WO2012072852A1 - Method for enriching nematodes with fatty acids - Google Patents

Abstract

The invention relates to a method for enriching nematodes with fatty acids for the use thereof in aquaculture. The invention also relates to a method consisting in feeding the nematodes with a bacteria that produces fatty acids and a method for feeding fish, molluscs or crustaceans with said nematodes.

Description

 METHOD OF ENRICHMENT OF NEMATODES WITH ACIDS

 FATS

FIELD OF THE INVENTION

The invention relates to a method for enriching nematodes with fatty acids for use in aquaculture. Said method consists of feeding the nematodes with a bacterium producing said fatty acids and subsequently feeding fish, molluscs or crustaceans with said nematodes. BACKGROUND OF THE INVENTION

Fatty acids, constituents of triacylglycerols, are necessary for all living beings, because they are not only a source of energy, but also essential for growth, development and reproduction. Those fatty acids that contain two or more double bonds are called polyunsaturated fatty acids (in English, PUF A). PUF As are constituents of cell membranes and are part of cellular signaling systems. Its deficiency can negatively affect cellular function and can eventually lead to death. In particular, the long-chain PUFA of the omega 3 (ω3) series, eicosapentaenoic acid (C20: 5co3, EPA), has been the subject of various investigations due to its beneficial effects observed in plasma lipids of people with coronary cardiomyopathies, in cancer and rheumatoid arthritis. From the physiological point of view, one of the most important functions of PUF As is to be precursors of eicosanoids, composed of twenty carbon atoms (C20). Eicosanoids correspond to prostaglandins, prostacyclines and leukotrienes (Simopoulos, 1991. J. Clin. Nutr. 54: 438-63). Eicosanoids derived from PUF As omega 6 (ω6) have different physiological properties than those originating from PUF As ω3. Prostaglandins are synthesized by the oxidation of γ-linolenic acids (C18: 3co6, GLA), arachidonic (C20: 4co6, ArA) and EPA, which regulate secretory, digestive, reproductive, immunological and circulation processes blood

Various investigations have also demonstrated the essentiality of PUF As ω3, especially ArA, EPA and docosahexaenoic acid (C22: 6co3, DHA), in the formulation of food used in aquaculture (fish and crustacean larvae). Normally, the diet for the cultivation of many marine species requires about 1 to 2% of long-chain PUF As (Rees et al., 1994. Aquaculture 122, 193-207). Currently, fish oils are the main source of PUF As of type ω3, such as EPA and DHA. However, there are some problems in obtaining the fish food from the fish themselves, such as the difficulty of obtaining very pure products and the unpleasant smell, as well as the low ecological sustainability. It is estimated that in the coming years the demand for PUFA ω3 will increase due to the growth of the aquaculture industry and the increase in demand for specific PUFs for the preparation of food supplements for humans and animals. Alternative sources for the production of PUF As ω3 are bacteria, fungi and microalgae.

The synthesis of -3 polyunsaturated fatty acids by bacteria depends not only on the microorganism but also on the environmental conditions in which these bacteria develop. However, production under controlled growth conditions allows to obtain consistent and reproducible products. The production of PUF As through bacteria is attractive since some species synthesize only one fatty acid, unlike the complex mixture of fatty acids produced by microalgae or accumulated in fish (Russell et al. 1999; Microbiology; 145: 767 -79). Marine bacteria that produce polyunsaturated fatty acids, particularly EPA and DHA, belong to the genera Shewanella, Pseudoalteromonas, Colwellia and Moritella (Russell, N. J. & Nichols, D. S. (1999). Microbiology 145, 767-779). These marine species are found in environments where high pressures and low temperatures prevail.

In most of the cultivated aquaculture species, in their first stages of development (larval stage), the feeding mainly requires three characteristics: that said feeding presents organisms that are alive so that they are attractive to the larva, that present small size (around 1 mm) and that are rich in PUF As. Marine zooplankton, which is the natural food of these species, is difficult to grow and the substitute that has been found most effective for larval culture is a crustacean known as Artemia that is captured in large salty lakes, mainly on the salty lake of Utah. The demand produced by Artemia at international level is enormous, therefore its availability fluctuates greatly, as well as its price, which is usually high. Artemia does not produce DHA, one of the essential PUF As, but there are protocols to enrich them in this fatty acid, for example, in WO0150880 a method is described to enrich with Artemia and rotifer DHA, culturing said organisms in a medium comprising DHA. Although some protocols have been developed to enrich Artemia in several compounds, being an organism that is not cultivated, it presents very little plasticity to modify its nutritional composition or serve as a vector for important compounds in the culture of fish larvae. Nematodes, in contrast, are cultivable and also naturally contain high concentrations of ArA and EPA, although not DHA, so they are good candidates for use in aquaculture.

