CN102154237A - Lipase capable of reducing cholesterol and blood fat levels in bodies of animals and use thereof - Google Patents
Lipase capable of reducing cholesterol and blood fat levels in bodies of animals and use thereof Download PDFInfo
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- CN102154237A CN102154237A CN2011100005355A CN201110000535A CN102154237A CN 102154237 A CN102154237 A CN 102154237A CN 2011100005355 A CN2011100005355 A CN 2011100005355A CN 201110000535 A CN201110000535 A CN 201110000535A CN 102154237 A CN102154237 A CN 102154237A
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Abstract
The invention applies for providing lipase capable of reducing cholesterol and blood fat levels in bodies of animals and use thereof. Researches on the influences of the addition of the lipase in daily rations on the growth property, apparent metabolizable energy, blood biochemical indexes and abdominal fat rate of commercial broiler chicken are performed. The results of the researches indicate that the addition of the lipase can improve the growth property and apparent metabolizable energy of boiler chicken and reduce the content of triglyceride, low-density lipoprotein and high-density lipoprotein in blood and abdominal fat rate and that the improvement on the e growth property and apparent metabolizable energy and the reduction in the triglyceride, low-density lipoprotein, high-density lipoprotein and abdominal fat rate reach the greatest extend in the middle term of a test.
Description
Technical field
The present patent application relates to a kind of lipase and application thereof that can effectively reduce animal body inner cholesterol, blood lipid level, belongs to biological technical field.
Background technology
Lipase, claim the triglyceride hydrolysis enzyme again, be the enzyme of class energy catalysis long-chain fat acid glyceride hydrolysis, also can catalysis be somebody's turn to do the reversed reaction of reacting, all right catalytic esterification of the lipase of many microorganism secretions, transesterification reaction, alcoholysis reaction, acidolysis reaction and ammonolysis reaction etc.
The activity of lipase is very low at birth in the poultry pancreas, the birth back increases gradually along with the rising of age in days, very poor during the fryer birth to the digestibility of animal tallow, increase gradually age in week at 1-8, than the animal innage, but the digestibility in two weeks of chick birth back is lower to the digestibility of vegetables oil for fryer.The lipase deficiency of poultry pancreatic secretion when birth just becomes the factor that the restriction fat digestion utilizes, and therefore, adds the digestibility that lipase can improve fat in corn-soybean meal diet, thereby influences the growth of animal.In feed, add lipase, can improve the fat digestibility and the capacity usage ratio of daily ration, thereby improve livestock and poultry production performance.
Summary of the invention
The present patent application promptly is at weak point of the prior art, and a kind of lipase and application thereof that can effectively reduce animal body inner cholesterol, blood lipid level is provided.
Specifically, the lipase of the described a kind of effective reduction animal body inner cholesterol of the present patent application, blood lipid level is characterized in that: described lipase is obtained by following method:
1, obtains hygromycin resistance expression cassette and being cloned on the target plasmid, obtain the recombinant plasmid of hygromycin resistance;
2, increase the respectively lipase gene (PEL) of mould, the strong promoter glyceraldehyde 3-phosphate dehydro-genase promotor (PgpdA) and the Aspergillus nidulans tryptophan synthetase terminator (TtrpC) of Aspergillus nidulans obtain the PEL expression casette that strong promoter drives;
3, this PEL expression casette is inserted in the recombinant plasmid of hygromycin resistance, has obtained to contain the PEL gene overexpression vector of hygromycin selection mark;
4, will contain the PEL gene overexpression vector conversion engineering Agrobacterium of hygromycin selection mark, and utilize the agrobacterium mediation converted method that the PEL expression casette is transformed in the penicillium bacterial strain, obtain the mould genetic engineering bacterium of high expression level lipase;
5, described mould genetic engineering bacterium is inserted seed culture medium with mycelium or spore suspension, under 25-35 ℃, 200-300rpm condition, cultivate after 24 hours, change fermention medium over to the 5%-20% inoculum size, after 2 days, obtain described lipase at 25-35 ℃, 150-300rpm condition bottom fermentation through separation and purification.
Wherein, the method for acquisition hygromycin resistance expression cassette comprises round pcr or Restriction Enzyme incision technology.
Obtain the lipase gene (PEL) of mould, the strong promoter glyceraldehyde 3-phosphate dehydro-genase promotor (PgpdA) of Aspergillus nidulans and the method for Aspergillus nidulans tryptophan synthetase terminator (TtrpC) and comprise pcr amplification or restriction enzyme clone or chemical synthesising technology clone.
The wherein said Penicillium notatum that is transformed is a penicillium expansum.
Described target plasmid is one of following:
PCAMBIA1300, pCAMBIA2300, pCAMBIA3300 or pHB.
Wherein, so the composition of substratum is as follows:
1, seed culture medium (%): soybean cake powder 4, W-Gum 0.8, Na NO
30.3, Na
2HPO
40.2, K
2SO
40.25, MgSO
40.03, FeSO
40.003;
2, fermention medium (%): soybean cake powder 6, W-Gum 1, NaNO
30.4, Na
2HPO
40.2, K
2SO
40.3, MgSO
40.035, FeSO
40.02, CaCO
30.5, Na
2CO
30.02.
The present patent application also requires to comprise the application of this lipase in reducing animal body inner cholesterol, blood lipid level.For example this lipase can be added in the daily feed of animal as animal feedstuff additive, improve the meat of animal.
Description of drawings
Fig. 1 is the structural representation of PV2+ carrier;
Fig. 2 is the structural representation of pCAMBIA2300 carrier;
Fig. 3 is the structural representation of pCAMBIA2302 carrier;
Fig. 4 is the structural representation of PMD18-T::gpdA-Lip-TtrpC carrier;
Fig. 5 is the structural representation of the final carrier of pCHAMBIA2302::PgpdA-Lip-TtrpC;
Fig. 6 is the structure route map of overexpression vector pCHAMBIA2302::PgpdA-PEL-TtrpC;
Fig. 7 identifies collection of illustrative plates for the PCR of the overexpression vector pCHAMBIA2302::PgpdA-PEL-TtrpC of structure;
Wherein, 1-6: independently Agrobacterium-mediated Transformation ,+: positive control ,-: negative control, M:DL-2000Marker;
Fig. 8 is that the PCR of mould transgenosis bacterial strain identifies collection of illustrative plates;
Wherein, 1-5: independently Agrobacterium-mediated Transformation ,+: positive control ,-: negative control, M:DL-2000Marker.
