CN103966106B - Preparation method for the mycete liquid spawn of shake flask fermentation contrast experiment - Google Patents
Preparation method for the mycete liquid spawn of shake flask fermentation contrast experiment Download PDFInfo
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- CN103966106B CN103966106B CN201410192861.4A CN201410192861A CN103966106B CN 103966106 B CN103966106 B CN 103966106B CN 201410192861 A CN201410192861 A CN 201410192861A CN 103966106 B CN103966106 B CN 103966106B
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Abstract
The invention provides the preparation method of a kind of mycete liquid spawn for shake flask fermentation contrast experiment, first mycotic spore is cultivated with potato sucrose agar culture medium test tube slant, then with physiological saline solution, spore is washed down, being inoculated in potato sucrose fluid medium cultivation after again spore suspension being diluted to finite concentration, obtained tiny bacterium grain is mycete liquid spawn.The inventive method is simple, and gained mycete liquid spawn size is uniform, character consistent, ensure that the concordance of each shaking flask inoculum concentration during inoculation, greatly strengthen comparability and the credibility of shake flask fermentation experiment.
Description
Technical field
The present invention relates to the preparation method of a kind of mycete liquid spawn, be specially adapted to prepare the mycete liquid spawn for shake flask fermentation contrast experiment.
Background technology
Mycete is the general designation of the fungus forming branch mycelia.Mycete and human being's production and life relation are extremely close, and in addition to people, animal, crops, industrial and agricultural products can being made ill and go mouldy, the compound such as its enzyme secreted, medicine, pigment is widely used in the industries such as agricultural, weaving, food, medicine, leather.In order to obtain new fungal strain or improve the yield of existing mycete metabolite, it is often necessary to carry out the experiments such as bacterial strain screening, mutation or fermentation condition optimization.During these experimental implementation, all refer to substantial amounts of contrast experiment yield of target metabolic product under the conditions of comparing different strains or different fermentations, in order to determine optimum strain or optimal conditions of fermentation.
In order to react between different strains really or the impact on target product yield of the different fermentations condition, when carrying out fermenting comparative experiments, must take measures to reduce experimental error to greatest extent, wherein, between reduction contrast experiment, inoculum concentration, the difference of minimizing strain character are exactly simple and effective measure.For mycete, typically being inoculated by two ways, one is inoculation mycelium pellet, this kind of method often cannot be carried out accurately quantitatively thus cause bigger experimental error；Two is inoculating spores, though the method energy accurate quantitative analysis, but cause spore character different owing to the time of spore generation there are differences, also can bring certain error to experiment.
Summary of the invention
It is an object of the invention to: provide the preparation method of a kind of mycete liquid spawn for shake flask fermentation contrast experiment, mycete seed prepared by the method can ensure that the inoculum concentration of each experiment shaking flask is consistent with thalline character to greatest extent.
The technical solution of the present invention is that this comprises the following steps for the preparation method of the mycete liquid spawn of shaking flask contrast experiment:
(1) a fritter mycete lawn is inoculated on potato sucrose agar (PDA) culture medium test tube slant, cultivates 96-192h in 25-30 DEG C of incubator, until solid slope covers with spore；
(2) mycotic spore normal saline is washed down, after being diluted to finite concentration, with 5mL pipettor, spore suspension is inoculated in the 500mL triangular flask equipped with 100mL potato sucrose fluid medium and a small amount of bead by a certain percentage, 25 DEG C-30 DEG C, cultivate 48-72h under 100-120rmp rotating speed, until growing the little bacterium grain of substantial amounts of uniformity in culture fluid, obtained tiny bacterium grain is mycete liquid spawn.
Wherein, the operation pipetting mycete liquid spawn is: be inoculated in corresponding liquid fermentation medium by little bacterium grain by the volume ratio of 3-5% with 5mL pipettor, 25 DEG C-37 DEG C, ferment under 120-150rmp rotating speed, obtains required mycete metabolite.
Wherein, in step (1), test tube specification used is 20mm × 200mm.
