CN105901336B - The method of enrichment antibacterial peptide and a kind of antibacterial peptide product from ferment product - Google Patents
The method of enrichment antibacterial peptide and a kind of antibacterial peptide product from ferment product Download PDFInfo
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- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 37
- 229910021536 Zeolite Inorganic materials 0.000 claims abstract description 50
- 239000010457 zeolite Substances 0.000 claims abstract description 50
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 33
- 239000000843 powder Substances 0.000 claims abstract description 30
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000000463 material Substances 0.000 claims abstract description 19
- 229920000858 Cyclodextrin Polymers 0.000 claims abstract description 15
- 239000001116 FEMA 4028 Substances 0.000 claims abstract description 15
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 15
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims abstract description 15
- 229960004853 betadex Drugs 0.000 claims abstract description 15
- 239000000203 mixture Substances 0.000 claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims description 27
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- 239000007788 liquid Substances 0.000 claims description 21
- 238000002360 preparation method Methods 0.000 claims description 11
- 241000193417 Brevibacillus laterosporus Species 0.000 claims description 4
- JYIBXUUINYLWLR-UHFFFAOYSA-N aluminum;calcium;potassium;silicon;sodium;trihydrate Chemical compound O.O.O.[Na].[Al].[Si].[K].[Ca] JYIBXUUINYLWLR-UHFFFAOYSA-N 0.000 claims description 3
- 229910001603 clinoptilolite Inorganic materials 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 230000002335 preservative effect Effects 0.000 claims description 3
- 241000193755 Bacillus cereus Species 0.000 claims description 2
- 241000194108 Bacillus licheniformis Species 0.000 claims description 2
- 229910052908 analcime Inorganic materials 0.000 claims description 2
- 239000000470 constituent Substances 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- 244000025254 Cannabis sativa Species 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 abstract description 17
- 239000003463 adsorbent Substances 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000000605 extraction Methods 0.000 abstract description 4
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- 230000003000 nontoxic effect Effects 0.000 abstract description 2
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- 238000001035 drying Methods 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 5
- 230000003385 bacteriostatic effect Effects 0.000 description 5
- 235000013339 cereals Nutrition 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 4
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
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- 235000014897 Streptococcus lactis Nutrition 0.000 description 3
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- 239000002245 particle Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000003828 vacuum filtration Methods 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
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- 230000015572 biosynthetic process Effects 0.000 description 2
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- 238000001914 filtration Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
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- 239000002609 medium Substances 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 235000015099 wheat brans Nutrition 0.000 description 2
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241000896292 Odontothrips loti Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001142 anti-diarrhea Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 231100000693 bioaccumulation Toxicity 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 244000144977 poultry Species 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000009461 vacuum packaging Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/358—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Inorganic Chemistry (AREA)
- Microbiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The method that the present invention provides a kind of to be enriched with antibacterial peptide from ferment product, material 1 is obtained the following steps are included: 1) ferment product to be carried out at 115-121 DEG C to first with beta-cyclodextrin and is contacted, wherein, relative to the ferment product of 100ml, the dosage of beta-cyclodextrin is 0.5-2.5g；2) material 1 is carried out second with zeolite powder and contacts acquisition material 2, wherein the material 1 relative to 100ml, the dosage of zeolite powder is 1-5g；3) acquisition antibacterial peptide-zeolite composition is filtered to material 2.Method extraction process of the invention is simple, nontoxic, low in cost and production cycle end, can improve adsorption rate and improve the adsorbance of Unit Weight adsorbent.
Description
Technical field
The invention belongs to field of biotechnology, are related to the quick separating preparation method of microbial antibacterial peptide, and in particular to one
The kind method of enrichment antibacterial peptide and a kind of product containing antibacterial peptide from ferment product.
Background technique
Antibacterial peptide (Antimicrobial peptide) abbreviation AMP, be otherwise known as antimicrobial peptide or peptide antibiotic, comes
It is the component part of natural defense system in nature biotechnology body derived from a variety of biologies.In terms of farming and animal husbandry, antibacterial peptide is as feeding
The problems such as feed additives can not only substitute the antibiotic in feed, solve medicament residue and drug resistance, also has livestock and poultry
Growth promotion, health care and the function for the treatment of disease.
