CN105519798A - Animal feed additive - Google Patents
Animal feed additive Download PDFInfo
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- CN105519798A CN105519798A CN201510791302.XA CN201510791302A CN105519798A CN 105519798 A CN105519798 A CN 105519798A CN 201510791302 A CN201510791302 A CN 201510791302A CN 105519798 A CN105519798 A CN 105519798A
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- mold removal
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Abstract
The present invention provides an animal feed additive comprising an antimicrobial peptide mold removal agent for feed, which is characterized in that the antimicrobial peptide mold removal agent for feed comprises yeast cell walls and antibacterial peptides. The antimicrobial peptide mold removal agent is used in the animal feed additive, the main component of the yeast cell walls is glucomannoglycan of which the chemical structure shows very excellent affinity with mycotoxin sharing the same organic class so that a relatively good affinity adsorption can be formed, and a better adsorption affinity capacity can be realized compared to inorganic adsorbents; and the combined use of antibacterial peptides can enhance sterilization at the same time of adsorption, enhancing the effect of the mold removal agent, and the antimicrobial peptides have repair function on the generated immunosuppression after the mold removal agent is used in livestock; and generally, the antimicrobial peptide mold removal agent has multiple effects in sterilization, mold removal, adsorption and repair at the same time.
Description
This case is the divisional application for CN201510095993.X patent.
Technical field
The invention belongs to animal feed additive technical field, be specifically related to a kind of animal feed additive.
Background technology
In livestock breed aquatics, long-standing problem is the germ endotoxin contamination in depositing of feed; After the contaminated feed of animal edible, cultivated animals is caused to occur the problems such as immunosupress, diarrhea, Sow abortion, piglet false heat.And in the face of this problem, due to relevant to grown climatic environment etc. in the plant development of feed, the pollution that completely cut off germ cannot be ensured from source; Therefore in this situation, the problem adopting the mode of adding this physical absorption of adsorbent effectively to solve food pollution more.Wherein, the many employings of mode " germ endotoxin adsorbent " of physical absorption carry out Adsorption to the germ toxin in feed, to reduce the above-mentioned murder by poisoning of germ toxin in cultivation.
" the germ endotoxin adsorbent " commonly used has water and alumina silicate, active carbon etc.The de-mould dose of energy of these absorption forms stable bonding with germ toxin under liquid phase, adsorbs; But above-mentioned conventional adsorbent, only can have adsorption effect for the germ toxin of part, such as: the adsorption effect of inorganic adsorbent to aflatoxin (AFL) is good, but but very low to the adsorption effect of other mycotoxins (FUM, F2, DON, T2 etc.).These adsorbents adsorb unilateral reason in using and are, the structure of the good and bad UI sorbing material of adsorption effect is correlated with on the one hand, for example the size, the distribution of charges mode of adsorption structure itself, effective adsorption area etc. in absorption cavity, cannot ensure to match with all germ toxin; Different adsorption structures has different compatibilities from different toxin on the other hand, and the differentia influence producing compatibility is because have hydrophily, hydrophobicity, structural formula and distribution of charges etc., therefore the restriction of self these character of hand absorption, also can not adsorb all mycotoxins all sidedly.
Therefore, existing " germ endotoxin adsorbent " is in use comparatively unilateral to the adsorption effect of multiple germ toxin, a large amount of demand removed germ and pollute can not be met, simultaneously in use these germ toxin can make the animal of cultivation produce immunosupress, and adopt adsorbent can not pollute to animal edible the immunosupress that feed produced after absorption to have good alleviation and the function of reparation.
Summary of the invention
It is unilateral and cannot repair the above-mentioned deficiency of the immune-suppression problems caused that the object of the embodiment of the present invention is to overcome existing de-mould dose of adsorption effect, provides a kind of.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
Feed antibacterial peptide takes off the animal feed additive of mould dose, and wherein feed antibacterial peptide takes off mould dose and comprises yeast cell wall and antibacterial peptide.
