CN108450667A - A kind of compound protease effectively improving feed protein utilization rate and its application - Google Patents
A kind of compound protease effectively improving feed protein utilization rate and its application Download PDFInfo
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Abstract
A kind of compound protease effectively improving feed protein utilization rate disclosed by the invention, it is characterized in that, including carrier and make an addition to compound protease in carrier, the compound protease, it is proportionally to prepare acid protease, neutral proteinase and alkali protease, wherein the enzyme activity of various list enzymes is as follows in every gram of compound protease：1000 10000U of acid protease, 1000 10000U of neutral proteinase, 1000 10000U of alkali protease.The invention also discloses application of the compound protease in animal feed, to effectively improve feed protein utilization rate.
Description
Technical field
The present invention relates to a kind of feed addictives, and in particular to be a kind of to effectively improve feed protein utilization rate
Compound protease and its application.
Background technology
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.High molecular weight protein in feed need to could be utilized after proteasome degradation is at peptide and amino acid by livestock and poultry.
Feed protein enzyme can be divided into animal-based protein enzyme, plant source protein enzyme, microorganism according to the difference in source
Endogenous binding protein enzyme.Animal-based protein enzyme is mostly extracted from the pancreases such as ox, sheep, pig, and production cost is higher.Plant source protein enzyme
It common are papain and bromelain.Microbial protease is existing to pass through bacterium (such as bacillus subtilis, lichens bud
Spore bacillus) culture extraction, also have by fungi (such as aspergillus niger, aspergillus oryzae, yeast) fermentation extraction.Due to microorganism egg
White enzyme and plant rennet production cost relative moderate, are used widely.
According to the optimum pH of effect, protease can be divided into again：Acid protease (pH2.5-4.0), neutral proteinase
(pH7.0 or so), alkali protease (pH8-11), plant animal protein are hydrolyzed in these types of protease under circumscribed effect
For small peptide and amino acid.Animal depends on pepsin (acid protease) and trypsase (alkali to the digestion of protein
Property protease).
Young animal itself can't secrete enough protease and be raised to digest since Development of Digestive Organs is still unsound
Protein in material.By taking pig as an example, 2 weeks after birth, pepsin activity was very low, and activity just gradually increases after two weeks；Wean
The development of gastric acid secretion and various enzyme activity to piglet has a negative impact, and apparent accumulation will occur in chyme in 12h after wean, with
After reduce, until 50 ages in days before ability rebound significantly.In this case, exogenous protease is added in feed, can effectively be mended
The deficiency of endogenous proteinase is filled, the digestive utilization ratio of the nutriments such as protein in feed is improved.
Exogenous protease is also possible to the digestive utilization ratio for improving animal to nutriment by following approach：1. enhancing endogenous
The activity of protease；2. certain exogenous proteases may be because of the difference of action site etc., by some animal endogenous proteinases
The protein for being difficult to digest is hydrolyzed to peptide and amino acid, and then improves digestibility of the animal to feed protein.
Protein content 6%~8% (dry matter basis) in corn, wherein alcohol soluble protein are the major proteins in endosperm,
The 60~80% of entire albumen are accounted for, hydrophobic residue is more, and dissolubility is poor, and digestibility is low, and exists in the form of combination with starch,
The enzymolysis for influencing starch, reduces the digestive utilization ratio of starch.Research is it has been shown that the high corn of content of prolamine, in vivo
With external starch digestibility be substantially reduced (philippeau, 2000；Ferraretto,2013；Allen, 2008), often increase
1% alcohol soluble protein, the total alimentary canal starch digestibility of ruminant reduce by 0.86% (lawton, 2002).
Sorghum is one of five big cereal crops, can be as a kind of basic type daily ration of animal feeding.The chemical group of sorghum
At similar with corn, it is rich in abundant starch and protein, therefore, sorghum has become potential energy feedstuff in Production of Livestock and Poultry.
But the feed conversion rate of actually sorghum is low, mainly due to being largely alcohol soluble protein in jowar, accounts for entire albumen
60%-80%, hydrophobic residue is more in alcohol soluble protein, and dissolubility is poor, and lacks lysine, and alcohol soluble protein is assembled by disulfide bond
At different proteosomes, and the starch granules in sorghum endosperm is embedded in closely knit proteosome, to influence starch
Digestion, reduces the digestive utilization ratio of starch.Studies have shown that complex enzyme of the external source addition containing protease can make sorghum starch
Percent hydrolysis increases by 26% (Lichtenwalner etc., 1978).
