CN105838762A - Biological feed containing subtilin and preparing method of biological feed - Google Patents
Biological feed containing subtilin and preparing method of biological feed Download PDFInfo
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- CN105838762A CN105838762A CN201610298651.2A CN201610298651A CN105838762A CN 105838762 A CN105838762 A CN 105838762A CN 201610298651 A CN201610298651 A CN 201610298651A CN 105838762 A CN105838762 A CN 105838762A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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Abstract
The invention discloses a biological feed containing subtilin and a preparing method of the biological feed. The preparing method comprises the steps that a fermentation culture solution is prepared, and the pH value is regulated to be 7.0-7.2, wherein the fermentation culture solution is prepared from, by mass, 0.1-0.2% of starch, 0.4-0.6% of glucose, 0.1-0.2% of urea, 0.2-0.4% of dipotassium phosphate, 0.1-0.2% of monopotassium phosphate, 0.04-0.06% of magnesium sulfate and 0.01-0.03% of yeast extract; the fermentation culture solution is sterilized for 10-20 min at 100-120 DEG C, after the fermentation culture solution is cooled to 30-40 DEG C, a compound Heteroclitin J is added first with the concentration of 30-50 micrograms/g in the culture solution, then bacillus subtilis is inoculated into the culture solution at the inoculation amount of 4-6%, fermentation culture is carried out for 18-24 h, and the pH value is controlled to be 7.0-7.2; after fermentation is finished, the fermentation culture solution is subjected to freezing drying, and active subtilin is obtained. The feed with the subtilin increases the IgM content, the IgA content and the IgG content of serum of dairy cow in the early perinatal stage, and thus the capability of resisting diseases of dairy cow in the early perinatal stage can be improved.
Description
Technical field
The invention belongs to biological feedstuff field, relate to antibacterial peptide, be specifically related to a kind of biological feedstuff containing subtilin and system thereof
Preparation Method.
Background technology
The Ministry of Agriculture of bacillus subtilis base China allows one of two kinds of bacillus as feed addictive, by more and more
It is developed into microorganism formulation.Its preparation is nontoxic, noresidue, free of contamination " green " additive, before having wide development
Scape, extensively applies in animal husbandry, feedstuff industry, shows huge social benefit and ecological benefits.Withered grass gemma
Bacillus has the strongest protease, lipase, amylase isoreactivity, can produce antibiotic, have relatively Johnson & Johnson in animal intestinal
Thing takes oxygen ability by force, and these characteristics, to promoting the zootrophic feed conversion rate digesting and assimilating, improving animal and diseases prevention, promote raw
Length plays an important role.At present in industrialized production, however it remains fermentation period length, spore forming rate are low, high in cost of production is asked
Topic.Fermentation of bacillus subtilis culture medium and fermentation condition are studied by the author, to improving the yield of bacillus subtilis
And reduce production cost.
Antibacterial peptide is a class to be produced through induction by animals and plants and microflora organisms, there is bactericidal activity and with body congenital immunity
The micromolecule polypeptide of the specific function being closely related.Through the research of nearly 40 years, existing 1500 kinds of peptides were proven to have efficiently
Antibacterial activity and immunoregulatory activity, and be used widely in fields such as medicine, food, agriculturals.Abroad, pharmaceutically have
Polymyxin and gramicidin S, Daptomycin and four kinds of medicines of enfuirtide come into the market, and separately have ten several medicines to enter clinic
Experimental stage.Streptococcus lactis peptide as food preservative is widely used in more than 50 countries；Antibacterial peptide gene is proceeded to agriculture
Crop, is the effective way cultivating crop disease-resistant new varieties.
Subtilin is a kind of antibacterial peptide being separated to from bacillus subtilis.Subtilin may be used for the interpolation of biological feedstuff
Agent, can improve the Anti-bacterium ability of livestock, improves immunity.
Summary of the invention
The first object of the present invention is to provide a kind of highly active subtilin, for the additive of biological feedstuff, significantly changes
The Anti-bacterium ability of kind livestock, improves immunity；
The second object of the present invention is to provide a kind of biological feedstuff containing above-mentioned high activity subtilin.
