HU202272B - Process for producing algae - Google Patents

Description

The present invention relates to a process for the production of improved biological algae.

Algae has been used by humans for food and feed since ancient times. Thus, seaweed is primarily consumed by Far Eastern peoples, but is also used more recently in dried form and in tableted form in developed countries. Algae are very valuable nutrient carriers, and their dried forms contain high levels of essential ingredients for healthy living, such as vitamins, proteins, protein-micronutrient complexes, saccharides, polyunsaturated fatty acids, lipids, and the like.

In recent years, pollution of the environment has taken on a global dimension, so that pollution of the seas and oceans is toxic to heavy metals (Pb, Hg, Cd, Cu) and carcinogens such as polycondensed cyclic aromatic compounds. Particularly problematic is the accumulation of these substances in the seaweed. Therefore, whole seaweed harvested from the sea or cultivated in other natural conditions should not be used for human, animal, animal, cosmetic or medicinal purposes, but only as a fraction thereof, which has been purged of toxic or harmful biological agents. Such a purification process is described, for example, in Carames de Gouvéa (Cosmetics and Toiletries 95: 47 (1980)). However, the purification operation is an intervention that destroys some of the biologically active substances in the algae and significantly reduces the biological value of the algae fraction thus produced (Zajic, Properties and Products of Algae, Edition Planum, New York, 1970).

For these reasons, the production of sterile algae, free from environmental damage, has become increasingly important. Several methods are known for artificially breeding algae. This can be done in an open pool under sunlight, under closed, asterile * conditions and natural or artificial lighting, or with the exclusion of lighting.

No. 56,96690. According to Japanese Patent Application, algae are cultivated in a solution of various nutrient salts in sterilized seawater using seaweed strains.

The commercial cultivation of single-celled Chlorella green algae is described in U.S. Patent No. 45.17146. Japanese Patent Publication according to which algae is produced in fresh water medium with the exclusion of light and carbon dioxide on sterile medium.

Freshwater single-cell green algae is also described in U.S. Patent No. 2,103,462. French patent specification, according to which industrial scale algae cultivation is carried out photosynthetically using a medium containing the appropriate nutrient salts.

Algae cultivation in mineral water of mineral origin, medicinal water, thermal water, or in mixtures thereof up to a concentration of 10 ² mol / l is described in U.S. Patent No. 4,613/84. and Nos. 4614/84. published Hungarian patent application.

Each of the described methods artificially produces algae for food, feed and / or medicine use under sterile conditions. According to these methods, the algae are bred under conditions such that the properties of the resulting algae are the same as those of the algae that are spontaneously grown in environmentally friendly conditions, and the characteristics of the produced algae differ only slightly depending on the method of artificial sterile cultivation.

Artificially bred algae are known to employ a variety of chemical elements such as selenium, zinc, silver, and the like. they contain no or only traces.

It is also known for its versatile physiological function. Thressa et al. (Nutrition Review 35, 7 (1977)), Shamberger (J. of Env. Path. And Tox. 4, 305 (1980)) and Masukawa et al. (Experientia 39, 405 (1983)). It is also known from these articles that selenium, by itself, is an effective antihypertensive agent that improves the ischemic, hypoxic and infarction state of the heart and prevents ceroid lipofuscinosis of the central nervous system, and has been shown to reduce the incidence of For example, deficiency of selenium causes the following disorders and diseases: hepatic necrosis, muscle necrosis, erythrocyte membrane destruction, connective tissue lesion, ST elevation as ECG lesion, kwashiorkor syndrome, multiple sclerosis.

The beneficial effect of selenium is mainly due to its activity as an activator of the glutathione peroxidase enzyme, the major endogenous inhibitor of harmful peroxidation processes. Selenium is an essential component of the prosthetic group of the glutathione peroxidase enzyme. Thus, selenium is one of the most important, essential substances of life, which does not accumulate in the body, so it must be constantly replaced. Until now, it has been administered exclusively to inorganic compounds (SeO2, Na2SeO3, etc.).

