CN110352920B - Method for artificially culturing white muscardine silkworms based on beauveria bassiana - Google Patents
Method for artificially culturing white muscardine silkworms based on beauveria bassiana Download PDFInfo
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- CN110352920B CN110352920B CN201910812863.1A CN201910812863A CN110352920B CN 110352920 B CN110352920 B CN 110352920B CN 201910812863 A CN201910812863 A CN 201910812863A CN 110352920 B CN110352920 B CN 110352920B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a white muscardine silkworm artificial culture method based on white muscardine fungi, which is characterized by comprising the following steps: artificially cultured white muscardine silkworm is cylindrical, is bent in a plurality of crimples, has a single silkworm body with the length of 4.30 +/-0.35 cm and the diameter of 0.5 +/-0.1 cm, weighs 7-8 kilograms of silkworms, has milky powdery hypha and conidia on the surface, is hard and crisp, has flat and smooth end surface and bright amber color, obviously sees a silk gland ring, has the ammonium oxalate content of 6.00-6.20 percent as measured by a high performance liquid chromatography, and is prepared by a spore suspension inoculation liquid preparation, a white muscardine fungus culture, a dip-dyeing spore liquid preparation and a white muscardine fungus spore preparation for artificially producing the white muscardine silkworm; the preparation process is simple, has low requirements on production environment and production equipment, greatly reduces the production cost, reduces the pollution problem of mixed bacteria in production, does not need to separate spores from culture materials after drying, directly subpackages finished products, almost completely recycles the spores, is very suitable for small enterprises and production units to rapidly produce the beauveria bassiana, and improves economic benefit and social benefit.
Description
Technical Field
The invention relates to a method for culturing white muscardine silkworms, in particular to a method for artificially culturing white muscardine silkworms based on beauveria bassiana.
Background
Bombyx Batryticatus (Bombyx Batryticatus)Bombyx batryticatus) The silkworm of Bombycidae is naturally infected (or artificially inoculated) Beauveria bassiana (Beauveria bassiana) by 4-5 instar larvaBeauveria) The dead dry whole insect and Bombyx Batryticatus as transmissionTraditional Chinese medicines, which are used for treating various diseases, have several thousand years of medicinal history in China and are also widely used in Japan and Korea. Modern pharmacological researches find that the white muscardine silkworm has various medicinal active ingredients and can inhibit bacteria, resist convulsion, resist coagulation, resist thrombosis, promote fibrinolysis, inhibit tumor, hypnotize, reduce blood fat, reduce blood sugar and the like. Modern medical practice research shows that the silkworm larva has a treatment effect on parotitis, epilepsy and encephalitis sequelae, and has different curative effects on esophagus cancer, lung cancer, gastric cancer, bladder cancer and the like. With the development of modern science and technology, scholars at home and abroad continuously and deeply research on the white muscardine silkworm, develop medicinal values in a plurality of new fields, and have clinical application reports on diseases such as psoriasis, chronic nasosinusitis-induced headache, chloasma, acne, cough, asthma, facial paralysis, viral infection, dysfunctional uterine bleeding, cyst, diabetes, peripheral neuropathy, infantile twitch and the like. According to statistics, 175 Chinese patent prescriptions contain white muscardine silkworm. In recent years, with the progress of pharmacological research on the white muscardine silkworm and the good curative effect of the white muscardine silkworm in clinical application, the medicinal value of the white muscardine silkworm is receiving more and more attention, and the market supply is short. Especially, in recent years, the research shows that the batryticated silkworm has the function of stimulating the adrenal cortex and the health care function, thereby greatly widening the application range of the batryticated silkworm, and causing the price to be continuously increased due to the sharp increase of the using amount. In China, besides a large amount of white muscardine silkworms are purchased from each silkworm area every year, the white muscardine silkworms are produced industrially and professionally, but the needs of medical departments are difficult to meet. At present, several domestic enterprises artificially infect and produce the white muscardine silkworm, and the problems in several aspects are mainly solved: the stability of strains is not high, spores are directly eluted from the naturally infected or artificially infected white muscardine silkworm for re-inoculation, the strains are not purified and optimized, the pathogenicity is influenced by a plurality of factors such as environment, climate and the