A method for reducing ammonia emission of aquatic animals

Abstract

The present invention provides a method for reducing ammonia emission of aquatic animals comprising administering to the animals one or more proteolytic enzymes, i.e., proteases. The present invention also provides a feed composition suitable for the above methods and use thereof.

Description

[0001] A METHOD FOR REDUCING AMMONIA EMISSION OF AQUATIC ANIMALS

[0002] Technical field

[0003] The present invention is related to a method for reducing ammonia emission of aquatic animals.

[0004] Background of the invention

[0005] Ammonia emissions in animal breeding industry represents one of several pain points as health or welfare issues affecting animal growth performance and farmers health. Techniques to reduce ammonia emission of animals while meeting nutritional needs of the animals take into consideration combination of nutritional management, reduction of dietary crude protein, addition of essential amino acids and / or addition of feed enzymes.

[0006] Protease enzymes are wildly used in animal feeds because they hydrolyze proteins in the feeds and break them down into more usable peptides. Alternative protein sources to soybean meal (SBM), such as Cottonseed Meal (CSM) and Corn Distillers Dried Grains with Solubles (DDGS) can have lower quality protein levels and reduced digestibility in animals. Supplementing protease helps the animals that lack adequate levels of endogenous enzymes to digest proteins in the diet, which reduces the flow of undigested protein and other anti-nutritionals entering the large intestine.

[0007] WO 95 / 02044 A discloses proteases derived from Aspergillus aculeatus, as well as the use in animal feed thereof. WO 01 / 58276 A discloses acid-stable proteases from the genus Nocardiopsis and their use in animal feed. WO 2019 / 043191 Al discloses an animal feed or animal feed additive comprising proteases which can improve animal performance and the nutritional value of the animal feed.

[0008] Surprisingly, the inventors of the present invention discovered that proteases provide benefit in reducing ammonia emission of aquatic animals, in particular of shrimps.

[0009] Summary of the invention

[0010] Accordingly, the present invention provides a method for reducing ammonia emission of aquatic animals comprising administering to the animal one or more proteolytic enzymes, i.e., proteases.

[0011] The present invention also provides a feed composition, a feed additive and an animal feed comprising one or more proteolytic enzymes, i.e., proteases, for reducing ammonia emission of an animal, and use thereof. Detailed description of the Invention

[0012] In the present invention, the term "aquatic animal" refers to crustaceans including but not limited to shrimps and prawns and fish including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish.

[0013] In the present invention, the term "animal feed" refers to any compound, preparation, or mixture suitable for or intended for intake by an animal and capable of maintaining life and / or promoting production of the animal without any additional substance being consumed except water.

[0014] In the present invention, the term "feed additive" refers to an ingredient or combination of ingredients added to the animal feed, usually used in micro quantities and requires careful handling and mixing. Such ingredient includes but is not limited to vitamins, amino acids, minerals, enzymes, eubiotics, colouring agents, growth improving additives and aroma compounds / flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds etc..

[0015] The present invention provides a method for reducing ammonia emission of an animal comprising administering to the animal one or more proteolytic enzyme(s), i.e., protease(s).

[0016] The present invention also provides use of one or more proteolytic enzyme(s), i.e., protease(s), for reducing ammonia emission of an animal.

[0017] In the present invention, the ammonia emission may be characterized by the amount of ammonia gas emitted from urine, faeces, manure (mixture of urine and feces), excreta and / or litter of animals.

[0018] In the present invention, the reduction is compared to the animals to which any protease is not administered (herein referred to as the control). Preferably, the ammonia emission of animals is reduced by at least 5%, such as by at least 8%, at least 10%, at least 12%, at least 15%, at least 18% or at least 20% compared to the control.

[0019] In the present invention, the proteolytic enzyme or protease catabolizes peptide bonds in proteins breaking them down into fragments of amino acid chains, or peptides. Proteases are classified according to their catalytic mechanism into the following groups: serine proteases (S), cysteine proteases (C), aspartic proteases (A), metalloproteases (M), and unknown, or as yet unclassified, proteases (U) (see Handbook of Proteolytic Enzymes, A. J. Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998)). The protease according to the present invention is a serine protease, preferably an acid stable serine protease, and more preferably a S8 protease, such as those disclosed in WO 2019 / 043191 Al.

