CN105859839B - A kind of biologically active peptide and its preparation method and application promoting piglet growth - Google Patents
A kind of biologically active peptide and its preparation method and application promoting piglet growth Download PDFInfo
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- CN105859839B CN105859839B CN201610347325.6A CN201610347325A CN105859839B CN 105859839 B CN105859839 B CN 105859839B CN 201610347325 A CN201610347325 A CN 201610347325A CN 105859839 B CN105859839 B CN 105859839B
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
The present invention provides a kind of biologically active peptides of promotion piglet growth, the biologically active peptide includes amino acid sequence shown in SEQ IDNo.1, the biologically active peptide can significantly improve piglet to the absorption rate of nutriment in feed, promote the growth and development of piglet, disease incidence is reduced, piglet survival rate is improved.Further, it provides the preparation method for promoting the biologically active peptide of piglet growth and its is promoting piglet growth, reduce the application in the piglet death rate, promote the biologically active peptide of piglet development by generating under the synergistic effect of a variety of producing enzyme probiotics, applied to piglet, promote body anabolism and lipolysis, protein is promoted to synthesize, reach the growth for promoting piglet, improve the purpose of its speed of growth, by the hormonal readiness for adjusting endocrine system, play the effect for promoting piglet development, the problem of overcoming hormone residues, achieve the purpose that healthy green cultivation.
Description
Technical field
The invention belongs to technical field of biological fermentation, effectively facilitate piglet development more particularly to one kind, improve it
The speed of growth promotes the biologically active peptide and its preparation method and application of piglet growth.
Background technique
The growth and development of piglet is to be related to a key link of live pig production, due to the unique digestive physiology knot of piglet
Structure, digestive organs are unsound, and the physiological function of glandula digestive is not perfect, lack innate immunity, and body heat regulation function is not good for
Entirely, poor to the resistance of cold stress, piglet is under general rearing conditions, and the disease incidence of a variety of diseases is high, and survival rate is low.It improves
Piglet promotes the growth and development of piglet to the absorption rate of nutriment in feed, reduces disease incidence, improves piglet survival
Rate is pig farm question of common concern.
Summary of the invention
In view of this, the present invention provides one kind to have safe and environment-friendly, noresidue, the speed of growth of piglet can be improved, is dropped
The biologically active peptide and its preparation method and application for promoting piglet growth of the advantages of death rate of low piglet.
First aspect present invention provides a kind of biologically active peptide of promotion piglet growth, and the biologically active peptide includes
Amino acid sequence shown in SEQ ID No.1.
The biologically active peptide that second aspect of the present invention provides above-mentioned promotion piglet growth is promoting piglet growth, is reducing son
Application in the pig death rate.
Third aspect present invention provides the preparation method of the biologically active peptide of above-mentioned promotion piglet growth, including walks as follows
It is rapid:
A, lactoenterococcus, bacillus licheniformis, bacillus coagulans, bacillus pumilus, Candida utilis are screened respectively
It is cultivated after yeast；
B, lactoenterococcus obtained by step A, bacillus licheniformis, bacillus coagulans, bacillus pumilus, production protein is false
Silk yeast is mixed fermentation, is concentrated after extraction fermented liquid supernatant liquid, is dry, biologically active peptide is obtained after separating-purifying.
The beneficial effects of the present invention are: the biologically active peptide provided by the invention for promoting piglet development, is applied to life
The piglet in long stage promotes protein to synthesize, reaches the life for promoting piglet by promoting body anabolism and lipolysis
It is long, improve the purpose of its speed of growth.Biologically active peptide, not instead of bio-hormone, one kind being capable of mimic hormone bioactivity
Polypeptide, by adjust endocrine system hormonal readiness, play promote piglet development effect, overcome hormone residues
The problem of, achieve the purpose that healthy green cultivation.Practical application the results show that promotion piglet development provided by the invention
Biologically active peptide piglet average daily gain can be made to improve 6.38% (P < 0.05), after weaned piglet, feed intake increases
4.16% (P < 0.05), the death rate that biologically active peptide is relatively not used in the death rate reduce by 50% (P < 0.05), and effect is obvious.
Specific embodiment
First aspect present invention provides a kind of biologically active peptide of promotion piglet growth, and the biologically active peptide includes
Amino acid sequence shown in SEQ ID No.1.
This kind of biologically active peptide is the polypeptide with 13 amino acid residues, amino acid sequence Arg-Pro-Cys-
Leu-Ser-Ala-Glu-Ile-Leu-Ser-Thr-Ser-Val, containing arginine, proline, cysteine, isoleucine,
A variety of essential amino acids such as leucine, valine, threonine and limiting amino acid, form certain spatial configuration of molecules.It is young
The physiological function ateliosis of swine alimentary canal, the value volume and range of product of the protease of secretion are few, it is difficult to this kind of bioactivity of degrading
Peptide.This kind of biologically active peptide enters the circulation of piglet by the intestinal cell of piglet ateliosis in piglet gastrointestinal tract
System plays similar hormone-like effect, promotes anabolism ability, accelerates N deposition, enhances the synthesis of protein, plays and promote
The effect of piglet development achievees the purpose that healthy green cultivation.
