CN110352920A - A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine - Google Patents
A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine Download PDFInfo
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- CN110352920A CN110352920A CN201910812863.1A CN201910812863A CN110352920A CN 110352920 A CN110352920 A CN 110352920A CN 201910812863 A CN201910812863 A CN 201910812863A CN 110352920 A CN110352920 A CN 110352920A
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/04—Silkworms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The present invention provides a kind of methods that the larva of a silkworm with batrytis is manually cultivated based on muscardine, it is characterized by: the larva of a silkworm with batrytis manually cultivated, it is cylindrical, more shrinkage bendings, single body grows 4.30 ± 0.35cm, diameter is 0.5 ± 0.1cm, ten thousand 7-8 kilograms of silkworm weights, surface is milky powdery mycelia and conidium, matter is hard and crisp, end face flat-satin, for bright amber color, it is clearly visible sericterium ring, it is 6.00-6.20% that high performance liquid chromatography, which measures ammonium oxalate content, it is prepared by spore suspension inoculation liquid, muscardine culture, spore liquid is disseminated to prepare, the preparation of artificial production larva of a silkworm with batrytis beauveria bassiana spore；Preparation process of the invention is simple, it is of less demanding to production environment and production equipment, substantially reduce production cost, reduce the pollution problem of miscellaneous bacteria in production, it is separated without by spore with compost after drying, direct packaging finished product, the recycling of spore almost all, it is very suitable to small business and production unit High-speed Production of Beauveria bassiana, is improved the economic and social benefits.
Description
Technical field
The present invention relates to the cultural method of the larva of a silkworm with batrytis more particularly to a kind of sides that the larva of a silkworm with batrytis is manually cultivated based on muscardine
Method.
Background technique
The larva of a silkworm with batrytis (Bombyx batryticatus), also known as bombyx batryticatus, stiff flying worm are Bombycidae insect silkworm 4-5 age childrens
Worm natural infection (or artificial infection) muscardine (Beauveria) and the full worm of ossified drying, the larva of a silkworm with batrytis answer as traditional Chinese medicine
For treating a variety of diseases, there are thousands of years medicinal histories in China, be also widely used in Japan and South Korea.Modern pharmacology
Learn research discovery the larva of a silkworm with batrytis have a variety of active pharmaceutical ingredients, can antibacterial, anticonvulsion, anticoagulant, antithrombotic, promote fibrinolytic, tumor suppression,
Hypnosis, lipid-loweringing, hypoglycemic etc..Modem medical practices are research shows that the larva of a silkworm with batrytis controls treatment parotitis, epilepsy and postencephalitis
Treatment effect, has different curative effects to cancer of the esophagus, lung cancer, gastric cancer, bladder cancer etc..With the development of modern science and technology, learning both at home and abroad
Person deepens continuously to the research of the larva of a silkworm with batrytis, develops the medical value of many frontiers, causes head in psoriasis, chronic nasosinusitis
Bitterly, chloasma, acne, cough, asthma, facial paralysis, viral infection, dysfunctional uterine bleeding, tumour, diabetes, surrounding
The illness such as neuropathy, infantile spasm have clinical application report.According to statistics, have in Chinese patent prescription at present 175 kinds containing white
Bombyx batryticatus.In recent years, with the pharmacological research progress and its good efficacy shown in clinical application to the larva of a silkworm with batrytis, so that white deadlock
The medical value of silkworm is of increasing concern, and supply falls short of demand in market.Especially research in recent years discovery bombyx batryticatus has stimulation adrenal gland skin
Matter effect and healthcare function have widened the use scope of bombyx batryticatus significantly, and dosage increases severely, its price is caused persistently to raise up.China is every
Year, also in the batch production professional production larva of a silkworm with batrytis, but this was also difficult to meet medical department in addition to largely purchasing the larva of a silkworm with batrytis from each silkworm area
Needs.Domestic some enterprise artificial challenges produce the larva of a silkworm with batrytis at present, and mainly have the problem of several respects: (one) strain stability is not
Height mostly directly elutes lower spore for inoculating from the larva of a silkworm with batrytis after natural infection or artificial challenge, strain do not purify and
It is preferred that, pathogenicity influenced by factors such as environment, weathers, metainfective silkworm body is not concentrated from the death time is inoculated into, family
Silkworm infection rate is low, bombyx batryticatus yield is low, and the time of ossifing is irregular, unstable quality, and yield is lower；(2) due to muscardine spore
