WO2014020226A1

WO2014020226A1 - Additives for feeding aquatic animals, containing probiotics

Info

Publication number: WO2014020226A1
Application number: PCT/ES2013/070573
Authority: WO
Inventors: Enrique PABLOS PÉREZ
Original assignee: Norel, S.A
Priority date: 2012-08-01
Filing date: 2013-08-01
Publication date: 2014-02-06
Also published as: ES2447090A1; ES2447090B1

Abstract

The invention relates to an additive for feeding aquatic animals, comprising the combination of two species pertaining to the genus Bacillus, said species being B. amyloliquefaciens and B. cereus, preferably the strains CECT 5940 of B. amyloliquefaciens and CECT 953 of B. cereus, in combination with an organic acid salt, preferably with the sodium salt of butanoic acid. The additives for feeding aquatic animals described in the present invention can also comprise other bacterial strains with a probiotic capacity, preferably pertaining to the species Pediococcus acidilactici.

Description

ADDITIVES FOR THE FEEDING OF AQUATIC ANIMALS CONTAINING PROBIOTICS.

FIELD OF THE INVENTION

The present invention discloses additives for feeding aquatic animals, additives containing at least one probiotic which, in addition, may be accompanied by at least one salt of an organic acid. These additives are used as promoters or growth stimulants and enhancers of the animal's health status. Therefore, the present invention is encompassed within the field of aquaculture and more specifically within animal production, food and health.

STATE OF THE TECHNIQUE Aquaculture has had significant progress in recent years in the production of a wide variety of aquatic organisms for both human consumption and ornamentation species. Due to the breeding conditions, such as high animal densities and variations in water quality, organisms are subject to constant stress that results in a high mortality rate, as well as low growth rates and of food efficiency, in addition to increasing the presence and incidence of diseases caused by opportunistic pathogens. The need for sustainable and profitable aquaculture has driven the search for acceptable solutions to optimize production in a natural and environmentally friendly way.

To overcome these problems, supplements and / or food additives have been used that prevent the onset of diseases and also operate as growth promoters. Among these supplements are hormones, antibiotics and some salts. However, its inappropriate use may have adverse effects on the animal or the final consumer, as well as giving rise to cross resistance in breeding animals, against pathogenic bacteria, causing environmental imbalances and economic losses of aquaculture farms. Therefore, the current trend in aquaculture is the search for a diet that not only covers the nutritional needs of animals, but also reinforces their immunological and sanitary status, in general and reduces pathological problems without using medications, in order to improve its production, avoid problems with food safety obtained from breeding animals and increase the economic yields of farms. For the achievement of these objectives, one of the ways of obtaining this has been the control of the intestinal flora of the breeding animals, using probiotics for this purpose (Robertson, PAW et al. Aquaculture 2000; 185: 235-243; Verschuere, L, G. et al. Microbiological Molecular 2000; 64: 655-671; Gullian,. Et al., Aquaculture 2004; 233: 1-14; Balcázar, JL, et al. Veterinary Microbioiogy 2006; 114: 173-186; Gatesoupe, FJ Journal of Molecular Microbiologlcal Bioiechnology. 2008; 14: 107-114) that can be defined as viable microorganisms that administered in the diet promote the welfare of breeding organisms through the stimulation of the immune system, as well as the establishment of intestinal microbial balance in such breeding animals, by excluding potentially pathogenic microorganisms (I rianto, A. and B. Journal of Fish Diseases 2002; 25: 333-342; Irianto, A. and B. Austin. Journal of Flsh Diseases 2003; 26: 59-62; Lara-Flores, M., et al. A quaculture 2003; 216: 193-201.). The use of probiotics in animal production is an effective way to control the intestinal microflora of breeding animals and has proven to be both a good substitute and a good complement to growth-promoting antibiotics. Probiotics are widely used in pig and poultry farming, but little has been done to incorporate them into aquaculture.

In this sense, aquatic animals differ from terrestrial animals in the level of interaction between the intestinal microbiota and the surrounding environment. The bacteria present in the aquatic environment influence the composition of the microflora of the intestine of aquatic animals and vice versa. The composition of the bacterial community of the intestinal tract of aquatic animals is different from what is found in terrestrial animals. Gram negative bacteria predominate in the marine environment and Gram positive in a smaller proportion; these usually constitute the majority of the bacteria that normally occur in the microflora associated with wild and cultivated shrimp. The vast majority of microbes present in the marine environment (bacteria, viruses, fungi or parasites) are harmless or are successfully controlled by the internal defense mechanisms of organisms in culture. The early stages of the life cycle of aquatic organisms are usually more susceptible to attacks by such agents, especially when larval cultures are carried out intensively, which, due to the stress caused by high densities, reduces the defense potential of the organisms The breakdown of the dynamic balance of the intestinal flora is caused by the stress generated by the intensive conditions of animal production, mentioned previously. When concentrations of beneficial bacteria decrease, the opportunity for less desirable bacteria or for potential pathogens to install and multiply appears.

There are different bacterial species used as probiotics in aquaculture, highlighting, above all, acid-lactic bacteria, preferably belonging to the genus Lactobacillus (Nikoskelainen, 8.A. et al. Fish Sellfísh Immunology 2003; 15: 443-452; Balcázar, JL et al. British Journal of Nutrition 2007; 97: 522-527; Gatesoupe, FJ. Journal of Molecular Microbioiogical Biotechnology. 2008; 14: 107-114). Bacteria of this genus are part of the natural microbial flora that are found accompanying shrimp in all their stages of life and part of these bacteria have potential to be used as probiotics in aquaculture. However, because this practice is still recent in aquaculture, few species of microorganisms are used, so it is currently necessary to continue selecting strains with probiotic characteristics. In addition, it may be beneficial to complement the immune system, particularly in the gastrointestinal tract of aquatic animals, to use, in combination with probiotics, other ingredients or additives that enhance the benefit of such probiotics in health and aquaculture production.

While probiotics are useful for promoting the health of an animal, they are often difficult to store, handle and administer to these animals, especially when such administration is done by dissolving the additive, along with the usual food of the animals, in The aquatic environment. In this sense, the food formulation not only has to complete the requirements of the animals, it must also produce a very stable food in the water because the aquatic animals are introduced continuously and continuously. Specifically, the shrimp detects the food at a distance by chemoreception, so that this food must also be attractive (that attracts the animal) and palatable (that likes the taste), and must also have a uniform shape and composition. In this sense, the use of attractive substances allows food to be located and consumed faster by animals. These compounds are very water soluble molecules and are of low molecular weight; as: amino acids, quaternary ammonium compounds, trimethylamine, betaine (trimethylglycine), nucleotides, biogenic amines and organic acids. Commercially available in the market soluble flours and fish oils, molluscs or crustaceans; They work as excellent attractants.

There is, therefore, a need for new additives for feeding aquatic animals, containing probiotics capable of strengthening the immune system thus preventing invasion by opportunistic pathogens and resulting in an increase in the longevity of animals reared in captivity by means of techniques Aquaculture Another objective, of the additives incorporated in the diet of aquatic animals is to achieve greater use of food and an increased activity of digestive enzymes that results in an increase in the growth of breeding animals and therefore results in the economic benefits for aquaculture farms.

DESCRIPTION OF THE INVENTION Brief description of the invention

The present invention describes a new food additive for aquatic animals comprising at least two bacterial strains, preferably in spore form, belonging to the genus Bacillus and selected from among Bacillus amyloliquefaciens and Bacillus cereus.

The bacterial strain Bacillus amyloliquefaciens CECT 5940 is a bacterial strain isolated by NOREL S.A., Spain deposited on April 25, 2001 in the Spanish Collection of Crops type sita in the Scientific Park of the University of Valencia. Calle Agustín Escardino Professor, 9 de Paterna (Valencia) and, which is marketed as the probiotic ECOBIOL.

ECOBIOL is a probiotic in the form of resistant spores capable of surviving even the process of granulation and / or pelletization of feed. Once in the animal, where the bacterium finds a suitable medium for its development in terms of temperature and humidity nutrients, the spores that make up the ECOBIOL germinate, then passing the bacteria to produce all the beneficial effects for the animal. Said probiotic is also characterized in that it has a better reproduction ratio compared to other Bacillus spp spores and is also capable of releasing chemical compounds that have an inhibitory effect on the pathogenic population. The bacterial strain Bacillus cereus CECT 953 is a bacterial strain isolated by NOREL SA, Spain deposited on October 25, 1988 in the Spanish Collection of Crops type sita in the Science Park of the University of Valencia. Calle Agustín Escardino Professor, 9 de Paterna (Valencia) and, which is marketed as the probiotic ESPORAFEED.

ESPORAFEED is a probiotic in the form of resistant spores capable of surviving even the process of granulation of feed for animal feed. Said probiotic is characterized by resisting high granulation temperatures, as well as physical and acidifying agents. In addition, ESPORAFEED produces enzymes that improve the digestibility of the diet.