Invention content
An object of the present invention is provided a kind of multiple for the above-mentioned technical problem present in existing independent protease
Hop protein enzyme, for the compound protease to supplement endogenous proteinase, enhancing animal is right under young age stage, disease or stress situation
The abilities of digestive and absorption of protein achievees the purpose that improve its intestinal health.
The second object of the present invention is to provide application of the above-mentioned compound protease in feed, degradable corn sorghum
In alcohol soluble protein, improve protein utilization, indirectly improve starch digestion utilization rate, save daily ration cost, improve productivity
The purpose of energy.
A kind of compound protease provided by the invention on the one hand can be insufficient by supplementing endogenous proteinase, and it is dynamic to reach enhancing
Object, to the digestibility and utilization ability of protein, improves the purpose of intestinal health, another party under young age stage, disease or stress situation
Face can reinforce the digestibility and utilization of protein with the alcohol soluble protein in degrading maize and sorghum, promote digesting and assimilating for starch, save
Daily ration cost improves production performance.
A kind of compound protease as first aspect present invention, it is characterised in that including carrier and make an addition in carrier
Compound protease, the compound protease is proportionally to match acid protease, neutral proteinase and alkali protease
System, wherein the enzyme activity of various list enzymes is as follows in every gram of compound protease：Acid protease 1000-10000U, neutral proteinase
1000-10000U, alkali protease 1000-10000U.
In a preferred embodiment of the invention, the acid protease, neutral proteinase and alkali protease by
Aspergillus niger, aspergillus oryzae, long handle trichoderma or producing bacillus subtilis, using liquid state fermentation and the preparation of solid-state deep layer fermenting process
At.
In a preferred embodiment of the invention, the compound protease by following weight percent raw material prepare and
At：20% acid protease, 20% neutral proteinase, 10% alkali protease, 50% carrier.
Application of a kind of compound protease in animal feed as the present invention, to effectively improve feed protein utilization
Rate.
In a preferred embodiment of the invention, the animal feed is in egg feedstuff, broiler fodder, pannage
It is a kind of.
In a preferred embodiment of the invention, the animal feed includes core material, concentrate feed or complete diet pellet.
In a preferred embodiment of the invention, the complete diet pellet is that the final additive amount of above-mentioned compound protease is
100-1000 g ton complete feeds.
The compound protease of the present invention has the beneficial effect that：
1, endogenous proteinase hyposecretion can be supplemented, enhancing animal is under young age stage, disease or stress situation to egg
The abilities of digestive and absorption of white matter improves intestinal health.
2, commercialization alcohol soluble protein degradation 30~60% can be made, content of prolamine in sorghum is made to reduce by 0.5%~2%
(dry matter).
3, meat chicken production performance can be improved, reduce feeding cost, increase economic benefit.It is raised through lab analysis and broiler chicken
Experiment is supported it is found that the effective enzyme activity of the protease of the present invention is high, internal body outer test effect is apparent, effectively facilitates the growth of animal,
Increase economic benefit.
Specific implementation mode
Below by specific example, the invention will be further described, but the present invention is not limited thereto.
If experimental method described in following embodiments is conventional method without specified otherwise, the experiment reagent and instrument
Device commercially obtains unless otherwise specified.
What is used in case study on implementation below can be improved the compound protease of protein digestibility utilization rate, including carrier, acidity
Protease, neutral proteinase and alkali protease, mass ratio 50:20:20:10.
The preparation method of the various protease used in case study on implementation below is as follows：Select the preferably black song of function and effect
It is mould, liquid state fermentation.Production procedure is as follows：Cryovial-shaking flask culture-seeding tank-fermentation tank-culture tank-spray drying-
Finished product.Concrete technology is common process, and details are not described herein.
Embodiment 1
A kind of protease effectively improving protein utilization of the present invention is with commercially available import protease to alcohol soluble protein
The comparison of degradation effect
1 materials and methods
1.1 material
Protease：Protease A (protease enzyme product of the invention), Cathepsin B (import enzyme sample 1), protease C (imports
Enzyme sample 2).
Zeins：It is bought in market.
Test reagent：The sodium tetraborate solution of 0.0125mol/L mercaptoethanols containing 1%SDS and 0.5%2-, 72% trichlorine
Acetic acid solution, KH2PO4 buffer solutions etc.；
Test apparatus：Oscillator, thermostat water bath, centrifuge, spectrophotometer etc..
1.1.2 degradation of the in vitro method research protease to zeins
(1) dissolving of zeins：A certain amount of jade is dissolved with the sodium tetraborate buffer solution (pH 10) containing 1%SDS
Rice alcohol soluble protein.