Adopt in animal feed additive of the present invention de-mould dose of adopting to take off mould dose for above-mentioned feed antibacterial peptide of the present invention, after it is added in feed, has lifting to the absorption affinity of germ toxin in feed, improve de-mould effect; And can have simultaneously kill mould and repair multi-efficiency.
Detailed description of the invention
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention provides a kind of feed antibacterial peptide to take off mould dose, comprises yeast cell wall and antibacterial peptide;
The chemical constitution of the composition of yeast cell wall wherein mainly glucomannoglycan own is very excellent with the same compatibility belonging to the mycotoxin of organic, can form good affine absorption; Compare inorganic adsorbent, the Adsorption on cell walls agent of this type is carried out for the surperficial organic structure that mycotoxin is common, relatively can make up the unilateral shortcoming of inorganic adsorbent.Simultaneously, de-mould dose mixes antibacterial peptide under yeast cell wall absorption, antibacterial peptide itself is utilized to have the feature of broad spectrum antibiotic activity, wide in conjunction with simple yeast cell wall relative adsorption, enhancing sterilization is carried out with antibacterial peptide while absorption, promote the effect of de-mould dose, and antibacterial peptide there is repair to the immunosupress produced after domestic animal uses; De-mould dose of the present invention can have simultaneously kill mould, de-mould, absorption and repair multi-efficiency on the whole.
Wherein, in above-mentioned de-mould dose of the present invention, the effector molecule of antibacterial peptide owing to being biological innate immune itself, content is very micro-in vivo, from natural resources, extraction cost is high, yield is low, operation is loaded down with trivial details, and in organism, the production of a large amount of antibacterial peptide of extracting directly does not meet the user demand added in a large amount of feed the present invention.Therefore, based on above-mentioned purpose, the present invention also proposes a kind ofly can meet the method that above-mentioned feed antibacterial peptide takes off mould dose of a large amount of preparation, comprises the steps:
S10, recombinates the encoding gene containing object antibacterial peptide and carrier, the recombinant expression carrier of construction expression object antibacterial peptide;
S20, the recombinant expression carrier obtained by step S10 imports in yeast cells, forms Yeast engineering bacteria;
S30, the Yeast engineering bacteria obtained by step S20 carries out fermented and cultured, obtains tunning;
S40, what step S30 fermented and cultured obtained carries out self-dissolving broken wall with the tunning of engineering bacterium expression product containing yeast cells thalline, is separated, can obtain product simultaneously containing yeast cells thalline and object antibacterial peptide after purifying, namely obtains above-mentioned de-mould dose of the present invention.
Under the conception of the method step of above-mentioned entirety preparation, the mode adopting structure Yeast engineering bacteria to carry out fermenting and producing is carried out, engineering bacteria great expression antibacterial peptide in the process of fermented and cultured, therefore has the expression product of saccharomycetic cell body and antibacterial peptide in final fermented and cultured product simultaneously; Then, after making oneself the appearance broken wall of saccharomycetic cell body in product, then carry out separation and purification and collect yeast cell wall and antibacterial peptide, de-mould dose of product can be obtained in a large number.
Further under the method conception of above-mentioned entirety, can carry out the characteristic of non-fusion expression in the present invention further according to yeast cells own, the genes of interest of expressing in the engineering bacteria wherein built in step S10 is antibacterial peptide IsCT encoding gene; IsCT own is a kind of natural alpha-helix antibacterial peptide, derive from the black pawl scorpion (Opisthacanthusmadagascariensis) of Madagascar, it is polypeptide the shortest in antibacterial peptide, only be made up of 13 amino acid residues (ILGKIWEGIKSLF), there is no cysteine, have activity to mammal and bacterial cell.Carrying out in the present invention adopting is [L6 in the IsCT derivative that designs based on this reason, K11] the same with IsCT in-IsCT (ILGKILKGIKKLF), to E.coli, P.aeruginosa, S.trphimurium, S.epidermidis, S.aureus has identical bacteriostatic activity, hemolytic activity simultaneously when [L6, K11]-IsCT concentration reaches 100 μMs is only 20%, can possess the ability of adding use in feed.
