CN105859839A - Biological active peptide for promoting growth of piglet and preparation method and application thereof - Google Patents
Biological active peptide for promoting growth of piglet and preparation method and application thereof Download PDFInfo
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- CN105859839A CN105859839A CN201610347325.6A CN201610347325A CN105859839A CN 105859839 A CN105859839 A CN 105859839A CN 201610347325 A CN201610347325 A CN 201610347325A CN 105859839 A CN105859839 A CN 105859839A
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- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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Abstract
The invention provides biological active peptide for promoting growth of a piglet. The biological active peptide comprises an amino acid sequence shown in SEQ IDNo.1, the absorption and utilization rate of nutrient substances in feed for the piglet can be obviously increased through the biological active peptide, growth and development of the piglet can be promoted, the morbidity can be reduced, and the survival rate of the piglet can be increased. Furthermore, the preparation method of the biological active peptide for promoting growth of the piglet and application of the biological active peptide to promoting growth of the piglet and reducing the death rate of the piglet are provided. The biological active peptide for promoting growth and development of the piglet is generated under the synergistic effect of multiple kinds of enzyme production probiotics and is applied to the piglet, constructive metabolism and lipolysis of the organism are promoted, protein synthesis is promoted, and the aims of promoting growth of the piglet and increasing the growth speed of the piglet are achieved. The function of promoting growth and development of the piglet is achieved by adjusting the hormone level of an endocrine system, the problem of hormone residues is solved, and the aim of healthy and environment-friendly breeding is achieved.
Description
Technical field
The invention belongs to technical field of biological fermentation, particularly relate to one and effectively facilitate Piglet Development, carry
High its speed of growth biologically active peptide promoting piglet growth and its preparation method and application.
Background technology
The growth promoter of piglet is to be related to the key link that live pig produces, and the digestion unique due to piglet is raw
Reason structure, its digestive organs are unsound, the physiological function imperfection of digestive gland, lack innate immunity, body
Temperature regulative mechanism unsound, poor to the resistance of cold stress, piglet under general rearing conditions, multiple disease
Sickness rate high, survival rate is low.Improve the piglet absorption rate to feedstuff Middle nutrition material, promote piglet
Growth promoter, reduces sickness rate, improves piglet survival rate, is pig farm question of common concern.
Summary of the invention
In view of this, the invention provides one and there is safe and environment-friendly, noresidue, the growth of piglet can be improved
Speed, reduce the advantage of the mortality rate of piglet the biologically active peptide and preparation method thereof promoting piglet growth and should
With.
First aspect present invention provides a kind of biologically active peptide promoting piglet growth, described biologically active peptide bag
Containing aminoacid sequence shown in SEQ ID No.1.
Second aspect present invention provides the biologically active peptide of above-mentioned promotion piglet growth and is promoting piglet growth, fall
Application in low piglet mortality rate.
Third aspect present invention provides the preparation method of the biologically active peptide of above-mentioned promotion piglet growth, including such as
Lower step:
A, screen lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein respectively
Cultivate after candida mycoderma；
B, by step A gained lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis mixed culture fermentation, concentrates after extracting fermented liquid supernatant liquid, is dried, must be raw after separating-purifying
Thing bioactive peptide.
The invention has the beneficial effects as follows: the biologically active peptide promoting Piglet Development that the present invention provides, application
In the piglet of growth stage, by promoting body anabolism and steatolysis, promote protein synthesis, reach
Promote the growth of piglet, improve the purpose of its speed of growth.Biologically active peptide, is not bio-hormone, but one
Kind can the bioactive polypeptide of mimic hormone, by the hormonal readiness of endocrine regulation system, play and promote son
The effect of pig growth and development, the problem overcoming hormone residues, reach the purpose of healthy green cultivation.Reality should
Result prove, the present invention provide promote Piglet Development biologically active peptide piglet can be made averagely to increase day by day
Bringing up again high by 6.38% (P ＜ 0.05), after weaned piglet, feed intake increases by 4.16% (P ＜ 0.05), dead
Rate does not uses the mortality rate of biologically active peptide to reduce by 50% (P ＜ 0.05), and effect is obvious.
Detailed description of the invention
First aspect present invention provides a kind of biologically active peptide promoting piglet growth, described biologically active peptide bag
Containing aminoacid sequence shown in SEQ ID No.1.
This kind of biologically active peptide is the polypeptide with 13 amino acid residues, and its aminoacid sequence is
Arg-Pro-Cys-Leu-Ser-Ala-Glu-Ile-Leu-Ser-Thr-Ser-Val, containing arginine, dried meat ammonia
Multiple essential amino acids such as acid, cysteine, isoleucine, leucine, valine, threonine and restricted
Aminoacid, forms certain spatial configuration of molecules.The physiological function ateliosis of piglet digestive, secretion
The value volume and range of product of protease is few, it is difficult to this kind of biologically active peptide of degrading.This kind of biologically active peptide is at piglet gastrointestinal
In road, entered into the blood circulation of piglet by the intestinal cell of piglet ateliosis, play similar hormonelike
Effect, promotes anabolism ability, accelerates N deposition, strengthens the synthesis of protein, plays and promotes that piglet is raw
The long effect grown, reaches the purpose of healthy green cultivation.