In recent years, how to be separated by the antimicrobial peptide preparation that various technological means efficiently obtain high-purity as antibacterial peptide
The research hotspot of extraction, in addition, the extensive extracting method of low cost is the key that realize antibacterial peptide industrialized production and mesh
The preceding principal element for restricting the antibacterial peptide market competitiveness.In terms of antibacterial peptide preparation, mainly pass through extraction, people from material liquid
Synthesis peptide chain is prepared with three kinds of fermentation technique in the synthesis of work method.Fermentation technique is that specific secretion of fermenting under certain condition is anti-
The test strain of bacterium peptide to achieve the purpose that largely to be enriched with, then extracts and antimicrobial peptide products is prepared.
Yang etc. establishes the method that thallus absorption method extracts streptococcus lactis peptide, reaches yield by changing fermentation liquid pH
93.3%；Coventry etc. is using calcium silicates antiplastering aid (trade name " Micro-Cel ") from the fermentation liquid of Lactococcus lactis
Adsorb streptococcus lactis peptide；Gao Zhao such as builds at a kind of process that antibacterial peptide is prepared using bacillus subtilis of patent report in 2013,
Fermentation liquid through and pretreatment, then obtains antibacterial peptide living solution with molecular sieve gel column filtration chromatography, then be freeze-dried
To high concentration antibacterial peptide powder, activity recovery reaches 43.5% or more；A kind of housefly of patent report point in 2007 such as Wu Jianwei
Separation method of type antibacterial peptide and products thereof and application are secreted, antibacterial peptide crude extract is dialysed, dialyzed solution extracts through solid phase
It taking, elution fraction is further purified into two antibacterial peptide components with reversed-phase high performance liquid chromatography through ultra-filtration and separation, active component,
Up to housefly secretion type antibacterial peptide.Once it reported within 1996 and antibacterial peptide is adsorbed on food-grade adsorbent surface；There is report again within 2010
Bacteriocin is adsorbed on two kinds of food-grade adsorbent surfaces by road.However, food-grade adsorbent price is costly, it is difficult to realize
Large-scale production is unfavorable for promoting and applying.
Therefore, it is necessary to the beneficiation technologies to antibacterial peptide improve, it is simple, at low cost and have to provide a kind of processing technology
There are the antibacterial peptide beneficiation technologies compared with high efficiency.
Summary of the invention
It is an object of the invention in view of the above defects of the prior art, provide a kind of simple process, at low cost
Antibacterial peptide enrichment method honest and clean and with high efficiency.
In order to reach the goals above, the method that the present invention provides a kind of is enriched with antibacterial peptide from ferment product, including
Following steps:
1) ferment product is carried out at 115-121 DEG C to first with beta-cyclodextrin and contacts acquisition material 1, wherein is opposite
In the ferment product of 100ml, the dosage of beta-cyclodextrin is 0.5-2.5g；
2) material 1 is carried out second with zeolite powder and contacts acquisition material 2, wherein the material 1 relative to 100ml, zeolite powder
Dosage be 1-5g；
3) acquisition antibacterial peptide-zeolite composition is filtered to material 2.
Wherein, the present invention is not particularly limited the type of fermentation liquid, and this field produces antibacterial using bacterial fermentation
Fermentation liquid obtained is enriched with using enrichment method provided by the present invention peptide after fermentation.Such as the bacterium
Fermentation liquid is Brevibacillus laterosporus, bacillus subtilis, bacillus licheniformis or the fermentation liquid of bacillus cereus.It is preferred that
, the ferment product is bacillus laterosporus fermentation liquid.
Preferably, in the ferment product, the concentration of antibacterial peptide is 1500-1800AU/ml.
In the present invention, the purpose of the first contact includes improving the heat resistance and stability of antibacterial peptide in fermentation liquid and keeping anti-
The bioactivity of bacterium peptide, meanwhile, after the first contact, the production bacterium in fermentation liquid can be killed, so that antibacterial peptide is from cell
In release, more conducively subsequent enrichment.