In the scenario above, for the expression of the antibacterial peptide IsCT adopted in the application step S10, the step of recombinant expression carrier can comprise as follows:
S11, according to the Preference of yeast codons, design the encoding gene that above-mentioned IsCT expresses, this gene is the IsCT gene of the base sequence with sequence table SEQ .ID.No.1;
| Title | Base sequence (5 '-3 ') | Length | Sequence table is numbered |
| IsCT gene | ATTTTGGGTAAGATTTGGGAAGGTATTAAGTCTTTGTTT | 39bp | SEQ.ID.No.1 |
S12, adopts plasmid pYES2 as carrier, and with the alpha signal peptide of pYES2 α plasmid for template carries out pcr amplification;
Adopt plasmid pYES2 as carrier in this step, build the recombinant expression carrier of antibacterial peptide IsCT;
Wherein in this step S12, adopt plasmid pYES2, its object is to through the amino acid structure to saccharomycetic non-fusion expression feature and IsCT own, through multiple checking repeatedly and after considering the final plasmid pYES2 that adopts carry out; Reason is: first, and plasmid pYES2 itself has yeast GAL1 promoter, and it can induce with galactolipin is high-caliber in saccharomyces cerevisiae, can be suppressed by glucose simultaneously; And the MCS region of plasmid pYES also has CYC1 terminator, effectively can stop the transcriptional expression of mRNA, adopt this plasmid to be also more easy to control while ability to express is stronger; Secondly, the MCS of plasmid pYES2 itself has multiple available restriction enzyme site, is convenient to reorganization operation; 3rd, plasmid itself has URA3 gene and ammonia benzyl resistant gene, may be used for screening respectively with ura3 genotypic yeast host bacterial strain transformant, and carry out vector selection in Escherichia coli.After the expression vector adopting this plasmid pYES2 as genes of interest, based on the employing of plasmid pYES2, alpha signal peptide for its pYES2 α plasmid is template design primer, and wherein the sequence of the alpha signal peptide of pYES2 α plasmid (932-1207 base section) has sequence table SEQ .ID.No.2 base sequence;
With above-mentioned alpha signal peptide for template, the primer designed respectively is have the upstream primer of sequence table SEQ .ID.No.3 base sequence and have the downstream primer of sequence table SEQ .ID.No.4 base sequence.
Wherein, in upstream primer, introduce Hind III restriction enzyme site, i.e. the base fragment " CAAGCTTG " of the 1st to 8 of upstream primer; And the base fragment " TTGCGGCCGCAA " of the 4th to 15 introduced in downstream primer in " Not I restriction enzyme site " i.e. downstream primer;
The genes of interest simultaneously directly will expressed in above-mentioned downstream primer is implanted in primer sequence, i.e. the base fragment of downstream primer the 41st to 79, directly by primer in the process in amplification, realize being connected of genes of interest and plasmid pYES2; Simultaneously in downstream primer, the base sequence of 27 to 40 is the terminator codon introduced, to genes of interest accurately expression sites carry out accurate localization and expression control.
In this step, with the sequence of alpha signal peptide for template increase time, directly introducing the encoding gene of antibacterial peptide IsCT in downstream primer, directly can obtain the recombination sequence α-IsCT containing IsCT gene and alpha signal peptide by increasing; Certain amplification product can carry out electrophoresis, cut glue reclaim, purification of Recombinant sequence α-IsCT is for subsequent use.
S13, is connected with DNA ligase after the recombination sequence α-IsCT of the PCR Successful amplification in step S12 is carried out double digestion with Hind III with Not I respectively with plasmid pYES2, forms recombinant plasmid pYES2 α-IsCT.