Many peptides as proteoclastic product, have biological activity and physical property that numerous protein do not possesses
Matter.The protein raw materials such as bean cake, fish flour, rich in multiple proteins, utilizes egg by the probiotic bacteria of multiple product protease
White raw material, carries out synchronizing compound criteria, and fermentative degradation produces the biologically active peptide that can promote piglet growth.This
Biologically active peptide, safe efficient, environmental protection, noresidue, can improve the speed of growth of piglet, reduce piglet
Mortality rate, can become the safe and effective substitute of hormones growth promotion medicine.
Second aspect present invention provides the biologically active peptide of above-mentioned promotion piglet growth and is promoting piglet growth, fall
Application in low piglet mortality rate.
In one embodiment of the invention, described application is to be diluted by biological activity peptide freeze-dried powder employment work gastric juice
Afterwards piglet is gavaged.The application mode of this biologically active peptide includes but not limited to that this is a kind of, it is also possible to pass through
It is formulated in the medium various ways of pig starter feed to apply.
Third aspect present invention provides the preparation method of the biologically active peptide of above-mentioned promotion piglet growth, including such as
Lower step:
A, screen lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein respectively
Cultivate after candida mycoderma；
B, by step A gained lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis mixed culture fermentation, concentrates after extracting fermented liquid supernatant liquid, is dried, must be raw after separating-purifying
Thing bioactive peptide.
The described biologically active peptide promoting Piglet Development is under the synergism of multiple product enzyme probiotic bacteria
Produce.Improve probiotic bacteria Enzymatic characteristic by induced mutations, yield of enzyme and enzyme are lived, by lactoenterococcus,
Clothing bacillus cereus, Bacillus coagulans, Bacillus pumilus, Candida utilis are according to corresponding different ratio
Carrying out synchronizing compound criteria, different probiotic bacteria strains expresses secretase class, degraded training under same condition of culture
The macromolecular substances such as protein-based, the polysaccharide in foster base, the metabolic activity for bacillus category strain provides ammonia
The multiple nutrients materials such as base acid, vitamin, oligosaccharide, organic acid, thus promote that its metabolism forms corresponding biology
Bioactive peptide.This kind of biologically active peptide is applied to the piglet of growth stage, piglet not yet fully-developed can be passed through
Gastrointestinal tract mucous barrier enters directly into piglet blood circulation, plays similar hormonal bioactivity effect, strengthens dynamic
Thing body anabolism, accelerates the absorption of the multiple nutrients material such as aminoacid, vitamin, promotes protein synthesis,
Promote the growth promoter of piglet, improve its speed of growth, reach the purpose of healthy green cultivation.
The preparation method of the biologically active peptide promoting piglet growth provided according to embodiments of the present invention, the method is also
Can have a following additional technical characteristic:
Preferably, described in step A, screening comprises the steps:
A, primary dcreening operation: by lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein
Candida mycoderma is added separately on the aseptic flat board of casein separation screening culture medium, and ultraviolet mutagenesis is chosen after cultivating
Transparent circle diameter and colony diameter ratio (HC) the bacterium colony subcultivation more than 1.5 are cultivated；
B, multiple sieve: after each dominant strain obtained through step a is carried out microwave irradiation, use casein bolter
Select constant temperature culture 2-3 days at culture medium, 30-34 DEG C, choose transparent circle diameter and colony diameter ratio (HC) is big
Bacterium colony subcultivation in 1.5 is cultivated；
C, the most again sieve: by molten with 180-220 μ g/mL nitrosoguanidine respectively for each dominant strain of obtaining through step b
Cultivate 20-30min at 30-35 DEG C after the mixing of liquid equal-volume, wash after terminating reaction, be formulated as bacteria suspension, will
Bacteria suspension coats casein separation screening culture medium flat plate, cultivates 34-38 hour, choose at 35-39 DEG C
Bright loop diameter and colony diameter ratio (HC) bacterium colony more than 1.5.
More preferably, the microwave irradiation step described in above-mentioned steps b includes: 850W peak power, specified
In the microwave oven of microwave frequency 2450MHz, microwave exposure 45-55s, and process every 5-10s ice bath, micro-
Amplitude is according to terminating rear lucifuge cold preservation 10-14 hour.
According to one embodiment of present invention, incubation step described in step A includes: by the lactic acid intestinal after screening
Coccus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, Candida utilis are in Carnis Bovis seu Bubali cream egg
White peptone culture medium culturing 30-40 hour, then be forwarded in nutrient agar, at 35-39 DEG C, cultivate 45-50
Hour.
Preferably, it is mixed fermentation step described in step B to include: by percentage to the quality, by lactic acid intestinal
Coccus 20-23%, Bacillus licheniformis 22-25%, Bacillus coagulans 26-30%, Bacillus pumilus
10-14%, the ratio of Candida utilis 14-17% are inoculated in same equipped with in the fermentation tank of fluid medium
Ferment is cultivated.
More preferably, lactoenterococcus described in step B, Bacillus licheniformis, Bacillus coagulans, short
Bacillus pumilus, Candida utilis are inoculated in seed liquor culture medium by the inoculum concentration of 8%-12% respectively, training
Domesticating carries out mixed culture fermentation after 10-14 hour.