like, the infected silkworm bodies are not concentrated from inoculation to death, the infection rate of the silkworm is low, the yield of the silkworm is low, the hardening time is irregular, the quality is unstable, and the yield is low; secondly, due to the restriction of the preservation method of the beauveria bassiana spore powder, the number of live spores and the activity of the spores are obviously reduced along with the increase of time, so that the conditions of low incidence rate, long incidence period and the like of the silkworm after inoculation often exist in the actual production, and the silkworm is caused to be on the mature silkwormThe silkworm can not be killed in the same period before cocooning, which causes great economic loss to the production of the silkworm
Therefore, in order to form the production technology integration of high-quality white muscardine silkworms and promote the sustainable development of the white muscardine silkworm industry, the invention discloses a method for artificially culturing white muscardine silkworms based on white muscardine fungi, the white muscardine silkworms prepared by the invention are cylindrical and are bent in a plurality of crimples, the length of a single silkworm body is 4.30 +/-0.35 cm, the diameter is 0.5 +/-0.1 cm, the weight of a ten thousand silkworms is 7-8 kg, the surface is provided with milky powdery hypha and conidia, the quality is hard and crisp, the end surface is flat and smooth, the color is bright amber, the silk gland ring is obviously seen, and the content of ammonium oxalate is 6.00-6.20 percent as measured by a high performance liquid chromatography.
Disclosure of Invention
Aiming at the problems, the invention provides a method for artificially culturing white muscardine silkworms based on beauveria bassiana, which comprises the following specific scheme:
a method for artificially culturing white muscardine silkworms based on beauveria bassiana is characterized by comprising the following steps: the artificially cultured white muscardine silkworm is cylindrical, is bent in a plurality of crimples, has a single silkworm body with the length of 4.30 +/-0.35 cm and the diameter of 0.5 +/-0.1 cm, weighs 7-8 kg of silkworms, has milky powdery hypha and conidia on the surface, is hard and crisp, has flat and smooth end surface and bright amber color, obviously shows a silk gland ring, and has the ammonium oxalate content of 6.00-6.20 percent as measured by a high performance liquid chromatography, and the culture method comprises the following steps:
(1) purifying beauveria bassiana:
producing white muscardine silkworm by using white muscardine silkworm spore powder on the market, and carrying out multiple flat plate streaking culture on the produced white muscardine silkworm by using a PDSA culture medium to obtain a white muscardine fungus pure strain, wherein the white muscardine fungus pure strain is beauveria bassiana;
(2) preparing a spore suspension inoculation liquid:
inoculating the pure strain obtained in the step (1) to a slant made of a PDSA culture medium in an eggplant bottle, and culturing at 26 +/-1 ℃ for 14 days until spores are fully distributed on the slant, wherein the total spore amount is 1.6 multiplied by 1010-1.8×1010Spore/bottle, and prepared into 5 × 10 concentration7-6×107Inoculating a spore suspension solution of/ml for later use;
(3) solid state fermentation beauveria bassiana spore
Preparing a solid fermentation culture medium, wherein the components of the solid fermentation culture medium comprise rice, water = 10: 9 (w: w), potassium nitrate 0.2% and soy sauce 2ml/100g rice, inoculating the spore suspension inoculation liquid in the step (2) into the solid fermentation culture medium according to the inoculation amount of 10% (v: w), culturing for 12 days at 26 +/-1 ℃, taking out, placing in an electric heating air blast drying oven for drying to obtain beauveria bassiana spores, bottling, sealing, and storing at 0-8 ℃;
(4) preparing a dip-dyeing spore solution:
taking the beauveria bassiana spores bottled in the step (3), immersing the spores in cold boiled water, fully eluting the conidia attached to the rice by oscillating the bottles, and finally eluting the spores in each bottle with the water consumption of 6000-7Filling each spore/ml into a spray bottle for later use;
(5) artificially culturing white muscardine silkworms:
s1: spraying the rice grains eluted with the spores in the step (4) on a silkworm bed, selecting silkworms which are all subjected to four-sleep waking and sleep eating the first mulberry leaves for 3 hours, inoculating the spore suspension prepared in the step (4) for two times, wherein the spore suspension is sprayed on the silkworm body for the first time to ensure that the silkworm body is wet, then feeding the mulberry leaves, and the spore suspension is sprayed on the mulberry leaves for the second time to allow the silkworms to freely eat;