The above-mentioned purpose of the present invention is achieved by techniques below scheme:
A kind of activity subtilin, is made by the steps and forms:
Step S1, prepares fermentation culture, starch-containing 0.1～0.2%, glucose 0.4～0.6%, urea 0.1～0.2%, phosphoric acid
Hydrogen dipotassium 0.2～0.4%, potassium dihydrogen phosphate 0.1～0.2%, magnesium sulfate 0.04～0.06%, yeast extract 0.01～0.03%, pH value is adjusted
It is 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 10～20 minutes at 100～120 DEG C, after being cooled to 30～40 DEG C, first adds
Add compound Heteroclitin J so that it is the concentration in nutrient solution is 30～50 μ g/g, then access withered grass by the inoculum concentration of 4～6%
Fermentation of bacillus is cultivated 18～24 hours, and control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Further, described active subtilin is made by the steps and forms:
Step S1, prepares fermentation culture, starch-containing 0.15%, glucose 0.5%, urea 0.15%, dipotassium hydrogen phosphate 0.3%,
Potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.05%, yeast extract 0.02%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 15 minutes at 110 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 40 μ g/g, then access fermentation of bacillus subtilis by the inoculum concentration of 5%
Cultivating 21 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Further, described active subtilin is made by the steps and forms:
Step S1, prepares fermentation culture, starch-containing 0.1%, glucose 0.4%, urea 0.1%, dipotassium hydrogen phosphate 0.2%,
Potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, yeast extract 0.01%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 20 minutes at 100 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 30 μ g/g, then access fermentation of bacillus subtilis by the inoculum concentration of 4%
Cultivating 24 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Further, described active subtilin is made by the steps and forms:
Step S1, prepares fermentation culture, starch-containing 0.2%, glucose 0.6%, urea 0.2%, dipotassium hydrogen phosphate 0.4%,
Potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.06%, yeast extract 0.03%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 10 minutes at 120 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 50 μ g/g, then access fermentation of bacillus subtilis by the inoculum concentration of 6%
Cultivating 18 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Above-mentioned activity subtilin is as the purposes of biology feed additive.
Further, above-mentioned activity subtilin addition in biological feedstuff is 80～120g/100kg.
Advantages of the present invention:
The active subtilin biologically active that the present invention provides is high, after adding in feed, can significantly improve pre-perinatal milk cow
Immunologic function.The present invention compared with prior art, has prominent substantive distinguishing features and significantly progress.
Detailed description of the invention
Further illustrate the essentiality content of the present invention below in conjunction with embodiment, but do not limit scope with this.To the greatest extent
The present invention is explained in detail by pipe with reference to preferred embodiment, it will be understood by those within the art that, can be to the present invention
Technical scheme modify or equivalent, without deviating from the spirit and scope of technical solution of the present invention.
In the present invention, do not do the test method emphasized especially and reagent is this area conventional methods and reagent.Compound
The preparation method of Heteroclitin J sees document: Compounds from Kadsura heteroclita and related anti-HIV
activity,Phytochemistry,69(2008)1266-1272。
Embodiment 1: the preparation of activity subtilin
Step S1, prepares fermentation culture, starch-containing 0.15%, glucose 0.5%, urea 0.15%, dipotassium hydrogen phosphate 0.3%,
Potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.05%, yeast extract 0.02%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 15 minutes at 110 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 40 μ g/g, then access fermentation of bacillus subtilis training by the inoculum concentration of 5%
Supporting 21 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Embodiment 2: the preparation of activity subtilin
Step S1, prepares fermentation culture, starch-containing 0.1%, glucose 0.4%, urea 0.1%, dipotassium hydrogen phosphate 0.2%,
Potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, yeast extract 0.01%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 20 minutes at 100 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 30 μ g/g, then access fermentation of bacillus subtilis training by the inoculum concentration of 4%
Supporting 24 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Embodiment 3: the preparation of activity subtilin
Step S1, prepares fermentation culture, starch-containing 0.2%, glucose 0.6%, urea 0.2%, dipotassium hydrogen phosphate 0.4%,
Potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.06%, yeast extract 0.03%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 10 minutes at 120 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 50 μ g/g, then access fermentation of bacillus subtilis training by the inoculum concentration of 6%
Supporting 18 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Embodiment 4: the contrast of embodiment 1, without compound Heteroclitin J
Step S1, prepares fermentation culture, starch-containing 0.15%, glucose 0.5%, urea 0.15%, dipotassium hydrogen phosphate 0.3%,
Potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.05%, yeast extract 0.02%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 15 minutes at 110 DEG C, after being cooled to 30～40 DEG C, by the inoculation of 5%
Amount accesses fermentation of bacillus subtilis and cultivates 21 hours, and control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Embodiment 5: effect example, the impact on pre-perinatal immunity of cow
One, materials and methods
1, material: prepare activity subtilin 1～4 according to the method for embodiment 1～4.
