CN102206263A - Tussah antibiotic extraction method based on tussah pupa in vivo induction - Google Patents
Tussah antibiotic extraction method based on tussah pupa in vivo induction Download PDFInfo
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- CN102206263A CN102206263A CN2011100776945A CN201110077694A CN102206263A CN 102206263 A CN102206263 A CN 102206263A CN 2011100776945 A CN2011100776945 A CN 2011100776945A CN 201110077694 A CN201110077694 A CN 201110077694A CN 102206263 A CN102206263 A CN 102206263A
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Abstract
The invention provides a tussah antibiotic extraction method based on a tussah pupa in vivo induction. The method is characterized by comprising the steps of: preserving tussah cocoons; removing cocoon shells and selecting diapause cocoons; cleaning tussah pupas and disinfecting the body surface thereof; culturing an escherichia coli E. coliK12D31vaccine; inducing; and extracting the tussah antibiotic, etc. The method has scientifically selected steps to realize high active value of inducted antibiotic, low tussah pupa corruption, pupa blood clearance and transparency, and the method has a high antibiotic yield and low costs, and is suitable for factory production. The extracted tussah antibiotic can be used as a new natural antiseptic for foodstuff, and has the characteristics of wide bactericidal spectrum, easy enzyme hydrolysis and digestion by internal protein, and no toxic and side effects. The tussah antibiotic can be added into beverage, bean product and dairy product, according to the tussah antibiotic property, to prolong shelf life, inhibit breeding of harmful bacteria in intestinal canal and enhance human body immunization.
Description
Technical field
The present invention relates to the antibiotic extraction process, specifically, is a kind of method of being induced and extracted antibiotic by the live body cocoon chrysalis.
Background technology
Since in Boman in 1980 is cherishing than ancient giant silkworm first, isolating cecropin, antibacterial peptide or antibiotic successively in many insects, have been found.The fundamental research of tussah antibiotic is relatively richer, and the applied research field is extensive.The contriver has consulted related data and has shown by literature search, and the research report of relevant antibacterial peptide is more both at home and abroad.
Usually research and development antiseptics for natural food from animal, plant, microorganism.In plant, many investigators find that the flavor compound in the pungent component in the volatile oil composition in wild peach and the almond, garlic, the onion, the non-pungent component in the cabbage juice and green tea, apple, the cashew nut all contains antibiotic effective ingredient.People such as Wang Nanzhou have studied the antibacterial preservative activity of glycyrrhiza preparation, capsule of weeping forsythia preparation, algae and garlic juice respectively, find that they have restraining effect widely to multiple common food spoilage bacterium.Mannosans is extensively to be present in natural a kind of polysaccharide.According to nearest the discovering of Japan, mannosans is a kind of efficient, economic antiseptics for natural food, can from natural products such as konjaku, coconut, marine alga, extract, can be used for the anti-corrosive fresh-keeping of numerous food products such as fruit, vegetables, bean product, eggs and fish.As the mannosans of interpolation 1% in bean product, at plum rain season, the quality guaranteed period is 4 days, and control group is only put 2 days with regard to mouldy fouling.Also contain abundant antimicrobial substance in animal body.People carry out antimicrobial spectrum research to the tussah antibacterial peptide, think that it has effect to food antiseptic.The chitosan that extracts from crab shell, shrimp shell claims chitosan that very strong mycostasis is also arranged again, when concentration is 0.4%, intestinal bacteria, streptococcus aureus is had germ resistance, and Gram-negative bacteria and positive bacteria are also had anti-microbial effect.Utilize Production by Microorganism Fermentation and preparation antiseptics for natural food to enjoy investigator's attention and concern, obtained the achievement that attracts people's attention.As the tennecetin that produces by streptomycete fermentation, the polylysine that produces by streptomyces albus, the nisin that produces by streptococcus acidi lactici etc.
But up to now, Shang Weijian is induced and extracts the report of the method extraction high reactivity tussah antibiotic of antibiotic by the live body cocoon chrysalis.
