PL224877B1 - Probiotic for fish, molluscs and fresh water crustaceans, containing the Carnobacterium divergens bacterial strain and its metabolites - Google Patents

Description

Description of the invention

The subject of the invention is the use of a bacterial strain Carnobacterium divergens and its metabolites in the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of the pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda.

The health of fish depends on many physiological and environmental factors. Among them, the digestive tract microflora plays an important role. Its source is the entire aquatic environment. The microorganisms that live in it, which are usually dominated by the bacteria Vibrio, Aeromonas, Flavobacterium, Corynebacterium, Micrococcus, Pseudomonas and Clostridium, colonize their not yet fully developed digestive tract as early as a few hours after the larvae start active food intake. Then it is also colonized by lactic acid bacteria. In the digestive tract of both fry and adult marine fish, they are usually found in small numbers. In many species of freshwater fish, especially rainbow trout, sea trout as well as cyprinid, perch, sturgeon and pike fish, they are the dominant group of microorganisms. Enterococcus, Lactobacillus, Lactococcus and Pediococcus have the largest share in the population of lactic acid bacteria colonizing the digestive tract of freshwater fish. Carnobacterium bacteria are also natural and usually completely safe components of the intestinal ecosystem of many freshwater fish. In the digestive tract of free-living brown trout and rainbow trout, they are represented by such species as: C. divergens, C. gallinarum, C. funditum, C. mobille and C. piscicola.

Carnobacterium bacteria are catalase negative, gram positive, non-spore forming relatively anaerobic bacilli closely related to Lactobacillus bacteria. They are unable to grow on acetic agar, contain mesodiaminopimelic acid in the cell wall, hydrolyze arginine to citrulline and, unlike many other LABs, grow even at 4 ° C. However, they do not develop at a temperature higher than 45 ° C. For proper growth, they require a diet rich in carbohydrates, amino acids, peptides, fatty acids and vitamins. Carnobacterium, like other lactic acid bacteria, are capable of producing lactic acid, which lowers the intestinal pH and, consequently, inhibits the growth of many microorganisms, e.g. pathogenic to fish. Other metabolites of Carnobacterium bacteria, i.e. hydrogen peroxide, acetaldehyde and bacteriocins, also play an important role in inhibiting the growth of pathogenic microorganisms for fish. Among the mentioned compounds, bacteriocins have a particularly strong antimicrobial activity. These compounds resemble antibiotics in many ways. Unlike them, however, they are completely safe for humans and animals and do not initiate the formation of resistant microorganisms. Due to the high specificity of bacteriocin activity, they do not disturb the natural balance of the intestinal ecosystem. Most bacteriocins have a narrow range of activity and only act on a few groups of microorganisms. Antibiotics do not show such selectivity.

However, only some strains of Carnobacterium bacteria have the ability to synthesize bacteriocins active against microorganisms pathogenic to fish. It should be emphasized that probiotic properties are rare and strain-specific. The probiotic effects of microorganisms have been known for years, but only selected and tested strains can be used for the production of probiotics. This means that in a given species of bacteria, only a few strains are distinguished by probiotic activity.

Water is an ideal environment for microbial growth. The multiplication of microorganisms is favored by the contamination of the water with the remains of feed and fish faeces. Floating microorganisms are absorbed by fish and easily penetrate their digestive system. Pathogenic microorganisms are often found among aquatic microorganisms. These microorganisms penetrate the digestive system of fish through water, especially highly polluted water, and cause serious fish diseases, often with high mortality. So far, the fight against fish pathogens has involved treating fish with antibiotics (erythromycin, josamycin, spiramycin, tetracyclines and gentamicin). However, antibiotic therapy is costly and environmentally disadvantageous. An alternative to treating fish with antibiotics is the use of probiotics. Unlike antibiotics, their action can be both curative and preventive, which is much more beneficial for practical reasons.