Certainly, after step s 13, to step S13 obtain with needing the successful recombinant plasmid of screening restructuring connecting in product after DNA ligase process, can adopt in the process of this screening and the connection product of DNA ligase process in step S13 is directed into the successful plasmid of screening restructuring in E. coli Top10.The method of screening can adopt the culture medium of ampicillin to carry out, because the plasmid pYES2 that itself adopts self has ammonia benzyl resistant gene, the Escherichia coli after transforming successfully can grow in the culture medium of ampicillin.
Be transformed in saccharomyces cerevisiae by the recombinant plasmid pYES2 α-IsCT that step S10 obtains further in step S20, the mode of importing adopts electricity conversion, chemistry forwarding etc. all passable, and screening afterwards transforms successful saccharomyces cerevisiae, for subsequent use as engineering bacteria.Wherein, transform success or not, the URA3 gene that plasmid pYES2 can be adopted to have carries out, anauxotrophic Wine brewing yeast strain cannot normal growth in Negative media, have the bacterial strain energy normal growth being transformed into URA3 gene after importing, therefore the URA3 gene itself had by plasmid pYES2 can realize the screening of Yeast engineering bacteria.
The Yeast engineering bacteria that step S30 adopts step S20 successfully to build afterwards carries out a large amount of cultivations, attentional manipulation growth conditions in the process of cultivation, induces antibacterial peptide IsCT to express in due course; After completing fermentation, zymotic fluid processes by step S40, first thalline self-dissolving broken wall wherein, inclusion is made to discharge, the cell membrane of yeast cells thalline is dissociated, then carry out separation and purification and retain cell membrane and antibacterial peptide product, the purified finally obtained can directly as de-mould dose of product of the present invention.
The engineering bacteria that the present invention adopts Wine brewing yeast strain to build carries out fermenting and producing, and in the process of de-mould dose of fermenting and producing, whole recombinant expressed adopted Host Strains and expression vector and production process all have no side effect; And this Wine brewing yeast strain is insensitive to antibacterial peptide, can tolerate the bactericidal activity of antibacterial peptide, the step realizing yeast cell wall and antibacterial peptide product prepares simultaneously; And the saccharomyces cerevisiae of this food-grade is as expressive host, alpha signal peptide gene in conjunction with plasmid is assisted, the process of expressed antibacterial peptide is nonfused secretion type expression, compares common amalgamation and expression and can further express the separation and purification being conducive to polypeptide products, cost-saving; Simultaneously saccharomyces cerevisiae expression does not need the harmful substance inductions such as methyl alcohol, it also avoid selective agents such as adding antibiotic, and product directly meets the standard as feed addictive, entirety can have and kill mould, de-mould, absorption and repair multi-efficiency simultaneously.
For making clearly complete, the enforcement reference that is easy to those skilled in the art of the implementation detail of the above-mentioned preparation of the present invention, and make the present invention take off the outstanding progressive effect of mould dose of product more significantly, by the following examples concrete example explanation is carried out to the enforcement of said process.
Embodiment 1
In this embodiment 1, adopt saccharomyces cerevisiae INVSc1 as the host of engineering bacteria, carry out structure and the fermented and cultured of engineering bacteria in accordance with the following steps:
S11, with the alpha signal peptide gene fragment of pYES2 α plasmid for template, adopts the upstream primer with sequence table SEQ .ID.No.3 base sequence and the downstream primer with sequence table SEQ .ID.No.4 base sequence to carry out pcr amplification, obtains pcr amplification product;
The standards system of 100 μ L can be adopted in pcr amplification process to carry out.