Product of the polypeptide as proteolysis, the bioactivity and physical property for thering are many protein not have.Beans
The protein raw materials such as the dregs of rice, fish meal are rich in multiple proteins, utilize protein raw materials by a variety of probiotics for producing protease, synchronize
Compound criteria, fermentative degradation generate the biologically active peptide that piglet can be promoted to grow.This biologically active peptide, safe and efficient, ring
It protects, noresidue, the speed of growth of piglet can be improved, reduce the death rate of piglet, the safety of steroids growth promotion drug can be become
Effective substitute.
The biologically active peptide that second aspect of the present invention provides above-mentioned promotion piglet growth is promoting piglet growth, is reducing son
Application in the pig death rate.
In one embodiment of the invention, the application be will bioactivity peptide freeze-dried powder manually gastric juice dilution after it is right
Piglet is gavaged.The application mode of the biologically active peptide includes but is not limited to this one kind, can also be raised by preparing in piglet
Expect that medium various ways are applied.
Third aspect present invention provides the preparation method of the biologically active peptide of above-mentioned promotion piglet growth, including walks as follows
It is rapid:
A, lactoenterococcus, bacillus licheniformis, bacillus coagulans, bacillus pumilus, Candida utilis are screened respectively
It is cultivated after yeast；
B, lactoenterococcus obtained by step A, bacillus licheniformis, bacillus coagulans, bacillus pumilus, production protein is false
Silk yeast is mixed fermentation, is concentrated after extraction fermented liquid supernatant liquid, is dry, biologically active peptide is obtained after separating-purifying.
The biologically active peptide of the promotion piglet development is generated under the synergistic effect of a variety of producing enzyme probiotics
's.Probiotics Enzymatic characteristic, yield of enzyme and enzyme activity are improved by induced mutations, by lactoenterococcus, bacillus licheniformis, condensation
Bacillus, bacillus pumilus, candida utili synchronize compound criteria, different benefits according to corresponding different ratio
Raw bacterium strain expresses secretase class under same condition of culture, the macromolecular complex such as protein-based, polysaccharide in culture medium of degrading
Matter provides a variety of nutriments such as amino acid, vitamin, oligosaccharides, organic acid for the metabolic activity of bacillus category strain, thus
Its metabolism is promoted to form corresponding biologically active peptide.This kind of biologically active peptide is applied to the piglet of growth phase, son can be passed through
The gastrointestinal tract mucous barrier that pig not yet reaches maturity enters directly into piglet blood circulation, plays similar hormonal bioactivity and makees
With, enhance animal body anabolism, accelerate the absorption of a variety of nutriments such as amino acid, vitamin, protein is promoted to synthesize,
The growth and development for promoting piglet, improves its speed of growth, achievees the purpose that healthy green cultivation.
What is provided according to embodiments of the present invention promotes the preparation method of the biologically active peptide of piglet growth, and this method can be with
With following additional technical characteristic:
Preferably, it screens and includes the following steps: described in step A
A, primary dcreening operation: by lactoenterococcus, bacillus licheniformis, bacillus coagulans, bacillus pumilus, Candida utilis ferment
Mother is added separately in the aseptic flat board of casein separation screening culture medium, and transparent loop diameter and bacterium are chosen after ultraviolet mutagenesis culture
Fall the bacterium colony culture transferring culture that diameter ratio (HC) is greater than 1.5；
B, it secondary screening: by after each dominant strain that step a is obtained carries out microwave irradiation, is trained using casein separation screening
Base is supported, constant temperature incubation 2-3 days at 30-34 DEG C, chooses the bacterium colony culture transferring training of transparent loop diameter and colony diameter ratio (HC) greater than 1.5
It supports；
C, secondary screening again: by each dominant strain obtained by step b respectively with 180-220 μ g/mL nitroso guanidine solution etc.
20-30min is cultivated after volume mixture at 30-35 DEG C, terminates and is washed after reacting, is formulated as bacteria suspension, bacteria suspension is coated on junket
Protein Separation screening and culturing medium plate is cultivated 34-38 hours at 35-39 DEG C, chooses transparent loop diameter and colony diameter ratio (HC)
Bacterium colony greater than 1.5.
More preferred, microwave irradiation step described in above-mentioned steps b includes: 850W maximum power, specified microwave frequency
In the micro-wave oven of 2450MHz, microwave irradiation 45-55s, and refrigeration is protected from light after 5-10s ice bath processing, microwave irradiation
10-14 hours.