Summary of the invention
The present invention is based on following design: tussah antibiotic is that raw material extracts with the cocoon chrysalis, especially the toothed oak woods of big area self-sow is arranged is the good base that abounds with cocoon chrysalis to China Changbai Mountain arteries and veins, the place of production is natural pollution-free, and cocoon chrysalis is classified as new resource food by country.Nutritious, the dietotherapeutic of cocoon chrysalis can provide high protein, lower fat, low-cholesterol and VITAMIN and some mineral substance for people.Simultaneously, often contain special activeconstituents in the insect body, human beings'health is had unique function.It is the senior nutrition of natural green.The extraction process process is pollution-free, does not use any pharmaceutical chemicals.Tussah antibiotic can directly be developed protective foods, also can be applicable to foodstuff additive, can prevent food spoilage, extends the shelf life, and can improve the immunizing power of human body after the people is edible.Tussah antibiotic has the fungicidal activity of wide spectrum, easily digested after the people is edible by intravital protease hydrolysis, and without any side effects.Can illustrate by above similar research comparative analysis: one, the extraction process of tussah antibiotic is similar with similar research, there is report to think and extracts active low the grade and the equipment requirements strictness of tussah antibiotic existence on producing in batches, cost is more high, this extractive technique has solved above-mentioned existing problems, has practicality.Two, the applied research of tussah antibiotic is more, but does not see system's report as yet as the research, the particularly application on food such as milk, cooling meat of foodstuff additive, has replenished this area blank.Has advance.Three, tussah antibiotic is a kind of novel biological preservatives as foodstuff additive, and favorable anti-corrosion effect, safe has a extensive future.Four, the various source of inducing is not quite similar to the inductive effect of the gene of cocoon chrysalis, and the synthetic antimicrobial substance quantitatively, have certain difference on the activity.In view of this to induce the system of replying will be a kind of rudimentary immune system of uniqueness to the antimicrobial substance of tussah silkworm chrysalis body.Therefore, when selecting to induce the source, select to induce anti-microbial activity height, fungus to be easy to cultivate, cost is low, the corrupt few source of inducing of silkworm chrysalis, is an important ring to the efficient that improves separation and Extraction antibiotic.
The objective of the invention is, prior art is carried out substantial improvements, a kind of method of being induced and extracted antibiotic by the live body cocoon chrysalis is provided, the step of its method is chosen scientific and reasonable, inductive antibiotic activity value height, cocoon chrysalis corruption are less, pupa blood keeps as clear as crystal, antibiotic yield height, cost is low, is easy to batch production production.
The objective of the invention is to realize: a kind ofly induce and extract the method for silkworm antibiotic, it is characterized in that it comprises following steps by the live body cocoon chrysalis by following technical scheme:
(1) tussah cocoon is preserved under 0～3 ℃ of condition, and test is prepended under 18～22 ℃ of room temperatures, 60～70% relative humidity conditions preserves 12～24h;
(2) remove the cocoon shell and take out the diapause pupa, select to remove dead pupa, growth pupa in the diapause pupa;
(3) cocoon chrysalis is cleaned with clear water, carries out the body surface sterilization with 65～75% alcohol;
(4) intestinal bacteria E.coliK is used in microorganism induction source
12D
31Vaccine is induced the source, and its LB culture medium prescription is: in the 1L solution, and peptone 10g, extractum carnis 5g, sodium-chlor 5g cultivates 24h, with same substratum switching, in the bacterium liquid of cultivating, add 36% formaldehyde solution and make it to become 3% formalin solution, place 24～168h at 4 ℃, centrifugal 2500rpm, 15min, abandoning supernatant, sedimentary thalline with 0.9% sodium chloride solution wash, 2500rpm, centrifugal repeatedly 3 times of 15min, the gained thalline is at intestinal bacteria E.coliK
12D
31On the solid medium, cultivate 24h for 37 ℃, confirm the aseptic length of being born, prove dead bacterium after, store in the refrigerator standbyly, the time spent transfers to 10 with vaccine concentration
8～10
5Cells/ml;
(5) induce, through the intestinal bacteria E.coliK of liquid culture
12D
31Vaccine is diluted to every ml with insect physiological saline and contains 10
8～10
5Cells, every pupa of concentration injection 40～60 μ l inject from intersegmental membrane place, pupal cell back with microsyringe, and the silkworm chrysalis after the processing places under 22～25 ℃, relative humidity 60～80% conditions and protects 72～120h;
(6) extraction of tussah antibiotic will be induced the cocoon chrysalis of 72～120h, extracts pupa blood plasma, centrifugal 15000rpm, 4～10 ℃, 20min removes blood cell and Oil globule, get supernatant liquor, centrifugal again 12000rpm, 4～6 ℃, 5min, remove soluble solid, 70～90 ℃ of temperature, 3～10min adds heat abstraction protein again, centrifugal 4000rpm, 10min get supernatant liquor and are tussah antibiotic aqueous solution finished product.
