CN111793594B - Preparation method of aspergillus conidium suspension - Google Patents
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Abstract
The invention relates to a preparation method of aspergillus conidium suspension, which comprises the following steps: inoculating the aspergillus strain preserved at low temperature to the inclined surface of the PDA test tube for activation, transferring to the inclined surface of the large PDA test tube, and culturing until the inclined surface is fully filled with conidium; adding the eluent into a test tube filled with conidium, and oscillating to separate the conidium from hypha; filtering the oscillated eluent rich in conidium, adding protease into the filtrate for treatment, and washing after the enzyme treatment is finished to obtain conidium precipitate; adding heavy suspension into the conidium precipitate for heavy suspension, and uniformly mixing through ultrasonic oscillation after heavy suspension to obtain the suspension with uniformly dispersed conidium. The aspergillus conidium suspension prepared by the method provided by the invention has uniform conidium dispersion and no aggregation, and is completely suitable for being used as a material for strain mutagenesis experiments and active substance bacteriostasis experiments.
Description
Technical Field
The invention relates to a preparation method of aspergillus conidium suspension, which is particularly suitable for being used as a material for strain mutagenesis or antibacterial material activity verification experiments.
Background
Aspergillus is a kind of mould widely distributed in nature, and its mycelium is very developed, has diaphragm and multiple branches, and is mainly propagated through conidium. Common aspergillus mainly include aspergillus niger, aspergillus flavus, aspergillus oryzae, aspergillus fumigatus and the like, and the aspergillus has important roles in industries such as brewing, food processing and the like. In the food industry, aspergillus has important roles, and part of strains are used for producing various food raw materials and auxiliary materials, such as citric acid, gluconic acid, kojic acid, haematochrome, amylase, cellulase and other enzymes. In industrial production, in order to fully exert the effect of aspergillus, strains are often required to be modified in a mutagenesis mode so as to improve the yield of beneficial products such as organic acid, enzyme and the like; however, aspergillus is a multicellular filamentous microorganism, hypha cannot be used as a mutagenesis starting material, and only a spore suspension which is uniformly dispersed can be used as a mutagenesis material so as to improve the mutagenesis success rate.
Aspergillus is also a major cause of spoilage of industrial and agricultural products and illness of human and animals. The aspergillus not only can cause the spoilage of industrial and agricultural products, but also can cause the toxins such as aflatoxin, fumagotoxin and the like produced by the aspergillus seriously threatens the health of human beings and causes great loss of animal breeding industry. In order to develop new mildew inhibiting substances, more and more natural or synthetic substances are subjected to mildew inhibiting activity measurement to evaluate the application potential in industrial and agricultural production. When the activity of natural or synthetic substances for inhibiting the mould is measured, the mould spores are required to be prepared into uniformly dispersed suspension, and then the suspension can be used for measuring indexes such as the inhibition rate, the minimum inhibitory concentration, the minimum bactericidal concentration and the like of the substances for inhibiting the spore germination.
The mould spores have higher hydrophobicity, are easy to aggregate and are not easy to be fully dispersed in water. Up to now, the preparation method of aspergillus conidium suspension has major defects: distilled water or Tween 80 water solution is added into a culture dish full of spores, and then spores are scraped by an inoculating loop, so that the spores are easy to dye due to the large opening of the culture dish, and more hyphae can be scraped into spore suspension; filtering spore suspension with absorbent cotton or 3-6 layers of degreasing gauze to remove mycelium, which is easy to cause a large amount of spores to be adsorbed on absorbent cotton or gauze, thereby reducing the spore yield; the spores are dispersed by a method of sucking by a pipette gun head or adding glass beads into spore liquid for oscillation, and the spores are not fully dispersed, and a large number of double spores, triple spores and even spore clusters often exist. The probability of obtaining false positive mutation by mutagenesis through the insufficiently dispersed spores is high, the mutagenesis efficiency is seriously influenced, and the method is also not suitable for the test of the spore germination inhibition rate of active substances.