According to one embodiment of present invention, incubation step described in step A include: by after screening lactoenterococcus,
Clothing bacillus, bacillus coagulans, bacillus pumilus, candida utili are in beef-protein medium culture 30-
40 hours, then be forwarded in nutrient agar, it is cultivated 45-50 hours at 35-39 DEG C.
Preferably, mixed culture fermentation step described in step B includes: by percentage to the quality, by lactoenterococcus 20-
23%, bacillus licheniformis 22-25%, bacillus coagulans 26-30%, bacillus pumilus 10-14%, candida utili
The ratio of 14-17% is inoculated in the same fermentation cylinder for fermentation culture equipped with fluid nutrient medium.
More preferred, lactoenterococcus described in step B, bacillus licheniformis, bacillus coagulans, short and small gemma bar
Bacterium, candida utili are inoculated in seed liquid culture medium by the inoculum concentration of 8%-12% respectively, after culture domestication 10-14 hours
Carry out mixed culture fermentation.
More preferred, the condition of the fermented and cultured is pH7.2-7.4, speed of agitator 180-220rpm, temperature 35-
39 DEG C, ventilatory capacity 1:0.5-1:0.7, fermentation time 22-24 hours.
Preferably, it is solution that separating-purifying step described in step B, which includes: by the products configuration after concentration, drying, is successively passed through
The ultrafiltration membrane of 10KDa, 5KDa, 3KDa, 1KDa separate step by step, and collection obtains biologically active peptide ultrafiltration segment, then carries out crosslinking Portugal
Polysaccharide gel chromatogram purification, elution flow rate 0.8-1.2mL/h, Detection wavelength 220nm are freeze-dried after collecting component.
Biologically active peptide and its preparation side below in conjunction with specific embodiment to promotion piglet growth provided by the invention
Method and application are further described.The embodiments described below is exemplary, and for explaining only the invention, and cannot be understood
For limitation of the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Reality as used in the following examples
It tests material unless otherwise specified, is that market is commercially available.
Embodiment 1
All strains used by the present embodiment are all the microorganism fungus kinds that can be added in feed, and lactoenterococcus,
Bacillus licheniformis, bacillus coagulans, bacillus pumilus, candida utili are by agromicrobiology state key
Laboratory provides, and commercially available lactoenterococcus, bacillus coagulans, bacillus pumilus, produces protein at bacillus licheniformis
Candida can also reach very approximate effect.
One, the preparation method of the biologically active peptide of the promotion piglet growth of the present embodiment includes the following steps:
Step 1: cultivating strain, each strain is carried out using mutation breeding technologies preferred.
In the case where power is the ultraviolet lamp of 20W, 30cm irradiation distance, by the lactoenterococcus of preservation, bacillus licheniformis, solidifying
Tie bacillus, bacillus pumilus, candida utili bacteria suspension 2mL difference casein separation screening culture medium it is sterile
On plate, irradiation culture in 2-3 minutes is counted, and observes the transparent circle on casein separation screening culture medium, and it is straight to choose transparent circle
Diameter and colony diameter ratio (HC) are cultivated on the bacterium colony culture transferring to test tube slant greater than 1.5.It is preferable to choose the advantage after ultraviolet mutagenesis
Bacterial strain, be made 106-108The bacteria suspension of/mL, 850W maximum power, specified microwave frequency 2450MHz micro-wave oven in, it is micro-
Amplitude shines 55s, and eliminates fuel factor every the processing of 10s ice bath, is protected from light 4 DEG C and refrigerates 14 hours, is coated on casein separation screening
Culture medium, 35 DEG C after constant temperature incubation 2 days, are observed the transparent circle on casein separation screening culture medium, choose transparent loop diameter
It is cultivated on bacterium colony culture transferring to test tube slant of the colony diameter ratio (HC) greater than 1.5.It is preferable to choose the advantage after microwave irradiation
Bacterial strain is made 10 with sterile water6-108It is placed in cleaning with 200 μ g/mL nitroso guanidine solutions by the bacteria suspension of/mL in equal volume
It is mixed in conical flask, is diluted to 50 times of terminations reactions, centrifuge washing 3 times removals with cold saline after processing 20min at 35 DEG C
Drug, then it is diluted to 106-108Times bacteria suspension, takes 0.2mL bacteria suspension to be spread evenly across casein separation screening culture medium flat plate
On, plate is inverted in 39 DEG C of constant incubators and is cultivated 34 hours, transparent loop diameter is chosen and colony diameter ratio (HC) is greater than
As production bacterium processed on 1.5 bacterium colony culture transferring to test tube slant.