Preferably, relative to the ferment product of 100ml, the dosage of beta-cyclodextrin can be obtained when being 1-2g and preferably be mentioned
In high fermentation liquid the heat resistance and stability of antibacterial peptide and keep antibacterial peptide bioactivity effect.It is described first contact when
Between preferably 20-30min.
Optionally, in step 2), the second contact carries out preferably in vacuum concentration pot, and the condition of the second contact includes:
60-120min is contacted under conditions of 40-60 DEG C of temperature, air pressure 0.07-0.09Mpa, revolving speed 100-200r/min.
Particularly preferred, the second contact enables to antibacterial peptide more when carrying out at 45-55 DEG C of temperature, air pressure 0.08Mpa
Add and be adequately adsorbed on zeolite granular surface, improves the bioaccumulation efficiency of antibacterial peptide.
Optionally, the zeolite powder is selected from and is made of clinoptilolite powder, modenite powder, analcime powder and chabasie powder
At least one of group, partial size is 80-180 mesh, and the effect that zeolite powder is played includes adsorbent and carrier.
Wherein, in step 3), the method for filtering is not particularly limited, as long as can be by the solution in material 2
Removal retains precipitating, it is preferred that is filtered using vacuum filtration machine, the aperture of the filter cloth utilized is 50-100 mesh.
Antibacterial peptide after step 3) in fermentation liquid is adsorbed to zeolite surface, forms antibacterial peptide-zeolite composition.
The method that invention further provides a kind of to be enriched with antibacterial peptide using aforementioned from ferment product prepares
Antibacterial peptide-zeolite composition.
The present invention also provides a kind of antibacterial peptide additive product, active constituent is the antibacterial peptide-zeolite composition.
The preparation method of the antibacterial peptide addition includes setting antibacterial peptide-zeolite composition by rotary flashing drying
Standby dry, drying temperature is 60 DEG C -90 DEG C；Or naturally dry or hot wind room dry.
Optionally, the material after drying is machined to particle with flat-die feed granules, die hole specification is 3-4mm；
Beta-cyclodextrin in the antibacterial peptide additive product.
Antibacterial peptide-zeolite composition provided by the invention can be used in preparing biological preservative.
Feed addictive provided by the invention can be used in preparing biological preservative.
Antibacterial peptide-zeolite composition provided by the invention can be used in preparing feed.
Feed addictive provided by the invention can be used in preparing feed.
The method provided by the invention that antibacterial peptide is enriched with from ferment product overcomes the deficiencies in the prior art, and use is low
The inorganic adsorbent zeolite powder of cost cooperates vacuum concentration technology, and antibacterial peptide molecule is directly adsorbed on zeolite granular, inhales
Attached rate is up to 88%-96%, rate of recovery 89%-94%, ratio of briquetting 88%-93%, reaches and utilizes cheap adsorbent from micro- life
The purpose that antibacterial peptide is prepared in object fermentation liquid realizes high antibacterial activity and high stability while improving the rate of recovery.The present invention
Provided method extraction process is simple, nontoxic, low in cost；The production cycle can be shortened, improve adsorption rate and raising
The adsorbance of Unit Weight adsorbent.
The inorganic zeolite powder cooperation of feed grade is concentrated in vacuo technical application in preparation antibacterial peptide addition for the first time by the present invention,
Zeolite powder not only has high adsorption rate, but also avoids that the stability that organic adsorbent has is low, perishable, Yi Yinqi livestock abdomen
The problems such as rushing down, zeolite powder itself have anti-oxidant, anti diar rhea, promotion growth of animal, raising production performance and other effects again.This method
The antibacterial peptide product safety prepared is stablized, and can be widely applied to feed industry, provides one for the large scale preparation of antibacterial peptide
Kind new method, has more extensive and practical application effect.
Specific embodiment
The present invention is described in detail With reference to embodiment.The following examples are intended to illustrate the invention, but
It is not intended to limit the scope of the invention.Unless otherwise specified, technological means used is ripe for those skilled in the art in embodiment
The conventional means known, it is raw materials used unless otherwise indicated, be commercially available.