s2: ensuring that the door and the window of the silkworm shed are closed within 24 hours after inoculation, keeping the humidity in the shed to be 90% by spraying clear water, ensuring ventilation and air exchange in the shed to be not less than 2 hours every day after 24 hours until disease attack, avoiding using lime to disinfect a silkworm bed after inoculation, and ensuring that the rest feeding methods are the same as those of conventional cocoon silk silkworms;
s3: after 5 days, morbid conditions are concentrated, mulberry feeding is stopped, concentrated death is carried out for about 6 days, the death rate is more than 95%, after silkworm bodies are completely dead, the closing of the doors and windows of the silkworm shed is ensured, the humidity in the shed is ensured to be 90% by spraying clear water, the temperature is 26-28 ℃, silkworm death occurs after 8 days of dead death, 11 days after inoculation, the silkworm bodies grow to be full of spores, the white death stage is finished, semi-finished products of the white muscardine silkworm are collected, and the white muscardine silkworm is placed in the sun and dried to obtain finished products of the white muscardine silkworm.
Further, after the white muscardine silkworm selected in the step (1) is sprayed and disinfected by 75% alcohol for 3-5s, the white muscardine silkworm is washed by sterile water for three times, then disinfected by 0.1% mercuric chloride water for 3-5min, rinsed by sterile water for three times again, cut off the white muscardine silkworm by using sterile tweezers, and smear a PDSA culture medium plate on the cross section or scrape surface hypha and spores by using a disposable inoculating loop to streak on the PDSA culture medium plate, and culture is carried out at the constant temperature of 26 ℃ for more than 5d, and colonies of suspected white muscardine silkworm are picked on the plate and streaked and purified by one or more plates until pure strains are obtained.
Further, in the step (2), spores are eluted by 300ml of 0.1% Tween-80, and poured into a sterile blue-covered glass bottle with glass beads, sufficiently and uniformly shaken, and spore concentration is calculated by a blood counting chamber to prepare a spore suspension inoculation liquid.
Further, the solid fermentation medium in the step (3) is prepared by weighing 130g of rice and adding 117ml of liquid by adopting a stainless steel plate with the specification of 17 × 23cm and the bottom area of 14 × 20cm, wherein the liquid comprises 10% KNO3Soaking 2.6ml of solution, 2.6ml of soy sauce and the balance of water for 2-3h, covering with a 35cm × 25cm PE fresh-keeping bag, and sterilizing with high-pressure steam at 121 deg.C for 20min to obtain solid fermentation culture medium.
Further, the inoculation amount of the spore suspension in the step (3) is 13ml per dish of the solid fermentation medium.
Further, in the step (3), the temperature of the air drying oven is 30-32 ℃, the drying time is not more than 48 hours until the rice is hard and not broken when being kneaded by hands, the rice is dried, the rice is quantitatively packaged in 2000ml PP big-mouth plastic bottles according to 500 g/bottle, and the caps are screwed and the batch numbers are marked.
Further, the components and contents of the PDSA culture medium in the step (1) and the step (2) are 250g of potatoes, 50-60g of white granulated sugar, 50mL of soy sauce, 20-25g of agar strips and 1000 mL of distilled water, and the pH is natural.
Further, the domestic silkworm variety in the step (5) is Haoyue multiplied by Pinna.
Compared with the prior art, the invention has the following beneficial effects:
the spore washed after the slant enlarged culture of the eggplant bottle is used as the beauveria bassiana inoculation liquid, the beauveria bassiana inoculation liquid is inoculated into a solid fermentation medium according to the inoculation amount of 10 percent for fermentation culture, the adopted fermentation medium has good moisture retention and air permeability, rich N source and C source nutrition can be provided for the proliferation of the beauveria bassiana, and the raw materials are economical and easy to obtain; the culture method provided by the invention is simple to operate, clear in flow, low in requirements on production environment and production equipment, low in production cost, free of special humidification on the culture environment in production, free of material mixing in the culture process, capable of reducing the pollution problem of infectious microbes in production, free of separation of spores from culture materials after drying, capable of directly subpackaging finished products, capable of almost completely recycling spores, very suitable for small enterprises and production units to rapidly produce the beauveria bassiana, simple and convenient to elute the spores, high in spore utilization rate, suitable for large-scale popularization, and capable of greatly improving economic benefits and social benefits.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the technical descriptions of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a photograph of a colony of a PDSA plate.
