CN106214704A - A kind of Antheraea pernyi Geurin Meneville extract, large-scale method for producing and application - Google Patents
A kind of Antheraea pernyi Geurin Meneville extract, large-scale method for producing and application Download PDFInfo
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- CN106214704A CN106214704A CN201610801095.6A CN201610801095A CN106214704A CN 106214704 A CN106214704 A CN 106214704A CN 201610801095 A CN201610801095 A CN 201610801095A CN 106214704 A CN106214704 A CN 106214704A
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- antheraea pernyi
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- pernyi geurin
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- 241000255978 Antheraea pernyi Species 0.000 title claims abstract description 103
- 239000000284 extract Substances 0.000 title claims abstract description 71
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- 238000000034 method Methods 0.000 claims abstract description 41
- 239000003814 drug Substances 0.000 claims abstract description 8
- 201000010099 disease Diseases 0.000 claims abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 235000013402 health food Nutrition 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 37
- 230000005058 diapause Effects 0.000 claims description 24
- 238000002347 injection Methods 0.000 claims description 24
- 239000007924 injection Substances 0.000 claims description 24
- 210000000087 hemolymph Anatomy 0.000 claims description 19
- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 10
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 10
- 239000002574 poison Substances 0.000 claims description 9
- 231100000614 poison Toxicity 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 7
- 241000186000 Bifidobacterium Species 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 10
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 8
- 241001597008 Nomeidae Species 0.000 abstract description 6
- 239000002994 raw material Substances 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 4
- 238000011084 recovery Methods 0.000 abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 238000012360 testing method Methods 0.000 description 9
- 241000588724 Escherichia coli Species 0.000 description 8
- 230000000844 anti-bacterial effect Effects 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 241000382353 Pupa Species 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 241000194017 Streptococcus Species 0.000 description 5
- 239000013543 active substance Substances 0.000 description 5
- 102000016943 Muramidase Human genes 0.000 description 4
- 108010014251 Muramidase Proteins 0.000 description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 4
- 239000002917 insecticide Substances 0.000 description 4
- 229960000274 lysozyme Drugs 0.000 description 4
- 235000010335 lysozyme Nutrition 0.000 description 4
- 239000004325 lysozyme Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 241000255789 Bombyx mori Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
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- 241000238631 Hexapoda Species 0.000 description 2
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- 229920001184 polypeptide Polymers 0.000 description 2
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000009366 sericulture Methods 0.000 description 2
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- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000191938 Micrococcus luteus Species 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 240000005049 Prunus salicina Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
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- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 235000009018 li Nutrition 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 231100001160 nonlethal Toxicity 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Epidemiology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Insects & Arthropods (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicinal Preparation (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a kind of Antheraea pernyi Geurin Meneville extract, large-scale method for producing and application.In the inventive method, with edible bacterium as derivant, therefore obtained extract safety is high, is adapted as raw material to prepare the medicine for disease treatment or health food.Meanwhile, the inventive method extract total recovery is high, Suitable commercial large-scale production；Further, the inventive method is also given priority to for the lifting of different activities component content, it is possible to meet different Production requirements.The inventive method have safe efficient, productivity is high, is suitable to the advantages such as large-scale production.
Description
Technical field
The present invention relates to Antherea pernyi Guerin-Meneville and extract field, in particular to a kind of Antheraea pernyi Geurin Meneville extract, large-scale method for producing and
Application.
Background technology
Insecticide does not has special immune system, does not the most produce the cell of immune protein, but insecticide innate immunity and after
It immunocompetence obtained but is surprising.In recent years, the research to insecticide defense mechanism finds, to Semen Ricini silkworm, giant silkworm, family
The larva of the insecticide such as silkworm and Sarcophga fuscicauda or pupa injection non-lethal antibacterial, vaccine bacterium etc., can induce insect hemolymph
Producing immuno active polypeptide and albumen, these albumen or polypeptide have stronger broad-spectrum bactericidal capacity.