The restriction of sub- powder store method, as time increases, spore count and conidium vitality living can all significantly reduce, therefore, in reality
Situations such as silkworm disease incidence is low, occurrent time is long after inoculation is often deposited in production, and silkworm is caused still cannot before matured silkworm is placed on small straw bundles to spin cocoons
Morbidity is dead as scheduled, causes very big economic loss to bombyx batryticatus production
Therefore, the present invention is integrated in order to form high-quality larva of a silkworm with batrytis production technology, pushes the sustainable development of larva of a silkworm with batrytis industry, discloses
A method of the larva of a silkworm with batrytis manually being cultivated based on muscardine, the present invention prepares the larva of a silkworm with batrytis obtained, and cylindrical, more shrinkages are curved
Song, the long 4.30 ± 0.35cm of single body, diameter are 0.5 ± 0.1cm, and ten thousand 7-8 kilograms of silkworm weights, surface is milky powdery mycelia
And conidium, matter is hard and crisp, end face flat-satin, for bright amber color, hence it is evident that see sericterium ring, high performance liquid chromatography
Measuring ammonium oxalate content is 6.00-6.20%.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine, concrete scheme
It is as follows:
A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine, it is characterised in that: the larva of a silkworm with batrytis manually cultivated, it is cylindrical,
More shrinkage bendings, the long 4.30 ± 0.35cm of single body, diameter are 0.5 ± 0.1cm, and ten thousand 7-8 kilograms of silkworm weights, surface is milky
Powdery mycelia and conidium, matter is hard and crisp, end face flat-satin, for bright amber color, hence it is evident that see sericterium ring, efficient liquid
It is 6.00-6.20% that phase chromatography, which measures ammonium oxalate content, and cultural method includes the following steps:
(1) muscardine purifies:
The larva of a silkworm with batrytis is produced using commercially available larva of a silkworm with batrytis beauveria bassiana spore powder, and multiple using PDSA culture medium from the larva of a silkworm with batrytis of production
Plate streaking culture, obtains muscardine pure culture, and the muscardine pure culture is beauveria bassiana；
(2) prepared by spore suspension inoculation liquid:
The pure culture that step (1) is obtained is seeded to the inclined-plane that PDSA in eggplant bottle cultivates matrix manufacturing, and cultivates in 26 ± 1 DEG C
14d, spore are covered with inclined-plane, and cultivating total spore amount is 1.6 × 1010-1.8×1010Spore/bottle, and it is 5 × 10 that concentration, which is made,7-6
×107The spore suspension inoculation liquid of/ml, it is spare；
(3) solid state fermentation beauveria bassiana spore
Prepare solid fermentation culture medium, ingredient includes rice: water=10: 9(w: w), potassium nitrate 0.2%, soy sauce 2ml/100g it is big
Rice, by step (2) spore suspension inoculation liquid by 10%(v: w) inoculum concentration is seeded in solid fermentation culture medium, in 26 ± 1 DEG C of items
After cultivating 12d under part, takes out and place drying in electric drying oven with forced convection, obtain beauveria bassiana spore, sealing of bottling, and in 0-8 DEG C
Under the conditions of store；
(4) dip dyeing spore liquid is prepared:
Bottled beauveria bassiana spore in above-mentioned steps (3) is taken, submerges spore with cold water, and attached sufficiently to elute by oscillation bottle
Conidium on rice, every bottle of final elution spore water consumption is 6000-8000ml, and obtaining spore liquid concentration is 5 × 107
A spore/ml is fitted into spare in spray bottle；
(5) larva of a silkworm with batrytis is manually cultivated:
S1: the grain of rice after the middle elution spore of step (4) is sprinkled upon on rearing shed, chooses all awake dormancy feeding first meal mulberry leaf 3 of four dormancies
Spore suspension prepared by step (4) is inoculated with by the silkworm after a hour in two times, wherein be sprayed on silkworm silkworm body for the first time,
Ensure that silkworm body is shown in wet, and then feeding mulberry leaf, second spore suspension is sprayed on mulberry leaf, allows the free feeding of silkworm；
S2: after inoculation for 24 hours in guarantee that silkworm canopy door and window is closed, humidity 90% in canopy is kept by sprinkling clear water, for 24 hours after to before the onset,
Guarantee that ventilation is not less than 2h in canopy daily, lime is not used after inoculation, rearing shed is sterilized, remaining feeding method and routine
Silk silkworm is the same；
It concentrating appearance ill after S3:5d, stops food mulberry, 6d or so concentrates death, and ossified rate is all ossified to silkworm body greater than 95%,
Ensuring that silkworm canopy door and window is closed, ensures that humidity is 90% in canopy by spraying clear water, temperature is 26-28 DEG C, and ossified silkworm whitens after 8d,
11d after inoculation, spore cover with silkworm body, and the stage of whitening terminates, collect larva of a silkworm with batrytis semi-finished product, be placed under the sun dry the larva of a silkworm with batrytis at
Product.
