WO2016037296A1 - Method for producing indigenous probiotocs with immunostimulant activity and use thereof in prophylaxis against flavobacteriosis in salmonids - Google Patents

Abstract

The invention relates to a method for identifying and producing in fish microbiological strains with immunostimulant capability for protection against pathogenic micro-organisms, comprising the steps of: isolating the microorganisms from the digestive tract of healthy hosts; genetically characterising the isolated micro-organisms; characterising the complementary beneficial properties; identifying immunostimulant capability in axenic models; and producing the probiotics. The invention also relates to the use of said probiotics in prophylaxis against flavobacteriosis in salmonids.

Description

"METHOD FOR THE PRODUCTION OF AUTONOMOUS PROBIOTICS WITH IMMUNOSTIMULATING ACTIVITY AND ITS USE IN PROFILAXIS AGAINST FLAVOBACTERIOSIS IN SALMONIDS"

FIELD OF THE INVENTION

 The field of the present invention relates to the control of microbiological diseases (such as Flavobacteriosis produced by the pathogen Flavobacterium psycrophilum) with the use of probiotics with immuno-stimulant capacity in underwater species such as salmonids, their identification and method to obtain them with the greatest immunostimulatory capacity.

BACKGROUND

 The use of antibiotics has been the main therapeutic tool used in the control of bacterial diseases in fish.

The development of antibiotics in the treatment of diseases in fish, has obtained as a counterpart a continuous development of resistance by the objective to be treated, not to mention its indiscriminate use.

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 The search for alternatives in treatment has led to the review of the probiotics tool as an element of immunity control, in order to improve it and prepare it for the environment where the fish is raised and developed.

The use of probiotics in the food industry is known and applied today in multiple uses. Its use in the displacement of bacterial or fungal cultures in fish is already known as presented for Vibrio sp. control and Saprolegnia sp. in documents (WO2012 / 105805) and (WO 2012/105804) respectively.

Some of the authors who started with the idea of using probiotics in aquaculture were Nikoskelainen et al. (2001) who investigated the properties of potential probiotics of five lactic acid bacteria used in humans such as Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus bulgaricus, Bifidobacterium lactis and Lactobacillus lactis johnsonii and a probiotic for animal use Enterococcus faecium. The researchers evaluated in vitro the ability of these strains to adhere and penetrate the mucous barrier, inhibit the growth and adhesion of pathogens such as Vij rio anguillarum, Aeromonas salmonicida and Flavobacterium psychrophylum and resistance to fish bile. They found that only two candidates (L. rhamnosus and L. bulgaricus) fulfilled these capabilities, could serve as possible probiotics for challenges at the in vivo level. Following this study, in later years the same author in (2003), decided to conduct a study with the L. rhamnosus bacteria, the probiotic was included and administered in the diet of Rainbow Trout (O. mykiss) in 5 different doses from 10 ⁴ to 10 ¹⁰ CFU / g, for two weeks observing that at the in vitro level the activity at the cellular level of the respiratory outbreak increased, at the humoral level of the complement system mediated by bactericidal activity and at the level of the adaptive immune system by the activation of Immunoglobulin ( Ig). The respiratory burst (also known as oxidative burst) is the rapid release of reactive oxygen species (ROS) such as superoxide radicals and hydrogen peroxide (H2O2), from different cell types such as granulocytes and phagocytes when they enter contact with different microorganisms, Its usefulness is the inactivation of microbial pathogens. Another author who decided to use the same bacteria (L. rhamnosus) was Panigrahi et al. (2004), observing that feeding Rainbow Trout (O. mykiss) with doses between 10 ⁹ and 10 ¹¹ CFU / g level in vitro induces the activation of some parameters of the innate immune system. This part of the immune system is the first defense barrier against invading microorganisms, which has no memory, is not specific and is based on general humoral (lysozyme, complement) and cellular (phagocytes, neutrophils) responses. In the study conducted by the authors Lara-Flores et al. (2003), decided to evaluate in fry of Tilapia Nilotica {Oreochromis niloticus), commercial probiotics for terrestrial vertebrates such as Streptococcus faecium and Lactobacillus acidophilus and a yeast Saccharomyces cerevisiae, and determined that adding 0.1% of these microorganisms in the diet improved growth and mitigated the effects of stressors. Salinas et al. (2008), worked with Lactobacillus delbrueckii subsp. Lactis strain that has had great attention in humans and veterinary medicine. These authors observed in vitro in the proximal intestine of Atlantic Salmon (S. salar), that this bacterium was able to adhere to the mucus and epithelial cells of the intestinal lumen, in the form of clusters on the surface of the villi and did not generate alterations in the morphology and integrity of the gastrointestinal barrier. This contrasted with what was observed for the pathogenic bacterium Aeromonas salmonicida, which caused epithelial damage and induced the migration of cells from the innate immune system to the adhesion site. Meanwhile, when the proximal intestine was incubated with the probiotic and then exposed to the pathogen, no tissue damage was observed, thus suggesting that the probiotic protects against the pathogen's action. In summary, the bacteria mentioned above were chosen for studies in aquaculture as possible candidates for probiotics because they have had beneficial effects on terrestrial organisms such as pigs, poultry, and humans. But this could represent a problem at medium or large scale in the future in the industry, since they are exogenous microorganisms, which when introduced into a habitat to which they do not belong can generate an imbalance in the environment and could become potential pathogens for the species of cultivation (Cahill, 1990; Ring0 et al., 2003, 2006 and 2008; Skrodenyte-Arbaciauskiene et al. 2006; Hovda et al. 2007 and 2012; Denev et al., 2009).

As a result of this, we begin to investigate candidates for probiotics in aquaculture from the microbiota of the gastrointestinal tract of healthy animals or bacteria from the same medium, an idea based on previously conducted studies, where it is established that the colonization of the normal microbiota on the surface of the intestinal mucosa provides resistance to the adhesion of pathogenic bacteria and mediates the stimulation of the immune response, in addition, it has a positive effect on the regulatory functions of nutrient metabolism

(Jóborn et al., 1997; Burr and Gatlin, 2005; Skrodenyte-Arbaciauskiene et al., 2006; Gómez and Balcázar, 2008; Denev et al., 2009; Pérez et al., 2010; Bezirtzoglou et al., 2011) . This challenge has increased research to improve the quality and competitiveness of production

(Planas et al., 2006; Denev et al., 2009; Bezirtzoglou et al., 2011).

In salmonids of commercial importance, bacteria have been isolated and identified from the tract Gastrointestinal, of these genera, have been studied for candidates as possible probiotics, to bacteria belonging to the so-called group of lactic acid bacteria (LAB), because they are found in the gastrointestinal tract of homeothermal animals such as mice, rats, pigs in pens and in humans, in dairy products and in some plant surfaces, they are also considered as "Generally Recognized as Safe" bacteria (GRAS) (Ring0 and Gatesoupe, 1998).

Jóborn et al. (1997), has been one of the researchers who has found in vitro that the strain Kl belonging to the genus Carnobacterium isolated from the intestine of Atlantic Salmon (Salmo salar), was tested in intestinal mucus and fecal culture of Rainbow Trout (O mykiss), finding that they produced substances that caused growth inhibition of the pathogenic bacteria Vibrio anguillarum and A. salmonicida. Also, at the in vivo level, the addition of lactic acid bacteria in the rotifers, food of sole larvae of Scophthalmus maximus, increased growth and resistance against pathogenic Vibrios (Gatesoupe, 1991 and 1994). Balcázar et al. (2008), studied three bacteria belonging to Lactococcus lactis (CLFP 101), Lactobacillus plantarum (CLFP 238) and Lactobacillus fermentu (CFLP 242) previously isolated and identified in Rainbow Trout, it was found that when tested in intestinal mucus under in vitro conditions, strains CLFP 101 and CFLP 242 inhibited the growth of pathogens such as Aeromonas hydrophila, A. salmonicida, Yersinia ruckeri and Vibrio anguillarum while CLFP 238 only reduced the growth of A. hydrophila and A. salmonicida. Other genera studied have been Carnobacterium, Bacillus, Lactobacillus, Lactococcus and Leuconostoc isolated from the gastrointestinal tract of Rainbow Trout, which at the level In vitro they have been able to induce the expression of the cytokines il-ΐβ and tnf-a by suggesting that these bacteria stimulate the innate immune system and at the cellular level they cause the increase in respiratory outburst and lysozyme activity, in addition to the production of antibacterial compounds such as bacteriocins and the improvement in the growth rate (Ring0 et al., 2000; Kim and Austin, 2006a and b; Gatesoupe, 2008; Pérez-Sánchez et al., 2011). At the in vivo level Vendrell et al., (2008) evaluated the beneficial effects that two bacteria (Leuconostoc mesenteroides and Lactobacillus plantarum) could produce, isolated from the intestine of healthy salmonids. These bacteria were included and administered in the Rainbow Trout feed, at a density of 10 ⁷ CFU / g, for a period of 30 days, subsequently challenged with a fish pathogen Lactococcus garvieae, finding that it reduced mortality by a lower percentage which only reached 30% with respect to the control group.

In addition, bacteria or yeasts isolated and identified in the gastrointestinal tract of salmonids, could be used in other species of fish such as Reyes-Becerril et al. (2008) who administered in the juvenile diet the yeast Debaryomyces hansenii strain CBS 8339, isolated from the intestine of rainbow trout. At the in vitro level in segments of digestive tract tissue, they observed that the yeast managed to adhere to the intestinal mucus. Tests were carried out to determine immunological activity in the blood of fish fed the diet containing the yeast, showing that at the humoral level there was activity of the complement and respiratory burst, but no activity of the peroxidase enzyme occurred. At the cellular level, the percentage of phagocytic and cytotoxic cells increased. In the liver, enzyme activity of superoxide dismutase was observed and catalase and in this same organ, the complement protein gene (c3) was significantly expressed, while in the intestine and renal cortex, the expression of the fccr-β, c3 and tnf-a genes was induced, after 4 weeks of feeding. However, no in vivo pathogen protection trial has been reported.