2, method
2.1 experimental animals and packet
Choose 30 be in the dry milk phase (antenatal 4 weeks), Body Condition Score close (3.25～3.75), parity close (3～4 tire),
The china holstein cows that the expected date of childbirth is close.It is randomly divided into 5 groups, often group 6.Control group uses Basic drawing to feed.Test
Group 1～4 adds the active subtilin 1～4 of embodiment 1～4 preparation of 100g/100kg respectively on the basis of Basic drawing respectively.
2.2 method for breeding
Milk cow is concentrated to raise and fastens in cowshed in same booth formula, and experimental period was 3 weeks, raises the phase in advance 1 week, from pre-antenatal 4 weeks
(antenatal 28d) starts, and lasts till the same day of giving a birth.Use routine to raise and immunity during test, free choice feeding, freely drink water.
The collection of 2.3 samples and process
Respectively at antenatal 14,7d and childbirth same day, tail venous collection blood 20ml, wherein take 5ml disposal vacuum and take a blood sample
Pipe (EDTA) is collected, and is used for measuring whole blood leucocyte and lymphocyte content；5ml is with disposal vacuum heparin tube (heparin)
Collect, for lymphocyte transformation test；Separately take 10ml disposal vacuum heparin tube (without anti-coagulants) to collect, 3000rpm
Centrifugal 15min, separates and prepares serum ,-20 DEG C of freezen protective.Measure Immunoglobulin in Serum M (IgM), immune globulin
White A (IgA) and the content of immunoglobulin G (IgG).
2.4 Testing index and method
Dry (DM) in feed, crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF),
Calcium (Ca), the assay of phosphorus (P) use " forage analysis and quality detection technology " method.Immunoglobulin in Serum
IgM, IgA and IgG use Immunity transmission turbidity detection.
3, data process
Test data uses SPSS20.0 software to carry out variance analysis, and the significance of difference carries out Duncans Multiple range test, number in table
Value is expressed as Mean ± SD.
Two, result
Activity subtilin is shown in Table 1 to the impact of pre-perinatal immunity of cow.
As shown in Table 1, test group 1～3 respectively before childbirth the serum IgM of 14,7,0 day, IgA and IgG be above comparison
Group, significant difference (P ＜ 0.05) compared with control group；Test group 4 before childbirth the serum IgM of 14,7,0 day, IgA and
There was no significant difference with control group for IgG (P ＞ 0.05), improves inconspicuous.
The table 1 impact on pre-perinatal immunity of cow
Result shows, the feed adding activity subtilin of the present invention improves pre-perinatal cow serum IgM, IgA and IgG
Content, and then the ability of pre-perinatal milk cow resist the disease can be improved.
The effect of above-described embodiment indicates that the essentiality content of the present invention, but does not limit protection scope of the present invention with this.
It will be understood by those within the art that, technical scheme can be modified or equivalent, and not take off
Essence and protection domain from technical solution of the present invention.
