WO2005015983A1

WO2005015983A1 - Method for the on-land production of red algae from the bonnemaisoniaceae family

Info

Publication number: WO2005015983A1
Application number: PCT/FR2004/001825
Authority: WO
Inventors: Vincent Lognone; Patrick Dion; Klaus Lüning; Rui Santos; Leonardo Filipe Rodrigues Da Mata; Andreas SCHÜNHOFF; Anne Bansemir; Ulrike Lindequist
Original assignee: Centre D'etude Et De Valorisation Des Algues
Priority date: 2003-07-17
Filing date: 2004-07-12
Publication date: 2005-02-24
Also published as: FR2857562A1; FR2857562B1

Abstract

The invention relates to an on-land method for the intensive, controlled production of red algae belonging to the Bonnemaisoniaceae family, using the tetrasporophytic form of said red algae. The invention further relates to the use of said method for the extraction of halogenated compounds having useful antibiotic properties.

Description

PROCESS FOR THE PRODUCTION ON THE GROUND OF RED ALGAE FROM THE BONNEMAISONIACEA FAMILY

The present invention relates to the field of algae production of industrial interest. In particular, the invention relates to methods of cultivation and production of red algae of the Bonnemaisoniacées family. More specifically, the present invention provides a process for intensive and controlled production, on land, of a red alga belonging to the Bonnemaisoniacées family, via the use of the tetrasporophytic form of said red alga. In addition, the invention relates to the application of such a method to the extraction of halogen compounds useful for their antibiotic properties. The antibacterial and antifungal activities of red algae extracts belonging to the Bonnemaisoniacées family, in particular extracts of Asparagopsis armata and its microscopic filamentous sporophyte Falkenbergia rufonalosa, are known. The compounds responsible for these activities have been partially identified and quantified (Comparative study of the halogen compounds of Falkenbergia rufolanosa and YAsparagopsis armata: Rhodophycées Bonnemaisoniales. Bruneau et al. Report of the Academy of Sciences of February 27, 1978). They are mainly brominated and / or chlorinated derivatives of propanone-2. Other halogenated compounds, such as bromoform and chloroform, as well as halogenated derivatives of acrylic acid or acetic acid (in free or ester form), are also present in lesser amounts. The techniques of propagation of Asparagopsis armata (Codomier et al, 1978) by cuttings are known. Patent FR 2 732 022 describes the cultivation in sea of the alga A. armata by microbouturage. The alga produced is intended for the extraction of organic silicon derivatives. Patent FR 2 753 101, and the corresponding patents (EP 0 926 956 and US 6 346 252), relate to the use of an extract of A armata having antibacterial and / or anti-fungal activity and containing halogenated molecules having a molecular weight greater than 10,000. According to these documents, the antibacterial activity of the extract is proven on the bacterial strains Vibrio anguillarum, Pseudomonas aeruginosa, Escherichia coli, Enterobacter gergoviae, Staphylococcus aureus as well as on the yeast strain Candida albicans . This activity depends in part on the presence of halogenated derivatives of molecular weight greater than 10,000. These macromolecules have not been characterized in the documents in question. Asparagopsis armata is described morphologically in its macroscopic form reaching a few tens of cm. It is organized morphologically, differentiated anatomically, and represents, in the reproductive cycle of the alga, the macroscopic or gametophytic generation. Currently, A. armata is produced on dies at sea. This production process has a number of drawbacks. On the one hand, it does not allow the control of the culture conditions which influence the biosynthesis by the alga of halogenated compounds. On the other hand, it only partially solves the difficulties of supplying this alga, given the small quantities produced. In addition, the morphology and physiology of A. armata do not allow it to be grown in suspension in onshore tanks. The present invention therefore aims to overcome the aforementioned drawbacks, by taking advantage of the fact that, unlike the macroscopic form of A. armata, its filamentous microscopic generation, called sporophytic and from which it is derived, is suitable for production in onshore tanks . This generation has been described separately as Falkenbergia rufalonosa by the first algologists who were unaware that the same reproductive cycle linked the two forms. Thus, the solution according to the present invention proposes to divert the culture of Asparagopsis armata by cultivating rather its filamentous phase which is maintained in dense suspensions in earthen basins, in the manner of a phytoplankton culture. It is therefore now possible, thanks to the invention, to produce the algal biomass on land, under intensive conditions, while controlling its content of halogen compounds and, consequently, to use this biomass for the production of an antibiotic extract. whose activity is standardized. Obviously, by extension, the method according to the invention can be applied to other species of the Bonnemaisoniacées family, such as Bonnemaisonia sp. and its tetrasporophyte, Trailliela sp. According to a first aspect, the subject of the present invention is a process for intensive and controlled production, on land, of a red alga belonging to the Bonnemaisoniaceae family, said process comprising at least: a) suspending the tetrasporophytic form said red alga in a culture medium containing sea water; b) culturing said tetrasporophytic form with stirring, at a temperature between about 10 ° C and about 29 ° C, with renewal of the culture medium; and c) harvesting said tetrasporophytic form from the culture obtained in step b). Contrary to the usual practice with regard to the production of red algae, the process according to the invention is carried out on land, and not at sea. This process can in particular be carried out in basins or in closed bioreactors . Closed bioreactors optimize the control of culture conditions, and in particular prevent external contamination. However, the implementation in basins is preferred. In this case, the basins can be of different sizes, shapes, capacities, depths, etc., so that one or more algae production conditions are controlled, as indicated below. The expression “intensive production” means a production in large quantities relative to the surface area or to the volume of culture, said production possibly reaching, for example, 80 g of dry matter per m ² and per day. In fact, according to the above method, the alga is kept in dense suspension in the culture medium. Under these conditions, it develops by growth and vegetative multiplication to give particularly advantageous productions per m ² . As shown in example 1.2 below, the production values observed are exceptional, in that they represent almost double what has been reported in the literature concerning other small macroalgae which have already subject of experiments in cultures in basins (see for example Neori and Shpigel. 