CN110628651A - Liquid submerged fermentation method for improving activity of paecilomyces lilacinus spores - Google Patents
Liquid submerged fermentation method for improving activity of paecilomyces lilacinus spores Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
Abstract
The invention discloses a liquid submerged fermentation method for improving the activity of paecilomyces lilacinus spores, which comprises the steps of inoculating paecilomyces lilacinus seed liquid into a liquid fermentation culture medium for fermentation, wherein the fermentation time is 36-48 h, the temperature is 26-28 ℃, a ventilation and dissolved oxygen two-stage control method is adopted in the fermentation process, the ventilation ratio is 3: 4-4: 4L/(L.min) in a thallus growth stage of 0-24h, the initial dissolved oxygen value of fermentation liquor is 45-50% by controlling the rotating speed, the stable growth of hyphae is promoted, the spore formation and maturation stage is carried out from 24h to 36-48 h, the dissolved oxygen value of the fermentation liquor is controlled to be 5-10%, the ventilation ratio is 2: 1-2.5: 1L/(L.min), the spore production and maturation are promoted, and the active spore rate is improved; the method solves the problem of low viable spore rate of the spores produced by liquid fermentation of the paecilomyces lilacinus; can obviously improve the live spore rate of the spores, and the live spore rate after 36 hours is more than 80 percent.
Description
Technical Field
The invention relates to a liquid submerged fermentation method for improving the activity of paecilomyces lilacinus spores, and belongs to the field of microbial fermentation.
Background
Paecilomyces lilacinus belongs to Deuteromycotina, hyphomycete, hyphomycetales, Moniliaceae and Paecilomyces, has outstanding treatment and prevention effects on plant root-knot nematode diseases, and is a strain mainly applied in the market at present.
The paecilomyces lilacinus is mainly characterized in that conidiophores are upright, 2-4 conidiophores arranged in a wheel shape are usually arranged on phialides and are in a bottle shape or a nearly spherical shape, conidiophores are single-spore cross-grown and are in a spindle shape or an oval shape, spore walls are smooth or slightly rough, single transparent colorless single conidiophores are formed, and the conidiophores are purple when being agglomerated.
Paecilomyces lilacinus (A)Paecilomyces lilacinus(Thom.)Samson) Belongs to endoparasitic fungi and can parasitize various plant parasitic nematodes. The domestic and foreign research shows that: the plant parasitic nematodes parasitized by Paecilomyces lilacinus mainly include root-knot nematodesMeloidogynespp), cyst nematode (Heteroderaspp), sweet potato stem nematode (Ditylenchusclestructor) And golden thread of potato (A)Globoderarostochiensis) And the like.
At present, a plurality of nematode experts in the world use paecilomyces lilacinus to prevent and control plant parasitic nematodes such as root-knot nematodes, cyst nematodes and the like, and obtain good prevention effect. Researches show that the paecilomyces lilacinus has prevention and treatment effects on parasitic nematodes of crops such as soybean, wheat, cotton, jute, papaya, pumpkin, young tea, chili, chrysanthemum, grape, sesame, potato, dementia, citrus reticulata, sweet potato, tobacco, peanut, banana, funori, potato, watermelon, melon, cassiabarktree, momordica grosvenori, named as peach, cucumber and the like.
Since 1961, the Czech scholars Sasinakova produced beauveria bassiana blastospores by submerged liquid fermentation for the first time, thereby arousing extensive interest. Although the liquid fermentation technology has the advantages of easy control of conditions, strong repeatability, less pollution, high yield, short production period and the like in fungus spore production fermentation, the liquid fermentation technology cannot be widely popularized due to the influence on the spore quality caused by low spore activity rate of produced spores.
At present, the research on the control conditions of the paecilomyces lilacinus liquid submerged fermentation sporulation technology at home and abroad is less, and the paecilomyces lilacinus liquid submerged fermentation sporulation technology is applied to the control of fermentation parameters based on different dissolved oxygen and ventilation requirements of a thallus growth stage, a spore formation stage and a maturation stage so as to improve the survival rate of spores, and no report is provided in the aspect.
Disclosure of Invention
In order to solve the problem of low viable spore rate of spores produced by liquid fermentation of paecilomyces lilacinus, the invention provides a liquid submerged fermentation method for improving the spore activity of the paecilomyces lilacinus; the aeration and dissolved oxygen in the fermentation process are controlled in a segmented mode on the control of a liquid submerged fermentation process, at the initial stage of fermentation, paecilomyces lilacinus is in a hypha growth stage, at the moment, the biomass is low in oxygen demand and low in air flux and 45-50% of high dissolved oxygen are adopted to promote the hypha to grow stably, dissolved oxygen is reduced to about 15% along with the fermentation, low-oxygen stress begins to form, the paecilomyces lilacinus enters a sporulation stage, in order to meet the dissolved oxygen requirement of sporulation and spore maturation, the oxygen acquisition amount of high-aeration enhanced bacteria is provided, and in order not to break the environmental factors of the low-oxygen stress, the dissolved oxygen is maintained at 5-10%, so that the spore survival rate is improved in the process of promoting the sporulation and the spore maturation; the paecilomyces lilacinus spores produced by the method can be used for preventing and treating root-knot nematode diseases of crops, and can also be used as a microbial fertilizer for improving plant growth.