In the rearing of aquatic organisms (fish, molluscs, arthropods), probiotic preparations have been used for several years in order to modify the quality of water as their living environment and to introduce beneficial microflora into their digestive tract. The modern concept of probiotics was developed 35 years ago by Parker, who defined probiotics as organisms or

Substances that contribute to the maintenance of the microbiological balance in the gastrointestinal tract. The development of research on the use of probiotics in the nutrition of aquatic organisms, including fish, is a direct consequence of the search for an alternative to antibiotic growth stimulants withdrawn from the feed industry. The action of many probiotics is similar to the effects of using feed antibiotics. Both probiotics and antibiotics can be used as tools to regulate the number of pathogenic bacteria. Probiotics, unlike antibiotics, however, do not destroy the native microflora of the gastrointestinal tract of aquatic organisms, but only stabilize it.

In recent years, in the production of feed, an ever wider range of feed additives has been used - substances or compounds whose task is to obtain the best production results in animal husbandry. The use of each feed additive must take into account the safety of human (consumer) health, animal health and animal welfare, as well as its impact on the environment. The adopted scheme of the authorization procedure for probiotics in aquaculture includes in vitro tests on: the production of inhibitory substances, competition for nutrients, resistance and adhesion factors, and in vivo tests for the ability to colonize the gastrointestinal tract and possible histopathological changes.

The next stage of research involves conducting experimental passages against pathogenic strains, which ultimately determine the confirmation of probiotic properties. The probiotic obtained according to the above scheme must be officially registered before being placed on the market, and in addition, the entity applying for registration carries out a profitability analysis of the production of the probiotic on a commercial scale.

Probiotic strains exhibit various mechanisms of action on their host - they act antagonistically against pathogens, colonize the surfaces of the gastrointestinal tract, increase fish resistance to diseases, increase the efficiency of digestion of nutrients and activate the immune system of animals.

The listed effects of probiotic microorganisms are only a part of their positive properties confirmed by experiments. Most commercial probiotic preparations for fish are based on the use of bacteria of the genus Bacillus, Pseudomonas, Yibrio and Lactobacillus.

The Carnobacterium divergens S1 strain, which is an active component of the probiotic according to the invention, is the subject of patent application P.369984 (published 20-03-2006). In accordance with the requirements of the Budapest Treaty on patent procedures, it has been deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p.

C. divergens bacteria belong to the naturally occurring microflora in the digestive tract of fish. The C. divergens S1 strain is also a component of the fish gut ecosystem. It has been isolated from the gastrointestinal tract of sea salmon. Detailed characteristics of the species Carnobacterium divergens were presented by Holzapfel and Gerber (1984) and Collins et al. (1987). The taxonomic lineage of this species is derived from the Bacilli class, the order Lactobacillales, the family Carnobacteriacae and the genus Carnobacterium.

An important feature of the C. divergens S1 strain is the ability to synthesize bacteriocin - divercin, which has a bactericidal effect on Listeria monocytogenes, Enterococcus fecalis and Clostidium, which are pathogenic for humans. It was surprisingly found that strain C. divergens SI also exhibits antagonistic activity against the pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum and Edwardsiella tarda. Antimicrobial activity is determined in bioassays and is measured in units of activity expressing the reciprocal of the lowest concentration of antimicrobial substances antagonizing the development of pathogenic organisms.

In farm freshwater fish farming, it is these bacteria that are the main cause of fry mortality. This discovery was the basis of the present invention and was used to develop a new probiotic based on the C. divergens S1 strain. Bacteria, which are an active component of the probiotic according to the invention, in the digestive tract of freshwater fish effectively produce lactic acid and bacteriocin-like extracellular metabolites, which clearly limit the growth of Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum and Edwardsiella tarda bacteria. Consequently, the probiotic according to the invention increases the survival of the fry and the resistance of juvenile fish to bacterial infections, thus limiting the serious losses that occur in the fish stock, especially in the first year of breeding. The use of the probiotic according to the invention also has a positive effect on the production effects of fish breeding: it increases daily body weight gain, improves the growth efficiency ratio of protein and lowers the nutritional ratio. The reported benefits are a consequence and / or effect of colonization of the gut by C. divergens S1

The antimicrobial activity of the metabolites of these bacteria. The probiotic according to the invention can therefore be used to protect freshwater fish, especially fry against pathogenic bacteria, increase their survival, improve growth rate and feed utilization. The probiotic containing C. divergens S1 and / or their metabolites can be administered to fish as a stand-alone preparation. This preparation can be put directly into water. It can also be an ingredient of feed, feed supplements and / or premixes. C. divergens S1 dead cells also have a pro-health effect, which in this case can be treated as a natural carrier of antimicrobial substances. These substances reduce the number of pathogenic bacteria in the intestinal ecosystem of freshwater fish.