Tovar-Ramírez et al. (2010) worked with the same yeast described above and administered it to larvae of 6 days post hatching of Dorada (Sparus aurata), for a period of 48 days. Subsequently, they measured on a mix of tissues that included segments of the pancreas, liver, heart, muscle, back and intestine, the expression of catalase (CAT), glutathione peroxidase (GPX) and super oxide dismutase (SOD). These authors found that yeast promoted greater larval growth and inhibited oxidative stress, by suppressing the expression of enzymes, compared to larvae that did not receive yeasts.

In view of the background, a large part of the studies use a strategy that can be considered in vitro, since they do their tests based on blood cells extracted from treated fish. These treatments generally consist of supplementing the fish's food with native bacteria from the fish's gastrointestinal tract. The observations indicate that by drawing blood and performing immunological analysis at the humoral level as a respiratory burst activity, complement, lysozyme and at the cellular level phagocytic activity, immunological stimulation was promoted. Similarly, they occurred when they extracted organs and mediated the gene expression of some genes related to innate immunity, observing that they modulated the expression (Gatesoupe, 1991 and 1994; Jóborn et al., 1997; Ring0 et al., 2000; Kim and Austin, 2006a and b; Balcázar et al. , 2008; Gatesoupe, 2008; Pérez-Sánchez et al., 2011). However, current research calls into question the relevance of these methods, since the handling of fish to extract the samples creates stress and can alter the results. Therefore, these tests should be strongly complemented with more modern and precise strategies that can assess the stimulation of the immune system without disturbing the fish. In vivo tests do not give a clear view of the type of interaction that occurs between bacteria-host, or the role that each bacterial component of the microbiota could play in the gastrointestinal tract. There is a history where they supplement the food with probiotic and then measure the expression of some genes of innate immunity and enzymes related to oxidative stress and have observed that they modulate the induction of the significant expression of some genes, but it is not known if it is produced by the probiotic bacterium or by another bacterium found in the gastrointestinal content (Gómez and Balcázar, 2008; Reyes-Becerril et al., 2008; Pérez et al., 2010; Tovar-Ramírez et al., 2010).

On the other hand, challenge studies against pathogens have shown that the inclusion of probiotics in food generates protection by reducing mortality (Nikoskelainen et al., 2001b; Sharifuzzaman and Austin, 2010). However, these studies do not establish for sure that protection is produced by that probiotic. In general, there are limited periods of persistence and sometimes failures in the detection of the probiotic in the host. Therefore, the protection observed may be due to the proliferation of another bacterium in the fish's gastrointestinal tract. Is This is why it is important that in order to generate a probiotic, prior studies in axenic organisms are carried out to verify its individual effect on the host.

The pathogenic bacterium Flavobacterium psycrophilum is the causative agent of bacterial cold water disease in salmon and juvenile syndrome in rainbow trout, pathological entities responsible for substantial economic losses in salmon farming.

Problems associated with epizootics include high mortality rates, increased susceptibility to other diseases, high labor costs of treatment and huge expenditure on antibiotic treatments. Despite the growing importance of the disease, the pathogenesis of F. psychrophilum infections has only been partially elucidated, making it difficult to develop preventive measures to effectively combat this disease (Nematollahi et al., 2003).

The state of the art indicates that Flavojbacterium strains are mainly used as bio-pesticides

(WO2013138398) or in processes of extraction of biological materials for the food industry (O2013087043) or

(EP2099906). There is no mention in the state of the art of its combat through the production of some microorganism that operates as an immuno-stimulant in some animal or human species.

SUMMARY OF THE INVENTION

In a first aspect of the present invention it presents a method of characterizing a probiotic contrasted in based on its immuno-stimulant capacity against infection caused by Flavobacterium psycrophilum.

A second aspect of the present invention protects the use of probiotics in the prevention and prophylaxis of infection caused by Flavobacterium psycrophilum.

A third aspect of the present invention protects the composition in which the probiotic to be used in the treatment, prevention and prophylaxis of infection caused by Flavobacterium psycrophilum resides.

A fourth aspect of the present invention protects specific mixtures of microorganisms that enhance the response in the treatment, prevention and prophylaxis of infection caused by Flavobacterium psycrophilum.

A fifth aspect of the present invention protects the bacterial microorganism Carnobacterium sp. A025 with biological deposit No. A025-B / 00069 to be used in the treatment, prevention and prophylaxis of infection caused by Flavojacteriujn psycrophilum.

A fifth aspect of the present invention protects the bacterial microorganism Lactococcus sp. B018 with biological deposit No. B018-B / 00070 to be used in the treatment, prevention and prophylaxis of infection caused by Flavobacterium psycrophilum.

A sixth aspect of the present invention protects the bacterial microorganism Pediococcus B022 with biological deposit No. B022-B / 00071 to be used in the treatment, prevention and prophylaxis of infection caused by Flavobacterium psycrophilum.

An eighth aspect of the present invention protects the microorganism of the Debaryomyces K002 yeast type with biological deposit No. RGM 2143 to be used in the treatment, prevention and prophylaxis of infection caused by Flavobacterium psycrophilum.

DETAILED DESCRIPTION OF THE INVENTION

 Some important definitions to understand the present invention begin with the clarification of the probiotic term. Within pro-biotics, live microorganisms that are considered healthy for the host organism are considered. According to the definition currently adopted by FAO / WHO, probiotics are "living microorganisms that when administered in adequate amounts confer a health benefit to the host." Probiotics are consumed as part of food with special added live active cultures encapsulated in a supporting protein matrix, without restricting this form of preparation to others existing in the state of the art. In this way, it is intended to administer live microorganisms, adding them in the food, in order to contribute to the balance of the intestinal microbiota and boost the immune system of farmed fish.

On the other hand, the term probiotic is intended to encompass any microorganism that manages to induce the host's immune system against a specific strain or family of strains that cause biological, competition or chemical damage to the host. 6

In the present invention, the host, host, host, host to that generally multicellular organism will be considered, which houses another in its interior or carries it on itself, whether in a parasitic symbiosis, a diner or a mutualist or another. In the case of the present invention, salmonid species such as Atlantic Salmon (Salmo salar), Rainbow Trout (Oncorhynchus mykiss) and Coho Salmon (Oncorhynchus kisutch) will be considered host.

In the present invention the term "axenic" refers to a multicellular organism that is completely free of all microorganisms. The use of axenic organisms is an important tool for the study of the pathogen-host, parasite-host or bacterium-host interaction.

Regarding sanitary regulations, with the purpose of exporting the product, it is not allowed to work with GMO bacteria (genetically modified), all have to be native to the species or similar species which do not involve artificial genetic modifications.

These sanitary limitations led to the development of the present technology in order to identify and test which microbial strains are the major immuno-stimulants for organisms of aquaculture interest, in this case salmonids, although the technique is not exclusive for other species.

After analyzing the known strategies in the use of probiotics in the market, it was detected that there were no tests with probiotics derived from the intestinal microbiota in axenic models in order to identify which bacteria stimulate the innate immune system of the model. This trial was tested through a prophylaxis strategy, managing to significantly reduce the mortality of the fish model against exposure to the pathogen, for this case but without excluding Flavobacterium psycrophilum in Salmonids.

Method of identification of a proven probiotic in favor of its immuno-stimulant capacity against infection caused by Flavobacteriwn psycrophilum.

The process of identifying the probiotic to be used in prophylaxis against the pathogen in salmon is described in the following stages: i.- isolation of microorganisms from the digestive tract of healthy hosts.

The identification of the optimal bacteria to be used as probiotics, were based on studies of the indigenous gastrointestinal microbial content of juveniles of the main salmonid species of Chilean farming centers, such as Atlantic Salmon (S. salar), Rainbow Trout (O. mykiss) and Coho Salmon (O. kisutch). To identify the bacterial species, the gastrointestinal tract of the individuals was aseptically removed by dissection under stereoscopic magnifying glass. Subsequently, each tissue was placed in a centrifuge tube containing cold 0.9% (w / v) NaCl solution and homogenized. This homogenate was seeded by serial dilutions in soy agarica tripticase (TSA) plates, incubated at a temperature of 17 ° C for 10 days under aerobic conditions (Romero and Navarrete, 2006; Navarrete et al. 2009 and 2010). Within the intestinal microbiota of salmonids, the following genera can be found: -Janthinobacterium, Rhodoferax, Shewanella, Aeromonas,

Pseudomonas, Bacillus, Photobacterium, Flavobacterium, Exiguobacterium, Vibrio, Haemophilus, Stenotrophomonas, Acinetobacter, Ilyobacter, Fusobacterium, Sporolactobacillus, Bifidobacterium, Stenotrophomonas, Vagococcus, Microbacterium, Enterococcus, Macrococcus, Staphylococcus, Rheínheimera, Ralstonia, Cupriavidus, Microbacterium, Cellulomonas, Serratia, Comamonas, Archaeglobus, Lactococcus, Citrobacter, Kluyvera, Obesumbacterium, Carnobacterium, Pediococcus, Debaryomyces, Rodhotorula (Romero and Navarrete, 2006; Navarrete et al. 2009 and 2010).