Of the present inventionly a kind ofly induce and extract the method for silkworm antibiotic by the live body cocoon chrysalis, its method is simple, and step is chosen scientific and reasonable, implement easily, inductive antibiotic activity value height, the silkworm chrysalis corruption is less, pupa blood keeps as clear as crystal, antibiotic yield height, cost is low, is easy to batch production production.The tussah antibiotic that extracts can be applicable to food as a kind of novel natural antiseptic agent, have fungicidal spectrum wide, edible after easily by body endoproteinase hydrolytic digestion, characteristics without any side effects, character according to tussah antibiotic is added it in beverage, bean product, the milk preparation to, can extend the shelf life, can also suppress the breeding of harmful intestinal tract bacterium, strengthen body immunity.
Embodiment
Utilize embodiment that the present invention is further described below.
Embodiment 1: a kind ofly induce and extract the method for silkworm antibiotic by the live body cocoon chrysalis, it is characterized in that it comprises following steps:
(1) tussah cocoon is preserved under 0～3 ℃ of condition, and test is prepended under 18 ℃ of room temperatures, the 60% relative humidity condition preserves 24h;
(2) remove the cocoon shell and take out the diapause pupa, select to remove dead pupa, growth pupa in the diapause pupa;
(3) cocoon chrysalis is cleaned with clear water, carries out the body surface sterilization with 65～75% alcohol;
(4) intestinal bacteria E.coliK is used in microorganism induction source
12D
31Vaccine is induced the source, its LB culture medium prescription is: in the 1L solution, and peptone 10g, extractum carnis 5g, sodium-chlor 5g, cultivate 24h,, in the bacterium liquid of cultivating, add 36% formaldehyde solution and make it to become 3% formalin solution with same substratum switching, place 24h at 4 ℃, centrifugal 2500rpm, 15min, abandoning supernatant, sedimentary thalline washs with 0.9% sodium chloride solution, centrifugal repeatedly 3 2500rpm, 5min, gained thalline cultivate 24h for 37 ℃ on the intestinal bacteria solid medium, confirm the aseptic length of being born, after proving dead bacterium, store in the refrigerator standbyly, the time spent transfers to 10 with vaccine concentration
8～10
5Cells/ml;
(5) induce, through the intestinal bacteria E.coliK of liquid culture
12D
31Vaccine is diluted to every ml with insect physiological saline and contains 10
8～10
5Cells, every pupa of concentration is injected 40 μ l, injects from intersegmental membrane place, pupal cell back with microsyringe, and the silkworm chrysalis after the processing places under 22 ℃, relative humidity 60% condition and protects 72h;
(6) extraction of tussah antibiotic will be induced the cocoon chrysalis of 72h, extracts pupa blood plasma, centrifugal 15000rpm, 4 ℃, 20min removes blood cell and Oil globule, get supernatant liquor, centrifugal again 12000rpm, 4 ℃, 5min removes soluble solid, again 70 ℃ of temperature, 3min adds heat abstraction protein, centrifugal 4000rpm, 10min gets supernatant liquor and is tussah antibiotic aqueous solution finished product.
