CN103966106B - Preparation method for the mycete liquid spawn of shake flask fermentation contrast experiment - Google Patents

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

Preparation method of mold liquid strains used in comparative experiment of shake flask fermentation

technical field

The invention relates to a method for preparing mold liquid strains, and is especially suitable for preparing mold liquid strains used in shake flask fermentation comparative experiments.

Background technique

Mold is a general term for fungi that form branched hyphae. Mold is closely related to human production and life. In addition to making people, livestock, crops, and industrial and agricultural products sick and mildewed, the enzymes, drugs, pigments and other compounds secreted by them are widely used in agriculture, textiles, food, medicine, In the leather and other industries. In order to obtain new mold strains or increase the yield of existing mold metabolites, experiments such as strain screening, mutagenesis, or optimization of fermentation conditions are often required. During these experimental operations, a large number of comparative experiments are involved to compare the yields of target metabolites under different strains or different fermentation conditions, so as to determine the best strains or the best fermentation conditions.

In order to truly reflect the influence of different strains or different fermentation conditions on the yield of the target product, measures must be taken to minimize the experimental error when conducting fermentation comparison experiments. Simple and effective measures. For molds, there are generally two ways to inoculate, one is to inoculate mycelium balls, which often cannot be accurately quantified and thus cause large experimental errors; the other is to inoculate spores, although this method can accurately quantify, but Due to the difference in the time of spore generation, the spore traits are different, which will also bring some errors to the experiment.

Contents of the invention

The purpose of the present invention is: provide a kind of preparation method of the mold liquid bacterial strain that is used in shake flask fermentation contrast experiment, the mold seed prepared by this method can guarantee the consistency of the inoculum amount and the thalline character of each experiment shake flask to the greatest extent.

Technical solution of the present invention is that the preparation method of the mold liquid bacterial classification that this is used for shake bottle comparative experiment comprises the following steps:

(1) Inoculate a small piece of mold lawn on the slant of a potato sucrose agar (PDA) medium test tube, and incubate in an incubator at 25-30°C for 96-192 hours until the solid slant is covered with spores;

(2) Wash the mold spores with physiological saline, dilute to a certain concentration, and use a 5mL pipette to inoculate the spore suspension in a certain proportion into a 500mL Erlenmeyer flask containing 100mL potato sucrose liquid medium and a small amount of glass beads. Cultivate for 48-72 hours at 25°C-30°C, 100-120rmp rotation speed, until a large number of uniform small bacterial particles grow in the culture medium, and the obtained small bacterial particles are mold liquid strains.

Among them, the operation of pipetting the mold liquid strain is: use a 5mL pipette to inoculate the small bacteria particles in the corresponding liquid fermentation medium at a volume ratio of 3-5%, at 25°C-37°C, 120-150rmp speed Fermentation is carried out to obtain the required mold metabolites.

Wherein, in step (1), the size of the test tube used is 20mm×200mm.

Among them, in step (1), potato sucrose agar (PDA) medium preparation: cut 200 g of peeled potatoes into small pieces of about 1 cm ³ , add 1000 mL of distilled water and boil for 30 min, filter the potato pieces with six layers of gauze, and add Add 20g of sucrose and 15g of agar powder to the medium, heat until the agar melts, make up to 1000mL with distilled water, then divide it into test tubes, plug it tightly with a silica gel stopper, wrap it with kraft paper, and sterilize it in an autoclave at 121°C for 20min. When the base is cooled to 80°C, place the test tube on an inclined plane until it cools and solidifies.

Wherein, in step (2), the concentration of the diluted mold spores is: the absorbance at 620nm wavelength is 0.5-0.7, and the inoculation ratio of the spore suspension is 3-5% of the volume of the potato sucrose liquid medium.

Among them, in step (2), the preparation of potato sucrose liquid medium: cut 200 g of peeled potatoes into small pieces of 1 cm ³ , add 1000 mL of distilled water and boil for 30 minutes, filter the potato pieces with six layers of gauze, add sucrose to the filtrate Dissolve 20g, make up to 1000mL with distilled water, dispense 100mL into a 500mL Erlenmeyer flask, add a small amount of glass beads, and finally wrap the mouth of the bottle with 6 layers of gauze, wrap it with kraft paper and sterilize in an autoclave at 121°C for 20min.

Among them, the 5mL pipette refers to: after cutting off the tip of the pipette tip, the tip aperture of the tip is 2-3mm.

The advantages of the present invention are: the obtained fungus granules are in the form of granules, the bacterium balls are small, the size and the thallus properties are consistent, and when it is inoculated into the fermentation medium, the amount of each experimental shaker flask and the thallus properties can be guaranteed Consistent, thereby improving the comparability of the experiment, enhancing the credibility of the experimental data, and finally improving the efficiency of the experiment.

detailed description

The technical solution of the present invention will be further described below in conjunction with specific examples, and these examples should not be construed as limitations on the technical solution.