Although the dominant bacteria in salmon correspond to Gram-negative bacteria of the phylum Proteobacteria, this type of microorganisms does not have the approval of regulatory agencies such as the FDA (with the administration of food and drugs) or consumers. In contrast, bacterial types generally associated with foods such as lactic bacteria (BAL) and some yeasts, are considered as Gras (Generally recognized as safe). For this reason, the search was focused on these types of microorganisms.

Now how to identify, isolate and test those that are of interest for the increase in innate immunity, in order to generate a formulation that decreases the mortality of specimens when exposed to a hostile environment, is part of the present invention patent. ii.- genetic characterization of isolated microorganisms.

The microorganisms previously isolated, the DNA was extracted and subsequently the 16SrRNA gene was amplified by PCR and this amplicon was sequenced (approximately 1500 bp) (PCR product). This sequence allowed to identify each microorganism through the RDPII® website, resulting in the strain B022 corresponds to Pediococcus sp, strain B018 corresponds to the bacterium Lactococcus sp., Strain A025 corresponds to the bacterium Carnobacterium sp. and strain K002 corresponds to the yeast De¿> aryo / nyces sp., as presented in Figure 1/8. iii.- characterization of the complementary beneficial properties.

The isolates were tested for their ability to produce enzymes that are important in nutrient degradation of foods formulated in aquaculture. Finding that there are strains containing amylase protease and a-glucosidase. In addition, they can grow in media containing salt concentrations of 0.5, 1.5, 2.5 and 3.5%. This indicates that these microorganisms have the ability to tolerate a wide range of salt concentrations. This is important and particular for salmon farming, since it is done in 2 phases, one with fresh water (without salts) and the other in salt water (high salt concentration). Therefore, this property of the selected microorganisms, this allows its use in phases of fresh water, estuary and seawater.

Specifically, Lactococcus sp. (B018) presented amylase and protease activity, but did not show glucosidase activity. Carnobacterium sp, (A025) presented protease activity and some glucosidase activity, but lacked amylase activity. While Pediococcus sp. (B022), did not show enzymatic activity of any of the 3 enzymes tested and the yeast Debaryomyees sp. (K002), presented protease activity, α-glucosidase and amylase, becoming a potential candidate as a probiotic, since they could contribute these enzymes in the digestive tract, helping digestion and compensating for the presence of anti-nutritional components, from the inclusion of Vegetable flours in the food. In addition, the isolates showed tolerance to grow in saline in concentrations of 0.5%, 1.5%, 2.5% and 3.5%, mentioned above and presented in the following Table I.

Table I. Enzymatic activity and tolerance to different salt concentrations of the isolates.

Therefore, the B018 bacteria can be complemented with the A025 bacteria, because both produce protease, but B018 produces amylase and A025 produces o-glucosidase, so the combination produces the 3 enzymes collaborating with the degradation and assimilation part of food. Something similar occurs when combined with the B022 bacteria and the K022 yeast. iv.- identification of the immuno-stimulant capacity in axenic models.

One of the fundamental steps in the development of this technology was to define the axenic models to be used, which for this technology were concentrated in two models, the first in zebrafish and the second in rainbow trout.

The zebrafish Danio rerio, has emerged as a very important biological model in studies of developmental and genetic biology, in different aspects of vertebrate biology, and even as an experimental model in human diseases and biomedical studies. The characteristics that make this fish so attractive and popular as a scientific model are: its small size, the ease of breeding and maintenance, its rapid development, the short time that passes from one generation to another, its optical transparency in the early stages of its development, among others.

Specifically, for the development of this patent, one of the fundamental characteristics of the zebrafish, is the amount of molecular tools that allow the study of innate immunity, as well as the ease to adapt protocols and obtain axenic zebrafish, with which facilitates the observation of the bacteria-host relationship.

A second axenic model used in later stages is rainbow trout (Oncorhynchus mykiss). This model is very useful because it behaves in a sanitary manner similar to the final objective, salmon. On the other hand, this similar sanitary behavior is based on the fact that in this species (trout) the response can be easily measured in the modulation of genes that have to do with the innate immune response with the salmonid microbiota, which makes it an ideal candidate to measure the optimal immune response against pathogens that affect salmon.

For the evaluation of the stimulation of innate immunity, the observation of the expression of certain genes that are affected in the interaction with the microorganisms was defined, these genes code for:

-Some pro-inflammatory cytokines, such as interleukin l-β (il 1- / 3)

 -Tumor necrosis factor alpha (tnf-a)

 -Myeloperoxidase [rapo)

 -Lisozyme (yz)

 -Acute phase protein, such as serum amyloid protein (saa)

 - Supplement Protein (c3)

 -Nitric oxide synthetase inducible (inos)

 -An antimicrobial peptide, Hepcidin (hep)

In zebrafish model:

The different isolates (A025, B022, B018 and K002) were tested separately in zebrafish (Danio rerio) axénico, determining that they promoted the expression of the genes mpo, lys and c3. Myeloperoxidase (mpo) codes for an enzyme that is present in neutrophils and macrophages (phagocytic cells). This enzyme is responsible for catalyzing the production of bactericidal compounds such as hydrogen peroxide involved in respiratory burst. Lysozyme (lys), this enzyme is found in phagocytes, is an enzyme with bactericidal properties, which has the ability to adhere to the cell wall of pathogens that have a cell wall composed of peptidoglycan, generating lysis in them. The Complement Protein (c3), involves a series of biological cascades that result in the amplification of defense responses against pathogens including phagocytosis, lysis of foreign cells, opsonization and formation of pathogen membrane attack complexes. In axenic rainbow trout fry (O. mykiss), original host it was observed that lactic bacteria induce the expression of lys, previously observed for being expressed in zebrafish and other genes such as il-ΐβ, tnf-a, inos and hep. Proinflammatory cytokines {il-ΐβ and tnf-a) are produced by phagocytic cells, mediate host defense responses and induce the expression of other genes such as acute phase proteins. These cytokines in turn have the ability to induce in the macrophages and neutrophils the production of the enzyme inducible nitric oxide synthetase (inos), which is responsible for transforming the amino acid L-arginine into nitric oxide, which when combined with molecular oxygen or with the superoxide anion, it generates reactive oxygen species (ROS) to inhibit the growth of pathogens. Hepcidin (hep) in rainbow trout, is an antimicrobial peptide that is part of the innate immune system and therefore, is the first line of defense against infections, it is also responsible for the control of iron homeostasis acting both at the cellular level as systemic (Crowhurst et al., 2002; Mathew et al., 2002; Magnadóttir, 2006; Alvarez-Pellitero, 2008; Castro et al., 2008; Lieschke and Trede, 2009; Rombout et al., 2011).

With regard to the exposure of specific strains and their genetic induction in the host, it can be said that: The A025 strain induced the expression of a large number of genes including cytokine il-ΐβ (5,803 times, p ≤ 0.05), mpo (81.7 times, p <0.05) and c3 (5.4 2 times , p <0.05).

The B022 strain belonging to the genus Pediococcus had a behavior similar to that observed in the A025 strain (Carnobacterium sp), therefore it induced the expression of il-ΐβ (5,468 times, p <0.05) and mpo (38.8 times, p < 0.05).

The strain B018, managed to induce the significant expression of myeloperoxidase (mpo), 8.2 times with respect to the control gene (p ≤ 0.05), lysozyme (lys), 1.6 times (p ≤ 0.05) and protein of complement (c3), 13.1 times (p ≤ 0.05).

Yeast K002, like the B018 bacterium, induced the expression of the myeloperoxidase {mpo) genes, 2.2 times with respect to the control gene (p ≤ 0.05) and lysozyme (lys), 2.6 times (p ≤ 0 , 05), as shown in Figure 3/8.

For the interpretation of the data, the level of significance of the expression of some genes of innate immunity modulated by the interaction of the salmonid microbiota bacteria (Lactococcus B018) inoculated in zebrafish larvae (D. rerio) was considered. ) axenic, from the p-value (<0.05).

According to the background and results obtained, the possibility of combining different types of microorganisms was postulated. In a first group, strain B022 and K002 would be found, as together they could promote in the original host the stimulation of the il-ΐβ, mpo, lis and c3 genes. In the second group would be found, the A025 strains and B018, which together could promote the same genes observed in the first group as shown in Figure 4/8 and Figure 5/8.

In rainbow trout model

According to what was observed in zebrafish, it was decided to analyze the interaction that could be carried out by the bacteria of the salmonid microbiota in rainbow trout (O. mykiss) in the original host, in order to verify if the response obtained is similar as for the induction of genes related to defense and the innate immune system. In this organism it was possible to observe a strong induction of the expression of the lys genes, which had previously been observed in zebrafish. In addition, the expression of some of the genes visualized in zebrafish was observed, such as: il l-β, tnf-, inos and hep; as presented in table III. For the interpretation, the level of significance of the expression of some genes of innate immunity modulated by the interaction of the salmonid microbiota bacteria (Lactococcus B018) inoculated in axenic rainbow trout (O. mykiss) fry was considered, from the p-value (<0.05).

Table III

tnf-a 18,7 3, 216-69, 376 0 OK

tnf-a 18.7 3, 216-69, 376 0 OK

Μρσ 1, 543 0, 135-23, 176 0, 421

Lyz 1/5 0, 898-2, 567 0, 001 OK

Saa 1, 314 0, 563-2, 511 0, 127 c3 3, 189 0.234-23, 993 0.273

Inos 4.2 2,371-7, 831 0 OK

Hep 1,4 0, 698-2, 250 0, 017 OK

This table presents the results of significant expression of quantitative RT-PCR for innate immunity genes il-ΐβ, tnf-a, mpo, lys, saa, c3, inos and hep modulated by the bacterium Lactococcus-B018, inoculated in fry of rainbow trout (Oncorhynchus mykiss) axenic. The binding factor (ef-lot) was used as a control gene (Housekeeping). v.- production of probiotics

It is important to take into account when working with probiotics, that these manage to overcome different conditions that could affect their viability. First they must overcome the different factors that can affect their physiology, when exposed to different industrial processes to look at the food. These processes can be freezing, drying oxygen exposure, drastic changes in temperatures, high concentrations of lactic acid in the culture medium among others. In addition, they must resist the passage through the digestive tract until they reach the place of activity (intestine), prevailing before acidity and digestive enzymes and in this way they can produce the aforementioned beneficial effects on the host.