Further, the larva of a silkworm with batrytis chosen in the step (1) is rushed after spraying disinfection 3-5s with 75% alcohol with sterile water
It washes three times, then bombyx batryticatus is truncated with aseptic nipper, is taken again three times with rinsed with sterile water with 0.1% mercuric chloride water sterilization 3-5min
It smears PDSA culture medium flat plate or is drawn on PDSA culture medium flat plate with disposable ring scraping surface mycelia and spore in section
Line, constant temperature incubation 5d or more at 26 DEG C, plate streaking purifies the doubtful muscardine bacterium colony of picking again or repeatedly on plate, directly
To obtaining pure culture.
Further, the step (2) elutes spore using 0.1% Tween-80 of 300ml, and is poured into glass
In the sterile blue cover glass bottle of pearl, abundant shaken well, the spore concentration in terms of blood counting chamber prepares spore suspension inoculation liquid.
Further, solid fermentation culture medium in the step (3), using stainless steel disc, specification is 17 × 23cm, bottom
14 × 20cm of area weighs 130g rice, 117ml liquid dosage is added, wherein liquid includes 10%KNO3Solution 2.6ml, soy sauce
2.6ml, surplus are water, impregnate 2-3h, put on 35cm × 25cmPE freshness protection package, in 121 DEG C of high pressure steam sterilization 20min, are made
Solid fermentation culture medium.
Further, step (3) the miospore suspension inoculum concentration is the every disk solid fermentation culture medium of 13ml.
Further, air dry oven temperature is 30-32 DEG C in the step (3), and drying time is not more than 48h, until
It is hard not broken that hand pinches rice, dry to complete, and according to 500g/ bottles of quantitative separatings in the big mouth plastic bottle of 2000mlPP, screws lid
And mark lot number.
Further, PDSA medium component and content are potato 250g, white sand in the step (1) and step (2)
Sugared 50-60g, soy sauce 50mL, agar strip 20-25g, distilled water 1000 mL, pH are natural.
Further, cultivated silkworm breed variety is bright moon × mountain valley with clumps of trees and bamboo pine in the step (5).
Compared with prior art, the device have the advantages that it is as follows:
The present invention will expand the spore after culture on eggplant bottle inclined-plane and wash lower conduct muscardine inoculation liquid, be inoculated with by 10% inoculum concentration
Fermented and cultured in solid fermentation culture medium, used fermentation medium have good moisture retention and gas permeability, can be white deadlock
Bacterium proliferation provides the source N abundant and the nutrition of the source C, raw material economics are easy to get；Cultural method of the invention is easy to operate, clear process,
Of less demanding to production environment and production equipment, lower production costs are not needed in production to the special humidification of culture environment,
Without spice in incubation, reduces the pollution problem of miscellaneous bacteria in production, is separated without by spore with compost after drying,
Direct packaging finished product, spore almost all recycling, is very suitable to small business and production unit High-speed Production of Beauveria bassiana, and wash
De- spore is simple and convenient, and spore utilization rate is high, is suitable for large-scale promotion, greatly improves economic benefit and social benefit.
