CN102613249A - Process for preparing board-spectrum antibiotics by immune induction biotechnology - Google Patents
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Abstract
The invention discloses a process for preparing board-spectrum antibiotics by immune induction biotechnology, and particularly relates to a process for preparing board-spectrum antibiotics by immune induction biotechnical synthesis, which employs super immunoprotein synthesis ability of silkworms and includes: allowing fifth-instar silkworms to synthetize high-quantity sericin antibacterial peptides from a middle division of silkgland and silk fibroin antibacterial peptides from a posterior division of the silkgland; regulating silk protein genes with highly bioactive agent; and performing synthesis and amplification to obtain board-spectrum antibiotics by solid-phase and liquid-phase biotechnology. By the process, biotechnical synthetic routes are simple and easy to learn, production cost is low, productivity is highly controllable, disease resistance reaches more than 80%-95%, and achievement is easy to transform and convenient for industrial production. The antibacterial peptides prepared by the process can be mixed with oil and used for soaking roots, and have a higher bacterial effect and efficient and broad-spectrum antibacterial advantages to infections caused by wilt fusarium, gram-positive bacterium, gram-negative bacterium or pathogens such as Bacillus subtilis, bacterial wilt and the like.
Description
Technical field
The present invention relates to a kind of preparation method of antibiotic, particularly a kind of immune induction biotechnology prepares the method for broad spectrum antibiotic.
Background technology
Antibacterial peptide (antibacterial peptides) is claimed peptide antibiotic (peptide antibiotics) or antimicrobial (antimicrobialpeptide) again; Be one type of active peptides of resisting extraneous biological infection source that Immune System produces, in insect, plant, animal and human body, extensively exist.It also has the effect of anti-cell variation, antitumor cell, and do not damage the human normal cell except that the effect that nonspecific antibacterium, fungi, virus, anti-infective, anti-disease are arranged, and is especially still effective to multiple antibiotic resistant strain.Antibacterial peptide all has stronger killing action to endurance strain such as kind, Pseudomonas aeruginosa and paratyphoid bacterium surplus Gram-positive and the negative bacteria about 40.Antibacterial peptide is the emphasis and the focus of current bio-pharmaceutical research and development.
Existing scholar to by the microbial tomato of bacterial wilt cause of disease with pepper ralstonia solanacearum (Bacterial Wilt), carried out research, test by worldwide bacterium destructiveness soil-borne diseases such as the microbial banana blights of withered reaping hook (Fusarium wilt); Proof is utilized antibacterial peptide; Through mix soil, execute filling, plant soaks methods such as root, seed dressing and can above disease be prevented and treated effectively; Especially in the wet environment of China Guangdong and Guangxi Provinces and Fujian isothermal warm current, the incidence of disease can reduce more than 80%.Through the extensive use of biologic antibiotic peptide, can prevent, alleviate agricultural pest effectively, reduce the amount of application of agricultural chemicals, reduce the agricultural production input, guarantee the agricultural product edible safety, realize agricultural production and income, thereby play get twice the result with half the effort, the effect of environmental protection.
Correlative study proves: silkworm has superpower immune protein synthesis capability, and a silkworm in less than one month vegetative period, utilizes the mulberry leaf of 22 ~ 25g can synthesize the silk-fibroin of justacrine 0.5 ~ 1.5g.The correlative study of existing at present cultivated silkworm antimicrobial peptide discloses a kind of method of inducing silkworms to produce antibacterial peptides like Chinese patent ZL200910116463.3, and said this method is to adopt virus or bacterium injection, perhaps can adopt the method that makes silkworm hungry; When employing virus or bacterium injection, to the bacterium or the virus of silkworm injection trace, when adopting the method that makes silkworm hunger; Do not feed mulberry leaf to silkworm; Let its hungry 48 hours, normally raised 24 hours again, the said abductive approach of this patent is simple; Can effectively induce more antibacterial peptide, the antibacterial peptide extraction efficiency is greatly improved; Chinese patent ZL200910193620.0 then discloses a kind of cultivated silkworm antimicrobial peptide BmcecE and cDNA sequence and application; The cultivated silkworm antimicrobial peptide of this invention all has antibacterial activity, has a broad antifungal spectrum to gram-positive bacteria (bacillus thuringiensis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum); Medicament research and development, prevention and treatment that cultivated silkworm antimicrobial peptide BmcecE can be used for people, animal, fowl cause causing a disease of people, animal, fowl by bacillus thuringiensis, Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum infection; Can also be used for the preparation of biological product additive, the biological product that prevents to cause putrid and deteriorated by bacillus thuringiensis, Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum.