[0020] There are no limitations on the origin of the protease according to the invention. Thus, the term protease includes not only natural or wild-type proteases, but also any mutants, variants, fragments etc. thereof exhibiting protease activity, as well as synthetic proteases, such as shuffled proteases, and consensus proteases. Such genetically engineered proteases can be prepared as is generally known in the art, e. g. by site-directed mutagenesis, by PCR (using a PCR fragment containing the desired mutation as one of the primers in the PCR reactions), or by random mutagenesis. The preparation of consensus proteins is described in e. g. EP 0 897 985.

[0021] Preferably, the protease according to the present invention is a microbial protease, the term microbial indicating that the protease is derived from, or originates from a microorganism, or is an analogue, a fragment, a variant, a mutant, or a synthetic protease derived from a microorganism. It may be produced or expressed in the original wild-type microbial strain, in another microbial strain, or in a plant; i.e. the term covers the expression of wild-type, naturally occurring proteases, as well as expression in any host of recombinant, genetically engineered or synthetic proteases.

[0022] Examples of the microorganism are bacteria, e. g. bacteria of the Family: Nocardiopsaceae, e. g. of the Genus: Nocardiopsis, e. g. Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba; bacteria of the family Bacillaceae, e.g. of the genus Bacillus, e.g. Bacillus horneckiae and Bacillus sp.; and bacteria of the families Pianococcaeae and Paenibacillaceae; and mutants or variants thereof.

[0023] Preferred protease according to the present invention is an acid stable serine protease obtained or obtainable from the Genus: Nocardiopsis, such as those derived from Nocardiopsis dassonvillei DSM 43235 (A1918L1), Nocardiopsis prasina DSM 15649 (NN018335L1), Nocardiopsis prasina (previously alba) DSM 14010 (NN18140L1), Nocardiopsis sp. DSM 16424 (NN018704L2), Nocardiopsis alkaliphila DSM 44657 (NN019340L2) and Nocardiopsis lucentensis DSM 44048 (NN019002L2); or the Genus: Bacillus, e.g. Bacillus horneckiae, Bacillus sp TY145, Bacillus sp-13380, Bacillus idriensis, Bacillus sp-62451 and Bacillus oceanisediminis; as well as homologous proteases. Commercially available serine proteases are Ronozyme®ProAct (DSM Nutritional Products AG, Switzerland), ProAct 360™ (DSM Nutritional Products

[0024] Ltd., Switzerland), and AxtraPro (Dupont, USA)

[0025] In the present invention, the protease, besides being acid-stable, may also be thermostable. The term thermostable means for proteases the temperature optimum is at least 50°C, 52°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, or at least 70°C.

[0026] According to the present invention, the protease may be provided in a dosage of between 1,000 units / kg animal feed and 1,000,000 units / kg animal feed, for example in one of the following amounts (dosage ranges): 1,000, 2,000, 4,000, 6,000, 8,000, 10,000, 15,000, 20,000, 30,000, 50,000, 80,000, 100,000, 150,000, 200,000, 250,000, 300,000, 500,000, 600,000, 800,000, 1,000,000 units / kg animal feed. One protease unit (PROT) is the amount of enzyme that releases 1 pmol of p-nitroaniline (pNA) from 1 mM substrate (such as N-succinyl-Ala-Ala-Pro-Phe-pNA) per minute at pH 9.0 and 37°C.

[0027] Protease activity can be measured using any assay, in which a substrate is employed, that includes peptide bonds relevant for the specificity of the protease in question. Examples of protease substrates are casein, and pNA-substrates, such as Suc-AAPF-pNA (available e.g. from Sigma S-7388) and N-succinyl-Ala-Ala-Pro- Phe-pNA (Bachem AG, Switzerland). WO2021 / 180539A1 describes suitable protease assays (see examples 1-4).

[0028] In the present invention, the one or more proteolytic enzyme(s), i.e., protease(s) may be formulated in the form of a feed composition or a feed additive (premix) for administering to aquatic animals.