It is an object of the present invention to provide an improved biological activity of algae that is capable of asserting the biological activity of selenium through its increased selenium content.

The present invention is based on the discovery that algae in selenium-containing medium are not only viable under certain conditions, that is, they do not die, but that they can grow in medium containing high concentrations of selenium, i.e. they can be grown. This recognition is surprising because the 3

HU 202272 Based on the state of the art, it was expected that algae would die of selenium toxic to it.

A further basis of the invention is the discovery that algae, when cultured on selenium-containing medium, under certain conditions, will incorporate selenium, which is otherwise toxic to it, into their body.

Accordingly, the present invention provides a process for the production of improved biological algae, wherein the selected algal strain is photosynthetically in a medium containing sterile conditions from fresh water and nutrients in the presence of light and carbon dioxide, or in a medium containing carbon, hydrogen, oxygen and nitrogen sources. and the resulting algae is isolated. According to the invention proceeds to a concentration between the medium ΙΟ ^'7 moles / liter and 2.10' ³ mol / L of compound (s) is added to the inorganic and / or organic selenium, and the medium was readily incorporate selenium capable algae inoculating it. The culture was obtained by inoculating the selected algal strain onto a liquid medium containing inorganic and optionally organic nutrients and treated with N-methyl-N'-nitro-N-nitrosoguanidine. Among the individuals of the strain thus obtained by mutation, those which, when cultured in a medium containing selenium after the mutation, incorporate the selenium well and have growth rates at least equal to those of the wild strain are highlighted. After inoculation with the resulting culture, the algae is isolated after the end of the culture period, dried to any moisture, if desired, and disrupted by ultrasound or milling in a manner known per se.

According to the invention, algal cultures capable of incorporating selenium well are preferably obtained from single-celled green and / or green algae. blue algae such as Chlorella sp., Scenedesmus sp., Spirulina sp. algae to carefully wash cells after treatment with N-methyl-N'-nitro-N-nitrosoguanidine and plated in a series of batch dilutions of solid medium supplemented with 10 ⁷ moles of 2.10 ' ³ molar concentration. From the fast-growing strains with good selenium incorporation, which are developed here, we select the most favorable strains and cultivate them in liquid medium, and then maintain the algae culture maintained in this way under industrial production.

Thus, the algae strain treated as above will incorporate selenium into its body during growth, at an average concentration of 10 ⁴ times the original algal strain.

For the commercial cultivation of an algal strain capable of incorporating selenium well, solutions in distilled water are used as nutrient solutions, which are supplemented with inorganic and / or organic selenium compounds in addition to the usual known nutrients. The thus prepared and sterilized culture medium is inoculated with algal strain which incorporates selenium in a well-described manner. As algae grows, selenium, along with other important trace elements, is gradually incorporated into the body without the toxic, unwanted contaminants described above.

The algae thus prepared is isolated from the culture medium and carefully dried under optimal conditions at 65 ° C, but not more than 80 ° C, under known conditions. Preferably, the dried algae is exposed to a milling of 1 µm. Alternatively, the resulting wet algae concentrate is sonicated and then carefully dried.

The resulting algae powder, having a selenium content of 250-4000 pg / g, may be directly consumed or used alone or in food, feed, cosmetics or with and / or added to biologically active pharmaceutical ingredients, in any form commercially available for such products. thus preferably in encapsulated, tableted or other forms.

The main advantages of the process according to the invention are as follows:

(a) Algae may be reared in a simple installation at an inexpensive process using a simple method.

b) After culturing, the process does not require purification or multi-step processing.

c) Algae are cultivated under sterile conditions, with the exclusion of environmental contamination, which results in clear algae suitable for human consumption.

(d) It allows the production of algae with a high selenium content and better biological and physiological activity than hitherto known algae.

e) The algae cultured by the process of the invention can be advantageously used in a wide variety of applications, including nutrition, cosmetics, medicine and other fields.