Wherein, in step (1), potato sucrose agar (PDA) culture medium is prepared: peeled potatoes 200g is cut into 1cm3The fritter of left and right, add after distilled water 1000mL boils 30min, filter off potato balls with six layers of gauze, with sucrose 20g, agar powder 15g in filtrate, be heated to after agar dissolves, 1000mL is complemented to distilled water, then be sub-packed in test tube, stopper with silica gel plug, kraft paper wrapping after in high-pressure sterilizing pot 121 DEG C of sterilizing 20min, when culture medium is cooled to 80 DEG C, it is put into test tube slant to cooled and solidified.
Wherein, in step (2), the concentration after mycotic spore dilution is: under 620nm wavelength, absorbance is 0.5-0.7, and the inoculative proportion of spore suspension is the 3-5% of potato sucrose fluid medium volume.
Wherein, in step (2), potato sucrose fluid medium is prepared: peeled potatoes 200g is cut into 1cm3Fritter, add after distilled water 1000mL boils 30 minutes, potato ball is filtered off with six layers of gauze, filtrate is dissolved with sucrose 20g, complement to subpackage 100mL in 1000mL, 500mL triangular flask with distilled water, add a small amount of bead, finally encase bottleneck with 6 layers of gauze, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min.
Wherein, 5mL pipettor refers to: after cutting suction pipette head tip, and the most advanced and sophisticated aperture of suction nozzle is 2-3mm.
The invention have the advantage that gained mycete little bacterium grain is in granular form, its fungus ball is less, size and thalline character are consistent, ensure that when being seeded to fermentation medium each experiment shaking flask access amount is consistent with thalline character, thus improve the comparability of experiment, enhance the credibility of experimental data, finally improve conventional efficient.
Detailed description of the invention
Further illustrate the technical solution of the present invention below in conjunction with specific embodiment, these embodiments are not to be construed as the restriction to technical scheme.
Embodiment 1: prepare the aspergillus oryzae liquid spawn generation amount in order to comparative starches enzyme according to following steps
(1) potato sucrose agar (PDA) culture medium preparation: peeled potatoes 200g is cut into 1cm3The fritter of left and right, add after distilled water 1000mL boils 30min, potato ball is filtered off with six layers of gauze, with sucrose 20g, agar powder 15g in filtrate, after being heated to agar fusing, complement to 1000mL with distilled water, it is sub-packed in test tube (20mm × 200mm) at 1/3rd volumes, stopper bottleneck with silica gel plug, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min, when culture medium is cooled to 80 DEG C, be put into test tube slant to cooled and solidified；
(2) mycete is inoculated in test tube solid slope: take the aspergillus oryzae strain of test tube slant preservation, superclean bench takes fritter lawn with disinfection inoculation shovel, it is inoculated on the fresh tube inclined-plane of step (1), it is stoppered silica gel plug and is placed on 25 DEG C of incubators cultivation 192h, till media surface covers with a large amount of spore；
(3) potato sucrose fluid medium preparation: peeled potatoes 200g is cut into 1cm3Fritter, add after distilled water 1000mL boils 30 minutes, potato ball is filtered off with six layers of gauze, filtrate is dissolved with sucrose 20g, 1000mL is complemented to distilled water, subpackage 100mL in 500mL triangular flask, encases bottleneck with 6 layers of gauze after adding a small amount of bead, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min；
(4) mycotic spore is inoculated in potato sucrose fluid medium: take the solid test tube slant seed of the fresh cultured of step (2), on superclean bench, draw 5mL sterile saline with 5mL sterilizing pipettor and join in test tube slant, test tube slant is gently scraped again with sterilizing spatula, until all mycotics spore are scraped, shake test tube gently, spore is made to be evenly distributed state, then in test tube, sterile saline is continuously added until spore suspension absorbance under 620nm wavelength is 0.5, draw the spore suspension prepared with sterilizing rifle head and add in potato sucrose fluid medium, each triangular flask containing 100mL culture medium accesses 3mL spore suspension；
(5) bacterium grain is cultivated: postvaccinal triangular flask is placed in constant temperature oscillator cultivation 48h, and rotating speed is 120rmp, and cultivation temperature is 30 DEG C, until growing the bacterium grain of uniform small rice grain size in triangular flask；
(6) aspergillus oryzae strain cultivation: take 5mL suction pipette head, the tip cutting suction nozzle with shears is a little, and making suction nozzle outlet diameter is 2mm, and high-pressure sterilizing pot sterilizing is standby；Quickly it is inoculated in fermentation medium after cultured bacterium grain is drawn certain volume with the suction pipette head processed, inoculum concentration is the 3% of fermentation medium volume, it is placed in constant incubator 25 DEG C with the wrapping of 8 layers of gauze, cultivates, in order to the generation amount of comparative starches enzyme under the conditions of 150rpm.