More preferably, the condition of described fermentation culture is pH7.2-7.4, speed of agitator 180-220rpm, temperature
Degree is 35-39 DEG C, ventilation 1:0.5-1:0.7, fermentation time 22-24 hour.
Preferably, described in step B, separating-purifying step includes: be molten by concentration, dried products configuration
Liquid, the ultrafilter membrane through 10KDa, 5KDa, 3KDa, 1KDa separates step by step successively, collects and obtains biological living
Property peptide ultrafiltration fragment, then carry out sephadex chromatogram purification, elution flow rate 0.8-1.2mL/h, detection
Wavelength is 220nm, collects component postlyophilization.
Below in conjunction with specific embodiment, offer of the present invention is promoted biologically active peptide and the system thereof of piglet growth
Preparation Method and application are further described.The embodiments described below is exemplary, is only used for explaining this
Bright, and be not considered as limiting the invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.Institute in following embodiment
Experiment material if no special instructions, be market and be commercially available.
Embodiment 1
All strains that the present embodiment is used are all the microorganism fungus kinds can added in feedstuff, and lactic acid intestinal ball
Bacterium, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, Candida utilis are all micro-by agricultural
National Key Laboratory's offer biology, the lactoenterococcus that market is buied, Bacillus licheniformis, condensation bud
Spore bacillus, Bacillus pumilus, Candida utilis also can reach the effect approximated very much.
One, the preparation method of the biologically active peptide promoting piglet growth of the present embodiment comprises the steps:
The first step: cultivate strain, utilize mutation breeding technologies that each strain is carried out preferably.
Under power is the uviol lamp of 20W, 30cm irradiation distance, by the lactoenterococcus preserved, lichens bud
Spore bacillus, Bacillus coagulans, Bacillus pumilus, the bacteria suspension 2mL cheese egg respectively of Candida utilis
On the aseptic flat board of white separation screening culture medium, irradiate 2-3 minute and cultivate counting, observe at casein bolter
Select the transparent circle in culture medium, choose transparent circle diameter and colony diameter ratio (HC) bacterium colony more than 1.5 and move
Plant and cultivate to test tube slant.Choose the preferable bacterial strain of advantage after ultraviolet mutagenesis, make 106-108/ mL's
Bacteria suspension, 850W peak power, specified microwave frequency 2450MHz microwave oven in, microwave exposure 55s,
And process elimination heat effect, 4 DEG C of cold preservations of lucifuge 14 hours every 10s ice bath, coat casein bolter
Selecting culture medium, 35 DEG C of constant temperature culture, after 2 days, observe the transparent circle in casein separation screening culture medium, choosing
Take transparent circle diameter and colony diameter ratio (HC) the bacterium colony subcultivation more than 1.5 to cultivate to test tube slant.Choosing
Learn from else's experience the preferable bacterial strain of advantage after microwave irradiation, make 10 with sterilized water6-108The bacteria suspension of/mL, by its with
200 μ g/mL nitroso guanidine solution equal-volumes are placed in mixing in the conical flask of cleaning, after processing 20min at 35 DEG C
Being diluted to 50 times with cold saline and terminate reaction, centrifuge washing is removed medicine 3 times, then is diluted to 106-108
Times bacteria suspension, takes 0.2mL bacteria suspension and is spread evenly across on casein separation screening culture medium flat plate, by flat board
It is inverted in 39 DEG C of constant incubators cultivation 34 hours, chooses transparent circle diameter and colony diameter ratio (HC)
Bacterium colony subcultivation more than 1.5 produces bacterium processed to conduct on test tube slant.
The preparation of described nitroso guanidine solution: accurately weigh 0.020g nitrosoguanidine, is placed in the capacity of 100mL
In Ping, add appropriate acetone soln hydrotropy, add the phosphate buffer constant volume of pH7.2, prepare 200 μ
G/mL nitroso guanidine solution, 4 DEG C save backup.
Described casein separation screening culture medium: casein 0.4-0.6%, Carnis Bovis seu Bubali cream 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
Sterilizing 18-22min.
Second step: produce bacterium processed
By the lactoenterococcus screened, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis is inoculated in beef-protein medium respectively, cultivates 40 hours, then is forwarded to equipped with battalion
Support in the Fructus Solani melongenae bottle of agar culture medium, cultivate 48 hours at 37 DEG C, put into 4 DEG C of Refrigerator stores.
Described beef-protein medium: glucose 5.0g, Carnis Bovis seu Bubali cream 5.0g, peptone 10.0g, chlorination
Sodium 5.0g, sterilized water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
Described nutrient agar: Carnis Bovis seu Bubali cream 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 20.0g,
Sterilized water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
3rd step: the synchronized culture of compound bacteria
By the lactoenterococcus preserved, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein
Candida mycoderma, the inoculum concentration by 12% is inoculated in seed liquor culture medium respectively, and domestication is cultivated 14 hours.
Each strain is weighed: lactoenterococcus 20%, Bacillus licheniformis 25%, solidifying by following percentage by weight
Knot bacillus cereus 30%, Bacillus pumilus 10%, Candida utilis 15%.