The additives described in the present invention are added to the diet of aquatic animals, preferably in those animals farmed by aquaculture and which are selected, among others, from any of the following list: crustaceans, molluscs, tilapia, gilthead sea bass and salmonids.

The additive of the invention acts as a natural growth promoter, showing an improvement in the feed conversion factor, an increase in the growth of animals and biomass, as well as an increase in the survival of the animals to which these additives are administered to animals fed without the additive.

The additive of the invention may also contain other ingredients capable of strengthening the immune system and improving the digestibility of the food resulting in an increase in the action of probiotics. Among these ingredients are salts of organic acids, with sodium salt of butyric acid being preferred.

Organic short-chain acids are capable of reducing the pH at the intestinal level, which limits the development of pathogens and helps in the digestion of proteins and, on the other hand, they have the ability to enter the bacteria, specifically those pathogens and block their metabolism. Its bactericidal action is exerted preferably between the genera: Escherichia, Campylobacter, Clostridium and Salmonella, among others. In the present invention the Norel SA Spain product marketed under the name of GUSTOR BP-70 is used, from here and, throughout the entire invention we will call it BP-70. The BP-70 is formed by the sodium salt of protected butyric acid, against early digestion, with a matrix of vegetable fats, which protect only a part of the active ingredient and allow the slow release of the product, thus ensuring its potentiating action growth and bactericide throughout the digestive tract of the animal. This partial protection ensures that a part of the butyrate reaches the farthest parts of the digestive tract of animals, thus exercising its action. On the one hand, unprotected sodium butyrate will exert its action on the nearest parts of the digestive tract. On the other, the protected sodium butyrate will exert its action on the most distal parts of the digestive tract, once the protective layer that prevents its action has hydrolyzed throughout the digestive process.

The additives described in the present invention further favor the specific activity of digestive enzymes, preferably of lipases, proteases, amylases and chymotrypsins, as well as the food conversion factor, which results in greater use of the food and consequently greater biomass of animals.

The concentrations of each of the ingredients purchased by the additives of the invention are incorporated in amounts sufficient to achieve the beneficial properties thereof, such as improving the food conversion factor, a better utilization of the food, an increase in its biomass. and an increase in the longevity of individuals.

Another object described in the present invention relates to the use of the additives of the invention as promoters of the growth of aquatic animals, as well as promoter of their health and well-being, by providing the additives of the invention to fed animals. with them from a reinforced immune system. In this sense, the white spot syndrome virus (WSSV) is the main pathogen of shrimp and outbreaks kill all shrimp populations in a few days, being by therefore responsible for large production / profit losses in this industry worldwide. In addition to infecting shrimp, the virus has a wide range of hosts, such as crustaceans such as crabs, spiny lobsters, crayfish, etc. which can be infected with varying severity depending on the vital stage of the crustacean and the presence of external stressors (temperature, salinity, bacterial diseases, contaminants, etc.). The clinical signs of "white spot" include a sudden reduction in food consumption, lethargy, cuticle loss, discoloration and presence of white spots of 0.5 to 2.0 mm in diameter on the inner surface of the shell, appendages and on the cuticle of the abdominal segments. Until now there is no effective treatment to control the infection caused by said virus, but aquatic animals fed with the different additives described here, having their immune system reinforced thanks to the additives of the invention, show no signs of the disease. therefore, the additives of the invention are capable of preventing infection by the white spot virus.

Another object of the present invention relates to a method for promoting the growth of aquatic animals by administering the additive described in the invention, since said additive is capable of giving greater use to the food consumed.

Other objects, features and advantages of the present invention will be readily apparent to those skilled in the art.

For the purposes of the present invention the following terms are defined:

The term "aquaculture" refers to the set of technical activities and knowledge of cultivation of aquatic plant and animal species. It is an important economic activity of food production, raw materials for industrial and pharmaceutical use, and living organisms for repopulation or ornamentation. Cultivation systems can be freshwater or seawater. The most common crops correspond to planktonic organisms (microalgae and Artemia), macroalgae, molluscs, crustaceans and fish.

The term "dietary additive", "food additive", "food additive" or "dietary supplement", for the purpose of the present invention refers to a composition intended to be ingested together with the normal diet of the animal. The additive described in the invention may comprise at least one probiotic, alone or in combination with other ingredients, preferably salts of organic acids.

For the purposes of the present invention, additives that are part of the animal diet and that fulfill the function of improving the animals' daily weight gain (GDP), as well as the factor of growth, are called "promoters or growth stimulants" food conversion In the present invention, promoters or stimulants of Growth can be administered in different ways, whether injected, in implants or as an additive in animal feed or feed. Administration as an additive in the food or feed is preferred. In the present invention, the effect on growth is measured as an increase in biomass.

For the purposes of the present invention, the term "organic acid" refers to compounds that contain in its formula one or more carboxylic groups (-COOH), proton generators, which may also have different functional groups such as hydroxy acids, keto acids, aromatic acids , heterocyclic compounds, as well as amides and lactones. The organic acids used in the present invention are preferably volatile fatty acids, preferably short chain and from which can be selected: butyric acid, propionic acid, formic acid, lactic acid, citric acid, lauric acid, cupric acid, caprylic acid, caproic acid, acetic acid, among others. The proportions in which the organic acid is found in the animal feed additive described in the present invention, usually vary between 30 and 70% by weight of total wet product, the preferred proportion being 50%.

In the context of the present invention, "probiotic" refers to any type of viable microorganism applied to an animal or human host, and which beneficially affects the host, due to its beneficial effects, on health or on the increase of the resistance to diseases, which it may have on its consumer. Probiotics may exhibit one or more of the following, non-limiting characteristics: non-pathogenic or non-toxic to the host; they are present as viable cells, capable of surviving in the intestinal environment (for example, at low pH and acids and gastrointestinal secretions), adhesion to epithelial cells, particularly to epithelial cells of the gastrointestinal tract, microbicidal or microbiostatic activity or effect towards pathogenic bacteria; anticancer activity; immune modulation activity; activity modulating the activity towards the endogenous flora; improvement of the health of the urogenital tract; antiseptic activity in or around wounds; diarrhea reduction; reduction of allergic reactions; reduction in neonatal necrotizing enterocolitis; reduction in inflammatory bowel disease and reduction of intestinal permeability.

For the purposes of the present invention, the term "viable" refers to a living microorganism capable of reproducing and / or colonizing. As used herein, the singular forms "a", "a", and "the" include plural reference unless the context clearly indicates otherwise.

Similarly, the words "understand" and "understand" must be interpreted in an inclusive and non-exclusive manner, encompassing, reciprocally, the terms "consist" and "consist."

Description of the figures Figure 1. Biomass of the Litopenaeus vannamei shrimp weighing approximately 0.85 g, analyzed every 10 days, for a total period of 60 days. The shrimp were fed with the standard food to which the different additives of the invention were added to the concentration of 2g / Kg standard diet: ECOBI OL containing the probiotic CECT 5940 (first column-grid bar), or ECOBIOL PLUS containing the CECT 5940 + BP70 probiotic (second column-white bar), or ECOBIOL SUPER containing the combination of CECT 5940 + CECT 953 + BP70 probiotics (third column-black bar) or exclusively the standard diet (control group) (fourth column- striped bar). The time expressed in days is shown on the X axis and the biomass expressed in grams on the Y axis. * p <0.05 vs control.

Figure 2. Litopenaeus vannamei survival percentage of approximately 0.85 g of weight, analyzed every 10 days, for a total period of 60 days feeding shrimp with a standard diet to which the different additives of the invention were added to the concentration of 2g / Kg standard diet: ECOBIOL containing the probiotic CECT 5940 (broken line with rhombus), or ECOBIOL PLUS containing the probiotic CECT 5940 + BP70 (continuous line with circle), or ECOBIOL SU PER containing the combination of probiotics CECT 5940 + CECT 953 + BP70 (dashed line with triangle) or exclusively the standard diet (continuous line with square). The time expressed in days is shown on the X axis and on the Y and Y the survival expressed as a percentage. * p <0.05 vs control.

Figure 3. Food conversion factor (grams of food per gram of biomass harvested) of Litopenaeus vannamei of approximately 0.85 g of weight, at the end of the 60 days of the crop, fed with its standard diet plus the additives described in the invention, ECOBIOL containing the CECT 5940 probiotic, or ECOBIOL PLUS containing the CECT 5940+ BP70 probiotic, or ECOB IOL SU PE R which contains the combination of the probiotics CECT 5940 + CECT 953 + BP70 or exclusively the standard diet (control group).