(2) degradation of the in-vitro simulated chicken pipe intestinal digesting Environmental Studies protease to zeins
The above zeins lysate (0.06g alcohol soluble proteins) 2mL is measured respectively, and 7mL is added to contain pepsin
KH2PO4Buffer solution, addition liquid of protease 1mL (by 1% addition of jowar), makees blank control with distilled water instead of liquid of protease,
PH value is adjusted to 3.0, and 120r/min vibrates in constant temperature oscillator, and 37 DEG C of reaction 1h add 0.3mL 2mol/ after reaction
LNaOH solution, pH are adjusted to 6.5, continue to be put into constant temperature oscillator reaction 3h, take out cooling immediately after reaction.It takes respectively
Reaction solution 1mL is stated, turbidity assay protein concentration is utilized.
(3) degradation of the in-vitro simulated pig stomach enteron aisle digestive environments research protease to zeins
The above zeins lysate (0.06g alcohol soluble proteins) 2mL is measured respectively, and 7mL is added to contain pepsin
KH2PO4Buffer solution, addition liquid of protease 1mL (by 1% addition of jowar), makees blank control with distilled water instead of liquid of protease,
PH value is adjusted to 2.0,120r/min oscillations, and 37 DEG C of reaction 5h, after reaction plus 2mol/L NaOH tune pH value is to 7.0, continues anti-
16h is answered, is cooled down immediately after reaction.Above-mentioned reaction solution 1mL is taken respectively, utilizes turbidity assay protein concentration.
1.1.3 degradation of the protease to kafirin
(1) preparation of kafirin lysate
A certain amount of jowar is weighed, 0.0125mol/L is added according to a certain percentage and contains 1%SDS and 0.5%2- sulfydryls
The sodium tetraborate buffer solution of ethyl alcohol, stirring extracting 2h, is made required kafirin solution at room temperature.
(2) degradation of the protease to kafirin
Take the extract 5mL of the above kafirin that the KH that 4mL pH are 7.0 is added respectively2PO4Buffer solution, preheating
5min, is added liquid of protease (1000g/t feeds) 1mL, and 37 DEG C of reaction 3h take above-mentioned reaction solution to carry out turbidity analysis.
(3) turbidity assay albumen concentration
Turbidity assay principle：First digestible protein, undigested protein are removed with pepsin
(i.e. alcohol soluble protein), at polypeptide chain, is used with the sodium tetraborate Solution Depolymerization of the 2 mercapto ethanol containing 1%SDS (w/v) and 0.5%
TCA condensing proteins, turbidity is measured under ultraviolet specrophotometer, and protein content is measured by protein standard curve.
In this experiment, to being improved on the basis of turbidity analytic approach principle, being removed with pepsin can digest first
Protein, suitable exogenous protease, external mould is added in undigested protein SDS sodium tetraborate buffer solutions
Quasi- animal gastrointestinal tract environment is hydrolyzed, and TCA is added in reaction solution, remaining indigestibility protein is reacted in concentration, in ultraviolet point
Turbidity is measured under light photometer, albumen concentration is calculated by albumen curve, albumen concentration is lower, illustrates protease to the molten egg of alcohol
White degradation rate is higher, and degradation effect is more apparent.
2 results
Degradation of the 2.1 in vitro method research protease to zeins
As shown in Table 1, it simulates under pig chicken digestive tract environment in vitro, protease A is high to the degradation rate of zeins
Up to 50%, and Cathepsin B and protease C to alcohol soluble protein degradation rate less than 20%.Illustrate the molten protein efficiency of protease A degrading alcohol
It is high.
Degradation of 1 protease of table to zeins
Degradation of 2.2 protease to kafirin
As shown in Table 2, after in-vitro simulated swine alimentary canal environment, protease A is 8% to the degradation rate of kafirin,
Slightly above Cathepsin B, protease C do not have degradation effect.
Degradation of 2 protease of table to kafirin
3 results and discussion
Alcohol soluble protein is the major storage albumen in feeding jowar and corn embryosperm, and with the starch granules in endosperm to tie
Fit form exists and is wrapped up, and has a negative impact to the starch digestibility of animal.Correlative study is it has been shown that often increase
1% content of prolamine, the total alimentary canal starch digestibility of ruminant will reduce by 0.86%.Alcohol soluble protein is not soluble in water, no
It is easily degraded and digests by endogenous proteinase.The structure feature of protein, which is not the presence of disulfide bond, can influence the hydrolysis of protein, grind
Study carefully and show that keratin, the alcohol soluble protein especially molten albumen of γ -ol are rich in cystine and a large amount of disulfide bond, protease can be prevented
Hydrolysis.Research has been pointed out, and pronase, the protease added with keratinase activity can hydrolyze alcohol soluble protein, increases
The percent hydrolysis of starch.