Embodiment
Below in conjunction with specific embodiment to the described a kind of effective reduction animal body inner cholesterol of the described the present patent application of the present patent application; the lipase of blood lipid level is described further; purpose is better to understand technology contents of the present invention for the public; rather than to the restriction of described technology contents; in fact; in spirit of the present invention; improvement to the described method of the present patent application; the replacement of target plasmid and restriction enzyme all is that persons skilled in the art need not performing creative labour and can obtain, therefore all within the present patent application technical scheme required for protection.
The structure of embodiment one, overexpression vector
Adopt following step:
(1) utilize restriction enzyme Sac I, Kpn I with the hygromycin resistance expression cassette from plasmid PV2
+Last enzyme cuts out (as shown in Figure 1), and fragment reclaims through gel, is cloned on the corresponding restriction enzyme site of pCAMBIA2300 plasmid (as shown in Figure 2), obtains to have the recombinant plasmid pCHAMBIA2302 (as shown in Figure 3) of hygromycin resistance; The Totomycin expression cassette also can contain any DNA of this sequence or directly synthetic from other, the plasmid that is used to make up PEL gene overexpression vector is gone back plasmid such as pCAMBIA serial carrier and pHB carriers such as pCAMBIA1300, pCAMBIA3300 that available energy is expressed in filamentous fungus except that pCAMBIA2300;
(2) according to the sequences Design primer (forward primer: TG of the lipase gene PEL (AF330635) of penicillium expansum P.expansium
ACTAGTThe sequence of ATGTTGTTCAACTACCAATCTTT underscore is Restriction Enzyme Spe I point of contact; Reverse primer: TGGATCC
GCGGCCGCThe sequence of TTATCAGCTCAGATAGC underscore is Restriction Enzyme Not I point of contact), utilize round pcr amplification complete genome sequence, the PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 90s, 35 circulations; 72 ℃ of 10min;
(3) according to the sequences Design primer (forward primer: GCTATCTGAGCTGATAA of plasmid pPAN7-1 (Punt et al.1987 Gene 56:117-124)
GCGGCCGCGGATCCACTTAACGTTA, the sequence of underscore is Restriction Enzyme Not I point of contact; Reverse primer:
GTTTAAACTCGAGTGGAGATGTGGAGTGGGCGC, the sequence of underscore is Restriction Enzyme PmeI point of contact), utilize round pcr to amplify tryptophan synthetase terminator TtrpC, the PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s, 35 circulations; 72 ℃ of 10min;
(4) each the 1 μ L of reaction solution that gets (2) and (3) is as template, with (TG
ACTAGTATGTTGTTCAACTACCAATCTTT) as forward primer; With (
GTTTAAACTCGAGTGGAGATGTGGAGTGGGCGC) as reverse primer, carry out pcr amplification, the reaction conditions of PCR is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 150s, 35 circulations; 72 ℃ of 10min.After reaction finishes, pcr amplification product is carried out gel column reclaim, and be cloned into PMD-18-T: carrier obtains intermediate carrier PMD-18-T::PEL-TtrpC;
(5) according to the plasmid pPAN7-1 (sequences Design primer (forward primer: GA of Punt et al.1987 Gene 56:117-124
GTCGACThe sequence of GAATTCCCTTGTATCTCTACACAC underscore is Restriction Enzyme Not I point of contact; Reverse primer: GA
ACTAGTThe sequence of CTGCTCAAGCGGGGTAGCTGTTAGT underscore is Restriction Enzyme PmeI point of contact) utilize round pcr amplification strong promoter PgpdA.The PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 150s, 35 circulations; 72 ℃ of 10min.The corresponding site that utilizes restriction enzyme Sal I and Spe I that PgpdA is cloned into carrier PMD-18-T::PEL-TtrpC obtains intermediate carrier PMD-18-T::PgpdA-PEL-TtrpC (as shown in Figure 4);
(6) utilize restriction restriction endonuclease Sal I and Pme I that carrier PMD-18-T::PgpdA-PEL-TtrpC is digested, reclaim expression cassette PgpdA-PEL-TtrpC, be cloned into the corresponding site of pCHAMBIA2302 then, obtain final carrier pCHAMBIA2302::PgpdA-PEL-TtrpC (as shown in Figure 5), whole building process as shown in Figure 6;
(7) bacterium that contains final carrier pCHAMBIA2302::PgpdA-PEL-TtrpC is carried out enlarged culturing, and extracting plasmid, utilize freeze-thaw method to transform engineering Agrobacterium EHA105, random choose 6 strain transformants, with (TGACTAGT ATGTTGTTCAACTACCAATCTTT) as forward primer;
As reverse primer, with the positive contrast of carrier PMD-18-T::PEL-TtrpC, the negative contrast of empty EHA105 is carried out PCR to this 6 strain bacterium and is identified that the result shows that this 6 strain bacterium is all positive, sees Fig. 7 with (TGGATCCGCGGCCGCTTATCAGCTCAGATAGC).
The acquisition of embodiment two, mould genetic engineering bacterium
The activity of described mould genetic engineering bacterium comprises following step:
(1) the wild-type mould after the picking separation and purification is inoculated on the PDA flat board, in 28 ℃ of cultivations about 20 days, washes ripe spore with sterilized water;
(2) the engineering Agrobacterium EHA105 that contains whole carrier pCHAMBIA2302::PgpdA-PEL-TtrpC in the example one is inoculated in the LB liquid nutrient medium that contains Streptomycin sulphate, kantlex (being 100 μ g/ml) 28 ℃, the 200rpm incubated overnight, reactivate with the MM substratum that contains Streptomycin sulphate and kantlex (being 100 μ g/ml), 28 ℃, 220rpm cultivated 48 hours.Draw the centrifugal supernatant that goes of an amount of culture 5000rpm, and, be diluted to OD600=0.15 with the IM liquid nutrient medium at last,, cultivated 6-8 hour under the condition of 220rpm, to OD600=0.5-0.6 then at 28 ℃ with the washing of IM liquid nutrient medium;
(3) the fresh spore that (1) step was obtained is mixed with 1 * 10
7The suspension of individual/mL concentration, getting above-mentioned spore suspension subsequently mixes with engineering Agrobacterium equal-volume in the step (2), getting 200 μ L evenly is applied on the IM solid medium (containing AS 200 μ g/mL) that is covered with glassine paper, cultivate 60h altogether for 28 ℃, then counter being taped against of glassine paper contained Totomycin (100 μ g/mL transform and select microbiotic), cynnematin (500 μ g/mL, inhibition Agrobacterium growth microbiotic) cultivated 2 days for 28 ℃ on the PDA substratum, take glassine paper off, under 28 ℃ of conditions, cultivated 1-3 days, with the transformant of moisture resistance mycin choose be inoculated into two the sieve substratum on, go down to posterity as stable, promptly be described mould genetic engineering bacterium, so obtained 30 strain mould genetic engineering bacteriums.Random choose 5 strain transformants carry out enlarged culturing, and difference extracting genomic dna, with (TG
ACTAGTATGTTGTTCAACTACCAATCTTT) as forward primer; With (
GTTTAAACTCGAGTGGAGATGTGGAGTGGGCGC) as reverse primer, with the positive contrast of carrier PMD-18-T::PEL-TtrpC, the negative contrast of wild-type mould genomic dna is carried out PCR to this 5 strain bacterium and is identified that the result shows that this 5 strain bacterium is all positive, sees Fig. 8.