Figure 4. Litopenaeus vannamei biomass of approximately 7.0 g weight, analyzed every 10 days, for a total period of 60 days. The shrimp were fed with the standard food to which the different additives of the invention were added to the concentration of 2g / kg of standard diet: ECOBIOL containing the probiotic CECT 5940 (first column-grid bar), or ECOBIOL PLUS containing the CECT 5940 + BP70 probiotic (second column-white bar), or ECOBIOL SUPER containing the combination of CECT 5940 + CECT 953 + BP70 probiotics (third column-black bar) or exclusively the standard diet (control group) (fourth column- striped bar). The time expressed in days is shown on the X axis and the biomass expressed in grams on the Y axis. * p <0.05. Figure 5. Litopenaeus vannamei survival percentage of approximately 7.0 g weight, analyzed every 10 days, for a total period of 60 days feeding shrimp with a standard diet to which the different additives of the invention were added to the concentration of 2g / kg of standard diet: ECOBIOL containing the probiotic CECT 5940 (broken line with rhombus), or ECOBIOL PLUS containing the probiotic CECT 5940 + BP70 (continuous line with circle), or ECOBIOL SU PER containing the combination of CECT 5940 + CECT 953 + BP70 probiotics (dashed line with triangle) or exclusively the standard diet (control group) (continuous line with a cross). The time expressed in days is shown on the X axis and the survival expressed as a percentage on the Y axis.

Figure 6. Food conversion factor (grams of food per gram of harvested biomass) of Litopenaeus vannamei of approximately 7.0 g of weight, at the end of the 60 days of the crop, fed with its standard diet plus the additives described in the invention, to the concentration of 2g / Kg of standard diet: ECOBIOL containing the probiotic CECT 5940, or ECOBIOL PLUS containing the probiotic CECT 5940 + BP70 or ECOBIOL SUPER containing the combination of the probiotics CECT 5940 + CECT 953 + BP70 or exclusively the diet standard (control group).

Figure 7. Response of the digestive enzyme activity in the hepatopancreas of the shrimp Litopenaeus vannamei, given the stimulation of diets containing the different types of probiotics: ECOBIOL containing the probiotic CECT 5940 (dashed line with rhombus), or ECOBIOL PLUS containing the probiotic CECT 5940 + BP70 (continuous line with circle), or ECOBIOL SUPER containing the combination of the probiotics CECT 5940 + CECT 953 + BP70 (dashed line with triangle) or exclusively the standard diet (control group) (continuous line with square). In time 0 the shrimp still did not receive the food; 1, 2, 3, 4 and 5h correspond to the time after the shrimp received the food. (a) = Lipases, (b) = Proteases, (c) Amylases and (d) = Chymotrypsins. The time expressed in hours is shown on the X axis and the specific enzymatic activity expressed as U / mg protein is shown on the Y axis. Figure 8. Study of the growth of Nile Tilapia Oreochromis niloticus (mean ± SD) fed with different concentrations of the additive of the invention ECOBIOL comprising the probiotic B. amyloliquefaciens CECT 5940. G1: control group fed with a basal diet. G2 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁹ cfu / kg. G3 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁷ cfu / kg. (A) The bars indicate the initial body weight (IBW), the final body weight after 30 days (FBW1), the final body weight after 60 days (FBW2), weight gain after 30 days (WG1), and Weight gain after 60 days (WG2). (B) The figure shows the specific growth rate (SGR) of each of the study groups in the two months of the study. (C) The figure shows the protein efficacy ratio (PER) for each of the study groups.

Figure 9. Intestinal morphology of Nile Tilapia O. niloticus after 2 months fed with the probiotic of the invention ECOBIOL (β. Amyloliquefaciens CECT 5940).

(A) Photograph of a cross-section of the proximal part of the intestine stained with hematoxylin & Eosin (H&E) showing the height of folding (double arrows), mucus-secreting cells (arrowheads) and intraepithelial lymphocytes (I EL) (arrows). Scale bar: 50 μηι. (B) The figure shows the height of the villi (mean ± SD) in the proximal, middle and distal parts of the intestine. The asterisk (*) indicates a significantly greater height of hairiness in the proximal part of the large intestine compared to other parts of the intestine, within the same group. G1: control group fed a basal diet. G2 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁹ cfu / kg. G3 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁷ cfu / kg. A cross section of the distal part of the intestine stained by staining of alcian blue showed few mucus-secreting cells (arrow) in the control group (C) and numerous mucus-secreting cells in the group supplemented with the probiotic ECOBIOL (β. amyloliquefaciens CECT 5940) (D). Scale bar: 50 μηι. (E) The figure shows the number of mucus-secreting cells (mean ± SD) in the proximal, middle and distal parts of the intestine. (F) The bars indicate the number of IEL (mean ± SD) in the proximal, middle and distal parts of the intestine. Figure 10. Hematological parameters of Nile Tilapia O. niloticus fed with the additive of the invention ECOBIOL containing the probiotic B. amyloliquefaciens CECT 5940), for 2 months.

(A) red blood cell count. (B) Hemoglobin content. (C)% PCV (Packed Cell Volume). (D) White blood cell count. (E) Glucose level. (F) Concentration of serum proteins (total proteins, albumin and globulin). G1: control group fed a basal diet. G2 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁹ cfu / kg. G3 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁷ cfu / kg.

Figure 11. Constituents of the body of Nile Tilapia O. niloticus fed with different concentrations of the ECOBIOL additive containing the probiotic B. amyloliquefaciens CECT 5940, for 2 months. The bars indicate% crude protein (A),% crude lipids (B), ash content (dust or impurities) (C) and% moisture (D). G1: control group fed a basal diet. G2 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁹ cfu / kg. G3 group fed a basal diet supplemented with the probiotic B. amyloliquefaciens CECT 5940 at a spore concentration of 1x10 ⁷ cfu / kg.

Figure 12. Biomass (expressed in Kg) of the rainbow trout Oncorhynchus mykiss analyzed 60 days after starting treatment. The experimental design is explained in Example 6. Each of the treatments analyzed comprises:

T1: 43% fishmeal (HP) and 25% soy cake (TS)

T2: 29% HP and 50% TS T3: HP + ECOBIOL

T4: TS + ECOBIOL

T5: HP + BP-70

T6: TS + BP-70

T7: HP + ECOBIOL + BP-70

T8: TS + ECOBIOL + BP-70

Figure 13. Length (expressed in cm) of the rainbow trout O.mykiss analyzed 60 days after starting treatment. The experimental design is explained in Example 6. Each of the treatments analyzed comprises:

T1: 43% fishmeal (HP) and 25% soy cake (TS)

T2: 29% HP and 50% TS

T3: HP + ECOBIOL

T4: TS + ECOBIOL

T5: HP + BP-70

T6: TS + BP-70

T7: HP + ECOBIOL + BP-70

T8: TS + ECOBIOL + BP-70 Figure 14. Food consumption (expressed in Kg) of the rainbow trout O.mykiss analyzed 60 days after starting treatment. The experimental design is explained in Example 6. Each of the treatments analyzed comprises:

T1: 43% fishmeal (HP) and 25% soy cake (TS)

T2: 29% HP and 50% TS

T3: HP + ECOBIOL

T4: TS + ECOBIOL

T5: HP + BP-70

T6: TS + BP-70

T7: HP + ECOBIOL + BP-70

T8: TS + ECOBIOL + BP-70

Figure 15. Conversion of the food (C.A.) of the rainbow trout O.mykiss analyzed 60 days after starting treatment. The experimental design is explained in Example 6. Each of the treatments analyzed comprises:

T1: 43% fishmeal (HP) and 25% soy cake (TS)

T2: 29% HP and 50% TS T3: HP + ECOBIOL

T4: TS + ECOBIOL

T5: HP + BP-70

T6: TS + BP-70

T7: HP + ECOBIOL + BP-70

T8: TS + ECOBIOL + BP-70

Figure 16. Survival (expressed as a%) of the rainbow trout O.mykiss analyzed 60 days after starting treatment. The experimental design is explained in Example 6. Each of the treatments analyzed comprises:

T1: 43% fishmeal (HP) and 25% soy cake (TS)

T2: 29% HP and 50% TS

T3: HP + ECOBIOL

T4: TS + ECOBIOL

T5: HP + BP-70

T6: TS + BP-70

T7: HP + ECOBIOL + BP-70

T8: TS + ECOBIOL + BP-70 Detailed description of the invention

One aspect of the present invention relates to an additive for feeding aquatic animals comprising the combination of at least two species belonging to the genus Bacillus, selected from B. amyloliquefaciens and Bacillus cereus.

In a preferred embodiment, the additive of the invention comprises the CECT 5940 strains of Bacillus amyloliquefaciens and the CECT 953 strain of Bacillus cereus.

In another preferred embodiment of the invention, the additive for feeding aquatic animals may further comprise other probiotics selected from any of the following genera: Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostcococus, , Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus. In preferred embodiments, the probiotics comprise at least one suitable strain or subspecies from among: Enterococcus, Streptococcus, Lactobacillus, Lactococcus, Bifidobacterium and Pediococcus and / or combinations thereof. In a more preferred embodiment of the invention the strain is selected from the genus Pediococcus, preferably from the species Pediococcus acidilactici, and in a more preferred embodiment, the most preferred probiotic is Pediococcus acidilactici strain CNCM MA 18/5 M, deposited in the Collection National Crops of Microorganisms (CNCM) of the Pasteur Institute in Paris. The additives described in the present invention include probiotics in an amount suitable to improve the immune system, to increase the beneficial effects of the probiotics and / or to increase the biomass of the animals and / or extend their life. In general, food additives comprise approximately 10 ⁵ to 10 ¹⁰ CFU / g of feed for each of the probiotics used, with a concentration of approximately 10 ⁶ CFU / g of feed being preferred, of each of the probiotics used.

