ES2447090B1 - ADDITIVES FOR THE FEEDING OF AQUATIC ANIMALS CONTAINING PROBIOTICS. - Google Patents

Abstract

Additives for feeding aquatic animals containing probiotics. # The present invention describes an additive for feeding aquatic animals comprising the combination of two species belonging to the genus Bacillus, said species being B. amyloliquefaciens and B. cereus, preferably strains CECT 5940 of B. amyloliquefaciens and CECT 953 of B. cereus, in combination with a salt of an organic acid, preferably with the sodium salt of butyric acid. The feed additives for aquatic animals described in the present invention may further comprise other bacterial strains with probiotic capacity, preferably belonging to the Pediococcus acidilactici species.

Description

DESCRIPTION

Feed additives for aquatic animals that contain probiotics.

FIELD OF THE INVENTION 5

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 stimulants of growth and animal survival. Therefore, the present invention is encompassed within the field of aquaculture and more specifically within animal production, food and health.

STATE OF THE TECHNIQUE

In recent years, aquaculture has made significant advances in the production of a wide variety of 15 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 nutritional efficiency, in addition to increasing the presence and incidence of diseases caused by opportunistic pathogens. The need for sustainable and profitable aquaculture 20 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 lead to cross resistance in breeding animals, against pathogenic bacteria, leading to 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 strengthens their immunological and sanitary status, in general and reduces pathological problems without using medications. , in order to improve its production, avoid problems with the safety of food obtained from livestock 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. 35 Microbiological Molecular 2000; 64: 655-671; Gullian, M. et al., Aquaculture 2004; 233: 1-14; Balcázar, JL, et al. Veterinary Microbiology 2006; 114: 173 -186; Gatesoupe, FJ Journal of Molecular Microbiological Biotechnology. 2008; 14: 107-114) that can be defined as viable microorganisms that administered in the diet promote the welfare of breeding organisms by stimulating the immune system, as well as of the establishment of the intestinal microbial balance in these breeding animals, by excluding 40 potentially pathogenic microorganisms (Irianto, A. and B. Journal of Fish Diseases 2002; 25: 333-342; Irianto, A. and B. Austin. Journal of Fish Diseases 2003; 26: 59-62; Lara-Flores, M., et al. Aquaculture 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 antibiotics 45 growth promoters. 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 that is 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, the 65 acid-lactic bacteria, preferably belonging to the genus Lactobacillus (Nikoskelainen, S.A. et al. Fish

Sellfish Immunology 2003; 15: 443-452; Balcázar, J.L. et al. British Journal of Nutrition 2007; 97: 522-527; Gatesoupe, F.J. Journal of Molecular Microbiological 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, there are few species of microorganisms that 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. 10

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 meet the requirements of the animals, it must also produce a very stable food in the water because aquatic animals feed slowly and continuously. Specifically, the shrimp detects the food at a distance by chemoreception, so that the food must also be attractive (attracting the animal) and palatable (which 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 20 are of low molecular weight; as: amino acids, quaternary ammonium compounds, trimethylamine, betaine (trimethylglycine), nucleotides, biogenic amines and organic acids. Commercially available soluble flours and fish oils, molluscs or crustaceans; They work as excellent attractants.

 25

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 translates into a 30 increase in the growth of breeding animals and therefore results in the economic benefits for aquaculture farms.

DESCRIPTION OF THE INVENTION

 35

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. 40

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 ECOBIOL probiotic. Four. Five

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 50 is further 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 S.A., Spain 55 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 ESPORAFEED probiotic.

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, dorado, 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. 5

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.

 10

Organic short-chain acids are capable of reducing the pH at the stomach 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 Your metabolism Its bactericidal action is exerted preferably between the genera: Escherichia, Campylobacter, Clostridium and Salmonella, among others. In the present invention the product of Norel S.A. is used. Spain marketed under the name of GUSTOR BP-15 70, 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 in the entire 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. 25

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. 30

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. 35

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) 40 is the main shrimp pathogen and outbreaks kill all shrimp populations in a few days, being 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 45 (temperature, salinity, bacterial diseases, contaminants, etc.). The clinical signs of the "white spot" include a sudden reduction in food consumption, lethargy, cuticle loss, discoloration and the presence of white spots of 0.5 to 2.0 mm in diameter on the inner surface of the shell, appendices and on the cuticle of the abdominal segments. So far 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 strengthened by 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. 60

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 species of plants and animals. It is an important economic activity of food production, raw materials for industrial and pharmaceutical use, and living organisms for repopulation or ornamentation. The systems of

Cultivation 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 purposes 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 daily weight gain of the 10 animals (GDP), as well as the factor, are called "promoters or growth stimulants" of food conversion. In the present invention, growth promoters or stimulants 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. fifteen

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%. 25

In the context of the present invention, "probiotic" refers to any type of viable micro-organism 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 disease resistance, which it may have on its consumer. Probiotics may exhibit one or more of the following, non-limiting characteristics: no pathogens 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 35; 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.