But do not see at present and adopt the immune induction biotechnology; Utilize the synthetic sericin antibacterial peptide of five silkworm middle division of silkgland a large amounts in age, synthesize the fibroin albumen antibacterial peptide at posterior division of silkgland; And then, amplification synthetic through solid phase or liquid phase biotechnology, preparation is used for the broad spectrum antibiotic of agriculture antibiotic production.
Summary of the invention
The purpose of this invention is to provide the method that a kind of immune induction biotechnology prepares broad spectrum antibiotic.
In order to address the above problem, the technical scheme that the present invention adopted is following: a kind of immune induction biotechnology prepares the method for broad spectrum antibiotic, with natural five age silkworm be raw material; Through the synthetic sericin antibacterial peptide of natural five silkworm middle division of silkgland a large amounts in age,, the silk-fibroin antifungal genes is regulated and control with biological high reactivity reagent at the synthetic fibroin albumen antibacterial peptide of posterior division of silkgland;, amplification synthetic with solid phase or liquid phase biotechnology; Make broad spectrum antibiotic, described regulation and control and solid phase or liquid phase biotechnology concrete steps synthetic, amplification are: natural five age silkworm grow into the 3rd day, when natural five synthetic sericins in silkworm middle division of silkgland 1/2 place in age or the synthetic fibroin albumen of natural five silkworm posterior division of silkglands in ages; Carry out immune induction; Then in immune peak period, with natural five age silkworm silkworm body through freeze drying to moisture be 70% ~ 75% or with natural five age silkworm silkworm body directly mill pulverize after, drop in the 50kg agitated reactor; And add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic.
The method that immune induction biotechnology of the present invention prepares broad spectrum antibiotic is a kind of in the following method:
(1) natural five age silkworm grow into the 3rd day, during synthetic sericin, inoculate hay bacillus at natural five silkworm body middle division of silkgland 1/2 places in ages with inoculator; Carry out immune induction; In immune peak period, be 70% ~ 75% with natural silkworm silkworm body freeze drying in five ages to moisture, then will through cryodesiccated natural five age silkworm silkworm body drop in the 50kg agitated reactor; And add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic;
(2) natural five age silkworm grow into the 3rd day, during synthetic sericin, inoculate hay bacillus at natural five silkworm body middle division of silkgland 1/2 places in ages with inoculator; Carry out immune induction, in immune peak period, with natural five age silkworm silkworm body directly mill in the liquid input 50kg agitated reactor that obtains after pulverizing and will pulverizing; Add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic;
(3) natural five age silkworm grow into the 3rd day, during synthetic fibroin albumen, inoculate hay bacillus at natural five silkworm body afterbody sericterium places in ages with inoculator; Carry out immune induction, in immune peak period, with natural silkworm silkworm body freeze drying in five ages to moisture 70% ~ 75%; Will through cryodesiccated natural five age silkworm silkworm body drop in the 50kg agitated reactor, and add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent, fermented at normal temperatures 6-8 days; After the freeze-drying, promptly obtain broad spectrum antibiotic;
(4) natural five age silkworm grow into the 3rd day, during synthetic fibroin albumen, inoculate hay bacillus at natural five silkworm body afterbody sericterium places in ages with inoculator; Carry out immune induction, in immune peak period, with natural five age silkworm silkworm body directly mill in the liquid input 50kg agitated reactor that obtains after pulverizing and will pulverizing; Add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic.
Said immune induction method is: with inoculator with hay bacillus be inoculated into natural five age silkworm silkworm body middle division of silkgland 1/2 place or be inoculated into natural five age silkworm silkworm body afterbody sericterium place, inoculation back 3 ~ 5 days natural five age silkworm silkworm body reach immune peak period.
Said biological high reactivity reagent is: biological mitogen, and for Zhanjiang Normal University's independent intellectual property right achievement in research is produced.
The condition of said freeze-drying is: in 2-4 hour, temperature is dropped to pre-freeze temperature-20 ℃ by normal temperature, keep-20 ℃ 1.5-3 hour, be warming up to-5 ~-8 ℃ by-20 ℃ again, kept 2 ~ 4 hours, drain with vavuum pump then.