The composition of MM substratum is as described below:
1M K
2HPO
4-KH
2PO
4(PH7.0) 10mL, M-N (MgSO
47H
2O 30g/L, NaCl 15g/L) 20mL, 1%CaCl
22H
2O 1mL, 20% glucose 10mL, 0.01%FeSO
410mL, Spore element (ZnSO
47H
2O 100mg/L, CuSO
45H
2O 100mg/L, H
3BO
3100mg/L, MnSO
4H
2O 100mg/L, Na
2MoO
42H
2O 100mg/L) 5mL, 20%NH
4NO
32.5mL, sterilized water 941.5mL.
The composition of IM substratum is as described below:
1M K-buffer (PH4.9) 10mL, M-N (MgSO
47H
2O 30g/L, NaCl 15g/L) 20mL, 1%CaCl
22H
2O 1mL, 20% glucose 10mL, 0.01%FeSO
410mL, Spore element (ZnSO
47H
2O 100mg/L, CuSO
45H
2O 100mg/L, H
3BO
3100mg/L, MnSO
4H
2O 100mg/L, Na
2MoO
42H
2O 100mg/L) 5mL, 20%NH
4NO
32.5mL, 50% glycerine 10mL, 1M MES (PH5.5) 40mL, 100mM AS (Syringylethanone) 2mL, sterilized water 898.7mL
The CM substratum: add the glucose amount of half IM substratum, add 1.5% agar, other composition is with the IM substratum.
PDA substratum (g/L): potato, 200; Sucrose, 20; Agar, 15; The PH nature.
Embodiment three, yielding lipase ability contrast experiment
The mould genetic engineering bacterium of random choose example 2 gained is inoculated on the PDA substratum, cultivate and insert respectively among the seed culture medium 50mL after 10 days, at 28 ℃, the 210rpm shaking table is cultivated 24h, change fermention medium (the bottled 30mL fermention medium of 250mL triangle) respectively over to 10% inoculum size then, at 28 ℃, 210rpm condition bottom fermentation 48h; Then that fermented liquid is centrifugal, get supernatant liquor and carry out the lipase activity detection.
Utilize acid base titration that lipase activity is detected.
Step:
Get 20 of 100mL triangular flasks, add Gly-NaOH damping fluid and the 5.0mL sweet oil emulsion of 4.0mL pH9.4 respectively; Put into concussion thermostat water bath 36 ℃ of water-bath preheatings 5 minutes. after enzyme liquid filters, dilute with the Gly-NaOH damping fluid of 0.05mol/L pH9.4 that to make enzyme work be to measure behind the 4-5u/mL.Toward wherein two respectively add 1mL and dilute good enzyme liquid, slowly vibration (60 times/min) 10 minutes (accurately timing). do blank for other two bottles. add 95% alcohol 20mL (blank adds 1mL and dilutes good enzyme liquid) immediately, shake up, add the sodium chloride solution of 10mL30%, shake up; Make its pH identical with 0.01mol/LNaOH drips of solution random sample product, write down the 0.01mol/LNaOH consumption with blank.
Calculate
X＝A×B×1/T×n
In the formula:
The enzyme activity of X---sample (u/g or u/mL);
A---the volume of quota of expenditure 0.01mol/LNaOH (mL) during the titration sample;
B---the titration concentration (μ mol/mL) of NaOH
T---time of enzymatic reacting (min),
N---extension rate;
Do simultaneously two parts parallel, results averaged, gained is the result represent to integer.The parallel test relative error must not surpass 5.0%.
PDA substratum (g/L): potato, 200; Sucrose, 20; Agar, 15; The PH nature
Seed culture medium (%): soybean cake powder 4, W-Gum 0.8, NaNO
30.3, Na
2HPO
40.2, K
2S0
40.25, MgSO
40.03, FeSO
40.003
Fermention medium (%): soybean cake powder 6, W-Gum 1, NaNO
30.4, Na
2HPO
40.2, K
2SO
40.3, MgSO
40.035, FeSO
40.02, CaCO
30.5, Na
2CO
30.02
Producing of sweet oil emulsion: 4%PVA solution and sweet oil are 2: 1 (v/v), are put into (outsourcing ice cube) in the little triangular flask, and being adjusted into rotating speed is 10000 rev/mins, an emulsification 3 minutes, and 3 minutes at interval, emulsification was 3 times altogether.
The enzyme activity determination of transgenosis mould and non-transgenic mould the results are shown in following table
Wherein, WT is the mould wild type strain, T1, and T2, T3, T4, T5: be mould transformant independently, as seen from the figure, the enzyme activity of transgenosis mould is significantly higher than the enzyme activity of non-transgenic mould.
Embodiment four, in animal-feed, add the experimentation on animals of above-mentioned lipase
The love denapon broiler chicken of 240 1 age in days health is selected in this test for use, is divided into 4 treatment group according to the random assignment principle, and each handles 4 repetitions, and each repeats 15 chickens.Trial period was 8 weeks.Whole feeding period is divided into three phases: 0-3wk is for growing early stage, and 4-6wk is for growing mid-term, and 7-8wk is for growing the later stage, and test design sees Table 1.
Table 1 test design
One, test daily ration
The disposable conventional raw material that uses in the required north of buying is pulverized the back and is formed by daily ration and mix with stirrer.According to the nutrient requirement preparation basal diet of national fryer standard (2004) as positive control group daily ration, negative control group, test I, test I I daily ration form with trophic level see Table 2, table 3 and table 4.All daily rations are powder, and are strict with the test design corresponding daily ration of feeding.
Table 2 0-3 week daily ration is formed and trophic level (%)
Table 3 4-6 week daily ration is formed and trophic level (%)
Table 4 7-8 week daily ration is formed and trophic level (%)
Wherein, 1) Preblend provides for every kilogram of basal diet: iron, 100mg, manganese, 45mg; Zinc, 105mg; Copper, 20mg; Iodine, 0.3mg; Selenium, 0.3mg.
2) Preblend provides for every kilogram of basal diet: vitamin A, 10000IU; Vitamin D3 500,000 I.U/GM, 135IU; Vitamin-E, 11mg; Vitamin B12,2.0ug; Riboflavin, 2.0mg; D-pantothenic acid, 7.4mg; Nicotinic acid, 7.0mg.
