CN106858134A - A kind of feed addictive containing separate sources lipase and its application in yellow-feather broiler fodder - Google Patents
A kind of feed addictive containing separate sources lipase and its application in yellow-feather broiler fodder Download PDFInfo
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- CN106858134A CN106858134A CN201710025167.7A CN201710025167A CN106858134A CN 106858134 A CN106858134 A CN 106858134A CN 201710025167 A CN201710025167 A CN 201710025167A CN 106858134 A CN106858134 A CN 106858134A
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- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
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Abstract
A kind of feed addictive containing separate sources lipase and its application in yellow-feather broiler fodder, wherein, the feed addictive is made up of the component of following weight percentage：Chicken pancreatic lipase 40% 60%, lipase from Aspergillus Niger 40% 60%.Therefore, the present invention has the advantages that substantially to increase the diversity of microbe species in yellow-feather broiler ileum, enhancing yellow-feather broiler to the digestibility of grease, so as to reach mitigation broiler chicken trophism diarrhea.
Description
Technical field
The present invention relates to feed additive field, more particularly, to a kind of feed addictive containing separate sources lipase and
Application of this feed addictive in yellow-feather broiler fodder.
Background technology
The characteristics of grease feed has metabolizable energy value high, it is 2.25 times of carbohydrate and protein that it can be worth, and is foster
Chicken specialist carries out when broiler chicken standardized production is raised one of indispensable important source material in daily ration, therefore, raiser can
A certain proportion of grease is added in broiler fodder, adding grease to meet the growth needs of broiler chicken, in livestock and poultry diet has turned into
Improve one of technological approaches of energetic nourishing.However, in recent years, the price of energy feed raw material especially grease is constantly being climbed
Rise, how to improve the utilization rate of Dietary Energy, reduce grease addition, reduce feed cost, culture benefit is improved, while subtracting
A series of influence and harm that few grease high brings on livestock and poultry are increasingly becoming the focus of breeding enterprise concern.
Lipase, as the most basic enzyme of fat metabolism, if lack fat indigestion body disorder etc. will be caused existing
As jeopardizing body health.Nonruminant itself being capable of the endogenous digestive ferment such as secreting amylase, protease, lipase, but young age
Animal digestion mechanism is not yet physically well developed, endogenous lipase hyposecretion.When endogenous pancreatic lipase and bile acid secretion amount are not enough
When, it is fatty then can not be made full use of by livestock and poultry body, so as to cause the waste and loss of fat, meanwhile, the incomplete profit of fat
With various ill symptomses, such as trophism diarrhea and fatty liver are also brought along, the healthy growth and cultivation comprehensive benefit of broiler chicken are influenceed.
To solve the above problems, there is scholar to attempt being added to lipase as feed addictive in daily ration of broiler, supplement
The endogenous lipase hyposecretion of young animal, to reach the purpose for improving broiler chicken to Lipid use rate.But, in repetition test mistake
Effect is not satisfactory in journey, and the species for returning intestinal microbial is not significantly increased, and poultry trophism diarrhea does not have and substantially changes
It is kind, analysis may the reason for one of be feeding dorking fatty enzyme source it is single, it is impossible to decompose utilize different types of oil completely
Fat, leads to not meet demand of the broiler chicken to aliphatic acid.How separate sources lipase is added to reach raising Lipid use rate,
Aquaculture cost is reduced, one of the purpose of cultivation comprehensive benefit, the focus as people's common concern is improved.
The content of the invention
To solve the above problems, the present invention provides one kind to society can substantially increase microbial species in yellow-feather broiler ileum
The diversity of class, improves yellow-feather broiler to the digestibility of grease, so as to reach mitigate broiler chicken trophism diarrhea containing separate sources
The feed addictive of lipase.
The present invention also provides a kind of multifarious, enhancing Huang that can substantially increase microbe species in yellow-feather broiler ileum
Plumage broiler chicken mitigates the broiler fodder of broiler chicken trophism diarrhea to the digestibility of grease, so as to reach.
The technical scheme is that：A kind of feed addictive containing separate sources lipase is provided, by following weight hundred
Divide the component composition of ratio：Chicken pancreatic lipase 40%-60%, lipase from Aspergillus Niger 40%-60%.
As improvement of the present invention, it is made up of the component of following weight percentage：Chicken pancreatic lipase 45%-55%, black song
Mould lipase 45%-55%.
Used as improvement of the present invention, the chicken pancreatic lipase is prepared by following methods：
（1）The preparation of thick chicken pancreatic lipase；
（2）Chicken pancreatic lipase is isolated and purified；
（21）Ammonium sulfate precipitation；
（22）It is concentrated by ultrafiltration；
（23）Sephadex（Sephadex G-100）Molecular filtration is chromatographed；
（24）SDS- polyacrylamide gel electrophoresises（SDS-PAGE）Detection；
Used as improvement of the present invention, the preparation of the thick chicken pancreatic lipase comprises the steps：
（11）Chicken pancreas is pounded pancreas slurry, buffer solution, magnetic agitation 2h under the conditions of 4 DEG C is added；
（12）Filtering, takes solution, and 1500rpm-2000rpm centrifugations removal first is precipitated, and solution adds acetone, continues magnetic agitation
25min-40min, then under the conditions of 4 DEG C, 1500rpm-2000rpm centrifugations 15min-30min, the second precipitation uses 75% acetone
Dissolving stirring, then be centrifuged, obtain the 3rd precipitation；
（13）Dry the 3rd to precipitate, obtain solid powder；
（14）The solid powder is added to 4 DEG C of chloroform-methanol（Chloroform is 2 with the volume ratio of methyl alcohol:1）In, magnetic force is stirred
15min-30min is mixed, then under the conditions of 4 DEG C, 1500rpm-2000rpm centrifugations, the 4th precipitation is obtained, the 4th precipitation is dissolved in ether
In, then under the conditions of 4 DEG C, 1500rpm-2000rpm centrifugations, the 5th precipitation being obtained, the 5th precipitation is dissolved in acetone, then the 5th is sunk
Shallow lake drying, obtains thick enzyme powder.
As improvement of the present invention, the step（21）Ammonium sulfate precipitation comprises the steps：
（211）, above-mentioned thick enzyme powder be dissolved in 50mL deionized waters be made crude enzyme liquid, the small beaker that will be equipped with 50mL crude enzyme liquids is put
In another large beaker equipped with a small amount of frozen water, it is placed in being slowly stirred on magnetic stirring apparatus；
（212）, while be slowly stirred, while being slowly added to 10.00g, 15.00g, 20.00g, 25.00g, 30.00g, 35.00g
Ammonium sulfate is to final concentration of 20%, 30%, 40%, 50%, 60%, 70%（w/v）, the step should complete in 10min；Then proceed to stir
Mix 15min-20min；
（214）, then 10000rpm centrifugation 8-12min；Abandoning supernatant, 1 ~ 2 times of sediment volume is suspended in by precipitation
0.05mol/LpH is 7.5 phosphate buffers, and 10000rpm is centrifuged away the insoluble substance of residual；
（216）, determine supernatant in enzyme activity and protein content, calculate concentration enzyme liquid Rate activity and purification；Its
In, Rate activity=enzyme activity unit/milligram protein；Purification=often walk Rate activity/first step Rate activity.
The present invention also provides a kind of broiler fodder, and the broiler fodder contains any one above-mentioned lipase containing separate sources
Feed addictive, feed addictive is 0.01%-0.02%, and base-material is 99.8%-99.9%.