Antherea pernyi Guerin-Meneville is a kind of to raise the economic insects in field, is often subject to the severe ring of various virus, antibacterial and nature
The invasion and attack in border, but Antherea pernyi Guerin-Meneville can adapt to the most severe ecological environment, and can survival and development.As can be seen here, Antherea pernyi Guerin-Meneville tool
There are defence and the immune system of uniqueness, and the strongest antiviral and antibacterial ability.
Many researchs it turned out, and in the hemolymph in Antherea pernyi Guerin-Meneville body, there is several disease-resistant active substance, but due to these
The concentration of material is relatively low, uses general laboratory facilities to be difficult to effectively and extracts, and existing extracting method the most only rests on reality
Test room conceptual phase, the extraction production technology of the most ripe industrial scale.
In order to solve industrialization cannot to extract the problem of active substance in Antherea pernyi Guerin-Meneville body, prior art is also made that substantial amounts of
Effort.Prior art (" extraction of Antherea pernyi Guerin-Meneville disease-resistant active substance and application ", Japanese plum English, Sericulture of China, the 36th phase volume 1, the
20-24 page), disclose one and carry out biotic induce by E. coli mutant strain, to excite Antherea pernyi Guerin-Meneville to defend body constitution, thus promote
Enter to generate sufficient active, the method for onestep extraction of going forward side by side.Meanwhile, prior art (" physiologically active in Antheraea pernyi Geurin Meneville growth course
Material and the Changeement of protein " Meng Nan etc., Sericulture of China, the 3rd phase of volume 36, the 24-27 page), also disclose that with Antherea pernyi Guerin-Meneville
Streptococcus induction Antherea pernyi Guerin-Meneville antibacterial substance, and the method extracted.
But, although these prior aries make some progress, but as Antheraea pernyi Geurin Meneville extract industrialized production
Early-stage Study for, there is also more problem.Such as: (1) existing extracting method easily occurs interference, has a strong impact on work
The quality of property material；(2) extracted, by what derivant carried out, the product yield that reaction obtains with tussah streptococcus and escherichia coli
Low；(3) escherichia coli and tussah streptococcus are all not belonging to edible bacterium, there is wind in functional food and pharmaceutical production are applied
Danger.
In view of this, the special proposition present invention.
Summary of the invention
The first object of the present invention is to provide a kind of Antheraea pernyi Geurin Meneville extract large-scale method for producing, in the inventive method,
With edible bacterium as derivant, therefore obtained extract safety is high；Meanwhile, the inventive method extract total recovery is high,
Suitable commercial large-scale production；Further, the inventive method is also given priority to for the lifting of different activities component content, energy
Enough meet different Production requirements.The inventive method have safe and efficient, productivity is high, is suitable to the advantages such as large-scale production.
The second object of the present invention is to provide a kind of Antheraea pernyi Geurin Meneville extract being extracted by the inventive method and obtaining.The present invention
Antheraea pernyi Geurin Meneville extract carries out inducing and extracting obtaining with edible bacterium, therefore has extract safety advantages of higher.
Third object of the present invention is to provide the application of a kind of described Antheraea pernyi Geurin Meneville extract.Antheraea pernyi Geurin Meneville of the present invention extracts
Thing safety is good, and active constituent content is high, is consequently adapted to as raw material to prepare the medicine for disease treatment or health care
Food.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
A kind of Antheraea pernyi Geurin Meneville extract large-scale method for producing, described method comprises the steps: to be in the toothed oak of duration of diapause
Pupa bombycis takes out after storing, and termination of diapause；Then, by bacterium solution, Antheraea pernyi Geurin Meneville is carried out injection treatment, and cultivate；Gather toothed oak after cultivating
The hemolymph of Pupa bombycis, lyophilization, and pulverize and sieve, obtain Antheraea pernyi Geurin Meneville extract；
Wherein, the mixture of one or more during described bacterium solution is PZ1, PZ2 or PZ3；
Further, PZ1 is animal bifidobacteria, and biological deposits number is CGMCC 1.3003；
PZ2 is bacillus acidophilus, and biological deposits number is CGMCC 1.3342；
PZ3 is bacillus acidophilus, and biological deposits number is CGMCC 1.2467.