FIG. 2 is a diagram of the slant of the eggplant bottle.
FIG. 3 is a schematic diagram of a stainless steel plate for a solid fermentation medium.
FIG. 4 is a schematic diagram of a process of solid fermentation of Beauveria bassiana.
FIG. 5 is a diagram of a spore entity of a fermentation product of beauveria bassiana.
FIG. 6 is a schematic diagram of a solid fermentation product spore finished product bottle.
FIG. 7 is a drawing of a semi-finished product of Bombyx Batryticatus.
FIG. 8 is a schematic illustration of a Bombyx Batryticatus product.
FIG. 9 is a schematic illustration of a silkworm rearing shed for rearing of Bombyx Batryticatus.
FIG. 10 is a graph showing the standard curve of ammonium oxalate.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without any inventive step, are within the scope of the present invention.
Example (b): the invention provides a high-quality silkworm larva for improving economic and social benefits, and the produced silkworm larva meets the following characteristics: the prepared white muscardine silkworm is cylindrical, is bent in a plurality of crimples, has a single silkworm body with the length of 4.30 +/-0.35 cm and the diameter of 0.5 +/-0.1 cm, weighs 7-8 kg of silkworms, has milky powdery hyphae and conidia on the surface, is hard and crisp, has flat and smooth end surface and bright amber color, obviously shows a silk gland ring, and has the ammonium oxalate content of 6.00-6.20 percent as measured by a high performance liquid chromatography.
Firstly, the method comprises the following steps: and (3) purifying strains:
preparing white muscardine silkworm by using commercial white muscardine fungus spore powder, taking a semi-finished product of the white muscardine silkworm which is not dried after white fermentation is finished, spraying and disinfecting for 3-5s by using 75% alcohol, washing for three times by using sterile water, disinfecting for 3-5min by using 0.1% mercuric chloride water, rinsing for three times by using the sterile water again, cutting off the white muscardine silkworm by using sterile tweezers, taking a cross section to be smeared on a PDSA culture medium flat plate or taking surface hypha and spores by using a disposable inoculating loop to be scribed on the PDSA culture medium flat plate, inverting the flat plate, culturing for more than 5 days at the constant temperature of 26 ℃, picking suspected white muscardine fungus colonies on the flat plate, and performing scribing and purifying for one time or more times on the PDSA flat plate until pure strains are obtained, wherein the strains are white muscardine fungi, and the colonies are shown in figure 1.
Secondly, preparing a spore suspension inoculation liquid:
inoculating the purified strain into PDSA culture medium, culturing at 26 + -1 deg.C for 14 days, and collecting the spores on slant surface of each bottle as shown in FIG. 2, and measuring the total amount of spores on slant surface of each bottle to be 1.6 × 1010-1.8×1010A bottle; pouring 300ml of 0.1% Tween-80 into eggplant bottle, scraping the surface with inoculating shovel or glass rod, washing off all spores as much as possible, pouring into sterile blue-cover glass bottle with glass beads, and shaking thoroughly to obtain Tween-80 and glass beadsIn order to sufficiently disperse spores, the spore concentration was measured on a hemocytometer at a concentration of 5X 107-6×107The spores/ml spore suspension is inoculated.
The components of the PDSA culture medium in the first step and the second step are 250g of potatoes, 50-60g of white granulated sugar, 50mL of soy sauce, 20-25g of agar strips and 1000 mL of distilled water, and the pH value is naturally prepared.