Detailed description of the invention
It to describe the technical solutions in the embodiments of the present invention more clearly, below will be to required in embodiment technical description
Attached drawing to be used does simple introduction, it should be apparent that, drawings in the following description are only some embodiments of the invention,
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings
Other attached drawings.
Fig. 1 is PDSA flat-plate bacterial colony photo.
Fig. 2 is eggplant bottle slant strains pictorial diagram.
Fig. 3 is solid fermentation culture medium stainless steel disc pictorial diagram.
Fig. 4 is muscardine solid fermentation process schematic diagram.
Fig. 5 is fermentation of white muscardine fungi product spore pictorial diagram.
Fig. 6 is the bottled schematic diagram of solid fermentation product spore finished product.
Fig. 7 is bombyx batryticatus semi-finished product object legend.
Fig. 8 is bombyx batryticatus finished product material object legend.
Fig. 9 is the silkworm canopy material object legend for cultivating bombyx batryticatus.
Figure 10 is ammonium oxalate canonical plotting.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art without creative labor it is obtained it is all its
His embodiment, belongs to protection scope of the present invention.
Embodiment: the present invention provides a kind of good larva of a silkworm with batrytis, produces to increase economic efficiency and social benefit
The larva of a silkworm with batrytis meet following feature: prepare the larva of a silkworm with batrytis of acquisition, cylindrical, more shrinkages bending, single body is long by 4.30 ±
0.35cm, diameter are 0.5 ± 0.1cm, 7-8 kilograms of ten thousand silkworms weight, and surface is milky powdery mycelia and conidium, matter it is hard and
It is crisp, end face flat-satin, for bright amber color, hence it is evident that see sericterium ring, high performance liquid chromatography measures ammonium oxalate content and is
6.00-6.20%。
One: the purifying of strain:
The larva of a silkworm with batrytis is prepared using commercially available beauveria bassiana spore powder, and takes to whiten and terminates undried larva of a silkworm with batrytis semi-finished product, the spray of 75% alcohol
After applying disinfection 3-5s, three times with aseptic water washing, then with 0.1% mercuric chloride water sterilization 3-5 min, rinsed with sterile water three is used again
It is secondary, with aseptic nipper be truncated bombyx batryticatus, take section smear PDSA culture medium flat plate or with disposable ring scraping surface mycelia and
Spore is crossed on PDSA culture medium flat plate, and plate is inverted, in 26 DEG C of constant temperature incubation 5d or more, the doubtful white deadlock of picking on plate
Bacterium bacterium colony, PDSA plate streaking purifies again or repeatedly, and until obtaining pure culture, which is beauveria bassiana, and bacterium colony is such as
Shown in Fig. 1.
Two, prepared by spore suspension inoculation liquid body:
Strain after purification is taken, PDSA culture medium eggplant bottle inclined-plane culture is inoculated with, 26 ± 1 DEG C of cultivation temperature, cultivates 14d,
Spore is covered with inclined-plane, as shown in Fig. 2, each eggplant bottle slant pore total amount about 1.6 × 10 after measured10-1.8×1010/ bottle；To
300ml0.1% Tween-80 is poured into eggplant bottle, scrapes surface with inoculation shovel or glass rod with gentle, as far as possible all by all spores
It washes down, and is poured into the sterile blue cover glass bottle with bead, abundant shaken well, the mesh of Tween-80 and bead
Be provided to spore can be allowed sufficiently to break up, blood counting chamber meter spore concentration, preparation concentration be 5 × 107-6×107A spore
Son/ml spore suspension inoculation liquid.
Above-mentioned one, PDSA medium component is potato 250g, white granulated sugar 50-60g, soy sauce 50mL, agar in two steps
20-25g, distilled water 1000 mL, pH are prepared naturally.
Three, muscardine is cultivated:
Firstly, prepare include rice: water=10: 9(w: w), potassium nitrate 0.2%, every 100g rice add the solid fermentation of soy sauce 2ml to train
Base is supported, specially uses specification for 17 × 23cm, 14 × 20cm of floor space stainless steel disc such as Fig. 3 weighs 130g rice in disk,
Add 2.6ml 10%KNO3Solution, 2.6ml soy sauce and water, wherein KNO3Solution, soy sauce and water inventory meter 117ml impregnate 2-
3h puts on 35cm × 25cmPE freshness protection package, 121 DEG C of high pressure steam sterilization 20min.