This is why it is important to establish procedural alternatives to maintain the viability of the microorganisms to be used, for this purpose microencapsulation has been used. Thus, one of the methodologies that it is proposed to use preferably in the present patent is that of spray-drying or spraying, without excluding other technologies that manage to maintain the viability of the same microorganisms. To achieve this specific process it was necessary:

* Growth of microorganisms:

According to the results obtained in zebrafish (D. rerio) and rainbow trout (O. mykiss) fry, it was decided to work with four microorganisms which were Lactococcus sp (B018), Carnobacterium sp (A025) and Pediococcus sp ( B022), these bacteria were also chosen because they belong to the so-called Lactic Acid Bacteria (BAL). On the other hand, it was also decided to work with a yeast, Debaryomyces sp (K002). These microorganisms were chosen because they are considered as Gras (Generally recognized as safe) according to the FDA (Food and Drugs Administration) international entity that regulates food quality, both for people and animals.

Bacteria A025 biological deposit No. A025-B / 00069, K002 biological deposit No. RGM 2143 and B018 biological deposit No. B018-B / 00070, were seeded in plates of TSA (soybean tripticasa agar), B022 biological deposit No. B022-B / 00071 was seeded in MRS (Man, Rogosa and Sharpe Agar). They were incubated at 28 ° C, under aerobic conditions, two days before inoculation in tubes. Prior to this to make sure that the cultures were pure, it was first observed that there were no different colony morphologies, having the certainty that the cultures were pure, an isolated colony was taken from each plate, DNA was extracted from the boiled bacteria, subsequently 16SrRNA and RFLP gene PCR was performed using restriction enzymes or restriction endonuclease ALUI (Navarrete et al., 2010). For the yeast, the ITS and RFLP were amplified using the restriction enzyme HaelII. Once the purity of each strain was verified, a colony of each culture was taken and allowed to grow in the liquid medium (TSB for A025, K002 and B018) and MRS broth (B022). They were left incubating overnight (overnight or ON). 2 tubes were left for each microorganism with 40 ml of each inoculated medium, under continuous stirring at room temperature. Subsequently, each tube served as inoculum to inoculate 2 2-liter flasks containing 1 liter of liquid medium for each microorganism. These flasks were left under continuous stirring for 42h at 30 ° C. After the time, under the hood the cultures were aliquoted in 50 ml tubes, in order to separate the liquid medium from the bacteria, for this purpose the tubes were centrifuged at 5000 gx 10 min at 17 ° C. The medium was discarded and the pellet was resuspended in 2 ml of previously sterile PBS. All aliquots are collected to reach a total volume of 70 ml.

To determine the concentration of microorganisms obtained, an aliquot was collected from the collection and the He performed a direct count under a microscope using a Petroff-Hausser camera. Upon reaching a concentration of 1 x 10 ¹² microorg / ml, as presented in Figure 8/8, the bacteria were sprayed on 1 kg of food.

To spray bacteria on 1 kg of food, the food must be previously divided into 200g portions to achieve a homogeneous distribution of particles on a flat surface. Every 200 g were distributed over the inn in order to generate a monolayer and that no lumps remain. Having spread the food evenly, 14 ml of the bacteria divided in two stages was sprinkled. The first was distributed 7 mi and homogenized by moving the pellets and stirring to promote mixing. For the second spray, the same procedure described above was performed to end the fixation of the bacteria by adding 5.3 ml of 100% soybean oil. Having inoculated the bacteria in the food, it was stored at 4 ° C for later administration to the fish.

Prior to the administration of the food with the probiotic, the microbial load per gram had to be determined, for this under hood, avoiding contamination, three aliquots of the food were taken each with 1 g and the food was macerated with a previously sterile mortar. The macerated food was placed in a 15 ml tube containing 10 ml of PBS, to later be homogenized using vortex, and serial dilutions were made up to - 3 in a 1:10 ratio. 100 ul of each sample was seeded by raking in triplicate on TSA plates and incubated at 28 ° C for 48 h. After the time the CFU of each plate was counted and an average was made, upon reaching A density of 1 x 10 cfu / g of feed was fed to the fish.

To determine the bacterial density and the presence of the probiotic in the intestine, the first week after starting the feeding period, we proceeded to take 3 fish group decade, the fish were anesthetized with BENZOCAINA ^® (BZ-20), in a 900 μΐ ratio in 6 L of water, each fish was subsequently taken and carefully massaged in the abdominal area until intestinal contents were obtained and deposited in a sterile 2 ml centrifuge tube previously weighed to remove the weight difference. Under hood, avoiding any external contamination, 1 ml of sterile PBS was added to each tube and homogenized the sample with the help of a vortex, then serial dilutions were made up to -3 and 100 μΐ of each sample were seeded in triplicate 1:10 They were seeded by raking in TSA plates and were incubated at 28 ° C for 48 h. It was observed that lactic bacteria (A025 and B018) achieve a load of 7 x 10 ⁷ CFU / g in intestinal content. While B022 achieves a load of 4 x 10 ⁸ CFU / g and K002 reaches 2 x 10 ⁸ CFU / g.

* Encapsulation:

10% w / v solutions of milk protein, maltodextrin, or in combination with protein and carbohydrate in distilled water were prepared and then homogenized. Subsequently, the bacterial suspension was added and kept under stirring (ice) in a flocculating system. Finally, they were dried in the spray-drying equipment, under different spray conditions. In the Mini Spray Dryer B-290 equipment, the atomization and obtaining of the particulates were carried out according to the parameters mentioned in Table II. Table II Atomization parameters

Parameter

Inlet temperature

 130

(° C)

Output temperature (° C) 70-80

Vacuum cleaner (m ³ / h) 30-35

Speed of

 2-5

feed (mL / min)

Atomization pressure

 40-54

(mbar)

Air flow (L / h) 500-1300

The morphology of the microparticles was determined by surface electron microscopy and optical microscopy, obtaining photographic records. For a more exact size determination, a zeta potential analyzer was used. The stability control was determined by microscopic visualization the change of appearance of the microparticles after 2 months at 23 ° C.

The microbial viability was determined by counting microorganisms by determining the colony forming units (CFU), before and after spray drying, using the micro drop technique on TSA agar plates (Tryptic Soya Agar). Briefly, dilutions -1 to -6 were prepared in PBS lx and seeded in The agar plates were then incubated for 48 h at 37 ° C to perform the final count.

To determine if the particles withstood the adverse intestinal conditions, controls were made to release the particles in gastric and intestinal buffers, for this purpose the buffers were prepared in distilled water acidified with HC1 or alkalinized with NaOH, until the required pH was achieved. In gastric (pH 3.0) and intestinal (pH 10.0) buffers, 2.5 g of microparticles containing the bacteria were added. It was kept under constant stirring for 2 hours and samples were taken at times 0, 10, 30 and 120 min. Then the microbial viability was verified as described above.

^♦ Inclusion in food:

To perform the microencapsulation of microorganisms spray, first the microencapsulate was resuspended in 70 ml of PBS (phosphate buffered saline) and then sprayed in 1 kg of food, which must be previously divided into 200g portions to achieve a homogeneous distribution of particles on a flat surface. Every 200 g were distributed over the inn in order to generate a monolayer and that no lumps remain. Having spread the food evenly, 14 ml of the bacteria divided in two stages was sprinkled. The first was distributed 7 mi and homogenized by moving the food pellets and stirring to promote mixing. For the second spray, the same procedure described above was performed to end the fixation of the bacteria by adding 5.3 ml of 100% soybean oil. Having inoculated the bacteria in the food, this It was stored at 4 ° C for later administration to the fish.

Prior to the administration of the food with the probiotic, the microbial load per gram had to be determined, for this under hood, avoiding contamination, three aliquots of the food were taken each with 1 g and the food was macerated with a previously sterile mortar. The macerated food was placed in a 15 ml tube containing 10 ml of PBS, to later be homogenized using vortex, and serial dilutions were made up to - 3 in a 1:10 ratio. 100 μΐ of each sample was seeded in triplicate in TSA plates and incubated at 28 ° C for 48 h. After the time, the CFU of each plate was counted and an average was made, when reaching a density of 1 x 10 ⁸ cfu / g in food, the fish were fed. The lower limit of the bacterial density required to achieve an immune response is 1 x 10 ⁷ cfu / g in food, the present invention encompasses this range and lower such as 1 x 10 ⁵ cfu / g of food. The upper limit has no ranges except for the toxic concentration for the fish which is not yet determined.

To determine the bacterial density and the presence of the probiotic in the intestine, the first week after starting the feeding period, we proceeded to take 3 fish group decade, the fish were anesthetized with BENZOCAINA ^® (BZ-20), in a 900 μΐ ratio in 6 L of water, each fish was subsequently taken and carefully massaged in the abdominal area until intestinal contents were obtained and deposited in a sterile 2 ml centrifuge tube previously weighed to remove the weight difference. Under hood, avoiding Any external contamination was added to each tube 1 ml of sterile PBS and homogenize the sample with the help of a vortex, then serial dilutions were made up to -3 and 100 μΐ of each sample were seeded in triplicate in a 1:10 ratio. They were seeded by raking in TSA plates and were incubated at 28 ° C for 48 h. It was observed that lactic bacteria (A025 and B018) achieve a load of 7 x 10 ⁷ CFU / g in intestinal content. While B022 achieves a load of 4 x 10 ⁸ CFU / g and K002 reaches 2 x 10 ⁸ CFU / g.