Specialized scientific literature mentions the species C. divergens as a component of probiotics for marine fish, mainly Atlantic salmon, rainbow trout and cod. Based on the results of nutritional tests, it has been shown that the addition of C. divergens to solid feed, produced on the basis of cod fish waste, reduces the mortality of cod fry. This effect is a consequence of the action of the probiotic on the pathogenic bacteria Vibrio anguillarum.

In the national literature, the results of research on the effect of the addition of Carnobacterium divergens bacteria to feed for carp were described by researchers from the University of Life Sciences in Poznań (Mazurkiewicz et al. 2007). However, they used strains of C. divergens bacteria that did not have probiotic properties, and thus did not show health-promoting effects. The same research group also tested the effects of the commercial Biosaf preparation and showed that carp given this probiotic had greater weight gain (Przybył et al. 2006). So far, there are no other descriptions of the production and use of probiotics containing Carnobacterium divergens in freshwater fish farming. There are, however, several patents relating to the use of other microorganisms as ingredients in probiotics intended for fish.

US Patent 7,708,988 describes a probiotic composition and describes its therapeutic use. The probiotic composition of this patent contained Bacillus coagulans bacteria and / or other lactic acid bacteria and was intended to combat enteric pathogens. It is claimed that the microbial components can be used as independent preparations or in combination with antibiotics, fungistatic, anti-yeast and anti-viral ingredients. Separate reservations were also made for the genetic improvement of the bacterial strains owned and the search for new strains.

A probiotic with a health-promoting effect on fish is also described in US patent 6,878,373. This invention relates to a new probiotic formula for the protection of fish against pathogens of the genus Vibrio, Pseudomonas, Streptococcus and Aeromonas. It describes a method of controlling the growth of pathogenic bacteria and reducing mortality in both freshwater and marine fish. He also claims the use of a developed probiotic formula to improve the hygienic condition of water in breeding tanks.

The new bacterial strain Roseobacter PP-154 was used in the Spanish patent 2 216 704 to obtain probiotics. The use of a probiotic based on it reduced the mortality of fish fry, as well as mussels and shrimps. The patent claims the strain and the method of its use for the production of a bacterial preparation. It was claimed that the preparation is intended for marine farms and is in the form of a lyophilisate, and its action is a consequence of activity against pathogenic bacteria of the genus Vibrio, Tenacibaculum maritimum, Photobacterium damsel and Pseudomonas anguilliseptica.

Bacillus coagulans bacteria were also used to control the amount of pathogens in the digestive tract of the fish. This solution is described in US Patent 7,708,988. It concerns the use of one or more strains of Bacillus coagulans. These strains show a therapeutic effect. They colonize the digestive tract of fish and inhibit the growth of pathogenic microorganisms, including antibiotic-resistant pathogens. This patent also covers antimicrobial compounds produced by Bacillus coagulans. It also stipulates the selective improvement and isolation of probiotic bacteria that produce lactic acid and antimicrobial metabolites.

The strain Bacillus subtilis VKM B2287 was also used as a probiotic, showing the ability to affect pathogenic bacteria Staphylococcus, Escherichia coli, Enterobacteria, Citrobacteria and Aeromonas. This use is described in patent RU2246537. On the basis of the B. subtilis VKM B2287 strain, a prophylactic preparation for farm animals, poultry and fish called Subtilis + was developed.

Another probiotic strain of Bacillus subtilis VKM V2250 D is described in the patent RU 2 223 643 C2. This strain was introduced into eggs, into young larvae and into fry feed. As a result, better survival of fry and greater resistance of young fish were obtained.

PL 224 877 B1

In another patent RU 2,186,576 C2, genetically modified bacteria Bacillus subtilis VKPM V-4759 were used as an active strain. The gene coding for alpha-interferon was cloned into these bacteria. The preparation (called subaline) containing such improved bacteria was administered to the fish along with the feed for 5 days at a dose of 700 trillion spores per 1 ton of feed. As a result, the appearance of fish skin and gills has been improved, the functioning of their digestive tract has been stabilized and the risk of pathogenic bacteria has been reduced.

