Use of Aspergillus niger aspergilloglutamic peptidase to improve animal performance
09986751 ยท 2018-06-05
Assignee
Inventors
- Maaike Johanna BRUINS (Kaiseraugst, CH)
- Luppo Edens (Kaiseraugst, CH)
- Helena Maria Nan (Kaiseraugst, CH)
Cpc classification
A61P1/00
HUMAN NECESSITIES
A61K38/488
HUMAN NECESSITIES
International classification
Abstract
This invention relates to the use of the Aspergillus niger aspergilloglutamic peptidase in a feed composition containing cereals, pulses, oilseeds, and/or tubers to improve animal performance, reduce nitrogen excretion and/or reduce intestinal inflammation. Furthermore, the present invention relates to a feed composition or a feed additive comprising the Aspergillus niger aspergilloglutamic peptidase. The present invention relates to the discovery that the Aspergillus niger aspergilloglutamic peptidase is capable of efficiently hydrolyzing alpha-amylase/trypsin inhibitors that are present in wheat, barley and related cereal species, as well as hydrolyzing trypsin inhibitors that are present in pulses, oilseeds, and tubers. Furthermore, the present invention relates to a method of improving animal performance comprising improving feed conversion ratio, and/or improving daily weight gain, and/or reducing intestinal inflammation, and/or reducing nitrogen excretion in an animal fed a feed stuff containing alpha-amylase and/or trypsin inhibitors, comprising orally administering a sufficient amount of the Aspergillus niger aspergilloglutamic peptidase. It also relates to the pretreatment of feedstuff with the Aspergillus niger aspergilloglutamic peptidase.
Claims
1. A method of improving animal performance comprising improving feed conversion ratio, and/or improving daily weight gain, and/or reducing intestinal inflammation, and/or reducing nitrogen excretion in an animal fed cereals, pulses, oilseeds, and/or tubers, wherein the method comprises orally administering to an animal in need of improved performance a sufficient amount of the Aspergillus niger aspergilloglutamic peptidase.
2. The method according to claim 1, wherein the cereals are selected from the group consisting of wheat, barley and maize, and wherein the pulse is soybean.
3. The method according to claim 1, wherein the animal is a monogastric animal.
Description
FIGURE LEGENDS
(1)
(2)
(3)
(4)
(5) The invention is further illustrated by the following examples.
EXAMPLES
Example 1
Aspergillus niger Aspergilloglutamic Peptidase Efficiently Cleaves Wheat Derived Alpha Amylase/Trypsin Inhibitors Under Simulated Stomach Conditions, While Other Acidic Endoproteases are not Efficient
(6) Materials & Methods
(7) Production of Aspergillopepsin I from Aspergillus niger
(8) The gene for aspergillopepsin I from Aspergillus niger (pepA; An14g04710) was over-expressed in an A. niger host using methods such as described in WO 98/46772. WO 98/46772 discloses how to select for transformants on agar plates containing acetamide, and to select targeted multicopy integrants. A. niger transformants containing multiple copies of the expression cassette were selected for further generation of sample material. The transformed A. niger strain was fermented in a modified CSM-fermentation medium, pH 6.2 (40 g/l Maltose, 30 g/l Bacto-soytone, 70 g/l Sodium citrate tribasic dihydrate, 15 g/l (NH.sub.4).sub.2SO.sub.4, 1 g/l NaH.sub.2PO.sub.4*2H.sub.2O, 1 g/l MgSO.sub.4*7H.sub.2O, 1 g/l L-Arg, 0.25 ml/l Clerol Antifoam). The culture broth obtained was filtered, sterile filtered and then concentrated by ultrafiltration. Chromatography was carried out by applying the enzyme to a Q-sepharose XK 26/10 column in 50 mmol/l Na-acetate, pH 5.6, followed by elution with a salt gradient. The presence of the aspergillopepsin I protein in the various fractions was quantified by judging the intensity of coloured protein bands after 4-12% SDS-PAGE (NuPAGE Bis-Tris Gel, Invitrogen).
(9) Enzymatic Assay
(10) Incubations were carried out in 50 mmol/l Na citrate at pH 4.0 for 90 minutes at 37 C. In all relevant incubations pepsin was present in an enzyme protein concentration of 0.2 mg/ml. The proline-specific endoproteinase was tested in a concentration of 0.5 mg enzyme protein/ml, the other acid endoproteinases in a concentration of 0.05 mg enzyme protein/ml. The amylase inhibitor was added last and present in a concentration of 2 mg/ml.
