FR2857562A1 - Intensive, controlled production of red algae on land, for use as a source of antimicrobial compounds, comprises growing the tetrasporophytic form in culture medium containing sea water - Google Patents

Abstract

Intensive and controlled production, on land, of red algae (A) of the family Bonnemaisoniaceae comprises: (a) suspending the tetrasporophytic form of (A) in a culture medium containing sea water; (b) culturing, with agitation, at 10-29 [deg]C with renewal of medium; and (c) harvesting the tetrasporophytic form. ACTIVITY : Antiseborrheic; Dermatological; Antibacterial; Antifungal. A propylene glycol extract of Falkenbergia rufalonosa was diluted 1:10 with water then inoculated with 105> - 107> cells/g of various bacteria. After 24 hours, the reduction in cell count was over 3 log for Staphylococcus epidermidis and over 4 log for Pityrosporum ovale, Propionibacterium acnes and S. aureus. MECHANISM OF ACTION : None given.

Description

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PROCESS FOR PRODUCING ALGAE EARTH
RED OF THE BONNEMAISONIACEES FAMILY
The present invention relates to the field of production of algae of industrial interest. In particular, the invention is concerned with the methods of cultivation and production of red algae of the family Bonnemaisoniacées.

 More specifically, the present invention provides a method of intensive production and controlled, on land, a red alga belonging to the Bonnemaisoniacées family, via the use of the tetrasporophytic form of said red algae.

 In addition, the invention relates to the application of such a process to the extraction of halogenated compounds useful for their antibiotic properties.

 The antibacterial and antifungal activities of red algae extracts belonging to the Bonnemaisoniaceae family, in particular extracts of Asparagopsis armata and its microscopic filamentous sporophyte Falkenbergia rufonalosa, are known.

 The compounds responsible for these activities have been partly identified and quantified (Comparative study of the halogenated compounds of Falkenbergia rufolanosa and Asparagopsis armata: Rhodophyceae Bonnemaisoniales, Bruneau et al., Proceedings of the Academy of Sciences of February 27, 1978). These 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 propagation techniques of Asparagopsis armata (Codomier et al., 1978) by cuttings are known.

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 Patent FR 2 732 022 describes the cultivation at sea of the seaweed A. armata by microbouturing. The alga produced is intended for the extraction of organic derivatives of silicon.

 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 exhibiting 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 and the yeast strain Candida albicans. This activity depends in part on the presence of halogenated derivatives with a 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 up to a few tens of cm. It is organized morphologically, differentiated anatomically, and represents, in the cycle of reproduction of the alga, the macroscopic or gametophytic generation. Currently, A. armata is produced on channels at sea.

This production process has a number of disadvantages.

On the one hand, it does not allow the control of culture conditions that influence the algal biosynthesis of halogenated compounds. On the other hand, it solves only part of the supply difficulties in 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 ground basins.

 The present invention therefore aims to obviate the above-mentioned drawbacks, taking advantage of the fact that, unlike the macroscopic form of A. armata, its microscopic filamentous generation, called sporophytic generation, from which it is derived, is suitable for production in the onshore basin . This generation has been described separately as

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 Falkenbergia rufalonosa by the first algologists who did not know that the same cycle of reproduction linked the two forms.

 Thus, the solution according to the present invention proposes to divert the cultivation of Asparagopsis armata by cultivating instead its filamentous phase which is maintained in dense suspensions in basins on land, in the manner of a phytoplankton culture.

 It is therefore now possible, thanks to the invention, to produce algal biomass on land, under intensive conditions, while controlling its content of halogenated compounds and, consequently, to use this biomass for the production of an antibiotic extract whose activity is standardized.

 Of course, by extension, the process according to the invention can be applied to other species of the Bonnemaisoniaceae family, such as Bonnemaisonia sp. and its tetrasporophyte, Trailliela sp.

 According to a first aspect, the subject of the present invention is a method for the intensive and controlled production, on land, of a red alga belonging to the Bonnemaisoniaceae family, said method comprising at least: a) suspending the tetrasporophytic form said red algae in a culture medium containing seawater; b) culturing said tetrasporophytic form with stirring, at a temperature of 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. . The closed bioreactors allow to optimize the control of the conditions of culture, and in particular to prevent the external contaminations. Nevertheless, the implementation

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 basins is preferred. In this case, the basins can be of different sizes, shapes, capacities, depths, etc., so that one or more algal production conditions are controlled, as indicated below.