To demonstrate its capacity as a probiotic of Lactococcus B0P1-8, salmon treated through food with the probiotic at a concentration for each strain of 1 x 10 ⁹ cfu / g of food were faced with a freshwater pathogen of interest in their control For the industry, this is the case of the Gram Negative F. psycrophylum bacteria (Figure 6/8).

Clearly the effect of food with the probiotic and without the probiotic generated a real difference in the mortality rate of the individuals. The mechanism of how the probiotic achieves its effect is based on the immunostimulatory effect of the probiotic on salmon and how it responds in a cross way to the exposed pathogen (the cross response corresponds that it can respond to other similar pathogens, under the same mechanism of the increase in the nonspecific immunostimulatory response), in this case F. psycrophylum. This could be due to the lactic acid bacteria (LAB), a group to which the Lactococcus sp. (B018); Pediococcus sp. (B022) and

Carnobacterium sp. (A025), have a high rate of colonization in the gastrointestinal tract, which infers a greater field of fixation or adherence to the epithelial surface of the intestine of the fish and thus greater exposure for the activation of the non-specific response such as complement activity and lysozyme, cross-inhibiting the growth of pathogens.

Food composition with probiotics

The route of inoculation of probiotics to salmon is orally through food.

This implies the possible use of any known food for salmon inoculated with the probiotic in any of its forms. This means that it can be inoculated as a solid powder as sprinkled in as an active microbiological suspension or in any form in which the bacteria can generate a non-specific response through the gastrointestinal system of salmon.

Among the salmon foods known and marketed in Chile, such as BIO AR®, SKRETTING®, EWOS®, among others, that can be inoculated, the commercial diet E OS®-transfer caliber 5 was used, with a rate of 2, 3% of the biomass per day.

The biochemical composition of this diet is as presented in Table IV.

Table IV Chemical composition of the EWOS®-transfer diet (% dry food), for Rainbow Trout, O. mykiss. % Dry material

 Crude Protein% min 51

 Lipids% min l8

 Humidity% Max 10

 Ashes% Max 12

 Crude Fiber% Max 1.5

 Use of pro-biotics in the treatment, prevention and prophylaxis of infection caused by Flavobacterium psycrophilvm.

The use of these probiotics against Flavobacterium psycrophilum is clearly seen in Figures 6/8, 7/8 and Table 5 of this specification.

On the other hand, this product can be widely used in species such as Atlantic Salmon (S. salar), Rainbow Trout (O. mykiss) and Coho Salmon (O. kisutch).

The different types of salmon culture from stages in lt to fattening can use this probiotic in their diet.

This probiotic can be used in open (fattening process) or closed (recirculation) crops, in fresh water, sea water and / or estuary.

The different strains of microorganisms can be mixed in ratios ranging from pure strains used individually, to mixtures 10% / 90%, 20% / 80%, 30% / 70%, 40% / 60%, 50% / 50% (1: 1) between 2 strains or mixtures between 3 and / or 4 strains in any proportion suitable for the best immune response against a pathogenic microorganism

DESCRIPTION OF FIGURES

Figure 1/8

The present figure presents two photographs, the photograph on the left depicts an agarose gel for an RFLP (restriction fragment length polymorphism of the 16SrRNA gene) of the A025 salmonella microbiota bacteria (Carnobacterium sp.), B018 {Lactococcus sp.), B022 {Pediococcus sp.) using restriction enzyme Alu I.

LB: molecular weight standard.

A025: corresponds to an electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA using Alu I restriction enzymes consisting of 5 bands, the first located at 800 bp, the second at 220 bp, the third at 200 bp; the fourth in 180 bp; and the fifth at 40 bp, for a total of 1440 bp.

B018: corresponds to an electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA using Alu I restriction enzymes consisting of 5 bands, the first located at 400 bp, the second at 250 bp, the third at 240 bp, the fourth at 230 bp and the fifth at 200 bp; giving a total of 1320 bp. B022: corresponds to an electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA using Alu I restriction enzymes consisting of 5 bands, the first located at 480 bp, the second at 340 bp, the third at 200 bp, the fourth in 180 bp and the fifth in 100 bp, for a total of 1300 bp.

The photograph on the right shows an agarose gel for an RFLP (restriction fragment length polymorphism) of the yeast strain K002 (Debaryomyees sp), using the restriction enzyme Hae III.

LB: molecular weight standard. K022: corresponds to an electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA using Hae III restriction enzymes consisting of 4 bands, the first located at 480 bp, the second at 150 bp, the third at 140 bp and the fourth at 80 bp; and it has a marker of resistance to gentamicin (5 μg / ml) and ampicilian (100 μg / ml).

Figure 2/8 This figure presents two diagrams, the diagram on the left represents the statistical results of the significant expression in a real-time quantitative RT-PCR, for the innate immunity genes ίΐ-ΐβ, tnf-α, mpo, lys , saa, c3, inos and hep, modulated by Lactococcus B0P18, bacterium of the microbiota of the digestive tract of salmonids, inoculated in zebrafish (D. rerio) axenic. Data were normalized with log 10. In the axis of the abscissa presents the relative expression of the different genes in logarithm 10, to improve the scale.

In the axis of the ordinates we can see the different types of genetic expressions and their statistical errors.

A. - MPO gene expression

 B. - Lys gene expression

 C- C3 gene expression

This was measured on the sixth day post-incubation.

The scheme on the right presents the statistical results of the significant expression in a quantitative RT-PCR in real time, for the innate immunity genes il-ΐβ, tnf-a, rapo, lys, saa, c3, inos and hep, modulated by Lactococcus B0P18, bacterium of the microbiota of the digestive tract of salmonids, inoculated in rainbow trout (O. mykiss) axenic. Data were normalized with log 10.

In the axis of the abscissa it presents the relative expression of the different genes in logarithm 10, to improve the scale.

In the axis of the ordinates we can see the different types of genetic expressions and their statistical errors.

D.- iNOS gene expression

 B.- Lys gene expression

 E.- HEP gene expression

F. - ^■ expression of IL 1-β genes

G.- ^■ TNF-OÍ gene expression

This was measured on the sixth day post-incubation. Figure 3/8

This figure presents the results of significant expression of quantitative RT-PCR for the innate immunity genes il-ΐβ, tnf-a, mpo, lys, and c3 modulated by Carnobacterium sp (A025), Lactococcus sp (B018), Pediococcus sp (B022) and Debaryomyces sp (K002) microorganisms of the microbiota of the salmonid digestive tract, inoculated in axenic zebrafish (Danio rerio) larvae, third day post inoculation. The data were normalized with logio-

In the axis of the abscissa it presents the relative expression of the different genes in logarithm 10, to improve the scale. In the axis of the ordinates we can see the different types of genetic expressions and their statistical errors.

Figure 4/8 Scheme of 1: 1 combination of K002 / B022 bacteria with concentrations according to their gene expression of C3, Lys, il-lb and mpo.

Figure 5/8

Scheme of 1: 1 combination of bacteria B018 / A025 according to their gene expression of c3 Lys, il-lb and mpo.

Figure 6/8

This figure presents the graphic results in the percentage of mortality of salmonids facing the Pathogenic bacterium Flavohacterium psycrophylum, in fish with and without treatment with the probiotic Lactococcus BOPl-8.

In the axis of the abscissa it presents the time scale in days of exposure of the salmon to Flavohacterium psycrophylum.

The percentage of mortality of salmon exposed to Flavohacterium psycrophylum can be seen in the ordinate axis. ti.-% of control diet mortality without the probiotic Lactococcus BOPl-8. I. -% dietary mortality with the probiotic Lactococcus

BOPl-8.

 Figure 7/8

This figure shows the percentage of survival of fish with diets containing probiotic candidates and that they can achieve in the face of the challenge with the pathogen Flavohacterium psycrophilum.

The dotted line with triangles represents the percentage of survival with the 1: 1 diet, B018 + A025.

The dotted line with crosses represents the percentage of survival with the 1: 1 diet, B022 + K002. The dotted line with squares represents the percentage of survival with the control diet. In the axis of the abscissa it presents the time scale in days of exposure of the salmon to Flavobacterium psycrophylum. The percentage of mortality of salmon exposed to Flavobacterium psycrophylum can be seen in the ordinate axis.

Figure 8/8

This figure represents a photograph of an agar plate showing the colony forming units (CFU) of the Lactococcus sp (B018) bacteria per gram of food.

APPLICATION EXAMPLE

Experimental tests i) Isolation and identification of bacteria from the gastrointestinal tract of salmonids:

The juveniles of Atlantic Salmon (Salmo salar), Coho Salmon (Oncorhynchus kisutch) and Rainbow Trout (Oncorhynchus ykiss) with an average weight between 15 and 30g, were obtained from different fish farms in Chile, the fish have never been treated with antibiotics, probiotics, immunostimulants or inhibitory agents. Each individual was aseptically removed by dissecting the gastrointestinal tract and each section (stomach, pyloric blind and intestine), placed separately in PBS (phosphate buffered saline IX, 8 g NaCl 0.2 g KC1 1.44 g Na ₂ HP0 ₄ 0.24 g KH ₂ P0 ₄ , 1 L). Each section was homogenized, the DNA was subsequently extracted with phenol / chloroform, the 16S rRNA region was amplified by PCR, subsequently the amplicons were sequenced. In addition, the counting and culture of bacteria was carried out, for this they were seeded in serial dilutions of the homogenate, 100 µΐ were seeded per rake in tryptic soy agar plates (TSA), the plates were incubated for 10 days at 17 ° C under conditions aerobics Colonies were counted and the number of colonies per gram of intestinal content was calculated.