The person skilled in the art can determine the appropriate amount of probiotics that is added to the additive of the invention. These factors that can be taken into account are the average consumption of certain types of compositions of different animals, if the animal that intends to ingest dietary supplements has a particular health problem, well-being or nutritional needs, or suffers of a disease or disorder, the age, sex, size or species of the animal. The concentrations of the probiotics to be added to the additive of the invention can be calculated based on the energy and nutrient requirements of the animal.

In another preferred embodiment of the invention the additive further comprises at least one salt of an organic acid. In another preferred embodiment, the organic acids are selected from any of the following: butyric, propionic, formic, lactic, citric, lauric, cupric, caprylic, caproic or acetic.

In another more preferred embodiment of the invention, the salts of the organic acids are selected from any of the list: sodium, calcium, cupric or potassium. The proportions in which the organic acid salt is found in the additive for animal feed described in the present invention, usually varies between 30 and 70% by weight of the final wet additive, the preferred proportion being 60%.

In another even more preferred embodiment, the organic acid salt comprising the additive of the invention is the sodium salt of butyric acid (sodium butyrate), although any of the salts of the acids described previously may be used.

In another preferred embodiment the additive of the invention is characterized in that it comprises a combination of the CECT 5940 probiotics of B. amyloliquefaciens and CECT 953 of B. cereus together with sodium salt of butyric acid.

The additives of the invention may additionally also comprise substances such as: minerals, vitamins, proteins, amino acids, dyes, preservatives, etc. Non-limiting examples of minerals include for example: calcium, phosphorus, potassium, sodium, iron, chlorine, copper, zinc, magnesium, manganese, selenium, etc. Non-limiting examples of vitamins include for example: Vitamin A, different types of Vitamin B (niacin, pantothenic acid, folic acid, biotin) Vitamin D and Vitamin K.

Another of the objects described in the present invention relates to the use of the additives described herein in the breeding of aquatic animals, as growth promoters of said animals, being able to favor the food conversion factor, as well as the specific activity of digestive enzymes, preferably of lipases, proteases, amylases and chymotrypsins.

In a preferred embodiment of the invention, the additives described herein are used to promote the health and welfare of aquatic animals.

In another preferred embodiment of the invention, the probiotics described herein, preferably the probiotics selected from B. amyloliquefaciens, B. cereus, B. subtilis and B. licheniformis and / or combinations thereof, are used in animal husbandry aquatic

In another more preferred embodiment, the probiotics used for the breeding of aquatic animals are preferably the CECT 5940 probiotic of B. amyloliquefaciens and the CECT 953 probiotic of B. cereus. In another more preferred embodiment, the probiotics used for the breeding of aquatic animals are characterized in that they can also include an additional probiotic of the genus Pediococcus, preferably the species Pediococcus acidilactici and more preferably the strain CNCM MA 18/5 M, deposited in the Collection National Crops of Microorganisms (CNCM) of the Pasteur Institute in Paris.

In another more preferred embodiment of the invention, the probiotics used for the breeding of aquatic animals are characterized in that they further comprise at least one salt of an organic acid, said salt of the organic acid being preferably in a proportion of between 30-70% in wet weight, a proportion of 60% being more preferable.

In another more preferred embodiment of the invention, the organic acids are selected from any of the following: butyric, propionic, formic, lactic, citric, lauric, cupric, caprylic, caproic or acetic.

In another more preferred embodiment of the invention, the salts of organic acids are selected from any of the list: sodium, calcium, cupric or potassium. In another even more preferred embodiment of the invention, the probiotics used for the breeding of aquatic animals are characterized in that the organic acid salt is the sodium salt of butyric acid.

In another more preferred embodiment of the invention, the probiotics used for the breeding of aquatic animals comprise a combination of the CECT 5940 probiotics of B. amyloliquefaciens and CECT 953 of B. cereus together with sodium salt of butyric acid.

In another preferred embodiment of the present invention, the probiotics used for the breeding of aquatic animals also have the ability to modulate the immune response and exert a bactericidal effect against pathogenic microorganisms. On the other hand, as demonstrated in the present invention, the probiotics described herein also have the ability to exert a bioremediating action, thus helping to maintain the environment of the surfaces on which they are located, preferably thanks to secretion. of exo-enzymes that break down organic matter and decrease the COD (chemical oxygen demand) accepted parameter as an indicator of water quality. In another preferred embodiment of the invention, aquatic animals are selected from any of the list: mollusks, crustaceans, tilapia, gilthead sea bass, and salmonids, with crustaceans being preferred, preferably shrimp. All percentages expressed herein are by weight of the additive composition based on dry matter unless specifically indicated otherwise.

The invention is not limited to the particular methodology, protocols and reagents described herein, as they may vary. In addition, the terminology used here is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Unless otherwise defined, all technical, scientific and any acronym terms used here have the same meaning as commonly understood by a person skilled in the art within the scope of the invention.

The examples described below are intended to illustrate the present invention, but without limiting the scope thereof. Example 1. Growth response and survival of the white shrimp Litopenaeus vannamei fed with a standard diet supplemented with the additives of the invention.

Shrimp of the species Litopenaeus vannamei, randomly selected, with an average weight of 0.85 g were used. These shrimp were previously acclimatized for 72 hours in a 2500 liter tank of seawater with 36.0 UPS (practical salinity units), constant aeration (> 4.0 mg / l oxygen), controlled temperature (30 to 34 ° C) and pH from 7.5 to 8.0. The shrimp were grown in 10 L aquariums, with sea water and keeping the quality variables mentioned above. The tests were carried out with a total of 8 shrimp per aquarium with six replications for each of the food additives used in the present invention. The experiment lasted 60 days, with biometrics every 10 to 10 days in order to assess growth, survival and biomass rates. Biomass refers to the sum of the individual weights of shrimp present in each of the treatments with the different dietary additives. For the preparation of the diets, a commercial feed / feed commonly used for feeding shrimp with 35% protein was used, which was ground and sieved at 200 microns. The different probiotics were added as an additive to the standard diet at a rate of 2g / kg of food. Once the additive was incorporated into the standard diet, the mixture was homogenized and passed through a meat grinder and finally by a drying oven at 40 ° C for 12 hours. The food was placed in feeders; food not consumed after two hours was removed and subsequently weighed for analysis. The four analyzed diets containing the dietary supplements or additives described in the present invention are described in Table 1.

Table 1. Shrimp were fed three times a day ad limitum according to the following feeding program.

Therefore, when the present description refers to food in the tests carried out, this term includes both the standard diet supplied to the controls, formed by the commercial food indicated above, and the mixture thereof with the different additives of the invention.

As previously indicated throughout the present document, the ECOBIOL probiotic comprises the bacteria B. amyloliquefaciens strain CECT 5940, the probiotic ECOBIOL PLUS containing the bacteria B. amyloliquefaciens strain CECT 5940 together with the sodium salt of the butyric acid BP70. As mentioned previously, the sodium salt of butyric acid, specifically the composition of Norel, SA called BP-70, is made up of 70% by weight of sodium butyrate and 30% by weight of vegetable fats. 30% of vegetable fat protects approximately 43% of total sodium butyrate, so the BP-70 product can be said to be made up of 30% protected sodium butyrate and 40% unprotected sodium butyrate . He ECOBIOL SUPER probiotic contains the combination of bacteria B. amyloliquefaciens strain CECT 5940, together with B. cereus strain 953 and together with BP-70.

The food was added to satiety three times a day using feeders. The food conversion factor (FCA) was calculated in grams of food per gram of biomass extracted; using the values of the food added daily, the unconsumed and the shrimp biomass obtained at the end of the 60 days of the experiment. Shrimp growth and survival were evaluated every 10 days by recording the average weight and the number of shrimp respectively.

The results of growth and survival show, as can be seen in Figures 1 and 2 respectively, that shrimp fed with the diet supplemented with the additive ECOBIOL SUPER generated significant differences (p <0.05) in weight and survival, starting from 40 days of the experiment, regarding the control diet. As can also be seen in Figure 1, shrimp fed with the additives ECOBIOL, ECOBIOL PLUS and ECOBIOS SUPER, at 60 days of treatment, show a significant increase (p <0.05) in their biomass with respect to the control group.

To demonstrate whether the initial weight of the animal influences the response to growth and survival of shrimp fed with the different dietary additives described in the invention, an experiment was designed with shrimp, randomly selected, with an average weight of 7.0 g. As can be seen in Figure 4, as of day 30 of treatment, shrimp fed with the ECOBIOL SUPER diet showed a significant increase in their biomass compared to shrimp fed with the control diet (p <0.05). On the other hand, as can be seen in Figure 5, shrimp fed the diets containing the additives of the invention, showed a higher survival rate compared to animals fed exclusively with the standard diet, control group and a better factor of feed conversion (Figure 6).