1999. World Aquaculture. Pages 46-47). The production of algae is "controlled" in that the production conditions liable to act on the chemical composition of the algae are both monitored and regulated in relation to reference values or levels which have been determined empirically within the framework of the present invention. The objective of this control is to obtain maximum surface production, while guaranteeing an optimal quality of the harvested material. In addition, it is used to prevent contamination of the basins by algae different from those which one seeks to produce. According to this definition, within the meaning of the invention, at least one of the following conditions is controlled: - the supply of nutritive salts; - the supply of carbon dioxide; - the temperature of the culture; - the agitation of the culture medium; - renewal of the culture medium; - exposure to light; - contamination by other algae; and - the frequency of harvest. Advantageously, the content of the algae harvested in halogen compounds of interest is also controlled, which makes it possible to produce extracts containing these compounds which have a standardized activity, in particular antibiotic (see below and examples). According to a first embodiment, the culture medium used in the context of the invention contains filtered sea water, enriched with nutritive salts and, where appropriate, carbon dioxide, so as to control the contributions in these elements. This control helps optimize the growth and chemical composition of algae. According to a second embodiment, during step b) of the above-mentioned process, the culture is advantageously maintained at a temperature of between approximately 10 ° C. and approximately 24 ° C. According to a third embodiment, during step b), the culture medium is renewed sequentially or continuously, preferably continuously, in order to avoid confinement of the algae culture medium and to help regulate the temperature of the cultures. In the context of the present invention, agitation is useful for several reasons: in the control of nutrient intakes, of temperature and of exposure to light, in the circulation of the medium for the purpose of its renewal, etc. In this regard, according to a fourth embodiment, during step b), the stirring is carried out by bubbling or by means of a mechanical device such as the impellers of the systems of so-called “raceway” tanks, propellers or circulation pumps. According to a fifth embodiment, the exposure to light is controlled by various means, namely the depth of the pools, the density of the culture and the agitation. The depth of the basins depends on the type of basins (shape and volume of the basins) and the level of agitation. It is most often between 50 cm and 1 m, but can reach several meters, especially when the basins are in the form of columns. As a general rule, depths of the order of a meter are preferred, or less. In addition, at start-up, it is advisable to protect the culture from an excess of sunlight, for example using a mesh, a canvas, a fabric, boards arranged so as to filter the light. . On the other hand, as the algae develop, the culture densifies and the self-shading by the algae themselves is enough to protect them from solar radiation (see examples below). Finally, the agitation helps control exposure to light by controlling the rate of passage of algae at the level of the lit surface of the pool. According to a sixth embodiment, the absence of contamination by algae other than those which it is desired to produce is controlled. To do this, both seawater filtration and crop density are involved. Indeed, at high density, the crops preserve themselves from possible contamination by other algae. According to a seventh embodiment, during step c) of the process which is the subject of the invention, the harvesting of the algae is carried out periodically, for example at one week intervals. Of course, those skilled in the art will retain from the various embodiments set out above that they all relate to the control of the conditions of production of algae. As such, each embodiment can be considered alone or in combination with one or more other embodiments. The method according to the invention is to preferably implemented by combining all the embodiments described above. It is obvious that a person skilled in the art can call on his general knowledge in the technical field of the invention, as well as on routine experiments, to determine, in the light of the present description and of the examples below. after, one or more algae production conditions. Any red algae of the Bonnemaisoniacées family can be used to implement the method according to the invention. In particular, a person skilled in the art can choose from the following algae: Asparagopsis armata and the corresponding tetrasporophytic form Falkenbergia rufalonosa, or Bonnemaisonia sp. and its tetrasporophytic form Trailliela sp. According to a second aspect, the present invention relates to the application of the production process described above, to the extraction of halogen compounds useful for their antibiotic properties. The term “antibiotic properties” is understood here to mean antibacterial and / or antifungal properties. The halogen compounds of interest have a low molecular weight. They are mainly propanones and alkyl acetates. There may also be, to a lesser extent, alkyl acrylates. All of these compounds are synthesized by algae at the level of specialized cells, in the form of ultrastructures called vesicles. These vesicles participate in the defense mechanisms of algae against various stresses (oxidations, epyphites, bacteria, etc.). The extraction of halogen compounds, insofar as they are small and have a relatively simple structure, can be carried out by classic way. In particular, a person skilled in the art can use the following extraction process, comprising at least: d) the dispersion of the tetrasporophytic form collected in propylene glycol; e) grinding said dispersion; f) extraction with stirring, at room temperature, for approximately 2 hours; g) elimination of the insoluble phase; and h) recovering the soluble phase containing said halogenated compounds. Advantageously, the above extraction method further comprises a step i) of purification of the compounds of interest by liquid-liquid separation, in particular using ether. The conditions for implementing the abovementioned extraction method form part of the general knowledge in the field of the invention and are, as such, perfectly known to those skilled in the art. For example, the elimination during step g) can be carried out by centrifugation and / or filtration. The halogenated antibiotic compounds extracted from red algae produced according to the process which is the subject of the present invention are intended to be used in very varied fields, comprising in particular: - pharmacy, in particular veterinary pharmacy; - cosmetics, in particular as microbial stabilizers or active principles to combat acne, dandruff, etc.; the environment, for example for the biological purification of polluted waters such as discharges from marine fish farms enriched with nutritive salts; and - anti-fouling coatings (paints, lacquers, coatings, that is to say “anti-fouling” coatings). The examples which follow serve to illustrate, without limiting, the present invention. EXAMPLES