Another probiotic based on bacteria of the Bacillus subtilis species is the preparation described in patent RU 2 376 755. The B. subtilis B-1895 strain was used for its development. This strain was added to high-protein feed in an amount of 0.05-0.15%, which was then used in the feeding of fish.

In the US patent US 6,797,266, the probiotic strain Lactobacillus casei strain KE01 was used. The preparation based on it limited the growth of the enteropathogenic bacteria Escherichia coli, Salmonella enetriditis, Yersinia and Listeria monocytogenes. The claims describe the composition of this preparation and its intended use, which included farm animals, birds and fish.

One of the forms of influencing the health of fish is also supplying water in breeding tanks with selected groups of microorganisms. Such an application is described in patent US7736509. More precisely, a solution was claimed consisting in introducing probiotic bacteria, yeast, fungi, daphnia, microalgae into the water, which on the one hand were food for the fish, and on the other hand, precipitated phosphate-containing suspensions. Descriptions of the beneficial effects of probiotics on fish can also be found in the scientific literature. For example, feeding the fish Epinephelus gigas (grouper, poi. Grouper - fish of the genus Epinephelus) with a feed containing the probiotic yeast strain Saccharomyces cerevisiae P13 in the amount of 10 ⁷ cfu / kg, increased daily weight gain, improved feed conversion, stimulated their immune system and increased immunity (Chiu et al. 2010). Similar changes were also observed after feeding the same fish with a feed containing the probiotic strain of Lactobacillus plantarum 7-40 (Son et al. 2009). The scientific literature also describes the effects of feeding carp with fodder with the addition of the Lactobacillus acidophilus LAD strain and Saccharomyces cerevisiae SCD brewer's yeast. Increased fry growth and an improvement in the composition of the intestinal microflora were achieved (Dhanaraj et al. 2010).

The use of the Carnobacterium divergens bacterial strain and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of the pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda is based on the fact that the strain of Carnobacterium divergens and its metabolites deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, in an amount of at least 10 ⁶ cells of C. divergens S1, most preferably more than 10 ⁸ , preferably in a live or dead form and in an amount such that in 1 grams of dry matter their amount exceeds 100 units of antimicrobial activity of metabolites of bacteria C. divergens SI, preferably they are placed on a carrier such as sand and / or gravel and / or organic carriers mixed with feed ingredients and / or complete feed mixtures or premixes, and biomes sa is preferably preserved by drying or freezing.

The obtained probiotic is used as a prophylactic, therapeutic or growth-stimulating agent for freshwater fish, especially carp fry, rainbow trout, European catfish and sturgeon fish as well as molluscs and crustaceans, especially freshwater mussels and crayfish.

The obtained probiotic can be used for the processing of eggs, or it is introduced into breeding pools, where it improves the sanitary condition of the water.

Preferably, the probiotic is in a form selected from: suspension, granulate, powder, shell, gel, paste or emulsion.

The probiotic reduces the activity, growth or survival of the pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella tarda, Listeria monocytogenes and some Clostridium sp.

The probiotic according to the invention is used as an anti-infection, therapeutic or growth stimulant for freshwater fish, especially carp fry, rainbow trout, European catfish and sturgeon fish, and molluscs and crustaceans, especially freshwater mussels and crayfish.

The probiotic increases the survival of juvenile fish and juvenile forms of fish by increasing the resistance to bacterial infections of juvenile fish, molluscs and crustaceans, while improving the rearing results and feed conversion.

PL 224 877 B1

The probiotic contains, in addition to the cells of the C. divergens S1 bacterial strain and / or their metabolites, other acceptable components, in particular carriers or biologically active substances.

