CN111793594A - 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 aspergillus strains preserved at low temperature to a PDA test tube inclined plane for activation, transferring the aspergillus strains to a large PDA test tube inclined plane, and culturing until the inclined plane is full of conidia; adding the eluent into a test tube full of conidia, and oscillating to separate the conidia from hyphae; filtering the oscillated eluent rich in conidia, adding protease into the filtrate for treatment, and washing after the enzyme treatment to obtain conidia precipitate; adding a resuspension solution into the conidium sediment for resuspension, and uniformly mixing the suspension solution after resuspension by ultrasonic oscillation to obtain the suspension solution with uniformly dispersed conidia. The aspergillus conidium suspension prepared by the method provided by the invention has the advantages of uniform dispersion of conidia, no aggregation and complete suitability 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 substance activity verification experiments.
Background
Aspergillus is a kind of mould widely distributed in nature, and its mycelium is very developed, has septum and many branches, and is mainly propagated by conidium. The common aspergillus is mainly aspergillus niger, aspergillus flavus, aspergillus oryzae, aspergillus fumigatus and the like, and the aspergillus has important functions in the industries of brewing, food processing and the like. In the food industry, aspergillus has the effect of playing a role, and part of strains are used for producing various food raw 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 function 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, hyphae cannot be used as a mutagenesis starting material, and only a uniformly dispersed spore suspension can be used as a mutagenesis material, so that the mutagenesis success rate is improved.
Aspergillus is also a significant cause of industrial and agricultural product spoilage and human and animal diseases. Besides the spoilage of industrial and agricultural products caused by aspergillus, the toxins such as aflatoxin, fumonisin and the like generated by the aspergillus seriously threaten the health of human beings and cause great loss of animal breeding industry. In order to develop new fungistatic substances, more and more natural or synthetic substances are subjected to a fungistatic activity assay to evaluate their potential for use in industrial and agricultural production. When the activity of natural or synthetic substances for inhibiting the mold is measured, the mold spores are prepared into uniformly dispersed suspension and then can be used for measuring the indexes of the substances, such as the inhibition rate of inhibiting the spore germination, the minimum inhibitory concentration, the minimum bactericidal concentration and the like.
The mold spores have high hydrophobicity, are easy to aggregate and are not easy to fully disperse in water. Up to now, the preparation method of the aspergillus conidium suspension has major defects: adding distilled water or Tween 80 aqueous solution into a culture dish full of spores, scraping the spores by using an inoculating loop, easily infecting the bacteria due to a large opening of the culture dish, and scraping more hyphae into spore suspension; the absorbent cotton or the 3-6 layers of the degreased gauze are used for filtering spore suspension to remove hyphae, so that a large number of spores are easily adsorbed on the absorbent cotton or the gauze, and the yield of the spores is reduced; spore dispersion is carried out by blowing and sucking with a pipette tip or adding glass beads into spore liquid for oscillation, the spore dispersion is insufficient, and a large amount of diplospores, trispores and even spore clusters exist. The probability of obtaining false positive mutation by adopting insufficiently dispersed spores for mutagenesis is higher, the mutagenesis efficiency is seriously influenced, and the method is also not suitable for the determination experiment of the spore germination inhibition rate by active substances.
Disclosure of Invention
The purpose of the invention is: the preparation method of the aspergillus conidium suspension is provided, spores of the spore suspension prepared by the method are uniformly dispersed and do not aggregate, and the method is suitable for strain mutagenesis experiments and bacteriostatic experiments of active substances.
The technical solution of the invention is as follows: a preparation method of aspergillus conidium suspension comprises the following steps:
(1) activating aspergillus strains: inoculating aspergillus strains preserved at low temperature to a PDA test tube slant for activation;
(2) culturing conidia: after activation, transferring the activated cells to a large-size PDA test tube inclined plane, and culturing until the inclined plane is full of conidia;
(3) and (3) eluting conidia: adding the eluent into a test tube full of conidia, and oscillating to separate the conidia from hyphae;
(4) and (3) filtering the eluent: filtering the eluate rich in conidia after oscillation;
(5) and (3) carrying out enzyme treatment on the filtrate: adding protease into the filtrate for treatment;
(6) washing conidia: washing after the enzyme treatment is finished to obtain conidium sediment;
(7) resuspending conidia: adding the resuspension liquid into the conidium sediment, and uniformly mixing the mixture by ultrasonic oscillation;
(8) counting and diluting: after the mixture is evenly mixed by ultrasonic oscillation, the suspension with evenly dispersed conidia is obtained by counting and diluting.