The in-vitro simulated animal gastrointestinal tract digestive environments of this experiment are to refer to protein degradation rate with turbidity analytic approach
Mark, the degradation rate of the protease of comparative evaluation separate sources to corn and kafirin.Test result discovery, protease A
Cathepsin B and protease C are better than to the degradation effect of alcohol soluble protein, 50% reached to the degradation rate of zeins, and
It is low compared with to kafirin degradation rate, this may be had differences with corn and kafirin structure it is related, such as sorghum
Content height of alcohol soluble protein disulfide bond etc..
Embodiment 2
A kind of protease effectively improving protein utilization of the present invention is with commercially available import protease to AA Broiler chicks
The influence of growth performance
1 materials and methods
1.1 test material
Using the consistent AA broiler chicken 96 of 1 age in days health, weight.
Protease：Protease A (protease enzyme product of the invention), Cathepsin B (import enzyme sample 1), protease C (imports
Enzyme sample 2).
1.2 experimental design
It chooses 1 consistent aa broiler chicken of health, weight and is randomly divided into 4 processing, each handle 4 repetitions, Mei Gechong
It is 6 multiple.Including control group (basal diet group), processing group T1 (basal diet+protease A), processing group T2 (basal diets+egg
White enzyme B), processing group T3 (basal diet+protease C), experimental period 25d, be divided to two stages of 1-10 ages in days and 11-25 ages in days into
Row.It respectively handles specific experimental design and is shown in Table 3.
3 experimental design of table
1.3 experiment daily rations
It tests daily ration and is raised and material nutrient component content standard preparation with reference to big victory AA commercial broiler flocks are pacified.
1.4 feeding managements and Testing index
1.4.1 feeding management
Test chicken carries out sub-cage rearing using the fowl of our company with positive/negative pressure isolator, is freely eaten and drinks water.Illumination for 24 hours,
Temperature and humidity requires to be controlled according to conventinal breeding management, observes the feed consumption rate of each group chicken in experimental period daily, tests
At the beginning and end of carry out chicken as unit of cage respectively and weigh, and calculate chicken and be averaged body weight gains, for accurate calculating feed intake,
Periodically collection sheds material and weighs.Feed consumption rate and feedstuff-meat ratio are calculated as unit of repeating cage.It records each repetition chicken and extremely washes in a pan situation.
1.4.2 Testing index
Growth performance index：Weight, average feed consumption rate, average daily gain, feedstuff-meat ratio
1.5 data processings and statistical analysis technique
Data are summarized with Excel tables, one-way analysis of variance is carried out to data using PASW18.0 softwares, is used
Duncan methods carry out Multiple range test, P<0.05 indicates significant difference, P>0.05 indicates significant difference.
2 results and analysis
Daily gain (P is significantly improved after adding protease in 1-25 age in days Broiler chicks diets known to the result of this experiment<
0.05) feedstuff-meat ratio (P, is significantly reduced<0.05), protease enzyme product of the invention is suitable with import protease enzyme product B effects.
Influence of 4 protease of table to 1-25 age in days broiler growth performances
Embodiment 3
A kind of protease effectively improving protein utilization of the present invention is with commercially available import protease to three way cross
Grow the influence of pig growth performance
1 materials and methods
1.1 test material
The healthy three way cross grower pigs 144 that initial average weight is 23.4kg.
Protease：Protease A (protease enzyme product of the invention), protease D (import enzyme sample)
1.2 experimental design
The healthy three way cross grower pigs 96 that initial average weight is 23.4kg are randomly divided into 3 processing, each handle 6
A repetition, it is each to repeat 8.Including control group (basal diet group), processing group T1 (basal diet+protease A), processing group T2
(basal diet+protease D) 70 days experimental periods, carries out in two stages, 35 days each stages.Respectively handle specific experimental design
It is shown in Table 5.
5 experimental design of table
1.3 experiment daily rations
Recommend trophic level to carry out sorghum-bean pulp type basal diet with reference to NRC (2012) pig nutritional need to prepare.