3) trophic level is a measured value.
Embodiment five, the detection of cholesterol, blood lipid level in animal body
Testing index and measuring method
One, growth performance index
Testing index: average daily ingestion amount (ADFI), average daily gain (ADG) and material anharmonic ratio (F/G).
Measuring method: the 21st, 42 and 56 age in days 8:00 in morning in test are that unit weighs (20 beginnings fasting at last night 12h is only for drinking-water) on an empty stomach to repeat respectively, claim clout heavy simultaneously.Early stage, mid-term, later stage with growth of meat chicken are Units of Account, and calculate 1-3,4-6 and 7-8 age in week average daily ingestion amount, average daily gain and material anharmonic ratio in conjunction with initial body weight.Formula is as follows:
Day weight gain (g/d)=(end weight-starting weight)/test fate
Material anharmonic ratio=daily ingestion amount/day weight gain
Two, the mensuration of apparent metabolizable energy
Testing index: energy in feed and the excrement (ME).
Measuring method: adopt the full excrement method of receiving, with reference to " animal feeding science experiment instruction ".Whenever, repeat to get two chickens and carry out metabolic test in finishing preceding 4 days in the 3rd, 6,8 weeks of test.Stopped material at 18 o'clock, change the excrement dish that is lined with plastic paper at 6 o'clock next day, fed intake simultaneously in 8 o'clock, complete continuously receipts excrement 4 days.Write down this free choice feeding amount of 4 days, collect 4 days movement.Mixing is weighed after choosing foreign material.Take out collection excrement dish, blow, choose feather and scurf on the movement with rubber suction bulb and tweezers, the feed of sorting out is wherein weighed, and deducts from the feeding capacity of this chicken, obtains the actual food consumption of this chicken.Movement is nondestructively scraped in the culture dish of known weight with scraper then, every repetition excrement sample is got after the sampling weighing and is placed 65 ℃ of drying in oven 48h to constant weight about 150g, and room temperature moisture regain 24h weighs.Pulverize, cross 40 mesh sieves, store to be equipped with and examine.To repeat is unit, once claims enough materials amounts in the phase of just trying, and claims clout heavy when off-test, and calculates the feed consumption rate of every repetition chicken in the phase of just trying.Energy adopts oxygen bomb formula calorimetric instrument to measure the apparent metabolizable energy of nutrient and presses following formula calculating in feed and the movement:
1 kilocalorie (kcal)=4.184 kilojoule (kJ),
Apparent metabolizable energy (Kcal/kg)=(fodder energy x food consumption-movement energy x movement quality)/food consumption
Apparent metabolizable energy (MJ/kg)=(fodder energy x food consumption-movement energy x movement quality)/food consumption
Nutrient apparent digestibility (%)=(eat and expect that nutrient content-excrement weighs nutrient content in the * excrement in the heavy * material)/(eat material and weigh nutrient content in the * material) * 100%
Three, the mensuration of blood parameters
Testing index: cholesterol (TC), very high density lipoprotein (VHDL), vldl (VLDL), triglyceride (TG) and glucose (GLU).
Measuring method: in test the 3rd, 6,8 all every group get an about 4mL of chicken venous blood collection immediately, behind 37 ℃ of about 10min of water-bath, centrifugal 2min under 3000r/min gets supernatant liquor and puts into refrigerator and cooled and freeze preservation.Cholesterol, triglyceride and glucose concn are measured with Fuller automatic clinical chemistry analyzer (Italy), and used kit all adopts Haifeng county favour biotech firm, and agriculture university's Animal nutrition research laboratory measures northeastward.
Four, the mensuration of abdomen fat rate
Testing index: abdomen fat is heavy, live-weight
Measuring method: get immediately and weigh after a chicken peels off the fat around belly and the muscular stomach and calculate abdomen fat rate for all every group in test the 6th, 8.Formula is as follows:
Abdomen fat rate=(abdomen fat weight/live-weight) * 100%
Embodiment six, result and analysis
Adopt the SAS9.0 statistical software to carry out statistical study, the result represents with mean value ± standard error.
One, lipase is to the broiler growth Effect on Performance
Table 5 lipase is to the influence (unit: g) of broiler chicken body weight
By table 5 as seen, at different growth phases, conventional daily ration+0.02% lipase group body weight is all the highest, compares with conventional daily ration group and improves 3.02g, 13.55g and 15.33g respectively, improve ratio and be respectively 0.46%, 0.70% and 0.52%, and it is the highest to improve ratio in growth body weight in mid-term; Compare with low-yield daily ration+0.02% lipase group and to improve 0.88g, 1.52g and 2.83g respectively, improve ratio and be respectively 0.42%, 0.08% and 0.10%.Low-yield daily ration+0.02% lipase group body weight all is higher than low-yield daily ration group, is respectively 2.79g, 14.75g and 22.88g, improve ratio and be respectively 0.42%, 0.77% and 0.78%, and it is the highest to improve ratio in growth middle and later periods body weight.Conventional daily ration group body weight all is higher than low-yield daily ration group, improves 0.65g, 2.72g and 10.38g respectively, improves ratio and is respectively 0.10%, 0.14% and 0.36%.
Table 6 lipase is to the influence (unit: g/d) of broiler chicken day weight gain
By table 6 as seen, at different growth phases, conventional daily ration+0.02% lipase group weight average that increases day by day is the highest, compares with conventional daily ration group and improves 0.14g/d, 0.50g/d, 0.13g/d and 0.27g/d respectively, and improve the highest in growth day weight gain in mid-term; Compare with low-yield daily ration+0.02% lipase group and to improve 0.03g/d, 0.03g/d, 0.10g/d and 0.05g/d respectively.Low-yield daily ration+0.02% lipase group weight average that increases day by day is higher than low-yield daily ration group, is respectively 0.13g/d, 0.57g/d, 0.58g/d and 0.41g/d, and improves the highest in growth middle and later periods day weight gain.The conventional daily ration group weight average that increases day by day is higher than low-yield daily ration group, improves 0.02g/d, 0.10g/d, 0.55g/d and 0.19g/d respectively.