Said broad spectrum antibiotic is biological organic products or the antibacterial biological additive that is used for agriculture antibiotic production.
Said broad spectrum antibiotic is solid antimicrobial organic manure or liquid antibiotic organic manure.
The present invention with respect to the beneficial effect of prior art is: the synthetic method for preparing broad spectrum antibiotic of immune induction biotechnology according to the invention; Its biotechnology synthetic route is simple and convenient and easy to study, low production cost, and the productive rate degree of controllability is high; The disease prevention ability is up to 95%, and is convenient to industrialization production; That adopts that the inventive method prepares mixes soil when agricultural; Soak root before planting; To gram-positive bacteria (bacillus thuringiensis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum) or by pathogens such as bacillus thuringiensis, Escherichia coli, Pseudomonas aeruginosa, clever bacterium sepsis germ or Ralstonia solanacearum, hay bacillus, bacterial wilt, the withered microbial infection of reaping hook, have stronger antibacterial action and antibiotic advantage efficient, wide spectrum.
Embodiment
Through embodiment the present invention is explained further details below, these embodiment only are used for explaining the present invention, do not limit the scope of the invention.
Embodiment 1Adopt the method for the invention, follow these steps to prepare a kind of broad spectrum antibiotic that is used for agriculture antibiotic production:
(1) antibody bacterial classification: hay bacillus; Biological high reactivity reagent: derive from the biological mitogen that Zhanjiang Normal University's independent intellectual property right achievement in research is produced;
(2) preparation process:
1. natural five age silkworm grow into the 3rd day; At natural five silkworm body middle division of silkgland 1/2 places in ages during synthetic sericin; With inoculator with hay bacillus be inoculated into natural five age silkworm silkworm body middle division of silkgland 1/2 place, immune induction, inoculate back 3 days natural five age silkworm silkworm body reach immune peak period;
2. in immune peak period; Natural silkworm silkworm body freeze drying in five ages to the moisture that will be in immune peak period is 70%; Then will through cryodesiccated natural five age silkworm silkworm body drop in the 50kg agitated reactor; And add account for natural five age silkworm silkworm physique amount 1/1000 biological mitogen, fermented at normal temperatures 7 days;
3. fermentation ends, the product freeze-drying after adopting following lyophilisation condition to ferment: 2 hours normal temperature drops to pre-freeze temperature-20 ℃, keep-20 ℃ 2.5 hours, be warming up to-6 ℃ by-20 ℃ again, kept 2 hours, drain with vavuum pump then;
4. promptly make after the freeze-drying and be used for agriculture antibiotic production organic-biological agricultural chemicals.
Embodiment 2Adopt the method for the invention, follow these steps to prepare a kind of broad spectrum antibiotic that is used for agriculture antibiotic production:
(1) antibody bacterial classification: hay bacillus; Biological high reactivity reagent: derive from the biological mitogen that Zhanjiang Normal University's independent intellectual property right achievement in research is produced;
(2) preparation process:
1. natural five age silkworm grow into the 3rd day; At natural five silkworm body middle division of silkgland 1/2 places in ages during synthetic sericin; With inoculator with hay bacillus be inoculated into natural five age silkworm silkworm body middle division of silkgland 1/2 place, immune induction, inoculate back 4 days natural five age silkworm silkworm body reach immune peak period;
2. in immune peak period, will be in immune peak period natural five age the silkworm silkworm body liquid that obtains after pulverizing and will pulverizing of directly milling drop in the 50kg agitated reactor, adding account for natural five age silkworm silkworm physique amount 1/1000 biological mitogen, fermented at normal temperatures 6 days;
3. fermentation ends, the product freeze-drying after adopting following lyophilisation condition to ferment: 2.5 hours normal temperature drops to pre-freeze temperature-20 ℃, keep-20 ℃ 1.5 hours, be warming up to-7 ℃ by-20 ℃ again, kept 2.5 hours, drain with vavuum pump then;
4. promptly make the liquid antibacterial biological additive that is used for agriculture antibiotic production after the freeze-drying.
