CN110093333A - A kind of application in compound protease and its low-protein diet - Google Patents
A kind of application in compound protease and its low-protein diet Download PDFInfo
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- CN110093333A CN110093333A CN201910338899.0A CN201910338899A CN110093333A CN 110093333 A CN110093333 A CN 110093333A CN 201910338899 A CN201910338899 A CN 201910338899A CN 110093333 A CN110093333 A CN 110093333A
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- 108091005804 Peptidases Proteins 0.000 title claims abstract description 75
- 239000004365 Protease Substances 0.000 title claims abstract description 71
- 150000001875 compounds Chemical class 0.000 title claims abstract description 54
- 235000020905 low-protein-diet Nutrition 0.000 title claims abstract description 25
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract 21
- 235000019419 proteases Nutrition 0.000 claims abstract description 69
- 230000000694 effects Effects 0.000 claims abstract description 24
- 108091005508 Acid proteases Proteins 0.000 claims abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims abstract description 15
- 239000003513 alkali Substances 0.000 claims abstract description 12
- 241000287828 Gallus gallus Species 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 21
- 230000008569 process Effects 0.000 claims description 10
- 238000009395 breeding Methods 0.000 claims description 5
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- 238000000855 fermentation Methods 0.000 claims description 5
- 230000004151 fermentation Effects 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 241000228245 Aspergillus niger Species 0.000 claims description 3
- 240000006439 Aspergillus oryzae Species 0.000 claims description 2
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims description 2
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 229920002261 Corn starch Polymers 0.000 claims description 2
- 241000235342 Saccharomycetes Species 0.000 claims description 2
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- 239000000654 additive Substances 0.000 claims description 2
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- 238000002360 preparation method Methods 0.000 claims description 2
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- 238000004519 manufacturing process Methods 0.000 abstract description 21
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- 239000000203 mixture Substances 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
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- 239000004382 Amylase Substances 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 4
- 108010065511 Amylases Proteins 0.000 description 4
- 108090000317 Chymotrypsin Proteins 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- 229960002376 chymotrypsin Drugs 0.000 description 4
- 238000013401 experimental design Methods 0.000 description 4
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 3
- 102000002322 Egg Proteins Human genes 0.000 description 3
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 235000019621 digestibility Nutrition 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 235000019800 disodium phosphate Nutrition 0.000 description 3
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- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- BDOYKFSQFYNPKF-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;sodium Chemical compound [Na].[Na].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O BDOYKFSQFYNPKF-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 101710089042 Demethyl-4-deoxygadusol synthase Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 108010010256 Dietary Proteins Proteins 0.000 description 1
- 102000015781 Dietary Proteins Human genes 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 101100100125 Mus musculus Traip gene Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
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- 235000019779 Rapeseed Meal Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
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- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- YUWBVKYVJWNVLE-UHFFFAOYSA-N [N].[P] Chemical compound [N].[P] YUWBVKYVJWNVLE-UHFFFAOYSA-N 0.000 description 1
- 102000004139 alpha-Amylases Human genes 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 229940024171 alpha-amylase Drugs 0.000 description 1
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- 230000003197 catalytic effect Effects 0.000 description 1
- 238000010959 commercial synthesis reaction Methods 0.000 description 1
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- 235000005822 corn Nutrition 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 235000021245 dietary protein Nutrition 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940124274 edetate disodium Drugs 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
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- 230000005802 health problem Effects 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009318 large scale farming Methods 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000021590 normal diet Nutrition 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000001175 peptic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000004456 rapeseed meal Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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Classifications
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- 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
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Animal Husbandry (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Birds (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Fodder In General (AREA)
Abstract
One kind is added in low-protein diet, and daily ration, which can be improved, can utilize the compound protease of protein content, acid protease enzyme activity 5000-15000U/g, neutral proteinase enzyme activity 20000-30000U/g, alkali protease enzyme activity 20000-30000U/g in the compound protease.The advantages that the invention also discloses application effect of the compound protease in low-protein diet, can obviously reduce production cost, improve production performance, and product of the present invention has green, environmentally friendly, safe, application value with higher.
Description
Technical field
The invention belongs to feed additive fields, and in particular to be it is a kind of added in livestock and poultry low-protein diet, can have
Effect improves compound protease and its application of forage protein utilization efficiency.