Table 7 lipase is to the influence (unit: g/d) of broiler chicken daily ingestion amount
By table 7 as seen, at different growth phases, low-yield daily ration group daily ingestion amount is all the highest, compares with conventional daily ration group and improves 0.38g/d, 0.83g/d, 2.03g/d and 0.72g/d respectively; Compare with low-yield daily ration+0.02% lipase group and to improve 0.22g/d, 0.58g/d, 0.94g/d and 0.78g/d respectively.Low-yield daily ration+0.02% lipase group daily ingestion amount all is higher than conventional daily ration+0.02% lipase group, improves 0.12g/d, 1.7g/d, 5.02g/d and 0.66g/d respectively; Conventional daily ration+0.02% lipase group is higher than conventional daily ration group at the daily ingestion amount of 1-3w, rises to 0.04g/d, and is lower than conventional daily ration group at the daily ingestion amount of 4-6w, 7-8w and 1-8w, is reduced to 1.45g/d, 3.93g/d and 0.72g/d respectively.
Table 8 lipase is to the influence of broiler chicken feedstuff-meat ratio
By table 8 as seen, at different growth phases, conventional daily ration+0.02% lipase group feedstuff-meat ratio is all minimum, compares with conventional daily ration group and reduces by 0.01,0.05,0.05 and 0.02 respectively, and reduce the highest at growth middle and later periods feedstuff-meat ratio; Compare and reduce by 0.01,0.03,0.06 and 0.01 respectively with low-yield daily ration+0.02% lipase group, and reduce the highest at growth middle and later periods feedstuff-meat ratio.Low-yield daily ration+0.02% lipase group all is lower than low-yield daily ration group at the feedstuff-meat ratio of different growth phases, is respectively 0.01,0.03,0.04 and 0.04, and improves the highest in apparent metabolizable energy of growth middle and later periods.Conventional daily ration group all is lower than low-yield daily ration group at the feedstuff-meat ratio of different growth phases, reduces by 0.01,0.01,0.03 and 0.03 respectively, and reduces the highest at growth middle and later periods feedstuff-meat ratio.
Two, lipase is to the influence of the apparent metabolizable energy of broiler chicken
Table 9 lipase is to the influence (unit: MC/kg) of the apparent metabolizable energy of broiler chicken
By table 9 as seen, at different days and different growth phases, conventional daily ration+the apparent metabolism energy rate of 0.02% lipase group is all the highest, compares with conventional daily ration group and improves 0.04MJ/kg, 0.19MJ/kg and 0.05MJ/kg respectively, and improve the highest in growth apparent metabolizable energy in mid-term; Compare with low-yield daily ration+0.02% lipase group and to improve 0.15MJ/kg, 0.10MJ/kg and 0.05MJ/kg respectively.Low-yield daily ration+apparent metabolizable energy of 0.02% lipase group all is higher than low-yield daily ration group and is respectively 0.01MJ/kg, 0.19MJ/kg and 0.08MJ/kg, and improves the highest in growth apparent metabolizable energy in mid-term.The apparent metabolism energy rate of conventional daily ration group all is higher than low-yield daily ration group, improves 0.12MJ/kg, 0.10MJ/kg and 0.07MJ/kg respectively.
Three, lipase is to the influence of broiler chicken blood parameters
Table 10 lipase is to the influence of broiler chicken 21 age in days blood parameters
By table 10 as seen, during 21 ages in days, low-yield daily ration+0.02% lipase group content of triglyceride in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.05mmo l/L (reduced rate is 8.06%), compare with low-yield daily ration group and to have reduced 0.16mmol/L (reduced rate is 21.92%), compare with conventional daily ration group and reduced 0.39mmol/L (reduced rate is 40.63%).Conventional daily ration+0.02% lipase group content of triglyceride is compared with conventional daily ration group and has been reduced 0.34mmol/L (reduced rate is 35.42%).Low-yield daily ration group content of triglyceride is compared with conventional daily ration group and has been reduced 0.23mmol/L (reduced rate is 23.96%).
Low-yield daily ration+0.02% lipase group low-density lipoprotein content in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.08mmol/L (reduced rate is 3.69%), compare with low-yield daily ration group and to have reduced 0.31mmol/L (reduced rate is 12.92%), compare with conventional daily ration group and reduced 0.53mmol/L (reduced rate is 20.23%).Conventional daily ration+0.02% lipase group low-density lipoprotein content is compared with convention amount daily ration group and has been reduced 0.45mmol/L (reduced rate is 17.18%).Low-yield daily ration group low-density lipoprotein content is compared with conventional daily ration group and has been reduced 0.22mmol/L (reduced rate is 8.40%).
It is conventional that daily ration+0.02% lipase group is the highest at different group middle-high density lipoprotein content, compare with conventional daily ration group and to have improved 0.22mmol/L (the raising rate is 16.18%), compare with low-yield daily ration+0.02% lipase group and to have improved 0.12mmol/L (the raising rate is 8.82%), compare with low-yield daily ration group and improved 0.32mmol/L (the raising rate is 23.53%).Low-yield daily ration+0.02% lipase group hdl concentration is compared with low-yield daily ration group and has been improved 0.20mmol/L (the raising rate is 16.13%).Low-yield daily ration group hdl concentration is compared with conventional daily ration group and has been reduced 0.10mmol/L (reduced rate is 8.77%).
Low-yield daily ration+0.02% lipase group total cholesterol level in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.37mmol/L (reduced rate is 7.43%), compare with low-yield daily ration group and to have reduced 0.67mmol/L (reduced rate is 12.69%), compare with conventional daily ration group and reduced 1.44mmol/L (reduced rate is 23.80%).Conventional daily ration+0.02% lipase group total cholesterol level is compared with convention amount daily ration group and has been reduced 1.07mmol/L (reduced rate is 17.69%).Low-yield daily ration group total cholesterol level is compared with conventional daily ration group and has been reduced 0.77mmol/L (reduced rate is 12.73%).
Conventional daily ration+0.02% lipase group is compared glucose content with conventional daily ration group and has been improved 0.40mmol/L, and low-yield daily ration+0.02% lipase group is compared and improved 0.39mmol/L low; Low-yield daily ration+0.02% lipase group glucose content is compared glucose with low-yield daily ration group and has been improved 0.51mmol/L; Low-yield daily ration group is compared glucose content with conventional daily ration group and has been reduced 0.50mmol/L.
Conventional daily ration+0.02% lipase group is compared total protein content with conventional daily ration group and has been reduced 0.32g/L, compares and reduced 0.07g/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group total protein content is compared with low-yield daily ration group and has been improved 0.92g/L; Low-yield daily ration group total protein content is compared with conventional daily ration group and has been reduced 0.17g/L.
Conventional daily ration+0.02% lipase group is compared albumin content with conventional daily ration group and has been improved 0.08g/L, compares and reduced 0.07g/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group albumin content is compared with low-yield daily ration group and has been reduced 0.08g/L; Low-yield daily ration group albumin content is compared with conventional daily ration group and has been improved 0.23g/L.