The preparation of the nitroso guanidine solution: 0.020g nitrosoguanidine accurately is weighed, is placed in the volumetric flask of 100mL, adds
Enter suitable acetone soln hydrotropy, the phosphate buffer constant volume of pH7.2 is added, obtained 200 μ g/mL nitroso guanidine solutions, 4 DEG C
It saves backup.
The casein separation screening culture medium: casein 0.4-0.6%, beef extract 0.2-0.4%g, peptone 0.5-
1.5%, sodium chloride 0.5-1.5%, agar 1.0-2.0%, pH7.0-7.2, distilled water 1000mL, 121 DEG C of sterilizing 18-22min.
Step 2: production bacterium processed
By the lactoenterococcus screened, bacillus licheniformis, bacillus coagulans, bacillus pumilus, produce protein vacation
Silk yeast is inoculated in beef-protein medium respectively, cultivates 40 hours, then be forwarded to the eggplant equipped with nutrient agar
It in sub- bottle, is cultivated 48 hours at 37 DEG C, is put into 4 DEG C of refrigerators and saves.
The beef-protein medium: glucose 5.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g,
Sterile water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
The nutrient agar: beef extract 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 20.0g, sterile water
1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
Step 3: the synchronous culture of compound bacteria
By the lactoenterococcus of preservation, bacillus licheniformis, bacillus coagulans, bacillus pumilus, Candida utilis ferment
Mother is inoculated in seed liquid culture medium respectively by 12% inoculum concentration, domestication culture 14 hours.
Each strain: lactoenterococcus 20%, bacillus licheniformis 25%, condensation gemma bar is weighed by following weight percent
Bacterium 30%, bacillus pumilus 10%, candida utili 15%.
All strains of culture are inoculated in by weight percentage in the same fermentor equipped with fluid nutrient medium and are cultivated,
PH7.2-7.4, speed of agitator 180-220rpm, cultivation temperature are 35-39 DEG C, ventilatory capacity 1:0.5-1:0.7, fermentation time 24
Hour.
The seed liquid culture medium: glucose 1-3%, yeast extract 0.5-1.0%, peptone 0.5-1.5%, beef extract
0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.05-0.15%, sterile water 94-96%, pH7.0-7.2,121
DEG C sterilizing 20-25min.
The fermentation liquid culture medium: dregs of beans 10-15%, fish meal 8-12%, wheat bran 4-10%, corn pulp 6-10%, grape
Sugared 1-3%, sodium chloride 0.5-1.5%, yeast extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.5-1.0%,
Ammonium sulfate 0.5-1.5%, manganese sulfate 0.005-0.01%, ferrous sulfate 0.01-0.03%, sterile water 62-65%, pH7.2-
7.4,121 DEG C of sterilizing 20-25min.
Step 4: the separation of biologically active peptide
After completing under the optimal conditions of fermentation of compound bacteria to the fermentation of compound protein raw material, by fermentation liquid in 4500-
It is centrifuged 25-35min under 5500rpm speed conditions, supernatant is taken to be concentrated in vacuo, is freeze-dried and the thick of biologically active peptide is made
Product.
The crude product of biologically active peptide is dissolved in deionized water, solution is configured to, is the super of 10KDa with molecular cut off
Filter membrane separates the crude product solution of biologically active peptide, permeate obtained by collecting, then the surpassing for 5KDa through molecular cut off
Filter membrane further separates, and the ultrafiltration membrane that 3KDa, 1KDa are then respectively adopted after the same method separates step by step, will finally collect
Biologically active peptide ultrafiltration segment is obtained, after being concentrated in vacuo, is freeze-dried spare at freeze-dried powder.Again by biologically active peptide ultrafiltration piece
Section freeze-dried powder be dissolved in be configured in deionized water mass-volume concentration be 50mg/mL solution, after mixing loading 2mL into
Row sephadex chromatogram purification, elution flow rate 0.8-1.2mL/h, Detection wavelength 220nm utilize automatic collector
The collection of separation component is carried out, repeatedly loading, is constantly enriched with each component and freeze-drying obtains the sterling of biologically active peptide,
It saves backup.Amino acid sequencing, amino acid sequence Arg-Pro-Cys-Leu-Ser- are carried out to the polypeptide of separating-purifying
Ala-Gln-Ile-Leu-Ser-Thr-Ser-Val, as shown in sequence table SEQ ID No.1.
Two, the biologically active peptide being prepared is applied to piglet, evaluation biologically active peptide promotees the effect of piglet growth, tool
Body method includes:
The preparation of simulated gastric fluid: it measures 234mL concentrated hydrochloric acid addition sterile water and is diluted to 1000mL, 10% dilute hydrochloric acid is made.
Dilute hydrochloric acid 16.4mL is taken, sterile water 800mL and pepsin 10g is added, stirs evenly, sterile water is added and is diluted to 1000mL
To obtain the final product.