The use of food, analyzed as a food conversion factor, was better in the diets containing the additives of the invention (Figures 3 and 6), indicating that animals have greater availability of nutrients for growth and for obtaining Energy. This in the long term, is reflected in achieving healthier animals and with sizes more suitable for commercialization, in a shorter time. Therefore, the data shown in the present invention show that the described additives function as growth promoters in aquatic animals, preferably crustaceans, with results superior to those obtained with the standard diet, not including additives.

Example 2. Digestive enzymatic activity of the white shrimp L. vannamei fed with the different additives described in the invention.

The expression and activity of digestive enzymes is affected by a series of limiting factors such as: physical-chemical parameters of water (pH, oxygen, salinity and temperature), age and size of shrimp, ontogenetic changes, fasting, diet ingredients , level and protein source, level and type of binders, growth promoting additives, quantity and frequency of feeding, circadian cycles, molting cycle and even the cultivation water.

To determine the response of digestive enzymatic activity to the stimulation of the different dietary additives used in the present invention: ECOBIOL, ECOBIOL PLUS and ECOBIOL SUPER, juvenile L vannamei shrimp were used, with an average weight of 6 g, which were maintained fasting for 12 hours and previously selected in intermuda stages. The intermuda is known as the sequence of transformations between two molts (elimination of the old exoskeleton and the formation of a new and generally larger tegument), in which period a complete cycle of morphological, physiological and biochemical modifications, responsible for the increase. The measurements of said enzymatic activity were carried out in intervals of one hour, for a time of 5 hours.

The activity and production of digestive enzymes in the hepatopancreas of crustaceans is mainly controlled by hormones. The hepatopancreas, unlike the intestine and stomach, has a greater enzymatic activity, suggesting the importance of this organ in the synthesis and secretion of digestive enzymes.

For the analysis of the digestive enzymatic activity, the protease, lipase, amylase and chymotrypsinase activity was determined in the enzymatic extracts prepared by the methodology proposed by Galgani F. (Galgani, F. PhD. Thesis Université dÁix-Marseille //. Etude des digestive proteases of crevettes peneides (Crustacean Pickled) France. 1983) in hepatopancreas extracted from shrimp, at the indicated intervals previously and that were stored, for said analysis, at a temperature of - 48 ° C. In addition, the concentration of proteins present in said enzymatic extracts was determined by the Bradford method. The method described by Hernández CM (Hernández, CM Crustacean protease characterization biochemical and molecular consideration. Comprehensive summary Ph D Thesis. 1983. Northwest Biological Research Center, La Paz, was followed to obtain the protease activity data present in the enzyme extracts. BCS Mexico.), Using azocasein as substrate. The lipase activity present in the extracts was determined according to Versaw WK et al. (Versaw, WK et al. Journal of Food Science. 1989; 54: 1557-1558), using R-naphthyl caprylate as substrate. Amylase activity was determined according to Vega-Villasante et al. , (Vega-Villasante, F. et al. Bulletin of Marine Science, 1999; 65: 1-9), using as a substrate 1% starch in TRIS-HCL (50 mM, pH 8.0). The chemotrypsin activity was evaluated according to Delmar EG et al. (Delmar EG et ai. Anal Biochem. 1979; 99: 318-320. The specific activity of each enzyme is represented in units of enzyme per milligram of protein (U / mg).

The results obtained showed that, the addition of the additives described in the invention to the diet of shrimp L. vannamei, increased digestive enzymatic activity, presenting a greater expression of the digestive enzymes analyzed (Figure 7) with respect to fed shrimp With the control diet. Shrimp that received the additive ECOBIOL SUPER showed greater evidence on the specific activity of the enzymes lipases and chymotrypsin (lipids and proteins).

It is concluded that the additives described in the present invention used in diets for aquatic animals, preferably crustaceans and particularly, the combination of probiotics CECT 5940 + CECT 953 + sodium butyrate (BP-70), favor the specific activity of digestive enzymes and the conversion factor in the food of the crustaceans which suggests a greater use of the food, as well as an increase in its biomass and survival, with respect to animals fed a control diet, not supplemented with the additives of the invention. Example 3. In vitro study of the bacteriocin release capacity of the additive of the invention B. amyloliquefaciens CECT 5940 and analysis of its biocidal activity against pathogenic organisms. Bacteriocins are small microbial peptides that are released extracellularly. Such peptides have a bactericidal or bacteriostatic effect on other bacterial species. The objective of this study was to evaluate the bacteriocin production capacity of the additive of the invention, B. amyloliquefaciens CECT 5940 (ECOBIOL).

To analyze the capacity of bacteriocins production by the additive of the invention B. amyloliquefaciens CECT 5940, said strains are grown in a flask with 50 ml of TSB medium (Bioser) for 24 hours and 2 flasks are inoculated with 50 ml of TSB medium in a 1: 100 ratio that is kept under stirring at 200 rpm and 30 ° C for 48 hours.

Samples are taken from these flasks at 7, 24 and 48 hours of culture. The cells are removed from the cultures by centrifugation for 20 minutes at 4000 rpm. Of the supernatants of the different samples collected, one part was neutralized at pH 6.2 with 5M HCI and the other was maintained at the original pH of the sample taken and shown in Table 2, in order to simulate the physiological conditions in the proximal and distal intestine. Both fractions were sterilized through a 0.22 μηι filter. These preparations were stored at -20 ° C until use. The biocidal activity of the above-mentioned supernatants was determined by the plate diffusion method (Tapes, LM et al. (1995). Applied & Environmental Microbiology. 61: 2643-2648), seeded with pathogenic microorganisms of different genera. It should be mentioned that all the microorganisms used in the present invention, either samples of them were available or were obtained from the Spanish Type Culture Collection (CECT), located in the Scientific Park of the University of Valencia. Calle Agustín Escardino Professor, 9 of Paterna (Valencia). 3.1 Biocidal activity of the additive of the invention ECOBIOL (B. amyloliciuefaciens CECT 5940) against organisms of the genus Clostridium Spp.

All strains of Clostridia were grown in agar-clostridium medium at an approximate concentration of 10 ⁶ CFU / plaque. With the exception of B. amyloliquefaciens CECT 5940, which was grown at 30 ° C, the rest of the strains were incubated at 37 ° C in the absence of oxygen, inside anaerobiosis bottles.

The following strains of Clostridium were used as indicators of the existence of bacteriocins.

Clostridium butyricum CECT 361 T

Clostridium perfringens CECT 376T

Clostridium perfringens CECT 486

Clostridium perfringens CECT 820

Clostridium perfringens C ECT 821

Clostridium perfringens CECT 822

Clostridium histolyticum CECT 4109T

50 μΙ of each culture supernatant of B. amyloliquefaciens CECT 5940 (ECOBIOL) were deposited in the wells (6mm in diameter) of Nutrient Agar plates (BioKar, pH 7 ± 0.2) supplemented with 3% v / v NaCI, previously planted with 200 of a culture that had been growing overnight of the pathogenic microorganisms to be tested against those intended to test the production of bacteriocins. The TSB medium is used as control.

After 2 hours at 4 ° C to allow diffusion of the product in the medium, the plates are incubated at the corresponding temperatures for each of the pathogenic microorganisms, to allow the growth of the target organism. After 24 hours, the presence or absence of a growth inhibition halo is determined. All sampling was done in duplicate, both in the collection of supernatants, and in the sowing of plates.

The biocidal activity of the supernatants of cultures of B. amyloliquefaciens CECT 5940 against different bacterial strains of the pathogenic Clostridium genus, are shown in Table 2. Table 2. Biocidal effect in vitro of the supernatants isolated from cultures of B. amyloliquefaciens CECT 5940 against different bacterial strains of the genus Clostridium.

+: Growth inhibition; -: no growth inhibition;

The data shown in Table 2 is explained as follows: (cm of inhibition halo) +: 1 cm of inhibition halo

+ +: 1.5 cm of inhibition halo

+ + +: 1.8 cm inhibition halo

+ + + +: 2.2 cm of inhibition halo

-: Absence of inhibition halo

Supernatants isolated from cultures of B. amyloliquefaciens CECT 5940 inhibited the growth of most strains of the Clostridium genus analyzed (Table 2). In this sense, the results shown in Table 2 show that B. amyloliquefaciens CECT 5940 shows a great capacity to release bacteriocins during the first 24 hours. It is convenient to comment that, the strains of the species Clostridium butyricum CECT 361 T have a slightly homogeneous growth in relation to the rest of the strains, so it is difficult to determine their inhibition halo. 3.2. Biocidal activity of the additive of the invention ECOBIOL (B. amyloliciuefaciens CECT 5940) against hemolytic and enterotoxic pathogens.