 By the term "intensive production" is meant a production in large quantities relative to the surface or the volume of culture, said production being able to reach, for example, 80 g of dry matter per square meter per day. Indeed, according to the process above, the algae is kept in dense suspension in the culture medium.

Under these conditions, it develops by growth and vegetative multiplication to give productions per m2 particularly advantageous. As shown in Example 1.2 below, the observed production values are exceptional in that they represent almost twice what has been reported in the literature for other small macroalgae that have already been reported. experimented with pond culture (see, for example, Neori and Shpigel, 1999. World Aquaculture, pp. 46-47).

 The production of algae is controlled in that the production conditions that may affect the chemical composition of the algae are both monitored and regulated in relation to reference values or levels that have been determined empirically within the framework of the present invention.

 The objective of this control is to obtain a maximum surface production, while guaranteeing an optimal quality of the harvested material.

In addition, it serves to prevent the 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 nutrient salts; - the supply of carbon dioxide; - the temperature of the crop;

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 agitation of the culture medium; - the renewal of the culture medium; - exposure to light; - contamination by other algae; and - the frequency of the harvest.

 Advantageously, the content of algae harvested in halogenated compounds of interest is also controlled, which makes it possible to produce extracts containing these compounds which exhibit an activity, in particular a standardized antibiotic activity (see infra and examples).

 According to a first embodiment, the culture medium used in the context of the invention contains filtered seawater, enriched with nutrient salts and, where appropriate, with carbon dioxide, so as to control the inputs in these forms. elements. This control helps to optimize the growth and chemical composition of algae.

 According to a second embodiment, during step b) of the aforementioned method, the culture is advantageously maintained at a temperature of between about 10 ° C. and about 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 nutrients, temperature and exposure to light, in the circulation of the medium for the purposes of its renewal, etc. .

 In this respect, 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 paddle wheels of so-called raceway tanks, propellers or circulation pumps.

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 According to a fifth embodiment, the exposure to light is controlled by various means, namely the depth of the basins, 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 one meter, or below, are preferred.

 In addition, when starting, it is necessary to protect the crop from an excess of sunlight, for example using a mesh, a cloth, a fabric, boards arranged so as to filter the light . On the other hand, as the algae grow, the culture becomes denser and self-shading by the algae themselves is enough to protect them from solar radiation (see examples below).

 Finally, agitation contributes to the control of exposure to light by controlling the rate of passage of algae at the illuminated surface of the basin.

 According to a sixth embodiment, the absence of contamination by algae other than those which one wants to produce is controlled. To do this, both the filtration of seawater and the density of crops are involved. Indeed, at high density, the crops themselves preserve themselves from possible contamination by other algae.

 According to a seventh embodiment, during step c) of the method which is the subject of the invention, the algae is harvested periodically, for example at a week's interval.

 Of course, those skilled in the art will retain the various embodiments set out above that they all concern the control of algae production conditions. As such, each embodiment may be considered alone or in combination with one or more other embodiments. The process according to the invention is

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 preferably implemented by combining all the embodiments described supra.

 It is obvious that one skilled in the art can use his general knowledge in the technical field of the invention, as well as routine experiments, to determine, in the light of the present description and the examples below. after, one or more conditions of production of algae.

 Any red algae of the family Bonnemaisoniacées can be used to implement the method according to the invention. In particular, those 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 method described above, to the extraction of halogenated compounds useful for their antibiotic properties.

 The term "antibiotic properties" here means antibacterial and / or antifungal properties.

 The halogenated compounds of interest have a low molecular weight. These are mainly propanones and alkyl acetates.

It may also be a question, to a lesser extent, of alkyl acrylates. All 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 the halogenated compounds, insofar as they are small and have a relatively simple structure, can be carried out in a conventional manner. In particular, those skilled in the art can use the following extraction process, comprising at least: d) the dispersion of the tetrasporophytic form harvested in propylene glycol;

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 e) grinding said dispersion; f) extraction with stirring at room temperature for about 2 hours; g) elimination of the insoluble phase; and h) recovering the soluble phase containing said halogenated compounds.

 Advantageously, the above extraction process further comprises a step i) of purifying the compounds of interest by liquid-liquid separation, in particular using ether.

 The conditions for carrying out the extraction process mentioned above are part of the general knowledge in the field of the invention and are, as such, perfectly well known to those skilled in the art. For example, the elimination in step g) can be carried out by centrifugation and / or filtration.