Background technique
Low-protein diet is proposed according to " ideal protein " is theoretical, before referring to that meet animal is to the requirement of amino acid
It mentions, by adding suitable commercial synthesis amino acid, daily ration CP level is reduced 2-4 percentage points by NRC proposed standard, is reduced
The amino acid balance daily ration of dietary protein level.Synthesizing amino acid is added in feed reduces the low-protein diet energy of daily ration CP
Livestock and poultry are enough improved to the utilization efficiency of forage protein raw material and other nutritional ingredients；Reduce the discharge of nitrogen phosphorus in excrement；Daily ration CP
The reduction of content can save production cost；Low-protein diet can effectively inhibit grice diarrhoea after wean simultaneously, maintain enteron aisle strong
Health, form and microbiota to enteron aisle have apparent regulating and controlling effect.But also some researches show that CP contents in livestock and poultry diet
Reduction can change carcass quality；Reduce reproduction of domesti animals potential；Reduce production performance etc..
Protease is the class of enzymes of catalytic proteins peptide bond hydrolysis, and breaks down proteins can be peptone, polypeptide and trip by it
From amino acid, the high molecular weight protein in feed is only degraded to ability after small peptide and free amino acid under the action of protease
It is utilized by livestock and poultry.And feed protein enzyme refers to the exogenous protease in animal and fowl fodder from microbial fermentation, according to it
The difference of the optimum pH of effect can be divided into: acid protease (pH2.5-4.0), neutral proteinase (pH7.0 or so) and alkalinity
Protease (pH8.0-11.0), acid protease mainly play a major role in the peptic digest stage, and neutral and alkali protease master
It to play a major role in small intestine digestion phase.Some researches show that add exogenous protease in feed, not only can effectively supplement
The deficiency of young animal endogenous proteinase, while certain exogenous proteases can be because of the difference of its action site etc., it will be some
The protein that endogenous proteinase is difficult to digest is hydrolyzed to peptide and amino acid, and then improves livestock and poultry to feed protein and amino acid
Digestion is horizontal.
The application of low-protein diet technical know-how is gradually expanded at present, but in actual production, especially in large-scale farming
Pig farm in, although its nitrogen discharged amount that can obviously improve livestock and poultry, its influence to production performance is it is clear that often cause
The nutritional deficiency of market pig, the reduction of meat, and such as DDGS, cotton dregs, rapeseed meal, corn germ cake, feather meal, when this kind of original
Material in formula using it is excessive when, although feed CP content can be improved, it is poor to will cause formulation stability, and feed quality reduces,
Livestock and poultry are lower to the protein and amino acid digestion utilization rate of this kind of feed, so that production performance be made to reduce, are easy to produce enteron aisle
Health problem.
Summary of the invention
Based on the anxiety of dregs of beans raw material currently on the market, the object of the present invention is to provide one kind to answer in low-protein diet
With the feed addictive and its application for improving forage protein utilization efficiency.Compound protease of the present invention can reduce breeding production at
This, and can achieve the purpose that improve production performance.
A kind of compound protease, including acid protease, neutral proteinase and alkali protease, every gram of complex enzyme formulation by
Acid protease, neutral proteinase and alkali protease are proportionally prepared, wherein various list enzymes in every gram of compound protease
Enzyme activity is as follows: acid protease 5000-15000U, neutral proteinase 20000-30000U, alkaline protease activity 20000-
30000U。
Preferably, the enzyme activity of various list enzymes is as follows in every gram of compound protease: acid protease 10000U, neutral proteinase
25000U, alkali protease 30000U.
Preferably, compound protease is formulated by the raw material of following weight percent: 20% acid protease, and 10%
Neutral proteinase, 25% alkali protease, 45% cornstarch.
Preferably, the acid protease, neutral proteinase and alkali protease of compound protease are formed by saccharomycete, black
Aspergillus, aspergillus oryzae, long handle trichoderma or fermentation of bacillus subtilis generate, and are prepared using liquid state fermentation and solid-state deep layer fermenting process
It forms.
An other technical solution of the invention is application of the compound protease in the breeding process of chicken.
Preferably, compound protease is applied in the low-protein diet formula of broiler chicken.
Preferably, the final additive amount of compound protease is 200-1000 g tons of complete feed in the breeding process of chicken.