[0029] Accordingly, the present invention also provides a feed composition or a feed additive comprising one or more proteolytic enzyme(s), i.e., protease(s), as defined above for reducing ammonia emission of an animal.

[0030] In the present invention, the feed composition, the feed additive and / or the components such as the protease contains therein may be formulated as a liquid formulation or a solid formulation, and thus may contains one or more formulating agents.

[0031] The formulating agents may be selected from the group consisting of polyol such as glycerol, sorbitol, ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, dipropylene glycol and polyethylene glycol (PEG); a salt such as organic or inorganic zinc, sodium, potassium, calcium or magnesium salts (for example, magnesium sulfate, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate and zinc sulfate); and starch or a sugar or sugar derivative such as sucrose, dextrin, glucose, lactose and sorbitol; small organic molecules, flour, cellulose and minerals and clay minerals (also known as hydrous aluminum phyllosilicates such as kaolinite or kaolin).

[0032] The feed composition or the feed additive according to the present invention may also comprise one or more emulsifying agents. The emulsifying agents may be selected advantageously from the group consisting of polyglycerol esters of fatty acids such as esterified ricinoleic acid or propylene glycol esters of fatty acids, saccharo-esters or saccharo-glycerides, polyethylene glycol, lecithins, etc..

[0033] Optionally, the feed composition or the feed additive of the present invention may further contain antimicrobial peptides; polyunsaturated fatty acids (PUFAs); reactive oxygen generating species; at least one enzyme, and fat- and water-soluble vitamins, as well as minerals.

[0034] Examples of antimicrobial peptides (AMP's) are CAP18, leucocin A, protegrin-1, thanatin, defensin, lactoferrin, lactoferricin, and ovispirin such as novispirin (Robert Lehrer, 2000), plectasins, and statins.

[0035] Examples of polyunsaturated fatty acids are Ci8-, C20- and C22- polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

[0036] Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

[0037] Examples of enzyme are phytase (EC 3.1.3.8 or 3.1.3.26), galactanase (EC 3.2.1.89), alpha-galactosidase (EC 3.2.1.22), phospholipase A 1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), phospholipase C (EC 3.1.4.3), and / or phospholipase D (EC 3.1.4.4).

[0038] Examples of fat-soluble vitamins include but are not limited to vitamin A, vitamin D3, and vitamin K, e.g. vitamin K3.

[0039] Examples of water-soluble vitamins include but are not limited to vitamin Bi2, biotin and choline, vitamin Bi, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.

[0040] Examples of minerals include but are not limited to calcium, phosphorus, sodium, potassium, magnesium, chlorine, iodine, iron, manganese, copper, molybdenum, cobalt and zinc. Common mineral supplements in feed are: limestone, Bone meal, oyster shell, sodium chloride, dicalcium phosphate, manganese sulphate, potassium iodide, and superphosphate. Sources of minerals include meat scraps, fish meal, milk products, ground limestone (calcium), ground oyster shells (calcium), dicalcium phosphate (calcium, phosphorus), defluorinated rock phosphate (phosphorus, calcium), steamed bone meal (phosphorus, calcium), salt (sodium, chlorine, iodine), manganese sulfate (manganese), manganese oxide (manganese), zinc carbonate (zinc), zinc oxide (zinc).

[0041] In the feed composition or the feed additive of the present invention, the one or more proteolytic enzyme(s), i.e., protease(s), may be contained in a total amount of from 0.5 wt% to 50 wt%, preferably from 1 wt% to 30 wt%, more preferably from 2 wt% to 20 wt%, and the most preferably from 5 wt% to 15 wt%, based on the weight of the feed composition or the feed additive of the present invention.

[0042] As anticipated by a person skilled in the art, the one or more proteolytic enzyme(s), i.e., protease(s), the feed composition or the feed additive according to the present invention may be finally incorporated into an animal feed.

[0043] Accordingly, the present invention further provides an animal feed for aquatic animals which comprises the one or more proteolytic enzyme(s), i.e., protease(s), the feed composition or the feed additive, as defined above, for reducing ammonia emission of an animal.