Embodiment 1: Prepare Aspergillus oryzae liquid strain in order to compare the production amount of amylase according to the following steps

(1) Potato sucrose agar (PDA) medium preparation: cut 200 g of peeled potatoes into small pieces of about 1 cm ³ , add 1000 mL of distilled water and boil for 30 minutes, filter the potato pieces with six layers of gauze, add 20 g of sucrose, Agar powder 15g, heat until the agar melts, make up to 1000mL with distilled water, divide into test tubes (20mm×200mm) to one-third of the volume, plug the bottle mouth with a silicone stopper, wrap it with kraft paper and put it in an autoclave Sterilize at 121°C for 20 minutes, and when the culture medium is cooled to 80°C, place it on an inclined plane of the test tube until it cools and solidifies;

(2) Inoculate the mold on the solid slope of the test tube: take the Aspergillus oryzae species preserved on the slope of the test tube, use a sterilized inoculation shovel on the ultra-clean workbench to pick up a small piece of mold lawn, and inoculate it on the slope of the fresh test tube in step (1). After plugging the silica gel plug, place it in a 25°C incubator and incubate for 192 hours until the surface of the medium is covered with a large number of spores;

(3) Preparation of potato sucrose liquid medium: Cut 200 g of peeled potatoes into small pieces of 1 cm ³ , add 1000 mL of distilled water to boil for 30 minutes, filter the potato pieces with six layers of gauze, add 20 g of sucrose to the filtrate to dissolve, and use distilled water Make up to 1000mL, divide into 100mL in a 500mL Erlenmeyer flask, add a small amount of glass beads, wrap the mouth of the bottle with 6 layers of gauze, wrap it with kraft paper, and sterilize in an autoclave at 121°C for 20 minutes;

(4) Inoculate mold spores into potato sucrose liquid medium: take the freshly cultured solid test tube slant seeds in step (2), and use a 5mL sterilized pipette to draw 5mL sterilized saline on the ultra-clean workbench and add to the inclined surface of the test tube, and then gently scrape the inclined surface of the test tube with a sterilized spatula until all the mold spores are scraped off, shake the test tube gently to make the spores evenly distributed, and then continue to add sterile saline to the test tube until the spore suspension The absorbance at 620nm wavelength is 0.5, absorb the prepared spore suspension with a sterilized pipette tip and add it to the potato sucrose liquid medium, and insert 3mL of the spore suspension into each Erlenmeyer flask containing 100mL of medium;

(5) Bacteria cultivation: place the inoculated triangular flask in a constant temperature oscillator for 48 hours, the rotation speed is 120rmp, and the cultivation temperature is 30°C, until uniform grains of the size of small grains grow in the conical flask;

(6) Aspergillus oryzae liquid strain culture: take a 5mL pipette tip, cut off a little of the tip of the tip with scissors, so that the diameter of the tip outlet is 2mm, and sterilize it in an autoclave for use; put the cultured bacteria The granules were quickly inoculated into the fermentation medium after absorbing a certain volume with the treated pipette tip, the inoculum amount was 3% of the volume of the fermentation medium, wrapped with 8 layers of gauze, and then placed in a constant temperature incubator at 25°C and 150rpm to compare the production of amylase.

Embodiment 2: According to the following steps, the liquid strain of Penicillium is prepared in order to compare the production amount of penicillin

(1) Potato sucrose agar (PDA) medium preparation: cut 200 g of peeled potatoes into small pieces of about 1 cm ³ , add 1000 mL of distilled water and boil for 30 minutes, filter the potato pieces with six layers of gauze, add 20 g of sucrose, Agar powder 15g, heat until the agar melts, make up to 1000mL with distilled water, divide into test tubes (20mm×200mm) to one-third of the volume, plug the bottle mouth with a silicone stopper, wrap it with kraft paper and put it in an autoclave Sterilize at 121°C for 20 minutes, and when the culture medium is cooled to 80°C, place it on an inclined plane of the test tube until it cools and solidifies;

(2) Inoculate the mold on the solid slant of the test tube: take the Penicillium species preserved on the slant of the test tube, use a sterilized inoculation shovel on the ultra-clean workbench to pick up a small piece of bacterial lawn, inoculate it on the slant of the fresh test tube in step (1), plug it Place the silica gel plug in a 27.5°C incubator for 144 hours until the surface of the medium is covered with a large number of spores;

(3) Preparation of potato sucrose liquid medium: Cut 200 g of peeled potatoes into small pieces of 1 cm ³ , add 1000 mL of distilled water to boil for 30 minutes, filter the potato pieces with six layers of gauze, add 20 g of sucrose to the filtrate to dissolve, and use distilled water Make up to 1000mL, divide into 100mL in a 500mL Erlenmeyer flask, add a small amount of glass beads, wrap the mouth of the bottle with 6 layers of gauze, wrap it with kraft paper, and sterilize in an autoclave at 121°C for 20 minutes;