I. Culture:

The alga Falkenbergia rufolanosa was introduced into the south of France around 1926, probably from Australia. It has been kept since 1978 in the collection of the Biologische Anstalt Helgoland (Germany). The cultures in external basin carried out in Faro in the South of Portugal were carried out starting from this sample, whose cultures were restarted at 10 ° C in sea water enriched with medium of Von Stosch (Stein, JR 1973. Culture methods and growth measurements. Handbook of Phycological methods. Cambridge University Press).

/.1.Starting of the initial stock: Several thalli of Falkenbergia rufonalosa were carefully cleaned, freed of other algae, then incorporated into a small culture tank under a continuous flow of filtered effluent from the fish farming tanks. At the start, the crop was protected from excess sunlight. When the culture became dense (about 5 g per liter), the self-shading of the algae was sufficient protection against solar radiation. At high density, the culture was preserved from contamination by other species of algae.

1.2. Production method: Circular polyethylene tanks with a surface area of 1.5 m ² and a depth of 1 m were used to cultivate Falkenbergia rufonalosa. An effluent filtered at 150 μm from the fish farming tanks continuously supplied the tanks with a pipe flush with their surface. The algae was kept in suspension by an annular perforated PVC pipe fixed to the bottom along the wall of the tank. A continuous bubbling of deoiled compressed air created the agitation of the middle. The drain line for the tanks was fitted with a grid (mesh diameter of 0.5 mm).

The algae were harvested weekly using a submersible pump discharging the water and the algae into a box covered with a net and located under the main drain. The algae were then introduced into nylon bags (mesh diameter of 0.1 mm) and centrifuged until constant fresh weight at 3600 rpm. After one week, the excess biomass compared to that initially corresponding to the optimal stock density represented weekly production. The raw material collected was packaged and frozen at -21 ° C. The optimal density of the algae stock usable to reach the highest productions (5 g of fresh matter per liter) was determined by testing different crop densities and measuring the production. To determine the dry mass production, a seaweed sample was dried in an oven at 60 ° C. The average dry matter of the algae was 25%. Production data from September 2002 to July 2003 showed seasonal fluctuations with a production peak of 738 +/- 25 g of dry matter per m ² and per week in May and a minimum value of 226 +/- 19 g of dry matter per m ² and per week in January (see Table I below). The influence of the ammonium ion content was also tested. By increasing the flow of ammonium ions to 300 μmol per liter per hour, production reached 800 g of dry matter per m ² and per week. The weekly production of red algae throughout the year in the south of Portugal is shown in Table I below. TABLE I