Compound protease of the present invention is applied in broiler chicken low-protein diet, can obviously reduce feed cost, and can improve meat
Chicken production performance, the external vivo efficacy of compound protease of the present invention in low-protein diet using there is remarkable result, have compared with
Strong Economic Application benefit, market popularization value.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments, based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
1 evaluating in vitro low-protein diet of embodiment adds compound protease to the influence using protein content
It is as described below that monogastric animal bionic digests operating process:
1, materials and methods
1.1 instrument and equipment
Plant sample crushing machine or mortar；Testing sieve: aperture 0.3mm；Assay balance: scale division value 0.0001g；PH meter: point
Angle value 0.01；Culture dish: diameter 90mm；Drier: anhydrous calcium chloride or the silica gel that changes colour is desiccant；Freeze drier；Low temperature
Centrifuge；Electrothermostat；Bionic digestive system for monogastric animals etc.；
1.2 reagents and material and processing mode
In addition to especially indicating person, all reagents are that analysis is pure.
Use for laboratory water should meet the specification of tertiary effluent in GB/T 6682-2008.
Pepsin (Sigma P7000)；Amylase (Sigma A3306)；Trypsase (Amresco 0785)；Rotten egg
White enzyme (Amresco 0164)；Hydrochloric acid (HCL)；Sodium chloride (NaCL)；Potassium chloride (KCL)；Anhydrous Disodium Phosphate
(Na2HPO4)；Anhydrous sodium dihydrogen phosphate (NaH2PO4)；Phosphoric acid (H3PO4)；Sodium hydroxide (NaOH)；Sodium bicarbonate
(NaHCO3)；Disodium ethylene diamine tetraacetate (C10H14N2O8Na2·2H2O, EDETATE DISODIUM)；Dehydrated alcohol (C2H6O)；Penicillin
(1,600,000 U)；Bag filter: the production of Viskase company, the U.S., model MEMBRA-CEL MD44-14, molecular cut off 14000
Er Dun.Pre-treatment is as follows: bag filter is cut into the segment of 25cm or so.In (W/V) sodium bicarbonate of 2000ml 2% and 1mmol/L
Bag filter is boiled 10 minutes in disodium ethylene diamine tetra-acetic acid solution (pH8.0).Bag filter is thoroughly cleaned with distilled water.It is placed in
It is boiled in 1mmol/L disodium ethylene diamine tetra-acetic acid solution 10 minutes.After cooling, it is stored in spare in 4 DEG C of refrigerators.Using preceding saturating
The built-in full water of bag is analysed, is then discharged out, thoroughly cleans bag filter.
Stomach buffer: weighing 10.36g sodium chloride, 0.965g potassium chloride, be put into 2000mL beaker, and 1800mL is added and goes
Ionized water dissolves, and adjusts the pH to 2.0 of solution at 39 DEG C with the hydrochloric acid of 2mol/L (HCL).Above-mentioned solution is turned after cooling
Enter 2000mL volumetric flask, and with deionized water constant volume.
Small intestine buffer: weighing 11.55g sodium chloride, 2.45g potassium chloride, 8.32g Anhydrous Disodium Phosphate, 40.96g without
Water sodium dihydrogen phosphate, 2g potassium sorbate, 1,600,000 U of penicillin.It is put into 2000mL beaker, 1800mL deionized water dissolving is added,
And the pH to 6.44 of solution is adjusted at 39 DEG C with the sodium hydroxide of the phosphoric acid of 1mol/L or 1mol/L.By above-mentioned solution after cooling
It is transferred in 2000mL volumetric flask, and with deionized water constant volume.
Simulate the gastric juice (pepsin activity 737.5U/ml: the pepsin activity described according to KLANS/X1001-2010
Measuring method measures the activity of pepsin in pepsin.Then, it is according to the concentration of pepsin in simulate the gastric juice
737.5U/ml, the pepsin for weighing 184.38kU are dissolved in the stomach buffer of 250ml pH2.0, are slowly stirred until molten
Solution.