In the step (1), the aspergillus strain activation comprises the following steps: taking an aspergillus strain inclined plane preserved at 4 ℃, digging a strain block with hypha by using an inoculating shovel, inoculating the strain block to an inclined plane of a common PDA test tube, tightly attaching the strain block to a culture medium with the hypha, plugging a plug, and placing the strain block in an incubator at 30 ℃ for dark culture for 3-5 days until conidia grow out.
In the step (2), the cultured conidia are as follows: scraping the conidium cultured in the step (1) by using an inoculating loop, streaking and inoculating the conidium on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 3-5 days at 30 ℃ in a dark place until the test tube inclined plane is full of the conidium.
In the step (3), the eluting conidia are as follows: and (3) adding 10-15mL of pre-sterilized eluent into the test tube inclined plane of the aspergillus full of conidia cultured in the step (2), inclining the test tube to enable the solid inclined plane full of conidia to face upwards, placing the test tube on a vortex mixer, oscillating at 300-500 rpm for 20-30s, and fully eluting the conidia on the aspergillus hyphae.
Wherein the pre-sterilized eluent is: 50mmol/L Tris-HCl with the mass concentration of 0.03-0.05% Tween 80, and pH7.5.
In the step (4), the eluent filtering is as follows: filtering the conidium eluate obtained in the step (3) by using sterilized 10cm multiplied by 15cm four-layer superposed microscope lens wiping paper, filtering out hypha fragments in the eluate, and collecting the filtrate containing conidia into a 50mL sterile triangular flask.
In the step (5), the filtrate is treated by enzyme: and (4) adding a 20mg/mL protease K solution for filtration and sterilization into the filtered conidium filtrate until the final enzyme concentration 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 the hydrophobin on the surface of the conidium.
In the step (6), the washed conidia are: and (5) placing the conidium filtrate after enzyme treatment in a refrigerated centrifuge for centrifugation for 6-10min at 4 ℃ and 8000rpm of 6000-.
In the step (7), the conidium resuspension is as follows: and (3) adding sterilized 20mL of sterile resuspension liquid with the mass concentration of 0.03-0.05% of Tween 80 and 20-30mmol/L of urea into the conidium precipitate obtained in the step (6) to resuspend the spores, transferring the spore suspension into a sterile 50mL triangular flask, putting the flask into an ultrasonic cleaner, and performing ultrasonic oscillation at room temperature of 30KHz-50KHz for 20-30s to fully disperse the spores.
In the step (8), the counting and diluting are as follows: observing and counting the conidium re-suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water6-107CFU/mL, and storing in a refrigerator at 4 ℃ for later use.
Compared with the prior art, the invention has the advantages that:
firstly, culturing mould spores by using a 32 x 200mm test tube inclined plane, and culturing enough spores due to the large area of the inclined plane of a culture medium; the opening of the test tube is smaller than that of the culture dish, so that the spore is not easy to be infected when being eluted;
secondly, adding an eluent into the test tube full of spores, and eluting the spores in an oscillation mode of a vortex mixer, so that not only can the conidia be thoroughly eluted into the eluent, but also mould hyphae can be prevented from being brought into the eluent;
thirdly, filtering the conidium suspension by using four layers of microscope lens wiping paper, not only removing a small amount of hyphae brought into the conidium suspension, but also avoiding the problems of large amount of conidium loss and low conidium yield caused by filtering by using absorbent cotton or absorbent gauze;
treating the conidium suspension by using protease K, so that part of hydrophobin on the surface of conidium can be removed, and the aggregation among spores caused by the existence of the hydrophobin on the surface of the conidium is avoided;
fifthly, oscillating the conidium suspension at low frequency by using an ultrasonic cleaner to disperse the accumulated diplospores, trispores or polysospores in the suspension;
and sixthly, the Tween 80 in the suspension can enable the conidia to be well dispersed in the suspension, and the urea in the suspension can avoid aggregation caused by hydrogen bond formed by spore wall polysaccharide molecules between spores, so that the conidia suspension is more stable under the dual action of the Tween 80 and the urea.
Drawings
FIG. 1 is a diagram showing the state of dispersion of conidia in a conidia suspension of Aspergillus flavus prepared by a conventional method;
FIG. 2 shows the dispersion state of conidia in the Aspergillus flavus conidia suspension prepared by the method of the present invention.