Disclosure of Invention
The purpose of the invention is that: the spore suspension prepared by the method is uniformly dispersed and free from aggregation, and is suitable for strain mutagenesis experiments and antibacterial experiments of active substances.
The technical scheme of the invention is as follows: a method for preparing aspergillus conidium suspension, comprising the following steps:
(1) Activation of aspergillus strain: inoculating the aspergillus strain preserved at low temperature to the PDA test tube slope for activating;
(2) Cultivating the nutrition conidium: transferring to the inclined plane of a large PDA test tube after activation, and culturing until the inclined plane is fully filled with conidium;
(3) Eluting conidium: adding the eluent into a test tube filled with conidium, and oscillating to separate the conidium from hypha;
(4) And (3) filtering eluent: filtering the oscillating eluent rich in conidium;
(5) Enzyme treatment of filtrate: adding protease into the filtrate for treatment;
(6) Washing the conidium: washing after enzyme treatment to obtain conidium precipitate;
(7) Conidium resuspension: adding heavy suspension into conidium precipitate for resuspension, and carrying out ultrasonic vibration and uniform mixing;
(8) Counting and diluting: after ultrasonic vibration and mixing, counting and diluting to obtain suspension with uniformly dispersed conidium.
In the step (1), the activation of the aspergillus strain is as follows: taking an aspergillus strain inclined plane preserved at 4 ℃, digging a mycelium-carrying fungus block by an inoculating shovel, inoculating the fungus block to an inclined plane of a common PDA test tube, tightly attaching the mycelium-carrying surface to a culture medium, plugging a plug, and placing the culture medium in a 30 ℃ incubator for light-shielding culture for 3-5 days until conidium grows out.
In the step (2), the cultured conidia are: scraping the conidium cultured in the step (1) by adopting an inoculating loop, streaking and inoculating the conidium onto the inclined surface of a PDA test tube with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 3-5 days at the temperature of 30 ℃ in a dark way until the inclined surface of the test tube is fully filled with the conidium.
In the step (3), the eluted conidium is: and (3) adding 10-15mL of pre-sterilized eluent into the inclined surface of the aspergillus test tube which is fully distributed with the conidia and cultured in the step (2), tilting the test tube to enable the solid inclined surface which is fully distributed with the conidia to be upwards, placing the solid inclined surface on a vortex mixer, oscillating at 300-500 rpm for 20-30s, and fully eluting the conidia on the aspergillus hypha.
Wherein, the pre-sterilization eluent is: 50mmol/L Tris-HCl, pH7.5 of Tween 80 with mass concentration of 0.03-0.05%.
In the step (4), the eluent is filtered: filtering the conidium eluent obtained in the step (3) by using sterilized 10cm multiplied by 15cm four layers of superimposed microscope mirror wiping paper, filtering hypha fragments in the eluent, and collecting filtrate containing conidium into a 50mL sterile triangular flask.
In the step (5), the filtrate enzyme treatment is as follows: and (3) adding 20mg/mL protease K solution which is subjected to filtration sterilization into the filtered conidium filtrate until the final concentration of the protease is 0.2-0.5mg/mL, and carrying out enzyme treatment for 20-40min by using a constant-temperature water bath oscillator at 37 ℃ at 60-100rpm to remove hydrophobin on the surface of the conidium.
In the step (6), the washing conidium is: and (3) centrifuging the enzyme treated conidium filtrate in the step (5) for 6-10min at the temperature of 6000-8000rpm in a refrigerated centrifuge, adding sterilized 20mL of aqueous solution with the mass concentration of 0.03-0.05% of Tween 80 into the precipitate to wash the conidium, and centrifuging for 6-10min under the refrigerated centrifugation condition to obtain the impurity-free conidium precipitate.