Once the larval stage is over, in the fattening process, the feeding of the organisms of interest in aquaculture is carried out by means of feed based on fishmeal that comes from the fishing activity, so the aquaculture activity is not sustainable since the environmental point of view, since it is finally based on extractive fishing. The main problem to develop feed from other sources is the need for fish to feed on diets rich in PUF As, which are produced mainly by marine organisms such as algae, phytoplankton and other microorganisms, which although they can be cultivated, are difficult to incorporate the majority of fish of interest in aquaculture into the diet, since these are carnivorous. Methods for enriching nematodes with fatty acids have been described in the state of the art, for example in WO03001909, where a process for preserving and storing nematodes having an increased food value for use in aquaculture is described. Specifically, nematodes are enriched by adding liposomes, among other additives, DHA. The drawback of the use of liposomes that include DHA to enrich nematodes is that the liposomes have to have an adequate size so that they can be ingested by the nematode and it is a complicated and expensive procedure to implement. Also, the amount of DHA that has to be included in the liposome must be quite high so that the nematode incorporates a sufficient amount that can later be used for feeding fish. Likewise, other methods have been developed to enrich organisms with DHA, such as algae, fungi and plants and have been included as fish feed (WO04080196). Recombinant bacteria have also been generated for the production of DHA, cloning a Moritella marina gene in E. coli (Orikasa, Y et al. Biotechnol. Lett. 28: 1841-47).

Although nematodes enriched with polyunsaturated fatty acids, in particular with DHA, have been described in the state of the art, there is a need to find a simple method of obtaining nematodes enriched with polyunsaturated fatty acids that do not come from extractive fishing, which has a high yield and where the nematodes serve as food in aquaculture, both for adult animals and for larval stage animals.

SUMMARY OF THE INVENTION The authors of the present invention have observed that nematodes fed with bacteria producing at least one fatty acid, in particular a polyunsaturated fatty acid, are capable of incorporating said fatty acid. In this way, they have obtained nematodes enriched with fatty acids, useful for example in aquaculture. Thus, in a first aspect, the invention relates to a method for obtaining a nematode enriched with at least one fatty acid comprising culturing said nematode with a bacterium producing said fatty acid.

In a second aspect, the invention relates to a method of feeding an animal selected from the group consisting of a fish, a mollusk and a crustacean, which comprises feeding said animal with a nematode enriched with at least one fatty acid. DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have observed that nematodes fed with fatty acid producing bacteria, in particular polyunsaturated fatty acids, are capable of incorporating said fatty acids. In this way, they have obtained nematodes enriched with fatty acids, useful for example in aquaculture, to feed larvae of fish, crustaceans or molluscs or to be used to be part of feed for fattening of adult fish. The tests carried out by the inventors have shown that the amount of DHA incorporated in the nematodes, with respect to the total fatty acids, is similar to the amount available in zooplankton, as can be seen in Example 2.

Therefore, in a first aspect, the invention relates to a method for obtaining a nematode enriched with at least one fatty acid (hereinafter the first method of the invention) comprising culturing said nematode with a bacterium producing said fatty acid.

The use of the first method of the invention to enrich nematodes with fatty acids, unlike the other methods described in the prior art, has the following advantages:

 1) It is very simple, since it does not need prior treatment, as is necessary in the extraction of lipids or the manufacture of liposomes.

 2) The fact that these bacteria can be cultivated means that nematodes enriched in this way are environmentally sustainable feed, since it avoids the use of products from extractive fishing.

 3) The culture media of these bacteria can be derived from by-products of the food industry so that their production is more profitable compared to Artemia or other methods of growing nematodes and more economically and environmentally sustainable.

4) Bacteria can be genetically modified or their culture conditions for a higher yield in the production of fatty acids. 5) Cultivated nematodes, especially those of the Caenorhabditis elegans species, unlike Artemia, have enormous plasticity. At present there are more than 3000 different strains and multiple protocols for their culture and genetic modification available, so the desired nematode can be used, depending on its composition in proteins, fats or other characteristics that facilitate the incorporation and accumulation in the nematode of the products that are desired.