Wherein in the first preferred embodiment of the sterile liquid medium placed in the bioreactor, is introduced into bacteria inoculum Carnobacterium divergens AI in an amount of 2x10 ⁶ cfu / ml. The bacterial cultivation process is carried out at the temperature of 36 ° C at pH 7.0, kept at a constant level with NaOH, with gentle agitation of the medium. The cultivation is carried out for 20 hours. The bacterial cells are then separated by centrifugation at 5,000 rpm for 15 minutes. The cell suspension is introduced into a solution containing mineral salt-free concentrated culture medium at an osmotic pressure of 1500 mOsm / kg, which is then incubated for 45 minutes with gentle agitation at 28 ° C to increase cell survival during drying. The cells are then centrifuged and incubated in a solution to which maltodextrin, milk protein, ascorbic acid and silicon dioxide are added. The whole is mixed and dried in a spray drier to obtain a probiotic preparation in a powdered and coated form, containing 10 ⁸ cfu / g. This preparation is introduced into the fodder made by the high-pressure pressing method in such an amount that in 1 gram of the final fodder the number of probiotic bacteria is 10 ⁶ cfu. The probiotic feed is fed to Cyprinus carpio fry. In clinical and breeding tests, it was found that the survival of carps in Aeromonas hydrophila and Vibrio angillarum contaminated water increased by 80%, and the daily gains were greater by 18%.

In a second preferred embodiment, the bacterial strain of Carnobacterium divergens S1 is introduced into the medium containing sucrose, hydrolysed corn flour, casein peptone, yeast extract and a set of mineral salts in an amount of 10 ⁴ cfu / ml. The medium is previously sterilized in the bioreactor at 121 ° C for 15 min and cooled down to 30 ° C. The process of bacterial cultivation is carried out at the temperature of 30 ° C at pH 6.7, maintained with Ca (OH) ₂ , with gentle agitation of the medium. The cultivation was carried out for 18 hours. Then, the bacterial cells are separated using a membrane microfilter with a porosity of 0.45 µm by cross-filtration. The cell suspension is mixed with cryoprotective agents and dried by lyophilization. As a result of drying, a probiotic preparation is obtained in a powdered form, containing 3x10 ⁹ cfu / g. This preparation is introduced into the feed mixture in such an amount that the number of probiotic bacteria in the finished feed is 7x10 ⁸ cfu / g. The fodder is pressed with high pressure, and after drying, the granulate is covered with a hydrophobic film consisting of a mixture of fish and rapeseed oils in the proportion of 50:50. The fry of Oncorchynhus mykiss rainbow trout are fed with the probiotic feed prepared in this way. In clinical and breeding tests it was found that trout survival in water contaminated with Aeromonas salmonicide, Edwardsiella tarda and Vibrio angillarum bacteria increased by 60%, and the daily fry gains were higher by 15%.

In a third preferred embodiment, the Carnobacterium divergens S1 bacterial strain is introduced into sterile MRS A medium enriched with 10 ⁶ cfu / ml of rye flour enzymatic hydrolyzate. The medium is previously sterilized by filtration in a membrane microfilter with a porosity of 0.45 µm and it is introduced into a steam-sterilized bioreactor. The bacterial culture process is carried out at 30 ° C at pH 7.0 with pH correction with NaOH, with gentle mixing of the medium. The cultivation is carried out for 15 hours, then the bacterial culture together with the fermented medium is concentrated on a membrane microfilter, reducing the volume by 60% in relation to the initial volume. The resulting bacterial slump contains 7x10 ⁹ cells / ml. The cell slurry along with the fermented medium is then sprayed onto the dried pelleted feed in a cell slurry to dry feed ratio of 1: 5. After thorough mixing, the feed is dried using the convection method with air at a temperature not exceeding 50 ° C, and then the pellets are greased with fish oil in the amount of 2% of the feed weight in the process of pressure lubrication. The analysis showed that the feed prepared in this way contained 8x10 ⁶ cfu / g and had an activity against pathogenic Aeromonas hydrophila bacteria at the level of 150 activity units / g. The finished feed is fed to the fry of the sterlet Acipenser ruthenus and the Siberian sturgeon Acipenser baeri. It was found in clinical and breeding tests that as a result of using probiotic feed, the survival of the fry increased and the feed conversion improved.

In a fourth preferred embodiment, the sterilized medium containing saccharides, amino acids, peptones, yeast extract and mineral salts is inoculated with a Carnobacterium dirergens S1 culture at 4x10 ⁶ cfu / ml and grown under constant stirring at 32 ° C for 18 hours by adjusting the pH of the medium with calcium carbonate (powdered chalk). After the end of the cultivation, the whole medium with the bacteria is concentrated by membrane microfiltration and added

Fish meal, shredded soybean meal, shredded rapeseed meal, wheat flour, vitamin complex and ascorbic acid are mixed thoroughly in a cubic mixer. The solution prepared in this way is dried in a fluid bed dryer equipped with a granulator. This results in a granular preparation containing 2.3 × ^{10 7} cells / g, which is directly administered to the fry of the Russian sturgeon Acipenser gueldenstaedti. As a result of its use, the survival of the fry was increased and the growth of the fish was accelerated.