Detailed Description
The technical solution of the invention is further illustrated below with reference to specific examples, which are not to be construed as limiting the technical solution.
Example 1: preparation of Aspergillus niger conidium suspension
(1) Activating aspergillus strains: taking an Aspergillus niger AS3.939 strain inclined plane preserved at 4 ℃, digging a strain block with hyphae by using an inoculating shovel, inoculating the strain block to an inclined plane of a common PDA test tube, tightly attaching the strain block to a culture medium with the hyphae, plugging a plug, and placing in an incubator at 30 ℃ for dark culture for 3 days until conidia grow out;
(2) culturing conidia: scraping the conidia of the Aspergillus niger AS3.939 cultured in the step (1) by using an inoculating loop, streaking and inoculating the conidia on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 3 days in a dark place at 30 ℃ until the test tube inclined plane is full of the conidia;
(3) and (3) eluting conidia: adding 10mL of pre-sterilized eluent into the slant surface of the test tube of the Aspergillus niger AS3.939 full of the conidia cultured in the step (2), inclining the test tube to enable the slant surface of the solid full of the conidia to be upward, placing the slant surface on a vortex mixer, oscillating at 300rpm for 30s, and fully eluting the conidia on the hyphae; wherein the pre-sterilized eluent is: 50mmol/L Tris-HCl with the mass concentration of 0.03 percent Tween 80, pH7.5;
(4) and (3) filtering the eluent: filtering the conidium eluate obtained in the step (3) by using sterilized 10cm multiplied by 15cm four-layer superposed microscope lens wiping paper, filtering out hypha fragments in the eluate, and collecting the filtrate containing conidia into a 50mL sterile triangular flask;
(5) and (3) carrying out enzyme treatment on the filtrate: adding 20mg/mL protease K solution for filtration and sterilization into the filtered conidium filtrate in the step (4) until the final enzyme concentration is 0.2mg/mL, and carrying out enzyme treatment for 40min by using a constant-temperature water bath oscillator at 37 ℃ at 60rpm to remove the hydrophobin on the surface of the conidium;
(6) washing conidia: placing the conidium filtrate after enzyme treatment in the step (5) in a refrigerated centrifuge for centrifugation for 10min at 4 ℃ and 6000rpm, adding sterilized 20mL of 0.03% Tween 80 aqueous solution with mass concentration into the precipitate to wash the conidium, and then centrifuging for 10min under the refrigerated centrifugation condition to obtain an impurity-free conidium precipitate;
(7) resuspending conidia: adding sterilized 20mL of sterile resuspension liquid with the mass concentration of 0.03% Tween 80 and 20mmol/L urea into the conidiospore sediment obtained in the step (6) to resuspend the spores, transferring the spore suspension into a sterile 50mL triangular flask, putting the flask into an ultrasonic cleaner, and performing ultrasonic oscillation at room temperature of 30KHz for 30s to fully disperse the spores;
(8) counting and diluting: observing and counting the conidium re-suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water6-107CFU/mL, stored in a refrigerator at 4 ℃ and used for mutagenesis of Aspergillus niger AS3.939 strain to improve citric acid yield.
