Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/14—Pretreatment of feeding-stuffs with enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
  • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/174—Vitamins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/22—Compounds of alkali metals
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/24—Compounds of alkaline earth metals, e.g. magnesium
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/26—Compounds containing phosphorus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/28—Silicates, e.g. perlites, zeolites or bentonites
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/20—Inorganic substances, e.g. oligoelements
  • A23K20/30—Oligoelements
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
  • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/002—Protozoa antigens
  • A61K39/012—Coccidia antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21014—Microbial serine proteases (3.4.21.14)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
  • A61K2039/543—Mucosal route intranasal
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)

Definitions

• the present invention relates to the use of acid-stable proteases in animal feed in combination with anti-coccidial agents, in particular to the use of such proteases for increasing the performance of vaccinated animals.
• proteases in animal feed is known from the following documents:
• W095/28850 discloses an animal feed additive comprising a phytase and a proteolytic enzyme.
• Various proteolytic enzymes are specified at p. 7.
• W096/05739 discloses an enzyme feed additive comprising xylanase and a protease. Suitable proteases are listed at p.25.W095/02044 discloses proteases derived from Aspergillus aculeatus, as well as the use in animal feed thereof.
• US 3966971 discloses a process of obtaining protein from a vegetable protein source by treatment with an acid phytase and optionally a proteolytic enzyme. Suitable proteases are specified in column 2.
• Coccidia is a generic name given to single cell protozoan organisms that are intestinal parasites that infect both vertebrates and invertebrates. The organisms cause coccidiosis, and usually settle in the small intestine, such as the colon. Infection with coccidia for farm animals can not only seriously reduce growth, but it can be lifethreatening. Symptoms from coccidial infection include loss of epithelial cells, the denuding of gut mucosa, and diarrhoea (often with a concomitant loss of blood). For some farm animals, such as poultry, coccidial infection can be fatal, if not seriously damaging to the animal's health.
• Poultry are particularly vulnerable for coccidiosis because of several reasons: (1 ) The parasitic cycle of 6 to 8 days hits them at a critical stage between weeks 2 and week 4, when maximum growth is usually expressed. Since the parasites virtually destroy the whole intestinal epithelium, the absorption of nutrients is dramatically reduced, which results in marked growth depression. Until slaughter at 5 or 6 weeks, there is not enough time to recover. (2) There are 7 species of Eimeria which can infect poultry, more than in any other animal category, and at least 4 of them are regularly seen in commercial operations. Thus, when one infectious cycle is concluded already another one can be at an early stage so that coccidiosis becomes chronic. (3) In poultry the most pathogenic species (Eimeria tenella, E.
• necatrix necatrix
• necatrix necatrix
• coccidiosis could easily ruin a poultry farmer.
• the intensive husbandry of poultry (100,000 chicks or more in one house) on deep litter facilitates access of poultry to the infectious stages of coccidia in the faeces via coprophagy and thus supports a fast spreading of the disease through a whole poultry flock. If the sanitary conditions are not rigorous, the disease will also transfer to other poultry houses on the same farm and stay on site for years.
• Coccidiostats that have been approved by the EEC for use with poultry (chickens, turkeys, broilers and laying hens) include sulphonimides, amprolium, decoquinate, and ionophores.
• cocci-vaccination causes decrease of the performance of broiler chicken.
• cocci-vaccination causes reduction in feed intake and feed efficiency.
• 20% of all broiler produced in US are vaccinated against coccidiocis. It is expected that the use of cocci-vacination will increase to 50% by 2020 in the broiler production in US. Therefore the use of cocci-vaccination has an enormous economic impact for the broiler producers.
• proteases are classified on the basis of 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
• proteases for use according to the invention are acid stable proteases.
• Preferred proteases according to the invention are acid stable serine proteases.
• serine protease refers to serine peptidases and their clans as defined in the above
• Serine proteases may be defined as peptidases in which the catalytic mechanism depends upon the hydroxyl group of a serine residue acting as the nucleophile that attacks the peptide bond.
• Examples of serine proteases for use according to the invention are proteases of Clan SA, e. g. Family S2 (Streptogrisin), e. g. Sub-family S2A (alpha-lytic protease), as defined in the above Handbook.
• 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.
• Assay-pH and assay-temperature are likewise to be adapted to the protease in question. Examples of assay-pH-values are pH 5, 6, 7, 8, 9, 10, or 1 1 . Examples of assay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65 or 70 °C.
• protease substrates examples include casein, and pNA-substrates, such as Suc-AAPF- NA (available e. g. from Sigma S7388).
• the capital letters in this pNA-substrate refers to the one-letter amino acid code.
• Protazyme AK azurine-dyed crosslinked casein prepared as tablets by Megazyme T-PRAK.
• the pNA-substrate is preferred, whereas for temperature activity studies, the Protazyme AK substrate is preferred.
• 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.
• acid-stable proteases for use according to the invention are a) the proteases derived from Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba ; b) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95% amino acid identity to any of the proteases of (i) ; c) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95% identity to any of SEQ ID NO : 1 , and/or SEQ ID NO : 2.