The all strains cultivated are inoculated in by weight percentage same equipped with training in the fermentation tank of fluid medium
Supporting, pH7.2-7.4, speed of agitator 180-220rpm, cultivation temperature is 35-39 DEG C, ventilation 1:0.5-1:0.7,
Fermentation time is 24 hours.
Described seed liquor culture medium: glucose 1-3%, yeast extract 0.5-1.0%, peptone 0.5-1.5%, cattle
Meat extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.05-0.15%, sterilized water 94-96%,
PH7.0-7.2,121 DEG C of sterilizing 20-25min.
Described fermentation liquid culture medium: bean cake 10-15%, fish flour 8-12%, Testa Tritici 4-10%, Semen Maydis pulp 6-10%,
Glucose 1-3%, sodium chloride 0.5-1.5%, yeast extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, biphosphate
Potassium 0.5-1.0%, ammonium sulfate 0.5-1.5%, manganese sulfate 0.005-0.01%, ferrous sulfate 0.01-0.03%, nothing
Bacterium water 62-65%, pH7.2-7.4,121 DEG C sterilizing 20-25min.
4th step: the separation of biologically active peptide
After completing the fermentation to compound protein raw material under the optimal conditions of fermentation of compound bacteria, fermentation liquid is existed
Under 4500-5500rpm speed conditions, centrifugal 25-35min, takes supernatant and is concentrated in vacuo, lyophilization system
Obtain the crude product of biologically active peptide.
By the dissolving crude product of biologically active peptide in deionized water, it is configured to solution, with molecular cut off is
The crude product solution of biologically active peptide is separated by the ultrafilter membrane of 10KDa, permeate obtained by collecting, then warp
Molecular cut off is that the ultrafilter membrane of 5KDa separates further, be respectively adopted the most after the same method 3KDa,
The ultrafilter membrane of 1KDa separates step by step, obtains biologically active peptide ultrafiltration fragment by finally collecting, after being concentrated in vacuo,
Lyophilization becomes lyophilized powder standby.Again biologically active peptide ultrafiltration fragment lyophilized powder is dissolved in deionized water configuration
Becoming mass body volume concentrations is the solution of 50mg/mL, and after mix homogeneously, loading 2mL carries out sephadex
Chromatogram purification, elution flow rate is 0.8-1.2mL/h, and detection wavelength is 220nm, utilizes automatic collector to carry out
The collection of separation component, repeatedly loading, be constantly enriched with each component and lyophilization obtain biologically active peptide
Sterling, saves backup.The polypeptide of separating-purifying is carried out amino acid sequencing, and its aminoacid sequence is
Arg-Pro-Cys-Leu-Ser-Ala-Gln-Ile-Leu-Ser-Thr-Ser-Val, such as sequence table SEQ ID No.1 institute
Show.
Two, the biologically active peptide prepared is applied to piglet, evaluates biologically active peptide and promote the effect of piglet growth
Really, concrete grammar includes:
The preparation of simulated gastric fluid: measure 234mL concentrated hydrochloric acid addition sterilized water and be diluted to 1000mL, prepare 10%
Dilute hydrochloric acid.Take this dilute hydrochloric acid 16.4mL, add sterilized water 800mL and pepsin 10g, stir,
Add sterilized water be diluted to 1000mL and get final product.
The preparation of need testing solution: accurately weigh biological activity peptide freeze-dried powder finished product 0.04g, artificial with 100.0g
Gastric juice dissolved dilution makes need testing solution, and 4 DEG C save backup.
Choose the piglet 100 of 10-15 age in days, be randomly divided into A, B two groups, often group 50.To every head
Ear's label of piglet numbering is identified respectively.As a control group, gavage 2mL every day supplies examination to A group
Product solution, fills continuously and feeds 20 days；B group as a control group, is freely drunk water, is searched for food, and the difference with A group is only
It is not gavage need testing solution.Observe the growth conditions of piglet every day, piglet is weighed, and make easy to remember
Record.The test of 20 days by a definite date, the piglet average daily gain of test group improves 6.38% (P ＜ 0.05), piglet
After wean, feed intake increases by 4.16% (P ＜ 0.05), and mortality rate relatively matched group reduces by 50% (P ＜ 0.05).
Embodiment 2
All strains that the present embodiment is used are all the microorganism fungus kinds can added in feedstuff, and lactic acid intestinal ball
Bacterium, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, Candida utilis are all on market
Buying, the strain of different manufacturers all can reach the effect of approximation.
One, the preparation method of the biologically active peptide promoting piglet growth of the present embodiment comprises the steps:
The first step: cultivate strain, utilize mutation breeding technologies that each strain is carried out preferably.