The concentration of pathogenic bacteria 10 ⁶ U FC / plaque was inoculated with the appropriate medium. Several notches of a size of 6 mm in diameter were made in the culture plates of the pathogenic microorganisms and the supernatant of B. amyloliquefaciens CECT 5940 was introduced in each (50 μΙ). The plates were incubated at 4 ° C for 2 hours to allow the different components of the supernatant to diffuse into the plate culture medium. After that, all plates were incubated at 37 ° C for 24 hours and the inhibition halo was determined in order to demonstrate the bactericidal effect.

The pathogenic strains evaluated were:

Enterococcus faecalis CECT 4176

Enterococcus gallinarum CECT 970

Escherichia coli CECT 352

Escherichia coli CECT 434

Escherichia coli CECT 504

Escherichia coli CECT 728

Escherichia coli CECT 735

Escherichia coli CECT 742

Escherichia coli CECT 816

Escherichia coli CECT 4076

Escherichia coli CECT 4267

Escherichia coli CECT 4782

Escherichia coli CECT 4783

Staphylococcus aureus CECT 957

Yersinia enterocolitica CECT 754 All strains of E. coli were selected according to the phenotype (0: 147 H: 7). The rest are pathogenic strains known according to the CECT (Spanish Crop Collection Type), except Enterococcus, which were selected as lactic acid bacterial strains (LAB).

The results obtained are shown in Table 3.

Table 3. In vitro bactericidal effect of the additive of the invention ECOBIOL (β. Amyloliquefaciens CECT 5940) against different pathogenic bacteria.

The results show that pathogenic E. coli strains, in addition to Yersinia, are inhibited with the additive supernatant ECOBIOL (β. Amyloliquefaciens CECT 5940).

It is fully demonstrated that pathogenic strains are inhibited by bacteriocins growth even at high pH, thus having a broad spectrum of action. Bacteriocins did not show positive interactions with LAB, the effects are only accused by pathogenic bacteria.

3.3 Biocidal activity of the additive of the invention ECOBIOL (B. amyloliciuefaciens CECT 5940) against pathogenic organisms of the Vibrio genus.

Vibrio genus bacteria are Gram-negative bacteria commonly found in estuaries and marine waters, usually in temperate and tropical climates. Some species, such as the V. parahaemolyticus species, can cause gastroenteritis in humans. Natural guests of V. parahemolyticus include crabs, shrimp, turtles, oysters, lobsters and fish. The species V. gastroenteritis is often associated with infection in shrimp.

Recent studies have linked the pathogen V. parahemolyticus to the EMS also known as AHPNS (acute hepatopancreatic necrosis syndrome), a pathology that causes high mortality of shrimp that has already produced losses of around 1 billion dollars in Southeast Asia. A study by Donald Lightner (University of Arizona) has found that this bacterium infected by a phage produces a toxin that affects the digestive organ of shrimp, the hepatopancreas causing the death of almost all animals grown at very early ages.

To demonstrate the bactericidal effect of the additive of the invention ECOBIOL (β. Amyloliquefaciens CECT 5940) on the bacteria of the genus Vibrio, particularly on the species V. parahaemolyticus CECT 51 1 and V. harveyi CECT 525, the inhibition halo technique was used . Briefly, after cultivation of the additive comprising B. amyloliquefaciens CECT 5940 (ECOBIOL), cell-free extracts of B. amyloliquefaciens were obtained by centrifugation and used to test the bactericidal effect of said supernatant on the aforementioned Vibrio strains, by the halo inhibition technique.

The two strains were grown in Nutrient Broth medium (Biokar) supplemented with 3% v / v NaCI, and incubated in an orbital shaker at the temperatures recommended in each case by the CECT for 16 hours. The results show that the supernatants obtained from the cultures of ECOBIOL (β. Amyloliquefaciens CECT 5940), as previously described, showed halos of inhibition around the traces made on cultures of the pathogenic species V. parahemolyticus CECT 511, but not, against the cultures of V. harveyi CECT 525. In addition, the results indicate that the strain V. parahaemolyticus CECT 511, which It has halo for all collected supernatants, such inhibition halos are more evident after 24 hours of incubation. Therefore, the supernatant of B. amyloliquefaciens CECT 5940 (ECOBIOL) contains some compound, bacteriocin type, that inhibits the growth of Vibrio parahaemolyticus CECT 511. The numbers shown in Table 4 indicate the diameter of the inhibition halo in corresponding millimeters to each of the supernatants tested (it should be noted that the well in which the sample is applied has a diameter of 6 mm which is also included in the size of the halo).

The demonstrated ability of B. amyloliquefaciens CECT 5940 to inhibit the growth of this pathogen could help with the control of EMS.

Table 4. In vitro bactericidal effect of B. amyloliquefaciens CECT 5940 (ECOBIOL) against pathogenic bacteria V. parahaemolyticus

3.4 Biocidal activity of the additive ECOBIOL (B. amyloliquefaciens CECT 5940) against pathogenic organisms of the species Aeromonas salmonicida and Yersinia ruckeri.

B. amyloliquefaciens CECT 5940 was incubated in TSA medium, which was adjusted to pH 5 and 8, in order to simulate physiological conditions in the proximal and distal intestine. In addition, two different salinity conditions (0.5 and 3%) were evaluated. The essays of activity (halo inhibition) were carried out in MHA-1 (Mueller-Hilton agar at 1% NaCl), in which the pathogens were inoculated and the soaked discs were placed with the probiotic. The results were expressed as inhibition (+) or lack of inhibition (-).

The results are shown below:

Table 5. In vitro bactericidal effect of B. amyloliquefaciens CECT 5940 (ECOBIOL) against the pathogenic bacteria A.salmonicida and Y.ruckeri.

The results (Table 5) show that at pH 5 and 8, as well as in high salinity conditions, B. amyloliquefaciens CECT 5940 (ECOBIOL) exhibits inhibitory activity against pathogenic bacteria that affect salmon and trout (A. salmonicida and Y. ruckeri).

Example 4. Production capacity of organic acids by B. amyloliquefaciens CECT 5940 under anaerobic conditions.

The objective of the present example is to evaluate the production capacity of lactic acid of the additive of the invention ECOBIOL (β. Amyloliquefaciens CECT 5940) under self-generated anaerobiosis conditions. Once B. amyloliquefaciens CECT 5940 reaches the intestine and begins to grow, the production of lactic acid excreted to the lumen of the intestine causes acidification of the medium resulting in a preferential growth of the acidophilic flora, having a beneficial effect on health intestinal. The preferential growth of this type of microorganisms displaces the pathogenic bacteria through the known mechanism of competitive exclusion. For all the above, the sanitary state of the animal experiences a strengthening. The growth and culture conditions of the strains of B. amyloliquefaciens CECT 5940 have already been described throughout this document. The composition of the experimental medium is as follows:

4% micronized soybeans

1% glucose syrup (equivalent to 5.1 g / L of reducing sugars)

1% yeast extract

The composition of the medium described above mimics the proportion of proteins and carbohydrates in the intestinal lumen of the animals.

Experimental cultures were carried out in closed 200 ml bottles, to avoid the transfer of oxygen to the culture medium. The oxygen initially dissolved in the culture medium, measured by probe, was approximately 0%, since it was eliminated in the sterilization process prior to inoculation. Traces of available oxygen were also eliminated in the early stages of microorganism growth, therefore, the conditions of self-generated anaerobiosis (same as the animal's intestinal lumen) were created. The culture conditions were: static cultures at 37 ° C for 24 hours. The following parameters were evaluated in the samples obtained throughout the fermentation:

1. CFU / ml: According to the technique of serum dilution and subsequent colony counting in the appropriate culture medium of B. amyloliquefaciens CECT 5940. 2. Lactic acid production: The methods used in the process. n spectophotometric, and the determination of the presence of NADH at an absorbance of A340 in centrifugal and cell-free fermentation extract samples, with the Boehringer Mannheim enzyme systems.

3. Acetic acid production: The method used was spectophotometric and the determination of the presence of NADH at A340 in samples of centrifugal and cell-free fermentation extracts, with Boehringer Mannheim enzymatic systems.

4. Reducer of sugar consumption: The method used was colorimetric, with the reactive DNS, to establish the residual amount of reducing sugars in centrifuged fermentation culture media. Appropriate formulas were used to calculate the acid production rate:

Yield (Y) = grams of acid / grams of reducing sugar consumed Acid production rate (R) = grams of acid / 10 ⁹ CFU biomass per hour

The results showed the acid production by the strains of B. amyloliquefaciens CECT 5940 under the conditions of self-generated anaerobiosis.

Table 6. Acid production by cultures of B. amyloliquefaciens CECT 5940 under anaerobic conditions.

Most of the acid production by the fermentative metabolism of B. amyloliquefaciens CECT 5940 results in L-lactic acid, but is also capable of producing acetic acid. The ability to convert acids regulated by intestinal pH is very high. The same mass of acids as the sugar assimilating bacteria can be achieved. Such acids can be used as pH controllers in the intestine (bio-control of pathogens) and / or as a source of carbon and available energy. In addition, sugar not converted to acid is used as biomass for the growth of microorganisms. This demonstrated ability to produce lactic acid exerts an effect of modulation of the intestinal bacterial flora, benefiting the growth of favorable species and inhibiting the growth of pathogens that could grow causing infections.