 The halogenated antibiotic compounds extracted from red algae produced according to the process of the present invention are intended to be used in a wide variety of fields, including in particular: - the pharmacy, in particular the veterinary pharmacy; cosmetics, especially as microbial stabilizers or active principles for combating acne, skin condition, etc .; - the environment, for example for the biological purification of polluted waters such as rejections of marine fish farms enriched with nutrients; and - anti-fouling coatings (paints, lacquers, coatings, ie so-called anti-fouling coatings).

 The following examples serve to illustrate, without limiting, the present invention.

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I. Cultivation: The alga Falkenbergia rufolanosa was introduced in the south of France around 1926, probably from Australia. It has been preserved since 1978 in the collection of the Biologische Anstalt Helgoland (Germany). The outdoor pool cultures carried out in Faro in southern Portugal were made from this sample, whose cultures were restarted at 10 C in seawater enriched with Von Stosch's medium (Stein, JR 1973 Culture methods and growth measurements, Cambridge University Press Handbook of Phycological Methods.

 I. 1. Starting up of the initial stock: Several thalli of Falkenbergia rufonalosa were carefully cleaned, freed of other algae, and then incorporated into a small continuous culture tank of filtered effluent from the fish ponds. At startup, the crop was protected from excess sunlight. When the crop became dense (about 5 g per liter), 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 process: Circular polyethylene pans with a surface area of 1.5 m2 and a depth of 1 m were used to cultivate Falkenbergia rufonalosa. A filtered effluent at 150 m from the fish ponds continuously supplied the tanks with a pipe flush with their surface. Algae suspension was maintained by a perforated PVC annular pipe attached to the bottom along the wall of the tank. Continuous bubbling of deoiled compressed air created the agitation of the

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 middle. The bins evacuation pipe was equipped with a grid (mesh size 0.5 mm).

The algae were harvested weekly using a submersible pump discharging water and algae into a net-covered box located under the main drain. The algae were then introduced into nylon bags (mesh size 0.1 mm) and centrifuged to constant fresh weight at 3600 rpm. After one week, the biomass in excess of that initially corresponding to optimal stocking density represented weekly production. The collected raw material was packaged and frozen at -21 C. The optimal density of the seaweed stock used to reach the highest yields (5 g of fresh material per liter) was determined by testing different crop densities and by measuring the production. To determine the dry mass production, a sample of seaweed was dehydrated 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 peak production of 738 +/- 25 g dry matter per m2 and per week in May and a minimum value of 226 +/- 19 g of material per m2 and per week in January (see Table # below). The influence of the ammonium ion content has also been tested. By increasing the flow of ammonium ions up to 300 moles per liter per hour, the production reached 800 g of dry matter per square meter per week.

Weekly production of red algae throughout the year in southern Portugal is shown in Table 1 below.

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BOARD

<Tb>
<tb> Month <SEP> Production <SEP> in <SEP> g <SEP> of <SEP> subject
<tb> dry <SEP> with <SEP> m2 <SEP> and <SEP> with <SEP> week
<tb> September <SEP> 479 <SEP> +/- <SEP> 19
<tb> October <SEP> 401 <SEP> +/- 36
<tb> November <SEP> 325 +/- 29
<tb> December <SEP> 294 +/- 49
<tb> January <SEP> 226 +/- 19
<tb> February <SEP> 383 <SEP> +/- 46 <SEP>
<tb> Mars <SEP> 590 <SEP> +/- 34 <SEP>
<tb> April <SEP> 529 +/- 45
<tb> May <SEP> 738 +/- 25
<tb> June <SEP> 616 +/- 34
<tb> July <SEP> 552 +/- 37
<Tb>
II. Extraction: 1. 46 kg of fresh algae were dispersed in 3. 82 kg of propylene glycol and then crushed with a colloid mill for 10 minutes. The extraction was carried out at room temperature for 2 hours.

The insoluble phase was removed by centrifugation at 8000 g for 15 minutes and then filtered with filtering soil. 3. 34 kg of hydropropyl extract (20% water) were stored at 4 C.

III. Analysis and characterization of the halogenated compounds: 300 g of fresh raw material were milled in the presence of 600 g of seawater for 10 minutes using a colloid mill. After a contact time of 20 minutes, the suspension was centrifuged at 8000 g for 15 minutes. The extraction was repeated twice. The three supernatants were extracted with 1060 ml of diethyl ether.

The diethyl ether was dehydrated 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 weight of algae. It was solubilized in methyl laurate for the analysis of halogenated compounds was performed by gas chromatography coupled with mass spectrometry.