Thirdly, culturing beauveria bassiana:
firstly, preparing a solid fermentation medium containing rice and water = 10: 9 (w: w), potassium nitrate 0.2%, and soy sauce 2ml per 100g of rice, specifically adopting a stainless steel dish with a specification of 17 × 23cm and a bottom area of 14 × 20cm as shown in figure 3, weighing 130g of rice in the dish, adding 2.6ml of 10% KNO3Solution, 2.6ml soy sauce and water, wherein KNO3The total amount of the solution, soy sauce and water is 117ml, soaking for 2-3h, covering with a 35cm × 25cm PE fresh-keeping bag, and sterilizing with high pressure steam at 121 deg.C for 20 min.
And (3) after cooling the solid fermentation culture medium, inoculating in a purification workbench, specifically, inoculating the spore suspension inoculation liquid eluted in the step two into the solid fermentation culture medium according to the inoculation amount of 10% (v: w), and inoculating 13ml of spore suspension in each disc, so that the ratio of the final water addition amount to the rice is 1: 1 (w: w), uniformly stirring and evenly spreading spores and rice, sealing the middle position of a fresh-keeping bag opening covered with a plate by a tape to a half to prevent microorganisms from falling into the air in the culture process, putting the inoculated fermentation plate into an illumination incubator for culture, and setting conditions as 26 ℃ and b: h = 12: 12, natural humidity and 12d of culture time; the humidity of spores of the beauveria bassiana produced by solid state fermentation is normally more than 80%, the whole stainless steel plate is sleeved with the freshness protection package, and the inner wall of the freshness protection package on the surface of the fermentation medium is always kept with uniform and fine water drops as shown in figure 4, so that the humidity requirement is ensured, the requirements on equipment input and setting conditions are reduced, cold boiled water can be filled in a household spray can every day, and the inner wall of the culture box is sprayed for 2 times to improve the humidity in the box body; and (3) dispersing rice particles of the culture after fermentation, fully spreading a thick layer of milky conidia as shown in figure 5, taking out a stainless steel fermentation disc, tearing off a freshness protection package, putting the freshness protection package into an electric heating air blowing drying box, covering a layer of sterilized newspaper on the arranged disc, drying for no more than 48 hours at 30-32 ℃, kneading rice by hand to be hard and not broken, easily dropping spore powder, drying, quantitatively subpackaging 500g of each bottle into 2000ml of PP (polypropylene) large-mouth plastic bottles, screwing covers, marking batch numbers, and storing at 0-8 ℃ to obtain finished spore products, wherein the bottled finished products are shown in figure 6.
Weighing 1 g of the fermented spore product, adding 9ml of 0.1% Tween-80, fully oscillating, eluting and scattering the spore, counting the spore concentration by a blood counting chamber to obtain the fermented spore product with concentration of (6.0-8.0) x 108Spores per gram.
Fourthly, preparing a dip-dyeing spore liquid:
taking 500g of bottled products obtained in the third step, directly pouring a proper amount of cold boiled water into the bottle for immersion, standing for 5min, oscillating the bottle to elute spores attached to the rice grains as much as possible, and pouring out spore suspension; in order to fully clean the spores, the spore elution step is repeated twice, the spore suspension eluted three times is diluted to 6000-8000ml with cold boiled water, and the concentration of the spore solution is about 5 multiplied by 107Mixing spores per ml, and filling into a spray bottle for later use. In the step, the rice grains serve as the glass beads, so that elution and scattering of spores are facilitated, and the spores are eluted without additionally adding the glass beads and without adding emulsifiers such as Tween and the like.
Fifthly, artificially culturing the white muscardine silkworms:
silkworm variety Haoyue × Pinus was sprayed with spore suspension about 3 hours after all four sleeps to wake up and feed the first mulberry leaf. The specific steps of spraying spore suspension are as follows: inoculating spore suspension in a sprayer twice, uniformly spraying half of the spore suspension on silkworm bodies for the first time, if the silkworm bodies are wet, ensuring that no residual leaves exist on a silkworm bed before spraying, and immediately feeding mulberry leaves after spraying; spraying the remaining half spore suspension on the folium Mori fed in the previous time for the second time to allow silkworm to freely take; and scattering the rest rice grains on a silkworm bed, keeping a silkworm shed door window closed after inoculation for 24 hours, spraying clear water to keep the humidity in the shed at about 90%, ensuring ventilation and air exchange in the shed not to be less than two hours every day after 24 hours until disease is encountered, not using lime to disinfect the silkworm bed after inoculation, performing the same feeding method as that of conventional cocoon silkworms, after the silkworms are completely dead, entering a white silkworm stage, closing the silkworm shed door window at the moment, spraying clear water to keep the humidity at about 90%, controlling the temperature to be between 26 and 28 ℃, finishing the white silkworm stage after spores are fully grown on silkworm bodies, collecting semi-finished white silkworm, and drying in the sun to obtain the finished white silkworm, wherein specific examples can be shown in figures 7-9.