Simulated intestinal fluid (amylase activity 221.43U/ml, tryptic activity 69.10U/ml, chymotrypsin activity
8.68U/ml): the alpha-amylase activity measuring method described according to KLANS/X 1002-2010, KLANS/X 1003-2010 are retouched
The determination of tryptic activity method stated, the chymotrypsin activity measuring method of KLANS/X 1004-2010 description, measures reagent
Grade amylase, trypsase, in chymotrypsin corresponding digestive ferment activity.Then, according to these three digestive ferments in simulated intestinal fluid
Activity, weigh amylase 41.41KU, trypsase 12.82KU respectively, chymotrypsin 1.62KU is dissolved in 22ml deionized water
In, and be slowly stirred until dissolution.
2, prepared by sample
The acquisition of 2.1 samples
It is sampled by GB/T 14699.1.
2.2 sample treatment
By the sample of sampling quartering point to 200g or so, with plant pulverizer or mortar by sample comminution to crossing aperture
0.3mm testing sieve encloses sample sack sealing storage, as sample.
3, determination step
3.1 preparations and loading
3.1.1 1000mL stomach buffer, 1000mL small intestine buffer are put into the corresponding of Bionic digestive system for monogastric animals
Position, and the pipeline of system and buffer bottle are connected.
3.1.2 in control software, the preheating time that Bionic digestive system for monogastric animals is arranged is 60min.Disappear to all
After the parameter input in change stage, running simulation digestion process.
3.1.3 during Bionic digestive system for monogastric animals preheating, following loading work are carried out.
3.1.4 the bag filter handled well is crossed into simulation digest tube, both ends turn up and are tightened bag filter with rubber band, Gu
It is scheduled on simulation digest tube.Then, with turned welt silica gel plug that one end plug is tight.
3.1.5 weighing 1-2g Feed Sample, (mixed feed and energy feed are 2g, and protein 1g is accurate to
0.0002g) it is placed in the simulation digest tube equipped with bag filter.The dry matter content of Simultaneous Determination sample.
3.2 simulation pig pipe intestinal digestings
3.2.1 stomach simulation digestion
3.2.1.1 20ml simulate the gastric juice is added into bag filter.
3.2.1.2 the other end for simulating digester is tight with the turned welt silica gel plug plug with digestive juice liquid-feeding tube.
3.2.1.3 simulation digester is placed in Bionic digestive system for monogastric animals, is intake according to simulation digester lower end,
The principle of upper end water outlet, connects pipeline.It is connected in series between every group of 5 simulation digesters.Digestive juice liquid-feeding tube and system successively with
Quick coupling is connected.
3.2.1.4 in Bionic digestive system for monogastric animals control software, the parameter of stomach step simulations digestion are as follows: temperature 39
DEG C, buffer flow rate 120ml/min, digestion time 4h, cleaning solution 1500ml/ times clean 40min every time, clean 3 times altogether.Its
He is operated control parameter by instrument specification.
3.2.2 small intestine simulation digestion
3.2.2.1 at the end of stomach is simulated and digested, it is imitative that 2.2ml simulated intestinal fluid (6.18) are accurately moved into nonruminant
In the small intestine digestive juice reserve room of raw digestive system.
3.2.2.2 in Bionic digestive system for monogastric animals control software, the parameter of small intestine step simulations digestion are as follows: temperature
41 DEG C, buffer flow rate 120ml/min, small intestine digestion time is 16h, and cleaning solution 1500ml/ times cleans 40min every time, clear altogether
It washes 6 times.Other control parameters are operated by instrument specification.
The processing of 3.3 slaking residues
3.3.1 after digesting, the non-slaking residue in bag filter is transferred to known oven dry weight without loss
In 90mm culture dish (this process needs to take out bag filter from simulation digester, longitudinally cuts off and is rinsed with deionized water).
3.3.2 the culture dish equipped with non-slaking residue is dried to after no washmarking at 65 DEG C (it is generally necessary to 8-10h), then
It dries to constant weight at 105 DEG C.
3.3.3 the slaking residue in culture dish is all scraped, is transferred in kjeldahl apparatus digest tube, uses dehydrated alcohol
Rinse residue 3 times (about 30mL every time), it is ensured that residue shifts completely, according to crude protein determining method GB/T in national feed
6432-2018 detects residue crude protein content.
4, result calculates
4.1 vitro Dry Matter Digestibilities and using protein content calculating be calculated according to the following formula respectively.