 Four. Five

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

 fifty

Figure 1. Litopenaeus vannamei shrimp biomass of approximately 0.85 g in 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 standard diet: ECOBIOL containing the probiotic CECT 5940 (first column-grid bar), or ECOBIOL PLUS containing the probiotic CECT 5940 + BP70 (second column-white bar), or ECOBIOL SUPER containing the combination 55 of the 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 60 every 10 days, for a total period of 60 days feeding the shrimp with a standard diet to which the different additives of the invention were added to the 2g / Kg standard diet concentration: ECOBIOL containing the CECT 5940 probiotic (broken line with rhombus), or ECOBIOL PLUS containing the CECT 5940 + BP70 probiotic (continuous circle line), or ECOBIOL SUPER containing the combination of probiotics CECT 5940 + CECT 953 + BP70 (dashed line with triangle) or exclusively the standard diet 65

(continuous line with square). The time expressed in days is shown on the X axis and the survival expressed as a percentage on the Y axis. * 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 ECOBIOL SUPER containing the combination of the CECT 5940 + CECT 953 + BP70 probiotics or exclusively the standard diet (control group).

Figure 4. Litopenaeus vannamei biomass of approximately 7.0 g in weight, analyzed every 10 days, 10 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 15 (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 2g / Kg concentration of standard diet: ECOBIOL containing the CECT 5940 probiotic (broken line with rhombus), or ECOBIOL PLUS containing the CECT 5940 + BP70 probiotic (continuous circle line), or ECOBIOL SUPER 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 on the Y axis the survival expressed as a percentage.

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: 30 ECOBIOL containing the CECT 5940 probiotic, or ECOBIOL PLUS containing the CECT 5940 + BP70 or ECOBIOL SUPER probiotic containing the combination of the CECT 5940 + CECT 953 + BP70 probiotics or exclusively the standard diet (control group).

Figure 7. Response of the digestive enzyme activity in the hepatopancreas of the Litopenaeus 35 vannamei shrimp, given the stimulation of diets containing the different types of probiotics: ECOBIOL containing the proctotic CECT 5940 (broken line with rhombus), or ECOBIOL PLUS containing the CECT 5940 + BP70 probiotic (continuous line with circle), or ECOBIOL SUPER containing the combination of CECT 5940 + CECT 953 + BP70 probiotics (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 40 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.

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.

 fifty

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

In another preferred embodiment of the invention, the feed additive for aquatic animals may further comprise other probiotics selected from any of the following genera: Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus.

In preferred embodiments, the probiotics comprise at least one suitable strain or subspecies of: 60 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 National Collection of Cultures of Microorganisms (CNCM) of the Pasteur Institute in Paris. 65

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 105 to 1010 CFU / g of feed for each of the probiotics used, with a concentration of approximately 106 CFU / g of feed being preferred, of each of the probiotics used. 5

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 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.

 twenty

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 animal feed additive described in the present invention, usually varies between 30 and 70% by weight of the wet final additive, the preferred proportion being 60%.

 25

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 probiotics B. amyloliquefaciens CECT 5940 and B. cereus CECT 953 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 refers 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. Four. Five

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 the breeding of aquatic animals.

 fifty

In another more preferred embodiment, the probiotics used for the breeding of aquatic animals are preferably the probiotic B. amyloliquefaciens strain CECT 5940 and the probiotic B. cereus strain CECT 953.

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 55 Pediococcus acidilactici and more preferably the strain CNCM MA 18/5 M, deposited in the National Microorganism Crops Collection (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 also comprise at least one salt of an organic acid, said salt of the organic acid being preferably in a proportion of between 30-70 % by 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. 65

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. 5

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

 10

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. twenty

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.

 25

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 30 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. Said shrimp were previously acclimatized for 72 hours in a 2500 liter seawater tank with 36.0 UPS (practical salinity units), constant aeration (> 4.0 mg / l oxygen), controlled temperature (30 to 34 ° C) and pH 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 the 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. 45 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. 50

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

 Group

 Diet Dietary supplement Dose / Way

 one

 ECOBIOL CECT 5940 Satiety / Oral

 2

 ECOBIOL PLUS CECT 5940 + BP-70 Satiety / Oral

 3

 ECOBIOL SUPER CECT 5940 + CECT 953 + BP-70 Satiety / Oral

 4

 Control Standard Satiety / Oral Diet

 55

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.