Used as improvement of the present invention, the base-material is basal diet or low energy daily ration.
As improvement of the present invention, wherein, the basal diet is made up of the component of following weight percentage：Corn
50%-65%, dregs of beans 15%-30%, corn protein powder 3%-6%, vinasse albumen feed (DDGS) 4%-6%, level Four soybean oil 2%-6%,
Wheat-middlings 0.1%-5%, stone flour 1.3%-1.6%, calcium monohydrogen phosphate 0.6%-1.5%, salt 0.3%-0.4%, lysine 0.3%-0.5%, Gu
Body methionine 0.4%-1.2%, threonine 0.2%-0.8%, the first premix 1%.
The present invention also provides application of the feed addictive containing separate sources lipase in yellow-feather broiler fodder.
The present invention also provides the feed containing separate sources lipase to yellow-feather broiler ileum microbial diversity aspect shadow
Loud application.
The present invention provides a kind of feed addictive containing separate sources lipase, separate sources fat in the feed addictive
Fat enzyme is made up of the component of following weight percentage：Chicken pancreatic lipase 40%-60%, lipase from Aspergillus Niger 40%-60%.When will by institute
State after chicken pancreatic lipase and lipase from Aspergillus Niger are prepared according to aforementioned proportion and be added to yellow-feather broiler daily ration as feed addictive
In, endogenous lipase hyposecretion in supplement Broiler Chicken is can reach, crude fat utilization rate is improved, and relative to prior art, by
Newly the combination that chicken lipase is chicken pancreatic lipase and lipase from Aspergillus Niger, chicken pancreas combined lipase are with the addition of in feed addictive
The effect played in yellow-feather broiler body is more stable, lasting, and activity is higher；Meanwhile, the chicken pancreatic lipase and black song of separate sources
Mould lipase can act synergistically because chicken pancreatic lipase and lipase from Aspergillus Niger source are different, and both can act synergistically, and can divide
Solution improves different type fat utilization rate using different types of grease in daily ration of broiler, hence it is evident that increase in yellow-feather broiler ileum
The diversity of microbe species, reduces the fatty liver and trophism diarrhea of yellow-feather broiler.Therefore, the present invention has and can substantially increase
Plus in yellow-feather broiler ileum microbe species diversity, improve yellow-feather broiler daily ration in fat utilization rate, so as to reach mitigation
The purpose of broiler chicken trophism diarrhea.
Brief description of the drawings
Fig. 1 is sephadex（Sephadex G-100）Filtration chromatography elution curve.
Fig. 2 is the SDS- polyacrylamide gel electrophoresises of purification step（SDS-PAGE）Detection figure.
Fig. 3 is the polymerase chain reaction of microorganism 16SrDNA gene V3 areas genetic fragment（ PCR）Figure.
Fig. 4 is the polymerase chain reaction of microorganism 16SrDNA gene V3 areas genetic fragment（ PCR）Figure.
Fig. 5 is microorganism 16SrDNA gene V3 areas genetic fragment denaturing gradient gel electrophoresis（DGGE）Finger-print.
Specific embodiment
In the present invention, special instruction, the source that " separate sources " refers to lipase is different, wherein, chicken pancreas
Lipase comes from chicken pancreas, and lipase from Aspergillus Niger comes from aspergillus niger.
In the present invention, the basal diet in feed is made up of the component of following weight percentage, and specific embodiment is as follows：
Embodiment 1
Corn 50%, dregs of beans 20%, corn protein powder 6%, vinasse albumen feed (DDGS) 6%, level Four soybean oil 6%, wheat-middlings 5%, stone
Powder 1.6%, calcium monohydrogen phosphate 1.5%, salt 0.4%, lysine 0.5%, solid methionine 1.2%, threonine 0.8%, the first premix
1%。
Embodiment 2
Corn 50%, dregs of beans 30%, corn protein powder 5%, vinasse albumen feed (DDGS) 4%, level Four soybean oil 6%, wheat-middlings 0.1%,
Stone flour 1.3%, calcium monohydrogen phosphate 0.6%, salt 0.3%, lysine 0.3%, solid methionine 1%, threonine 0.4%, the first premix
1%。
Embodiment 3
Corn 65%, dregs of beans 15%, corn protein powder 3%, vinasse albumen feed (DDGS) 6%, level Four soybean oil 2%, wheat-middlings 4.9%,
Stone flour 1.3%, calcium monohydrogen phosphate 0.6%, salt 0.3%, lysine 0.3%, solid methionine 0.4%, threonine 0.2%, the first premix
Material 1%.
In the present invention, the low energy daily ration in feed includes the component of following weight percentage, and specific embodiment is as follows：
Embodiment 4
Corn 58.95%, dregs of beans 28%, corn protein powder 4%, vinasse albumen feed (DDGS) 5%, level Four soybean oil 0.4%, wheat-middlings
0.1%, stone flour 1.3%, calcium monohydrogen phosphate 0.6%, salt 0.3%, lysine 0.3%, solid methionine 0.03%, threonine 0.02%, the
Two premixes 1%.
Embodiment 5
Corn 59.82%, dregs of beans 14%, corn protein powder 5%, vinasse albumen feed (DDGS) 6%, level Four soybean oil 4%, wheat-middlings 5%,
Stone flour 1.5%, calcium monohydrogen phosphate 1.5%, salt 0.4%, lysine 0.5%, solid methionine 1.2%, threonine 0.08%, the second premix
Material 1%.
Embodiment 6
Corn 55%, dregs of beans 25%, corn protein powder 4%, vinasse albumen feed (DDGS) 5%, level Four soybean oil 1%, wheat-middlings 5%, stone
Powder 1.5%, calcium monohydrogen phosphate 1.5%, salt 0.4%, lysine 0.5%, solid methionine 0.05%, threonine 0.05%, the second premix
Material 1%.
Embodiment 7
Corn 65%, dregs of beans 15%, corn protein powder 4%, vinasse albumen feed (DDGS) 5%, level Four soybean oil 1%, wheat-middlings 5%, stone
Powder 1.5%, calcium monohydrogen phosphate 1.5%, salt 0.4%, lysine 0.5%, solid methionine 0.05%, threonine 0.05%, the second premix
Material 1%.
In the present invention, the embodiment of feed addictive is specific as follows：
Embodiment 8
Chicken pancreatic lipase 40%, lipase from Aspergillus Niger 60%.
Embodiment 9
Chicken pancreatic lipase 60%, lipase from Aspergillus Niger 40%.
Embodiment 10
Chicken pancreatic lipase 50%, lipase from Aspergillus Niger 50%.
Embodiment 11
Chicken pancreatic lipase 45%, lipase from Aspergillus Niger 55%.
Embodiment 12
Chicken pancreatic lipase 55%, lipase from Aspergillus Niger 45%.
In the present invention, the chicken pancreatic lipase is the institute by obtained in following " isolating and purifying for chicken pancreatic lipase " techniques
It is powdered to state lipase from Aspergillus Niger, and mesh number scope is the mesh of 30 mesh -50.