Some of the bacteria that were able to identify and prevail in the gastrointestinal tract of the three salmonid species were three strains belonged to the genus Carnobacterium whose assigned codes were A025, 2A7 and 39L, two strains belonging to the genus Lactococcus whose codes were A0P1-7 and B018 In addition, a strain belonging to the genus Pediococcus, whose code is B022. In addition, 6 Gram negative bacteria such as: Providencia sp. (Dll), Psycro £> acter sp. (D13), Pectobacteríu sp. (B10), Shewanella sp. (18), Aeromonas hp. (Ae hp) and Pseudomona sp. (Pl). ii) Modulation of gene expression of innate immunity in zebrafish (D. rerio) axenic induced by gastrointestinal microbiota of salmonids:

Obtaining axenic zebrafish.

To generate and investigate axenic zebra (D. rerio) fish, 2 adult males and one female were placed, in order to ensure the success of the fertilization of the eggs. The fish were placed in 1.5 L acrylic aquariums, these contain a basket inside which separates the breeders from the eggs, avoiding cannibalism of the adults towards them (Mullins et al. 1994; Lawrence 2007). The aquarium contained fresh water and constant aeration, at a temperature of 28 ° C. The breeding fish were fed twice a day to satiety with Artemia and SUPERVIT ^® , at the time of generating the reproduction the feeding was suspended for a period of 6-8 hours.

In order to have the greatest amount of eggs, 8 aquariums with breeders were placed and kept in an incubator with controlled photoperiod (14 light hours / 10 dark hours). The breeders were placed during the night and in the morning hours, the aquariums containing ova were separated from the rest and the breeders were removed. The aquariums containing the eggs were transferred to the laboratory. With a disposable plastic pasteur pipette, the eggs were carefully removed and placed in a Petri dish, which should contain 30 ml of sterile E3 medium (Reference medium for zebrafish). The eggs were washed with consecutive changes of medium, avoiding the presence of visible particles such as feces and other debris.

On the second day, after fertilization (dpf), the eggs were washed with 0.1% iodine for 30 sec, washed with abundant E ₃ medium. Next, the eggs were washed twice in 0.1% iodine solution. The first wash should contain 0.1% iodine for 1.5 min and the second iodine 0.1% 40 sec. Between each wash the eggs were washed with abundant E3 medium. Next, the eggs were left in an antibiotic mix which had 200 μς / τΐ ampicillin, 200] ig ceftazidime, 5 μg / ml kanamycin, 20 g / ml chloramphenicol and 2 g / ml methylene blue. The eggs were left in the antibiotic mix for a period of 4 hours, at a temperature of 28 ° C, after the time the antibiotic mix was changed, for a mix of Fresh antibiotic to avoid the presence of resistant bacteria. They were left incubating at 28 ° C until the next day.

On the third day, hatching was induced, where zebrafish larvae were first passed through a 0.1% iodine wash, then a wash with antibiotic mix. Subsequently, the larvae were left incubating in antibiotic mix at 28 ° C for a period of 4 hours. After the time, this same procedure was washed again with iodine and antibiotic mix. Finally, the larvae were washed with E3 medium, eliminating the antibiotic or some chemical component that could alter the immune response. Then, the larvae were divided into groups in 6 - well plates that contained 5 ml of sterile medium E _3.

Preparation of microorganisms.

The microorganisms chosen to be nominated as candidates for probiotics were selected because their families are recognized as generally safe (GRAS) since they have been proposed for use in food. In addition, because they belong to the so-called lactic acid bacteria (LAB) or beneficial yeasts, which have been of great interest as potential probiotics in various production systems (Lara-Flores et al., 2003; Vázquez et al., 2005). A third reason is because its total safety was tested in Atlantic Salmon fry (Salmo salar) determining that it did not induce mortality and were recognized as safe for the host at a concentration of 10 ⁹ microorganisms per me.

Bacteria A025, B0P18 and K002 were seeded in TSA and B022 was seeded in MRS, incubated at 28 ° C, two days before from induction to hatching. An inoculum from a single colony of each crop was taken and allowed to grow in liquid medium for 16h, with continuous stirring and at 28 ° C. After the time, an aliquot of each medium was removed by observing the microotg / ml concentration that they presented under a microscope and using Petroff-Hausser plates. When obtaining the volume that was needed from each of the plates with the microorganisms that should reach a concentration between 10 ⁷ and 10 ⁸ microorg / ml, the zebrafish larvae were inoculated with the microorganisms.

Monoassociation trials.

Monoassociation assays consist in obtaining an axenic organism which is experimentally inoculated with a known microorganism. The mono association is understood as: 1 metazoan with 1 microorganism.

The larvae were divided into 3 groups of 15 individuals each, then they were placed in each well with 5 ml of sterile E ₃ medium. Each treated group was inoculated with 100 μΐ of each microorganism (Lactococcus sp. (B018); Carnobacterium sp. (A025); Pediococcus sp. (B022) and Debaryomyces sp (K002).

In parallel, a control group was incubated, which was not inoculated and remained axenic (Germ Free-GF). The experience extended to the sixth dpf. On that day all the larvae were sacrificed leaving at least 3 larvae per group to determine if the fish were axenic (Germ free) and the bacterial load in each inoculated group was verified. For this, the bacterial count was carried out, using serial dilutions and plating. Gene expression analysis.

 Trizol® was used for total RNA extraction. An amount of 6 μΐ of RNA was used for reverse transcription for cDNA synthesis. The starting points for the innate immunity genes to be studied are given in the following table V.

 ^ Splitters used for the detection of innate immunity genes in axenic zebrafish (Danio rerio) larvae.

The 18S ribosomal subunit was used as a control gene (Housekeeping). No amplification was observed in the negative control (mix without cDNA). The data obtained were analyzed using the REST ^® program. Gene expression is generated with respect to the comparison between the control group and the treated group.

As a result in strain B018 {Lactococcus sp.), The expression of the mpo, lys and c3 genes was obtained (Figure 7/8). Myeloperoxidase {mpo) encodes an enzyme that is present in neutrophils and macrophages (cells phagocytic). This enzyme is responsible for catalyzing the production of bactericidal compounds such as hydrogen peroxide involved in respiratory burst. Lysozyme (lys), this enzyme is found in phagocytes, is an enzyme with bactericidal properties, which has the ability to adhere to the cell wall of pathogens that have a cell wall composed of peptidoglycan, generating lysis in them. The Complement Protein (c3), involves a series of biological cascades that result in the amplification of defense responses against pathogens including phagocytosis, lysis of foreign cells, opsonization and formation of pathogen membrane attack complexes ( Crowhurst et al., 2002; Mathew et al., 2002; agnadóttir, 2006; Alvarez-Pellitero, 2008; Castro et al., 2008; Lieschke and Trede, 2009; Rombout et al., 2011).

• Modulation of gene expression of innate immunity in rainbow trout (O. ykiss) induced by microbiota:

Obtaining axenic rainbow trout.

All manipulations were performed under laminar flow hood, using previously sterilized materials. The 500 rainbow trout eggs (O. mykiss) with 290 UTAS (accumulated thermal units, or accumulated temperature degrees per day), prior to acclimatization were disinfected by washing them with Buffodine®, in a 1: 100 ratio of water treated to reduce hardness at 60 mg / 1 of CaCC> 3 and autoclaved. Subsequently, the eggs were washed with plenty of treated water. Following disinfection, the eggs were placed in an axenic mouse maintenance box previously adapted to place a metal bat resembling the trout culture. There they were left with continuous aeration, and incubated at a temperature of 10 ± 1 ° C in the dark. This same washing and sanitation procedure was performed for 4 days in a row. On the fifth day, the hatching process began to occur naturally. The fry were removed from the rest of the group and underwent a wash with Buffodine® in a 1: 1000 ratio. They were left in this solution for 1 min and subsequently washed with plenty of treated water. Next, they were placed in a mix of antibiotics containing 400 g / ml of ampicillin, 400 g of ceftazidime, 10 μg / ml kanamycin, 20 μg / ml chloramphenicol.

The fry were maintained with iodine-antibiotic treatment for a period of 30 days. The absence of microorganisms was verified based on daily sampling and plate count; in addition every third day fry were sampled, which were homogenized in PBS for subsequent bacterial count analysis. In this way, the bacterial load was completely eliminated and the inoculation of the specific bacteria could be performed in the mono-association assays.

Monoassociation Assays

Before inoculating each microorganism, the fry were washed with iodine in a 1: 1000 ratio for 1 min and washed with plenty of water, left incubating for a period of 6 hours at 10 ° C. The fry were divided into 2 groups, leaving one control group (GF) and the other to inoculate separately the microorganisms to be tested (belonging to the salmonid microbiota). After the time and following the protocol of preparation of the bacteria of the previous point. The strain B018 was inoculated, in a concentration of 10 bact / ml. On the sixth day after the bacteria were inoculated, the sacrifice was made where 16 fry were taken for each group. The fry were placed in 50 ml tubes, containing 30 ml of previously treated water and 3 μΐ of BENZOCAINA ^® (BZ-20) was added as an anesthetic. Subsequently, when the fry did not show movement, they were taken with previously sterilized metal tweezers, 4 fry which were placed on a petri dish. With a scalpel, a first gill cut was made and then a second cut was made at anus level. The logs were placed in a tube containing 4 ml of TRIZOL. The heads and caudal fins were eliminated. In this way, 4 groups of logs were obtained from the two study groups (control and B018). The samples were stored at -80 ° C.