In order for a molecule of a fatty acid to be incorporated into the fat granules that the nematodes present, it must compete with the fatty acids of the nematode's own synthesis and with the mixture of fatty acids present in the diet. Therefore, the expectations that when a nematode is fed with a fatty acid producing microorganism, the nematode incorporates said fatty acid at sufficient levels to consider the nematode suitable for use in aquaculture (3% of total fatty acids ), are quite low. In the case of M. marina, DHA levels are around 10% of the total fatty acids present in the bacteria (one of the microorganisms with the highest level of this fatty acid), in turn the total fatty acids of a Bacteria is about 10% of the total biomass of a bacterium. This indicates that in a bacterium the total amount of DHA is 1% of the total bacterial biomass, which makes it very unlikely that a nematode that ingested this bacterium would be enriched in this fatty acid. However, the inventors have succeeded in enriching nematodes with fatty acids reaching amounts of DHA of about 3% of the total fatty acids. The term "nematode," as used in the present invention, includes any nematode. Although the first method of the invention can be used to enrich any existing nematode, it will preferably be used to enrich nematodes belonging to non-parasitic free-living species suitable for aquaculture. Examples of nematodes that can be used to practice the first method of the invention include, but are not limited to, organisms of the Rhabditidae and Panagrolaimidae families. More preferably genus nematodes Panagrellus and Caenorhabditis. Even more preferably, the species is selected from Caenorhabditis elegans and Panagrellus redivivus.

Nematode culture is performed following conventional methods described in the prior art, such as the protocol described in Eisenmann, DM (Wnt signaling 2005, WormBook, ed. The C. elegans Research Community), in which it is incubated the nematode for a week in the medium indicated in said protocol, is subsequently subcultured 1/10 of said culture in a medium whose saline concentration is approximately 0.2 M, preferably NaCl. The culture is incubated again for one week and the culture is subcultured 1/10 in a medium whose saline concentration is approximately 0.4 M, preferably NaCl. This process not only manages to cultivate the nematode, but also adapts to these organisms, which are originally terrestrial, to live in a liquid medium in saline concentrations similar to those of seawater, allowing their survival in seawater. The necessary salt concentration in the medium is variable according to the use that is going to be given to the nematodes, if the destination is going to be feeding freshwater animals it is not convenient to increase the salt content of the original protocol.

The term "nematode enriched with at least one fatty acid" refers to a nematode that has levels of at least one fatty acid higher than those of a conventional nematode. Said fatty acid is provided to the nematode through the diet or the medium, so that the levels of fatty acid in the nematode are increased. Some species of nematodes, such as the P. redivivus nematode lack eicosapentaenoic acid (EPA) and do not contain high levels of docosahexaenoic acid (DHA). Other nematodes, such as C. elegans, naturally contain high concentrations of ArA and EPA, but not other fatty acids, such as DHA. An enrichment with at least one fatty acid indicates an increase in said fatty acid with respect to the levels that the nematode naturally presents. Said enrichment or increase may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or even greater than 10%, depending on the applications for which said nematode is to be used, with respect to the non-grown nematode in the presence of the fatty acid producing bacteria. In a particular embodiment, said fatty acid is selected from the group of myristic acid (C14: 1), docosahexanoic acid (C22: 6co3, DHA), eicosapentaenoic acid (C20: 5co3, EPA), palmitic acid (C 16: 0), oleic acid (C 18: lco9), linoleic acid (C18: 2co6), dihomo-gamma-linolenic acid (C20: 3co6, DGLA) and arachidonic acid (C20: 4co6). In an even more preferred embodiment, the nematode is enriched with at least docosahexanoic acid (C22: 6co3, DHA). Preferably, after carrying out the first method of the invention, the nematode is enriched with at least one fatty acid. In a particular embodiment, the enrichment of at least one fatty acid corresponds to a percentage of DHA between 1% and 5% of the total fatty acids, of myristic acid between 0.5% and 3% of the total fatty acids, of eicosapentaenoic acid between 1% and 20% of the total fatty acids, of palmitic acid between 1% and 5% of the total fatty acids, of oleic acid between 1% and 5% of the total of fatty acids, of linoleic acid between 1% and 10% of the total fatty acids, of dihomo-gamma-linolenic acid between 1% and 10% of the total of fatty acids and of arachidonic acid between 1% and 5% of the total fatty acids.

Suitable methods to know if the nematode has been enriched with a certain fatty acid are widely known to those skilled in the art. There are several methods from which it can be known if the nematodes have been enriched with a fatty acid and the proportion of the fatty acid obtained with respect to the total fatty acids. Such methods include, without limitation, chromatographic methods, chemical titration methods (by titration for example with KOH or NaOH or following the method described in Dole VP et al, J Clin Invest 1956, 35, 150 or the method described in Ke PJ et al., Anal Chim Acta 1978, 99, 387), thermometric titration methods (such as that described in Smith TK, J Am Oil Chem Soc 2003, 80, 21-24), measurement of metal-fatty acid complexes (method described in Ho RJ et al., Anal Biochem 1969, 3 1, 426-436, for cobalt complexes), enzymatic methods (described for example in Jebens E et al. Scand J Clin Lab Invest 1992, 52, 717), methods using a fatty acid binding protein (such as those described in Spector AA et al, Biochemistry 1971, 10, 3229, Richieri GV et al. Biochemistry 1993, 32, 7574 and Richieri GV et al., J Lipid Res 1995, 36, 229) and spectroscopic methods (Yoshida S, Lipid Technol 2008, 20, 184 and Wu D et al, Anal Chim cta 2009, 634, 1 66). The person skilled in the art will use the most convenient method to determine the amount of fatty acids generated in the nematode.