The enrichment of 1 antibacterial peptide of embodiment and the preparation of antibacterial peptide addition
Antibacterial peptide is extracted from Brevibacillus laterosporus fermentation liquid, prepares antibacterial peptide addition, steps are as follows:
(1) antibacterial peptide is enriched with
Fermentation liquor pretreatment: (concentration of antibacterial peptide is 1600AU/ml when fermentation ends) addition β-ring paste in fermentation liquid
Smart protective agent, the dosage relative to every 100ml ferment product beta-cyclodextrin is 1g, mixes well, is then heated to 121 DEG C of guarantors
Hold 20min.
Vacuum concentration absorption: clinoptilolite powder (partial size is 100 mesh) and fermentation liquid, zeolite powder are added into vacuum concentration pot
Amount be upper step obtain volume of material 4% (m/v), adsorption temp be 50 DEG C, revolving speed 200r/min, stir 120min, very
Reciprocal of duty cycle is maintained at 0.08Mpa, and antibacterial peptide is made sufficiently to be adsorbed on zeolite granular surface, and antibacterial peptide-zeolite compound is made；
Vacuum filtration: being filtered using vacuum filtration machine, and filter cloth is 100 mesh, removes supernatant, retains precipitating, and what is filtered out is heavy
It forms sediment and obtains feed grade biologic antibiotic peptide-zeolite compound.
(2) antibacterial peptide addition is prepared
Dry: biologic antibiotic peptide-zeolite composition is dry by rotary flashing drying equipment, and drying temperature is 60 DEG C；
Granulating and forming: the powder after drying is machined to particle with flat-die feed granules, die hole specification is 4mm；
Vacuum packaging: granular antibacterial peptide-zeolite composition is vacuum-packed and obtains the production of antibacterial peptide additive
Product.
Adsorption rate, the rate of recovery and the grain forming rate of zeolite adsorption antibacterial peptide in antibacterial peptide addition product are surveyed
Fixed, the results are shown in Table 2.
Adsorption rate %=(A-B)/A × 100%
A is absorption primary fermentation liquid potency in formula；
B is supernatant potency after adsorption equilibrium.
Rate of recovery %=t/T × 100%
T is to obtain zeolite powder weight (kg) after drying in formula；
T is zeolite powder additive amount (kg).
Grain forming rate %=(1-r/t) × 100%
T is to obtain zeolite powder weight (kg) after drying in formula
R is the zeolite powder particles (kg) that can be sieved by No. 5
Embodiment 2-13
Antibacterial peptide-zeolite compound and antibacterial peptide additive product are prepared according to the condition that table 1 is listed, other processing
Process is same as Example 1.
The adsorption rate, the rate of recovery and grain forming rate of zeolite adsorption antibacterial peptide in embodiment 2-13 are measured, as a result
As shown in table 2.
Comparative example 1-5
Antibacterial peptide-zeolite compound and antibacterial peptide additive product are prepared according to the condition that table 1 is listed, other processing
Process is same as Example 1.
Adsorption rate, the rate of recovery and the grain forming rate for measuring zeolite adsorption antibacterial peptide in comparative example 1-5, as a result such as 2 institute of table
Show.
1 product preparation condition of table
Comparative example 6
Antibacterial peptide-zeolite compound and antibacterial peptide additive product are prepared using method same as Example 1.Area
It is not only that the wheat bran that zeolite powder is changed to etc. to quality, the adsorption rate of wheat bran adsorption antibacterial peptide is measured, as a result such as table 2
It is shown.
Comparative example 7
Antibacterial peptide-zeolite compound and antibacterial peptide additive product are prepared using method same as Example 1.Area
It is not only that and does not add beta-cyclodextrin, the adsorption rate of zeolite adsorption antibacterial peptide is measured, the results are shown in Table 2.