In a fifth preferred embodiment, a probiotic strain of Carnobacterium divergens S1 bacteria is grown in a sterile liquid medium. At the end of the logarithmic growth phase, the medium is centrifuged at 6,000 rpm for 15 minutes to obtain a bacterial paste containing 10 ⁹ bacteria / g. This paste is stored in a refrigerator at 4 ° C for three days, after which a cell suspension is prepared and mixed with the fertilized and decontaminated Esox lucius pike roe in such an amount that the mixture with roe contains 3x10 ⁸ CFU / ml. The bacterial paste is mixed with the pike roe also during its incubation at two-day intervals. As a result of this action, it was shown that the amount of live brood obtained with this method in relation to the brood obtained from the eggs not treated with bacteria was significantly higher.

In a sixth embodiment, the probiotic strain of Carnobacterium divergens S1 bacteria is grown in a sterile liquid medium, and after the end of the cultivation, after 24 hours, the cells are centrifuged at 4,000 rpm for 10 minutes, and the resulting cell density is mixed with sand and gravel. After 6 hours, sand and gravel with adsorbed bacteria are placed at the bottom of the rearing tank for two species of clams: Anodonta anatina and Pseudoanodonta complananta. It was found in clinical and breeding tests that this effect increased the survival rate of freshwater mussels.

In a seventh preferred embodiment, the probiotic bacterial strain Carnobacterium divergens S1 is grown in a sterile liquid medium, after the end of the cultivation, after 24 hours, the cells are centrifuged at 4,000 rpm for 10 minutes and the resulting cell density is added directly to water. In clinical and breeding tests, it was found that the sanitary condition of the water in the breeding tank was significantly improved.

Most of the probiotics described in the literature and subject to patent applications are intended for marine fish. Breeding freshwater fish, due to the limited volume of water reservoirs, is much more difficult. It is associated with many sanitary and hygienic problems. High contamination of breeding tanks with pathogenic microorganisms causes significant losses, especially in the fry, which is extremely sensitive, and additionally slows down the growth of the fish. These unfavorable phenomena can be eliminated or limited by the use of probiotic preparations.

The present invention therefore has a profound practical justification. It is fully in line with the strategy of improving the efficiency of fish farming, health and welfare.

The probiotic of the composition according to the invention can be introduced directly or via various carriers into fish feed and into water and meets all the criteria set for bacterial preparations:

- it is capable of immediate implantation in the gastrointestinal tract,

- colonizes the digestive tract by colonizing intestinal villi and the walls of the digestive tract or may be able to grow directly in the digestive content,

- competes with pathogenic bacteria and synthesizes antagonistic substances - bacteriocins,

- maintains the pH in the digestive tract at an optimal level,

- it is resistant to elevated temperature used in the fodder production technology.

The invention is described in detail in the following examples, which, however, do not exhaust all its possible embodiments.

Example d 1

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, in the amount of 10 ⁸ cfu / g, is introduced into the sterile liquid medium placed in the bioreactor, the inoculum of Carnobacterium divergens S1 bacteria in the amount of 2x10 ⁶ cfu / ml was introduced. The bacterial cultivation process was carried out at the temperature of 36 ° C with the pH of 7.0 and maintained at

The mixture was kept constant with NaOH, with gentle agitation of the medium. The cultivation was carried out for 20 hours. Then, bacterial cells were separated by centrifugation at 5,000 rpm for 15 minutes. The cell suspension was introduced into a solution containing mineral-free concentrated culture medium at an osmotic pressure of 1500 mOsm / kg, which was then incubated for 45 minutes with gentle agitation at 28 ° C to increase cell survival during drying. The cells were then centrifuged and incubated in a solution to which was added maltodextrin, milk protein, ascorbic acid and silicon dioxide. Everything was mixed well and dried in a spray dryer. As a result of drying, a probiotic preparation was obtained in a powdered and coated form, containing 10 ⁸ cfu / g. This preparation was introduced into the fodder made by the high-pressure pressing method in such an amount that in 1 gram of the final fodder the number of probiotic bacteria was 10 ⁶ cfu. Probiotic fodder to stimulate the growth of freshwater fish was fed to fry of Cyprinus carpio. It was found that the survival of carps in contaminated water with Aeromonas hydrophila and Vibrio angillarum increased by 80%, and the daily gains were greater by 18%.