Table 11 lipase is to the influence of broiler chicken 42 age in days blood parameters
By table 11 as seen, during 42 ages in days, low-yield daily ration+0.02% lipase group content of triglyceride in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.03mmol/L (reduced rate is 6.98%), compare with low-yield daily ration group and to have reduced 0.06mmol/L (reduced rate is 13.04%), compare with conventional daily ration group and reduced 0.12mmol/L (reduced rate is 23.08%).Conventional daily ration+0.02% lipase group content of triglyceride is compared with conventional daily ration group and has been reduced 0.09mmol/L (reduced rate is 17.31%).Low-yield daily ration group content of triglyceride is compared with conventional daily ration group and has been reduced 0.06mmol/L (reduced rate is 11.54%).
Low-yield daily ration+0.02% lipase group low-density lipoprotein content in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.03mmol/L (reduced rate is 1.67%), compare with low-yield daily ration group and to have reduced 0.06mmol/L (reduced rate is 3.28%), compare with conventional daily ration group and reduced 0.10mmol/L (reduced rate is 5.35%).Conventional daily ration+0.02% lipase group low-density lipoprotein content is compared with conventional daily ration group and has been reduced 0.07mmol/L (reduced rate is 3.74%).Low-yield daily ration group low-density lipoprotein content is compared with conventional daily ration group and has been reduced 0.04mmol/L (reduced rate is 2.14%).
It is conventional that daily ration+0.02% lipase group is the highest at different group middle-high density lipoprotein content, compare with conventional daily ration group and to have improved 0.08mmol/L (the raising rate is 8.70%), compare with low-yield daily ration+0.02% lipase group and to have improved 0.05mmol/L (the raising rate is 5.43%), compare with low-yield daily ration group and improved 0.16mmol/L (the raising rate is 17.39%).Low-yield daily ration+0.02% lipase group hdl concentration is compared with low-yield daily ration group and has been improved 0.11mmol/L (the raising rate is 12.64%).Low-yield daily ration group hdl concentration is compared with conventional daily ration group and has been reduced 0.08mmol/L (reduced rate is 9.52%).
Low-yield daily ration+0.02% lipase group total cholesterol level in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.11mmol/L (reduced rate is 3.47%), compare with low-yield daily ration group and to have reduced 0.26mmol/L (reduced rate is 7.83%), compare with conventional daily ration group and reduced 0.40mmol/L (reduced rate is 11.56%).Conventional daily ration+0.02% lipase group total cholesterol level is compared with conventional daily ration group and has been reduced 0.29mmol/L (reduced rate is 8.38%).Low-yield daily ration group total cholesterol level is compared with conventional daily ration group and has been reduced 0.14mmol/L (reduced rate is 4.05%).
Conventional daily ration+0.02% lipase group is compared glucose content with conventional daily ration group and has been improved 0.29mmol/L, compares and improved 0.28mmol/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group glucose content is compared with low-yield daily ration group and has been improved 0.19mmol/L; Low-yield daily ration group glucose content is compared with conventional daily ration group and has been reduced 0.18mmol/L.
Conventional daily ration+0.02% lipase group is compared total protein content with conventional daily ration group and has been reduced 0.81g/L, compares and reduced 0.41g/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group total protein content is compared with low-yield daily ration group and has been improved 0.15g/L; Low-yield daily ration group total protein content is compared with conventional daily ration group and has been reduced 0.55g/L.
Conventional daily ration+0.02% lipase group compare with conventional daily ration group albumin fall content low 0.61g/L, compare and reduced 0.26g/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group albumin content is compared with low-yield daily ration group and has been improved 0.20g/L; Low-yield daily ration group albumin content is compared with conventional daily ration group and has been reduced 0.55g/L.
Table 12 lipase is to the influence of broiler chicken 56 age in days blood parameters
By table 12 as seen, during 56 ages in days, low-yield daily ration+0.02% lipase group content of triglyceride in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.07mmol/L (reduced rate is 11.48%), compare with low-yield daily ration group and to have reduced 0.15mmol/L (reduced rate is 21.74%), compare with conventional daily ration group and reduced 0.24mmol/L (reduced rate is 30.77%).Conventional daily ration+0.02% lipase group content of triglyceride is compared with conventional daily ration group and has been reduced 0.17mmol/L (reduced rate is 21.79%).Low-yield daily ration group content of triglyceride is compared with conventional daily ration group and has been reduced 0.09mmol/L (reduced rate is 11.54%).
Low-yield daily ration+0.02% lipase group low-density lipoprotein content in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.06mmol/L (reduced rate is 3.28%), compare with low-yield daily ration group and to have reduced 0.08mmol/L (reduced rate is 4.32%), compare with conventional daily ration group and reduced 0.10mmol/L (reduced rate is 5.35%).Conventional daily ration+0.02% lipase group low-density lipoprotein content is compared with conventional daily ration group and has been reduced 0.04mmol/L (reduced rate is 2.14%).Low-yield daily ration group low-density lipoprotein content is compared with conventional daily ration group and has been reduced 0.02mmol/L (reduced rate is 1.07%).
It is conventional that daily ration+0.02% lipase group is the highest at different group middle-high density lipoprotein content, compare with conventional daily ration group and to have improved 0.18mmol/L (the raising rate is 9.63%), compare with low-yield daily ration+0.02% lipase group and to have improved 0.15mmol/L (the raising rate is 8.02%), compare with low-yield daily ration group and improved 0.42mmol/L (the raising rate is 22.46%).Low-yield daily ration+0.02% lipase group hdl concentration is compared with low-yield daily ration group and has been improved 0.27mmol/L (the raising rate is 15.70%).Low-yield daily ration group hdl concentration is compared with conventional daily ration group and has been reduced 0.24mmol/L (reduced rate is 14.20%).
Low-yield daily ration+0.02% lipase group total cholesterol level in different groups is minimum, compare with conventional daily ration+0.02% lipase group and to have reduced 0.36mmol/L (reduced rate is 9.89%), compare with low-yield daily ration group and to have reduced 0.38mmol/L (reduced rate is 10.38%), compare with conventional daily ration group and reduced 0.56mmol/L (reduced rate is 14.58%).Conventional daily ration+0.02% lipase group total cholesterol level is compared with conventional daily ration group and has been reduced 0.20mmol/L (reduced rate is 5.21%).Low-yield daily ration group total cholesterol level is compared with conventional daily ration group and has been reduced 0.18mmol/L (reduced rate is 4.69%).
Conventional daily ration+0.02% lipase group is compared total cholesterol level with conventional daily ration group and has been reduced 0.02mmol/L, compares and reduced mmol/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group total cholesterol level is compared with low-yield daily ration group and has been improved 0.47mmol/L; Low-yield daily ration group total cholesterol level is compared with conventional daily ration group and has been reduced 0.11mmol/L.