In the present invention, by use the edible strain such as animal bifidobacteria and bacillus acidophilus as derivant, from
And so that the extract obtained by extracting further has good safety, and it is corresponding with preparation to be adapted as raw material
Medicine；Meanwhile, the present invention induces with edible strain, it is also possible to obtain higher productivity, additionally it is possible to a certain extent
Control the content of different activities material in extract, thus be suitable to industrialized large-scale production, be also suitably for active matter in product
Matter requires in different large-scale production.
Optionally, in the present invention, termination of diapause can be carried out by the method controlling illumination.
Optionally, in the present invention, described in save as the Antheraea pernyi Geurin Meneville by being in duration of diapause under the conditions of 0～4 DEG C, store 10～
70d；Such as, in the present invention, can will be in the Antheraea pernyi Geurin Meneville of duration of diapause under conditions of 1,2 or 3 DEG C, store 15,30,45,
50,55,60 or 65d.
In the present invention, by duration of diapause Antheraea pernyi Geurin Meneville storage temperature to be extracted with store the selection of time and adjustment, from
And so that in the present invention Antheraea pernyi Geurin Meneville to be extracted there is good biological activity, and further biological bacterium stimulate with
And the high efficiency extraction of active component.
Optionally, in the present invention, described injection treatment is that each Antheraea pernyi Geurin Meneville injects 40～60 μ L bacterium solution, and bacterial concentration is 1
×105～1 × 108cells/ml；Such as, in the present invention, each Antheraea pernyi Geurin Meneville can inject 45,50 or 55 μ L bacterium solution, bacterium solution
Concentration can be 1 × 106Or 1 × 107Cells/ml etc..
In the present invention, by the bacterium solution consumption in order to induce and the selection of concentration and adjustment, such that it is able to Antheraea pernyi Geurin Meneville
Carry out effective biostimulation.
Optionally, in the present invention, described cultivation is by the Antheraea pernyi Geurin Meneville after injection treatment under the conditions of 18～24 DEG C, cultivates 3
～7d；Such as, in the present invention, the Antheraea pernyi Geurin Meneville after injection treatment can process 4,5 under the conditions of 19,20,21,22 or 23 DEG C
Or 6d.
Optionally, in the present invention, after described collection cultivation, the hemolymph of Antheraea pernyi Geurin Meneville is collection hemolymph under condition of ice bath.
In the present invention, by gathering hemolymph under condition of ice bath, such that it is able to effectively keep active substance in extract
Activity.
Optionally, in the present invention, the described cryodesiccated time is 12～24h；Such as, of the present invention cryodesiccated
Time can be, but is not limited to 13,15,17,20,22 or 23h etc..
In the present invention, by the selection of sublimation drying and adjustment, such that it is able to pure to extract efficient drying
While change, additionally it is possible to avoid the reduction of long the caused overall preparation efficiency of sublimation drying and to active component
Structural deterioration.
Optionally, in the present invention, described method still further comprises the step sterilized by Antheraea pernyi Geurin Meneville extract.
In the present invention, by increasing the step of sterilization the most after extraction such that it is able to improve toothed oak of the present invention further
The safety of Pupa bombycis extract.
Optionally, in the present invention, described sterilization is Co60Irradiation sterilization.
Further, extracting method step of the present invention is as follows:
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, the mixture of one or more during described bacterium solution is PZ1, PZ2 or PZ3；
Further, PZ1 is animal bifidobacteria, and biological deposits number is CGMCC 1.3003；PZ2 is bacillus acidophilus,
Biological deposits number is CGMCC 1.3342；PZ3 is bacillus acidophilus, and biological deposits number is CGMCC 1.2467.