P r z k ł a d 2

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, is introduced in the amount of 10 ⁴ cfu / ml to the medium containing sucrose, corn flour hydrolyzate, casein peptone, yeast extract and a set of mineral salts. This medium was previously sterilized in a bioreactor at 121 ° C for 15 min and cooled to 30 ° C.

The process of bacterial cultivation was carried out at the temperature of 30 ° C at pH 6.7, maintained with Ca (OH) ₂ , with gentle agitation of the medium. The cultivation was carried out for 18 hours. Then, the bacterial cells were separated using a membrane microfilter with a porosity of 0.45 µm by cross-filtration. The cell suspension was mixed with cryoprotective agents and dried by lyophilization. As a result of drying, a probiotic preparation was obtained in a powdered form, containing 3x10 ⁹ cfu / g. This preparation was introduced into the feed mixture in such an amount that the number of probiotic bacteria in the finished feed was 7x10 ⁸ cfu / g. The forage was shaped in the process of high-pressure pressing, and after drying, the granulate was covered with a hydrophobic film consisting of a mixture of fish and rapeseed oils in the proportion of 50:50. The fry of Oncorchynhus mykiss rainbow trout were fed with the prophylactic fodder prepared in this way. It was found that trout survival in water contaminated with Aeromonas salmonicide, Edwardsiella tarda and Vibrio angillarum bacteria increased by 60%, and daily fry gains were higher by 15%.

P r z k ł a d 3

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, 10 ⁶ cfu / ml is added to the sterile% MRS medium enriched with enzymatic hydrolyzate of rye flour. This medium was previously sterilized by filtration in a 0.45 µm porosity membrane microfilter and introduced into a steam-sterilized bioreactor. The bacterial cultivation process was carried out at 30 ° C at pH 7.0 with pH correction with NaOH, with gentle mixing of the medium. The cultivation was carried out for 15 hours, after which the bacterial culture together with the fermented medium concentrated on a membrane microfilter reducing the volume by 60% in relation to the initial volume. The resulting bacterial slump contained 7x10 ⁹ cells / ml. Then the cell slurry along with the fermented medium was sprayed onto the dried pelleted feed in a cell slurry to dry feed ratio of 1: 5. After thorough mixing, the feed was dried using the convection method with air at the temperature not exceeding 50 ° C, and then the granules were greased with fish oil in the amount of 2% of the feed weight in the process of pressure lubrication. The analysis showed that the feed prepared in this way contained 8x10 ⁶ cfu / g and had an activity against pathogenic Aeromonas hydrophila bacteria at the level of 150 activity units / g. The finished fodder was fed to the fry of the sterlet Acipenser ruthenus and the Siberian sturgeon

PL 224 877 B1

Acipenser baeri to improve howu results. Studies have shown that as a result of feeding probiotic feed, the survival of the fry has increased and the feed conversion has improved.

Example 4

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, in the amount of 4x10 ⁶ cfu / ml was added to the sterilized medium containing saccharides, amino acids, peptones, yeast extract and mineral salts and cultured with constant stirring at 32 ° C for 18 hours by adjusting the pH of the medium with calcium carbonate (powdered chalk). After the end of the cultivation, the whole medium with the bacteria was concentrated by membrane microfiltration, fishmeal, ground soybean meal, ground rapeseed meal, wheat flour, vitamin kit and ascorbic acid were added, and then the whole was thoroughly mixed in a cubic mixer. The solution prepared in this way was dried in a fluid bed drier equipped with a granulator. This resulted in a granular preparation containing 2.3 × ^{10 7} cells / g, which was directly administered to the fry of the Russian sturgeon Acipenser gueldenstaedti. As a result of its use, the survival of the fry was increased and the growth of the fish was accelerated.