Wherein, white muscardine silkworm is artificially cultured, and the death condition of silkworm is inoculated by different spore concentrations, as shown in table 1:
| inoculation concentration | 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% |
| Clean water | 0% | 0% | 0% | 0% | 0% | 0% | 0% |
TABLE 1 Bombyx mori mortality by inoculation of Beauveria bassiana spores at different concentrations
As can be seen from Table 1: the production of the white muscardine silkworm is greatly influenced by inoculating spores with different concentrations, and the inoculation concentration is less than 5 multiplied by 107When the number of spores is one (mL), the pathogenicity of the spores of the strains to the silkworms is not ideal, a large number of silkworms cannot be attacked and died in time before mounting on the silkworm, the yield of the stiff silkworms is low, and large economic loss is caused to the production of the stiff silkworms; when the inoculation concentration is 5X 107When each spore is contained in the silkworm, the pathogenicity of the spores to the silkworms reaches 96% in 6 days, the diseases are concentrated, and the death rate is high; but when the inoculation concentration reaches 1X 108When the spore is per mL, the incidence rate of the silkworm reaches 63-85% at 4-5 days, the silkworm larva does not fully develop and is died prematurely, so that the finished product of the silkworm is poor in quality, and the economic benefit is greatly reduced, therefore, the concentration of the spore selected and inoculated by the white silkworm artificially produced in the base is higher than 1 x 107spore/mL concentration at 5X 107Preferably about one spore/mL.
Sixthly, measuring the content of ammonium oxalate in the white muscardine silkworm:
the two samples are white muscardine silkworm products produced by cooperative society of 10 months in 2018 and 6 months in 2019 respectively, and the specific method comprises the following steps:
(1) sample treatment and sample liquid preparation: taking and marking two dry samples of white muscardine silkworm, crushing the dry samples, sieving the samples by a 30-mesh sieve, putting the samples into an electric heating constant temperature blast drying oven for drying to remove water, weighing 0.5000 g of each sample, putting the samples into a small beaker, adding 50mL of double distilled water, treating the samples for 10 min at 30 ℃ by using an SB-5200DTD ultrasonic cleaner (settings: 99% power, 40 KHz and 300W), then carrying out vacuum filtration on the cleaning solution by using a suction filtration device, filtering the solution by using a 0.45 mu m filter membrane, transferring the filtrate into a glass wide-mouth bottle, removing 10 mL of sample solution from the wide-mouth bottle, fixing the volume in the 50mL bottle, sucking 200 mu L of sample solution by using a micro-pipetting gun, and adding the sample solution into a 1.5 mL EP tube for standby.
(2) Determination of ammonium oxalate content
Chromatographic conditions: a high performance liquid chromatography ultraviolet detector (HPLC-VWD) with a maximum absorption wavelength of 204 nm; column Diamonsil 5 μm C18 (250 × 4.6 mm, 5 μm) dima Diamonsil C18 diamond generation column, 4.6 mm × 250 mm × 5 u; mobile phase: 0.5% NH4H2PO4Aqueous solution (A)// methanol (B) (90/10, V/V), flow rate 0.4 mL/min, sample size 20. mu.L, total sample injection time 10 min.
Cleaning: the column was washed with 10% methanol solution for 60 min.
Drawing a standard curve: placing the EP tube filled with the ammonium oxalate standard solution with different concentrations into a high-speed centrifuge for centrifugation before column loading, reserving for sampling, sucking 20 mu L of the standard solution by using a sample injector for sample loading, measuring peak areas under different concentrations, and drawing a standard curve with the abscissa as the concentration of the ammonium oxalate standard substance and the ordinate as the peak area.