(11) At t=0, 100 microliter of the reaction mixture was transferred into 400 microliter 25% TCA. After 90 minutes of incubation at 37 C., another 100 microliter was transferred into 400 microliter of fresh TCA solution. After 2 hours at 4 C., the samples were centrifuged for 10 minutes at 14,000 rpm. After centrifugation, 65 microliter of phosphate buffer pH 7, 25 microliter of lithium dodecyl sulfate (LiDS) and 10 microliter of sample reducing agent were added. The samples were stored at 4 C. overnight and then prepared for SDS-PAGE following the Invitrogen protocol (Invitrogen, www.lifetechnologies.com)
(12) Determination of A. niger Aspergilloglutamic Peptidase Activity (HPU)
(13) 20.0 g haemoglobin from bovine blood (Sigma product H2625) was suspended in approximately 700 mL water by stirring for 10 minutes at room temperature. After the addition of 3.73 g potassium chloride (KCl) the pH was adjusted to 1.75 with 0.5 mol/L hydrochloric acid. The volume of the haemoglobin suspension was adjusted to 1 L with water. The pH was checked again and adjusted to pH 1.75.
(14) Enzyme solutions were prepared by dissolving purified aspergilloglutamic peptidase produced as disclosed above in a KCl/HCl buffer containing 3.73 g/I KCl adjusted to pH 1.75 with 2.0 mol/L HCl. To test aspergilloglutamic peptidase activity, 5 ml of the haemoglobin solution was heated at 40 C. and subsequently 1 mL enzyme solution with an activity between 5 and 25 Histidine Protease Units (HPU/mL) was added to start the reaction. After 30 minutes the reaction was stopped by adding 5 mL trichloro acetic acid solution (140 g/L) to precipitate larger peptide fragments. A blank measurement was done by adding 1.0 mL enzyme sample to a mixture of 5 mL haemoglobin solution and 5 mL trichloro acetic acid solution. The tubes were incubated at 40 C. for 30 minutes to complete the precipitation. After centrifugation, the optical density of the clear supernatant containing small peptides was measured at 275 nm. The result was compared to an L-tyrosine solution of 1 g/mL.
(15) One HPU is the amount of enzyme that hydrolyzes an amount of haemoglobin per minute, giving a solution with an optical density at 275 nm equal to the optical density of a solution containing 1 g L-tyrosine per mL in 0.1 mol/L HCl solution. Conditions of the test are: pH 1.75, temperature 40 C., haemoglobin concentration during incubation 16.7 g/L.
Activity (HPU/mL)=(OD.sub.sampleOD.sub.blank/S)11/30
(16) Where:
(17) OD.sub.sample: Optical density of the sample filtrate (275 nm)
(18) OD.sub.blank: Optical density of the sample blank filtrate (275 nm)
(19) S: OD of a L-tyrosine standard solution of 1.1 g/mL (mL/g)
(20) 30: incubation time (minutes)
(21) 11: total volume reaction mixture (mL)
(22) LC-MS/MS Analysis
(23) In-vitro Digestion
(24) The sample was dissolved to 1 mg/ml in MilliQ water. The solution was 10 diluted in 100 mM NH.sub.4HCO.sub.3 (pH7.8). The sample was reduced by addition of DTT, 5 mM, 30 minute incubation at room temperature and alkylated by addition of iodoacetamide (IAA), 5.5 mM, 30 minute incubation at room temperature in the dark. Digestion with trypsin was performed at 37 C. overnight.
(25) In Gel Digestion
(26) Gel bands were cut out of the gel using the ExQuest spot cutter (Biorad, Hercules, Calif., USA) and transferred into a lo-protein bind MTP (Eppendorf, Hamburg Germany). The gel pieces were washed by adding 75 l 50 mM NH.sub.4HCO.sub.3 to swell and 75 l Acetronitrile to shrink, total 3 washes. The washed gel pieces were digested with trypsin digestion was performed by incubation at 37 C. overnight. The samples were sonicated for 1 minute and the supernatant was collected into an injection-vial.
(27) LC-MS/MS Analysis
(28) The samples were acidified to 1% formic acid and analyzed on the Accela-LTQ-Velos (Thermo Scientific, San Diego, Calif. USA). The chromatographic separation was achieved with a 2.1100 mm 1.8 micrometer particle size, 80 pore size, C-18 Eclipse XDB Zorbax column (Agilent Santa Clara, Calif. USA), using a gradient elution with (A) LC-MS grade water containing 0.1% formic acid B) LC-MS grade acetonitrile containing 0.1% formic acid solution (Biosolve BV, the Netherlands) as mobile phases. The gradient was from 5 to 40% B in 83 minutes. The flow rate was kept at 0.4 ml/min, using an injection volume of 25 l and the column temperature was set to 50 C. MS data acquisition was performed using a top 10 data-dependent acquisition with mass range 400-2000 m/z, using Dynamic exclusion and including charge states 2 and 3 only. MS/MS experiments were performed with an isolation width set at 3.0, and the normalized collision energy was set to 35. Database searches were performed using the Sorcerer 2 (Sorcerer-SEQUEST) search engine and the Trans Proteome Pipeline (TPP), using trypsin as preferred enzyme. Only proteins identified with a confidence >90% were considered. The data was searched against the Swissprot database.