The preparation of test solution: accurately weighing bioactivity peptide freeze-dried powder finished product 0.04g, molten with 100.0g simulated gastric fluid
Test solution is made in solution dilution, and 4 DEG C save backup.
Piglet 100 for choosing 10-15 age in days, are randomly divided into two groups of A, B, every group 50.Every piglet is numbered
Ear's label is identified respectively.A group as a control group, gavages the test solution of 2mL daily, continuously feeds 20 days；B group is made
For control group, free water, feeding, the difference with A group, which is only that, does not gavage test solution.The growth shape of observation piglet daily
State weighs to piglet, and performs record.The piglet average daily gain of test in 20 days by a definite date, test group improves 6.38%
(P < 0.05), after weaned piglet, feed intake increases by 4.16% (P < 0.05), and the death rate reduces by 50% (P < compared with control group
0.05)。
Embodiment 2
All strains used by the present embodiment are all the microorganism fungus kinds that can be added in feed, and lactoenterococcus,
Bacillus licheniformis, bacillus coagulans, bacillus pumilus, candida utili are the different manufacturers in buying in the market
Strain can reach approximate effect.
One, the preparation method of the biologically active peptide of the promotion piglet growth of the present embodiment includes the following steps:
Step 1: cultivating strain, each strain is carried out using mutation breeding technologies preferred.
In the case where power is the ultraviolet lamp of 20W, 30cm irradiation distance, by the lactoenterococcus of preservation, bacillus licheniformis, solidifying
Tie bacillus, bacillus pumilus, candida utili bacteria suspension 2mL difference casein separation screening culture medium it is sterile
On plate, irradiation culture in 2-3 minutes is counted, and observes the transparent circle on casein separation screening culture medium, and it is straight to choose transparent circle
Diameter and colony diameter ratio (HC) are cultivated on the bacterium colony culture transferring to test tube slant greater than 1.5.It is preferable to choose the advantage after ultraviolet mutagenesis
Bacterial strain, be made 106-108The bacteria suspension of/mL, 850W maximum power, specified microwave frequency 2450MHz micro-wave oven in, it is micro-
Amplitude shines 45s, and eliminates fuel factor every the processing of 5s ice bath, is protected from light 4 DEG C and refrigerates 10 hours, is coated on the training of casein separation screening
Support base, 30 DEG C after constant temperature incubation 3 days, are observed the transparent circle on casein separation screening culture medium, choose transparent loop diameter and
Colony diameter ratio (HC) is cultivated on the bacterium colony culture transferring to test tube slant greater than 1.5.Choose the preferable bacterium of advantage after microwave irradiation
Strain, is made 10 with sterile water6-108It is placed in clean cone with 220 μ g/mL nitroso guanidine solutions by the bacteria suspension of/mL in equal volume
It is mixed in shape bottle, is diluted to 50 times of terminations reactions, 3 removal medicines of centrifuge washing with cold saline after processing 30min at 30 DEG C
Object, then it is diluted to 106-108Times bacteria suspension, takes 0.2mL bacteria suspension to be spread evenly across on casein separation screening culture medium flat plate,
Plate is inverted in 35 DEG C of constant incubators and is cultivated 38 hours, transparent loop diameter is chosen and colony diameter ratio (HC) is greater than 1.5
Bacterium colony culture transferring to test tube slant on as producing bacterium processed.
The preparation of the nitroso guanidine solution: 0.020g nitrosoguanidine accurately is weighed, is placed in the volumetric flask of 100mL, adds
Enter suitable acetone soln hydrotropy, the phosphate buffer constant volume of pH7.2 is added, obtained 200 μ g/mL nitroso guanidine solutions, 4 DEG C
It saves backup.
The casein separation screening culture medium: casein 0.4-0.6%, beef extract 0.2-0.4%g, peptone 0.5-
1.5%, sodium chloride 0.5-1.5%, agar 1.0-2.0%, pH7.0-7.2, distilled water 1000mL, 121 DEG C of sterilizing 18-22min.
Step 2: production bacterium processed
By the lactoenterococcus screened, bacillus licheniformis, bacillus coagulans, bacillus pumilus, produce protein vacation
Silk yeast is inoculated in beef-protein medium respectively, cultivates 30 hours, then be forwarded to the eggplant equipped with nutrient agar
It in sub- bottle, is cultivated 50 hours at 35 DEG C, is put into 4 DEG C of refrigerators and saves.
The beef-protein medium: glucose 5.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g,
Sterile water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
The nutrient agar: beef extract 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 20.0g, sterile water
1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
Step 3: the synchronous culture of compound bacteria
By the lactoenterococcus of preservation, bacillus licheniformis, bacillus coagulans, bacillus pumilus, Candida utilis ferment
Mother is inoculated in seed liquid culture medium respectively by 8% inoculum concentration, domestication culture 10 hours.