In a particular embodiment, in order to know if the nematode has been enriched in fatty acids, the nematode culture is collected, centrifuged and a pellet is obtained, in which the concentration of fatty acids is determined by gas chromatography . For this, the Bligh & Dyer method is followed (Can. J. Biochem. Physiol, 37, 9 1 1-917 (1959)), as described in Example 2. Finally, the fatty acid levels are compared in the culture of nematodes grown in the presence of fatty acid producing bacteria with respect to the levels of nematode fatty acid that have been grown in the presence of non-producing fatty acid bacteria of interest, determining whether the nematode has been enriched in at least one fatty acid. The term "bacteria producing at least one fatty acid" refers to a bacterium that during its growth is capable of generating at least one fatty acid. The production of fatty acids can occur naturally from their metabolism, without said bacteria being modified, or it can be a genetically modified bacterium to produce fatty acids. In a particular embodiment, the bacterium produces at least one fatty acid. In a particular embodiment, said fatty acid is selected from the group of myristic acid (C14: 1), docosahexanoic acid (C22: 6co3, DHA), eicosapentaenoic acid (C20: 5co3, EPA), palmitic acid (C16: 0), acid oleic (C18: lco9), linoleic acid (C 18: 2co6), dihomo-gamma-linolenic acid (C20: 3co6, DGLA) and arachidonic acid (C20: 4co6). In a preferred embodiment, the bacterium producing at least one fatty acid produces at least docosahexanoic acid (C22: 6co3, DFÍA). In a preferred embodiment, the bacterium producing at least one fatty acid is a marine bacterium or a functionally equivalent mutant. Said marine bacterium can present some mutation in its genome as long as its ability to produce fatty acids is maintained. Examples of non-genetically modified bacteria that produce naturally occurring fatty acids, in particular polyunsaturated fatty acids and more particularly, DHA, include, without limitation, marine bacteria of the genera Moritella, Shewanella, Pseudoalteromonas and Colwellia. Preferably, the bacterium producing at least one fatty acid is Moritella marina, in which the level of DHA is approximately 10% of the total fatty acids in the cell. The genus Moritella, as used in the present invention, refers to a member of the relatively new Moritellaceae family (Ivanova et al. 2004; International Journal of Systematic and Evolutionary Microbio lo gy, 54: 1773-88), previously a genus of the order Alteromonadales, which covered other heterotrophic marine Gram-negative bacteria of various genus s within the Gammaproteobacteria class. These genera included Alteromonas, Pseudoalteromonas, Shewanella, Colwellia, Psychromonas, Glacicola and others. Based on the sequence analysis of the 16S rRNA gene, Ivanova et al. they proposed to form new families with these genera, since they separated into different groups in the phylogenetic tree. This resulted in new families that included Moritellaceae, Alteromonadaceae, Pseudoalteromonadaceae, Shewanellaceae and others (Ivanova et al. 2004, ad supra). The species Moritella marina was the first one described in the genus Moritella, and so far six other species belonging to the same genus have been identified: M. japónica, M. yayanosii, M.viscosa, M.profunda, M.abyssi and M dasanensis The genus Shewanella, as used in the present invention, refers to the only genus included in the family of marine bacteria Shewanellaceae (MacDonell & Colwell 1985. Syst Appl Bacteriol 6: 171-182). Currently this genus comprises more than twenty species. All of them are Gram-negative, anaerobic and facultative aerobic associated mainly with aquatic habitats. They are also capable of producing high proportions of polyunsaturated fatty acids (Myers et al. 1988. Science 240: 1319-1321).