Under power is the uviol lamp of 20W, 30cm irradiation distance, by the lactoenterococcus preserved, lichens bud
Spore bacillus, Bacillus coagulans, Bacillus pumilus, the bacteria suspension 2mL cheese egg respectively of Candida utilis
On the aseptic flat board of white separation screening culture medium, irradiate 2-3 minute and cultivate counting, observe at casein bolter
Select the transparent circle in culture medium, choose transparent circle diameter and colony diameter ratio (HC) bacterium colony more than 1.5 and move
Plant and cultivate to test tube slant.Choose the preferable bacterial strain of advantage after ultraviolet mutagenesis, make 106-108/ mL's
Bacteria suspension, 850W peak power, specified microwave frequency 2450MHz microwave oven in, microwave exposure 45s,
And process elimination heat effect, 4 DEG C of cold preservations of lucifuge 10 hours every 5s ice bath, coat casein separation screening
Culture medium, 30 DEG C of constant temperature culture, after 3 days, are observed the transparent circle in casein separation screening culture medium, are chosen
Transparent circle diameter and colony diameter ratio (HC) the bacterium colony subcultivation more than 1.5 are cultivated to test tube slant.Choose
After microwave irradiation, the preferable bacterial strain of advantage, makes 10 with sterilized water6-108The bacteria suspension of/mL, by it with 220
μ g/mL nitroso guanidine solution equal-volume is placed in mixing in the conical flask of cleaning, uses after processing 30min at 30 DEG C
Cold saline is diluted to 50 times and terminates reaction, and centrifuge washing is removed medicine 3 times, then is diluted to 106-108
Times bacteria suspension, takes 0.2mL bacteria suspension and is spread evenly across on casein separation screening culture medium flat plate, by flat board
It is inverted in 35 DEG C of constant incubators cultivation 38 hours, chooses transparent circle diameter and colony diameter ratio (HC)
Bacterium colony subcultivation more than 1.5 produces bacterium processed to conduct on test tube slant.
The preparation of described nitroso guanidine solution: accurately weigh 0.020g nitrosoguanidine, is placed in the capacity of 100mL
In Ping, add appropriate acetone soln hydrotropy, add the phosphate buffer constant volume of pH7.2, prepare 200 μ
G/mL nitroso guanidine solution, 4 DEG C save backup.
Described casein separation screening culture medium: casein 0.4-0.6%, Carnis Bovis seu Bubali cream 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
Sterilizing 18-22min.
Second step: produce bacterium processed
By the lactoenterococcus screened, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis is inoculated in beef-protein medium respectively, cultivates 30 hours, then is forwarded to equipped with battalion
Support in the Fructus Solani melongenae bottle of agar culture medium, cultivate 50 hours at 35 DEG C, put into 4 DEG C of Refrigerator stores.
Described beef-protein medium: glucose 5.0g, Carnis Bovis seu Bubali cream 5.0g, peptone 10.0g, chlorination
Sodium 5.0g, sterilized water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
Described nutrient agar: Carnis Bovis seu Bubali cream 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 20.0g,
Sterilized water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
3rd step: the synchronized culture of compound bacteria
By the lactoenterococcus preserved, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein
Candida mycoderma, the inoculum concentration by 8% is inoculated in seed liquor culture medium respectively, and domestication is cultivated 10 hours.
Each strain is weighed: lactoenterococcus 22%, Bacillus licheniformis 22%, solidifying by following percentage by weight
Knot bacillus cereus 26%, Bacillus pumilus 13%, Candida utilis 17%.
The all strains cultivated are inoculated in by weight percentage same equipped with training in the fermentation tank of fluid medium
Supporting, pH7.2-7.4, speed of agitator 180-220rpm, cultivation temperature is 35-39 DEG C, ventilation 1:0.5-1:0.7,
Fermentation time is 22 hours.
Described seed liquor culture medium: glucose 1-3%, yeast extract 0.5-1.0%, peptone 0.5-1.5%, cattle
Meat extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.05-0.15%, sterilized water 94-96%,
PH7.0-7.2,121 DEG C of sterilizing 20-25min.
Described fermentation liquid culture medium: bean cake 10-15%, fish flour 8-12%, Testa Tritici 4-10%, Semen Maydis pulp 6-10%,
Glucose 1-3%, sodium chloride 0.5-1.5%, yeast extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, biphosphate
Potassium 0.5-1.0%, ammonium sulfate 0.5-1.5%, manganese sulfate 0.005-0.01%, ferrous sulfate 0.01-0.03%, nothing
Bacterium water 62-65%, pH7.2-7.4,121 DEG C sterilizing 20-25min.
4th step: the separation of biologically active peptide
After completing the fermentation to compound protein raw material under the optimal conditions of fermentation of compound bacteria, fermentation liquid is existed
Under 4500-5500rpm speed conditions, centrifugal 25-35min, takes supernatant and is concentrated in vacuo, lyophilization system
Obtain the crude product of biologically active peptide.
By the dissolving crude product of biologically active peptide in deionized water, it is configured to solution, with molecular cut off is
The crude product solution of biologically active peptide is separated by the ultrafilter membrane of 10KDa, permeate obtained by collecting, then warp
Molecular cut off is that the ultrafilter membrane of 5KDa separates further, be respectively adopted the most after the same method 3KDa,
The ultrafilter membrane of 1KDa separates step by step, obtains biologically active peptide ultrafiltration fragment by finally collecting, after being concentrated in vacuo,
Lyophilization becomes lyophilized powder standby.Again biologically active peptide ultrafiltration fragment lyophilized powder is dissolved in deionized water configuration
Becoming mass body volume concentrations is the solution of 50mg/mL, and after mix homogeneously, loading 2mL carries out sephadex
Chromatogram purification, elution flow rate is 0.8-1.2mL/h, and detection wavelength is 220nm, utilizes automatic collector to carry out
The collection of separation component, repeatedly loading, be constantly enriched with each component and lyophilization obtain biologically active peptide
Sterling, saves backup.The polypeptide of separating-purifying is carried out amino acid sequencing, and its aminoacid sequence is
Arg-Pro-Cys-Leu-Ser-Ala-Gln-Ile-Leu-Ser-Thr-Ser-Val, such as sequence table SEQ ID No.1 institute
Show.