Embodiment 2: prepare penicillium liquid spawn in order to compare the generation amount of penicillin according to following steps
(1) potato sucrose agar (PDA) culture medium preparation: peeled potatoes 200g is cut into 1cm3The fritter of left and right, add after distilled water 1000mL boils 30min, potato ball is filtered off with six layers of gauze, with sucrose 20g, agar powder 15g in filtrate, after being heated to agar fusing, complement to 1000mL with distilled water, it is sub-packed in test tube (20mm × 200mm) at 1/3rd volumes, stopper bottleneck with silica gel plug, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min, when culture medium is cooled to 80 DEG C, be put into test tube slant to cooled and solidified；
(2) mycete is inoculated in test tube solid slope: take the penicillium sp strain of test tube slant preservation, superclean bench takes fritter lawn with disinfection inoculation shovel, it is inoculated on the fresh tube inclined-plane of step (1), it is stoppered silica gel plug and is placed on 27.5 DEG C of incubators cultivation 144h, till media surface covers with a large amount of spore；
(3) potato sucrose fluid medium preparation: peeled potatoes 200g is cut into 1cm3Fritter, add after distilled water 1000mL boils 30 minutes, potato ball is filtered off with six layers of gauze, filtrate is dissolved with sucrose 20g, 1000mL is complemented to distilled water, subpackage 100mL in 500mL triangular flask, encases bottleneck with 6 layers of gauze after adding a small amount of bead, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min；
(4) mycotic spore is inoculated in potato sucrose fluid medium: take the solid test tube slant seed of the fresh cultured of step (2), on superclean bench, draw 5mL sterile saline with 5mL sterilizing pipettor and join in test tube slant, test tube slant is gently scraped again with sterilizing spatula, until all mycotics spore are scraped, shake test tube gently, spore is made to be evenly distributed state, then in test tube, sterile saline is continuously added until spore suspension absorbance under 620nm wavelength is 0.6, draw the spore suspension prepared with sterilizing rifle head and add in potato sucrose fluid medium, each triangular flask containing 100mL culture medium accesses 4mL spore suspension；
(5) bacterium grain is cultivated: postvaccinal triangular flask is placed in constant temperature oscillator cultivation 60h, and rotating speed is 110rmp, and cultivation temperature is 27.5 DEG C, until growing the bacterium grain of uniform small rice grain size in triangular flask；
(6) penicillium strain cultivation: take 5mL suction pipette head, the tip cutting suction nozzle with shears is a little, and making suction nozzle outlet diameter is 2.5mm, and high-pressure sterilizing pot sterilizing is standby；Quickly it is inoculated in fermentation medium after cultured bacterium grain is drawn certain volume with the suction pipette head processed, inoculum concentration is the 4% of fermentation medium volume, it is placed in constant incubator 31 DEG C with the wrapping of 8 layers of gauze, cultivates, in order to compare the generation amount of penicillin under the conditions of 135rpm.