II. Extraction: 1.46 kg of fresh algae were dispersed in 3.82 kg of propylene glycol and then ground in a colloid mill for 10 min. The extraction was carried out at room temperature for 2 h. The insoluble phase was removed by centrifugation at 8000 g for 15 min then filtration using filter earth. 3.34 kg of hydro-propyl extract (20% water) were stored at 4 ° C.

III. Analysis and characterization of the halogen compounds: 300 g of fresh raw material were ground in the presence of 600 g of sea water for 10 min using a colloid mill. After a contact time of 20 min, the suspension was centrifuged at 8000 g for 15 min. The extraction was repeated twice. The three supernatants were extracted with 1060 ml of diethyl ether. The diethyl ether was dried with anhydrous magnesium sulfate and then removed under a stream of nitrogen. The oil obtained was weighed. The oil yield was 0.18% relative to the dry mass of algae. It was solubilized in methyl laurate for the analysis of the halogen compounds was carried out by gas chromatography coupled with mass spectrometry. The following Table II identifies the halogen compounds which have thus been highlighted:

TABLE II

The compounds whose index of similarity is indicated are those which exist in the library. The other compounds were found thanks to the specific ions resulting from the various fragmentations of the molecules.

IV. Determination of antibiotic properties: IV.1. Bactericidal and fungicidal activity on human pathogenic microorganisms: A distilled water control and a solvent control were used in each test. The hydropropylic extract was tested at the dilution 1/10, dilution for which there is no longer any effect of propylene glycol. The extracts were inoculated with an initial density of 10 ⁵ to 10 ⁷ germs / g and kept in contact for 24 h for Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis and 72 h for Candida albicans, Propionibacterium acnes, Aspergillus niger and Pityrosporum ovale. They were neutralized by adding a mixture of lecithin, polysorbate and triton in order to stop the action of the antibacterial and anti-fungal molecules (final mixture content of 10 ^"2 ). The extracts were incubated and the number of micro -organisms present determined (final density) Table III below shows the antibiotic activity of the hydropropylic extract.

TABLE III

^a : the "reduction" is the decrease in density compared to the initial density N ₀ . As can be seen from Table III, the algal extract tested at 1/10 ^th revealed a bactericidal and fungicidal activity allowing a reduction in germs of 3 to 4 log compared to the initial control (distilled water).

IV.2. Antibiotic activity on pathogenic microorganisms with respect to fish and humans (diffusion test on agar medium): The extracts of Falkenbergia rufolanosa (freeze-dried biomass) were prepared from different organic solvents, of increasing polarity ( dichloromethane, methanol, water). The extraction was carried out in each case by a 24-hour treatment using a Soxhiet extractor. After evaporation under vacuum, the extracts obtained were stored at -20 ° C before use.

The antibacterial activity was tested by diffusion on agar medium, according to the method recommended by the European Pharmacopoeia, against 5 fish pathogenic bacteria: Vibrio anguillarum DSMZ 11323; Aeromonas salmonicida ssp salmonicida ATCC 51413; Aeromonas hydrophila ssp hydrophila DSMZ 6173; Pseudomonas anguilliseptica DSMZ 12111; Yersinia ruckeri ATCC 29493; and against 6 strains pathogenic for humans: Staphylococcus aureus ATCC 6538 Bacillus subtilis ATCC 6051 Micrococcus flavus SBUG Pseudomonas aeruginosa ATCC 27853 Escherichia coli ATCC 11229 Candida maltosa (yeast) SBUG.

ATCC = American Type Culture Collection

DSMZ = Deutsche Sammlung fur Mikroorganismen und Zellkulturen SBUG = Strain collection of the Institute of Biology of the University Greifswald, Germany.