Two, the biologically active peptide prepared is applied to piglet, evaluates biologically active peptide and promote the effect of piglet growth
Really, concrete grammar includes:
The preparation of simulated gastric fluid: measure 234mL concentrated hydrochloric acid addition sterilized water and be diluted to 1000mL, prepare 10%
Dilute hydrochloric acid.Take this dilute hydrochloric acid 16.4mL, add sterilized water 800mL and pepsin 10g, stir,
Add sterilized water be diluted to 1000mL and get final product.
The preparation of need testing solution: accurately weigh biological activity peptide freeze-dried powder finished product 0.05g, artificial with 100.0g
Gastric juice dissolved dilution makes need testing solution, and 4 DEG C save backup.
Choose the piglet 100 of 10-15 age in days, be randomly divided into A, B two groups, often group 50.To every head
Ear's label of piglet numbering is identified respectively.As a control group, gavage 2mL every day supplies examination to A group
Product solution, fills continuously and feeds 20 days；B group as a control group, is freely drunk water, is searched for food, and the difference with A group is only
It is not gavage need testing solution.Observe the growth conditions of piglet every day, piglet is weighed, and make easy to remember
Record.The test of 20 days by a definite date, the piglet average daily gain of test group improves 6.50% (P ＜ 0.05), piglet
After wean, feed intake increases by 4.31% (P ＜ 0.05), and mortality rate relatively matched group reduces by 50% (P ＜ 0.05).
Embodiment 3
All strains that the present embodiment is used are all the microorganism fungus kinds can added in feedstuff, and lactic acid intestinal ball
Bacterium, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, Candida utilis are all on market
Buying, the strain of different manufacturers all can reach the effect of approximation.
One, the preparation method of the biologically active peptide promoting piglet growth of the present embodiment comprises the steps:
The first step: cultivate strain, utilize mutation breeding technologies that each strain is carried out preferably.
Under power is the uviol lamp of 20W, 30cm irradiation distance, by the lactoenterococcus preserved, lichens bud
Spore bacillus, Bacillus coagulans, Bacillus pumilus, the bacteria suspension 2mL cheese egg respectively of Candida utilis
On the aseptic flat board of white separation screening culture medium, irradiate 2-3 minute and cultivate counting, observe at casein bolter
Select the transparent circle in culture medium, choose transparent circle diameter and colony diameter ratio (HC) bacterium colony more than 1.5 and move
Plant and cultivate to test tube slant.Choose the preferable bacterial strain of advantage after ultraviolet mutagenesis, make 106-108/ mL's
Bacteria suspension, 850W peak power, specified microwave frequency 2450MHz microwave oven in, microwave exposure 45s,
And process elimination heat effect, 4 DEG C of cold preservations of lucifuge 10 hours every 5s ice bath, coat casein separation screening
Culture medium, 32 DEG C of constant temperature culture, after 3 days, are observed the transparent circle in casein separation screening culture medium, are chosen
Transparent circle diameter and colony diameter ratio (HC) the bacterium colony subcultivation more than 1.5 are cultivated to test tube slant.Choose
After microwave irradiation, the preferable bacterial strain of advantage, makes 10 with sterilized water6-108The bacteria suspension of/mL, by it with 200
μ g/mL nitroso guanidine solution equal-volume is placed in mixing in the conical flask of cleaning, uses after processing 25min at 33 DEG C
Cold saline is diluted to 50 times and terminates reaction, and centrifuge washing is removed medicine 3 times, then is diluted to 106-108
Times bacteria suspension, takes 0.2mL bacteria suspension and is spread evenly across on casein separation screening culture medium flat plate, by flat board
It is inverted in 37 DEG C of constant incubators cultivation 36 hours, chooses transparent circle diameter and colony diameter ratio (HC)
Bacterium colony subcultivation more than 1.5 produces bacterium processed to conduct on test tube slant.
The preparation of described nitroso guanidine solution: accurately weigh 0.020g nitrosoguanidine, is placed in the capacity of 100mL
In Ping, add appropriate acetone soln hydrotropy, add the phosphate buffer constant volume of pH7.2, prepare 200 μ
G/mL nitroso guanidine solution, 4 DEG C save backup.
Described casein separation screening culture medium: casein 0.4-0.6%, Carnis Bovis seu Bubali cream 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
Sterilizing 18-22min.
Second step: produce bacterium processed
By the lactoenterococcus screened, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis is inoculated in beef-protein medium respectively, cultivates 35 hours, then is forwarded to equipped with battalion
Support in the Fructus Solani melongenae bottle of agar culture medium, cultivate 47 hours at 37 DEG C, put into 4 DEG C of Refrigerator stores.