In step (7), the conidium resuspension is: adding sterilized 20mL of sterile Tween 80 with the mass concentration of 0.03-0.05% and 20-30mmol/L of urea into the conidium precipitate obtained in the step (6), resuspending the spores, transferring the spore suspension into a sterile 50mL triangular flask, placing into an ultrasonic cleaner, and carrying out ultrasonic oscillation for 20-30s at the room temperature of 30KHz-50KHz to enable the spores to be fully dispersed.
In the step (8), the counting and diluting steps are as follows: observing and counting the conidium heavy suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water 6 -10 7 CFU/mL is put into a refrigerator with the temperature of 4 ℃ for standby.
Compared with the prior art, the invention has the advantages that:
1. mould spores are cultivated by using a 32X 200mm test tube slant, and enough spores can be cultivated due to the large slant area of the culture medium; the test tube port is smaller than the opening of the culture dish, so that the spore is not easy to be infected when the spore is eluted;
2. adding eluent into the test tube full of spores, and eluting the spores in a vortex mixer oscillation mode, so that the conidium can be eluted into the eluent more thoroughly, and the fungus silk can be prevented from being brought into the eluent;
3. the conidium suspension is filtered by using four layers of microscope paper, so that not only can a small amount of hyphae brought into the conidium suspension be removed, but also the problem of low yield of spores caused by a large amount of loss of spores due to filtration by using absorbent cotton or absorbent gauze can be avoided;
4. treating the conidium suspension with proteinase K to remove part of hydrophobin on the surface of conidium and avoid aggregation between spores caused by the existence of hydrophobin on the surface of conidium;
5. oscillating the conidium suspension at low frequency by using an ultrasonic cleaner to disperse the aggregated bispores, trispores or polyspora in the suspension;
6. tween 80 in the suspension can enable the conidium to be well dispersed in the suspension, meanwhile, urea in the suspension can prevent aggregation caused by hydrogen bond formation of spore wall polysaccharide molecules, and the conidium suspension is more stable under the double functions of Tween 80 and urea.
Drawings
FIG. 1 shows the dispersion of conidia in a suspension of conidia of Aspergillus flavus prepared by conventional methods;
FIG. 2 shows the dispersion of conidia in a conidia suspension of Aspergillus flavus prepared by the method of the invention.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples, which should not be construed as limiting the technical solution.
Example 1: preparation of Aspergillus niger conidium suspension
(1) Activation of aspergillus strain: taking an Aspergillus niger AS3.939 strain inclined plane preserved at 4 ℃, digging a mycelium-carrying fungus block by an inoculating shovel, inoculating the fungus block to an inclined plane of a common PDA test tube, tightly attaching the fungus-carrying surface to a culture medium, plugging a plug, and placing the culture medium in a 30 ℃ incubator for light-proof culture for 3 days until conidia grow;
(2) Cultivating the nutrition conidium: scraping the conidium of the aspergillus niger AS3.939 cultured in the step (1) by adopting an inoculating loop, streaking and inoculating the conidium on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, and culturing the conidium in a dark place at the temperature of 30 ℃ for 3 days after a plug is plugged until the test tube inclined plane is fully filled with the conidium;
(3) Eluting conidium: adding 10mL of pre-sterilization eluent into the inclined surface of the Aspergillus niger AS3.939 test tube which is fully distributed with conidia and is cultured in the step (2), tilting the test tube to enable the solid inclined surface which is fully distributed with conidia to be upwards, placing the solid inclined surface on a vortex mixer, and oscillating at 300rpm for 30s to fully elute the conidia on hyphae; wherein, the pre-sterilization eluent is: 50mmol/L Tris-HCl, pH7.5 of Tween 80 with mass concentration of 0.03%;
(4) And (3) filtering eluent: filtering the conidium eluent obtained in the step (3) by using sterilized 10cm multiplied by 15cm four layers of superimposed microscope mirror wiping paper, filtering hypha fragments in the eluent, and collecting filtrate containing conidium into a 50mL sterile triangular flask;