[0044] In the animal feed according to the present invention, the protease may be provided in a dosage of between 1,000 units / kg animal feed and 1,000,000 units / kg animal feed, for example in one of the following amounts (dosage ranges): 1,000, 2,000, 4,000, 6,000, 8,000, 10,000, 15,000, 20,000, 30,000, 50,000, 80,000, 100,000, 150,000, 200,000, 250,000, 300,000, 500,000, 600,000, 800,000, 1,000,000 units / kg animal feed.

[0045] Preferably, the animal feed according to the present invention is an animal diet based on soy-bean meal, corn and / or wheat.

[0046] As also anticipated by any person skilled in the art, the animal feed according to the present invention may further include any number of components typical for an animal feed, such as proteins, fats and additional additives.

[0047] Examples of suitable types of proteins that can be included in the feed include, but are not limited to, meat scraps (lysine), fish meal (lysine, methionine), poultry by-product meal (tryptophan, lysine), blood meal, liver and glandular meal, feather meal (hydrolyzed), animal tankage, milk products, cottonseed meal, peanut meal, soybean meal, sesame meal, sunflower seed meal.

[0048] Most feed ingredients (maize, barley, safflower, milo, wheat, rice, bran, etc.) contain approximately 2-5% fat and linoleic acid. Sources of fats include animal tallow (beef), lard, corn oil, and other vegetable oils. Any person skilled in the art are familiar with the particular recipes for making the feed for particular types of animals and can be prepared in similar formulations when adding an effective amount of the composition or the animal feed additive according to the present invention.

[0049] The present invention will be further illustrated by the following examples.

[0050] EXAMPLES

[0051] Example 1: Nitrogen excretion in whiteleg shrimp (Penaeus vannamei) fed diets supplemented with graded doses of protease (ProAct 360)

[0052] In an expert, the effect of graded supplemental doses of ProAct 360 (in liquid form) on nitrogen excretion in whiteleg shrimp was assessed.

[0053] The experimental species under testing was whiteleg shrimp (Penaeus vannamei). A stock of post-larvae shrimp (± 30000 individuals) originated from Shrimp Improvement Systems, LLC (Florida, USA). No pathological signs were observed in association to transport. Prior to start of the trial, the shrimp stock was kept in four 500 L tanks supplied with recirculated seawater (salinity 25%o, water-flow 2.3 L / min, temperature 26 ± 1QC, dissolved oxygen kept above 6 mg / L) for approximately 1 month. During this period shrimp were fed a commercial shrimp diet. Shrimp post-larvae were fed continuously (hourly meals) with automatic feeders at approximately 12% biomass / day. Prior to the start of the trials, shrimp were manually sorted to constitute a sub-stock with a homogenous weight range.

[0054] The trial comprised four dietary treatments, all based on a single basal formulation mimicking a commercial shrimp feed (Table 1). The sole variable within the feeds was the supplementation with protease (ProAct 360) at 0, 100, 200 and 300 mg / kg (diets S-CTRL, S-PROIOO, S-PR0200 and S-PR0300, respectively). All diets comprised the inclusion of yttrium oxide (1000 mg / kg feed) as an inert marker for digestibility measurements. Test enzyme was applied post-extrusion by top-coating. Diets were isonitrogenous (crude protein: 34.4%), isolipidic (crude fat: 8%), and isoenergetic (gross energy: 18.3 MJ / kg).

[0055] At the end of the growth performance trial (after 62 days of feeding) and subsequently to all associated samplings, post-prandial daily total ammonia nitrogen (TAN) excretion was measured by a standardized collection of water from the rearing tanks. To this purpose, shrimp from the various replicate tanks were manually sorted to discard outlier individuals and reconstitute sub-groups of 20 shrimp, with an IBW of 14.6 ± 0.3 g. The water volume in the rearing tanks was reduced to 200L. Measurements of ammonia excretion were made under aerated static water conditions, on three non-consecutive days. To reduce the basal nitrogen level in the rearing system, a complete replacement of the rearing water was made prior to measurements. On measurement days, shrimp were fed a fixed ration for 2 hours, corresponding to the minimum feed intake recorded during the growth study (3.4% of biomass). No leftovers of feed were observed after the 2 hours meal. One tank with unfed shrimp was used as a blank. Twenty-four hours after the meal, samples of water from each tank were collected, using a multichannel peristaltic pumps (Minipuls® 3, Gilson, Middleton, Wl, USA) into 2L glass flasks, and immediately shipped on ice for analysis of total ammonia nitrogen (NH4+ and NH3) by an external laboratory (CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, Porto, Portugal). As mentioned before, this procedure was repeated on 3 non-consecutive days.