Described beef-protein medium: glucose 5.0g, Carnis Bovis seu Bubali cream 5.0g, peptone 10.0g, chlorination
Sodium 5.0g, sterilized water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
Described nutrient agar: Carnis Bovis seu Bubali cream 3.0g, peptone 10.0g, sodium chloride 5.0g, agar 20.0g,
Sterilized water 1000mL, pH7.0-7.2,121 DEG C of sterilizing 20-25min.
3rd step: the synchronized culture of compound bacteria
By the lactoenterococcus preserved, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein
Candida mycoderma, the inoculum concentration by 10% is inoculated in seed liquor culture medium respectively, and domestication is cultivated 12 hours.
Each strain is weighed: lactoenterococcus 23%, Bacillus licheniformis 23%, solidifying by following percentage by weight
Knot bacillus cereus 28%, Bacillus pumilus 11%, Candida utilis 15%.
The all strains cultivated are inoculated in by weight percentage same equipped with training in the fermentation tank of fluid medium
Supporting, pH7.2-7.4, speed of agitator 180-220rpm, cultivation temperature is 35-39 DEG C, ventilation 1:0.5-1:0.7,
Fermentation time is 23 hours.
Described seed liquor culture medium: glucose 1-3%, yeast extract 0.5-1.0%, peptone 0.5-1.5%, cattle
Meat extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, potassium dihydrogen phosphate 0.05-0.15%, sterilized water 94-96%,
PH7.0-7.2,121 DEG C of sterilizing 20-25min.
Described fermentation liquid culture medium: bean cake 10-15%, fish flour 8-12%, Testa Tritici 4-10%, Semen Maydis pulp 6-10%,
Glucose 1-3%, sodium chloride 0.5-1.5%, yeast extract 0.5-1.0%, magnesium sulfate 0.05-0.1%, biphosphate
Potassium 0.5-1.0%, ammonium sulfate 0.5-1.5%, manganese sulfate 0.005-0.01%, ferrous sulfate 0.01-0.03%, nothing
Bacterium water 62-65%, pH7.2-7.4,121 DEG C sterilizing 20-25min.
4th step: the separation of biologically active peptide
After completing the fermentation to compound protein raw material under the optimal conditions of fermentation of compound bacteria, fermentation liquid is existed
Under 4500-5500rpm speed conditions, centrifugal 25-35min, takes supernatant and is concentrated in vacuo, lyophilization system
Obtain the crude product of biologically active peptide.
By the dissolving crude product of biologically active peptide in deionized water, it is configured to solution, with molecular cut off is
The crude product solution of biologically active peptide is separated by the ultrafilter membrane of 10KDa, permeate obtained by collecting, then warp
Molecular cut off is that the ultrafilter membrane of 5KDa separates further, be respectively adopted the most after the same method 3KDa,
The ultrafilter membrane of 1KDa separates step by step, obtains biologically active peptide ultrafiltration fragment by finally collecting, after being concentrated in vacuo,
Lyophilization becomes lyophilized powder standby.Again biologically active peptide ultrafiltration fragment lyophilized powder is dissolved in deionized water configuration
Becoming mass body volume concentrations is the solution of 50mg/mL, and after mix homogeneously, loading 2mL carries out sephadex
Chromatogram purification, elution flow rate is 0.8-1.2mL/h, and detection wavelength is 220nm, utilizes automatic collector to carry out
The collection of separation component, repeatedly loading, be constantly enriched with each component and lyophilization obtain biologically active peptide
Sterling, saves backup.The polypeptide of separating-purifying is carried out amino acid sequencing, and its aminoacid sequence is
Arg-Pro-Cys-Leu-Ser-Ala-Gln-Ile-Leu-Ser-Thr-Ser-Val, such as sequence table SEQ ID No.1 institute
Show.
Two, the biologically active peptide prepared is applied to piglet, evaluates biologically active peptide and promote the effect of piglet growth
Really, concrete grammar includes:
The preparation of simulated gastric fluid: measure 234mL concentrated hydrochloric acid addition sterilized water and be diluted to 1000mL, prepare 10%
Dilute hydrochloric acid.Take this dilute hydrochloric acid 16.4mL, add sterilized water 800mL and pepsin 10g, stir,
Add sterilized water be diluted to 1000mL and get final product.
The preparation of need testing solution: accurately weigh biological activity peptide freeze-dried powder finished product 0.04g, artificial with 100.0g
Gastric juice dissolved dilution makes need testing solution, and 4 DEG C save backup.
Choose the piglet 100 of 10-15 age in days, be randomly divided into A, B two groups, often group 50.To every head
Ear's label of piglet numbering is identified respectively.As a control group, gavage 2mL every day supplies examination to A group
Product solution, fills continuously and feeds 20 days；B group as a control group, is freely drunk water, is searched for food, and the difference with A group is only
It is not gavage need testing solution.Observe the growth conditions of piglet every day, piglet is weighed, and make easy to remember
Record.The test of 20 days by a definite date, the piglet average daily gain of test group improves 6.2% (P ＜ 0.05), and piglet is broken
After milk, feed intake increases by 4.2% (P ＜ 0.05), and mortality rate relatively matched group reduces by 50% (P ＜ 0.05).
