamination, so that the alga can be used without problem for human consumption.

d) It is possible to prepare algae having a high selenium content and having improved physiological and biological effects compared to those of known acute.

e) The high frequencies obtained by the process of the invention can be widely and preferably used for example for aliaentatIon, in the cosmetic industry, for therapeutic purposes and in other fields.

It is more preferred in therapy, since the selenlu is an activating agent of the prosthetic group of the enzyme glutathione peroxidase. Selenium is not accumulated in the organism, so that its coapleaental contribution is made possible with the acute effects obtained by the process of the invention and that it is thus possible to treat all the health disorders resulting from a insufficiency in selection.

The process of the invention is illustrated in detail by the following non-binding examples.

Exeiple 1

A culture of Scenedesaus obtisiuscuius is maintained in a 250 μl flask in a culture shaken on a place of liquid Bold culture containing 100 to 500 μg / al of N-aethyl-H'-nitro-H-nitrosoguanidine at 25- 27 ° C for 30 minutes. After this treatment, the cells are carefully washed with water and dispersed on a series of Bold 15 culture media solidified with agar. This series of ai cultivation sites contains selenium, the concentration of which is doubled regularly from 3.125 pg / al to 400 pg / al each time. The colonies formed from cells which develop and survive on this series of culture sites are isolated and the 20 selected cell lines are reproduced on a laboratory scale in a Bold nutrient solution containing at least 20 pg / al of seléniua. The colonies which develop, incorporate more advantageously selenium and have a growth rate practically identical to that of a wild strain (téaoin) in a culture site containing at least 20 pg / al of selenium.

The selenium content of the alga is determined by drying at 65 ° 0 the cells previously isolated and carefully washed with water, deconposing them by ultrasound and then measuring the selenium content by an absorption method. atoaic.

The selenium content of the seaweed powder thus obtained is indicated below: 8

Strain Selenium content (Mg / g of seaweed powder)

Untreated wild strain I 50 ^ Untreated wild strain I 30

Strain Code No. Fil-1-120 1300

Strain Code No. FN-1-1871 2400

Strain Code No. FH — 441/87 1600 10 Strain Code No. F11-449 / 87 1800

Example 2 40 mg of sodium selenite is added to 8 liters of Knop-Pringsheim culture medium introduced into a glass vial of algae culture having a volume of 10 liters. The resulting nutrient solution is sterilized at 121 ° C under a pressure of 1 bar for 30 minutes. Then the sterile solution is cooled and inoculated with a pure culture of Scenedesmus 28 obtîsiusculus which is able to easily incorporate selenium. Sterile air containing 5% by volume of carbon dioxide is bubbled through the culture medium at 25 ° C., while lighting the system by an electric discharge tube working with 4000 lux, at a wavelength from 440 to 520 and from 640 to 25,700 pm.

After a culture period of 14 days, the alga is separated from the culture medium, then washed with water. The mass of algae thus obtained is decomposed by ultrasound and carefully dried at a temperature below 65 ° C. The selenium content of the algae powder thus obtained is 1200 pg / g.

EXAMPLE 3 8.0 g of sodium nitrite, 0.8 g of magnesium sulphate heptahydrate, 0.8 g of dipotassic acid phosphate, 2.5 55 ml of solution of trace mineral elements as well as 5 g of glucose, 9 0.1 g of cysteine and 0.1 g of methionine are dissolved in 8 liters of distilled water introduced into an acute culture fermenter having a volume of 10 liters. The nutritive solution thus obtained is supplemented with 50 mg of sodium selenite, then the solution is poured through a sterilizing filter and inoculated under sterile conditions carefully maintained, with a culture of pure algae of Scenedesmus obtisiuscuius capable of incorporating easily selenium. After a culture period of 4 days at 25-28 ° Ü in the dark, the algae is separated from the culture medium 10 and washed with water. The mass of algae thus obtained is decomposed by ultrasound and finally dried at a temperature below 65 ° C. The selenium content of the algae powder thus obtained is 1380 pg / g.

15 Example 4

The process described in Example 2 is repeated, except that instead of being treated hot, the culture medium is sterilized by flow through a filter sterilizing bacteria of type 6-5. The selenium content of the algae powder 20 thus obtained is 1300 pg / g.

E2êi & lg-5

The process described in example 2 or 3 is repeated, except that the strain Scenedesmus obtisiuscuius is replaced by a strain of alga Chlorella vulgaris capable of easily incorporating selenium and which has been subjected to the mutation treatment described in example 1.

The selenium content of the acute powder thus obtained is indicated below *.

10

Strain Level of 3éléniui (pg / g of seaweed powder)

"Wild" strain i 140 5 "Wild" strain II 140

DU-35-42 3200 strain

DU-78-20 2300 strain

Strain DÜ-104-21 1500 10 __

Exeiple 6

The process described in exiple 2 or 3 is repeated, except that the strain Scenedesaus obtlsiusculus is replaced by a strain of alga Chlorella ainitissiaa capable of easily incorporating selenium. The selenium content of the algae powder thus obtained is 1400 pg / g.