Particularly advantageous is the use of algae bred by the process of the invention in medicine, since selenium is an activator of the prosthetic group of the glutathione peroxidase enzyme, which plays an important role in physiological processes. Selenium does not accumulate in the body, so the algae according to the invention also enables the replacement of selenium. Through this, there is a good opportunity to remedy any health damage that can be attributed, among other things, to selenium deficiency.

The invention is further illustrated by the following non-limiting examples.

HU 202272 A

Example 1

On a Bold liquid medium containing 100-500 mg / ml of N-methyl-N'-nitro-N-nitrosoguanidine, the culture of Scenedesmus obtisiusculus was shaken for 30 minutes in a 250 ml flask at 25-27 ° C. After treatment, the cells were washed thoroughly with water and plated on a series of agar-solidified Bold media. The medium series contains increasing amounts of selenium from 3.125 mg / ml to 400 pg / ml respectively. Colonies of surviving cells cultivated on the culture broth are isolated and the selected cell lines are plated on a laboratory scale in Bold's medium containing at least 20 pg / ml selenium. The colonies that incorporate the most selenium are grown and exhibit the same growth potential as the control wild-type strain in medium containing at least 20 Mg / ml selenium.

The isolated and carefully washed cells were dried at 65 ° C and sonicated. The selenium content of algae powder is determined by atomic absorption.

Algae strains thus obtained and their selenium content:

Selenium content Mg / g algae powder Untreated wild strain I. 50 Untreated wild II. tribe. 30 FM-I - 120 strain 1300 Strain FM-I - 1871 2400 Strain FM-441/87 1600 Strain FM-449/87 1800

EXAMPLE 2 8 L of Knop-Pringsheim's medium was added to a liter of algal culturing bottle and 40 mg of sodium selenite was added. The resulting medium was sterilized at 121 ° C for 30 minutes at 1 bar. The sterile solution was plated and inoculated with Scenedesmus obtisiusculus algae inoculation with selenium. The medium was bubbled through sterile air containing 5 volumes of CO2 at 25 ° C, and the system was illuminated with a fluorescent lamp of 4000 lux at 440-520 and 640-700 mm.

After a day's culture cycle, the algae was separated from the culture medium and washed with water. The resulting algae mass is sonicated and carefully dried at temperatures below 65 ° C. Selenium content of algae powder is 1200 mg / g ·

Example 3

Dissolve 8.0 g of NaNO2, 0.8 g of MgSO4 .7H2O, 0.8 g of lHPCM in 8 liters of distilled water in an algal culture fermentor 1

2.5 ml of Arnon's trace element solution, 5 g of solo sugar, 0.1 g of cysteine and 0.1 g of methionine. The medium is supplemented with 50 mg of sodium selenite and, after passing through a sterile filter, inoculated with Scenedesmus obtisiusculus algae, which is capable of incorporating selenium, under strictly sterile conditions. After culturing in the dark for 4 days at 25-28 ° C, the algae was separated from the culture medium, washed with water, and the resulting algae mass was sonicated and finally dried below 65 ° C. The algae powder obtained had a selenium content of 1380 Mg / g.

Example 4

All proceed as in Example 2, except that sterilization of the culture medium is accomplished by passing it through a sterile bacterial strainer G-5 instead of heat treatment. The algae powder thus obtained has a selenium content of 1300 Mg / g.

Example 5

All proceed as in Example 2 or Example 3, with the exception that a selenium-capable Chlorella vulgaris algal strain capable of incorporating selenium was used in place of the Scenedesmus obtisiusculus strain, which was subjected to the mutation treatment of Example 1.