Each strain: lactoenterococcus 22%, bacillus licheniformis 22%, condensation gemma bar is weighed by following weight percent
Bacterium 26%, bacillus pumilus 13%, candida utili 17%.
All strains of culture are inoculated in by weight percentage in the same fermentor equipped with fluid nutrient medium and are cultivated,
PH7.2-7.4, speed of agitator 180-220rpm, cultivation temperature are 35-39 DEG C, ventilatory capacity 1:0.5-1:0.7, fermentation time 22
Hour.
The seed liquid culture medium: glucose 1-3%, yeast extract 0.5-1.0%, peptone 0.5-1.5%, beef extract
0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.05-0.15%, sterile water 94-96%, pH7.0-7.2,121
DEG C sterilizing 20-25min.
The fermentation liquid culture medium: dregs of beans 10-15%, fish meal 8-12%, wheat bran 4-10%, corn pulp 6-10%, grape
Sugared 1-3%, sodium chloride 0.5-1.5%, yeast extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.5-1.0%,
Ammonium sulfate 0.5-1.5%, manganese sulfate 0.005-0.01%, ferrous sulfate 0.01-0.03%, sterile water 62-65%, pH7.2-
7.4,121 DEG C of sterilizing 20-25min.
Step 4: the separation of biologically active peptide
After completing under the optimal conditions of fermentation of compound bacteria to the fermentation of compound protein raw material, by fermentation liquid in 4500-
It is centrifuged 25-35min under 5500rpm speed conditions, supernatant is taken to be concentrated in vacuo, is freeze-dried and the thick of biologically active peptide is made
Product.
The crude product of biologically active peptide is dissolved in deionized water, solution is configured to, is the super of 10KDa with molecular cut off
Filter membrane separates the crude product solution of biologically active peptide, permeate obtained by collecting, then the surpassing for 5KDa through molecular cut off
Filter membrane further separates, and the ultrafiltration membrane that 3KDa, 1KDa are then respectively adopted after the same method separates step by step, will finally collect
Biologically active peptide ultrafiltration segment is obtained, after being concentrated in vacuo, is freeze-dried spare at freeze-dried powder.Again by biologically active peptide ultrafiltration piece
Section freeze-dried powder be dissolved in be configured in deionized water mass-volume concentration be 50mg/mL solution, after mixing loading 2mL into
Row sephadex chromatogram purification, elution flow rate 0.8-1.2mL/h, Detection wavelength 220nm utilize automatic collector
The collection of separation component is carried out, repeatedly loading, is constantly enriched with each component and freeze-drying obtains the sterling of biologically active peptide,
It saves backup.Amino acid sequencing, amino acid sequence Arg-Pro-Cys-Leu-Ser- are carried out to the polypeptide of separating-purifying
Ala-Gln-Ile-Leu-Ser-Thr-Ser-Val, as shown in sequence table SEQ ID No.1.
Two, the biologically active peptide being prepared is applied to piglet, evaluation biologically active peptide promotees the effect of piglet growth, tool
Body method includes:
The preparation of simulated gastric fluid: it measures 234mL concentrated hydrochloric acid addition sterile water and is diluted to 1000mL, 10% dilute hydrochloric acid is made.
Dilute hydrochloric acid 16.4mL is taken, sterile water 800mL and pepsin 10g is added, stirs evenly, sterile water is added and is diluted to 1000mL
To obtain the final product.
The preparation of test solution: accurately weighing bioactivity peptide freeze-dried powder finished product 0.05g, molten with 100.0g simulated gastric fluid
Test solution is made in solution dilution, and 4 DEG C save backup.
Piglet 100 for choosing 10-15 age in days, are randomly divided into two groups of A, B, every group 50.Every piglet is numbered
Ear's label is identified respectively.A group as a control group, gavages the test solution of 2mL daily, continuously feeds 20 days；B group is made
For control group, free water, feeding, the difference with A group, which is only that, does not gavage test solution.The growth shape of observation piglet daily
State weighs to piglet, and performs record.The piglet average daily gain of test in 20 days by a definite date, test group improves 6.50%
(P < 0.05), after weaned piglet, feed intake increases by 4.31% (P < 0.05), and the death rate reduces by 50% (P < compared with control group
0.05)。
Embodiment 3
All strains used by the present embodiment are all the microorganism fungus kinds that can be added in feed, and lactoenterococcus,
Bacillus licheniformis, bacillus coagulans, bacillus pumilus, candida utili are the different manufacturers in buying in the market
Strain can reach approximate effect.