A kind of Antheraea pernyi Geurin Meneville extract, described extract is extracted by the method for the invention and obtains.
In the present invention, obtain Antheraea pernyi Geurin Meneville extract by extracting with the method for the invention, therefore extract the Antherea pernyi Guerin-Meneville obtained
Pupa extract safety is high, is adapted as preparing medicine and health food raw material.
The Antheraea pernyi Geurin Meneville extract of the present invention application in preparation treatment disease medicament or health food.
Compared with prior art, the invention have the benefit that
(1) present invention is with edible bacterium as derivant, and therefore obtained extract safety is high；Meanwhile, side of the present invention
Method extract total recovery is high, Suitable commercial large-scale production, additionally it is possible to meet different Production requirements；
(2), in the inventive method, by adjusting further and optimizing each step condition, thus this is further increased
The extraction efficiency of invention Antheraea pernyi Geurin Meneville extract；
(3) Antheraea pernyi Geurin Meneville extract safety of the present invention is good, and is adapted as preparing medicine and health food is raw materials used.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but those skilled in the art will
Understanding, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.In embodiment unreceipted specifically
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument unreceipted production firm person, be
Can be by the commercially available conventional products bought and obtain.
Embodiment 1
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is PZ1, i.e. animal bifidobacteria, and biological deposits number is CGMCC 1.3003；
Further, the yield of embodiment 1 method Antheraea pernyi Geurin Meneville extract is 2.8%.
Embodiment 2
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is PZ2, i.e. bacillus acidophilus, and biological deposits number is CGMCC 1.3342；
Further, the yield of embodiment 2 method Antheraea pernyi Geurin Meneville extract is 2.6%.
Embodiment 3
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is PZ3, i.e. bacillus acidophilus, and biological deposits number is CGMCC 1.2467；
Further, the yield of embodiment 3 method Antheraea pernyi Geurin Meneville extract is 2.5%.
Embodiment 4
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is the mixture of PZ1 and PZ2, and wherein, PZ1 is animal bifidobacteria, and biological deposits number is
CGMCC 1.3003；PZ2 is bacillus acidophilus, and biological deposits number is CGMCC1.3342；
Further, the yield of embodiment 4 method Antheraea pernyi Geurin Meneville extract is 2.2%.
Comparative example 1
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is escherichia coli E.eoliKl2D31；
Further, the yield of comparative example 1 method Antheraea pernyi Geurin Meneville extract is 0.5%.
Comparative example 2
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is tussah streptococcus (1010)；
Further, the yield of comparative example 2 method Antheraea pernyi Geurin Meneville extract is 2.2%.
Comparative example 3
The Antheraea pernyi Geurin Meneville of duration of diapause will be under the conditions of 0～4 DEG C, take out after storing 10～70d, and termination of diapause；Then,
By bacterium solution, Antheraea pernyi Geurin Meneville being carried out injection treatment, every Antheraea pernyi Geurin Meneville injection 40～60 μ L concentration are 1 × 105～1 × 108cells/ml
Bacterium solution；And under the conditions of 18～24 DEG C, cultivate 3～7d；Under condition of ice bath, gather the hemolymph of Antheraea pernyi Geurin Meneville after cultivating, cold
Lyophilizing is dry is 12～24h, and pulverizes, and obtains Antheraea pernyi Geurin Meneville extract, then gained Antheraea pernyi Geurin Meneville extract is carried out Co60Irradiation disappears
Poison；
Wherein, described bacterium solution is tussah streptococcus (1212)；
Further, the yield of comparative example 3 method Antheraea pernyi Geurin Meneville extract is 2.0%.
Experimental example 1
Further, the Antheraea pernyi Geurin Meneville extract obtained by equivalent embodiment 1-4 and comparative example 1-3, and difference are taken respectively
Antibacterial Peptide and the content of Antherea pernyi Guerin-Meneville lysozyme isoreactivity material in test difference group Antheraea pernyi Geurin Meneville extract, detecting step is concrete
As follows:
(1) Activity determination of antibacterial peptide
1. material
(1) bacterial isolates escherichia coli (E colikl2D31)。
(2) glass culture dish (diameter 7.5cm) sterilizing is standby.