Conventional daily ration+0.02% lipase group is compared glucose content with conventional daily ration group and has been improved 0.64mmol/L, compares and improved 1.66mmol/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group glucose content is compared with low-yield daily ration group and has been improved 0.09mmol/L; Low-yield daily ration group glucose content is compared with conventional daily ration group and has been reduced 0.01mmol/L.
Conventional daily ration+0.02% lipase group is compared total protein content with conventional daily ration group and has been reduced 0.83g/L, compares and reduced 1.56g/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group total protein content is compared with low-yield daily ration group and has been improved 2.33g/L; Low-yield daily ration group total protein content is compared with conventional daily ration group and has been reduced 1.60g/L.
Conventional daily ration+0.02% lipase group is compared albumin content with conventional daily ration group and has been reduced 0.21g/L, compares and reduced 0.13g/L with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group albumin content is compared with low-yield daily ration group and has been improved 0.07g/L; Low-yield daily ration group albumin content is compared with conventional daily ration group and has been reduced 0.15g/L.
By table 10,11,12 as seen, at different days and different growth phases, conventional daily ration+0.02% lipase group content of triglyceride is compared all minimum with conventional daily ration group, low-yield daily ration group, low-yield daily ration+0.02% lipase group, and minimum at growth content in mid-term; Low-yield daily ration+0.02% lipase group content of triglyceride all is lower than low-yield daily ration group, and minimum at growth content in mid-term; Low-yield daily ration group content of triglyceride all is lower than conventional daily ration group, and minimum at growth content in mid-term.
At different days and different growth phases, conventional daily ration+0.02% lipase group high-density lipoprotein (HDL), low-density lipoprotein content are compared all minimum with conventional daily ration group, low-yield daily ration group, low-yield daily ration+0.02% lipase group, and minimum at growth content in mid-term; Low-yield daily ration+0.02% lipase group high-density lipoprotein (HDL), low-density lipoprotein content all are lower than low-yield daily ration group, and minimum at growth content in mid-term; Low-yield daily ration group low-density lipoprotein content all is lower than conventional daily ration group, and minimum at growth content in mid-term; Low-yield daily ration group high-density lipoprotein (HDL) all is higher than conventional daily ration group, and the highest at growth content in mid-term.
At different days and different growth phases, conventional daily ration+0.02% lipase group total cholesterol level is lower than conventional daily ration, low-yield daily ration+0.02% lipase, and minimum at growth content in mid-term, but than low-yield daily ration height, and minimum at growth content in mid-term.
At different days and different growth phases, conventional daily ration+0.02% lipase group glucose content is with conventional daily ration group, low-yield daily ration group, low-yield daily ration+0.02% lipase group is compared all the highest; Low-yield daily ration+0.02% lipase group glucose content all is lower than low-yield daily ration group; Low-yield daily ration group glucose content all is lower than conventional daily ration group.
At different days and different growth phases, conventional daily ration+0.02% lipase group total protein, albumin content are lower than conventional daily ration, low-yield daily ration+0.02% lipase, but than low-yield daily ration height.
Four, lipase is to the influence of broiler chicken abdomen fat rate
Table 13 lipase is to the influence (%) of broiler chicken abdomen fat rate
By table 13 as seen, at different days and different growth phases, conventional daily ration+0.02% lipase group abdomen fat rate is compared with conventional daily ration group and is reduced by 0.259% and 0.260% respectively; Compare and improve 0.067% and 0.087% respectively with low-yield daily ration+0.02% lipase group; Low-yield daily ration+0.02% lipase group abdomen fat rate all is lower than low-yield daily ration group and is respectively 0.133% and 0.091%, and reduces the highest in growth abdomen fat rate in mid-term; Conventional daily ration group abdomen fat rate all is higher than low-yield daily ration group, improves 0.059% and 0.082% respectively.
The efficiency analysis of embodiment seven, lipase
One, lipase is to the growth of meat chicken Effect on Performance
The a series of variations of performance of growing of broiler chicken for body weight gain, physique increase and body tissue's composition, these change the control of genetic mechanism in the principal recipient, present certain rules, also be subjected to all multifactor influences such as nutritional status, external environment simultaneously].At different growth phases, conventional daily ration+0.02% lipase group body weight, the weight average that increases day by day are the highest, and it is the highest to improve ratio in growth body weight in mid-term; Low-yield daily ration+0.02% lipase group is higher than low-yield daily ration group at the body weight of different growth phases, the weight average that increases day by day; Conventional daily ration group is higher than low-yield daily ration group at the body weight of different growth phases, the weight average that increases day by day.
At different growth phases, low-yield daily ration group daily ingestion amount is all the highest; Low-yield daily ration+0.02% lipase group daily ingestion amount all is higher than conventional daily ration+0.02% lipase group; Conventional daily ration+0.02% lipase group is higher than conventional daily ration group at the daily ingestion amount of 1-3w, and is lower than conventional daily ration group at the daily ingestion amount of 4-6w, 7-8w and 1-8w.
At different growth phases, conventional daily ration+0.02% lipase group feedstuff-meat ratio is all minimum, and it is the highest to improve ratio in growth body weight in mid-term; Low-yield daily ration+0.02% lipase group all is lower than low-yield daily ration group at the feedstuff-meat ratio of different growth phases; Conventional daily ration group all is lower than low-yield daily ration group at the feedstuff-meat ratio of different growth phases.
On the whole, the daily ration that adds lipase can improve the growth performance of fryer, increases broiler weight and day weight gain, reduces feedstuff-meat ratio, obtains economic benefit, and can reach maximum value in growth economic benefit in mid-term.
Two, lipase is to the influence of the apparent metabolizable energy of fryer
Apparent metabolizable energy is that the reflection animal body is for one of metabolism situation index of daily ration, the metabolic situation and the vigorous degree of vitality that can well reflect animal body, at different days and different growth phases, conventional daily ration+the apparent metabolism energy rate of 0.02% lipase group is all the highest, and improves the highest in growth apparent metabolizable energy in mid-term.Low-yield daily ration+apparent metabolism energy rate of 0.02% lipase group all is higher than low-yield daily ration group, and improves the highest in growth apparent metabolizable energy in mid-term.The apparent metabolism energy rate of conventional daily ration group all is higher than low-yield daily ration group.
On the whole, the daily ration that adds lipase can improve the apparent metabolizable energy of fryer, improves metabolism of human body energy metabolic rate and body and the vigorous degree of vitality.