One, the preparation method of the biologically active peptide of the promotion piglet growth of the present embodiment includes the following steps:
Step 1: cultivating strain, each strain is carried out using mutation breeding technologies preferred.
In the case where power is the ultraviolet lamp of 20W, 30cm irradiation distance, by the lactoenterococcus of preservation, bacillus licheniformis, solidifying
Tie bacillus, bacillus pumilus, candida utili bacteria suspension 2mL difference casein separation screening culture medium it is sterile
On plate, irradiation culture in 2-3 minutes is counted, and observes the transparent circle on casein separation screening culture medium, and it is straight to choose transparent circle
Diameter and colony diameter ratio (HC) are cultivated on the bacterium colony culture transferring to test tube slant greater than 1.5.It is preferable to choose the advantage after ultraviolet mutagenesis
Bacterial strain, be made 106-108The bacteria suspension of/mL, 850W maximum power, specified microwave frequency 2450MHz micro-wave oven in, it is micro-
Amplitude shines 45s, and eliminates fuel factor every the processing of 5s ice bath, is protected from light 4 DEG C and refrigerates 10 hours, is coated on the training of casein separation screening
Support base, 32 DEG C after constant temperature incubation 3 days, are observed the transparent circle on casein separation screening culture medium, choose transparent loop diameter and
Colony diameter ratio (HC) is cultivated on the bacterium colony culture transferring to test tube slant greater than 1.5.Choose the preferable bacterium of advantage after microwave irradiation
Strain, is made 10 with sterile water6-108It is placed in clean cone with 200 μ g/mL nitroso guanidine solutions by the bacteria suspension of/mL in equal volume
It is mixed in shape bottle, is diluted to 50 times of terminations reactions, 3 removal medicines of centrifuge washing with cold saline after processing 25min at 33 DEG C
Object, then it is diluted to 106-108Times bacteria suspension, takes 0.2mL bacteria suspension to be spread evenly across on casein separation screening culture medium flat plate,
Plate is inverted in 37 DEG C of constant incubators and is cultivated 36 hours, transparent loop diameter is chosen and colony diameter ratio (HC) is greater than 1.5
Bacterium colony culture transferring to test tube slant on as producing bacterium processed.
The preparation of the nitroso guanidine solution: 0.020g nitrosoguanidine accurately is weighed, is placed in the volumetric flask of 100mL, adds
Enter suitable acetone soln hydrotropy, the phosphate buffer constant volume of pH7.2 is added, obtained 200 μ g/mL nitroso guanidine solutions, 4 DEG C
It saves backup.
The casein separation screening culture medium: casein 0.4-0.6%, beef extract 0.2-0.4%g, peptone 0.5-
1.5%, sodium chloride 0.5-1.5%, agar 1.0-2.0%, pH7.0-7.2, distilled water 1000mL, 121 DEG C of sterilizing 18-22min.
Step 2: production bacterium processed
By the lactoenterococcus screened, bacillus licheniformis, bacillus coagulans, bacillus pumilus, produce protein vacation
Silk yeast is inoculated in beef-protein medium respectively, cultivates 35 hours, then be forwarded to the eggplant equipped with nutrient agar
It in sub- bottle, is cultivated 47 hours at 37 DEG C, is put into 4 DEG C of refrigerators and saves.
The beef-protein medium: glucose 5.0g, beef extract 5.0g, peptone 10.0g, sodium chloride 5.0g,
Sterile water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
The nutrient agar: beef extract 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 20.0g, sterile water
1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
Step 3: the synchronous culture of compound bacteria
By the lactoenterococcus of preservation, bacillus licheniformis, bacillus coagulans, bacillus pumilus, Candida utilis ferment
Mother is inoculated in seed liquid culture medium respectively by 10% inoculum concentration, domestication culture 12 hours.
Each strain: lactoenterococcus 23%, bacillus licheniformis 23%, condensation gemma bar is weighed by following weight percent
Bacterium 28%, bacillus pumilus 11%, candida utili 15%.
All strains of culture are inoculated in by weight percentage in the same fermentor equipped with fluid nutrient medium and are cultivated,
PH7.2-7.4, speed of agitator 180-220rpm, cultivation temperature are 35-39 DEG C, ventilatory capacity 1:0.5-1:0.7, fermentation time 23
Hour.
The seed liquid culture medium: glucose 1-3%, yeast extract 0.5-1.0%, peptone 0.5-1.5%, beef extract
0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.05-0.15%, sterile water 94-96%, pH7.0-7.2,121
DEG C sterilizing 20-25min.
The fermentation liquid culture medium: dregs of beans 10-15%, fish meal 8-12%, wheat bran 4-10%, corn pulp 6-10%, grape
Sugared 1-3%, sodium chloride 0.5-1.5%, yeast extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.5-1.0%,
Ammonium sulfate 0.5-1.5%, manganese sulfate 0.005-0.01%, ferrous sulfate 0.01-0.03%, sterile water 62-65%, pH7.2-
7.4,121 DEG C of sterilizing 20-25min.