For the diffusion test on agar medium, the content of an inoculation loop was suspended in 3 ml of 0.9% NaCl solution, for each strain. The suspensions thus prepared were each inoculated on an agar nutrient medium. Sterile paper discs previously impregnated with seaweed extract (2 mg) were placed on the inoculated petri dishes. The control discs respectively contained oxytetracycline and the extraction solvent used. The inhibition diameters around these disks were measured after 18 or 48 hours of incubation at 26 ° C., excluding the diameter of the disks (6 mm). In parallel, the minimum inhibitory concentration (MIC) was determined by successive dilutions of the extract, against the bacteria most sensitive to the previous test: Vibrio anguillarum, Pseudomonas anguilliseptica and Staphylococcus aureus.

a) Preparation of the bacterial suspensions: The content of an inoculation loop was suspended in 3 ml of 0.9% NaCl solution, for each strain. The suspensions thus prepared were each inoculated on a liquid nutritive medium. After 18 hours of incubation at 26 ° C (in a water bath, with shaking), the bacterial suspensions were deposited in microplate wells containing the algae extract at different concentrations: Column 1 (well A to H): solution containing the algae extract to be tested Column 1 to 11 (wells A to H): successive dilutions of this solution (concentrations 1/1, 5). The microplates were incubated: - 24 hours at 26 ° C, for Vibrio anguillarum,

- 48 hours at 26 ° C, for Pseudomonas anguilliseptica, - 24 hours at 37 ° C, for Staphylococcus aureus. The plates were read at 620 nm using an ELISA microplate reader, then at 550 nm after revelation with iodonitrotetrazolium violet. b) Results: The antibacterial activities were determined 10 times for the tests on fish pathogenic strains and twice for human pathogenic strains. Table IV below shows the results of the diffusion test on agar medium on fish pathogenic strains.

TABLE IV

Table V below shows the results of the diffusion test on agar medium on human pathogenic strains.

TABLE V

The minimum inhibitory concentrations were determined on the dichloromethane extract. The results are 105.5 μg / ml for Vibrio anguillarum, for Pseudomonas anguilliseptica and 222.5 μg / ml for Staphylococcus aureus. The minimum inhibitory concentrations for pure antibiotic molecules such as OTC are 0.08 μg / ml for Vibrio anguillarum and 0.08 μg / ml for Pseudomonas anguilliseptica. For ampicillin, the minimum inhibitory concentration is 20 μg / ml for Staphylococcus aureus. The complex composition of the algal mixes explains the deviations from the pure control molecules.

Claims

1. A method of intensive and controlled production, on land, of a red alga belonging to the Bonnemaisoniacées family, said method comprising at least: a) suspending the tetrasporophytic form of said red alga in a culture medium containing sea water; b) culturing said tetrasporophytic form with stirring, at a temperature between about 10 ° C and about 29 ° C, with renewal of the culture medium; and c) harvesting said tetrasporophytic form from the culture obtained in step b).

2. Method according to claim 1, characterized in that it is implemented in a basin.

3. Method according to claim 1, characterized in that at least one of the following conditions is controlled:

- the supply of nutritive salts; - the supply of carbon dioxide;

- the temperature of the culture;

- the agitation of the culture medium;

- renewal of the culture medium;

- exposure to light; - contamination by other algae; and

- the frequency of the harvest.

4. Method according to claim 1, characterized in that said culture medium contains filtered sea water, preferably enriched in nutritive salts and, optionally, in carbon dioxide.

5. Method according to claim 1, characterized in that, during step b), said culture is maintained at a temperature between about 10 ° C and about 24 ° C.

6. Method according to claim 1, characterized in that, during step b), said culture medium is renewed sequentially or continuously.

7. Method according to claim 1, characterized in that, during step b), said stirring is carried out by bubbling or by means of a device such as impellers, propellers or circulation pumps.

8. Method according to claim 1, characterized in that, during step c), said harvest is carried out periodically, for example at one week intervals.

9. Method according to claim 1, characterized in that said red alga is Asparagopsis armata, and said tetrasporophytic form is Falkenbergia rufalonosa.

10. Method according to claim 1, characterized in that said red alga is Bonnemaisonia sp., And said tetrasporophytic form is Trailliela sp.

11. Application of a production process according to any one of claims 1 to 10, to the extraction of halogen compounds useful for their antibiotic properties.

12. Application according to claim 11, characterized in that said antibiotic properties are antibacterial and / or antifungal properties.

13. Application according to claim 11, characterized in that said extraction is carried out by means of a process comprising at least: a) the dispersion of the tetrasporophytic form collected in propylene glycol; b) grinding said dispersion; c) extraction with stirring, at room temperature, for approximately 2 hours; d) elimination of the insoluble phase; and e) recovering the soluble phase containing said halogenated compounds.

14. Application according to claim 13, characterized in that said elimination during step d) is carried out by centrifugation and / or filtration.

15. Application according to any one of claims 11 to 14, characterized in that said halogen compounds are intended to be used in a field chosen from the fields of pharmacy, cosmetics, the environment and anti-fouling coatings.