First, chicken pancreatic lipase is isolated and purified
The preparation of 1.1 thick chicken pancreatic lipases
（111）, take the good chicken pancreas of Liquid nitrogen storage, with tissue mashing machine stir into pancreas starch, add buffer solution（Used in the present embodiment
Be phosphate buffer, naturally it is also possible to be other buffer solutions of phase same-action, other buffer solutions belong to prior art, herein
No longer repeat one by one）, 4 DEG C of magnetic agitation 2h；
（112）, filtered with double gauze, take solution, 1500rpm-2000rpm centrifugation the first sediments of removal, the present embodiment is
With 1500rpm, solution adds the acetone for being cooled to 4 DEG C, is continued to stir 25min-40min with magnetic stirring apparatus, and the present embodiment is
30min, then under the conditions of 4 DEG C, 2000rpm centrifugation 20min, the second sediment is stirred with 75% acetone solution, then is centrifuged, and is obtained
3rd sediment；
（113）, the 3rd sediment be placed on freeze-drying in cryogenic vacuum, obtain fixed powder；
（114）, lyophilized solid powder be added to 4 DEG C of chloroform-methanol（Chloroform is 2 with the volume ratio of methyl alcohol:1）In, magnetic force
Stirring 20min, then under the conditions of 4 DEG C, 2000rpm centrifugations, obtains the 4th sediment, and the 4th sediment is dissolved in ether, then 4
Under the conditions of DEG C, 2000rpm centrifugation, obtain the 5th sediment, the 5th sediment is dissolved in acetone, then the 5th sediment is placed on into low temperature
Freeze-drying in vacuum, obtains thick enzyme powder.
1.2 chicken pancreatic lipases are isolated and purified
1.2.1 ammonium sulfate precipitation
（1211）, above-mentioned thick enzyme powder be dissolved in 50mL deionized waters be made crude enzyme liquid, will be equipped with the small beaker of 50mL crude enzyme liquids
It is placed in another large beaker equipped with a small amount of frozen water, is placed in being slowly stirred on magnetic stirring apparatus；
（1212）, while be slowly stirred, while being slowly added to 10.00g, 15.00g, 20.00g, 25.00g, 30.00g, 35.00g
Ammonium sulfate is to final concentration of 20%, 30%, 40%, 50%, 60%, 70%（w/v）, the step should complete in 10min；Then proceed to stir
Mix 15min；
（1213）, then 10000rpm centrifugation 10min；Abandoning supernatant, 1 ~ 2 times of sediment volume is suspended in by precipitation
0.05mol/LpH is in 7.5 phosphate buffers, 10000rpm is centrifuged away the insoluble substance of residual；
（1214）, determine supernatant in enzyme activity and protein content, calculate concentration enzyme liquid Rate activity and purification；Its
In, Rate activity=enzyme activity unit/milligram protein；Purification=often walk Rate activity/first step Rate activity
1.2.2 it is concentrated by ultrafiltration
（1221）, using it is preceding by 15mL molecular cut offs for 10kDa super filter tube（Millipore, the U.S.）, filled it up with water ,-
Precooling 10min in 20 DEG C of refrigerators, water is poured out；
（1222）, by enzyme liquid add super filter tube inner tube, under the conditions of 4 DEG C, 6000rpm centrifugation 20min；
（1223）, remove the waste liquid of outer tube, inner tube is tipped upside down in outer tube；
（1224）, under the conditions of 4 DEG C, 3000rpm centrifugation 10min；
（1225）, outer tube liquid be enzyme liquid after ultrafiltration, be transferred in clean centrifuge tube；
（1226）, determine ultrafiltration after enzyme liquid enzyme activity and protein content, calculate Rate activity value and purification.
1.2.3 sephadex（Sephadex G-100）Molecular filtration is chromatographed
1.2.3.1 sephadex（Sephadex G-100）Pretreatment
（12311）, weigh 20 grams of sephadexes（Sephadex G-100）Particle adds 500mL deionized waters in beaker,
Soak swelling 48h；
（12312）, by sephadex（Sephadex G-100）The aqueous solution is contained in bottle,suction, and cup is clogged with ear washing bulb
Mouthful, decompression pumping 30min removes the bubble in gel solution, and the gel solution after degassing is gently poured into beaker；
1.2.3.2 post is filled
（12321）, chromatographic column（1.2×90cm）Cleaned up with deionized water, two ends are tightened, be perpendicularly fixed at fixed mount
On.Post upper port injection water is opened, makes to be retained in post the water for having 1/4-1/5；
（12322）, slowly stir gel solution, and pour into chromatography pipe along glass rod immediately, make the gel of addition natural in post
Sedimentation, after after the gel of the long-pending about 1-2cm that gets up on post bottom surface, opens column outlet current.With the outflow of water in post, above constantly
Coagulant liquid is added, makes there is gel continuously to decline on the gel bed surface to be formed；
（12323）, gel deposit at the top about 3cm of post, stop dress post, make the water surface in post left higher than gel bed interface 1cm
It is right；
（12324）, observation post inner gel it is whether uniform, whether " lines " or bubble.If chromatographic column heterogeneity, it is necessary to fill again
Post；
（12325）, after chromatographic column installs, make gel bed stabilization 30min, then the outlet of connection wash-out bottle and chromatographic column top, use
The 3-5 times of 0.05mol/LpH7.5 phosphate buffers balance chromatographic column of volume.Control the pressure of column-loading buffer to keep constant, protect
Flow velocity 0.3mL/min is held, until the pH of efflux is identical with column-loading buffer.
1.2.3.3 sample elution
（12331）, sample elution loading front opening chromatographic column upper end, first check gel bed interface it is whether smooth, if incline not
It is smooth, after gel bed interface can gently be stirred with glass rod, then make gel natural subsidence, form the gel bed circle of formation state
Face.Then allow liquid level to decline naturally, expose gel bed interface；
（12332）, with pipettor enzyme liquid is added drop-wise on gel bed surface, open constant flow pump, the mL/min of coutroi velocity 0.3 treats enzyme
After liquid all flows into gel bed, with buffer solution to 2cm higher than bed surface；
（12333）, with 0.05mol/LpH7.5 phosphate buffers elute, control elution flow rate 0.3mL/min, use part collector
Collect, one is collected per 2min and is managed；
（12334）, using the optical density A values of the every pipe collection liquid of nucleic acid-protein analyzer the real time measure, be horizontal seat with test tube numbering
Mark, optical density A values are ordinate, draw elution curve；The test tube near each crest is selected, enzyme activity is determined, and SDS- is poly-
Acrylamide coagulates（SDS-PAGE）Detect the purity of enzyme.
1.2.4 SDS- polyacrylamide gel electrophoresises（SDS-PAGE）Detection
1.2.4.1 spacer gel is prepared
Add various reagents to prepare 10% separation gel by table 1, mix, in pouring into the good glass sheet separation of sealing, Ran Houjia
Enter a certain amount of hydraulic pressure glue, stand 30min.Water is outwelled, the liquid remained on glue plate is blotted with filter paper, added respectively according to table 2
The concentration glue of preparation of reagents 5% is planted, is mixed, poured into rapidly above the separation gel solidified in rubber moulding, comb is plugged immediately, stood
30min。
1.2.4.2 preparation of samples
Each sample takes 20.0 μ L, adds 5.0 μ L5 × sample-loading buffer, is vortexed and mixes, 90 DEG C of metal bath 10.0min, cold on ice
But, concussion is vortexed, of short duration centrifugation, adds comb hole, last hole to add 5.0 μ L protein marks 15.0 μ L samples with loading pipette tips
Note thing（Protein Marker）.