P r z k ł a d 5

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, is grown in a sterile liquid medium. At the end of the logarithmic growth phase, the medium was centrifuged at 6,000 rpm for 15 minutes to obtain a bacterial paste containing 10 ⁹ bacteria / g. The paste was stored in a refrigerator at 4 ° C for three days, and then cell suspension was prepared and stirred with fertilized roe free from tackiness and pike Esox lucius in an amount such that the mixture of fish eggs contained 3x10 ⁸ cfu / ml. The bacterial paste was mixed with the pike roe also during its incubation at two-day intervals to obtain the form of a gel. As a result of this action, it was shown that the amount of live brood obtained by this method in relation to the brood obtained from the eggs not treated with bacteria was significantly higher.

Example 6

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, cultivated in a sterile liquid medium, and after the end of the culture, which took place after 24 hours, the cells were centrifuged at 4,000 rpm for 10 minutes and the resulting density cellular mixture was mixed with sand and gravel. After 6 hours, the sand and gravel with adsorbed bacteria were placed on the bottom of the rearing tank for two species of clams: Anodonta anatina and Pseudoanodonta complananta. It was shown that this effect increased the survival rate of freshwater mussels.

Example 7

The use of a strain of Carnobacterium divergens bacteria and its metabolites for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic bacteria Aeromonas hydrophila, Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, which is based on the fact that a strain of Carnobacterium divergens bacteria deposited in the Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2012p, grown in a sterile liquid medium, and after the end of the culture, which took place after 24 hours, the cells were centrifuged at 4,000 rpm for 10 minutes and the obtained cell density put directly into the water, mixing part of the slurry with rapeseed oil to form an emulsion. The research showed that the sanitary condition of the water in the breeding tank improved significantly.

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Claims (12)

1. The use of the Carnobacterium divergens bacterial strain and its metabolites deposited in the Collection of Industrial Microbial Cultures under the number KKP 2012p for the production of a probiotic for fish, molluscs and freshwater crustaceans to reduce the activity, growth or survival of pathogenic hydrophilic bacteria, Aeromonas hydrophilic bacteria. Aeromonas salmonicida, Vibrio angillarum, Edwardsiella Tarda, wherein the probiotic comprises a Carnobacterium divergens strain and / or its metabolites in an amount of at least 10 ⁶ C. divergens S1 bacterial cells, most preferably greater than 10 ⁸ . 2. The use according to claim 1 The method of claim 1, characterized in that the Carnobacterium divergens bacteria and / or its metabolites contained in the probiotic are in a live or dead form and in an amount such that in 1 gram of dry matter their amount exceeds 100 units of antimicrobial activity of the metabolites of bacteria C. divergens S1. 3. Use according to claim 1 1 or 2, characterized in that the Carnobacterium divergens bacteria and / or its metabolites are placed on a carrier which is sand and / or gravel and / or organic carriers mixed with feed ingredients and / or complete feed mixtures or premixes, and the biomass is fixed by the method drying or freezing. 4. Use according to claim 1 A method according to claim 3, characterized in that the obtained probiotic is used as a prophylactic, therapeutic or growth-stimulating agent for freshwater fish, especially carp fry, rainbow trout, European catfish and sturgeon fish, and molluscs and crustaceans, especially freshwater mussels and crayfish. 5. Use according to claim 1 A method according to any of the preceding claims, characterized in that the probiotic is used to treat eggs. 6. Use according to claim 1 Any one of the preceding claims, characterized in that the probiotic used is in the form of a suspension. Use according to claim 1 Any of the claims 1 or 2 or 3 or 4 or 5, characterized in that the probiotic used is in the form of granules. PL 224 877 B1 8. Use according to claim 1 the probiotic is in the form of a powder. Use according to claim 1 The new probiotic is in an encapsulated form. Use according to claim 1 The new probiotic is in the form of a gel. 11. The use according to claim 1 The new probiotic is in the form of a paste. 12. Use according to p. Another probiotic is in the form of an emulsion. 1 or 2 or 3 or 4 or 5, 1 or 2 or 3 or 4 or 5, 1 or 2 or 3 or 4 or 5, 1 or 2 or 3 or 4 or 5, 1 or 2, or 3, or 4, or 5, characterized by using the variables by using the variables by using the variables by using the variables by using the UPRP Publishing Department Price PLN 4.92 (including 23% VAT)