[0056] Overall data supports a beneficial effect of supplemental protease (ProAct 360) in shrimp, supported by an increase of protein and amino acid digestibility, which resulted in a significant improvement of feed utilization criteria and a lower excretion of TAN into the water (Fig.l).

[0057] Example 2: Nitrogen excretion in Nile tilapia (Oreochromis niloticus) fed diets supplemented with graded doses of protease

[0058] In a further study, effects of graded supplemental doses of a protease enzyme in liquid form (ProAct 360 L) on the growth performance, nutrient digestibility and nitrogen excretion in Nile tilapia (Oreochromis niloticus) was assessed.

[0059] The study comprised three dietary treatments, all based on a single basal formulation mimicking a typical commercial, practical tilapia feed (Table 1). The sole variable within the feeds was the supplementation with protease (ProAct 360 L) at 0, 100, and 200 mg / kg (diets CTRL, PRO100T, and PRO200T, respectively; for an activity of approximately 20600, and 44700 NFP / kg, wherein 1 protease unit (NFP) is defined as the amount of enzyme that releases 1 pmol of p-nitroaniline from 1 mM substrate (N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide) per minute at pH 9.0 and 37 °C, according to the colorimetric method described by European Commission Joint Research Centre for Protease (Subtilisin) LU.B 3.4.21.62 (FAD-2021-0025; CRL / 200088)). All diets comprised the inclusion of yttrium oxide (200 mg / kg feed) as an inert marker for digestibility measurements. Test enzyme was applied post-extrusion by top-coating. Diets were isonitrogenous (crude protein: 27.6%), isolipidic (crude fat: 7.3%), and isoenergetic (gross energy: 18.6 MJ / kg). Table 1: Formulation and composition of experimental diets.

[0060] * Values are means ± standard deviation (n=2).1Poultry meal 65: 65% CP, 12% CF;2Poultry blood meal: 90% CP, 1%CF;3Solvent extracted soybean meal: 43% CP, 2.7% CF;4Solvent extracted rapeseed meal: 34% CP, 3.5% CF;5Solvent extracted sunflower meal: 28% CP, 1.8% CF;6Wheat bran: 14.8% CP, 4.7% CF;7Rice bran full fat: 13.3% CP; 16.3% CF;8Corn meal: 8.6% CP; 4.3% CF.9Vitamins (III or mg / kg diet): DL-alphatocopherol acetate, 600mg; sodium menadione bisulphate, 33mg; retinyl acetate, 26600IU; DL- cholecalciferol, 1900IU; thiamine, 40mg; riboflavin 40mg; niacin 530mg; calcium pantothenate 130mg; pyridoxine 40mg; folic acid 25mg; cyanocobalamin 0.3mg; ascorbic acid 667mg; inositol 666mg; biotin 2mg; betaine lOOOmg. Minerals (g or mg / kg diet): copper sulphate 7 mg; ferric sulphate 25 mg; potassium iodide 3mg; manganese sulphate 17mg; selenium (selenised yeast) 0.13mg; zinc sulphate 80mg; calcium carbonate 1.9g; excipient wheat middling's. Quadruplicate groups of 40 fish, with a mean initial body weight (IBW) of 16.2 ± 1.1 g were fed either one of the experimental diets during 93 days. Fish were grown in fiberglass rectangular tanks (volume: 350 L) supplied with recirculated freshwater (water flow rate: 4.7 L / min). During the growth performance trial, the average water temperature was 27.9 ± 0.4QC (ranging between 27.2 and 28.9QC), dissolved oxygen levels were kept above 6.2 mg / L and pH varied between 6.7 and 7.5. During the trial, fish were subjected to a photoperiod regime of 16 hours light and 8 hours dark.