Embodiment 2: a kind ofly induce and extract the method for silkworm antibiotic by the live body cocoon chrysalis, it is characterized in that it comprises following steps:
(1) tussah cocoon is preserved under 0～3 ℃ of condition, and test is prepended under 20 ℃ of room temperatures, the 65% relative humidity condition preserves 18h;
(2) remove the cocoon shell and take out the diapause pupa, select to remove dead pupa, growth pupa in the diapause pupa;
(3) cocoon chrysalis is cleaned with clear water, carries out the body surface sterilization with 65～75% alcohol;
(4) intestinal bacteria E.coliK is used in microorganism induction source
12D
31Vaccine is induced the source, and its LB culture medium prescription is: in the 1L solution, and peptone 10g, extractum carnis 5g, sodium-chlor 5g cultivates 24h, with same substratum switching, in the bacterium liquid of cultivating, add 36% formaldehyde solution and make it to become 3% formalin solution, place 96h, centrifugal 2500rpm at 4 ℃, 15min, abandoning supernatant, sedimentary thalline with 0.9% sodium chloride solution wash, centrifugal 3 2500rpm 10min repeatedly, the gained thalline is at intestinal bacteria E.coliK
12D
31On the solid medium, cultivate 24h for 37 ℃, confirm the aseptic length of being born, prove dead bacterium after, store in the refrigerator standbyly, the time spent transfers to 10 with vaccine concentration
8～10
5Cells/ml;
(5) induce, through the intestinal bacteria E.coliK of liquid culture
12D
31Vaccine is diluted to every ml with insect physiological saline and contains 10
8～10
5Cells, every pupa of concentration is injected 50 μ l, injects from intersegmental membrane place, pupal cell back with microsyringe, and the silkworm chrysalis after the processing places under 23.5 ℃, relative humidity 70% condition and protects 96h;
(6) extraction of tussah antibiotic will be induced the cocoon chrysalis of 96h, extracts pupa blood plasma, centrifugal 15000rpm, 7 ℃, 20min removes blood cell and Oil globule, get supernatant liquor, centrifugal again 12000rpm, 5 ℃, 5min, remove soluble solid, 80 ℃ of temperature, 6min adds heat abstraction protein again, and centrifugal 4000rpm 10min gets supernatant liquor and is tussah antibiotic aqueous solution finished product.
Embodiment 3: a kind ofly induce and extract the method for silkworm antibiotic by the live body cocoon chrysalis, it is characterized in that it comprises following steps:
(1) tussah cocoon is preserved under 0～3 ℃ of condition, and test is prepended under 22 ℃ of room temperatures, the 70% relative humidity condition preserves 12h;
(2) remove the cocoon shell and take out the diapause pupa, select to remove dead pupa, growth pupa in the diapause pupa;
(3) cocoon chrysalis is cleaned with clear water, carries out the body surface sterilization with 65～75% alcohol;
(4) intestinal bacteria E.coliK is used in microorganism induction source
12D
31Vaccine is induced the source, and its LB culture medium prescription is: in the 1L solution, and peptone 10g, extractum carnis 5g, sodium-chlor 5g cultivates 24h, with same substratum switching, in the bacterium liquid of cultivating, add 36% formaldehyde solution and make it to become 3% formalin solution, place 168h, centrifugal 2500rpm at 4 ℃, 15min, abandoning supernatant, sedimentary thalline with 0.9% sodium chloride solution wash, centrifugal 3 2500rpm 10min repeatedly, the gained thalline is at intestinal bacteria E.coliK
12D
31On the solid medium, cultivate 24h for 37 ℃, confirm the aseptic length of being born, prove dead bacterium after, store in the refrigerator standbyly, the time spent transfers to 10 with vaccine concentration
8～10
5Cells/ml;
(5) induce, through the intestinal bacteria E.coliK of liquid culture
12D
31Vaccine is diluted to every ml with insect physiological saline and contains 10
8～10
5Cells, every pupa of concentration is injected 60 μ l, injects from intersegmental membrane place, pupal cell back with microsyringe, and the silkworm chrysalis after the processing places under 25 ℃, relative humidity 60～80% conditions and protects 120h;
(6) extraction of tussah antibiotic will be induced the cocoon chrysalis of 120h, extracts pupa blood plasma, centrifugal 15000rpm, 10 ℃, 20min removes blood cell and Oil globule, get supernatant liquor, centrifugal again 12000rpm, 6 ℃, 5min, remove soluble solid, 90 ℃ of temperature, 10min adds heat abstraction protein again, centrifugal 2500rpm 10min gets supernatant liquor and is tussah antibiotic aqueous solution finished product.
Tussah antibiotic aqueous solution finished product can be directly used in foodstuff additive, also can make oral liquid according to a conventional method or further adopt general lyophilize to make dry powder for using.
Of the present inventionly a kind ofly induce and extract the embodiment of method of silkworm antibiotic and non exhaustive by the live body cocoon chrysalis, those skilled in the art all belong to the scope that claim of the present invention is protected without any conversion of creative work.