F: the weight (g) of Feed Sample
DMF: diet dry matter content (two-decimal)
NF: the over dry content (two-decimal) of Feed Sample nutrient
R: the oven dry weight (g) of food-residue=(sample weighting bottle over dry weight+filter paper over dry weight+residue over dry weight)-(sample weighting bottle
Over dry weight+filter paper over dry weight)
NR: the over dry content (two-decimal) of residue sample nutrient
CPF: Feed Sample over dry crude protein content (two-decimal)
CPR: residue over dry crude protein content (two-decimal)
5, daily ration is tested
Before on-test, feeds utilized raw material is acquired, analyzes its conventional nutrient and amino acid content, with actually detected result
Feed formula production is carried out, each other nutrient composition contents of raw material are referring to " Chinese feed ingredient and nutritive value table 2017 ", battalion
It supports requirement and refers to NRC (1994) broiler feeding standard.Wherein daily ration one is CP:20.5%, and daily ration two is CP:19.5%, day
Grain three is CP:18.5%, and daily ration four is CP:18%, and it is that compound protease group, group is not added in daily ration one that group, which is designed as group one,
Two be daily ration two plus compound protease 0.5g/kg, and group three is daily ration three plus compound protease 0.5g/kg, and group four is daily ration
Four plus compound protease 0.5g/kg, group five is daily ration four plus compound protease 0.8g/kg, 5 repetitions of each group, each
1 digest tube is repeated, experimental design is as shown in table 1:
Compound protease of the present invention is fermented by yeast and aspergillus niger etc. through liquid or solid, and production procedure is normal
Rule technique, this will not be repeated here.Compound protease 10000U/g containing acid protease, neutral proteinase 25000U/g, alkaline egg
White enzyme 30000U/g.
1 experimental design of table
| Group | Test daily ration |
| Group one | Daily ration CP:20.5% |
| Group two | Daily ration CP:19.5%+0.5g/kg compound protease |
| Group three | Daily ration CP:18.5%+0.5g/kg compound protease |
| Group four | Daily ration CP:18%+0.5g/kg compound protease |
| Group five | Daily ration CP:18%+0.8g/kg compound protease |
Data analysis is analyzed using SPSS17.0 software ANOVO module single factor test, and P value < 0.05 indicates that difference is aobvious
It writes.Test result is as shown in table 2:
As shown in Table 2, daily ration dry can be significantly improved by compound protease of the present invention being added in different low-protein diets
Matter digestibility (P < 0.05)；The crude protein content addition compound protease that normal daily ration reduces by 1% is remarkably improved daily ration can benefit
With protein content (P < 0.05), raising amount is up to 7.27%；It is compound in the crude protein content addition that normal daily ration reduces 2-3.5%
For protease compared with normal diet group, daily ration can utilize protein content no significant difference (P > 0.05).
Table 2 adds compound protease to the influence using protein content in low-protein diet
| Group | Dry matter digestibility (%) | Using protein content (%) |
| Group one | 68.72±0.35a | 13.07±0.28a |
| Group two | 70.12±0.16b | 13.92±0.13b |
| Group three | 70.43±0.25b | 13.51±0.30ab |
| Group four | 69.84±0.33b | 12.91±0.26a |
| Group five | 70.02±0.45b | 13.02±0.39a |
Significant difference (P < 0.05) is indicated between different letters.
Case study on implementation 2 adds influence of the compound protease to meat chicken production performance in low-protein diet
1, experimental animal
Influence of the compound protease to meat chicken production performance is added in low-protein diet in order to study, and selects 1 age in days meat
Chick 648,3 processing, 12 repetitions of each processing are randomly divided by male and female and weight, 18 chickens of each repetition test rank
Section is 42 days, is fed in two stages.
2, daily ration is tested
Before on-test, feeds utilized raw material is acquired, analyzes its conventional nutrient and amino acid content, with actually detected result
Feed formula production is carried out, each other nutrient composition contents of raw material are referring to " Chinese feed ingredient and nutritive value table 2017 ", battalion
It supports requirement and refers to NRC (1994) broiler feeding standard.Wherein control group uses normal protein daily food；Handle one group for nutrition at
Divide content and normal daily ration essentially identical, but CP content reduces by 1.5%；Two groups are handled as nutrient composition content and normal daily ration base
This is identical, but CP content reduces by 1.5%, adds compound protease of the present invention by 0.5g/kg；Three groups are handled as nutrient composition content
It is essentially identical with normal daily ration, but CP content reduces by 1.5%, adds compound protease of the present invention by 0.8g/kg；Experimental design is such as
Shown in table 3:
Compound protease of the present invention is fermented by yeast and aspergillus niger etc. through liquid or solid, and production procedure is normal
Rule technique, this will not be repeated here.Compound protease 10000U/g containing acid protease, neutral proteinase 25000U/g, alkaline egg
White enzyme 30000U/g.