The polyacrylamide gel electrophoresis of table 1 10%（PAGE）Separation gel is formulated
The polyacrylamide gel electrophoresis of table 2 5%（PAGE）Concentration glue formula
| Composition | Addition |
| 1.13mL | |
| 30% polyacrylamide | 0.33mL |
| 1.5mol/L tri- (methylol) aminomethane（Tris-HCl）（pH8.8） | 0.5mL |
| 10% anionic detergent（SDS） | 20.0μL |
| 10% ammonium persulfate | 20.0μL |
| Tetramethylethylenediamine（TEMED） | 4.0μL |
1.2.4.3 electrophoresis
1 × electrophoretic buffer is filled it up with electrophoresis tank.About 20 ~ 30min is first run with 60V voltages, until protein band is condensed to
One line；Then adjustment voltage runs about 90min to 100V, until blue bromophenol blue goes to blob of viscose bottommost.
1.2.4.4 dyeing-decolorzing
After electrophoresis is complete, blob of viscose is carefully removed, be put into the culture dish equipped with coomassie brilliant blue staining liquid, culture dish is placed on shaking table
Upper constantly vibration, dyes 40min；Dyeing liquor is outwelled, destainer is changed into, decolourized 4 ~ 5 times, the every 30 ~ 60min of destainer changes one
It is secondary.
1.2.4.5 photographic analysis
After the completion of decolouring, taken pictures with digital camera.
1.3 ammonium sulfate precipitation methods are to chicken pancreatic lipase extraction effect
Effect of the ammonium sulfate precipitation method of table 3 in extraction at the beginning of zymoprotein
Effect of the ammonium sulfate precipitation method in extraction at the beginning of zymoprotein is shown in Table 3, as shown in Table 3, in 30% ammonium sulfate saturation degree
When, Lipase protein starts to separate out, and during to 50% saturation degree, the zymoprotein in crude enzyme liquid is extracted completely substantially, and now enzyme activity is returned
Yield reaches 53.76%, and Rate activity is 6.14U/mg rot, and purification is maximum, 43.89 is reached, with ammonium sulfate saturation degree
Continue to increase, foreign protein increases, occur Rate activity and purification reduction on the contrary, therefore, the ammonium sulfate that this experiment chooses 50% is satisfied
With degree as zymoprotein in crude enzyme liquid initial concentration extracting method.
1.4 sephadexes（Sephadex G-100）Filtration chromatography isolates and purifies chicken pancreatic lipase result of the test
Fig. 1 sephadexes（Sephadex G-100）Filtration chromatography elution curve
The zymoprotein that 50% ammonium sulfate saturation degree chromatographs out is redissolved into buffer solution, 10kDa super filter tube ultrafiltration, after ultrafiltration
Solution carries out sephadex（Sephadex G-100）Filtration chromatography, elution curve such as Fig. 1 carries out enzyme activity detection and finds,
The eluting peak occurred in 100 ~ 110 pipe has stronger lipase activity.Each eluting peak separation condition preferably, is suitable as chicken
Pancreatic lipase albumen is isolated and purified.
The purity detecting result of 1.5 chicken pancreatic lipases
The purity detecting of chicken pancreatic lipase is shown in Fig. 2
The SDS- polyacrylamide gel electrophoresises of Fig. 2 purification steps（SDS-PAGE）Detection
Note：1, protein label is followed successively by from left to right（Protein Marker）2nd, sephadex（Sephadex G-
100）Purification of Lipase 3, lyophilized enzyme powder 4, ammonium sulfate precipitation enzyme liquid 5, ultrafiltration enzyme liquid
As can be seen from Figure 2, this experiment has finally given the lipase from chicken pancreas with single protein band, and molecular weight is
32kDa, has lipolytic activity through Enzyme activity assay, can carry out subsequent experimental research.
The purification result of 1.6 chicken pancreatic lipases
Table 4 chicken pancreatic lipase isolates and purifies result
Chicken pancreatic lipase powder is redissolved in buffer solution, through ammonium sulfate precipitation, ultrafiltration and sephadex（Sephadex G-100）
Filtration chromatography, as shown in table 4, the pancreatic lipase Rate activity for finally giving is 8.70U/mg prot, purification to the result for obtaining
It is 61.93, the rate of recovery is 4%.
Hydrolysis selectivity of the 1.7 chicken pancreatic lipases to different length carbochain
The chicken pancreatic lipase of table 5 hydrolyzes selectivity to different length carbochain
| pNPB（C4） | pNPO（C8) | pNPL（C12） | pNPP（C16） | |
| With respect to enzyme activity（%） | 10.86 | 100.00 | 90.80 | 62.55 |
Chicken pancreatic lipase is shown in Table 5 to different length carbochain hydrolysis selectivity, and chicken pancreatic lipase after purification is to pNPO（C8）Have
Highest hydrolysing activity, enzyme hydrolysis chain length selectivity is pNPO（C8）>pNPL(C12)>pNPP(C16)>PNPB (C4), to pNPB
（C4）Relative enzyme activity it is minimum, only 10.86%.
2nd, the reaction of separate sources lipase hydrolysis fish oil and its product analysis
2.1 hydrolysis fish oil reactions
1.0000g fish oil, 2mL phosphate buffer solutions are added in shellfish plug bottle（0.05mol/L, pH7.5）, 1mL lipase solutions,
Gas bath concussion reaction 12h under the conditions of 40 DEG C.
2.2 fatty acid extractions
5.0mL petroleum ethers are added in fish oil enzymolysis mixed reaction solution, fully 10min is stood after vibration, 50.0mL is then transferred to
Centrifuge tube 5000rpm is centrifuged 10min.
2.3 methyl esterification of fatty acid
Aspirating adipose acid extraction is centrifuged supernatant solution 1.0mL, adds 5.0mL1% sulfuric acid（H2SO4）- methanol solution, 70 DEG C of water-baths
Reaction 60min, treats that solution reaction terminates, and adds 2.5mL isooctane and 0.5mL inner mark solutions to mix after cooling, is subsequently added
7.0mL saturated nacl aqueous solutions vibrate 3min, and it is standby to draw upper solution after 5000rpm centrifugations 10min.
2.4 gas chromatography-mass spectrographies
According to GB《The gas chromatographic analysis of GBT17377-2008 animal and plant fat fatty acid methyl esters》Step operation, chromatographic column：
DB-5 fused-silica capillary columns（30 m*0.32 mm*0.25 um）；Column temperature：Temperature programming, 150 DEG C of holdings of initial temperature
5min, 220 DEG C are warming up to 5 DEG C/min speed, keep 1min, then are warming up to 260 DEG C with 10 DEG C/min speed, keep 5min；
Injector temperature：260℃；Detector temperature：280℃；Carrier gas is high pure nitrogen；Mass Spectrometry Conditions：Ion gun is 230 DEG C, electronics
Energy 70eV, MS level Four bar temperature is 150 DEG C.