[0061] Quantification of total ammonia nitrogen (NH4+ and NH3) in the water was performed according to the indophenol blue method proposed by Grasshoff & Johannsen (1972. ICES Journal of Marine Science, 34(3), 516-521) and adapted from Koroleff (1970. ICES Information on Techniques and Methods for Sea Water Analysis Interlab. Rep. No. 3, pp 19-22).

[0062] At the end of the growth performance trial and subsequently to all associated samplings, post-prandial daily total ammonia nitrogen (TAN) and non-ionized ammonia (NH3) excretion were measured by a standardized collection of water from the rearing tanks. To this purpose, fish from the various replicate tanks were manually sorted to discard outlier individuals and reconstitute sub-groups of 25 fish. The water volume in the rearing tanks was adjusted to 330L. Measurements of ammonia excretion were made under aerated static water conditions, on three non-consecutive days. To reduce the basal nitrogen level in the rearing system, a complete replacement of the rearing water was made prior to measurements. On measurement days, fish were fed a fixed ration for 30 minutes, corresponding to the minimum feed intake recorded during the growth study (1.8% of biomass). No feed leftovers were observed after the meal. One tank with unfed fish was used as a blank. Twenty-four hours after the meal, samples of water from each tank were collected, using a multi-channel peristaltic pump (Minipuls® 3, Gilson, Middleton, Wl, USA) into 2L glass flasks, and immediately shipped on ice for analysis of total ammonia nitrogen (NH4+ and NH3). This procedure was repeated on 3 non-consecutive days.

[0063] After 93 days of feeding, overall survival was 99.7% and was not affected by dietary treatments (P>0.05). Final body weight (FBW) varied between 119.9 and 138.9 grams, which in the best performing treatment (PRQ200T) represented an 8.5-fold increase of initial body weight. In comparison to the CTRL treatment (non-supplemented diet), diet supplemented with ProAct 360 L at 200 mg / kg (PRQ200T) led to a significant increase of weight gain criteria such as FBW and SGR (P<0.05). Moreover, and again in comparison to the CTRL treatment, diets supplemented with ProAct 360 L at 100 and 200 mg / kg (PROIOOT and PR0200T) resulted on a significant reduction of FCR and feed intake and a significantly increase of PER (P<0.05). The whole-body composition of fish was not affected by dietary treatments (P<0.05). However, fish fed with the diets supplemented with ProAct 360 L at 100 and 200 mg / kg (PROIOOT and PR0200T) showed significantly higher whole-body protein, fat and energy retention than those fed with the CTRL diet. Diets supplemented with ProAct 360 L at 100 and 200 mg / kg (PROIOOT and PR0200T) showed a significantly higher apparent digestibility coefficients (ADC) of protein, several amino acids and energy than the CTRL diet (P<0.05). Moreover, diet PR0200T showed a significantly higher ADC of protein than diet PROIOOT (P<0.05). Diets supplemented with ProAct 360 L at 100 and 200 mg / kg (PROIOOT and PR0200T) resulted on a significantly reduction of TAN, NH4+ and NH3 excretion than the CTRL treatment (Table 2).

[0064] Table 2: Daily ammonia nitrogen excretion. Values are means ± standard deviation (n=3 measuring days; made on 4 replicate tanks / day). Values within a row with different superscripts, denote a statistical difference (P<0.05).

[0065] Concluding, data supports that ProAct 360 L supplementation at 100 and particularly at 200 mg / kg, is an effective strategy to enhance the growth rate, protein and energy digestibility, whole-body nutrient retention and reduce FCR, and to reduce ammonia nitrogen excretion in Nile tilapia.

[0066] Example 3: Nitrogen excretion in European seabass (Dicentrarchus labrax) fed fed diets supplemented with graded doses of protease

[0067] In an additional study, effects of graded supplemental doses of a protease enzyme in liquid form (ProAct 360 L) on the growth performance, nutrient digestibility and nitrogen excretion in European seabass (Dicentrarchus labrax) was assessed.