3 experimental design of table
| Group | Test daily ration |
| Control group | Normal daily ration |
| Handle one group | Low-protein diet |
| Handle two groups | Low-protein diet+0.5g/kg compound protease |
| Handle three groups | Low-protein diet+0.8g/kg compound protease |
3, feeding management
Feeding experiment carries out in Beijing Challenge Biotechnologies Co., Ltd., animal experiment base, Zunhua, and test process is conventional
Raising, is freely eaten and drinking-water, immune and medication are operated according to chicken house " immune programme " and " medicine guide ".
Feeding experiment is weighed at the end of on an empty stomach in the morning respectively at on-test, the daily feeding of record during test
Amount, calculating the broiler chicken full stage after the test be averaged feed intake, daily gain and feedstuff-meat ratio, and data analysis is using SPSS17.0 software
ANOVO module single factor test is analyzed, and P value < 0.05 indicates significant difference.Test result is as shown in table 4.
As shown in Table 4, average daily gain and average daily gain difference is not significant (P > 0.05) between each processing, place
Manage one group compared with the control group feedstuff-meat ratio improve 1.71%, two groups of test with test three groups can reduce meat compared with the control group
Chicken feedstuff-meat ratio reduces by 0.57% respectively, and 2.86% (P > 0.05), feed efficiency has reduction in broiler chicken low-protein diet
Trend, but add protease and meat chicken production performance had no significant effect, and have the tendency that reduction to broiler chicken feedstuff-meat ratio, to drop
Low production cost.
Table 4 adds influence (1-42d) of the compound protease to meat chicken production performance in low-protein diet
| Group | Control group | Handle one group | Handle two groups | Handle three groups |
| Average weight increasing a day ADG (g) | 44.16±0.73 | 45.62±0.91 | 42.08±0.32 | 45.43±0.51 |
| Average daily feed intake ADFI (g) | 77.32±1.00 | 81.27±1.42 | 73.50±1.73 | 77.42±1.21 |
| Feedstuff-meat ratio FCR | 1.75±0.02 | 1.78±0.02 | 1.74±0.03 | 1.70±0.02 |
Result of study of the invention shows that compound protease of the present invention is added in low-protein diet can be obviously improved livestock and poultry
To daily ration using protein content, improves broiler chicken feedstuff-meat ratio, save production cost.
Claims (7)
1. a kind of compound protease, including acid protease, neutral proteinase and alkali protease, which is characterized in that every gram multiple
Synthase preparation is proportionally prepared by acid protease, neutral proteinase and alkali protease, wherein in every gram of compound protease
The enzyme activity of various list enzymes is as follows: acid protease 5000-15000U, neutral proteinase 20000-30000U, basic protein enzyme activity
Property 20000-30000U.
2. compound protease according to claim 1, which is characterized in that the enzyme activity of various list enzymes in every gram of compound protease
It is as follows: acid protease 10000U, neutral proteinase 25000U, alkali protease 30000U.
3. compound protease according to claim 1, which is characterized in that compound protease by following weight percent original
Material is formulated: 20% acid protease, 10% neutral proteinase, 25% alkali protease, 45% cornstarch.
4. compound protease according to claim 1, which is characterized in that form compound protease acid protease, in
Property protease and alkali protease by saccharomycete, aspergillus niger, aspergillus oryzae, long handle trichoderma or fermentation of bacillus subtilis generate,
It is prepared using liquid state fermentation and solid-state deep layer fermenting process.
5. application of the compound protease described in claim 1 in the breeding process of chicken.
6. application according to claim 5, which is characterized in that compound protease is applied to the low-protein diet formula of broiler chicken
In.
7. application according to claim 5, which is characterized in that the final additive amount of compound protease in the breeding process of chicken
For 200-1000 g tons of complete feed.
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