Embodiment 3Adopt the method for the invention, follow these steps to prepare a kind of broad spectrum antibiotic that is used for agriculture antibiotic production:
(1) target antibody bacterial classification: hay bacillus; Biological high reactivity reagent: derive from the biological mitogen that Zhanjiang Normal University's independent intellectual property right achievement in research is produced;
(2) preparation process:
1. natural five age silkworm grow into the 3rd day; At natural five silkworm body afterbody sericterium places in ages during synthetic fibroin albumen; With inoculator with hay bacillus be inoculated into natural five age silkworm silkworm body afterbody sericterium place, immune induction, inoculate back 5 days natural five age silkworm silkworm body reach immune peak period;
2. in immune peak period; Natural silkworm silkworm body freeze drying in five ages to the moisture that will be in immune peak period is 75%; Then will through cryodesiccated natural five age silkworm silkworm body drop in the 50kg agitated reactor; And add account for natural five age silkworm silkworm physique amount 1/1000 biological mitogen, fermented at normal temperatures 8 days;
3. fermentation ends, the product freeze-drying after adopting following lyophilisation condition to ferment: 4 hours normal temperature drops to pre-freeze temperature-20 ℃, keep-20 ℃ 2 hours, be warming up to-5 ℃ by-20 ℃ again, kept 4 hours, drain with vavuum pump then;
4. product is solid antimicrobial organic manure finished product after the freeze-drying, is used for agriculture antibiotic production after the packing.
Embodiment 4Adopt the method for the invention, follow these steps to prepare a kind of broad spectrum antibiotic that is used for agriculture antibiotic production:
(1) antibody bacterial classification: hay bacillus; Biological high reactivity reagent: derive from the biological mitogen that Zhanjiang Normal University's independent intellectual property right achievement in research is produced;
(2) preparation process:
1. natural five age silkworm grow into the 3rd day; At natural five silkworm body afterbody sericterium places in ages during synthetic fibroin albumen; With inoculator with hay bacillus be inoculated into natural five age silkworm silkworm body afterbody sericterium place, immune induction, inoculate back 3 days natural five age silkworm silkworm body reach immune peak period;
2. in immune peak period, will be in immune peak period natural five age the silkworm silkworm body liquid that obtains after pulverizing and will pulverizing of directly milling drop in the 50kg agitated reactor, adding account for natural five age silkworm silkworm physique amount 1/1000 biological mitogen, fermented at normal temperatures 7 days;
3. fermentation ends, the product freeze-drying after adopting following lyophilisation condition to ferment: 3 hours normal temperature drops to pre-freeze temperature-20 ℃, keep-20 ℃ 3 hours, be warming up to-8 ℃ by-20 ℃ again, kept 3 hours, drain with vavuum pump then;
4. product is liquid antibiotic organic manure finished product after the freeze-drying, is used for agriculture antibiotic production after the packing.
Claims (7)
1. an immune induction biotechnology prepares the method for broad spectrum antibiotic, it is characterized in that: this method be with natural five age silkworm be raw material, through the synthetic sericin antibacterial peptide of natural five silkworm middle division of silkgland a large amounts in age; At the synthetic fibroin albumen antibacterial peptide of posterior division of silkgland; With biological high reactivity reagent the silk-fibroin antifungal genes is regulated and control,, amplification synthetic with solid phase or liquid phase biotechnology make broad spectrum antibiotic; Described regulation and control and solid phase or liquid phase biotechnology concrete steps synthetic, amplification are: natural five age silkworm grow into the 3rd day; When natural five synthetic sericins in silkworm middle division of silkgland 1/2 place in age or the synthetic fibroin albumen of natural five silkworm posterior division of silkglands in ages, carry out immune induction, then in immune peak period; With natural five age silkworm silkworm body through freeze drying to moisture be 70% ~ 75% or with natural five age silkworm silkworm body directly mill pulverize after; Drop in the 50kg agitated reactor, and add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent, fermented at normal temperatures 6-8 days; After the freeze-drying, promptly obtain broad spectrum antibiotic.