2 product result of implementation of table
| Antibacterial peptide-zeolite composition | Adsorption rate (%) | The rate of recovery (%) | Grain forming rate (%) |
| Embodiment 1 | 96 | 94 | 92 |
| Embodiment 2 | 92 | 94 | 90 |
| Embodiment 3 | 95 | 91 | 91 |
| Embodiment 4 | 92 | 92 | 91 |
| Comparative example 1 | 86 | 84 | 87 |
| Embodiment 5 | 94 | 93 | 92 |
| Comparative example 2 | 81 | 80 | 82 |
| Embodiment 6 | 92 | 90 | 90 |
| Embodiment 7 | 93 | 93 | 93 |
| Comparative example 3 | 80 | 85 | 84 |
| Embodiment 8 | 95 | 93 | 90 |
| Embodiment 9 | 95 | 92 | 89 |
| Comparative example 4 | 82 | 80 | 80 |
| Embodiment 10 | 92 | 93 | 90 |
| Embodiment 11 | 90 | 93 | 91 |
| Embodiment 12 | 88 | 89 | 88 |
| Embodiment 13 | 95 | 93 | 90 |
| Comparative example 5 | 84 | 80 | 81 |
| Comparative example 6 | 71 | 75 | 72 |
| Comparative example 7 | 78 | 76 | 75 |
1 biologic antibiotic peptide additive product minimum bactericidal concentration research of test case
Bacteriocidal concentration research is carried out to antibacterial peptide addition prepared by embodiment 1, by indicator bacteria S.aureus ATCC
14458 add feed grade antibacterial peptide feed addictive when cultivating two generation 4h under the conditions of 37 DEG C, revolving speed 200r/min, work as culture medium
When middle antibacterial peptide concentration is 50AU/ml, the culture medium gradually clear after 4h, OD value is close with aseptic culture medium when 6h,
Therefore feed grade antibacterial peptide feed addictive minimum bactericidal concentration is 50AU/ml.
The biologic antibiotic peptide additive product minimum bactericidal concentration research of 2 comparative example 6 of test case preparation
Bacteriocidal concentration research is carried out to antibacterial peptide addition prepared by comparative example 6, by indicator bacteria S.aureus ATCC
14458 add feed grade antibacterial peptide feed addictive when cultivating two generation 4h under the conditions of 37 DEG C, revolving speed 200r/min, work as culture medium
When middle antibacterial peptide concentration is 65AU/ml, the culture medium gradually clear after 4h, OD value is close with aseptic culture medium when 6h,
Therefore feed grade antibacterial peptide feed addictive minimum bactericidal concentration is 65AU/ml.
The biologic antibiotic peptide additive product minimum bactericidal concentration research of 3 comparative example 7 of test case preparation
Bacteriocidal concentration research is carried out to antibacterial peptide addition prepared by comparative example 7, by indicator bacteria S.aureus ATCC
14458 add feed grade antibacterial peptide feed addictive when cultivating two generation 4h under the conditions of 37 DEG C, revolving speed 200r/min, work as culture medium
When middle antibacterial peptide concentration is 64AU/ml, the culture medium gradually clear after 4h, OD value is close with aseptic culture medium when 6h,
Therefore feed grade antibacterial peptide feed addictive minimum bactericidal concentration is 64AU/ml.
4 biologic antibiotic peptide additive product enteron aisle enzyme effect stability of test case
Embodiment 1, comparative example 6-7 the antibacterial peptide addition prepared are subjected to simulation chicken intestinal environmental test, studying enzyme pair
The influence of stability carries out bacteriostatic test research, calculation processing group and control group antibacterial circle diameter using AGP test punch method,
It is as shown in table 3 to compare the loss late being calculated.Cellulase influences maximum as the result is shown, but also only there was only 9% or so, institute
With antibacterial peptide addition, influence of the enzyme to bacteriostatic activity be not significant in enteron aisle, still is able to give expression to apparent antibacterial work
With high stability.
The loss late of the different enzymes of table 3
| Antibacterial peptide addition | Enzyme (3% (E/S)) | Pepsin 1:10000 | Lipase | Cellulase |
| Embodiment 1 | Loss late (%) | 7.55 | 1.75 | 9.01 |
| Comparative example 6 | Loss late (%) | 20.21 | 19.53 | 27.13 |
| Comparative example 7 | Loss late (%) | 17.64 | 21.88 | 19.42 |
5 biologic antibiotic peptide additive product THERMAL STABILITY of test case
The antibacterial peptide addition of embodiment 1 is passed through into 100 DEG C of boiling water water-baths, water bath time 120min utilizes AGP test
Punch method carries out bacteriostatic test research, and compared with control group antibacterial circle diameter, time-triggered protocol is longer, and loss late is higher, in 120min
When loss late stablize 10% or so, lose smaller, and the zeolite after handling still has apparent bacteriostasis, so
Short time heat treatment will not influence antibacterial peptide feed addictive antibacterial activity, and thermal stability is high.