After solid fermentation culture medium is cooling, it is inoculated in clean work station, will be specially eluted in step 2
Spore suspension inoculation liquid, by 10%(v: w) inoculum concentration be inoculated with solid fermentation culture medium, every disk inoculating spores suspension 13ml so that
Whole amount of water and rice ratio are 1:1(w:w), spore and rice are mixed thoroughly and are divided, will be among the fresh-keeping sack of covered plate with adhesive tape
Half is sealed in position, prevents from falling into incubation microbes in air, the fermentation plate after inoculation is put into illumination box
Culture is arranged 26 DEG C of condition, and b:h=12:12, humidity is naturally, incubation time 12d；The spore that solid state fermentation produces muscardine is normal
Humidity is needed to entangle entire stainless steel disc with freshness protection package in 80% or more, the present invention, in the freshness protection package of fermented and cultured primary surface
It is always maintained at uniformly tiny droplet such as Fig. 4 on wall, ensure that the requirement of humidity, reduce to equipment investment and setting condition
Requirement, available household watering can is packed into cold boiling water daily, cultivates chamber interior wall 2 times by spraying, to improve humidity in cabinet；Fermentation is completed
Culture, rice pellets dispersion is covered with a thick layer of milky conidium such as Fig. 5, takes out stainless steel and ferments disk, shuts down
Freshness protection package is put into electric drying oven with forced convection, and one layer of sterilized newspaper can be blocked by arranging above the plate put, and 30-32 DEG C
Drying not more than 48 hours, hand pinched that rice is not hard broken, and spore powdery is easily fallen, dry to complete, then every bottle of quantitative separating of 500g
In the big mouth plastic bottle of 2000ml PP, lid is screwed, lot number is marked, is placed in 0-8 DEG C of preservation to get spore finished product, bottled finished product is such as
Shown in Fig. 6.
1 gram of spore finished product after weighing above-mentioned fermentation is added 9ml0.1% Tween-80, sufficiently vibrates, elute and break up spore
Son, blood counting chamber count spore concentration, show that the concentration of the spore finished product of fermentation is (6.0-8.0) × 108A spore/gram.
Four, dip dyeing spore liquid is prepared:
The 500g bottled product for taking above-mentioned steps three directly pours into appropriate cold water submergence in bottle, stands 5min, vibrate bottle
It elutes the spore adhered on the grain of rice as far as possible, pours out spore suspension；In order to sufficiently clean spore, above-mentioned elution spore is repeated
Twice, by the spore suspension eluted three times cold water constant volume 6000-8000ml, this spore liquid concentration is about 5 × 10 to sub-step7
A spore/ml is fitted into spare in spray bottle after mixing.Since the big grain of rice acts as the effect of bead in this step, help spore
It the elution of son and breaks up, spore elution is not required to additionally to add bead also not need to add the emulsifiers such as tween.
Five, the larva of a silkworm with batrytis is manually cultivated:
About 3 hours sprinkling spore suspensions after cultivated silkworm breed variety bright moon × mountain valley with clumps of trees and bamboo pine sleeps all four and wakes up dormancy and feeding first meal mulberry leaf.Spray
It spills spore suspension specific steps: sprayer endospore suspension is connect into bacterium in two times, uniformly sprayed using half spore suspension for the first time
Be sprinkled upon on silkworm body, with silkworm body see it is wet be advisable, should ensure that before sprinkling without remaining residual leaf on rearing shed, feeding immediately after this time spraying
Mulberry leaf；Second is sprayed at remaining half spore suspension on the mulberry leaf of previous feeding, allows the free feeding of silkworm；And it will be remaining
The grain of rice is sprinkling upon on rearing shed, and 24 after connecing bacterium hour should keep silkworm canopy door and window to close, and keeps humidity in canopy to exist by sprinkling clear water
To before the onset, guaranteeing that ventilation is not less than two hours in canopy daily after 90% or so, 24 hours, lime is not used after connecing bacterium
Rearing shed is sterilized, remaining feeding method after silkworm is all ossified, into the stage of whitening, needs to close at this time as routine silk silkworm
Silkworm canopy door and window, sprinkling clear water keep humidity 90% or so, and temperature controls between 26-28 DEG C, after spore covers with silkworm body, hair
The white stage terminates, be larva of a silkworm with batrytis semi-finished product, the semi-finished product larva of a silkworm with batrytis is collected, be placed under the sun dry to obtain the larva of a silkworm with batrytis at
Product, the visible Fig. 7-9 of specific example.