The genus Pseudoalteromonas, as used in the present invention, refers to a genus of large negative marine bacteria comprising more than thirty species. These are facultative aerobic or anaerobic bacteria that are capable of producing polyunsaturated fatty acids. The genus Colwellia, as used in the present invention, originally included two facultative anaerobic bacteria, Colwellia psychrerythraea and C. hadaliensis (Deming et al. 1998. Syst. Appl. Microbiol. 10: 152-60). Subsequently, Bowman et al. (Int. J. Syst. Bacteriol. 1998; 48: 1 171-80) described four new psychrophilic species of this genus, C. demingiae, C. hornerae, C. rossensis and C. psychrotropica, and new strains of C. psychrerythraea . None of these Antarctic isolates are barophilic and all of them synthesize docosahexanoic acid in amounts of up to 8% of the total cellular content of fatty acids. In a particular embodiment, the fatty acid producing bacterium is a genetically modified recombinant bacterium that does not produce said fatty acid naturally. As used in the present invention, the term "recombinant bacteria" refers to any bacteria that has been genetically modified by the introduction of heterologous DNA, such that said heterologous nucleic acid sequences allow the bacteria to be able to generate less a fatty acid, preferably a polyunsaturated fatty acid and more preferably DHA. Recombinant bacteria producing at least one polyunsaturated fatty acid would be those in which the desired gene or a vector containing the desired gene is introduced, by conventional recombinant techniques (Sambrook et al., 2001. "Molecular cloning: a Laboratory Manual ", 3rd ed., Cold Spring Harbor Laboratory Press, NY, Vol. 1-3). It is known that EPA, ArA and DHA polyunsaturated fatty acids can be generated in different ways. For example, they can be produced by marine bacteria from the polyketide pathway (Yu R et al. Lipids 2000; 35: 1061-64). An alternative strategy is a route, where the alternate activity of desaturases and elongases is used (Zank TK et al. Plant Journal 2002; 3 1: 255-68). A modification of this route by the A6-desaturase, A6-elongasa, A5-desaturase, Δ5-elongasa and A4-desaturase is the so-called Sprecher route (Sprecher 2000. Biochim. Byophis. Act 1486: 219-31) in mammals. Recombinant bacteria may include at least one gene from one of the enzymes described above, such as an elongase and / or a desaturase, so that the resulting bacterium has the ability to produce polyunsaturated fatty acids, such as DHA. Examples of recombinant bacteria include, but are not limited to, bacteria selected from the group of the families Bacillaceae, Enterobacteriacae or Rhizobiaceae, such as bacteria of the selected genera of Bacillus, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Klebsiella, Salmonella, Serratia, Agrobacterium, Carbophilus, Chelatobacter, Ensifel, Rhizobium. Rhizobium. Rhizobium. Rhizobium. Rhizobium. Rhizobium. In a preferred embodiment, the recombinant bacterium is Escherichia coli.

The culture of the bacterium producing at least one fatty acid is carried out by conventional methods described in the state of the art and depends on the bacterium that is to be cultivated. A person skilled in the art knows extensively the means and conditions according to the bacteria used to enrich the nematodes. In a particular embodiment, the bacteria is M. marina. Preferably the growth of M. marina is carried out in Luria-Bertani (LB) medium supplemented with NaCl at a culture temperature between 4 and 20 ° C, preferably approximately 16 ° C for a period of time between 24 and 72 hours More preferably, the culture is carried out with stirring.

The enrichment of the nematode with the fatty acid producing bacteria is carried out in a conventional manner for the growth of nematodes in the presence of said bacteria. In a particular embodiment, a culture of bacteria is centrifuged and the pellet or centrifuge of said culture is added to the culture medium of the nematodes, known as "Medium S". On the other hand, the nematode is inoculated from a culture grown in solid NGM medium (2.5 g peptone, 17 g agar, 3 g NaCl, 975 ml distilled water, 1 ml cholesterol (5 mg / ml) (stock in ethanol), 1 ml of 1 M CaCl ₂ , 1 ml of MgSO ₄ 4 M, 25 ml of KH ₂ P0 ₄ 1 M). The composition of the nematode culture medium, "Medium S", is preferably as follows (to prepare 1 liter of medium):

NaCl 5.85 g

K ₂ HP0 ₄ ig

 10 ml potassium citrate

 pH 6 1 M

 Disodium EDTA 0.0186 g

FeS0 ₄ 0.0069 g

MnCl ₂ 0.002 g

ZnS0 ₄ 0.0029 g

CuS0 ₄ 0.00025 g CaCl ₂ 1M 3 ml

The invention contemplates the growth of the nematodes with the bacterium producing at least one fatty acid at a temperature suitable for the enrichment of said nematodes and which will be determined by one skilled in the art according to the nematode that is intended to be enriched. In a particular embodiment, the growth temperature of the nematode for enrichment to occur is between 15 ° C and 25 ° C. Preferably said temperature is between 16 ° C and 20 ° C. The growth temperature of the nematode is variable according to the fatty acid with which you want to enrich the nematode, since, although fatty acids are generated at virtually any temperature, the production of some fatty acids is optimal at certain temperatures. For example, a temperature of about 16 ° C is preferred if it is desired to enrich a nematode with oleic acid, linoleic acid, dihomo-gamma-linolenic acid and arachidonic acid. However, a temperature of about 20 ° C is preferred if it is desired to enrich a nematode with myristoleic acid and docosahexanoic acid (DHA).