(3) card punch overall diameter 2.7mm.
2. the preparation of reagent
(1) LB fluid medium
PH to 7.4,121 DEG C of autoclaving 20min are adjusted with 5moL/L NaOH, standby.
(2) E+B buffer (50 ×)
Add 50ml distilled water to dissolve, be settled to 100ml, 121 DEG C of autoclaving 20min.
(3) liquid applied culture base
(4) LB solid medium:
LB fluid medium 10ml
Agar 1.0g
121 DEG C of autoclaving 20min.
3. the process of sample
Take to induce pupa hemolymph (hemolymph lyophilized products, by 1:10, is dissolved in normal saline) under ice bath, then put 100 DEG C
Heating 5min in water-bath, take supernatant and be centrifuged 5min in 3 000r/min, taking centrifuged supernatant is test sample.
4. Determination of Antibacterial Activity
(1) preparation of Escherichia coli bacteria liquid
With inoculating loop picking E.coliKl2D31Slant strains is inoculated in liquid applied culture base, 37 DEG C of static gas wave refrigerator
16h.Therefrom draw 0.2ml cultivation bacterium solution and transfer again in fresh 5ml liquid applied culture base, 37 DEG C of shaken cultivation 4～6h,
Make every milliliter of bacterium solution containing about escherichia coli 106Individual left and right.
(2) preparation of flat board is detected
Be cooled to 55 DEG C after being dissolved by 100ml LB solid medium, by final concentration 2% add E+B buffer (50 ×),
1.0% glucose solution (20%), 0.1% streptomycin sulfate (add people's Escherichia coli bacteria liquid after 250 000U/ml, fully mixing
50μl.After rapid oscillation fully mixes, incline in people's glass culture dish, 6.5ml/ ware, horizontal positioned, solidify latter 4 DEG C and save backup.
(3) vitality test
On detection flat board, get the small sircle hole of diameter 2.7mm with card punch, and indicate testing sample, control sample volume
Number.Drawing testing sample and each 5 μ l of reference substance respectively, inject in aperture the most one by one, each sample sets 3 repetitions.Often
On block flat board all with reference substance as internal control.Build ware lid, be inverted in 37 DEG C of calorstats after standing 10min cultivation 12～
18h, it is seen that inhibition zone clearly.Its diameter, respectively record testing sample and the antibacterial circle diameter of reference substance is measured with vernier cursor.
(2) Antheraea pernyi Geurin Meneville antalzyme activity assay method
With Beckman DU-7 type spectrophotometric determination and automatically record and scan.Concrete grammar is as follows:
1. the modulation of substrate
With 0.1moL/L, pH6.2 phosphate buffer, micrococcus lysodeikticus thalline is modulated into 0.72 OD450nmSuspension,
It is placed in 18%～20% water-bath, is used while allocating.
2. the modulation of lysozyme standard liquid
Accurately weigh lysozyme formulation, be dissolved in 0.1mol/L, pH6.2 phosphate buffer, become 1mg/ml's
Mother solution, draws 0.1ml immediately and injects in the little centrifuge tube containing 0.9ml buffer in advance, and the used time dilutes 25 times again, becomes 4mg/ml
Titer.
3. determination step
2.5ml substrate suspension is moved in cuvette (1cm diameter), constant temperature 18～20 DEG C, add standard lysozyme liquid
O.5ml, it is sufficiently mixed 1min, automatically records and scan the change of 450nm OD value immediately with spectrophotometer.Record continuously
3min.In the case of average minute clock declines 0.02～is O.04OD worth, the response time declines linear with OD value.