Step 4: the separation of biologically active peptide
After completing under the optimal conditions of fermentation of compound bacteria to the fermentation of compound protein raw material, by fermentation liquid in 4500-
It is centrifuged 25-35min under 5500rpm speed conditions, supernatant is taken to be concentrated in vacuo, is freeze-dried and the thick of biologically active peptide is made
Product.
The crude product of biologically active peptide is dissolved in deionized water, solution is configured to, is the super of 10KDa with molecular cut off
Filter membrane separates the crude product solution of biologically active peptide, permeate obtained by collecting, then the surpassing for 5KDa through molecular cut off
Filter membrane further separates, and the ultrafiltration membrane that 3KDa, 1KDa are then respectively adopted after the same method separates step by step, will finally collect
Biologically active peptide ultrafiltration segment is obtained, after being concentrated in vacuo, is freeze-dried spare at freeze-dried powder.Again by biologically active peptide ultrafiltration piece
Section freeze-dried powder be dissolved in be configured in deionized water mass-volume concentration be 50mg/mL solution, after mixing loading 2mL into
Row sephadex chromatogram purification, elution flow rate 0.8-1.2mL/h, Detection wavelength 220nm utilize automatic collector
The collection of separation component is carried out, repeatedly loading, is constantly enriched with each component and freeze-drying obtains the sterling of biologically active peptide,
It saves backup.Amino acid sequencing, amino acid sequence Arg-Pro-Cys-Leu-Ser- are carried out to the polypeptide of separating-purifying
Ala-Gln-Ile-Leu-Ser-Thr-Ser-Val, as shown in sequence table SEQ ID No.1.
Two, the biologically active peptide being prepared is applied to piglet, evaluation biologically active peptide promotees the effect of piglet growth, tool
Body method includes:
The preparation of simulated gastric fluid: it measures 234mL concentrated hydrochloric acid addition sterile water and is diluted to 1000mL, 10% dilute hydrochloric acid is made.
Dilute hydrochloric acid 16.4mL is taken, sterile water 800mL and pepsin 10g is added, stirs evenly, sterile water is added and is diluted to 1000mL
To obtain the final product.
The preparation of test solution: accurately weighing bioactivity peptide freeze-dried powder finished product 0.04g, molten with 100.0g simulated gastric fluid
Test solution is made in solution dilution, and 4 DEG C save backup.
Piglet 100 for choosing 10-15 age in days, are randomly divided into two groups of A, B, every group 50.Every piglet is numbered
Ear's label is identified respectively.A group as a control group, gavages the test solution of 2mL daily, continuously feeds 20 days；B group is made
For control group, free water, feeding, the difference with A group, which is only that, does not gavage test solution.The growth shape of observation piglet daily
State weighs to piglet, and performs record.The piglet average daily gain of test in 20 days by a definite date, test group improves 6.2% (P
< 0.05), after weaned piglet, feed intake increases by 4.2% (P < 0.05), and the death rate reduces by 50% (P < compared with control group
0.05)。
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (2)
1. a kind of biologically active peptide for promoting piglet growth, it is characterised in that: the biologically active peptide is shown in SEQ ID No.1
Amino acid sequence.
2. the biologically active peptide of promotion piglet growth described in claim 1 is preparing the growth of promotion piglet, is reducing the piglet death rate
Feed in application.
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| CN107736296A (en) * | 2017-10-11 | 2018-02-27 | 浙江青莲食品股份有限公司 | Cherish the cultural method of son phase black pig |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1766088A (en) * | 2004-10-26 | 2006-05-03 | 中国农业大学 | Bacillus of high proteinase yield and its induction mutation breeding method and uses |
| CN101209088A (en) * | 2006-12-28 | 2008-07-02 | 上海创博生态工程有限公司 | Microorganism polyzyme additive agent for improving pigling growth and development |
| CN105410337A (en) * | 2015-12-11 | 2016-03-23 | 上海邦成生物科技有限公司 | Biological feed additive rich in bioactive peptides and probiotics as well as preparation method of additive |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766088A (en) * | 2004-10-26 | 2006-05-03 | 中国农业大学 | Bacillus of high proteinase yield and its induction mutation breeding method and uses |
| CN101209088A (en) * | 2006-12-28 | 2008-07-02 | 上海创博生态工程有限公司 | Microorganism polyzyme additive agent for improving pigling growth and development |
| CN105410337A (en) * | 2015-12-11 | 2016-03-23 | 上海邦成生物科技有限公司 | Biological feed additive rich in bioactive peptides and probiotics as well as preparation method of additive |
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