In a preferred embodiment, the nematode culture is incubated with stirring at the chosen temperature. The culture is frequently monitored under a microscope, so that if the culture does not appear cloudy under the microscope, it is indicative that the nematodes have finished the food, that is, the bacteria producing at least one fatty acid have run out. At this time, it is necessary to add more pellet of fatty acid producing bacteria to the medium. Preferably on the fourth or fifth day, the culture is collected, placing the flask on ice to allow the nematodes to deposit in the bottom. Subsequently, the nematode culture is centrifuged and the nematodes enriched in at least one fatty acid are recovered.

In a second aspect, the invention relates to a method of feeding an animal selected from the group consisting of a fish, a mollusk and a crustacean, hereinafter second method of the invention, comprising feeding said animal with a nematode enriched with the Less a fatty acid. The second method of the invention developed by the inventors implies several advantages over other methods of feeding aquatic animals. One of the advantages is that the use for aquatic animals of enriched nematodes from fatty acid producing bacteria results in a very high efficiency of incorporation of said fatty acids, in particular DHA. In the case of fish, these levels of incorporation of DHA into the nematode reach levels of around 3%, similar to that obtained with zooplankton, a natural food for fish larvae. On the other hand, the second method of the invention can be used both to feed adult animals and to feed larvae of fish, crustaceans or molluscs. The feeding of fish, molluscs and crustaceans in larval stage presents several problems, solved by the second method of the invention; One of them is that the size of the food has to be around 1 mm and in addition the food must be mobile, so that it is more attractive to the larva. Likewise, the use of nematodes provides arachidonic acid and eicosapentaenoic acid to the animal, while the bacterium provides, among other fatty acids, DHA, essential for the proper development of said animal, both in the larval stage and in the adult stage.

In a particular embodiment, the second method of the invention can be used to feed adult fish, that is, as feed for fattening for said animals, be they fish, crustaceans or mollusks.

The term "larval stage", as used in the present invention, refers to a distinctive juvenile form of mollusk, crustacean or fish before its metamorphosis occurs in an adult animal. The duration of said larval stage depends on the species of fish, crustacean or mollusk, thus, in fish, for example, it is relatively short, between 3 and 8 weeks. The larval stage ends at the moment when the larva changes its appearance and becomes an adult animal.

The specific details of the culture of the nematodes enriched with at least one fatty acid are found in the first method of the invention, so reference is made to them for the second method of the invention and will not be explained in more detail. The fish contemplated in the second method of the invention include, without limitation, fish of the following classes: Conodonta, Hyperoartia, Petromyzontidae (lampreas), Pteraspidomorphi, Thelodonti, Anaspida, Cephalaspidomorphi, Galeaspida, Pituriaspida, Osteostrata (Gnathostíbta) (Gnathostibula) , Placodermi, Chondrichthyes (cartilaginous fish), Acanthodii, Osteichthyes (bony fish). In a preferred embodiment said fish belongs to the Cyprinidae family, more preferably it is zebrafish (Danio rerio). In another particular embodiment, said fish belongs to the Moronidae family and to the genus Dicentrarchus. More preferably, said fish is sea bass (Dicentrarchus labrax). In another particular embodiment, said fish belongs to the Sparidae family and more preferably, to the genus Sparus. Even more preferably, said fish is Sparus aurata or dorado.

Crustaceans contemplated in the second method of the invention include, without limitation, crustaceans of the following classes: Branchiopoda, Remipedia, Cephalocarida, Maxillopoda, Ostr acoda and Malacostraca.

Molluscs contemplated in the second method of the invention include, without limitation, mollusks of the following classes: Caudofoveata, Solenogastres, Polyplacophora, Monoplacophora, Bivalvia, Scaphopoda, Gastropoda and Cephalopoda.

In order to carry out the second method of the invention, nematodes are first enriched by any conventional method, preferably following the first method of the invention described above. Subsequently, the animals (fish, crustaceans or mollusks) are fed with enriched nematodes. The amount of nematodes that is administered to the animal depends on the method of culture used, the stage of development of said animal and the species of cultured animal. A person skilled in the art is able to determine the feeding conditions and will administer different amount of nematodes according to said conditions. Suitable methods to know if the animal is feeding properly with the nematodes include, but is not limited to, (a) observation under the magnifying glass of the main morphometric parameters of said animal (size, thickness, physiological state, development of organic structures, such as bone structures, eyes, pigmentation); (b) measurement of the levels of fatty acids and proteins of animals fed with the nematode; and (c) use of a transgenic nematode strain that expresses the green fluorescent protein (GFP), in this way it is observed by fluorescence microscopy if the nematodes are being ingested or not by the larvae of the different species.

Also, nematodes enriched with at least one fatty acid can be combined together with other organisms used to feed fish, such as Artemia or with feed produced from fishmeal.