(5) Enzyme treatment of filtrate: adding 20mg/mL protease K solution subjected to filtration sterilization into the filtered conidium filtrate until the final concentration of the protease is 0.2mg/mL, and carrying out enzyme treatment for 40min by a constant-temperature water bath oscillator at 37 ℃ at 60rpm to remove hydrophobin on the surface of the conidium;
(6) Washing the conidium: centrifuging the enzyme-treated conidium filtrate in the step (5) for 10min at the temperature of 4 ℃ and the speed of 6000rpm in a refrigerated centrifuge, adding sterilized 20mL of 0.03% Tween 80 aqueous solution into the precipitate to wash the conidium, and centrifuging for 10min under the refrigerated centrifugation condition to obtain the conidium precipitate without impurities;
(7) Conidium resuspension: adding sterilized 20mL of sterile 0.03% Tween 80 and 20mmol/L urea heavy suspension to the conidium precipitate obtained in the step (6), transferring the spore suspension to a sterile 50mL triangular flask, and then placing the triangular flask into an ultrasonic cleaner, and performing ultrasonic oscillation for 30s at room temperature of 30KHz to enable the spores to be fully dispersed;
(8) Counting and diluting: observing and counting the conidium heavy suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water 6 -10 7 CFU/mL was stored in a refrigerator at 4deg.C for mutagenesis of Aspergillus niger AS3.939 strain to increase citric acid yield.
Example 2: preparation of Aspergillus oryzae conidium suspension
(1) Activation of aspergillus strain: taking an aspergillus oryzae AS3.863 strain inclined plane preserved at 4 ℃, digging a mycelium-carrying fungus block by an inoculating shovel, inoculating the fungus block to an inclined plane of a common PDA test tube, tightly attaching the fungus-carrying surface to a culture medium, plugging a plug, and placing the culture medium in a 30 ℃ incubator for light-proof culture for 4 days until conidia grow;
(2) Cultivating the nutrition conidium: scraping the conidium of the aspergillus oryzae AS3.863 cultured in the step (1) by adopting an inoculating loop, streaking and inoculating the conidium on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, and culturing for 4 days at the temperature of 30 ℃ in a dark place after a plug is plugged until the test tube inclined plane is fully filled with the conidium;
(3) Eluting conidium: adding 12.5mL of pre-sterilized eluent into the inclined surface of the test tube which is cultivated in the step (2) and is full of conidium, tilting the test tube to enable the solid inclined surface which is full of conidium to be upward, placing the test tube on a vortex mixer, and oscillating at 400rpm for 25s to fully elute the conidium on the mycelium; wherein, the pre-sterilization eluent is: 50mmol/L Tris-HCl, pH7.5 of Tween 80 with mass concentration of 0.04%;
(4) And (3) filtering eluent: filtering the conidium eluent obtained in the step (3) by using sterilized 10cm multiplied by 15cm four layers of superimposed microscope mirror wiping paper, filtering hypha fragments in the eluent, and collecting filtrate containing conidium into a 50mL sterile triangular flask;
(5) Enzyme treatment of filtrate: adding 20mg/mL protease K solution subjected to filtration sterilization into the filtered conidium filtrate until the final concentration of the protease is 0.35mg/mL, and performing enzyme treatment for 30min by using a constant-temperature water bath oscillator at 37 ℃ at 80rpm to remove hydrophobin on the surface of the conidium;
(6) Washing the conidium: centrifuging the enzyme-treated conidium filtrate in the step (5) for 8min at 4 ℃ and 7000rpm in a refrigerated centrifuge, adding sterilized 20mL of 0.04% Tween 80 aqueous solution into the precipitate to wash the conidium, and centrifuging for 8min under the refrigerated centrifugation condition to obtain the impurity-free conidium precipitate;
(7) Conidium resuspension: adding sterilized 20mL of sterile 0.04% Tween 80 and 25mmol/L urea heavy suspension to the conidium precipitate obtained in the step (6), transferring the spore suspension to a sterile 50mL triangular flask, and then placing the triangular flask into an ultrasonic cleaner, and performing ultrasonic oscillation at room temperature of 40KHz for 25s to enable the spores to be fully dispersed;
(8) Counting and diluting: observing and counting the conidium heavy suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water 6 -10 7 CFU/mL was stored in a refrigerator at 4deg.C for mutagenesis of Aspergillus oryzae strain AS3.863 to increase protease yield.