Example 2: preparation of Aspergillus oryzae conidium suspension
(1) Activating aspergillus strains: collecting Aspergillus oryzae AS3.863 strain slant preserved at 4 deg.C, digging with inoculating shovel to collect the block with mycelium, inoculating the block to slant of common PDA test tube, tightly adhering the surface with mycelium to culture medium, plugging plug, and culturing in 30 deg.C incubator in dark for 4 days until conidia grow out;
(2) culturing conidia: scraping conidia of Aspergillus oryzae AS3.863 cultured in step (1) by using an inoculating loop, streaking and inoculating on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 4 days in a dark place at 30 ℃ until the test tube inclined plane is full of the conidia;
(3) and (3) eluting conidia: adding 12.5mL of pre-sterilized eluent into the test tube slant covered with the conidia cultured in the step (2), inclining the test tube to enable the solid slant covered with the conidia to be upward, placing the test tube on a vortex mixer, and oscillating at 400rpm for 25s to fully elute the conidia on the hyphae; wherein the pre-sterilized eluent is: 50mmol/L Tris-HCl with the mass concentration of 0.04 percent Tween 80, and the pH value is 7.5;
(4) and (3) filtering the eluent: filtering the conidium eluate obtained in the step (3) by using sterilized 10cm multiplied by 15cm four-layer superposed microscope lens wiping paper, filtering out hypha fragments in the eluate, and collecting the filtrate containing conidia into a 50mL sterile triangular flask;
(5) and (3) carrying out enzyme treatment on the filtrate: adding 20mg/mL protease K solution for filtration and sterilization into the filtered conidium filtrate in the step (4) until the final enzyme concentration is 0.35mg/mL, and carrying out enzyme treatment for 30min by using a constant-temperature water bath oscillator at 37 ℃ and at 80rpm to remove the hydrophobin on the surface of the conidium;
(6) washing conidia: placing the conidium filtrate after enzyme treatment in the step (5) in a refrigerated centrifuge for centrifuging for 8min at 4 ℃ and 7000rpm, adding sterilized 20mL of 0.04% Tween 80 aqueous solution with mass concentration into the precipitate to wash the conidium, and centrifuging for 8min under the refrigerated centrifugation condition to obtain an impurity-free conidium precipitate;
(7) resuspending conidia: adding sterilized 20mL of sterile resuspension liquid with the mass concentration of 0.04% Tween 80 and 25mmol/L urea into the conidiospore sediment obtained in the step (6) to resuspend the spores, transferring the spore suspension into a sterile 50mL triangular flask, putting the flask into an ultrasonic cleaner, and carrying out ultrasonic oscillation at room temperature of 40KHz for 25s to fully disperse the spores;
(8) counting and diluting: observing and counting the conidium re-suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water6-107CFU/mL, stored in a 4 ℃ refrigerator, was used for mutagenesis of Aspergillus oryzae AS3.863 strain to increase protease production.
Example 3: preparation of Aspergillus flavus conidia suspension
(1) Activating aspergillus strains: taking an Aspergillus flavus AS3.3950 strain inclined plane preserved at 4 ℃, digging a strain block with hypha by using an inoculating shovel, inoculating the strain block to an inclined plane of a common PDA test tube, tightly attaching the strain block to a culture medium, plugging a plug, and placing in an incubator at 30 ℃ for 5 days in a dark place until conidia grow out;
(2) culturing conidia: scraping conidia of the aspergillus flavus AS3.3950 cultured in the step (1) by using an inoculating loop, streaking and inoculating the conidia on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 5 days in a dark place at 30 ℃ until the test tube inclined plane is full of the conidia;
(3) and (3) eluting conidia: adding 15mL of pre-sterilized eluent into the test tube slant covered with the conidia cultured in the step (2), inclining the test tube to enable the solid slant covered with the conidia to be upward, placing the test tube on a vortex mixer, and oscillating at 500rpm for 20s to fully elute the conidia on the hyphae; wherein the pre-sterilized eluent is: 50mmol/L Tris-HCl with the mass concentration of 0.05 percent Tween 80, pH7.5;
(4) and (3) filtering the eluent: filtering the conidium eluate obtained in the step (3) by using sterilized 10cm multiplied by 15cm four-layer superposed microscope lens wiping paper, filtering out hypha fragments in the eluate, and collecting the filtrate containing conidia into a 50mL sterile triangular flask;
(5) and (3) carrying out enzyme treatment on the filtrate: adding a 20mg/mL protease K solution for filtration and sterilization into the filtered conidium filtrate in the step (4) until the final enzyme concentration is 0.5mg/mL, and carrying out enzyme treatment for 20min by using a constant-temperature water bath oscillator at 37 ℃ at 100rpm to remove the hydrophobin on the surface of the conidium;
(6) washing conidia: placing the conidium filtrate after enzyme treatment in the step (5) in a refrigerated centrifuge for centrifugation for 6min at 8000rpm and 4 ℃, adding sterilized 20mL of Tween 80 aqueous solution with the mass concentration of 0.05% into the precipitate to wash the conidium, and then centrifuging for 6min under the refrigerated centrifugation condition to obtain an impurity-free conidium precipitate;
(7) resuspending conidia: adding sterilized 20mL of sterile resuspension liquid with the mass concentration of 0.05% Tween 80 and 30mmol/L urea into the conidiospore sediment obtained in the step (6) to resuspend the spores, transferring the spore suspension into a sterile 50mL triangular flask, putting the flask into an ultrasonic cleaner, and performing ultrasonic oscillation at room temperature of 50KHz for 20s to fully disperse the spores;
(8) counting and diluting: observing and counting the conidium re-suspension obtained in the step (7) by using an optical microscope, and diluting the conidium concentration of the suspension to 10 by using sterile water6-107CFU/mL, and storing in a refrigerator at 4 ℃ for inhibiting Aspergillus flavus AS3.3950 activity experiment.