2. an immune induction biotechnology as claimed in claim 1 prepares the method for broad spectrum antibiotic, it is characterized in that: a kind of in the following method:
(1) natural five age silkworm grow into the 3rd day, during synthetic sericin, inoculate hay bacillus at natural five silkworm body middle division of silkgland 1/2 places in ages with inoculator; Carry out immune induction; In immune peak period, be 70% ~ 75% with natural silkworm silkworm body freeze drying in five ages to moisture, then will through cryodesiccated natural five age silkworm silkworm body drop in the 50kg agitated reactor; And add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic;
(2) natural five age silkworm grow into the 3rd day, during synthetic sericin, inoculate hay bacillus at natural five silkworm body middle division of silkgland 1/2 places in ages with inoculator; Carry out immune induction, in immune peak period, with natural five age silkworm silkworm body directly mill in the liquid input 50kg agitated reactor that obtains after pulverizing and will pulverizing; Add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic;
(3) natural five age silkworm grow into the 3rd day, during synthetic fibroin albumen, inoculate hay bacillus at natural five silkworm body afterbody sericterium places in ages with inoculator; Carry out immune induction, in immune peak period, with natural silkworm silkworm body freeze drying in five ages to moisture 70% ~ 75%; Will through cryodesiccated natural five age silkworm silkworm body drop in the 50kg agitated reactor, and add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent, fermented at normal temperatures 6-8 days; After the freeze-drying, promptly obtain broad spectrum antibiotic;
(4) natural five age silkworm grow into the 3rd day, during synthetic fibroin albumen, inoculate hay bacillus at natural five silkworm body afterbody sericterium places in ages with inoculator; Carry out immune induction, in immune peak period, with natural five age silkworm silkworm body directly mill in the liquid input 50kg agitated reactor that obtains after pulverizing and will pulverizing; Add account for natural five age silkworm silkworm physique amount 1/1000 biological high reactivity reagent; Fermented at normal temperatures 6-8 days, and after the freeze-drying, promptly obtained broad spectrum antibiotic.
3. a kind of immune induction biotechnology according to claim 1 and 2 prepares the method for broad spectrum antibiotic; It is characterized in that: said immune induction method is: with inoculator with hay bacillus be inoculated into natural five age silkworm silkworm body middle division of silkgland 1/2 place or be inoculated into natural five age silkworm silkworm body afterbody sericterium place, inoculation back 3 ~ 5 days natural five age silkworm silkworm body reach immune peak period.
4. a kind of immune induction biotechnology according to claim 1 and 2 prepares the method for broad spectrum antibiotic, it is characterized in that: said biological high reactivity reagent is: biological mitogen.
5. the method for preparing broad spectrum antibiotic according to claim 1 or 2 described a kind of immune induction biotechnologys; It is characterized in that: the condition of said freeze-drying is: in 2-4 hour; Temperature is dropped to pre-freeze temperature-20 ℃ by normal temperature, keep-20 ℃ 1.5-3 hour, be warming up to-5 ~-8 ℃ by-20 ℃ again; Kept 2 ~ 4 hours, and drained with vavuum pump then.
6. prepare the method for broad spectrum antibiotic according to claim 1 or 2 described a kind of immune induction biotechnologys, it is characterized in that: said broad spectrum antibiotic is biological organic products or the antibacterial biological additive that is used for agriculture antibiotic production.
7. prepare the method for broad spectrum antibiotic according to claim 1 or 2 described a kind of immune induction biotechnologys, it is characterized in that: said broad spectrum antibiotic is solid antimicrobial organic manure or liquid antibiotic organic manure.
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| CN102584452A (en) * | 2012-02-22 | 2012-07-18 | 湛江师范学院 | Method for preparing biological antibacterial organic fertilizer by using immune induced biological technology |
| CN103539843A (en) * | 2013-10-25 | 2014-01-29 | 承德医学院 | Method for producing sericin protein |
| CN103621564A (en) * | 2013-12-17 | 2014-03-12 | 湛江师范学院 | Preparation method of biological antibacterial agent |
| CN104042636A (en) * | 2014-06-23 | 2014-09-17 | 南通惠然生物科技有限公司 | Method for preparing broad spectrum antibiotic by use of immunity induction technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102584452A (en) * | 2012-02-22 | 2012-07-18 | 湛江师范学院 | Method for preparing biological antibacterial organic fertilizer by using immune induced biological technology |
| CN103539843A (en) * | 2013-10-25 | 2014-01-29 | 承德医学院 | Method for producing sericin protein |
| CN103621564A (en) * | 2013-12-17 | 2014-03-12 | 湛江师范学院 | Preparation method of biological antibacterial agent |
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Application publication date: 20120801 |