(4) Inoculate mold spores into potato sucrose liquid medium: take the freshly cultivated solid test tube slant seeds in step (2), and use a 5mL sterilized pipette to draw 5mL sterilized saline on the ultra-clean workbench and add to the inclined surface of the test tube, then gently scrape the inclined surface of the test tube with a sterilized spatula until all the mold spores are scraped off, shake the test tube gently to make the spores evenly distributed, and then continue to add sterile saline to the test tube until the spores are suspended The absorbance at 620nm wavelength is 0.6, absorb the prepared spore suspension with a sterilized pipette tip and add it to the potato sucrose liquid medium, and insert 4mL of the spore suspension into each Erlenmeyer flask containing 100mL of medium;

(5) Bacteria cultivation: Place the inoculated Erlenmeyer flask in a constant temperature oscillator for 60 hours, the rotation speed is 110rmp, and the culture temperature is 27.5°C, until uniform grain-sized bacteria grow in the flask;

(6) Penicillium liquid strain culture: take a 5mL pipette tip, cut off a little tip of the tip with scissors, so that the diameter of the tip outlet is 2.5mm, and sterilize it in an autoclave for use; put the cultured Use the treated pipette tip to absorb a certain volume and quickly inoculate it in the fermentation medium. The inoculation amount is 4% of the volume of the fermentation medium. Wrap it with 8 layers of gauze and place it in a constant temperature incubator at 31°C and 135rpm conditions to compare the production of penicillin.

Embodiment 3: Prepare Trichoderma liquid strain in order to compare the production amount of cellulase according to the following steps

(1) Potato sucrose agar (PDA) medium preparation: cut 200 g of peeled potatoes into small pieces of about 1 cm ³ , add 1000 mL of distilled water and boil for 30 minutes, filter the potato pieces with six layers of gauze, add 20 g of sucrose, Agar powder 15g, heat until the agar melts, make up to 1000mL with distilled water, divide into test tubes (20mm×200mm) to one-third of the volume, plug the bottle mouth with a silicone stopper, wrap it with kraft paper and put it in an autoclave Sterilize at 121°C for 20 minutes, and when the culture medium is cooled to 80°C, place it on an inclined plane of the test tube until it cools and solidifies;

(2) Inoculate Trichoderma on the solid slant of the test tube: Take the Trichoderma strains preserved on the slant of the test tube, use a sterilized inoculation shovel on the ultra-clean workbench to pick up a small piece of bacterial lawn, and inoculate it on the fresh test tube slant in step (1) , plugged with a silica gel plug and placed in a 30°C incubator for 96 hours, until the surface of the medium is covered with a large number of spores;

(3) Preparation of potato sucrose liquid medium: Cut 200 g of peeled potatoes into small pieces of 1 cm ³ , add 1000 mL of distilled water to boil for 30 minutes, filter the potato pieces with six layers of gauze, add 20 g of sucrose to the filtrate to dissolve, and use distilled water Make up to 1000mL, divide into 100mL in a 500mL Erlenmeyer flask, add a small amount of glass beads, wrap the mouth of the bottle with 6 layers of gauze, wrap it with kraft paper, and sterilize in an autoclave at 121°C for 20 minutes;

(4) Inoculate Trichoderma spores into potato sucrose liquid medium: Take the freshly cultivated solid test tube slant seeds in step (2), and use a 5 mL sterilized pipette to draw 5 mL of sterilized saline on the ultra-clean workbench Add it to the slope of the test tube, then gently scrape the slope of the test tube with a sterilized spatula until all the mold spores are scraped off, shake the test tube gently to make the spores evenly distributed, and then continue to add sterile saline to the test tube until the spores are suspended. The absorbance of the solution at a wavelength of 620nm is 0.7, and the prepared spore suspension is drawn into the potato sucrose liquid medium with a sterilized pipette tip, and 5mL of the spore suspension is inserted into each Erlenmeyer flask containing 100mL of medium;

(5) Bacteria cultivation: place the inoculated triangular flask in a constant temperature shaker for 72 hours, the rotation speed is 100rmp, and the cultivation temperature is 25°C, until uniform grains of the size of small grains grow in the triangular flask;

(6) Trichoderma liquid strain culture: Take a 5mL pipette tip, cut off a little tip of the tip with scissors, so that the diameter of the tip outlet is 3mm, and sterilize it in an autoclave for use; The granules were quickly inoculated into the fermentation medium after absorbing a certain volume with the treated pipette tip, the inoculum amount was 5% of the volume of the fermentation medium, wrapped with 8 layers of gauze, and then placed in a constant temperature incubator at 37°C and 120rpm to compare the production of cellulase.

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.