Claims (6)
1. an active subtilin, it is characterised in that be made by the steps and form:
Step S1, prepares fermentation culture, starch-containing 0.1～0.2%, glucose 0.4～0.6%, urea 0.1～0.2%, phosphoric acid
Hydrogen dipotassium 0.2～0.4%, potassium dihydrogen phosphate 0.1～0.2%, magnesium sulfate 0.04～0.06%, yeast extract 0.01～0.03%, pH value is adjusted
It is 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 10～20 minutes at 100～120 DEG C, after being cooled to 30～40 DEG C, first adds
Add compound Heteroclitin J so that it is the concentration in nutrient solution is 30～50 μ g/g, then access withered grass by the inoculum concentration of 4～6%
Fermentation of bacillus is cultivated 18～24 hours, and control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Activity subtilin the most according to claim 1, it is characterised in that be made by the steps and form:
Step S1, prepares fermentation culture, starch-containing 0.15%, glucose 0.5%, urea 0.15%, dipotassium hydrogen phosphate 0.3%,
Potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.05%, yeast extract 0.02%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 15 minutes at 110 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 40 μ g/g, then access fermentation of bacillus subtilis training by the inoculum concentration of 5%
Supporting 21 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Activity subtilin the most according to claim 1, it is characterised in that be made by the steps and form:
Step S1, prepares fermentation culture, starch-containing 0.1%, glucose 0.4%, urea 0.1%, dipotassium hydrogen phosphate 0.2%,
Potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.04%, yeast extract 0.01%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 20 minutes at 100 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 30 μ g/g, then access fermentation of bacillus subtilis training by the inoculum concentration of 4%
Supporting 24 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
Activity subtilin the most according to claim 1, it is characterised in that be made by the steps and form:
Step S1, prepares fermentation culture, starch-containing 0.2%, glucose 0.6%, urea 0.2%, dipotassium hydrogen phosphate 0.4%,
Potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.06%, yeast extract 0.03%, pH value is adjusted to 7.0～7.2；It is weight/mass percentage composition；
Step S2, by above-mentioned fermentation culture sterilizing 10 minutes at 120 DEG C, after being cooled to 30～40 DEG C, first adds compound
Heteroclitin J so that it is the concentration in nutrient solution is 50 μ g/g, then access fermentation of bacillus subtilis training by the inoculum concentration of 6%
Supporting 18 hours, control ph is 7.0～7.2；
Step S3, after fermentation ends, obtains activity subtilin by fermentation culture freeze-drying.
5. the arbitrary described activity subtilin of Claims 1 to 4 is as the purposes of biology feed additive.
Purposes the most according to claim 5, it is characterised in that: addition is 80～120g/100kg.
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Citations (3)
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|---|---|---|---|---|
| CN101116676A (en) * | 2006-08-01 | 2008-02-06 | 复旦大学 | Method for preparing compound milettia reticulata particles |
| CN102168045A (en) * | 2010-12-24 | 2011-08-31 | 北京科为博生物科技有限公司 | Bacillus subtilis preparation and preparation method thereof |
| CN104109191A (en) * | 2014-07-02 | 2014-10-22 | 北京龙科方舟生物工程技术有限公司 | Separation and purification method for subtilin antibacterial peptide |
-
2016
- 2016-05-06 CN CN201610298651.2A patent/CN105838762A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101116676A (en) * | 2006-08-01 | 2008-02-06 | 复旦大学 | Method for preparing compound milettia reticulata particles |
| CN102168045A (en) * | 2010-12-24 | 2011-08-31 | 北京科为博生物科技有限公司 | Bacillus subtilis preparation and preparation method thereof |
| CN104109191A (en) * | 2014-07-02 | 2014-10-22 | 北京龙科方舟生物工程技术有限公司 | Separation and purification method for subtilin antibacterial peptide |
Non-Patent Citations (3)
| Title |
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| 付雯等: "伊枯草菌素研究进展", 《安徽农学通报》 * |
| 李建平等: "五味子、柠檬酸对断奶仔猪血清生化指标和免疫功能的影响", 《中国畜牧杂志》 * |
| 王宗伟等: "五味子在动物营养上的研究进展", 《饲料博览》 * |
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Application publication date: 20160810 |