S12, carries out abstraction and purification by pcr amplification product agarose gel electrophoresis after amplification, and film band genes of interest fragment gone out scales off, and recovery is dissolved for subsequent use; Recombination sequence α-the IsCT of IsCT gene and alpha signal peptide for the purpose of the DNA band reclaimed;
S13, after the recombination sequence α-IsCT reclaimed by electrophoresis in step S12 carries out double digestion with plasmid pYES2 Hind III and Not I, then adopts DNA ligase to carry out connection handling, obtains connecting product;
S14, the connection product obtained by step S13 is directed in competent Escherichia coli Top10, and the mode of operation of the wherein colibacillary preparation of competence and importing can adopt the means of laboratory routine to carry out, and does not limit at this;
And the product through importing process is screened with containing ampicillin medium, the thalline collecting normal growth is the E. coli Top10 importing correct recombinant plasmid pYES2 α-IsCT.
S20, by the successful Escherichia coli of screening restructuring in step S14, recombinant plasmid pYES2 α-IsCT is extracted with plasmid extraction kit, then the mode that the recombinant plasmid electricity consumption of extraction transforms is directed in saccharomyces cerevisiae INVSc1 bacterial strain, then adopt the Negative media of 5-FOA (5-fluororotic acid) to cultivate, 5-FOA can be converted into noxious material (Suicide mortifier) and cannot cultivate by normal growth by the orotidine 5-phosphate decarboxylase of normal prototrophy yeast cells; Can normal growth be the saccharomycete that successful conversion has recombinant plasmid pYES2 α-IsCT;
S30, then fermented and cultured, need to detect zymotic fluid at Initial stage of culture and measure the growth that can IsCT suppress E.coliATCC8739 and S.aureusATCC25923; Continue after normal to cultivate;
Meanwhile, after the density of OD reaches and usually meets standard, induce, when directly reaching the decline phase, collect tunning;
S40, can obtain yeast cell wall and antibacterial peptide IsCT by tunning respectively after self-dissolving broken wall, complex enzyme degradation, separation, purification, drying, the enzyme that goes out.
On the basis of the above, the application proposes a kind of animal feed additive further, includes above-mentioned de-mould dose of the present invention in additive; Include the animal feed additive of above-mentioned de-mould dose of the present invention, after it is added in feed, has lifting to the absorption affinity of germ toxin in feed, improve de-mould effect; And can have simultaneously kill mould and repair multi-efficiency.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. an animal feed additive, comprises feed antibacterial peptide and takes off mould dose, it is characterized in that, described feed antibacterial peptide takes off mould dose and comprises yeast cell wall and antibacterial peptide.
2. animal feed additive as claimed in claim 1, it is characterized in that, described antibacterial peptide is IsCT antibacterial peptide.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201510095993.XA CN104782909A (en) | 2015-03-04 | 2015-03-04 | Antibacterial peptide mold removal agent for feed, preparation method thereof and animal feed additive |
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| CN201510095993.XA Division CN104782909A (en) | 2015-03-04 | 2015-03-04 | Antibacterial peptide mold removal agent for feed, preparation method thereof and animal feed additive |
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| CN201510791302.XA Pending CN105519798A (en) | 2015-03-04 | 2015-03-04 | Animal feed additive |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105963688A (en) * | 2016-06-23 | 2016-09-28 | 四川英格瑞生物科技有限公司 | Powder for curing intestinal diseases of piglet and enhancing immunity and preparation method thereof |
| CN108018222A (en) * | 2017-12-07 | 2018-05-11 | 浙江大学 | For suppressing the preparation GS115/Ac-AMP2 of pear fruit Postharvest Penicillium |
| CN112262916A (en) * | 2020-09-30 | 2021-01-26 | 四川特驱农牧科技集团有限公司 | Complex microbial inoculum fermentation product and preparation method and application thereof |
| CN116850267A (en) * | 2023-07-13 | 2023-10-10 | 山东宝来利来生物工程股份有限公司 | Yeast cell wall-antibacterial peptide A3 composite preparation and preparation method and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636176B (en) * | 2016-12-20 | 2018-05-04 | 广州格拉姆生物科技有限公司 | A kind of prebiotic feeding saccharomyces cerevisiae for producing xylo-oligosaccharide and antibacterial peptide |
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| CN104782909A (en) | 2015-07-22 |
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Application publication date: 20160427 |