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The following Table II identifies the halogenated compounds which have thus been highlighted:
TABLE II

<Tb>
<tb> Time <SEP> of <SEP>
<tb> Compounds <SEP> Retention <SEP> in <SEP> Index <SEP> of <SEP> similarity
<tb> mn
<tb> CH2BrCI <SEP> 3:13 <SEP> 680
<tb> Bromochloromethane
<tb> BrCH2CHO <SEP> 4 <SEP>: <SEP> 62 <SEP>
<tb> 2-bromoethanal
<tb> CH21CI <SEP> 6 <SEP>: <SEP> 56 <SEP>
<tb> Chloroiodomethane
<tb> BrCH2COCH3 <SEP> 10:26 <SEP> 802
<tb> 1-bromo-propanone
<tb> Br2CHCHO <SEP> 12:42
<tb> 2,2-dibromoethanal
<tb> Br3CH <SEP> 15:15 <SEP> 677
<tb> Bromoform
<tb> Br2CHCOCH3 <SEP> 15:66
<tb> 1,1-Dibromo-propanone
<tb> CICH2COCHCI2 <SEP> 17:19 <SEP> 558
<tb> 1,1,3-trichloro-2-propanone
<tb> CHBr21 <SEP> 18 <SEP>: <SEP> 79 <SEP>
<tb> Dibromoiodomethane
<tb> BrCH2COCHCI2 <SEP> 18 <SEP>: <SEP> 90 <SEP>
<tb> 1-bromo-3,3-dichloro-2propanone
<tb> BrCH2COCH2Br <SEP> 19:02 <SEP> 559
<tb> 1,3-dibromo-2- <SEP> propanone
<tb> Br2CHCOOC2H5 <SEP> 19:18 <SEP> 407
<tb> Ethyldibromoacetate
<tb> Br2CHCH (OH) CHBr2 <SEP> 26:53
<Tb>
The compounds whose similarity index 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:

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 IV. 1. Bactericidal and fungicidal activity on human pathogenic microorganisms: A control distilled water and a solvent control were used in each test. The hydropropyl extract was tested at 1/10 dilution, dilution for which there is no effect of propylene glycol. The extracts were inoculated with an initial density of 105 to 107 seed / g and maintained 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 microorganisms present determined (final density).

Table III below shows the antibiotic activity of the hydropropyl extract.

TABLE III

<Tb>
<tb> Strain <SEP> tested <SEP> Density <SEP> final <SEP> / <SEP> mL <SEP> Activity <SEP> biocide <SEP> from <SEP> extract
<tb> Staphylococcus <SEP> epidermidis <SEP><<SEP> 102 <SEP> Abattementa <SEP>><SEP> 3 <SEP> log <SEP> / <SEP> NO
<tb> Pityrosporum <SEP> oval <SEP><<SEP> 1.0 <SEQ> 102 <SEP> Abatement <SEP>><SEP> 4 <SEP> log <SEP> / <SEP> NO
<tb> Propiombacterium <SEP> acne <SEP><<SEP> 1.0 <SEP> 102 <SEP> Abatement <SEP>><SEP> 4 <SEP> log <SEP> / <SEP> NO
<tb> Eschenchia <SEP> colt <SEP> 1.5 <SEP> 10 <SEP> 3 <SEP> Abatement <SEP> of <SEP> 3 <SEP> log <SEP> / <SEP> NO
<tb> Staphylococcus <SEP> aureus <SEP><<SEP> 1.102 <SEP> Abatement <SEP>><SEP> 4 <SEP> log <SEP> / <SEP> NO
<tb> Pseudomonas <SEP> aerugmosa <SEP> 6.0 <SEP> 10 <SEP> 2 <SEP> Abatement <SEP> = <SEP> 3.5 <SEP> log <SEP> / <SEP> NO
<Tb>
a: the reduction is the decrease in density from the original density No.

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As shown in Table III, the algal extract tested at 1 / 10th revealed a bactericidal and fungicidal activity allowing a reduction of germs by 3 to 4 log compared to the initial control (distilled water).

 IV.2.Antibiotic activity on pathogenic micro-organisms with respect to fish and man (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 Soxhlet 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 pathogenic strains 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 für Mikroorganismen und Zellkulturen

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 SBUG = Strain Collection of the Institute of Biology of the University Greifswald, Germany.