The following examples illustrate the invention and should not be considered in a limiting sense thereof.

EXAMPLE 1

 Nematode growth with Moritella marina

250 ml of culture medium (S medium) inoculated with Caenorhabditis elegans recovered from 4 petri dishes were grown for 7 days. Subsequently, 1/10 of said culture was subcultured in the same medium with a 0.2 M NaCl salt concentration. It was incubated for a week and 1/10 of said culture was subcultured again in a medium with a NaCl salt concentration. 0.4 m.

The medium S has the following composition:

- Basal S medium [5.85 g NaCl, 1 g K ₂ HP0 ₄ , 6 g KH ₂ P0 ₄ , 1 ml cholesterol (5 mg / ml in ethanol), H ₂ 0 to 1 1. Sterilize].

- 1 M potassium citrate pH 6.0 [20 g citric acid monohydrate, 293.5 g potassium citrate monohydrate, H ₂ 0 to 1 1. Sterilize].

Trace solution of metals [1.86 g EDTA disodium, 0.69 g FeS0 ₄ ^» 7 H ₂ 0, 0.2 g MnCl ₂ ^» 4 H ₂ 0, 0.29 g ZnS0 ₄ ^» 7 H ₂ 0, 0.025 g CuS0 ₄ ^» 5 H ₂ 0, H ₂ 0 to 1 1. Sterilize. Store in darkness].

- 1 M CaCl ₂ [55.5 g CaCl ₂ in 1 liter H ₂ 0. Sterilize]

Medium S [1 1 S Basal, 10 ml 1 M potassium citrate pH 6, 10 ml trace metal solution, 3 ml 1 M CaCl ₂ , 3 ml 1 M MgSO ₄ . It does not autoclave]. The culture of Moritella marina was incubated with stirring for 72 h in 2 1 of LB supplemented with 3% NaCl. The culture was centrifuged, so that a "pellet" of bacteria was obtained. The culture comprising C. elegans was inoculated with the M. marina culture pellet. The culture was incubated with shaking at 20 ° C or 16 ° C, according to the experiment. The cultures were monitored under a microscope to check the growth of the nematode. If the nematode no longer has food, that is, if the culture medium is not cloudy (no bacteria present), more bacteria pellets were added. The moment was observed microscopically that sufficient nematodes in a drop, the culture was harvested at approximately 5 ^or day. To allow the collection of the nematodes, the culture was placed on ice for 15 minutes for the nematodes to deposit. Most of the liquid in the flask was aspirated. The remaining culture liquid was transferred to a sterile centrifuge tube and centrifuged for at least 2 minutes at 1150 g to collect the nematodes.

As a control, Caenorhabditis elegans were grown using a non-fatty acid producing bacteria, such as E. coli, as food, under the same conditions in which the culture with Moritella marina was carried out. EXAMPLE 2

 Fatty acid production

The determination of the amount of fatty acids in the nematode pellet obtained in Example 1 was performed by gas chromatography. For this, the Bligh & Dyer method (Can. J. Biochem. Physiol, 37, 91 1-917 (1959)) was followed, in which 3.75 ml of 1: 2 (v / v) CHC1 are added ₃ : methanol and stir, then 1.25 ml of CHCI ₃ is added, and it is stirred again, finally adding 1.25 ml of H ₂ 0. It is centrifuged at 1000 rpm for 5 minutes at room temperature and the phase is recovered lower. Then, to determine the fatty acid composition, the lipid extract obtained is transmethylated for further analysis by gas chromatography, adding 1 ml of 2.5% H ₂ S0 ₂ diluted in methanol and incubating it 1 hour at 80 ° C. Once cold, it will Add 0.2 ml of hexane and 1.5 ml of H ₂ 0, centrifuge at low speed, recovering the upper phase for subsequent injection in the gas chromatograph.

Table 1 shows the percentage of fatty acids with respect to the total fatty acids present in the crop. The first row of Table 1 shows the amounts of different fatty acids in M. marina. As can be seen, the most abundant fatty acids in Moritella are, in this order: palmitoleic acid (16: ln7); palmitic acid (16: 0), octadecenoic acid (18: ln7) and finally docosahexanoic acid (22: 6) (DHA). The levels of incorporation of the most abundant fatty acid (16: ln7) in M. marina, which represents 55.7% of the total fatty acids is really very low (it goes from 3.01% in the nematode grown with E. coli 3.32% in the nematode grown with M. marina at 20 ° C). The same goes for the following two types of more abundant fatty acids. However, 22: 6 (DHA) fatty acid levels are increased to levels similar to those of zooplankton (about 3%) (Koven et al. 2001. Aquaculture 193: 107), with respect to the levels of said fatty acid of a nematode grown in a standard medium (without Moritella), Therefore, it can be concluded that a preferential incorporation of DHA occurs.