Exeaole 7 20 The process described in exeapie 2 or 3 is repeated, except that the nutritive solution is allowed to warm to a temperature of taxiBBua 50 ° C. and that a strain of alga Bphanocapsa theraalis is used which eet theraodurique and able to incorporate easily seléniua. The selenium content of the algae powder 25 thus obtained is indicated below:

Strain selenium content 2Q (pg / g seaweed powder)

"Wild" strain 130

DU-12-220 strain 1100

Strain DU-12-340 1550 35 __

Example 8 11

The process described in Example 2 is repeated, except that the strain Scenedesmus obtisiuscuIus is replaced by a strain of alga Splrulina ep, belonging to blue algae, capable of easily incorporating selenium. The selenium content of the algae powder thus obtained is indicated below; jg Strain Selenium content (pg / g of seaweed powder)

"Wild" strain I 50

Strain II "wild * 30 15 Strain 87-104 1100

Strain 89-241 1200

Strain 89-302 1500 20 Example 9

The process described in Example 2 is repeated, except that the temperature of the nutrient solution is lowered to 8 ° C. and that a common threadlike blue Nostoc algae strain is used which is cold resistant and capable of easily incorporate selenium. The selenium content of the algae powder thus obtained is indicated below; on Strain Selenium content (pg / g of seaweed powder)

"Wild" strain 140

Strain BK-1218-2 1140 35 _: -

Exeaple 10 12 5 x 10-¾ of sodium seiénite · is added to 8 liters of Bold culture medium sterilized by filtration and introduced into a vial of seaweed culture with a volume of 10 liters. The culture medium thus obtained is inoculated with a pure culture of Chlorella fusca prepared according to the procedure described in Example 1 and capable of easily incorporating selenium. The alga is cultivated for H days according to the procedure of example 2. The mass of algae is then filtered, carefully washed with water and dried at less than 80 ° C. The selenium content of the powder algae thus obtained is 2700 pg / g.

Exeiple 11

A strain Scenedeseus obliquus cultivated as described in Example 1 and capable of easily incorporating selenium is inoculated in a culture flask containing 8 liters of Bold nutrient solution and 10-¾ of sodium selenite. The process of Example 10 is then followed to obtain an algae powder having a selenium content of 300 pg / g.

20

Exeeole 12

The process described in example 2 is repeated, except that the separated alga is dried at 65 ° C. and then decoeposed by grinding to a particle size of 1 μ ·. The selenium content of the algae powder thus obtained is 1200 pg / g.

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Claims (10)

13 REUEND I CATIONS 1. Process for the preparation of algae having an improved biological effect according to which the selected algae strain is cultivated under sterile conditions on a culture medium consisting of fresh water and nutritive salts in the presence of carbon dioxide and Light in a photosynthetic way, or excluding it on a · ί ii had of culture also containing a source of carbon, hydrogen, oxygen and nitrogen, then the alga thus obtained is separated, characterized in what it includes the addition to the place of culture of one or more inorganic and / or organic coaposes of the selenium at a concentration of NT7 at 2x1 r3 aole / liter, inoculation of the place of culture with a cultivation of pure seaweed capable of incorporating selenium easily, if desired, drying the seaweed obtained after culture and decoaposition thereof by ultrasound or grinding, and in this pureβ the pure culture is obtained by inoculation of the selected algae strain on an ai li had a liquid culture containing inorganic nutritive matter and possibly organic, treat it with H-nethyl-N'-nitro-20 H-nitrosoguanidine, then culture the strain on a place of culture also containing selenium and finally selection of individuals who incorporate selenium and who have a growth rate identical to that of the wild strain. 2. Method according to claim 1, characterized in that it comprises the use of sodium selenite and / or selenium dioxide coaae inorganic components of selenium in the culture medium. 3. Method according to claim 1, characterized in that it comprises the use of selenocysteine and / or selenonethionine coaae organic coaposants of selenium in the culture medium. 4. Method according to claim 1, characterized in that it comprises the use of a strain of alga Chlorella or Scenedesnus coaae strain of selected alga. 5. Method according to claim 1, characterized in 14> that it comprises the use of Rphanocapsa thermalis as selected algae strain. 6. Method according to claim 1, characterized in that it comprises the use of common Nostoc as a selected strain of algae. - 7. Method according to claim 1, characterized in that it comprises the use of Chlorella minitlssima or Chlorella fusca as selected seaweed strain. 8. Method according to claim 1, characterized in that it comprises the use of a strain of freshwater blue alga as the selected algal strain. 9. Method according to claim 1, characterized in that it comprises the use of Spirulina sp. as a blue algae strain. 10. Method according to claim 1, characterized in that it comprises the use of Scenedesmus obtisiusculus or Scenedesmus obiiquus as selected seaweed strain.