For the agar medium diffusion assay, the content of an inoculation loop was suspended in 3 ml of 0.9% NaCl solution for each strain. The suspensions thus prepared were inoculated each on an agaric nutrient medium. Sterile paper discs previously impregnated with the algae extract (2 mg) were placed on the inoculated petri dishes. The control discs contained, respectively, oxytretracycline and the extraction solvent used. The diameters of inhibition 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 minimal 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 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 inoculated each on a liquid nutrient medium. After incubation for 18 hours at 26 ° C. (in a water bath, with stirring), the bacterial suspensions were deposited in microplate wells containing the extract of algae at different concentrations: Column 1 (wells A to H) ): Containing the test algae extract Column 1 to 11 (wells A to H): successive 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,

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 - 24 hours at 37 C, for Staphylococcus aureus.

The plates were read at 620 nm using an ELISA microplate reader and then at 550 nm after revelation with iodonitrotetrazolium violet. b) Results: The determinations of antibacterial activities were carried out 10 times for the tests on pathogenic strains of the fish and twice for the pathogenic strains of the man.

Table IV below shows the results of the diffusion test on agar medium on pathogenic strains of fish.

TABLE IV

<Tb>
<tb>#.<SEP>#.<SEP>#<SEP> P
<tb> Strain <SEP> anguillarum <SEP> ruckeri <SEP> salmonicida <SEP> hydrophila <SEP> anguilliseptica
<tb> extracts <SEP> Zone <SEP> inhibition <SEP> [mm]
<tb> dichloromethane <SEP> 10.7 <SEP> 8.9 <SEP> 10.3 <SEP> 6.3 <SE> 13.5 <SEP>
<tb> methanol <SEP> 3.9 <SEP> 0 <SEP> 0 <SEP> 0 <SEP>
<tb> water <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP>
<Tb>
The following Table V shows the results of the diffusion test on agar medium on pathogenic strains of man.

TABLE V

<Tb>
<tb> S <SEP> B. <SEP> M. <SEP> P <SEP> E. <SEP> C
<tb> Strain <SEP> aureus <SEP> subtilis <SEP> falvus <SEP> aeruginosa <SEP> coli <SEP> maltosa
<tb> extracts <SEP> Zone <SEP> inhibition <SEP> [mm]
<tb> dichloromethane <SEP> 12 <SEP> 10.5 <SEP> 18 <SEP> 8.5 <SEP> 6.5 <SEP> 11.5 <SEP>
<tb> methanol <SEP> 3.5 <SEP> 1 <SEP> 6 <SEP> 0 <SEP> 0 <SEP> 11.5 <SEP>
<tb> water <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP>
<Tb>
The minimum inhibitory concentrations were determined on the dichloromethane extract. The results are 105.5 g / ml for

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 Vibrio anguillarum, for Pseudomonas anguilliseptica and 222.5 g / ml for Staphylococcus aureus. The minimum inhibitory concentrations for pure antibiotic molecules such as OTC are 0.08 g / ml for Vibrio anguillarum and 0.08 g / ml for Pseudomonas anguilliseptica.

For ampicillin, the minimum inhibitory concentration is 20 g / ml for Staphylococcus aureus. The complex composition of the algal mixtures explains the deviations from the pure control molecules.

Claims (15)

 CLAIMS 1. A method of intensive and controlled production, on land, of a red alga belonging to the Bonnemaisoniaceae family, said method comprising at least: a) suspending the tetrasporophytic form of said red algae in a culture medium containing seawater; b) culturing said tetrasporophytic form with stirring, at a temperature of 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 basin. 3. Method according to claim 1, characterized in that at least one of the following conditions is controlled: - the supply of nutrients; - the supply of carbon dioxide; - the temperature of the crop; agitation of the culture medium; - the 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 seawater, preferably enriched with nutrient salts and, optionally, with carbon dioxide. <Desc / Clms Page number 19> 5. Method according to claim 1, characterized in that, in 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, in step b), said culture medium is renewed sequentially or continuously. 7. Method according to claim 1, characterized in that, in step b), said agitation is carried out by bubbling or by means of a device such as paddle wheels, propellers or circulation pumps. 8. Method according to claim 1, characterized in that, in step c), said harvest is carried out periodically, for example one week apart. 9. Process according to claim 1, characterized in that said red algae is Asparagopsis armata, and said tetrasporophytic form is Falkenbergia rufalonosa. 10. Process 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 method according to any one of claims 1 to 10, the extraction of halogenated compounds useful for their antibiotic properties. <Desc / Clms Page number 20> 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 harvested in propylene glycol; b) grinding said dispersion; c) extraction with stirring at room temperature for about 2 hours; d) the 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 removal in 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 halogenated compounds are intended to be used in a field chosen from the fields of pharmacy, cosmetics, environment and antifouling coatings.