Example 5. Growth response, intestinal morphology, hematology and body composition of Nile Tilapia (Oreochromis niloticus (L.)) fed a standard diet supplemented with the additives of the invention. In the present example, the use of the additive of the invention ECOBIOL (β. Amyloliquefaciens CECT 5940) as a probiotic in Nile tilapia is evaluated. A total of 120 O. niloticus (L.) fry (average weight of 9.4 ± 0.3 g) They were divided into 3 groups (20 fish / group) in duplicate. The group called G1 was fed a basal diet as the control group; while the groups called G2 and G3 were fed the basal diet to which the additive ECOBIOL (β. amyloliquefaciens CECT 5940) was added in the final levels at a spore concentration of 1x10 ⁹ and 1x10 ⁷ cfu / kg respectively.

After 30 days of feeding, the G2 group showed significant increases in final body weight (FBW) (Figure 8), in weight gain (WG). English) (Figure 8) and the specific growth rate (SGR) (Figure 8) compared to groups G1 and G3. At the end of the feeding period, groups G2 and G3 had significant increases in FBW, WG, SGR (Figure 8A and B) and in the protein efficacy ratio (PER) compared to the control group (G1) (Figure 8C).

In addition, the use of a higher concentration of probiotics in the animals' diet increased the height of the intestinal villi, especially in the proximal part of the intestine. Thus, the G2 group presented a significantly higher number of mucus-secreting cells (goblet cells), in the proximal and distal parts of the intestine, than the G3 or G1 groups (Figure 9A-E).

The results obtained also showed that the animals of the G2 group had a higher significant concentration of intra-epithelial lymphocytes (IEL) in the intestine compared to the control group G1, in all parts of the intestines, while the G3 group had significantly more LELs than the G1 control group, but only in the distal part of the intestine (Figure 9F).

The addition to the diet of the Nile tilapia of the additive ECOBIOL (β. Amyloliquefaciens CECT 5940) had no harmful effects on the total red blood cell count or glucose levels, but it did increase the hemoglobin content, hematocrit values and the total white blood cell count (Figure 10).

The addition of high concentration of B. amyloliquefaciens CECT 5940 in the diet of Nile tilapia (group G2) produced better effects than a lower concentration of said additive (group G3) on the total serum protein concentration and on the levels of globulin The protein content throughout the body of the animals included in groups G2 and G3 were higher than the levels of proteins found in the control group (G1). The highest fat concentration was recorded in group G2, followed by group G3 and then group G1 (Figure 11).

In other experiments performed in tilapia grown in the presence of the pathogenic strain A. hydrofila, it was observed that the animals that received the additive of the invention ECOBIOL (β. Amyloliquefaciens CECT 5940) in the diet had higher lysozyme activity (enzyme with antibacterial activity) in blood (p <0.05) and a numerical increase in white blood cell count. These parameters indicate a better predisposition to resist infections.

These results show, therefore, that the addition of the additive ECOBIOL (β. Amyloliquefaciens CECT 5940) to the diet of Nile tilapia (O. niloticus), shows a beneficial effect on these animals, both in the potentiation of their growth, as of your immune system (immunostimulator).

Example 6. Evaluation of the additive of the invention ECOBIOL (B. amyloliquefaciens CECT 5940) and Gustor BP-70 as growth promoters in diets with fishmeal or soy cake for rainbow trout fry (Oncorhynchus mykiss).

The objective of the present example is to evaluate the addition to a diet of rainbow trout fry (O. mykiss), two growth promoters, ECOBIOL (β. Amyloliquefaciens CECT 5940) and Gustor BP-70, alone or in combination, at the level 0.1%, in diets based on fishmeal or soy cake, on the productive behavior, taking into account the parameters of growth (weight gain and height), food conversion and survival of these animals. The facilities of the Vinchos Fish Production Center, located in the district of Canchayllo, belonging to the SAIS "Tupac Amaru", in the department of Junín (Peru) were used. The experimental diets were prepared at the Balanced Food Plant of the Social Research and Projection Program in Food, School of Zootechnics, of the National Agrarian University La Molina (Lima, Peru), during the months of March, April and May of 2012, with a duration of 60 days.

170000 rainbow trout fry were installed in 30 ponds of 5 x 0.60 meters (experimental units), with capacity for 5000 fish each. Three entries were made, in periods of 15 days (replicas); the first with fish with an average weight of 2.2 g and 6.0 cm in size; after 15 days, fish with an average weight of 0.15 g and 2.25 cm in size were installed and after 15 days, fish with an average weight of 0.08 g and 1.90 cm in size. Two commercial diets were formulated, one based on fishmeal (43%) and one based on soybean cake (50%), linear programming was used at minimum cost, according to the recommendations of the NRC (1993). To each experimental diet 0.1% of

ECOBIOL (β. Amyloliquefaciens CECT 5940) or BP-70 or the mixture of both, giving rise to 8 treatments. Each of the treatments includes:

T1: 43% fishmeal (HP) and 25% soy cake (TS)

T2: 29% HP and 50% TS

T3: HP + ECOBIOL

T4: TS + ECOBIOL

T5: HP + BP-70

T6: TS + BP-70

T7: HP + ECOBIOL + BP-70

T8: TS + ECOBIOL + BP-70

The data of the results were subjected to a statistical analysis, considering the Design of Blocks Completely Random (DBCA), with the purpose of expressing the differences between the treatments, by means of the analysis of variance (ANVA) and differences of the averages in the parameters evaluated using the DUNCAN statistical test. Table 7 shows the composition of the food used and its nutritional value. Table 7. Formulas used in experimental diets and their nutritional value.

Basal Diet Flour Basal Diet Cake

Ingredients (%) of Soy Fish

Prime Flour 43.00 29.00

Wheat flour 30.10 15.10

Soy cake, 47 25.00 50.00

Vegetable oil 1.00 3.00

Semi refined fish oil 1.92

Salt 0.58 0.66

Vitamin and mineral premix 0.30 0.30

Antioxidant 0.02 0.02

Total 100.00 100.00

Calculated Nutritional Value

Total protein,% 45.27 45.00

Fiber,% 2.81 2.78

Fat,% 7.84 9.60

ED. Trout, Mcal / kg 3.70 3.70

Lysine,% 3.20 3.15

Methionine,% 1.08 0.95

Cystine,% 0.51 0.59

Arginine,% 2.87 3.11

Histidine,% 1.12 1.14

Leucine,% 3.40 3.45

Isoleucine,% 2.10 2.10

Phenialanine,% 2.02 2.14

Tyrosine,% 1.53 1.60

Threonine,% 1.85 1.85

Tryptophan,% 0.55 0.59

Valine,% 2.37 2.41

Met + Cist,% 1.59 1.53

Fen-tir,% 3.53 3.72

Ac Gr n-3,% 1.79 1.97 Basal Diet Flour Basal Diet Cake

Ingredients (%) of Soy Fish

Ac Gr n-6,% 0.62 1.61

Total match,% 1.49 1.17

Calcium,% 1.71 1.24

Sodium,% 0.70 0.60

Potassium,% 1.26 1.50

Chlorine,% 0.75 0.72

Hill, ppm 2957.75 2846.29

Prot Dig,% 41.80 42.43

6.1 Results of the weight and size of O. mykiss fed with the diets described in Table 7 together with the additives of the invention.

The results of the parameters of productive behavior, whose values correspond to the average of three replicates, are shown in Figures 12 to 16. Fish fed with diets containing a higher percentage of fishmeal as the main source of protein, showed greater growth in weight and size compared to those who received diets containing soy cake, at 5.34 and 4.77%, respectively; experiencing smaller difference smaller fish.

The addition of the additives ECOBIOL (B. amyloliquefaciens CECT 5940) or BP-70 or the mixture of both to the diets with fishmeal or soy cake, did not statistically improve weight gain and height (Figures 12 and 13) . However, there is a numerical trend towards a greater increase in size with diets based on soy cake with the addition of ECOBIOL (B. amyloliquefaciens CECT 5940) or BP-70. On the other hand, the addition of ECOBIOL (B. amyloliquefaciens CECT 5940) to the diet with soy cake generated greater growth of weight and size in smaller fish, 10 and 16% respectively in relation to the control diet without ECOBIOL (B. amyloliquefaciens CECT 5940) (Figures 12 and 13). 6.2. Results of food consumption and food conversion of O.mykiss fed with the diets described in Table 7 together with the additives of the invention.

The results indicate that there are no statistical differences in food consumption. However, fish fed with diets containing fishmeal showed a tendency to higher feed consumption (Figure 14).