To determine the presence of the inoculated microorganism, fry from the control group (GF) and fry from the treated group were sacrificed, for plate count analysis. Having the certainty that the control group preserved their axenic condition and that the treated groups retained their mono-associated status, the RT-PCR analyzes were carried out.

Gene Expression Analysis

The binding factor (EF-la) was used as a control gene (Housekeeping) and no amplification was observed in the negative control (mix without cDNA). The data obtained were analyzed using the REST ^® program.

It was observed that in rainbow trout, he was able to induce the expression of the lys genes, previously observed for being expressed in zebrafish and other genes such as il-ΐβ, tnf-ot, inos and hep (Figure 2/8). The proinflammatory cytokines {il-ΐβ, tnf- a) that were expressed by interacting with the host with B018, are produced by phagocytic cells and induce the expression of other genes such as acute phase proteins or anti-inflammatory cytokines. These cytokines in turn have the ability to induce in the macrophages and neutrophils the production of the enzyme inducible nitric oxide synthetase (inos), which is responsible for transforming the amino acid L-arginine into nitric oxide, which when combined with molecular oxygen or with the superoxide anion, it generates reactive oxygen species (ROS) to kill or inhibit the growth of pathogens.

In addition, B018 managed to induce Hepcidin expression in rainbow trout. This gene, in addition to being an antimicrobial peptide that is part of the innate immune system and therefore constitutes the first line of defense against infections, is also responsible for the control of iron homeostasis acting both at the cellular and systemic levels (Cro hurst et al., 2002; Mathe et al., 2002; Magnadóttir, 2006; Alvarez-Pellitero, 2008; Castro et al., 2008; Lieschke and Trede, 2009; Rombout et al., 2011).

Therefore, it is inferred that this strain manages to stimulate the innate immune system by promoting protection against the possible arrival of a pathogen.

• Prevention of infections caused by the pathogenic agent Flavóbacterlvim psycrophilvun by being inoculated in a controlled manner in the food, Lactococcus sp. (B018) and CamoJacterium sp (A025) (Diet 1) and

Pediococcus sp. (B022) in combination with Debaryomyces sp. (K002) (Diet 2), microorganisms of the gastrointestinal microbiota that were able to modulate the innate immune system in rainbow trout (.O.zaykiss) axenic

Persistence of the microorganism in the digestive tract The persistence of the selected microorganisms in the digestive tract of salmon was evaluated, for this, diets supplemented with the chosen microorganisms and control diet (without added microorganisms) were administered, for a period of 7 days. Samples of fish feces were then taken every 3-4 days from day 0 to day 28.

This trial was performed by administering three different diets to salmon (salmo salar) of 25 gr. 150 fish were required which were divided into three groups of 50 fish each.

• Group 1 was given commercial food added with Carnobacterlum strain 2A-5 (10 x ⁹ bacteria / gr food) and Lactococcus, strain B018 (10 x ⁹ bacteria / gr food).

• Group 2 was given commercial food added with Pedlococcus B022 (10 x ⁹ bacteria / g food) and Debaryomyces, strain K002 (10 x ⁹ yeasts / g food).

• Group 3 was administered commercial food without the addition of microorganisms.

The amount of food to be administered daily corresponded to 2% of the weight of the fish. For the 10 consecutive days, the above-mentioned diets were administered to groups 1, 2 and 3. After this period everyone began to eat with the normal diet (commercial diet).

Stool samples were taken on days 0, 4, 8, 11, 14, 17, 22 and 28. These samples were collected in sterile form and plated on plates with TSA medium in serial dilutions for subsequent colony count. The feces extracted from the fish with diet 2 were plated on MRS plates (to favor the growth of Pediococcus) and plates with TSA with the addition of antibiotics (to favor the growth of yeasts). The feces were also subjected to DNA extraction with MOBIO kit to then perform PCR of the 16SrRNA gene for bacteria, and STI for the case of yeast. RFLP was then performed for each marker, digesting the samples with AluI enzyme for bacteria and HaelII in the case of yeasts.

Challenge test of probiotic bacteria against a pathogen (Flavobacterium psycrophilum)

The ability of probiotic candidate microorganisms to protect salmon from an induced infection of Flavobacterium psycrophilum was evaluated.

The design was as follows: 150 salmon fry (Salmo salar) of 25 gr. These fish were separated into 3 groups of 50 individuals. Group 1 was given a diet supplemented with probiotics, group 2 a diet with and group 3 a diet without added bacteria (control) during the 21 days of the trial. On day 8, 15 fish from each of the 3 groups were infected by intraperitonial injection with the pathogen Fiavojacterium psycrophilum (1x10 ^to bacteria per fish). Also as a negative control, a pond was maintained with fish under control diet to which only the culture medium was injected. Throughout the trial, a daily mortality record was made in the different groups, to determine the level of protection against the pathogen.

Fish death was confirmed due to Flavojbacteriujn infection with a visual analysis of dead individuals.

Statistical analysis

With the mortality and survival data, proportions to the final day of the study were calculated. Differences in survival rates were analyzed using the Chi-square test (X ² ) in 2x2 contingency tables comparing each diet with the control group.

To assess the protective effect, the survival of the group treated with probiotics versus a control group (not treated) was observed. It was observed that the fish fed with Diets 1 and 2, had a notable increase in survival (87 and 73%) being these statistically significant differences of the control, p = 0.00287 and p = 0.02811, respectively. BIBLIOGRAPHIC REFERENCES