Three, lipase is to the influence of fryer blood parameters
The natural substrate of lipase is a glyceride type, and at different days and different growth phases, low-yield daily ration+0.02% lipase group triglyceride level, low-density lipoprotein and total cholesterol level are all minimum, and minimum at growth content in mid-term; Conventional daily ration+0.02% lipase group triglyceride level, high-density lipoprotein (HDL) and low-density lipoprotein content is a little more than low-yield daily ration+0.02% lipase group, and minimum at growth content in mid-term, but far below low-yield daily ration group and conventional daily ration group.
At different days and different growth phases, conventional daily ration+0.02% lipase group hdl concentration is all the highest, and low-yield daily ration+0.02% lipase group is taken second place, and conventional daily ration group is higher than low-yield group.It is conventional that daily ration+0.02% lipase group glucose content is all the highest.Conventional daily ration+0.02% lipase group total protein, albumin content are lower than conventional daily ration, low-yield daily ration+0.02% lipase, but than low-yield daily ration height.
On the whole, the daily ration that adds lipase can improve the blood parameters of fryer, reduce triglyceride level, low-density lipoprotein and total cholesterol level in the blood, improve hdl concentration, though total protein and albumin content slightly reduce, it is not remarkable that glucose content is influenced rule, but blood parameters has improved generally.
Four, lipase is to the influence of fryer abdomen fat rate
Abdomen fat rate raises with the rising of daily ration metabolizable energy, and at different days and different growth phases, low-yield daily ration+0.02% lipase group abdomen fat rate is all minimum; Low-yield daily ration group abdomen fat rate all is lower than conventional day daily ration group.Conventional daily ration+0.02% lipase group abdomen fat rate all is lower than low-yield daily ration group and conventional daily ration group, and reduces the highest in growth abdomen fat rate in mid-term.
On the whole, consider from the angle that reduces the fryer fatty deposits, should adopt low-yield daily ration+0.02% lipase, suitably improve the dietary digestibility of energy level and can improve feed efficiency, therefore, in actual production, should adopt moderate energy daily ration+0.02% lipase and conventional daily ration+0.02% lipase.
Interpolation lipase can improve the growth performance and the apparent metabolizable energy of fryer, reduce triglyceride level, low-density lipoprotein, hdl concentration in the blood, reduce abdomen fat rate, and phase growth performance and apparent metabolizable energy improve maximum in test, and triglyceride level, low-density lipoprotein, hdl concentration and abdomen fat rate reduce maximum.
Claims (8)
1. lipase that effectively reduces animal body inner cholesterol, blood lipid level, it is characterized in that: described lipase is obtained by following method:
(1) obtains hygromycin resistance expression cassette and being cloned on the target plasmid, obtain the recombinant plasmid of hygromycin resistance;
(2) increase respectively the lipase gene PEL of mould, the strong promoter glyceraldehyde 3-phosphate dehydro-genase promotor PgpdA and the Aspergillus nidulans tryptophan synthetase terminator TtrpC of Aspergillus nidulans obtain the PEL expression casette that strong promoter drives;
(3) this PEL expression casette is inserted in the recombinant plasmid of hygromycin resistance, has obtained to contain the PEL gene overexpression vector of hygromycin selection mark;
(4) the PEL gene overexpression vector that will contain the hygromycin selection mark transforms the engineering Agrobacterium, utilizes the agrobacterium mediation converted method that the PEL expression casette is transformed in the penicillium bacterial strain, obtains the mould genetic engineering bacterium of high expression level lipase;
(5) described mould genetic engineering bacterium is inserted seed culture medium with mycelium or spore suspension, under 25-35 ℃, 200-300rpm condition, cultivate after 24 hours, change fermention medium over to the 5%-20% inoculum size, after 2 days, obtain described lipase at 25-35 ℃, 150-300rpm condition bottom fermentation through separation and purification.
2. the lipase of a kind of effective reduction animal body inner cholesterol according to claim 1, blood lipid level is characterized in that: the method that obtains the hygromycin resistance expression cassette in step (1) comprises round pcr or Restriction Enzyme incision technology.
3. the lipase of a kind of effective reduction animal body inner cholesterol according to claim 1, blood lipid level is characterized in that: obtain the lipase gene PEL of mould, the strong promoter glyceraldehyde 3-phosphate dehydro-genase promotor PgpdA of Aspergillus nidulans and the method for Aspergillus nidulans tryptophan synthetase terminator TtrpC and comprise pcr amplification or restriction enzyme clone or chemical synthesising technology clone in step (2).
4. the lipase of a kind of effective reduction animal body inner cholesterol according to claim 1, blood lipid level, it is characterized in that: the Penicillium notatum described in the step (4) is a penicillium expansum.
5. the lipase of a kind of effective reduction animal body inner cholesterol according to claim 1, blood lipid level is characterized in that: described target plasmid is one of following:
PCAMBIA1300, pCAMBIA2300, pCAMBIA3300 or pHB.
6. the lipase of a kind of effective reduction animal body inner cholesterol according to claim 1, blood lipid level, it is characterized in that: the composition of substratum is as follows described in the step (5):
(1) seed culture medium (%): soybean cake powder 4, W-Gum 0.8, Na NO3 0.3, Na2HPO40.2, K2SO4 0.25, and MgSO4 0.03, and FeSO4 0.003;
(2) fermention medium (%): soybean cake powder 6, W-Gum 1, NaNO3 0.4, and Na2HPO4 0.2, and K2SO4 0.3, and MgSO4 0.035, and FeSO4 0.02, and CaCO3 0.5, and Na2CO3 0.02.
7. the application of the described lipase of claim 1 in reducing animal body inner cholesterol, blood lipid level.
8. lipase according to claim 7 is as the application of additive in animal-feed.
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| CN104278054A (en) * | 2013-07-04 | 2015-01-14 | 中国农业大学 | Application of kinase Lats2 in regulation of precursor fat cell proliferation |
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| CN104714038A (en) * | 2014-12-24 | 2015-06-17 | 东北农业大学 | Serum biochemical marker for auxiliary screening of low-fat meat chickens and application of serum biochemical marker |
| CN104714038B (en) * | 2014-12-24 | 2016-09-14 | 东北农业大学 | The serum biochemistry labelling of a kind of assisting sifting low fat broiler and application |
| CN105039385A (en) * | 2015-05-22 | 2015-11-11 | 天津科技大学 | T10,c12-conjugated linoleic acid engineering bacterial strain, recombinant expression plasmid thereof, construction method and application thereof |
| CN105039385B (en) * | 2015-05-22 | 2019-04-19 | 天津科技大学 | T10, c12- conjugated linoleic acid engineered strain and its recombinant expression plasmid and construction method and application |
| CN112136979A (en) * | 2020-09-17 | 2020-12-29 | 福建傲农生物科技集团股份有限公司 | Feed premix for reducing pig blood fat and preparation method and application thereof |
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Application publication date: 20110817 |