1.4 feeding managements and Testing index
1.4.1 feeding management
It is flat foster to close cement flooring in column home, is freely eaten, automatic drinking bowl supplies water, and each column environment is consistent, routinely manages
Program is managed to carry out expelling parasite and be immunized.Group is carried out as unit of experimental period, 35d and 70d were by repetition to weigh and count.
1.4.2 Testing index
Growth performance index：Weight (BW), average daily gain (ADFI), average daily gain (ADG), feedstuff-meat ratio (FCR).
1.5 data processings and statistical analysis technique
Data are summarized with Excel tables, one-way analysis of variance is carried out to data using PASW18.0 softwares, is used
Duncan methods carry out Multiple range test, P<0.05 indicates significant difference, P>0.05 indicates significant difference.
2 results and discussion
The influence that protease grows three way cross pig growth performance is shown in Table 6.As shown in Table 6, no matter experimental period 0-
35d, 36-70d or 0-37d, addition protease enzyme product of the present invention can be adopted day in reduction in grower pigs sorghum-soybean meal based diets
Daily gain is improved while appetite, to effectively reduce feedstuff-meat ratio.The effect of grower pigs to feeding sorghum-soybean meal based diets is remote
It is better than certain import protease enzyme product.
6 protease of table grows three way cross the influence of pig growth performance
Claims (7)
1. a kind of compound protease, which is characterized in that including carrier and compound protease in carrier is made an addition to, it is described compound
Protease is proportionally to prepare 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 1000-10000U, neutral proteinase 1000-10000U, alkali protease
1000-10000U。
2. a kind of compound protease as described in claim 1, which is characterized in that the acid protease, neutral proteinase and
Alkali protease is sent out by aspergillus niger, aspergillus oryzae, long handle trichoderma or producing bacillus subtilis using liquid state fermentation and solid-state deep layer
Ferment technique is prepared.
3. a kind of compound protease as described in claim 1, which is characterized in that the compound protease is by following weight percent
The raw material of ratio is formulated：20% acid protease, 20% neutral proteinase, 10% alkali protease, 50% carrier.
4. application of the compound protease described in a kind of any one of claims 1 to 3 claim in animal feed, with effective
Improve feed protein utilization rate.
5. application as claimed in claim 4, which is characterized in that the animal feed is egg feedstuff, broiler fodder, pannage
In one kind.
6. application as claimed in claim 4, which is characterized in that the animal feed includes core material, concentrate feed or complete diet pellet.
7. application as claimed in claim 6, which is characterized in that the complete diet pellet is the final additive amount of above-mentioned compound protease
For 100-1000 g ton complete feeds.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109480056A (en) * | 2018-11-12 | 2019-03-19 | 济南百斯杰生物工程有限公司 | A method of degradation alcohol soluble protein |
| CN110093333A (en) * | 2019-04-25 | 2019-08-06 | 天津挑战博德生物技术有限公司 | A kind of application in compound protease and its low-protein diet |
| CN110973371A (en) * | 2019-12-31 | 2020-04-10 | 河南科技大学 | Livestock and poultry egg white enzyme feed additive and application thereof |
| CN111000052A (en) * | 2019-12-31 | 2020-04-14 | 河南科技大学 | Piglet enzyme bacterium feed additive and application thereof |
| BE1030865B1 (en) * | 2022-09-12 | 2024-04-09 | Nu3Guts Bv | FEED ADDITIVE FOR IMPROVING INTESTINAL HEALTH AND/OR DIGESTIBILITY OF FEED IN LIVESTOCK |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
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2016
- 2016-12-09 CN CN201611131732.XA patent/CN108450667A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779423A (en) * | 2015-11-17 | 2016-07-20 | 济南诺能生物工程有限公司 | Compound protease as well as production method and applications thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109480056A (en) * | 2018-11-12 | 2019-03-19 | 济南百斯杰生物工程有限公司 | A method of degradation alcohol soluble protein |
| CN110093333A (en) * | 2019-04-25 | 2019-08-06 | 天津挑战博德生物技术有限公司 | A kind of application in compound protease and its low-protein diet |
| CN110973371A (en) * | 2019-12-31 | 2020-04-10 | 河南科技大学 | Livestock and poultry egg white enzyme feed additive and application thereof |
| CN111000052A (en) * | 2019-12-31 | 2020-04-14 | 河南科技大学 | Piglet enzyme bacterium feed additive and application thereof |
| BE1030865B1 (en) * | 2022-09-12 | 2024-04-09 | Nu3Guts Bv | FEED ADDITIVE FOR IMPROVING INTESTINAL HEALTH AND/OR DIGESTIBILITY OF FEED IN LIVESTOCK |
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