Claims (10)
1. A preparation method of aspergillus conidium suspension is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) activating aspergillus strains: inoculating aspergillus strains preserved at low temperature to a PDA test tube slant for activation;
(2) culturing conidia: after activation, transferring the activated cells to a large-size PDA test tube inclined plane, and culturing until the inclined plane is full of conidia;
(3) and (3) eluting conidia: adding the eluent into a test tube full of conidia, and oscillating to separate the conidia from hyphae;
(4) and (3) filtering the eluent: filtering the eluate rich in conidia after oscillation;
(5) and (3) carrying out enzyme treatment on the filtrate: adding protease into the filtrate for treatment;
(6) washing conidia: washing after the enzyme treatment is finished to obtain conidium sediment;
(7) resuspending conidia: adding the resuspension liquid into the conidium sediment, and uniformly mixing the mixture by ultrasonic oscillation;
(8) counting and diluting: after the mixture is evenly mixed by ultrasonic oscillation, the suspension with evenly dispersed conidia is obtained by counting and diluting.
2. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (1), the aspergillus strain activation comprises the following steps: taking an aspergillus strain inclined plane preserved at 4 ℃, digging a strain block with hypha by using an inoculating shovel, inoculating the strain block to an inclined plane of a common PDA test tube, tightly attaching the strain block to a culture medium with the hypha, plugging a plug, and placing the strain block in an incubator at 30 ℃ for dark culture for 3-5 days until conidia grow out.
3. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (2), the cultured conidia are as follows: scraping the conidium cultured in the step (1) by using an inoculating loop, streaking and inoculating the conidium on a PDA test tube inclined plane with the diameter of 32 multiplied by 200mm, plugging a plug, and culturing for 3-5 days at 30 ℃ in a dark place until the test tube inclined plane is full of the conidium.
4. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (3), the eluting conidia are as follows: and (3) adding 10-15mL of pre-sterilized eluent into the test tube inclined plane of the aspergillus full of conidia cultured in the step (2), inclining the test tube to enable the solid inclined plane full of conidia to face upwards, placing the test tube on a vortex mixer, oscillating at 300-500 rpm for 20-30s, and fully eluting the conidia on the aspergillus hyphae.
5. The method for preparing a suspension of Aspergillus conidia according to claim 4, wherein the method comprises the following steps: the pre-sterilized eluent is: 50mmol/L Tris-HCl with the mass concentration of 0.03-0.05% Tween 80, and pH7.5.
6. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (4), the eluent filtering is as follows: filtering the conidium eluate obtained in the step (3) by using sterilized 10cm multiplied by 15cm four-layer superposed microscope lens wiping paper, filtering out hypha fragments in the eluate, and collecting the filtrate containing conidia into a 50mL sterile triangular flask.
7. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (5), the filtrate is treated by enzyme: and (4) adding a 20mg/mL protease K solution for filtration and sterilization into the conidium filtrate obtained after filtration in the step (4) until the final enzyme concentration 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 surfaces of the conidia.
8. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (6), the washed conidia are: and (5) placing the conidium filtrate after enzyme treatment in a refrigerated centrifuge for centrifugation for 6-10min at 4 ℃ and 8000rpm of 6000-.
9. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (7), the conidium resuspension is as follows: and (3) adding sterilized 20mL of sterile resuspension liquid with the mass concentration of 0.03-0.05% of Tween 80 and 20-30mmol/L of urea into the conidium precipitate obtained in the step (6) to resuspend the spores, transferring the spore suspension into a sterile 50mL triangular flask, putting the flask into an ultrasonic cleaner, and performing ultrasonic oscillation at room temperature of 30KHz-50KHz for 20-30s to fully disperse the spores.
10. The method for preparing a suspension of Aspergillus conidia according to claim 1, wherein the method comprises the following steps: in the step (8), the counting and diluting are as follows: observing and counting the conidium resuspension obtained in step (7) with an optical microscope, and diluting the conidium concentration of the suspension to 10 with sterile water6-107CFU/mL, and storing in a refrigerator at 4 ℃ for later use.
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