(29) Results
(30) In the present Example we demonstrate (See
(31) The results (cf.
(32) To confirm the nature of the different proteins present in each one of these bands, samples of gel bands were cut out, extracted and the proteins present were identified using LC-MS/MS analysis as described in the Materials & Methods above.
(33) In this case the 10 mg/ml of the Sigma alpha amylase inhibitor solution was diluted 10 times with water. Then 65 microliter of this solution was mixed with 25 microliter of LiDS sample buffer and 10 microliter of the sample reducing agent, heated for 10 minutes at 70 C., after which the proteins were separated by SDS-PAGE according to the Invitrogen protocol. Then the gel was fixed for 1 hour with 50% methanol/7% acetic acid, rinsed twice with demineralized water and stained with Sypro Ruby overnight. Gel samples were obtained of the three, presumably alpha amylase inhibitor, bands as illustrated in
(34) This data demonstrates that the Aspergillus niger aspergilloglutamic peptidase is surprisingly the most efficacious in degrading wheat derived alpha amylase inhibitors under stomach conditions and most notably, wheat alpha amylase inhibitors: CM 2, CM 3, CM 16, and 0.19.
Example 2
Animal Feed Additive
(35) An animal feed additive is prepared by adding 100,000 HPU of Aspergillus niger aspergilloglutamic peptidase to the following premix (per kilo of premix):
(36) TABLE-US-00001 1100000 IE Vitamin A 50004 mg Cholin chloride 300000 IE Vitamin D3 6000 mg Fe 4000 IE Vitamin E 3000 mg Cu 250 mg Vitamin B1 5400 mg Zn 800 mg Vitamin B2 8000 mg Mn 1200 mg Ca-D-Panthothenate 124 mg I 500 mg Vitamin B6 60 mg Co 2.5 mg Vitamin B12 29.7 mg Se 5000 mg Niacin 9000 mg Lasalocid 10000 mg Vitamin C Sodium (Avatec) 300 mg Vitamin K3 17.3% Ca 15 mg Biotin 0.8% Mg 150 mg Folic acid 11.7% Na
Example 3
Animal Feed
(37) A broiler grower diet having the following composition (%, w/w) is prepared by mixing the ingredients. Wheat, rye and SBM 48 are available from Moulin Moderne Hirsinque, Hirsingue, France. After mixing, the feed is pelleted at a desired temperature, e.g. about 70 C. (325 mm).
(38) TABLE-US-00002 Wheat 46.00 Rye 15.00 Soy Bean Meal (SBM 48) 30.73 Soybean oil 4.90 DL-Methionine 0.04 DCP (Di-Calcium Phosphate) 1.65 Limestone 0.43 Salt 0.15 TiO2 0.10 Animal feed additive (above) 1.00
(39) The resulting animal feed comprises 1000 HPU Aspergillus niger aspergilloglutamic peptidase per kg.
Example 4
Piglet Feed
(40) A piglet feed containing Aspergillus niger aspergilloglutamic peptidase can be prepared by mixing the following ingredients together using a conventional mixing apparatus at room temperature.
(41) TABLE-US-00003 Ingredient Amount (Weight %) Wheat 32.6 Maize 18.7 Rice 5.0 Wheat bran 9.0 Soybean meal 23.0 Soy oil 2.0 Wheat starch 4.5 Minerals* 2.9 Synthetic amino acids premix** 0.8 Vitamins and trace elements premix*** 1.0 Aspergillus niger aspergilloglutamic 0.5 peptidase premix (10% in wheat starch) In principle the Aspergillus niger aspergilloglutamic peptidase premix may contain 1-20% of the Aspergillus niger aspergilloglutamic peptidase. *Sea salt, dicalcium phosphate and calcium carbonate; **Lysine, methionine and threonine; ***Vitamins A, E, D3, K3, B1, B2, B6, B12, C, biotin, folic acid, niacin, pantothenic acid, choline chloride, copper sulphate, iron sulphate, manganese oxide, zinc oxide, cobalt carbonate, calcium iodide and sodium selenite.
Example 5
Growing Pig Feed
(42) A growing pig feed containing Aspergillus niger aspergilloglutamic peptidase can be prepared by mixing the following ingredients together using a conventional mixing apparatus at room temperature.
(43) TABLE-US-00004 Ingredient Amount (Weight %) Soybean meal 18.0 Maize 52.3 Barley 13.0 Oat meal 6.0 Wheat bran 5.2 Soy oil 2.0 Minerals* 1.5 Synthetic amino acids premix** 0.5 Vitamins and trace elements premix*** 1.0 Aspergillus niger aspergilloglutamic peptidase 0.5 premix (10% in wheat starch)
Example 6
Broiler Chicken Starter Feed
(44) A broiler chicken feed (starter) containing Aspergillus niger aspergilloglutamic peptidase can be prepared by mixing the following ingredients together using a conventional mixing apparatus at room temperature.