Example 3: preparation of Aspergillus flavus conidium suspension
(1) Activation of aspergillus strain: taking an aspergillus flavus AS3.3950 strain inclined plane preserved at 4 ℃, digging a mycelium-carrying fungus block by an inoculating shovel, inoculating the fungus block to an inclined plane of a common PDA test tube, tightly attaching the fungus-carrying surface to a culture medium, plugging a plug, and placing the culture medium in a 30 ℃ incubator for light-proof culture for 5 days until conidium grows out;
(2) Cultivating the nutrition conidium: scraping the conidium of the aspergillus flavus AS3.3950 cultured in the step (1) by adopting an inoculating loop, streaking and inoculating the conidium on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, and culturing for 5 days at the temperature of 30 ℃ in a dark place after a plug is plugged until the test tube inclined plane is fully filled with the conidium;
(3) Eluting conidium: adding 15mL of pre-sterilization eluent into the inclined surface of the test tube which is cultivated in the step (2) and is full of conidium, tilting the test tube to enable the solid inclined surface which is full of conidium to be upward, placing the test tube on a vortex mixer, oscillating at 500rpm for 20s, and fully eluting the conidium on the mycelium; wherein, the pre-sterilization eluent is: 50mmol/L Tris-HCl, pH7.5 of Tween 80 with mass concentration of 0.05%;
(4) And (3) filtering eluent: filtering the conidium eluent obtained in the step (3) by using sterilized 10cm multiplied by 15cm four layers of superimposed microscope mirror wiping paper, filtering hypha fragments in the eluent, and collecting filtrate containing conidium into a 50mL sterile triangular flask;
(5) Enzyme treatment of filtrate: adding 20mg/mL protease K solution subjected to filtration sterilization into the filtered conidium filtrate until the final concentration of the protease is 0.5mg/mL, and performing enzyme treatment for 20min by using a constant-temperature water bath oscillator at 37 ℃ at 100rpm to remove hydrophobin on the surface of the conidium;
(6) Washing the conidium: centrifuging the enzyme-treated conidium filtrate in the step (5) for 6min at the temperature of 4 ℃ and the rpm of 8000rpm in a refrigerated centrifuge, adding sterilized 20mL of 0.05% Tween 80 aqueous solution into the precipitate to wash the conidium, and centrifuging for 6min under the refrigerated centrifugation condition to obtain the impurity-free conidium precipitate;
(7) Conidium resuspension: adding sterilized 20mL of sterile 0.05% Tween 80 and 30mmol/L urea heavy suspension to the conidium precipitate obtained in the step (6), transferring the spore suspension to a sterile 50mL triangular flask, and then placing the triangular flask into an ultrasonic cleaner, and performing ultrasonic oscillation for 20s at room temperature and 50KHz to enable the spores to be fully dispersed;
(8) Counting and diluting: observing and counting the conidium heavy suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water 6 -10 7 CFU/mL was stored in a refrigerator at 4deg.C for use in inhibiting Aspergillus flavus AS3.3950 activity.