Claims (1)
1. induce and extract the method for silkworm antibiotic by the live body cocoon chrysalis for one kind, it is characterized in that it comprises following steps:
(1) tussah cocoon is preserved under 0～3 ℃ of condition, and test is prepended under 18～22 ℃ of room temperatures, 60～70% relative humidity conditions preserves 12～24h;
(2) remove the cocoon shell and take out the diapause pupa, select to remove dead pupa, growth pupa in the diapause pupa;
(3) cocoon chrysalis is cleaned with clear water, carries out the body surface sterilization with 65～75% alcohol;
(4) intestinal bacteria E.coliK is used in microorganism induction source
12D
31Vaccine is induced the source, and its LB culture medium prescription is: in the 1L nutrient solution, and peptone 10g, extractum carnis 5g, sodium-chlor 5g cultivates 24h, with same substratum switching, in the bacterium liquid of cultivating, add 36% formaldehyde solution and make it to become 3% formalin solution, place 24～168h at 4 ℃, centrifugal 2500rpm, 15min, abandoning supernatant, sedimentary thalline with 0.9% sodium chloride solution wash, 2500rpm, under the 15min condition centrifugal repeatedly 3 times, the gained thalline is at intestinal bacteria E.coliK
12D
31On the solid medium, cultivate 24h for 37 ℃, confirm the aseptic length of being born, prove dead bacterium after, store in the refrigerator standbyly, the time spent transfers to 10 with vaccine concentration
8～10
5Cells/ml;
(5) induce, through the intestinal bacteria E.coliK of liquid culture
12D
31Vaccine is diluted to every ml with insect physiological saline and contains 10
8～10
5Cells, every cocoon chrysalis of concentration injection 40～60 μ l inject from intersegmental membrane place, pupal cell back with microsyringe, and the cocoon chrysalis after the processing places under 22～25 ℃, relative humidity 60～80% conditions and protects 72～120h;
(6) extraction of tussah antibiotic will be induced the cocoon chrysalis of 72～120h, extracts pupa blood plasma, centrifugal 15000rpm, 4～10 ℃, 20min removes blood cell and Oil globule, get supernatant liquor, centrifugal again 12000rpm, 4～6 ℃, 5min, remove soluble solid, 70～90 ℃ of temperature, 3～10min adds heat abstraction protein again, centrifugal 2500～4000rpm 10min gets supernatant liquor and is tussah antibiotic aqueous solution finished product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103549195A (en) * | 2013-10-31 | 2014-02-05 | 辽宁省农业科学院大连生物技术研究所 | Stichopus japonicus feed and preparation method and application thereof |
| CN103704178A (en) * | 2013-12-19 | 2014-04-09 | 中国农业科学院草原研究所 | Method for adjusting diapause of Exorista civilis Rond |
| CN106387885A (en) * | 2016-09-05 | 2017-02-15 | 丹东欣龙生物科技有限公司 | Tussah chrysalis extract, as well as scale production method and application thereof |
| CN107365814A (en) * | 2017-06-06 | 2017-11-21 | 太仓新亚逊生物科技有限公司 | A kind of method that immune induction biotechnology prepares broad-spectrum antibiotic |
-
2011
- 2011-03-23 CN CN2011100776945A patent/CN102206263A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103549195A (en) * | 2013-10-31 | 2014-02-05 | 辽宁省农业科学院大连生物技术研究所 | Stichopus japonicus feed and preparation method and application thereof |
| CN103549195B (en) * | 2013-10-31 | 2016-02-10 | 辽宁省农业科学院大连生物技术研究所 | A kind of extra large apostichopus japonicus Selenka feed and its preparation method and application |
| CN103704178A (en) * | 2013-12-19 | 2014-04-09 | 中国农业科学院草原研究所 | Method for adjusting diapause of Exorista civilis Rond |
| CN103704178B (en) * | 2013-12-19 | 2016-04-13 | 中国农业科学院草原研究所 | A kind of regulate and control full skirt chase after the method for posting fly diapause |
| CN106387885A (en) * | 2016-09-05 | 2017-02-15 | 丹东欣龙生物科技有限公司 | Tussah chrysalis extract, as well as scale production method and application thereof |
| CN107365814A (en) * | 2017-06-06 | 2017-11-21 | 太仓新亚逊生物科技有限公司 | A kind of method that immune induction biotechnology prepares broad-spectrum antibiotic |
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Application publication date: 20111005 |