Influence of the 2.5 separate sources lipase hydrolysis products to microbial growth
2.5.1 separate sources lipase hydrolysis fish oil product component
The separate sources lipase hydrolysis fish oil product component analysis of table 6
| Chicken pancreatic lipase | Lipase from Aspergillus Niger | Embodiment 10（Chicken pancreatic lipase：Lipase from Aspergillus Niger=1:1） | |
| C6 | - | - | - |
| C8 | - | - | - |
| C10 | - | - | - |
| C12 | 0.03 | 0.04 | 0.03 |
| C14 | 0.32 | 0.54 | 0.39 |
| C16：1 | 0.83 | 0.11 | 0.08 |
| C16 | 10.5 | 19.93 | 18.31 |
| C18：2 | 4.01 | 5.52 | 4.03 |
| C18：1 | 7.32 | 9.64 | 9.18 |
| C18 | 1.17 | 2.37 | 1.85 |
| C20：1 | 0.11 | 0.16 | 0.12 |
| C20 | 0.06 | 0.16 | 0.12 |
| Amount to | 24.35 | 38.47 | 34.11 |
Chicken pancreatic lipase, lipase from Aspergillus Niger, and combinations thereof hydrolysis fish oil composition analysis be shown in Table 6, as shown in Table 6, chicken pancreas
Lipase and lipase from Aspergillus Niger hydrolysate based on 16 carbon and 18 carbon, lipase from Aspergillus Niger and combined lipase
（In the present invention, combined lipase is to refer to chicken pancreatic lipase and lipase from Aspergillus Niger according to 1:Mixture after 1 combination）To 16
Carbon, 18 carbon and total percent hydrolysis apparently higher than only addition chicken pancreatic lipase is hydrolyzed when percent hydrolysis, it is seen then that chicken pancreatic lipase and black
Aspergillus niger lipase enzyme is according to 1:After the combination of 1 ratio, to fatty hydrolysis effect more preferably.
2.5.2 separate sources lipase hydrolysis fish oil product regulation and control Bacillus acidi lactici is bred
The influence that the separate sources lipase of table 7 is bred to Bacillus acidi lactici
| Blank | Chicken pancreatic lipase | Lipase from Aspergillus Niger | Embodiment 10（Chicken pancreatic lipase：Lipase from Aspergillus Niger=1:1） | |
| 3h | ||||
| 6h | ||||
| 12h | 1.951±0.032 | 1.929±0.011 | 1.905±0.003 | 1.928±0.013 |
Note：N=5, numeral represents average in table（M）± standard error（SE）, colleague's shoulder mark difference lowercase letter indication difference is notable
（P<0.05）, shoulder mark is without letter or has same letter to represent that difference is not notable（P>0.05）, similarly hereinafter.
Chicken pancreatic lipase, lipase from Aspergillus Niger, and combinations thereof hydrolysis fish oil product the influence that Bacillus acidi lactici is bred is shown in Table
7, as shown in Table 7, chicken pancreatic lipase and combined lipase hydrolyze fish oil product and can significantly improve the propagation of 3h, 6h Bacillus acidi lactici（P
<0.05）, it is seen then that for only addition lipase from Aspergillus Niger, the product of the hydrolysis fish oil of combined lipase can be carried significantly
The propagation of 3h, 6h Bacillus acidi lactici high, but all lipase hydrolysis fish oil products are not notable to Bacillus acidi lactici 12h proliferative effects（P
>0.05）, because now Bacillus acidi lactici has reached stable state.
2.5.3 separate sources lipase hydrolysis fish oil product regulation and control Escherichia coli breed
The influence that the separate sources lipase hydrolysis fish oil product of table 8 is bred to Escherichia coli
| Blank | Chicken pancreatic lipase | Lipase from Aspergillus Niger | Embodiment 10（Chicken pancreatic lipase：Lipase from Aspergillus Niger=1:1） | |
| 3h | ||||
| 6h | ||||
| 9h | 1.578±0.003 | 1.438±0.074 | 1.613±0.102 | 1.393±0.066 |
Chicken pancreatic lipase, lipase from Aspergillus Niger, and combinations thereof hydrolysis fish oil product be shown in Table 8 to the influence that Escherichia coli breeds, try
Test and show, chicken pancreatic lipase and combined lipase hydrolyze fish oil product and can significantly improve the propagation of 3h, 6h Escherichia coli（P<
0.05）, three kinds of lipase hydrolysis fish oil products can significantly improve Escherichia coli 6h propagation（P<0.05）, but all lipase hydrolysis
Fish oil product is not notable to the Escherichia coli equal difference of 9h proliferative effects（P>0.05）, because now Escherichia coli have reached stabilization shape
State.
3rd, feeding experiment
3.1 experimental animals and packet
Choose emerging yellow II broiler chicken of 1 age in days（Cock）2100 plumages be divided into first group, second group, the 3rd group, the 4th group, the 5th
Group totally 5 treatment groups, every group of 6 repetitions, each repeats 70 chickens.First group of feeding basal diet for the treatment of group, treatment group second
Group feeding low energy daily ration（Basal diet level 0.29 MJ/kg metabolizable energies of reduction）, the 3rd group for the treatment of group, the 4th group, BSA
200g/T chickens pancreatic lipase, 200g/T lipase from Aspergillus Niger and 200g/T embodiments 10 are not added on the basis of low energy daily ration.Examination
Test packet situation and be shown in Table 9：
Table 9 tests packet situation
| Treatment group | Packet |
| First group | Basal diet |
| Second group | Low energy daily ration（Reduce 0.29MJ/kg metabolizable energies） |
| 3rd group | Low energy daily ration+200g/T chicken pancreatic lipases |
| 4th group | Low energy daily ration+200g/T lipase from Aspergillus Niger |
| 5th group | The embodiment 10 of low energy daily ration+200g/T |
3.2 metabolic tests
3.2.1 metabolic test packet
9 points of table is 5 treatment groups（It is grouped with pilot production）, 6 repetitions of each treatment group, each repeats 4 chickens, same to support one
In individual metabolic cage, free water.
3.2.2 metabolism testing method
Endogenous indicator method is used, each treatment group feeds basal diet 4d, free choice feeding；Then prohibit and raise 17h, free water；Prohibit and raise
After end, each group chicken feeding alignment processing group experiment diet 55h, fracture, cervical dislocation execution after 4h, collect ileum immediately after
End excrement, 10% hydrochloric acid fixed nitrogen is added by weight volume.
3.2.3 testing index and method
After the completion of metabolic test, energy, dry, crude protein, crude fat and ash insoluble in hydrochloric acid in measure feed and excrement sample
Content.Calculate effective metabolizable energy value.
Dry：Determine with reference to National Standard of the People's Republic of China GB/T 6435-2006.
Energy：The WGR-1A micro computer calorimeter measurements produced with Changsha Instruments Factory.
Crude protein：Using the micro nitriding of kelvin, with the KJELTEC ANALYZER of full-automatic azotometer FOSS 2300
UNIT is determined.
Crude fat：Using Soxhlet extraction, determine with reference to National Standard of the People's Republic of China GB/T 6433-2006.
Ash insoluble in hydrochloric acid：Determine with reference to National Standard of the People's Republic of China GB/T 23742-2009.
3.3 slaughter experiments
At the end of each feeding experiment stage, each repeats to choose 2 chickens close to average weight, is butchered.Chicken is butchered
Afterwards, chest muscle and leg flesh are gathered, and claims live-weight, slaughter traits, half net thorax weight, complete net thorax weight, chest muscle weight, leg flesh weight, abdominal fat before carcass
Weight, calculates dressing percentage, half net thorax rate, complete net thorax rate, chest muscle rate, leg flesh rate and abdominal fat.
Live-weight before carcass：Stop to raise the weight after 12h before butchering.
Slaughter traits：Bloodletting is molted the weight after hair（Wet method epilation is weighed after need to draining）.
Half net thorax weight：Slaughter traits remove tracheae, esophagus, crop, intestines, spleen, pancreas, reproductive organs；Retain the heart, lung, courage（Remove courage
Capsule）, kidney, muscular stomach, glandular stomach（Exenterate and cutin membrane）And abdominal fat（Belly leaf fat and muscular stomach peripheral adipose）Weight.