[0068] The trial comprised four dietary treatments, all based on a single basal formulation mimicking a common commercial, practical seabass feed. The sole variable within the feeds was the supplementation with protease (ProAct 360 L) at 0, 100, 200 and 300 mg / kg (diets CTRL, PROIOOSB, PR0200SB and PR0300SB, respectively). Diets were isonitrogenous (crude protein: 44.4%), isolipidic (crude fat: 17.4%), and isoenergetic (gross energy: 21.9 MJ / kg).

[0069] The trial comprised four dietary treatments, all based on a single basal formulation mimicking a practical seabass feed. The sole variable among the feeds was the supplementation with protease (ProAct 360 L) at 0, 100, 200 and 300 mg / kg (diets CTRL, PROIOOSB, PR0200SB and PR0300SB, respectively). All diets comprised the inclusion of yttrium oxide (200 mg / kg feed) as an inert marker for digestibility measurements. Test enzyme was applied post-extrusion by top-coating. Diets were isonitrogenous (crude protein: 44.4%), isolipidic (crude fat: 17.4%), and isoenergetic (gross energy: 21.9 MJ / kg).

[0070] Quadruplicate groups of 45 fish, with a mean initial body weight (IBW) of 26.6 ± 1.7 g were fed either one of the four experimental diets during 90 days. Fish were grown in fiberglass circular tanks (volume: 500 L) supplied with recirculated saltwater (water flow rate: 5.5 L / min). During the growth performance trial, the average water temperature was 20.3 ± 0.3 °C (ranging between 19.3 and 20.8 °C), salinity 35.8 ppt, dissolved oxygen levels were kept above 6.3 mg / L and pH varied between 7.0 and 7.3. During the trial, fish were subjected to a photoperiod regime of 14 hours light and 10 hours dark.

[0071] At the end of the growth performance trial and subsequently to all associated samplings, post-prandial daily total ammonia nitrogen (TAN) and non-ionized ammonia (NH3) excretion were measured by a standardized collection of water from the rearing tanks. To this purpose, fish from the various replicate tanks were manually sorted to discard outlier individuals and reconstitute sub-groups of 35 fish. The water volume in the rearing tanks was adjusted to 450 L. Measurements of ammonia excretion were made under aerated static water conditions, on three non-consecutive days. To reduce the basal nitrogen level in the rearing system, a complete replacement of the rearing water was made prior to measurements. On measurement days, fish were fed a fixed ration for 30 minutes, corresponding to the minimum feed intake recorded during the growth study (1.5% of biomass). No feed leftovers were observed after the meal. One tank with unfed fish was used as a blank. Twenty-four hours after the meal, samples of water from each tank were collected, using a multi-channel peristaltic pump (Minipuls® 3, Gilson, Middleton, Wl, USA) into 2 L glass flasks, and immediately shipped on ice for analysis of total ammonia nitrogen (NH4+ and NH3) as described above. This procedure was repeated on 3 non-consecutive days.

[0072] After 90 days of feeding, overall survival was 99.9% and was not affected by dietary treatments (P>0.05). The final body weight (FBW) varied between 88.8 and 100.1 grams, which in the best performing treatment (PR0300SB) represented a 3.8-fold increase of initial body weight. In comparison to the CTRL (non-supplemented diet) and PROIOOSB treatments, diets supplemented with ProAct 360 L at 200 and 300 mg / kg (PR0200SB and PR0300SB) led to a significant increase of weight gain criteria such as FBW and SGR (P<0.05). Moreover, in comparison to the CTRL treatment, diets supplemented with ProAct 360 L at 200 and 300 mg / kg (PR0200SB and PR0300SB) resulted on a significant reduction of FCR (P<0.05) and a significantly increase of PER (P<0.05). Feed intake was not affected by dietary treatments (P<0.05). The whole-body composition of fish was not affected by dietary treatments (P<0.05). Fish fed with the diets supplemented with ProAct 360 L at 200 and 300 mg / kg (PR0200SB and PR0300SB) showed significantly higher whole-body protein, fat and energy retention than those fed with the CTRL diet (P<0.05). Diets supplemented with ProAct 360 L, irrespective of the dose, showed a significantly higher ADC of protein and energy than the CTRL diet (P<0.05). Moreover, dietary ProAct 360 L supplementation, irrespective of the dose, resulted on a significant enhancement on the ADC of Arg, Lys, Thr, Vai, Met, Asx, Ala and Pro (P<0.05). The apparent digestibility of His, Cys, Phe, Tyr and Ser was not significantly affected by dietary treatments (P>0.05). Fish fed diets supplemented with ProAct 360 L at 200 and 300 mg / kg (PR0200SB and PR0300SB) showed a significantly lower excretion of NH3 that those fed the CTRL diet (P<0.05). Additionally, fish fed diets supplemented with ProAct 360 L, irrespective of the dose, showed significantly lower excretion of NH4+ and TAN than those fed the CTRL diet (P<0.05), see Table 3.