Wherein, the larva of a silkworm with batrytis is manually cultivated, is inoculated with different spore concentrations to the lethal cases of silkworm, as shown in table 1:
| Inoculum density | 1 d | 2 d | 3 d | 4 d | 5 d | 6 d | 7d |
| 1×105 | 0% | 0% | 0% | 6% | 23% | 50% | 62% |
| 1×106 | 0% | 0% | 0% | 9% | 25% | 53% | 70% |
| 5×106 | 0% | 0% | 0% | 8% | 32% | 60% | 78% |
| 1×107 | 0% | 0% | 1% | 12% | 45% | 82% | 85% |
| 5×107 | 0% | 0% | 1% | 20% | 56% | 96% | 98% |
| 1×108 | 0% | 0% | 3% | 63% | 85% | 100% | 100% |
| Clear water | 0% | 0% | 0% | 0% | 0% | 0% | 0% |
The ossified rate of silkworm of the inoculation various concentration beauveria bassiana spore of table 1
As shown in Table 1: inoculation various concentration spore is larger to the Influence of production of the larva of a silkworm with batrytis, in inoculum density less than 5 × 107A spore
When son/mL, bacterial strain spore is undesirable to the pathogenicity of silkworm, and a large amount of silkworms cannot still fall ill death as scheduled before matured silkworm is placed on small straw bundles to spin cocoons,
Bombyx batryticatus yield is low, causes larger economic loss to bombyx batryticatus production；When inoculum density is 5 × 107When a spore/mL, spore is to family
The pathogenicity of silkworm reached 96% at 6 days, and morbidity is concentrated, and ossified rate is high；But work as inoculum density up to 1 × 108When a spore/mL,
Silkworm disease incidence in 4-5 d has just reached 63-85%, and the silkworm young does not reach full growth just to fall ill ossified too early, leads to bombyx batryticatus
The small matter of finished product is poor, and economic benefit is also had a greatly reduced quality, and therefore, base manually produces larva of a silkworm with batrytis selection inoculating spores concentration and should be higher than that
1×107A spore/mL concentration, with 5 × 107A spore/mL or so is advisable.
Six, larva of a silkworm with batrytis ammonium oxalate assay:
Two samples are respectively in October, 2018, the larva of a silkworm with batrytis product that in June, 2019, cooperative society produced twice, and specific method step is such as
Under:
(1) preparation of the processing of sample and sample liquid: the drying sample and label of two kinds of larva of a silkworm with batrytis are taken, 30 mesh are smashed it through
Sieve is put into electric heating constant-temperature blowing drying box and dries to remove moisture removal, and every part of sample weighs 0.5000 g, is placed in small beaker,
50 mL distilled waters are added, handle 10 min(settings:99% under the conditions of 30 DEG C using SB-5200DTD supersonic wave cleaning machine
Power, 40 KHz, 300 W), then its cleaning solution is filtered by vacuum with Suction filtration device, crosses 0.45 μm of filter membrane, filtrate
It is transferred in glass jar, 10 mL sample liquids is removed from wide-mouth bottle, the constant volume in 50 mL volumetric flasks uses micropipette rifle
200 μ L sample liquid are drawn, are added in 1.5 mL EP pipes, it is spare.