4. antalzyme activity unit calculates
20 DEG C, it is an active unit that the 450nm of making wavelength digestion value per minute reduces the enzyme amount of 0.001OD.Experiment proves
Blank extinction value A0It is 0.Sample extinction value, A/min means the mean concentration drop-out value of action time 1～3min, specifically
Computing formula is as follows:
Antheraea pernyi Geurin Meneville extract activity substance content testing result is as follows:
From above-mentioned testing result, the present invention extract the obvious activity substance content of Antheraea pernyi Geurin Meneville extract obtained more
High.
The present invention produces active substance with edible bacterium for derivant induction Antheraea pernyi Geurin Meneville, and further will induction gained activity
Material extracts, and therefore obtained extract safety is high, and can be as medicine and the raw materials for production of health food；With
Time, the inventive method extract total recovery is high, Suitable commercial large-scale production, additionally it is possible to meet different Production requirements.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that without departing substantially from the present invention's
May be made that in the case of spirit and scope many other change and amendment.It is, therefore, intended that in the following claims
Including all such changes and modifications belonged in the scope of the invention.
Claims (10)
1. an Antheraea pernyi Geurin Meneville extract large-scale method for producing, it is characterised in that described method comprises the steps: being in stagnant
Take out after educating the Antheraea pernyi Geurin Meneville storage of phase, and termination of diapause；Then, by bacterium solution, Antheraea pernyi Geurin Meneville is carried out injection treatment, and cultivate；Gather
The hemolymph of Antheraea pernyi Geurin Meneville, lyophilization after cultivation, and pulverize and sieve, obtain Antheraea pernyi Geurin Meneville extract；
Wherein, the mixture of one or more during described bacterium solution is PZ1, PZ2 or PZ3；
PZ1 is animal bifidobacteria, and biological deposits number is CGMCC 1.3003；PZ2 is bacillus acidophilus, and biological deposits number is
CGMCC 1.3342；PZ3 is bacillus acidophilus, and biological deposits number is CGMCC 1.2467.
Method the most according to claim 1, it is characterised in that described in save as the Antheraea pernyi Geurin Meneville by being in duration of diapause 0～4
Under the conditions of DEG C, store 10～70d.
Method the most according to claim 1, it is characterised in that described injection treatment is that each Antheraea pernyi Geurin Meneville injects 40～60 μ L
Bacterium solution, bacterial concentration is 1 × 105～1 × 108cells/ml。
Method the most according to claim 1, it is characterised in that described cultivation be by the Antheraea pernyi Geurin Meneville after injection treatment 18～
Under the conditions of 24 DEG C, cultivate 3～7d.
Method the most according to claim 1, it is characterised in that after described collection cultivation, the hemolymph of Antheraea pernyi Geurin Meneville is at ice bath
Under the conditions of gather hemolymph.
Method the most according to claim 1, it is characterised in that the described cryodesiccated time is 12～24h.
Method the most according to claim 1, it is characterised in that described method still further comprises and disappeared by Antheraea pernyi Geurin Meneville extract
The step of poison.
Method the most according to claim 7, it is characterised in that described sterilization is Co60Irradiation sterilization.
9. an Antheraea pernyi Geurin Meneville extract, it is characterised in that described extract is to be carried by method according to any one of claim 1-8
Acquirement is arrived.
10. the application in preparation treatment disease medicament or health food of the Antheraea pernyi Geurin Meneville extract described in claim 9.
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Cited By (1)
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| CN108477082A (en) * | 2018-03-09 | 2018-09-04 | 北京中农富通园艺有限公司 | The instant termination of diapause of silkworm heat shock and the method for refrigeration delay hatching |
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| CN108477082A (en) * | 2018-03-09 | 2018-09-04 | 北京中农富通园艺有限公司 | The instant termination of diapause of silkworm heat shock and the method for refrigeration delay hatching |
| CN108477082B (en) * | 2018-03-09 | 2019-12-17 | 北京中农富通园艺有限公司 | Method for instantly relieving diapause by heat shock of silkworms and delaying incubation by refrigeration |
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