Complete net thorax weight：Half net thorax removes the heart, lung, liver, kidney, glandular stomach, muscular stomach, abdominal fat and head again（Before atlas）, pin
Weight.
Chest muscle weight：The weight that carcass chest muscle is stripped down.
Leg flesh weight：By the peeling of chicken leg portion, the muscle weight boned.
Abdominal fat weight：Including abdominal fat（Belly leaf fat）And muscular stomach fat.
3.4 result of the tests
3.4.1 influence of the daily ration containing separate sources lipase to yellow-feather broiler Slaughter
Influence (%) of daily ration of the table 11 containing separate sources lipase to yellow-feather broiler Slaughter
| First group（Basal diet） | Second group（Low energy daily ration） | 3rd group（Low energy daily ration+200g chicken pancreatic lipases） | 4th group（Low energy daily ration+200g lipase from Aspergillus Niger） | 5th group（The embodiment 10 of low energy daily ration+200g/T） | |
| Dressing percentage | 0.930±0.009 | 0.920±0.005 | 0.920±0.003 | 0.926±0.004 | 0.939±0.002 |
| Half net thorax rate | 0.842±0.007 | 0.820±0.008 | 0.838±0.014 | 0.840±0.006 | 0.841±0.005 |
| Complete net thorax rate | 0.692±0.009 | 0.686±0.014 | 0.693±0.017 | 0.690±0.005 | 0.690±0.006 |
| Chest muscle rate | 0.075±0.001 | 0.072±0.002 | 0.072±0.002 | 0.079±0.003 | 0.073±0.001 |
| Leg flesh rate | 0.109±0.002 | 0.107±0.001 | 0.106±0.003 | 0.105±0.002 | 0.108±0.002 |
| Abdominal fat | 0.039±0.003 | 0.036±0.005 | 0.036±0.006 | 0.032±0.004 | 0.030±0.003 |
Dressing percentage aspect, first group is compared with basal diet group, and the 3rd group, the 4th group change of test group less, tests the 5th group
0.97% is improved, difference is not notable（P>0.05）.Second group is compared with low energy diet group, and the change of each test group is little.
Half net thorax rate aspect, first group of basal diet group, second group of low energy diet group, the 3rd group of test group, the 4th group, the
Five groups of this 5 groups are maintained between 0.820-0.850, and change is little.0.680- is maintained between complete net thorax rate aspect, 5 groups
Between 0.700, change is little.
Chest muscle rate aspect, first group of basal diet group, second group of low energy diet group, the 3rd group of test group, the 4th group, the 5th
The data for organizing this 5 groups are maintained between 0.070-0.080, and change is little.
Leg flesh rate aspect, first group of basal diet group, second group of low energy diet group, the 3rd group of test group, the 4th group, the 5th
The data for organizing this 5 groups are maintained between 0.100-0.110, and change is little.
Abdominal fat aspect, first group is compared with basal diet group, the 3rd group of test group, the 4th group, BSA do not reduce
7.69%th, 17.95%, 23.08%, wherein the 5th group of highest, the 5th group and first group of significant difference（P<0.05）.With low energy daily ration
Second group of group compares, and the 3rd group of test group maintains an equal level, and the 4th group of experiment, BSA do not reduce by 11.11%, 16.67%, respectively with the
One group of significant difference（P<0.05）.
On the whole, after adding separate sources lipase in daily ration, to dressing percentage, half net thorax rate, complete net thorax rate, chest muscle
Rate, the influence of leg flesh rate substantially, do not have rule.Abdominal fat aspect, reduces substantially after addition separate sources lipase and difference is aobvious
Write（P<0.05）, abdominal fat is can obviously reduce after adding separate sources lipase in this explanation daily ration.
3.5 Biochemical Indices In Serums
3.5.1 blood serum sample collection
At the end of feeding experiment, each 2 chicken for repeating to choose close to average weight carries out wing venous blood sampling, stands, 3000rpm
Centrifugation 10min, serum is sub-packed in centrifuge tube after centrifugation, and -30 DEG C of preservations are to be measured.
3.5.2 Serum Indexes result of the test
Venous blood collection, determines T-CHOL, low-density lipoprotein（LDL）, HDL（HDL）And glucose content.Institute
There is index to build up bioengineering kit measurement using Nanjing.
Influence of daily ration of the table 12 containing separate sources lipase to yellow-feather broiler Biochemical Indices In Serum
| First group（Basic day Grain） | Second group（Low energy Daily ration） | 3rd group（Low energy daily ration+200g chickens Pancreatic lipase） | 4th group（The black songs of low energy daily ration+200g Mould lipase） | 5th group（Low energy daily ration+200g/T's Embodiment 10） | |
| Glucose | |||||
| Cholesterol | |||||
| HDL（HDL） | |||||
| Low-density lipoprotein（LDL） | |||||
| HDL（HDL）/ low close Degree lipoprotein（LDL） |
Glucose aspect, first group is compared with basal diet group, the 3rd group of test group, the 4th group, the 5th group of change less, do not have
It is regular；Second group is compared with low energy diet group, the 3rd group of test group, the 4th group, BSA do not raise 20.73%,
16.97%th, 11.85%, wherein the 3rd group, the 4th group and second group of significant difference of low energy diet group（P<0.05）, each group difference
It is not notable（P>0.05）.
Cholesterol aspect, first group is compared with basal diet group, the 3rd group of test group, the 4th group, BSA do not reduce
8.12%th, 4.19%, 8.38%, difference is not notable（P>0.05）；Second group is compared with low energy diet group, the 3rd group of test group, the 4th
Group, BSA do not reduce by 17.80%, 14.29%, 18.03%, and difference is notable（P>0.05）, it is right after this explanation addition lipase
Reduce cholesterol substantially, wherein, it is most obvious with the 5th group of reduction.
HDL（HDL）, first group is compared with basal diet group, the 3rd group of test group, the 4th group, the 5th group of change
Change less, there is elevated trend, but difference is not obvious（P>0.05）；Second group is compared with low energy diet group, the 3rd group of test group,
4th group, BSA do not raise 4.57%, 12.57%, 9.71%, the 4th group, the 5th group and second group of comparing difference it is notable（P<
0.05）, each group difference is notable（P>0.05）.
Low-density lipoprotein（LDL）, first group is compared with basal diet group, the 3rd group of test group, the 4th group, BSA
Jiang Di not by 3.93%, 6.18%, 12.36%, the 5th group of test group and first group of significant difference（P<0.05）, group difference is notable
（P>0.05）；Second group is compared with low energy diet group, the 3rd group of test group, the 4th group, BSA do not reduce by 8.06%,
10.22%th, 16.13%, the 5th group of test group and second group of significant difference（P<0.05）, each group difference is notable（P>0.05）.
HDL（HDL）/ low-density lipoprotein（LDL）Aspect, first group is compared with basal diet group, test group
3rd group, the 4th group, BSA you can well imagine it is high by 3.88%, 14.56%, 19.42%；4th group, the 5th group and first group is compared, poor
It is different notable（P<0.05）, other each group differences are notable（P>0.05）；Second group is compared with low energy diet group, test group the 3rd
Group improve 13.83%, the 4th group of test group, BSA you can well imagine it is high by 25.53%, 30.85%, difference is notable（P<0.05）, each group
Between difference it is not notable（P>0.05）.