Embodiment 3: prepare trichoderma liquid strain in order to compare the generation amount of cellulase according to following steps
(1) potato sucrose agar (PDA) culture medium preparation: peeled potatoes 200g is cut into 1cm3The fritter of left and right, add after distilled water 1000mL boils 30min, potato ball is filtered off with six layers of gauze, with sucrose 20g, agar powder 15g in filtrate, after being heated to agar fusing, complement to 1000mL with distilled water, it is sub-packed in test tube (20mm × 200mm) at 1/3rd volumes, stopper bottleneck with silica gel plug, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min, when culture medium is cooled to 80 DEG C, be put into test tube slant to cooled and solidified；
(2) trichoderma is inoculated in test tube solid slope: take the trichoderma strain of test tube slant preservation, superclean bench takes fritter lawn with disinfection inoculation shovel, it is inoculated on the fresh tube inclined-plane of step (1), it is stoppered silica gel plug and is placed on 30 DEG C of incubators cultivation 96h, till media surface covers with a large amount of spore；
(3) potato sucrose fluid medium preparation: peeled potatoes 200g is cut into 1cm3Fritter, add after distilled water 1000mL boils 30 minutes, potato ball is filtered off with six layers of gauze, filtrate is dissolved with sucrose 20g, 1000mL is complemented to distilled water, subpackage 100mL in 500mL triangular flask, encases bottleneck with 6 layers of gauze after adding a small amount of bead, after kraft paper wrapping in high-pressure sterilizing pot 121 DEG C of sterilizing 20min；
(4) by trichoderma spore inoculating in potato sucrose fluid medium: take the solid test tube slant seed of the fresh cultured of step (2), on superclean bench, draw 5mL sterile saline with 5mL sterilizing pipettor and join in test tube slant, test tube slant is gently scraped again with sterilizing spatula, until all mycotics spore are scraped, shake test tube gently, spore is made to be evenly distributed state, then in test tube, sterile saline is continuously added until spore suspension absorbance under 620nm wavelength is 0.7, draw the spore suspension prepared with sterilizing rifle head and add in potato sucrose fluid medium, each triangular flask containing 100mL culture medium accesses 5mL spore suspension；
(5) bacterium grain is cultivated: postvaccinal triangular flask is placed in constant temperature oscillator cultivation 72h, and rotating speed is 100rmp, and cultivation temperature is 25 DEG C, until growing the bacterium grain of uniform small rice grain size in triangular flask；
(6) trichoderma liquid spawn culture: take 5mL suction pipette head, the tip cutting suction nozzle with shears is a little, and making suction nozzle outlet diameter is 3mm, and high-pressure sterilizing pot sterilizing is standby；Quickly it is inoculated in fermentation medium after cultured bacterium grain is drawn certain volume with the suction pipette head processed, inoculum concentration is the 5% of fermentation medium volume, it is placed in constant incubator 37 DEG C with the wrapping of 8 layers of gauze, cultivates, in order to compare the generation amount of cellulase under the conditions of 120rpm.
Claims (1)
1. it is used for the preparation method of the mycete liquid spawn of shake flask fermentation contrast experiment, it is characterised in that the method comprises the steps:
(1) first a fritter mycete lawn is inoculated on potato sucrose agar PDA culture medium test tube slant, culture medium test tube a size of 20mm × 200mm, cultivates 96-192h in 25-30 DEG C, until solid slope covers with spore；
(2) mycotic spore normal saline is washed down, it is diluted to after under 620nm wavelength, absorbance is 0.5-0.7, with 5mL pipettor, spore suspension is inoculated in the 500mL triangular flask equipped with 100mL potato sucrose fluid medium and a small amount of bead in the ratio of 3-5%, 25 DEG C-30 DEG C, cultivate 48-72h under 100-120rpm rotating speed, until growing the little bacterium grain of substantial amounts of uniformity in culture fluid, obtained tiny bacterium grain is mycete liquid seeds；This liquid seeds pipettor is pressed after the volume ratio of 3-5% accesses and cultivate in fermentation medium, 25 DEG C-37 DEG C, ferment under 120-150rpm rotating speed, i.e. obtains corresponding metabolite；5mL pipettor refers to: after cutting suction pipette head tip, and the most advanced and sophisticated aperture of suction nozzle is 2-3mm.
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