• any computer program known in the art can be used. Examples of such computer programs are the Clustal V algorithm (Higgins, D. G., and Sharp, P. M. (1989), Gene (Amsterdam), 73, 237-244 ; and the GAP program provided in the GCG version 8 program package (Program Manual for the Wisconsin Package, Version 8, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453.
• the protease for use according to the 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. Examples of microorganisms are bacteria, e. g.
• bacteria of the phylum Actinobacteria phy. nov. e. g. of class I : Actinobacteria, e. g. of the Subclass V: Actinobacteridae, e. g. of the Order I : Actinomycetales, e. g. of the Suborder XII : Streptosporangineae, e. g. of the Family II: Nocardiopsaceae, e. g. of the Genus I : Nocardiopsis, e. g.
• Nocardiopsis sp. NRRL 18262, and Nocardiopsis alba e.g. of the species Bacillus or mutants or variants thereof exhibiting protease activity.
• This taxonomy is on the basis of Berge's Manual of Systematic Bacteriology, 2nd edition, 2000, Springer (preprint:
• microorganisms are fungi, such as yeast or filamentous fungi.
• the protease can be fed to the animal before, after, or simultaneously with the diet of the cocci-vaccinated bird.
• the latter is preferred.
• Example 1 herein (substrate : Suc-AAPF-pNA, pH 9. 0, 25 °C).
• the protease activity is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 97% of the reference activity.
• Residual activity of aliquot II is measured (pH 9.0 and 25 °C);
• the step b) buffer pH-value may be 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.1 , 3.2, 3.3, or 3.4.
• the residual protease activity as compared to the reference is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or at least 97%.
• pH values of 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5 can be applied for the step d) buffer.
• the term pure protease refers to a sample with a A 2 8o A 2 6o ratio above or equal to 1 .70.
• the protease for use according to the invention besides being acid-stable, is also thermostable.
• thermostable means one or more of the following : That 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.
• the protease should of course be applied in an effective amount, i. e. in an amount adequate for improving the feed conversion ratio in birds vaccinated with an anti- coccidial vaccine.
• the intended dosage of the protease is 0.01 -200 mg protease enzyme protein per kg final feed.
• feed conversion ratio is determined on the basis of a growth trial comprising a first treatment in which the composition according to the invention is added to the animal feed in a suitable concentration per kg feed, and a second treatment (control) with no addition of the composition to the animal feed.
• an improved FCR is lower than the control FCR.
• the FCR is improved (i.e. reduced) as compared to the control by at least 1 .0% or 5%.
• the protease need not be that pure; it may e. g. include other enzymes, even other acid stable proteases, in which case it could be termed am enzyme or protease preparation. Nevertheless, a well-defined enzyme / protease preparation is advantageous. For instance, it is much easier to dose correctly to the feed a protease that is essentially free from interfering or contaminating other proteases. The term dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimising dosage based upon the desired effect.
• the protease is used in form of a feed additive.
• the incorporation of the composition of feed additives as exemplified herein above to poultry feeds is in practice carried out using a concentrate or a premix.
• a premix designates a preferably uniform mixture of one or more micro-ingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix.
• a premix according to the invention can be added to feed ingredients or to the drinking water as solids (for example as water soluble powder) or liquids.
• animal feed additives of the invention contain at least one fat-soluble vitamin, and/or at least one water soluble vitamin, and/or at least one trace mineral, and/or at least one macro mineral.
• feed-additive ingredients are coloring agents, e.g.
• carotenoids such as beta-carotene, astaxanthin, canthaxanthin, apoester and lutein; aroma compounds; stabilisers; antimicrobial peptides; polyunsaturated fatty acids (PUFAs); reactive oxygen generating species; and/or at least one enzyme selected from amongst phytase (EC 3.1 .3.8 or 3.1 .3.26); xylanase (EC 3.2.1 .8); galactanase (EC 3.2.1 .89); alpha-galactosidase (EC 3.2.1 .22); protease (EC 3.4., phospholipase A1 (EC
• phospholipase C (EC 3.1 .4.3); phospholipase D (EC 3.1 .4.4); amylase such as, for example, alpha-amylase (EC 3.2.1 .1 ); and/or beta-glucanase (EC 3.2.1 .4 or EC 3.2.1 .6).
• amylase such as, for example, alpha-amylase (EC 3.2.1 .1 ); and/or beta-glucanase (EC 3.2.1 .4 or EC 3.2.1 .6).
• antimicrobial peptides are CAP18, Leucocin A, Protegrin-1 ,
• Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins.
• polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
• reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.
• chemicals such as perborate, persulphate, or percarbonate
• enzymes such as an oxidase, an oxygenase or a syntethase.
• fat- and water-soluble vitamins, as well as trace minerals form part of a so- called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.
• fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.
• water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D- panthothenate.
• trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.
• macro minerals are calcium, phosphorus and sodium.
• a premix can contain, for example, per ton of poultry feed, 50 to 200 g of a propylene glycol solution of the mixture of the active compounds, 20 to 1000 g of an emulsifying agent, 50 to 900 g of cereals and by-products, 20 to 100 g of a proteinic support (milk powder, casein, etc) and 50 to 300 g of a mineral component (expanded silica, feed quality lime, bi-calcium phosphate, etc).