On the whole, after addition separate sources lipase, glucose content change is without evident regularity, and cholesterol reduction is bright
It is aobvious, significant difference；HDL（HDL）Improve notable；Low-density lipoprotein（LDL）Reduce obvious；HDL
（HDL）/ low-density lipoprotein（LDL）Improve notable（P<0.05）, especially in the 5th group, add chicken pancreatic lipase and aspergillus niger
After the combination of lipase, due to chicken pancreatic lipase and the synergy of lipase from Aspergillus Niger, cholesterol reduction is obvious, significant difference
（P<0.05）；HDL（HDL）It is more notable than second group of raising；Low-density lipoprotein（LDL）With first group and second group
Compare, reduce obvious；Compared with first group and second group, HDL（HDL）/ low-density lipoprotein（LDL）Improve aobvious
Write（P<0.05）.
3.6 intestine microbial diversity detection methods
3.6.1 microbial DNA is extracted
Using Tiangeng company's T IANamp StoolDNA Kit kits, main operational steps are as follows：
（1）In weighing fecal sample 200mg to 2mL centrifuge tubes, and pipe is placed on ice；
（2）Mixed to addition 1.4mL buffer solution GSL, intermittent oscillation 1min in sample to sample；
（3）70 DEG C of incubation 5min；
（4）Vortex 15s, 12,000rpm are centrifuged 1min；Shift supernatant 1.2mL to new 2mL centrifuge tubes；
（5）An inhibitor suction sheet InhibitEX is added, vibration thoroughly opens resuspended to suction sheet.Incubation at room temperature 1min, makes
Suction sheet can be acted on fully；
（6）12,000rpm is centrifuged 3min；
（7）Previous step gained supernatant is transferred to new 1.5mL centrifuge tubes, repeat step 6；
（8）The transfer gained μ L of supernatant 200 add 15 μ L protease k to new 1.5mL centrifuge tubes（Proteinase K）.
（9）Add 200 μ L buffer solution GB, vortex 15s；
（10）70 DEG C of incubation 10min.Brief centrifugation is collecting the drop in tube wall and lid；
（11）200 μ L absolute ethyl alcohols are added, is vortexed and is mixed.Brief centrifugation is collecting the drop in tube wall and lid；
（12）Previous step resulting solution is added in an adsorption column CR2（Adsorption column is put into collecting pipe）, 12,000rpm from
Heart 30s, outwells waste liquid, and adsorption column CR2 is put into collecting pipe；
（13）To adding 500 μ L buffer solutions GD in adsorption column CR2（Please first checked whether before and added absolute ethyl alcohol）, 12,
000rpm is centrifuged 30s, outwells waste liquid, and adsorption column CR2 is put into collecting pipe；
（14）To adding 600 μ L rinsing liquids PW in adsorption column CR2（Please first checked whether before and added absolute ethyl alcohol）, 12,
000rpm is centrifuged 30s, outwells waste liquid, and adsorption column CR2 is put into collecting pipe.
（15）Repeat step 14；
（16）Adsorption column CR2 is put back in collecting pipe, 12,000rpm centrifugation 2min outwell waste liquid.Adsorption column CR2 is placed in room
Temperature places several minutes, thoroughly to dry the rinsing liquid of remnants in sorbing material；
（17）Adsorption column CR2 is transferred in a clean centrifuge tube, 50 μ L are vacantly added dropwise to the middle part of adsorbed film elutes
Buffer solution TB, room temperature places 2-5min, 12,000rpm centrifugation 2min, and solution is collected into centrifuge tube；
（18）To be centrifuged during the solution that obtains adds adsorption column CR2, room temperature places 2min, 12,000rpm centrifugation 2min；DNA
Product should be stored in -20 DEG C, in case degraded.
3.6.2 the polymerase chain reaction of microorganism 16SrDNA genes V3 areas genetic fragment（PCR）Amplification
The polymerase chain reaction of target fragment is carried out to the DNA solution of sample microbial using the universal primer of bacterium（PCR）Expand
Increase, as shown in table 13, the primer is synthesized primer sequence used by Shanghai Sheng Gong bio tech ltd.
Using 25 μ L polymerase chain reactions（PCR）Amplification reaction system, including 2.5 μ LDNA templates, 12.5 μ L heat-resistant dnas
Polymerase（Taq enzyme）, 0.6 μ L sense primers（10μM）, 0.6 μ L anti-sense primers（10μM）, plus the μ of ddH2O to 25 L.
Polymerase chain reaction（PCR）Program is：94 DEG C of predegeneration 300s；94 DEG C of denaturation 30s；60 DEG C of 60 s of annealing；72℃
Extend 120s；30 circulations, last 72 DEG C re-extend 600s.
The polymerase chain reaction of gained（PCR）Product uses 1% agarose gel electrophoresis 20min under 120V voltages, so
Polymerase chain reaction is observed by gel imaging system afterwards（PCR）Product, and Taking Pictures recording.
The universal primer of the bacterium of table 13
Note：5 ' one 40bpGC hair clip of increase of F357 primers, are beneficial to follow-up denaturing gradient gel electrophoresis（DGGE）Analysis,
GC hairpins are CGCCCGCC GCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC.
3.6.3 polymerase chain reaction（PCR）The denaturing gradient gel electrophoresis of product（DGGE）Analysis
Using detection in Gene Mutation system（Bio-Rad DCode TM Universal Mutation Detection
System）To the polymerase chain reaction of bacterium（PCR）Product carries out denaturing gradient gel electrophoresis（Denaturing Gradient
Gel Electrophoresis, abbreviation DGGE）Analysis, denaturing gradient gel electrophoresis（DGGE）Operating procedure is as follows：
（1）1 × nucleic acid electrophoresis buffer solution of 6.50L is poured into electrophoresis tank（TAE）, design temperature is 60 DEG C, preheating；
（2）Respectively prepare 16mL40% denaturant and 16mL60% denaturant, then be separately added into 150 μ L10% ammonium persulfates and
15 μ L tetramethylethylenediamines（TEMED）；
（3）During various concentrations to suck into glue corresponding special syringe respectively, by the slow two pieces of glass of injection at the uniform velocity of glue
In the middle of glass plate, encapsulating is finished, and is rapidly inserted into comb, and it is solidifiable that glue is denatured after 2h；
（4）Gel glass is put into and is preheated in 60 DEG C of electrophoresis tank, then by 20 μ L polymerase chain reactions（PCR）Product is moved
Liquid device is added in loading wells, finally with 80V voltages, 60 DEG C of electrophoresis 16h；
（5）After electrophoresis is finished, gel is peeled off, 2min is rinsed with ultra-pure water；Ultra-pure water is outwelled, 350 mL fixers are added, placed
15min；Fixer is outwelled, continuation rinses 2min, outwells ultra-pure water with ultra-pure water；
（6）Pour into 350mL silver staining liquid, low speed lucifuge sways 10min；Silver staining liquid is outwelled, with deionized water rinsing twice, every time
5 ~ 10 s, add the colour developing of 350mL nitrite ions, treat that DNA bands show clear stopping；
（7）Nitrite ion is outwelled, fixer is poured into, dyeing is completed；The gel of silver staining is placed on white light plate, is clapped with digital camera
According to preservation picture.
3.6.4 Shannon ' s diversity indices is calculated
Use Quantity-One（Bio-Rad）Software analysis denaturing gradient gel electrophoresis（DGGE）Collection of illustrative plates, and calculated according to formula 1
Shannon ' s diversity indices H ＇.