This process of high incorporation of DHA does not occur in all culture conditions. As can be seen in the last row of Table 1, at a lower temperature of the nematode culture (16 ° C), the enrichment in DHA is lower and the levels of 3% presented by zooplankton (natural food of fish larvae). However, at a growth temperature of 20 ° C, the incorporation of DHA is much more efficient.

As for enrichment with other fatty acids, as shown in Table 1, some fatty acids, both saturated and polyunsaturated, are efficiently incorporated into the nematode when grown with M. marina, with respect to levels when grown with E. coli. Said fatty acids with which C. elegans is enriched are the following: myristic acid (C 14: 1), palmitic acid (C 16: 0), oleic acid (C18: lco9), linoleic acid (C18: 2co6), acid dihomo-gama-linolenic (C20: 3co6, DGLA) and arachidonic acid (C20: 4co6), apart from enrichment with docosahexanoic acid (C22: 6co3, DHA), already mentioned. The optimum temperature of Nematode growth for the production of said fatty acids is variable, according to the fatty acid that is desired to be produced, as observed in Table 1, comparing both growth temperatures. The temperature will preferably be 16 ° C if it is desired to enrich the nematode with oleic acid, linoleic acid, dihomo-gamma-linolenic acid and arachidonic acid. However, the temperature will preferably be 20 ° C if it is desired to enrich the nematode with myristoleic acid and docosahexanoic acid (DHA).

Table 1: Comparison of the concentration of fatty acids (% of total fatty acids) present in Moritella marina (row 1), in C. elegans in the presence of E. coli, grown at 20 ° C (row 2) and in C. legumes in the presence of Moritella marina grown at 20 ° C and 16 ° C (rows 3 and 4).

 [The fatty acids are named by numbers, the first indicates the total number of carbons, the second followed by two points the number of double bonds and the nx indicates the position of the first double bond]

Claims

A method of obtaining a nematode enriched with at least one fatty acid comprising culturing said nematode with a bacterium producing said fatty acid.  Method according to claim 1, wherein the fatty acid is a polyunsaturated fatty acid.  Method according to claim 1, wherein the fatty acid is selected from the group of myristic acid (C14: 1), docosahexanoic acid (C22: 6co3, DHA), eicosapentaenoic acid (C20: 5co3, EPA), palmitic acid (C16: 0 ), oleic acid (C18: lco9), linoleic acid (C 1 8: 2co6), dihomo-gamma-linolenic acid (C20: 3co6, DGLA) and arachidonic acid (C20: 4co6).  Method according to any of claims 1 to 3 wherein the bacterium producing said fatty acid is a marine bacterium.  Method according to claim 4 wherein the marine bacterium is a bacterium selected from the genera Moritella, Shewanella, Photobacterium, Pseudoalteromonas and Colwellia.  Method according to any one of claims 1 to 3 wherein the bacterium producing said fatty acid is a recombinant bacterium that produces said fatty acid.  Method according to any one of claims 1 to 6, wherein the nematode is selected from Caenorhabditis elegans and Panagrellus redivivus. A method of feeding an animal selected from the group consisting of a fish, a mollusk and a crustacean, which comprises feeding said animal with a nematode enriched with at least one fatty acid.  Method according to claim 8, wherein the fatty acid is a polyunsaturated fatty acid. Method according to claim 9, wherein the fatty acid is selected from the group of myristic acid (C14: 1), docosahexanoic acid (C22: 6co3, DHA), eicosapentaenoic acid (C20: 5co3, EPA), palmitic acid (C16: 0 ), oleic acid (C18: lco9), linoleic acid (C 1 8: 2co6), dihomo-gamma-linolenic acid (C20: 3co6, DGLA) and arachidonic acid (C20: 4co6). 11. Method according to any of claims 8 to 10 wherein the nematode enriched with at least one fatty acid has been obtained by culturing said nematode with a bacterium producing said fatty acid.  12. Method according to claim 11, wherein the bacterium producing said fatty acid is a marine bacterium.  13. Method according to claim 12, wherein the marine bacterium is a bacterium selected from the genera Moritella, Shewanella, Photobacterium, Pseudoalteromonas and Colwellia.  14. A method according to claim 11, wherein the bacterium producing at least one fatty acid is a recombinant bacterium that produces said fatty acid. 15. Method according to any of claims 8 to 14, wherein the nematode is selected from Caenorhabditis elegans and Panagrellus redivivus.  16. Method according to any of claims 8 to 15, wherein the animal is in a larval stage of its development.  17. Method according to any of claims 8 to 15, wherein the animal is an adult animal.