Selenium content of algae powder:

Selenium content Mg / g algapor .Vad 'I. 140Vad ^- II. 140

DV-35-42 strain 3200 DV-78-20 strain 2300 DV-104-21 strain 1500

Example 6

All were carried out according to Example 2 or 3, except that the strain Chlorella minitissima, which had good selenium incorporation, was used in place of the strain Scenedesmus obtisiusculus. The algae powder thus obtained has a selenium content of 1400 Mg / g ·

HU 202272 A

Example 7

All were carried out according to Example 2 or 3, except that the medium was allowed to warm to a maximum of 50 ° C and a heat-pure Aphanocapsa thermalis strain was incorporated which was well-incorporated in selenium. Selenium content of algae powder:

.Wad 'Strain Selenium content Pg / g algae powder 130 Strain DV-12-220 1100 Strain DV-12-340 1550 Example 8 We do everything in Example 2, except that Scenedesmus ob- tisiusculus strain selenium is well incorporated spiny blue algae Spirulina sp. sub- gatewash is used. Selenium from algae powder content of: Selenium content pg / g algapor .I lead I strain 50 .Dead Strain II 30 Strain HE-87-104 1100 Strain HE-89-241 1200 Strain HE-89-302 1500 Example 9 We do everything in Example 2, except that the medium was thermally

The temperature was reduced to + 5 ° C and a cold-tolerant filamentous strain of Nostoc commune blue, capable of increased selenium incorporation, was used. Selenium content of algae powder:

Selenium content Pg / g algapor .Vad 'strain 140

Strain BK-1218-2 1140

Example 10 A 8 liter sterile filtered Bold's medium was added to a liter of algal culturing bottle and 5.10 * ³ M sodium selenite was added. The medium was inoculated with a culture of Chlorella fusca capable of incorporating selenium well, prepared as in Example 1, and the algae was grown for 14 days in the same manner as in Example 2. The algal mass is then filtered, rinsed thoroughly with water and dried below 80 ° C. The algae powder thus obtained has a selenium content of 2700 pg / g.

Example 11

The Scendesmus obliquus strain capable of incorporating selenium cultured as described in Example 1 was inoculated into a 10 L culture flask containing 8 L of sterile filtered Bold's medium and 10 * ¹ M sodium selenite. In the following, all the procedures of Example 10 are followed. The algae powder thus obtained has a selenium content of 300 pg / g.

Example 12

All proceed as in Example 2, except that the isolated algae is dried at 65 ° C and the digestion is carried out by milling to 1 µm. The selenium content of algae powder is 1200 pg / g.

Claims (9)

PATENT CLAIMS A process for the production of algae, wherein the selected algal strain is cultivated under sterile conditions on photosynthetically, or in the absence of light, medium containing hydrocarbon, oxygen and nitrogen sources in a medium of fresh water and nutrients, and the resulting algae is produced. characterized in that the medium is 10 'to ⁷ mol / liter and 2.10 Inorganic and / or organic selenium compound (s) are added at a concentration of ³ mol / liter, and the medium is inoculated with an algae culture capable of incorporating selenium well, the algae obtained after 15 cultures is dried if desired and sonicated. algae cultures are obtained by inoculating the selected algal strain onto a liquid medium containing inorganic and optionally organic nutrients, then treating with the strain containing N-methyl-N'-nitro-N-nitrosoguanidine. followed by isolation of individuals with good selenium incorporation and at least the same growth rate as the parent strain. 2. A process according to claim 1, wherein the inorganic selenium compound in the medium is sodium selenite and / or 30 or selenium dioxide. 3. The process according to claim 1, wherein the organic selenium compound is selenocysteine and / or selenomethionine. 35 4. The method of claim 1, wherein the selected algae is selected from the group consisting of Chlorella or Scenedesmus. The method of claim 4, wherein the Chlorella Algae strain is selected from the group consisting of Chlorella Minitissima and Chlorella Fusca. 6. The method of claim 4, wherein the Scenedesmus alginate strain is 45 Scenedesmus obtisiusculus or Scenedesmus obliquus strain. The method of claim 1, wherein the selected algae is Aphanocapsa thermalis. 50 The method of claim 1, wherein the selected algae is Nostoc commune strain. 9. The method of claim 1, wherein the selected algae is freshwater blue algae, preferably Spirulina ssp. algae strain. .