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Claims

A method to identify and produce microbiological strains with immuno-stimulating capacity in fish for defense against pathogenic microorganisms, CHARACTERIZED because it comprises the stages of: (i) isolation of microorganisms from the digestive tract of healthy hosts;  (ii) genetic characterization of isolated microorganisms;  (iii) characterization of the complementary beneficial properties; (iv) identification of immuno-stimulatory capacity in axenic models; Y (v) production of probiotics. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 1, CHARACTERIZED because step i) comprises aseptic extraction of the gastrointestinal tract from the fish, and then homogenizing it in NaCl, sowing it under serial dilutions in Soy tripticase agar (TSA) and subsequent incubation. A method for identifying and producing microbiological strains with immuno-stimulatory capacity in fish, according to claim 1, CHARACTERIZED because step ii) comprises the extraction of DNA from microorganisms, PCR amplification of the 16 srRNA subunit gene, and identification of the microorganism A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 1, CHARACTERIZED because step iii) comprises testing the enzymatic production capacity for degradation of foods formulated in fish feed. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 1, CHARACTERIZED because step iv) comprises: a) preparing the axenic model for the zebrafish test; b) stimulation of innate immunity in the zebrafish model, with candidate microorganisms; c) prepare the axenic model for the rainbow trout test; d) stimulation of innate immunity in the rainbow trout model with the microorganisms selected from the test with the zebrafish model and; e) verification of the response capacity in the target fish fed with each of the strains previously identified in step d) or their mixtures, against exposure of the target fish to a pathogenic microorganism. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 5, CHARACTERIZED because in sub-stages a) and c) the eggs of the axenic models in fish are disinfected, washed and treated. with a mixture of antibiotics to eliminate the presence of any native or exogenous microorganism. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 5, CHARACTERIZED because in sub-stages b) and d) the greatest expression of a group of genes such as mpo, saa, lyz, is sought. c3, il-1β, tnf-α, inos and hep, in zebrafish and rainbow trout respectively, in order to verify the innate immune response against exposure of the axenic fish to a pathogenic microorganism. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 1, CHARACTERIZED in that stage v) comprises the following sub-stages: f) incubation of pure strains separately, with identification of bacterial microorganisms by PCR of the 16srRNA and RFLP gene, and identification of the yeast microorganism by PCR of the ITS gene and RFLP; g) micro-encapsulation of the microbiological suspension in cold, individually by separating and / or mixing of seeds; h) spray drying; and i) inclusion in fish feed. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 8, CHARACTERIZED because in sub-stage f) the final concentration of Microorganisms have to reach a range of 10 x ⁵ bact / ml to ²⁰ x ²⁰ bac / ml. 0. A method for identifying and producing microbiological strains with immuno-stimulatory capacity in fish according to claim 9, CHARACTERIZED because in sub-stage f) the final concentration is 10 x ¹² bact / ml. 1. A method for identifying and producing microbiological strains with immuno-stimulatory capacity in fish according to claim 8, CHARACTERIZED because in sub-stage g) suitable excipients are used for feeding fish. 2. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 8, CHARACTERIZED because in sub-stage h) it is atomized with pressure ranges ranging from 20 to 75 nabar, with a air flow from 200 to 2200 L / h and a feed rate of the equipment in a range of 1 to 10 ml / min, without excluding other feeding ranges depending on the equipment. 3. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 8, CHARACTERIZED because in sub-stage i) the food provides at least 1% of biomass per day to the crop. 14. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 13, CHARACTERIZED because in sub-stage i) the food provides 2.3% of biomass per day to the crop. 15. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 8, CHARACTERIZED because in sub-stage i) the final microbiological concentration must be in a range of lxlO ⁵ bact / ml to lxlO ¹² bact / ml 16. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 15, CHARACTERIZED because in sub-stage i) the final microbiological concentration is 10 x ⁸ bact / ml. 17. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish according to claim 5, CHARACTERIZED because the microorganisms identified and isolated in sub-stage d) are: Carnobacterium sp. A025 with biological deposit No. A025-B / 00069; Lactococcus sp. B018 with biological deposit No. B018-B / 00070; Pediococcus B022 with biological deposit No. B022-B / 00071; and Debaryomyces K002 with biological deposit No. RG 2143. 18. A method to identify and produce microbiological strains with immuno-stimulant capacity in Fish, according to claims 1 and 5, CHARACTERIZED because the pathogenic strain against which the innate immune response is to be increased is Flavovacterium psycrophilum. 9. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claims 1 and 5, CHARACTERIZED because the target fish against which they are to increase the innate immune response comprise: Atlantic Salmon (Salt Psalm) , Rainbow trout (0. mykiss) and Coho Salmon (0. kisutch). 0. A method for identifying and producing microbiological strains with immuno-stimulant capacity in fish, according to claim 19, CHARACTERIZED because the target fish against which they want to increase the innate immune response is Atlantic Salmon (Salt Psalm). 1. - Bacterial strain for the generation of immune response in fish against the bacteria F. psycrophylum through the method described in claim 1, CHARACTERIZED for being strain B018 of the bacterial species Lactococcus sp., Deposited in the Polish collection No. B018-B / 00070, or an extract or active derivative thereof or a mutant of the wild strain, all of which possess essentially the same specific specific molecular and phenotypic markers thereof. Bacterial strain for the generation of immune response in fish against the bacteria F. psycrophylum, according to claim 21, CHARACTERIZED because it has the following microbiological and biochemical properties: It produces colonies with white pigmentation, translucent in color when looking at light, small colonies, grown in a medium of Trysticasa Soya Agar agar (TSA), at an optimum temperature of between 28-30 ° C; the cultures, at the microscopic level, consist of Gram-positive cocci, not mobile, alone or in clusters; metabolism characterized by the enzymatic activity of amylase (+), protease (+), -glucosidase (-); grows in Tripticasa Soya Caldo (TSB) with different salt concentrations such as 0.5%, 1.5%, 2.5% and 3.5%; its electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA using Alu I restriction enzymes consisting of 5 bands, the first located at 400 bp, the second at 250 bp, the third at 240 bp, the fourth at 230 bp and the fifth at 200 bp; giving a total of 1320 bp. Bacterial strain according to claim 21, CHARACTERIZED because it has an effective dose range average between 10 ⁷ to 10 ¹⁰ cfu / g in food versus F. psycrophylum. 24- Bacterial strain according to claim 23, CHARACTERIZED because it has an average effective dose of 10 ⁹ cfu / g in food against F. psycrophylum. 25- Bacterial strain according to claim 21,  CHARACTERIZED because it shows antagonisms against bacterial pathogens generating halos of growth inhibition of pathogens, which are indicated in millimeters against the following bacteria: Vibrio ordalii 15 mm; Aeromonas salmonicida 9 mm; Y  Fiavobacterium psycrophylum 12 mm. 26.- Bacterial strain for the generation of immune response in fish against the F. psycrophylum bacterium through the method described in claim 1, CHARACTERIZED for being strain A025 of the bacterial species Carnobacterium sp., Deposited in the Polish collection No. A025-B / 00069, or an extract or active derivative thereof or a mutant of the wild strain, all of which possess essentially the same specific specific molecular and phenotypic markers thereof. 27- Bacterial strain for the generation of immune response in fish against the bacterium F. psycrophylum, according to claim 26, CHARACTERIZED because it has the following microbiological and biochemical properties: It produces colonies with white pigmentation, dense color when looking at light, small colonies, grown in a medium of Tripsticasa Soya Agar agar (TSA), at an optimum temperature of between 28-30 ° C; the cultures, at the microscopic level, consist of Gram-positive bacilli, not mobile, alone or in clusters; metabolism characterized by the enzymatic activity of amylase (-), protease (+), -glucosidase (+); grows in Tripticasa Soya Caldo (TSB) with different salt concentrations such as 0.5%, 1.5%, 2.5% and 3.5%; and its electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of the genomic DNA using Alu I restriction enzymes consisting of 5 bands, the first located at 800 bp, the second at 220 bp, the third at 200 bp; the fourth in 180 bp; and the fifth at 40 bp, for a total of 1440 bp. Bacterial strain according to claim 26, CHARACTERIZED because it has an average effective dose range of 10 ⁷ to 10 ¹⁰ cfu / g in food versus F. psycrophylum. Bacterial strain according to claim 28, CHARACTERIZED because it has an average effective dose of 10 ⁹ cfu / g in food against F. psycrophylum. 30- Bacterial strain according to claim 26,  CHARACTERIZED because it shows antagonisms against bacterial pathogens generating halos of growth inhibition of pathogens, which are indicated in millimeters against the following bacteria: Vibrio ordalii 12 mm; Aeromonas salmonicida 12 mm; Y  Flavobacterium psycrophylum 11 mm. 31.- Bacterial strain for the generation of immune response in fish against the bacteria F. psycrophylum through the method described in claim 1, CHARACTERIZED for being strain B022 of the bacterial species Pediococcus sp., Deposited in the Polish collection No. B022-B / 00071, or an extract or active derivative thereof or a mutant of the wild strain, all of which possess essentially the same specific specific molecular and phenotypic markers thereof. 32- Bacterial strain for the generation of immune response in fish against the bacteria F. psycrophylum, according to claim 31, CHARACTERIZED because it has the following microbiological and biochemical properties: It produces colonies with white pigmentation, small colonies, grown in a medium of Man, Rogosa and Sharpe agar (MRS), at an optimum temperature of between 28-30 ° C; the cultures, at the microscopic level, consist of Gram-positive, non-mobile, coconut or tetrad coconuts; metabolism characterized by the enzymatic activity of amylase (-), protease (-), a-glucosidase (-); it grows in a medium of Man, Rogosa and Sharpe agar (MRS), with different salt concentrations such as 0.5%, 1.5%, 2.5% and 3.5%; and its electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA using Alu I restriction enzymes consisting of 5 bands, the first located at 480 bp, the second at 340 bp, the third at 200 bp, the fourth in 180 bp and the fifth in 100 bp, for a total of 1300 bp. Bacterial strain according to claim 31, CHARACTERIZED because it has an average effective dose range of 10 ⁷ to 10 ¹⁰ cfu / g in food versus F. psycrophylum. Bacterial strain according to claim 33, CHARACTERIZED because it has an average effective dose of 10 ⁹ cfu / g in food against F. psycrophylum. Bacterial strain according to claim 31, CHARACTERIZED because it shows antagonisms against bacterial pathogens generating halos of growth inhibition of pathogens, which are indicated in millimeters against the following bacteria: Vibrio anguillarum 10 mm; Vi¿> rio ordalii 15 mm; Aeromonas salmonicida 13 mm; and Flavobacterium psycrophylum 18 mm; and Yersinia ruckeri 8 mm. Yeast strain for the generation of immune response in fish against the bacteria F. psycrophylum through the method described in claim 1, CHARACTERIZED as the strain K002 of the bacterial species Debaryomyces sp. , deposited in the Chilean collection (INIA) No. RG 2143, or an extract or active derivative thereof or a mutant of the wild strain, all of which possess essentially the same specific molecular and phenotypic markers distinguishing it. Yeast strain for the generation of immune response in fish against the bacteria F. psycrophylum, according to claim 36, CHARACTERIZED because it has the following microbiological and biochemical properties: It produces colonies with cream pigmentation, large colonies, grown in a medium of Tripticasa Soya Caldo (TSB) at an optimum temperature of between 28-30 ° C; the cultures, at the microscopic level, are constituted by unicellular, mobile yeasts; metabolism characterized by the enzymatic activity of amylase (+), protease (+), a-glucosidase (+); It grows in a medium of Tryticasa Soya Caldo (TSB) with different salt concentrations such as 0.5%, 1.5%, 2.5% and 3.5% its electrophoretic pattern of restriction fragment length polymorphisms (RFLP) of genomic DNA by restriction enzymes Hae III consisting of 4 bands, the first located at 480 bp, the second at 150 bp, the third at 140 bp and the fourth at 80 bp; and it has a marker of resistance to gentamicin (5 g / ml) and ampicilian (100 μς / πιΐ). 38- Yeast strain according to claim 36, CHARACTERIZED because it has an average effective dose range of 10 ⁷ to 10 ¹⁰ cfu / g in food versus F. psycrophylum. 39- Yeast strain according to claim 38, CHARACTERIZED because it has an average effective dose of 10 ⁹ cfu / g in food against F. psycrophylum. 40. Mixture of microorganisms selected by the method of claim 1, CHARACTERIZED in that it comprises the mixture of strains of microorganisms in ratios ranging from 10% / 90%, 20% / 80%, 30% / 70%, 40% / 60%, 50% / 50%, between 2 strains or mixtures between 3 and / or the 4 strains (B018, A025, K002 and B022) in any proportion suitable for the best immune response against a pathogenic microorganism. 41. Mixture of microorganisms according to claim 40, CHARACTERIZED in that the mixture of two strains is 50% / 50% or 1: 1. 42. Mixture of microorganisms according to claim 40, CHARACTERIZED because the mixture of two strains is K002 / B022. 43. Mixture of microorganisms selected by the method of claim 40, CHARACTERIZED because the mixture of two strains is B018 / A025. 44. Food composition including the microorganisms selected by the method of claim 1, CHARACTERIZED in that it comprises the microorganisms B018, A025, K002 and B022 and / or their mixture with food nutrients or processed foods, suitable for feeding fish. 45. Use of the microorganisms selected by the method of claim 1, CHARACTERIZED because they are useful for treating infections by the following pathogenic organisms: Vibrio anguillarum Vibrio ordalii, Aeromonas salmonicida, Flavobacterium psycrophylum, and Yersinia ruckeri. Use of the microorganisms selected by the method of claim 45, CHARACTERIZED because they are useful for treating the infection of the following pathogenic organism: Flavobacterium psycrophylum.