With the thermal stability of the antibacterial peptide addition of same method test comparison example 6-7, the loss late point in 120min
It Wei 17% and 15%.
6 biologic antibiotic peptide additive product preservation Journal of Sex Research of test case
Antibacterial peptide addition prepared by embodiment 1 utilizes fine jade in room temperature and 0 DEG C of long period under refrigeration storage every two weeks
Rouge spreads punch method measurement bacteriostatic activity variation, and experimental period is 10 weeks.As a result, it has been found that in room temperature storage and 0 DEG C of freezing conditions
Storage bacteriostasis does not have significantly different, and keeps higher bacteriostatic activity.
The experiment of 7 beta-cyclodextrin heat resistance of test case
The beta-cyclodextrin of 5,10,20,40,60g/l is added respectively in microbial fermentation solution, is then heated to fermentation liquid
121 DEG C of holding 20min, by Odontothrips loti calculate potency discovery when beta-cyclodextrin additive amount be 5g/l, 10g/l, 20g/l,
When 40g/l, 60g/l, potency difference is high by 35.04%, 39.92%, 27.08%, 21.98%, 40.24% compared with the control group,
Therefore, illustrate that beta-cyclodextrin can be improved the heat resistance of antibacterial peptide in fermentation liquid, and keep higher antibacterial activity, beta-cyclodextrin
Additive amount be 10g/l when effect it is best.
The research of 8 feeding effect of test case
The antibacterial peptide additive product that embodiment 1 is prepared is applied in broiler feeding, and additive amount is respectively
500AU/g, 400AU/g, 300AU/g, experimental result are shown in Table 4, the results showed that high, medium and low dosage group after broiler feeding 42 days
Chicken average weight and the rate of animals delivered to the slaughter-house are higher than blank group, and high, medium and low dosage group feedstuff-meat ratio and the death rate are lower than blank group.
This illustrates that this antibacterial peptide product can actually promote the growth and development of broiler chicken, can substitute antibiotics use, enhancing animal exempt from
Epidemic disease.
The experiment of 4 feeding effect of table
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a kind of method for being enriched with antibacterial peptide from ferment product, which comprises the following steps:
1) ferment product is carried out at 115-121 DEG C to first with beta-cyclodextrin and contacts acquisition material 1, wherein relative to
The ferment product of 100ml, the dosage of beta-cyclodextrin are 0.5-2.5g；
2) material 1 is carried out second with zeolite powder and contacts acquisition material 2, wherein the material 1 relative to 100ml, the use of zeolite powder
Amount is 1-5g；Second contact carries out in vacuum concentration pot, and the condition of the second contact includes: in 40-60 DEG C of temperature, air pressure
60-120min is contacted under conditions of 0.07-0.09Mpa, revolving speed 100-200r/min；
3) acquisition antibacterial peptide-zeolite composition is filtered to material 2.
2. the method according to claim 1, wherein the concentration of antibacterial peptide is 1500- in the ferment product
1800AU/ml。
3. according to the method described in claim 2, it is characterized in that, the time of first contact is 20-30min.
4. method according to claim 1 to 3, which is characterized in that the zeolite powder is selected from by clinoptilolite
At least one of group composed by powder, modenite powder, analcime powder and chabasie powder, partial size are 80-180 mesh.
5. according to the method described in claim 4, it is characterized in that, the ferment product is Brevibacillus laterosporus, withered grass
The fermentation liquid of bacillus, bacillus licheniformis or bacillus cereus.
6. antibacterial peptide-the zeolite composition prepared using method described in any one of claim 1-5.
7. a kind of antibacterial peptide addition, which is characterized in that its active constituent is that antibacterial peptide-zeolite as claimed in claim 6 is compound
Object.
8. antibacterial peptide-zeolite composition as claimed in claim 6 or antibacterial peptide addition as claimed in claim 7 are in preparation biology
Application in preservative.
9. antibacterial peptide-zeolite composition as claimed in claim 6 or antibacterial peptide addition as claimed in claim 7 are preparing feed
In application.
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