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement etc. made, should be included in the protection of the present invention
Within the scope of.
Claims (10)
1. the biologically active peptide promoting piglet growth, it is characterised in that: described biologically active peptide comprises SEQ
Aminoacid sequence shown in ID No.1.
2. the biologically active peptide promoting piglet growth described in claim 1 is promoting piglet growth, is reducing piglet
Application in mortality rate.
3. described in claim 1, promote the preparation method of the biologically active peptide of piglet growth, it is characterised in that:
Comprise the steps:
A, screen lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein respectively
Cultivate after candida mycoderma；
B, by step A gained lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis mixed culture fermentation, concentrates after extracting fermented liquid supernatant liquid, is dried, must be raw after separating-purifying
Thing bioactive peptide.
4. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 3, its feature exists
In: screening described in step A comprises the steps:
A, primary dcreening operation: by lactoenterococcus, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus, product protein
Candida mycoderma is added separately on the aseptic flat board of casein separation screening culture medium, and ultraviolet mutagenesis is chosen after cultivating
Transparent circle diameter and colony diameter are cultivated than the bacterium colony subcultivation more than 1.5；
B, multiple sieve: after each dominant strain obtained through step a is carried out microwave irradiation, use casein bolter
Select constant temperature culture 2-3 days at culture medium, 30-34 DEG C, choose transparent circle diameter and colony diameter ratio more than 1.5
Bacterium colony subcultivation is cultivated；
C, the most again sieve: by molten with 180-220 μ g/mL nitrosoguanidine respectively for each dominant strain of obtaining through step b
Cultivate 20-30min at 30-35 DEG C after the mixing of liquid equal-volume, wash after terminating reaction, be formulated as bacteria suspension, will
Bacteria suspension coats casein separation screening culture medium flat plate, cultivates 34-38 hour, choose at 35-39 DEG C
Bright loop diameter and colony diameter are than the bacterium colony more than 1.5.
5. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 4, its feature exists
In: the microwave irradiation step described in step b includes: 850W peak power, specified microwave frequency 2450MHz
Microwave oven in, microwave exposure 45-55s, and every 5-10s ice bath process, it is cold that microwave exposure terminates rear lucifuge
Hide 10-14 hour.
6. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 3, its feature exists
In: described in step A, incubation step includes: by the lactoenterococcus after screening, Bacillus licheniformis, condensation
Bacillus cereus, Bacillus pumilus, Candida utilis are cultivated 30-40 hour in beef-protein medium,
It is forwarded to again in nutrient agar, cultivates 45-50 hour at 35-39 DEG C.
7. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 3, its feature exists
In: it is mixed fermentation step described in step B and includes: by percentage to the quality, lactoenterococcus 20-23%,
Bacillus licheniformis 22-25%, Bacillus coagulans 26-30%, Bacillus pumilus 10-14%, product protein are false
The ratio of silk yeast 14-17% is inoculated in same equipped with cultivation and fermentation in the fermentation tank of fluid medium.
8. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 7, its feature exists
In: lactoenterococcus described in step B, Bacillus licheniformis, Bacillus coagulans, Bacillus pumilus,
Candida utilis is inoculated in seed liquor culture medium by the inoculum concentration of 8%-12% respectively, cultivates domestication 10-14
Mixed culture fermentation is carried out after hour.
9. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 7, its feature exists
In: the condition of described cultivation and fermentation is pH7.2-7.4, speed of agitator 180-220rpm, and temperature is 35-39 DEG C,
Ventilation 1:0.5-1:0.7, fermentation time 22-24 hour.
10. the preparation method of the biologically active peptide promoting piglet growth as claimed in claim 3, it is special
Levy and be: described in step B, separating-purifying step includes: be solution by concentration, dried products configuration,
Ultrafilter membrane through 10KDa, 5KDa, 3KDa, 1KDa separates step by step successively, collects and obtains biologically active peptide
Ultrafiltration fragment, then carry out sephadex chromatogram purification, elution flow rate 0.8-1.2mL/h, detects wavelength
For 220nm, collect component postlyophilization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN107736298A (en) * | 2017-10-11 | 2018-02-27 | 浙江青莲食品股份有限公司 | The cultural method of the black pig of children phase |
| CN107736297A (en) * | 2017-10-11 | 2018-02-27 | 浙江青莲食品股份有限公司 | Grow up the cultural method of black pig |
| CN107743918A (en) * | 2017-10-11 | 2018-03-02 | 浙江青莲食品股份有限公司 | The cultural method of nursing period black pig |
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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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| 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 |
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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 |
| CN107736298A (en) * | 2017-10-11 | 2018-02-27 | 浙江青莲食品股份有限公司 | The cultural method of the black pig of children phase |
| CN107736297A (en) * | 2017-10-11 | 2018-02-27 | 浙江青莲食品股份有限公司 | Grow up the cultural method of black pig |
| CN107743918A (en) * | 2017-10-11 | 2018-03-02 | 浙江青莲食品股份有限公司 | The cultural method of nursing period black pig |
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