(45) TABLE-US-00005 Ingredient Amount (Weight %) Soybean meal 34.50 Maize 20.00 Wheat 37.80 Soy oil 3.13 Minerals* 2.90 Synthetic amino acids premix** 0.17 Vitamins and trace elements premix*** 1.00 Aspergillus niger aspergilloglutamic 0.50 peptidase premix (10% in wheat starch)
Example 7
Broiler Chicken Grower Feed
(46) A broiler chicken food (grower) containing Aspergillus niger aspergilloglutamic peptidase can be prepared by mixing the following ingredients together using a conventional mixing apparatus at room temperature.
(47) TABLE-US-00006 Ingredients Amount (Weight %) Soybean meal 31.2 Maize 20.0 Wheat 41.3 Soy oil 3.4 Minerals* 2.5 Synthetic amino acids premix** 0.1 Vitamins and trace elements premix*** 1.0 Aspergillus niger aspergilloglutamic peptidase 0.5 premix (10% in wheat starch)
Example 8
Aspergillus niger Aspergilloglutamic Peptidase Efficiently Cleaves Soybean Derived Trypsin-Chymotrypsin Inhibitors Under Simulated Stomach Conditions, While Other Acidic Endoproteases are not Efficient
(48) Materials & Methods
(49) Enzymatic Assay
(50) Incubations were carried out in 50 mmol/l Na citrate at pH 4.0 for 60 minutes at 37 C. In all relevant incubations pepsin was present in an enzyme protein concentration of 0.2 mg/ml. All peptidases were tested in a concentration of 0.5 mg enzyme protein/ml. The trypsin-chymotrypsin inhibitors inhibitor (purchased from Sigma T9777) was added last and present in a concentration of 2 mg/ml.
(51) At t=0, 100 microliter of the reaction mixture was transferred into 400 microliter 25% TCA. After 60 minutes of incubation at 37 C., another 100 microliter was transferred into 400 microliter of fresh TCA solution. After 18 hours at 4 C., the samples were centrifuged for 30 minutes at 14,000 rpm. After centrifugation, 65 microliter of phosphate buffer pH 7, 25 microliter of lithium dodecyl sulfate (LiDS) and 10 microliter of sample reducing agent were added and prepared for SDS-PAGE following the Invitrogen protocol (Invitrogen, www.lifetechnologies.com)
(52) Results
(53) In the present Example we demonstrate that, under simulated gastric conditions, only Aspergillus niger aspergilloglutamic peptidase among a number of acidic endoproteinases is capable to efficiently degrade a purified preparation incorporating Trypsin-Chymotrypsin from soybean, Sigma). In the experiment the efficacies of the following enzymes were compared in the presence of pepsin (control): pepsin (porcine gastric mucosa, Sigma), proline-specific endoproteinase from Aspergillus niger (MaxiPro PSP, DSM Food Specialities, Delft, The Netherlands) Aspergillus niger aspergilloglutamic peptidase also called aspergillopepsin II (MaxiPro HSP,DSM Food Specialities, Delft, The Netherlands), aspergillopepsin I (see Materials & Methods), Multifect PR 15 L (aspergillopepsin I-like protease from Trichoderma reesei; http//biosciences.dupont.com).
(54) The results (cf.
Example 9
Aspergillus niger Aspergilloglutamic Peptidase Cleaves Alpha Amylase/Trypsin Inhibitors in a Dose Dependent Manner
(55) In the present Example we determine the quantity of A. niger aspergilloglutamic peptidase enzyme protein required to hydrolyze under simulated stomach conditions the alpha amylase/protease inhibitors present in 1 gram of wheat gluten. To that end gluten from wheat (Sigma) was solubilized in 50 mmol/l citric acid pH 4.0 in a concentration of 9.35 mg/ml. To this thoroughly stirred mixture pepsin enzyme protein was added to reach an end concentration of 0.2 mg/ml and then six one ml samples were taken. To these six samples increasing quantities of pure A. niger aspergilloglutamic peptidase enzyme were added. To sample 1: no AGP was added, to sample 2: 0.09 mg, to sample 3: 0.19 mg, to sample 4: 0.28 mg, to sample 5: 0.37 mg and to the last sample: 0.47 mg. The different samples were then incubated for 60 minutes at 37 degrees Celcius and from each sample aliquots for SDS-PAGE analysis were taken at t=0 minutes and t=60 minutes. SDS-PAGE analysis was carried out according to the Invitrogen protocol.
(56) The results (cf.