[0073] Table 3: Daily ammonia nitrogen excretion. Values are means ± standard deviation (n=3 measuring days; made on 4 replicate tanks / day). Values within a row with different superscripts, denote a statistical difference (P<0.05).

[0074] Concluding, data supports that protease supplementation is an effective strategy to enhance the growth rate, protein, amino acid and energy digestibility, whole-body nutrient retention and reduce FCR, and to reduce ammonia emission in European seabass.

Claims

CLAIMS1. A method for reducing ammonia emission of aquatic animals comprising administering to the animal one or more proteolytic enzyme(s), i.e., protease(s).

2. The method of claim 1, wherein the protease is a serine protease, preferably an acid stable serine protease, and more preferably a S8 protease.

3. The method of claim 1 or 2, wherein the protease is a microbial protease.

4. The method of claim 1 or 2, wherein the protease is derived from a microorganism such as bacteria, e. g. bacteria of the Family: Nocardiopsaceae, e. g. of the Genus: Nocardiopsis, e. g. Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba; bacteria of the family Bacillaceae, e.g. of the genus Bacillus, e.g. Bacillus horneckiae and Bacillus sp.; and bacteria of the families Pianococcaeae and Paenibacillaceae; and mutants or variants thereof.

5. The method of claim 1 or 2, wherein the protease is an acid stable serine protease obtained or obtainable from the Genus: Nocardiopsis, such as those derived from Nocardiopsis dassonvillei DSM 43235 (A1918L1), Nocardiopsis prasina DSM 15649 (NN018335L1), Nocardiopsis prasina (previously alba) DSM 14010 (NN18140L1), Nocardiopsis sp. DSM 16424 (NN018704L2), Nocardiopsis alkaliphila DSM 44657 (NN019340L2) and Nocardiopsis lucentensis DSM 44048 (NN019002L2); or the Genus: Bacillus, e.g. Bacillus horneckiae, Bacillus sp TY145, Bacillus sp-13380, Bacillus idriensis, Bacillus sp- 62451 and Bacillus oceanisediminis; as well as homologous proteases.

6. The method of claim 1 or 2, wherein the protease is acid-stable and thermostable.

7. The method of claim 1 or 2, wherein the protease is provided in a dosage of between 1,000 units / kg animal feed and 1,000,000 units / kg animal feed, for example in one of the following amounts (dosage ranges): 1,000, 2,000, 4,000, 6,000, 8,000, 10,000, 15,000, 20,000, 30,000, 50,000, 80,000, 100,000, 150,000, 200,000, 250,000, 300,000, 500,000, 600,000, 800,000, 1,000,000 units / kg animal feed.

8. The of any of claims 1 to 7, wherein aquatic animal refers to crustaceans including but not limited to shrimps and prawns and fish including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole,spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish.

9. A feed composition, a feed additive or an animal feed comprising one or more proteolytic enzymes, i.e., proteases, for reducing ammonia emission of aquatic animals.

10. The feed composition, a feed additive or an animal feed of claim 9, wherein the protease is contained in a total amount of from 0.5 wt% to 50 wt%, preferably from 1 wt% to 30 wt%, more preferably from 2 wt% to 20 wt%, and the most preferably from 5 wt% to 15 wt%, based on the weight of the feed composition, the feed additive or the animal feed.

11. Use of one or more proteolytic enzymes, i.e., proteases, and a carbohydrase in an animal feed for reducing ammonia emission of aquatic animals.

Images