(2) measurement of ammonium oxalate content
1. chromatographic condition: HPLC with detection (HPLC-VWD), a length of 204 nm of maximum absorption wave；Chromatographic column
5 μm of Diamonsil C18(250 × 4.6 mm, 5 μm) enlightening horse Diamonsil C18 diamond generation chromatographic column, 4.6 mm ×
250 mm×5 u；Mobile phase: 0.5% NH4H2PO4Aqueous solution (A) // methanol (B) (90/10, V/V), 0.4 mL/ of flow velocity
Min, sample volume 20 μ L, total 10 min of sample injection time.
2. cleaning: cleaning 60 min of chromatographic column with 10% methanol solution.
3. the drafting of standard curve: the EP pipe equipped with various concentration ammonium oxalate titer being put into high speed centrifugation before upper prop
It is centrifuged in machine, remains to sample, drawn 20 μ L titer loadings with sample injector, measure the peak area under various concentration, draw standard
Abscissa is ammonium oxalate standard concentration when curve, and ordinate is peak area.
4. the measurement of sample: the EP pipe equipped with different samples being put into supercentrifuge before upper prop and is centrifuged, remains to take
Sample.20 μ L sample liquid loadings are drawn with sample injector, the peak area of sample liquid is measured, is calculated in sample liquid using standard curve
Ammonium formiate concentration finally calculates larva of a silkworm with batrytis sample mesoxalic acid ammonium content again.
(3) result: the peak area by measuring various concentration ammonium oxalate titer under maximum absorption wavelength 204nm draws standard
Curve is as shown in Figure 10, calibration curve formula are as follows: Y=0.5725x+3.5182(R2=0.9983)；Measure the peak area of sample liquid
And it finally calculates larva of a silkworm with batrytis sample mesoxalic acid ammonium content and is shown in Table 2:
| Sample | Peak area (204 nm) | Ammonium oxalate (%) |
| 2018-10 | 72.2188 | 5.99% |
| 2019-06 | 74.5085 | 6.20% |
The peak area and ammonium oxalate content of 2 two batches larva of a silkworm with batrytis product of table
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any to be familiar with
Those skilled in the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all cover
Within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (8)
1. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine, which is characterized in that the larva of a silkworm with batrytis manually cultivated is in cylinder
Shape, more shrinkage bendings, the long 4.30 ± 0.35cm of single body, diameter are 0.5 ± 0.1cm, and ten thousand 7-8 kilograms of silkworm weights, surface is cream
White powder mycelia and conidium, matter is hard and crisp, end face flat-satin, for bright amber color, hence it is evident that see sericterium ring, it is high
It is 6.00-6.20% that effect liquid phase chromatogram method, which measures ammonium oxalate content, and artificial culture method includes the following steps:
(1) muscardine purifies:
The larva of a silkworm with batrytis is produced using commercially available larva of a silkworm with batrytis beauveria bassiana spore powder, and multiple using PDSA culture medium from the larva of a silkworm with batrytis of production
Plate streaking culture, obtains muscardine pure culture, and the muscardine pure culture is beauveria bassiana；
(2) prepared by spore suspension inoculation liquid:
The pure culture that step (1) is obtained is seeded to the inclined-plane that PDSA in eggplant bottle cultivates matrix manufacturing, and cultivates in 26 ± 1 DEG C
14d, spore are covered with inclined-plane, and cultivating total spore amount is 1.6 × 1010-1.8×1010Spore/bottle, and it is 5 × 10 that concentration, which is made,7-6
×107The spore suspension inoculation liquid of/ml, it is spare；
(3) solid state fermentation beauveria bassiana spore
Prepare solid fermentation culture medium, ingredient includes rice: water=10: 9(w: w), potassium nitrate 0.2%, soy sauce 2ml/100g it is big
Rice, by step (2) spore suspension inoculation liquid by 10%(v: w) inoculum concentration is seeded in solid fermentation culture medium, in 26 ± 1 DEG C of items
After cultivating 12d under part, takes out and place drying in electric drying oven with forced convection, obtain beauveria bassiana spore, sealing of bottling, and in 0-8 DEG C
Under the conditions of store；
(4) dip dyeing spore liquid is prepared:
Bottled beauveria bassiana spore in above-mentioned steps (3) is taken, submerges spore with cold water, and attached sufficiently to elute by oscillation bottle
Conidium on rice, every bottle of final elution spore water consumption is 6000-8000ml, and obtaining spore liquid concentration is 5 × 107