Formula 1
In formula, s is each swimming lane band number, and pi is the ratio that each band intensity accounts for total band intensity, and H ＇ are that Shannon ' s are more
Sample sex index, the diversity level of the bigger explanation sample of its value is higher.
3.6.5 data statistics processing
Data statistical product and the software of service solution 18.0（SPSS）It is analyzed, as a result using average ± standard error
Method represent, one-way analysis of variance is carried out the data between different disposal group（one way ANOVE）, carried out with Duncan
Multiple range test,P<0.05 is significant difference.
Influence of the 3.7 separate sources lipase and combinations thereof to yellow-feather broiler ileum microbial diversity
The polymerase chain reaction of Fig. 3 and Fig. 4 microorganism 16SrDNA gene V3 areas genetic fragment（ PCR）
The agarose electrophoresis of product 1%
Fig. 5 microorganism 16SrDNA gene V3 areas genetic fragment denaturing gradient gel electrophoresis（DGGE）Fingerprint image spectrogram
Influence of daily ration of the table 14 containing separate sources lipase to yellow-feather broiler ileum microbial diversity
Fig. 3 and Fig. 4 represent microorganism 16SrDNA gene V3 areas genetic fragment polymerase chain reaction（PCR）The agarose of product 1% electricity
Swimming figure.Band is single and clear, in 200bp or so, can be used for carrying out denaturing gradient gel electrophoresis（DGGE）.
Fig. 5 is 16SrDNA gene V3 areas genetic fragment polymerase chain reaction（PCR）The denaturing gradient gel electrophoresis of product
（DGGE）The addition of finger-print, separate sources lipase and combinations thereof is shown in Table 14 to yellow-feather broiler ileum microbiological effect, leads to
Analysis is crossed to find difference and be applied in combination come lipase of originating that yellow-feather broiler ileum microbial diversity can be dramatically increased（P<0.05）.
Claims (10)
1. a kind of feed addictive containing separate sources lipase, it is characterised in that be made up of the component of following weight percentage：
Chicken pancreatic lipase 40%-60%, lipase from Aspergillus Niger 40%-60%.
2. the feed addictive containing separate sources lipase according to claim 1, it is characterised in that：By following weight hundred
Divide the component composition of ratio：Chicken pancreatic lipase 45%-55%, lipase from Aspergillus Niger 45%-55%.
3. the feed addictive containing separate sources lipase according to claim 1 and 2, it is characterised in that：The chicken pancreas
Lipase is prepared by following methods：
The preparation of thick chicken pancreatic lipase；
Chicken pancreatic lipase is isolated and purified；
（21）Ammonium sulfate precipitation；
（22）It is concentrated by ultrafiltration；
（23）Sephadex（Sephadex G-100）Molecular filtration is chromatographed；
（24）SDS- polyacrylamide gel electrophoresises（SDS-PAGE）Detection.
4. the feed addictive containing separate sources lipase according to claim 3, it is characterised in that：The thick chicken pancreas fat
The preparation of fat enzyme comprises the steps：
（11）Chicken pancreas is pounded pancreas slurry, buffer solution, magnetic agitation 2h under the conditions of 4 DEG C is added；
（12）Filtering, takes solution, and 1500rpm-2000rpm centrifugation the first sediments of removal, solution adds acetone, continues magnetic force and stir
25min-40min is mixed, then under the conditions of 4 DEG C, 1500rpm-2000rpm centrifugation 15min-30min, the second sediment uses 75%
Acetone solution is stirred, then is centrifuged, and obtains the 3rd sediment；
（13）The 3rd sediment is dried, solid powder is obtained；
（14）The solid powder is added to 4 DEG C of chloroform-methanol（Chloroform is 2 with the volume ratio of methyl alcohol:1）In, magnetic force is stirred
15min-30min is mixed, then under the conditions of 4 DEG C, 1500rpm-2000rpm centrifugations, the 4th sediment is obtained, the 4th sediment is dissolved in
In ether, then under the conditions of 4 DEG C, 1500rpm-2000rpm centrifugations, the 5th sediment is obtained, the 5th sediment is dissolved in acetone, then
By the 5th drying precipitate, thick enzyme powder is obtained.
5. the feed addictive containing separate sources lipase according to claim 3, it is characterised in that：The step（21）
Ammonium sulfate precipitation comprises the steps：
（211）, above-mentioned thick enzyme powder be dissolved in 50ml deionized waters be made crude enzyme liquid, will be equipped with the small beaker of 50 mL crude enzyme liquids
It is placed in another large beaker equipped with a small amount of frozen water, is placed in being slowly stirred on magnetic stirring apparatus；
（212）, while be slowly stirred, while being slowly added to 10.00g, 15.00g, 20.00g, 25.00g, 30.00g, 35.00g
Ammonium sulfate is to final concentration of 20%, 30%, 40%, 50%, 60%, 70%（w/v）, the step should complete in 10 min；Then proceed to
Stir 15 min-20min；
（213）, then 10000 rpm centrifugations 8-12 min；Abandoning supernatant, 1 ~ 2 times of sediment volume is suspended in by precipitation
0.05 mol/L pH are 7.5 phosphate buffers, and 10000 rpm are centrifuged away the insoluble substance of residual；
（214）, determine supernatant in enzyme activity and protein content, calculate concentration enzyme liquid Rate activity and purification；Its
In, Rate activity=enzyme activity unit/milligram protein；Purification=often walk Rate activity/first step Rate activity.
6. a kind of broiler fodder, it is characterised in that：The broiler fodder contains any one in claim 1 to claim 5
Feed addictive containing separate sources lipase, feed addictive is 0.01%-0.02%, and base-material is 99.8%-99.9%.
7. broiler fodder according to claim 6, it is characterised in that：The base-material is basal diet or low energy daily ration.
8. broiler fodder according to claim 7, it is characterised in that：Wherein, the basal diet is by following weight percentage
The component composition of ratio：Corn 50%-65%, dregs of beans 15%-30%, corn protein powder 3%-6%, vinasse albumen feed (DDGS) 4%-6%,
Level Four soybean oil 2%-6%, wheat-middlings 0.1%-5%, stone flour 1.3%-1.6%, calcium monohydrogen phosphate 0.6%-1.5%, salt 0.3%-0.4%, rely
Propylhomoserin 0.3%-0.5%, solid methionine 0.4%-1.2%, threonine 0.2%-0.8%, the first premix 1%.
9. application of a kind of feed addictive containing separate sources lipase in yellow-feather broiler fodder.
10. application of a kind of feed containing separate sources lipase on the aspect influence of yellow-feather broiler ileum microbial diversity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN103125765A (en) * | 2011-12-05 | 2013-06-05 | 深圳市绿微康生物工程有限公司 | Chicken feed containing lipase and preparation method of lipase |
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| CN103125765A (en) * | 2011-12-05 | 2013-06-05 | 深圳市绿微康生物工程有限公司 | Chicken feed containing lipase and preparation method of lipase |
Non-Patent Citations (2)
| Title |
|---|
| 冯定远、左建军著: "《饲料酶制剂技术体系的研究与实践》", 31 March 2011, 中国农业大学出版社 * |
| 王政等: "海洋脂肪酶及单细胞蛋白对白羽肉鸡生产性能和肠道微生物区系的影响", 《中国畜牧杂志》 * |
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Application publication date: 20170620 |