In relation to food conversion, the fish that received the diets containing fishmeal, showed better feed conversion per unit of weight gain, being statistically different from the fish that received the diets with soy cake. The addition of ECOBIOL (β. Amyloliquefaciens CECT 5940) to the diet based on soybean cake, significantly improved the utilization of the food in the growth of the fish, being more efficient in 4%, compared with the fish that received the diet soy cake control (Figure 15). Similar behavior is observed in fish of different sizes (replicas).

6.3. Survival results of O.mykiss fed the diets described in Table 7 together with the additives of the invention. Fish that received BP-70 in the diet based on soy cake, achieved lower mortality (2.6%), statistically significant compared to those who received diets with fishmeal (4%), with or without the addition of BP -70 or ECOBIOL (β. Amyloliquefaciens CECT 5940) to the diet with soy cake (3.6%). Fish survival was very similar in all treatments (Figure 16).

The results obtained in the present example show that the diets containing fishmeal produced greater growth in weight and size of trout fry in relation to the diet with soy cake. In addition, the addition of ECOBIOL (β. Amyloliquefaciens CECT 5940) or BP-70 to diets containing fishmeal or soy cake did not improve growth in relation to weight, although if there is a tendency to increase growth in size in Diets with soy cake.

The addition of the additive ECOBIOL (β. Amyloliquefaciens CECT 5940) to the diet containing soy cake statistically improved food conversion, while the addition of BP-70 to the diet containing soy cake, significantly reduced mortality, achieving greater survival of animals. With the results shown, the use of diets based on soy cake as a source of protein plus the addition of ECOBIOL (β. Amyloliquefaciens CECT 5940) to reduce the cost of feeding due to economic savings due to greater efficiency in Trout diets

Example 7. Use of ECOBIOL (B. amyloliquefaciens CECT 5940) as a bioremediation agent.

Water quality and soil quality management is considered one of the most important aspects of pond aquaculture. While water quality parameters, such as dissolved oxygen, pH, or ammonia concentrations affect essential functions of the body, the state of the bottom of the ponds and the exchange of substances between the soil and water greatly affects water quality The accumulation of waste will increase the demand for oxygen by bacteria in order to perform their metabolism and degrade organic matter, favoring the development of anaerobic areas. These anoxic sediments will be in contact with the oxygen-rich water column, producing an alteration of the environmental conditions suitable for a healthy development of the organisms that inhabit these ponds. In addition, ammonia can diffuse in the water column not only as a result of fish excretion, but also after microbial decomposition of organic matter. Under high temperature and pH conditions, the accumulation of ammonia can cause sub-lethal effects such as growth retardation or disease propensity. Therefore, it is of key importance to keep the bottom of the pond in aerobic conditions and prevent organic matter and silts from accumulating. In this sense, Bacillus spp. They are considered as bioremediation agents, a concept that refers to the use of microorganisms that help in maintaining the environment of the appropriate pond thanks to the secretion of exo-enzymes that break down organic matter. The purpose of the present example is to evaluate the in vitro capacity of ECOBIOL (β. Amyloliquefaciens CECT 5940) as a bioremediation agent.

First, the chemical oxygen demand (COD) was evaluated, which is defined as a measure of the ability of water to consume oxygen during the decomposition of organic matter and the oxidation of inorganic chemicals, such as ammonia and nitrite. Water is obtained from a fish farm and added in trays (300 cm ² ). The depth reached by the layer of water and mud was 5 cm. ECOBIOL (β. Amyloliquefaciens) and other probiotic bacteria were added in the treatment trays and evaluated in duplicate as follows:

Two COD values were determined: 1) soluble and 2) total.

The soluble COD measurements in the supernatant were made after 15 min of sedimentation after homogenization of the media, while the total COD measurements were made immediately after homogenization. The measurements were carried out on day 0 (initial), day 3 and day 7. The temperature was maintained at 25 ° C. The results revealed that the total COD was progressively reduced in the treatment groups, especially in those treated with ECOBIOL (β. Amyloliquefaciens CECT 5940). In addition, the layer of water and mud was reduced to 4 cm deep. This indicates lower levels of waste, and consequently, better environmental conditions, due to the bioremediation action of treatment with ECOBIOL (β. Amyloliquefaciens CECT 5940) (See Table 8).

According to the results shown, the use of ECOBIOL (B. amyloliquefaciens CECT 5940) as a bioremediation agent can help maintain the proper pond environment thus favoring the healthy growth of the organisms that live in it. Table 8. Results of the chemical oxygen demand (COD) analyzed throughout the treatment.

COD mg0 ₂ / L Initial 3 days 7 days

Variation

Soluble Total Soluble Total Soluble Total

Control 89.0 1677.5 <30 1848.5 <30 1873.5 + 10.43%

ECOBIOL 89.5 2145.5 <30 1602.0 <30 1559.0 -37.25%

B. cereus 86 2694.0 <30 2143.5 <30 2057.5 -30.85%

B. licheniformis 95.5 1692.5 <30 1678.5 <30 1653.5 -2.35%

Claims

1. Use of Bacillus amyloliquefaciens, in the breeding of aquatic animals. 2. Use according to claim 1 characterized in that the strain CECT 5940 of B. amyloliquefaciens is used in the breeding of aquatic animals.

3. Use according to any one of claims 1 to 2 characterized in that at least the Bacillus cereus species is also used in the breeding of aquatic animals.

4. Use according to claim 3 characterized in that in the breeding of aquatic animals the strain CECT 953 of B. cereus is used.

5. Use according to any one of claims 1 to 4 characterized in that at least the Pediococcus acidilactici species is also used in the breeding of aquatic animals.

6. Use according to claim 5 characterized in that in the breeding of aquatic animals the strain CNCM MA 18/5 M of P. acidilactici is used.

7. Use according to any of claims 1 to 6, characterized in that at least other species selected from any of the following are used in the breeding of aquatic animals: B. subtilis and B. licheniformis. 8. Use according to any one of claims 1 to 7 characterized in that at least one salt of an organic acid is used in the breeding of aquatic animals.

9. Use according to claim 8 characterized in that the organic acid salt is preferably in a proportion of between 30-70% by wet weight, a proportion of 60% being more preferable.

10. Use according to any of claims 8 or 9 characterized in that the organic acids are selected from any of the following: butyric, propionic, formic, lactic, citric, lauric, cupric, caprylic, caproic or acetic.

1 1. Use according to any of claims 8 to 10 characterized in that the salts of organic acids are selected from any of the list: sodium, calcium, cupric or potassium.

12. Use according to claims 8 to 11 characterized in that the organic acid salt is the sodium salt of butyric acid.

13. Use according to claims 8 to 12 characterized in that a combination of the CECT 5940 strains of B. amyloliquefaciens and CECT 953 of B. cereus is used in the breeding of aquatic animals together with sodium salt of butyric acid.

14. Use according to any of claims 1-2 characterized in that it is also able to modulate the immune response and exert a bactericidal effect against pathogenic microorganisms.

15. Use according to any of claims 1 to 14 wherein the aquatic animals are preferably molluscs, crustaceans, tilapia, gilthead sea bass, sea bass and salmonids.

16. Additive for feeding aquatic animals comprising the combination of at least two species belonging to the genus Bacillus, selected from: B. amyloliquefaciens and B. cereus.

17. Additive according to claim 16 characterized in that it comprises the combination of at least the CECT 5940 strain of B. amyloliquefaciens and the CECT 953 strain of B. cereus.

18. Additive according to any of claims 16 to 17 characterized in that it can also comprise the species, P. acidilactici.

Additive according to claim 18 characterized in that it comprises the CNCM MA 18/5 M strain of P. acidilactici.

20. Additive according to claims 16 to 19 characterized in that it further comprises at least one salt of an organic acid.

21. Additive according to claim 20 characterized in that the organic acid salt is preferably in a proportion of between 30-70% by wet weight, a proportion of 60% being more preferable.

Additive according to any of claims 20 to 21 characterized in that the organic acids are selected from any of the following: butyric, propionic, formic, lactic, citric, lauric, cupric, caprylic, caproic or acetic.

Additive according to claims 20 to 22 characterized in that the salts of organic acids are selected from any of the list: sodium, calcium, cupric or potassium.

24. Additive according to claims 20 to 23 characterized in that the organic acid salt is the sodium salt of butyric acid.

25. Additive according to claims 16 to 24 characterized in that it comprises a combination of the CECT 5940 strains of B. amyloliquefaciens and CECT 953 of B. cereus together with sodium salt of butyric acid. 26. Additive according to claims 16 to 25 characterized in that the aquatic animals are selected from any of the list: mollusks, crustaceans, tilapia, gilthead sea bass, sea bass and salmonids.

Use of the additives of claims 16 to 26 in the breeding of aquatic animals.

28. Use of the additives according to claim 27 characterized in that they also act as modulators of the immune response. 29. Use of the additives according to claim 27 characterized in that they also have bactericidal action against pathogenic microorganisms.

30. Use of the additives according to claim 27 characterized in that they also have bioremediating action. Use of the additives according to claims 27 to 30, characterized in that the aquatic animals are selected from any of the list: molluscs, crustaceans, tilapia, gilthead sea bass, sea bass and salmonids.