A spore/ml is fitted into spare in spray bottle；
(5) larva of a silkworm with batrytis is manually cultivated:
S1: the grain of rice after the middle elution spore of step (4) is sprinkled upon on rearing shed, chooses all awake dormancy feeding first meal mulberry leaf 3 of four dormancies
Spore suspension prepared by step (4) is inoculated with by the silkworm after a hour in two times, wherein be sprayed on silkworm silkworm body for the first time,
Ensure that silkworm body is shown in wet, and then feeding mulberry leaf, second spore suspension is sprayed on mulberry leaf, allows the free feeding of silkworm；
S2: after inoculation for 24 hours in guarantee that silkworm canopy door and window is closed, humidity 90% in canopy is kept by sprinkling clear water, for 24 hours after to before the onset,
Guarantee that ventilation is not less than 2h in canopy daily, lime is not used after inoculation, rearing shed is sterilized, remaining feeding method and routine
Silk silkworm is the same；
It concentrating appearance ill after S3:5d, stops food mulberry, 6d or so concentrates death, and ossified rate is all ossified to silkworm body greater than 95%,
Ensuring that silkworm canopy door and window is closed, ensures that humidity is 90% in canopy by spraying clear water, temperature is 26-28 DEG C, and ossified silkworm whitens after 8d,
11d after inoculation, spore cover with silkworm body, and the stage of whitening terminates, collect larva of a silkworm with batrytis semi-finished product, be placed under the sun dry the larva of a silkworm with batrytis at
Product.
2. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, it is characterised in that: the step
Suddenly the larva of a silkworm with batrytis chosen in (1), after spraying disinfection 3-5s with 75% alcohol, three times with aseptic water washing, then with 0.1% mercuric chloride water
Sterilize 3-5min, again three times with rinsed with sterile water, with aseptic nipper be truncated bombyx batryticatus, take section smear PDSA culture medium flat plate or
Surface mycelia is scraped with disposable ring and spore is crossed on PDSA culture medium flat plate, constant temperature incubation 5d or more at 26 DEG C,
Plate streaking purifies the doubtful muscardine bacterium colony of picking again or repeatedly on plate, until obtaining pure culture.
3. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, the step (2) is utilized
0.1% Tween-80 of 300ml elutes spore, and is poured into the sterile blue cover glass bottle with bead, and sufficiently oscillation is equal
Even, the spore concentration in terms of blood counting chamber prepares spore suspension inoculation liquid.
4. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, it is characterised in that: the step
Suddenly solid fermentation culture medium in (3), using stainless steel disc, specification is 17 × 23cm, and it is big to weigh 130g by 14 × 20cm of floor space
117ml liquid dosage is added in rice, and wherein liquid includes 10%KNO3Solution 2.6ml, soy sauce 2.6ml, surplus are water, impregnate 2-
3h puts on 35cm × 25cmPE freshness protection package, and in 121 DEG C of high pressure steam sterilization 20min, solid fermentation culture medium is made.
5. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, it is characterised in that: the step
Suddenly (3) miospore suspension inoculum concentration is the every disk solid fermentation culture medium of 13ml.
6. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, it is characterised in that: the step
Suddenly air dry oven temperature is 30-32 DEG C in (3), and drying time is not more than 48h, until to pinch rice hard not broken for hand, has been dried
At screwing lid and mark lot number according to 500g/ bottles of quantitative separatings in the big mouth plastic bottle of 2000mlPP.
7. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, it is characterised in that: the step
Suddenly PDSA medium component and content are potato 250g, white granulated sugar 50-60g, soy sauce 50mL, agar strip in (1) and step (2)
20-25g, distilled water 1000 mL, pH are natural.
8. a kind of method for manually cultivating the larva of a silkworm with batrytis based on muscardine according to claim 1, it is characterised in that: the step
Suddenly cultivated silkworm breed variety is bright moon × mountain valley with clumps of trees and bamboo pine in (5).
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| CN115586286A (en) * | 2022-12-13 | 2023-01-10 | 天地恒一制药股份有限公司 | Method for measuring content of ammonium oxalate in fried stiff silkworm |
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