Claims (2)
1. A preparation method of aspergillus conidium suspension is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) Activation of aspergillus strain: inoculating the aspergillus strain preserved at low temperature to the PDA test tube slope for activating; the activation of the aspergillus strain is as follows: taking an aspergillus strain inclined plane preserved at 4 ℃, digging a mycelium-carrying fungus block by an inoculating shovel, inoculating the fungus block to an inclined plane of a common PDA test tube, tightly attaching the fungus-carrying surface to a culture medium, plugging a plug, and placing the culture medium in a 30 ℃ incubator for light-shielding culture for 3-5 days until conidium grows out;
(2) Cultivating the nutrition conidium: transferring to the inclined plane of a large PDA test tube after activation, and culturing until the inclined plane is fully filled with conidium; the culture conidium is: scraping the conidium cultured in the step (1) by adopting an inoculating loop, streaking and inoculating the conidium onto the inclined surface of a PDA test tube with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 3-5 days at the temperature of 30 ℃ in a dark way until the inclined surface of the test tube is fully filled with the conidium;
(3) Eluting conidium: adding the eluent into a test tube filled with conidium, and oscillating to separate the conidium from hypha; the eluted conidia are: adding 10-15mL of pre-sterilized eluent into the inclined surface of the aspergillus test tube which is fully distributed with conidia and cultured in the step (2), tilting the test tube to enable the solid inclined surface which is fully distributed with conidia to be upwards, placing the solid inclined surface on a vortex mixer, oscillating at 300-500 rpm for 20-30s, and fully eluting the conidia on the aspergillus hypha;
(4) And (3) filtering eluent: filtering the oscillating eluent rich in conidium; the eluent is filtered: filtering the conidium eluent obtained in the step (3) by using sterilized 10cm multiplied by 15cm four layers of superimposed microscope mirror wiping paper, filtering hypha fragments in the eluent, and collecting filtrate containing conidium into a 50mL sterile triangular flask;
(5) Enzyme treatment of filtrate: adding protease into the filtrate for treatment; the filtrate enzyme treatment is as follows: adding 20mg/mL protease K solution subjected to filtration sterilization into the filtered conidium filtrate in the step (4) until the final concentration of the enzyme is 0.2-0.5mg/mL, and performing enzyme treatment for 20-40min by using a constant-temperature water bath oscillator at 37 ℃ at 60-100rpm to remove hydrophobin on the surface of the conidium;
(6) Washing the conidium: washing after enzyme treatment to obtain conidium precipitate; the washing conidium is: centrifuging the enzyme-treated conidium filtrate in the step (5) for 6-10min at the temperature of 6000-8000rpm in a refrigerated centrifuge, adding sterilized 20mL of aqueous solution with the mass concentration of 0.03-0.05% of Tween 80 into the precipitate to wash the conidium, and centrifuging for 6-10min under the refrigerated centrifugation condition to obtain the conidium precipitate without impurities;
(7) Conidium resuspension: adding heavy suspension into conidium precipitate for resuspension, and carrying out ultrasonic vibration and uniform mixing; the conidium resuspension is as follows: adding sterilized 20mL of sterile Tween 80 with the mass concentration of 0.03-0.05% and 20-30mmol/L of urea into the conidium precipitate obtained in the step (6), resuspending the spores, transferring the spore suspension into a sterile 50mL triangular flask, and then placing the sterilized 50mL triangular flask into an ultrasonic cleaner, and carrying out ultrasonic oscillation for 20-30s at the room temperature of 30KHz-50KHz to enable the spores to be fully dispersed;
(8) Counting and diluting: after ultrasonic oscillation and mixing, counting and diluting to obtain suspension with uniformly dispersed conidia; the counting and dilution steps are as follows: observing and counting the conidium heavy suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water 6 -10 7 CFU/mL is put into a refrigerator with the temperature of 4 ℃ for standby.
2. The method for preparing aspergillus conidium suspension according to claim 1, wherein the method comprises the following steps: in the step (3), the pre-sterilized eluent is: 50mmol/L Tris-HCl, pH7.5 of Tween 80 with mass concentration of 0.03-0.05%.
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