• a feed additive or premix as described above is finally added the animal feed composition. It is prepared and added such that the amount of the protease
• An animal feed composition according to the invention has a crude protein content of 50-800 g/kg, and furthermore comprises at least one protease as claimed herein.
• the animal feed composition of the invention has a content of metabolisable energy of 10 - 30 MJ/kg; and/or a content of calcium of 0.1 - 200 g/kg; and/or a content of available phosphorus of 0.1 - 200 g/kg; and/or a content of methionine of 0.1 - 100 g/kg; and/or a content of methionine plus cysteine of 0.1 - 150 g/kg; and/or a content of lysine of 0.5-50 g/kg.
• the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 as disclosed in Table B of WO 01 /58276.
• the protease is purified from the feed composition, and the specific activity of the purified protease is determined using a relevant assay (see under protease activity, substrates, and assays).
• the protease activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg protease protein per kg feed is calculated.
• the protease of Nocardiopsis sp. NRRL 18262 according to the invention can be prepared using conventional methods, as generally described in WO01 /58276.
• a feed additive comprising the protease of Nocardiopsis sp. NRRL 18262 is also
• Nutritional Products can easily be prepared by a skilled person using processes and methods well-known in the prior art.
• Example 1 pH-stability assay Suc-AAPF-pNA (Sigma S-7388) was used for obtaining pH stability profiles.
• Assay buffer 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCI 2 , 150mM KCI, 0.01 % Triton ® X-100 adjusted to pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6 0, 7.0, 8.0, 9.0, 10.0 or 1 1 .0 with HCI or NaOH.
• protease samples were diluted in 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCI 2 , 150 mM KCI, 0.01 % Triton ® X- 100, pH 9.0, bringing the pH of all samples to pH 9.0.
• the temperature was 25 °C.
• 300 ⁇ diluted protease sample was mixed with 1 .5ml of the pH 9.0 assay buffer and the activity reaction was started by adding 1 .5ml pNA substrate (50mg dissolved in 1 .0 ml DMSO and further diluted 45x with 0.01 % Triton ® X-100) and, after mixing, the increase in A 40 5 was monitored by a spectrophotometer as a measurement of the (residual) protease activity.
• 1 .5ml pNA substrate 50mg dissolved in 1 .0 ml DMSO and further diluted 45x with 0.01 % Triton ® X-100
• the 37 C incubation was performed at the different pH-values and the activity measurements were plotted as residual activities against pH.
• the protease samples were diluted prior to the activity measurement in order to ensure that all activity
• Example 2 Feeding of vaccinated birds with acid stable proteases
• a floor pen trial was conducted using day old male birds Ross 708. A total of 27 pens were used in the trial and 10 birds were placed per pen on day 1 of age. The pens were randomly allocated across 3 treatments with 9 replicates/ treatment. The birds were fed nutritionally-adequate basal diets based on corn/soybean meal and adjusted to the bird age by feeding a starter diet from day 1 to 21 and a grower diet from day 22 to 42.
• Cocci Vac® B on day 1 of age + dietary Monteban® (coccidiostat) 80 ppm. All birds received Cocci Vac® B on day 1 of age administered nasally in the hatchery.
• Cocci-Vac induces immunity to coccidiosis by causing a low level of the disease.
• the enzyme blend comprised:
• RONOZYME®ProAct acid stable protease
• RONOZYME WX + 50 ppm RONOZYME A + 50 ppm ROXAZYME G2 enzyme mixture containing xylanase, amylase, cellulose, beta-glucanase and others
• Feed Conversion Ration was calculated by dividing the feed intake by the weight gain for each treatment.
• table 1 lists body weight at day 42 and table 2 lists the FCR at day 42.
• Example 3 Feeding of vaccinated birds with acid stable proteases
• a floor pen trial was conducted using day old male birds Cobb X Cobb chicks. A total of 2,160 were allocated to the study. The experiment consisted of 48 pens of 45 male broiler chickens. Treatments were replicated in eight (8) blocks with six (6) treatments randomized in each. The birds were fed nutritionally-adequate basal diets based on corn/soybean meal and adjusted to the bird age by feeding a starter diet from day 1 to 16 and a grower diet from day 17 to 32 and a finisher diet from day 33 to 42. The 6 treatment were as follow:
• NC Cocci Vac® B
• NC Cocci Vac® B
• NC Cocci Vac® B
• NC Cocci Vac® B
• RONOZYME enzyme products supplied by DSM Nutritional Products
• Ronozyme® AX 80% Ronozyme®WX + 20% Ronozyme® A
• Ronozyme® ProAct pure protease product.
• Salinomcyin is the most used coccidiostate that suppress development of the very common decease coccidiosis.
• Cocci Vac® B induces immunity to coccidiosis by causing a low level of the disease.
• CVB ProAct 200 ppm 187.85 be 1 .760 b 2.412 b
• CVB ProAct 200 ppm+ Rono WX 200 ppm 181 .27 d 1 .747 be 2.393 b

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