 60

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 previously mentioned, the sodium salt of butyric acid, specifically the composition of Norel, S.A. called BP-70 is formed by 70% wet weight of sodium butyrate and 30% wet weight of vegetable fats. 30% 5 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% sodium butyrate without to protect. The probiotic ECOBIOL SUPER contains the combination of bacteria B. amyloliquefaciens strain CECT 5940, together with B. cereus strain 953 and together with BP70.

 10

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. fifteen

The growth and survival results show, as can be seen in Figures 1 and 2 respectively, that shrimp fed 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 20 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 randomly selected shrimp, 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 food conversion (Figure 6).

The use of food, analyzed as a food conversion factor, was better in the diets containing the additives of the invention (Figure 6), indicating that animals have a greater availability of 35 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.

 Four. Five

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. fifty

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 55 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 of growth The measurements of said enzymatic activity were carried out in intervals of one hour, for a time of 5 hours.

 60

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 65 chymotrypsinase activity was determined in the enzyme extracts prepared by the methodology proposed by Galgani F.

(Galgani, F. PhD. Thesis Université dÁix-Marseille II. Etude des digestive proteases of crevettes peneides (Crustacea Decapoda) France. 1983) in hepatopancreas extracted from shrimp, at the intervals indicated 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. 5

To obtain the protease activity data present in the enzyme extracts, the method described by Hernández C.M. (Hernández, C.M. Crustacean protease characterization biochemical and molecular consideration. Comprehensive summary Ph D Thesis. 1983. Northwest Biological Research Center, La Paz, B.C.S. Mexico.), Using azocasein as a substrate. The lipase activity present in the extracts was determined according to Versaw W.K. et al. (Versaw, W.K. 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 chymotrypsin activity was evaluated according to Delmar E.G. et al. (Delmar E.G. et al. Anal Biochem. 1979; 99: 316-320. 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. The 20 shrimp that received the additive ECOBIOL SUPER showed greater evidence in 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 the 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 enzymes digestive factors and the conversion factor in the food of crustaceans which suggests a greater use of the food, as well as an increase in its biomass and survival, compared to animals fed with a control diet, not supplemented with the additives of the invention.

 30

Claims (27)

1. Use of Bacillus amyloliquefaciens, in the breeding of aquatic animals.  5 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. 10 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 of claims 1 to 4, characterized in that at least the Pediococcus acidilactici species is used in the breeding of 15 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.  twenty 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 of claims 1 to 7 characterized in that at least one salt of an organic acid is used in the breeding of 25 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. 30 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.  35 11. 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. 40 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.  Four. Five 14. Use according to claims 1 to 13 wherein the aquatic animals are preferably mollusks, crustaceans, tilapia, gilthead sea bass, sea bass and salmonids. 15. Additive for feeding aquatic animals comprising the combination of at least two species belonging to the genus Bacillus, selected from: B. amyloliquefaciens 50 and B. cereus. 16. Additive according to claim 15 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.  55 17. Additive according to any of claims 15 to 16 characterized in that it can also comprise the species P. acidilactici. 18. Additive according to claim 17 characterized in that it comprises the CNCM MA 18/5 M strain of Pediococcus acidilactici. 60 19. Additive according to claims 15 to 18 characterized in that it further comprises at least one salt of an organic acid. 20. Additive according to claim 19, 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. 21. Additive according to any of claims 19 to 20 characterized in that the organic acids are selected from any of the following: butyric, propionic, formic, lactic, citric, lauric, cupric, caprylic, caproic or acetic. 22. Additive according to claims 19 to 21 characterized in that the salts of organic acids are selected from any of the list: sodium, calcium, cupric or potassium. 10 23. Additive according to claims 19 to 22, characterized in that the organic acid salt is the sodium salt of butyric acid. 24. Additive according to claims 15 to 23 characterized in that it comprises a combination 15 of the CECT 5940 strains of B. amyloliquefaciens and CECT 953 of B. cereus together with sodium salt of butyric acid. 25. Additive according to claims 15 to 24 characterized in that the aquatic animals are selected from any of the list: mollusks, crustaceans, tilapia, gilthead sea bass, sea bass and 20 salmonids. 26. Use of the additives of claims 15 to 25 in the breeding of aquatic animals. 27. Use of the additives according to claim 26, characterized in that the aquatic animals are selected from any of the list: molluscs, crustaceans, tilapia, gilthead sea bass, sea bass and salmonids.