And (4) measuring a sample: and placing the EP pipe filled with different samples into a high-speed centrifuge for centrifugation before the upper column, and reserving for sampling. And (3) sucking 20 mu L of sample liquid by using a sample injector for sample loading, measuring the peak area of the sample liquid, calculating the concentration of ammonium oxalate in the sample liquid by using a standard curve, and finally calculating the content of ammonium oxalate in the white muscardine silkworm sample.
(3) Results: a standard curve is drawn according to the peak areas of the ammonium oxalate standard solution with different concentrations under the maximum absorption wavelength of 204nm, and is shown in FIG. 10, wherein the formula of the standard curve is as follows: y =0.5725x +3.5182 (R)2= 0.9983); the peak area of the sample liquid is measured, and the content of ammonium oxalate in the white muscardine silkworm sample is finally calculated and shown in the table 2:
| sample (I) | Peak area (204 nm) | Ammonium oxalate (%) |
| 2018-10 | 72.2188 | 5.99% |
| 2019-06 | 74.5085 | 6.20% |
TABLE 2 Peak area and ammonium oxalate content of two batches of Bombyx Batryticatus products
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (7)
1. A method for artificially culturing white muscardine silkworms based on beauveria bassiana is characterized in that the artificially cultured white muscardine silkworms are cylindrical and are bent in a plurality of crimps, a single body is 4.30 +/-0.35 cm long, the diameter is 0.5 +/-0.1 cm, the weight of each silkworm is 7-8 kg, the surface of each silkworm is milky powdery hypha and conidiospore, the silkworm is hard and crisp, the end surface is flat and smooth, the end surface is bright amber, a silk gland ring is obviously seen, the content of ammonium oxalate is 6.00-6.20% as measured by high performance liquid chromatography, and the artificial culturing method comprises the following steps:
(1) purifying beauveria bassiana:
utilizing commercially available white muscardine silkworms to produce white muscardine silkworms by using white muscardine fungus spore powder, and taking the produced white muscardine silkworms to smear a PDSA culture medium flat plate or streaking and culturing the white muscardine silkworms to obtain a white muscardine fungus pure strain, wherein the white muscardine fungus pure strain is beauveria bassiana;
(2) preparing a spore suspension inoculation liquid:
inoculating the pure strain obtained in the step (1) to a slant made of a PDSA culture medium in an eggplant bottle, and culturing at 26 +/-1 ℃ for 14 days until spores are fully distributed on the slant, wherein the total spore amount is 1.6 multiplied by 1010-1.8×1010Spore/bottle, and prepared into 5 × 10 concentration7-6×107Inoculating a seed solution into a spore suspension per mL for later use, wherein the components and contents of the PDSA culture medium in the step (1) and the step (2) are 250g of potatoes, 50-60g of white granulated sugar, 50mL of soy sauce, 20-25g of agar strips, 1000 mL of distilled water and natural pH;
(3) solid state fermentation of beauveria bassiana spores:
preparing solid fermentation culture medium, weighing 130g rice according to rice: water = 10: 9 (w: w) and soy sauce 2ml/100g rice, adding 117ml liquid, wherein the liquid comprises 10% KNO3Soaking 2.6ml of solution, 2.6ml of soy sauce and the balance of water for 2-3h, covering a 35cm multiplied by 25cm PE fresh-keeping bag, sterilizing by high-pressure steam at 121 ℃ for 20min to prepare a solid fermentation culture medium, inoculating the spore suspension inoculation liquid in the step (2) into the solid fermentation culture medium according to the inoculation amount of 10% (v: w), culturing at 26 +/-1 ℃ for 12d, taking out, placing in an electrothermal blowing drying oven for drying to obtain beauveria spores, bottling, sealing and storing at 0-8 ℃;
(4) preparing a dip-dyeing spore solution:
taking the beauveria bassiana spores bottled in the step (3), and immersing the beauveria bassiana spores in cold boiled waterThe conidium attached to the rice is fully eluted by oscillating the bottles, the water consumption for finally eluting the conidium in each bottle is 6000-7Filling each spore/ml into a spray bottle for later use;
(5) artificially culturing white muscardine silkworms:
s1: spraying the rice grains eluted with the spores in the step (4) on a silkworm bed, selecting all silkworms which are subjected to four-sleep waking and sleep eating the first mulberry leaves for 3 hours, inoculating the spore liquid prepared in the step (4) for two times, wherein the spore liquid is sprayed on the bodies of the silkworms for the first time to ensure that the bodies are wet, then feeding the mulberry leaves, and the spore liquid is sprayed on the mulberry leaves for the second time to allow the silkworms to freely take the food;
s2: ensuring that the door and the window of the silkworm shed are closed within 24 hours after inoculation, keeping the humidity in the silkworm shed to be 90% by spraying clear water, ensuring ventilation in the silkworm shed to be not less than 2 hours every day after 24 hours until disease attack, avoiding using lime to disinfect a silkworm bed after inoculation, and ensuring that other feeding methods are the same as those of conventional cocoon silk silkworms;
s3: after 5 days, morbid conditions are concentrated, mulberry feeding is stopped, concentrated death is carried out for about 6 days, the death rate is more than 95%, after all silkworm bodies are dead, the closing of doors and windows of a silkworm shed is ensured, the humidity in the silkworm shed is ensured to be 90% by spraying clear water, the temperature is 26-28 ℃, the silkworm which is dead after 8 days is white, 11 days after inoculation, spores are grown to be full of the silkworm bodies, the white-off stage is finished, semi-finished products of the white muscardine silkworm are collected, and the semi-finished products of the white muscardine silkworm are placed in the sun and dried to obtain finished products of the white muscardine silkworm.
2. The method for artificially culturing muscardine silkworm based on muscardine fungi according to claim 1, wherein the culture medium comprises: selecting the white muscardine silkworm produced by using the white muscardine silkworm sold in the step (1) and using white muscardine fungus spore powder, spraying and disinfecting the white muscardine silkworm with 75% alcohol for 3-5s, washing the white muscardine silkworm with sterile water for three times, disinfecting the white muscardine silkworm with 0.1% mercuric chloride water for 3-5min, rinsing the white muscardine silkworm with sterile water for three times, cutting off the white muscardine silkworm with sterile tweezers, smearing a PDSA culture medium plate on a cross section of the white muscardine silkworm, scraping surface hypha and spores with a disposable inoculating loop on the PDSA culture medium plate, culturing the white muscardine silkworm at the constant temperature of 26 ℃ for more than 5d, picking suspected white muscardine fungus colonies on the plate, and carrying out streak purification on the plate for one time or more times until a pure strain is obtained.
3. The method for artificially culturing muscardine silkworm based on beauveria bassiana according to claim 1, wherein the spore suspension is prepared in the step (2), the spores are eluted by using 300ml of 0.1% tween-80, and poured into a sterile blue-top glass bottle with glass beads, and the solution is sufficiently shaken to be uniform, and the concentration of the spores is measured by a hemocytometer.
4. The method for artificially culturing muscardine silkworm based on muscardine fungi according to claim 1, wherein the culture medium comprises: the solid fermentation culture medium in the step (3) adopts a stainless steel disc.
5. The method for artificially culturing muscardine silkworm based on muscardine fungi according to claim 1, wherein the culture medium comprises: and (3) inoculating the spore suspension in the step (3) with 13ml of solid fermentation culture medium per dish.
6. The method for artificially culturing muscardine silkworm based on muscardine fungi according to claim 1, wherein the culture medium comprises: and (3) in the step (3), the temperature of the air drying oven is 30-32 ℃, the drying time is not more than 48 hours until the rice is hard and not broken when being kneaded by hands, the rice is dried, the rice is quantitatively packaged in 2000ml PP big-mouth plastic bottles according to 500 g/bottle, the caps are screwed, and batch numbers are marked.
7. The method for artificially culturing muscardine silkworm based on muscardine fungi according to claim 1, wherein the culture medium comprises: and (5) in the step (5), the variety of the silkworm is Haoyue multiplied by pinus.
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