The method comprises the following specific steps:
A. preparation of Strain plates
Inoculating the paecilomyces lilacinus strain on a PDA (Potato dextrose agar) solid culture medium, putting the PDA solid culture medium into an incubator at 28 ℃ for culturing for 3-5 days until hyphae grow over a flat plate to produce spores;
B. liquid shake flask seed culture
Filling a liquid culture medium into a triangular conical flask, sterilizing the triangular conical flask at the temperature of 121 ℃ for 20min under high pressure, after cooling the triangular conical flask to room temperature, beating a bacterium block by using a puncher, inoculating the bacterium block into the liquid culture medium, and culturing the bacterium block for 24 to 30 hours in a constant-temperature incubator at the temperature of 28 ℃ and at the speed of 200r/min to obtain paecilomyces lilacinus seed liquid;
C. fermenting in a fermentation tank
Filling a liquid culture medium into a fermentation tank, sterilizing at 121 ℃ for 20min under high pressure, cooling to 26-28 ℃, inoculating paecilomyces lilacinus seed liquid into the fermentation tank, performing liquid submerged fermentation at 26-28 ℃ for 36-48 h, and harvesting spores after fermentation is completed;
in the process of liquid submerged fermentation, a two-stage control method of aeration and dissolved oxygen is adopted, the aeration ratio is 3: 4-4: 4L/(L.min) in a thallus growth stage of 0-24h, and the initial dissolved oxygen value of fermentation liquor is 45-50% higher by controlling the rotating speed, so that the smooth growth of hyphae is promoted; and (3) as the fermentation continues, the dissolved oxygen is reduced to about 15% and the fermentation starts to enter a sporulation period, the spore formation and maturation stage is from 24h to 36-48 h, the dissolved oxygen value of the fermentation liquor is controlled to be 5-10%, and the aeration ratio is 2: 1-2.5: 1L/(L.min), so that the sporulation and the maturation of spores are promoted, and the sporulation rate is improved.
The method of the invention has the following characteristics:
the aeration and dissolved oxygen are controlled in two stages in the fermentation process control, at the initial stage of fermentation, the paecilomyces lilacinus is in a hypha growth stage, the biomass is low in oxygen demand and small in amount, so that the hypha is promoted to grow stably by adopting low air flux and 45-50% of higher dissolved oxygen, hypoxia stress begins to form as the dissolved oxygen drops to about 15%, the paecilomyces lilacinus enters a sporulation stage, the oxygen acquisition amount of high-aeration enhanced bacteria is provided in order to meet the requirement of dissolved oxygen for sporulation and spore maturation, and the dissolved oxygen is maintained to be 5-10% in order to not break the environment of the hypoxia stress, so that the spore survival rate is also improved in the process of promoting the sporulation and the spore maturation; the method is simple and easy to implement, the obtained spores have high survival rate, and the method is suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is a control diagram of fermentation parameters in example 1;
FIG. 2 is a control chart of aeration amount in the fermentation process in example 1;
FIG. 3 is a comparison graph of viable count results of the process of the present invention and other control processes;
FIG. 4 is a graph comparing the results of the process of the present invention and other control processes for the rate of spores;
FIG. 5 is a normal uninfected egg;
FIG. 6 shows the invasion of paecilomyces lilacinus hypha into insect eggs;
FIG. 7 is a drawing of a Paecilomyces lilacinus hypha protruding from an egg;
FIG. 8 is a schematic diagram showing the egg disintegration of Paecilomyces lilacinus infected insects.
Detailed Description
The technical scheme of the present invention is further described in detail by the following examples, but the content of the present invention is not limited thereto, and the methods in the examples are all conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available unless otherwise specified.
Example 1: the liquid submerged fermentation method for improving the activity of paecilomyces lilacinus spores comprises the following steps:
(1) preparation of Strain plates
Boiling peeled potato 100g with 500mL of water for 20min, collecting the juice, adding glucose 10g, agar 10g and water to obtain 500mL solution, adjusting pH to 6.8-7.0, placing into 2L triangular conical flask, sterilizing, and pouring into flat plate; then inoculating the paecilomyces lilacinus strain on a PDA (personal digital assistant) plate, putting the PDA plate into an incubator at 28 ℃ for culturing for 5 days until hyphae grow over the PDA plate to produce spores;
(2) liquid shake flask seed culture
Putting a 500mL conical flask into 100mL liquid culture medium, carrying out autoclaving at 121 ℃ for 20min, cooling to room temperature, punching a bacterium block by using a puncher, inoculating the bacterium block into the liquid culture medium, and culturing for 25h in a constant temperature incubator at 28 ℃ and 200r/min to obtain paecilomyces lilacinus seed liquid; wherein the formula of the liquid culture medium is as follows: 2.25g of glucose, 1g of soybean meal, 0.1g of monopotassium phosphate, 0.015g of anhydrous calcium chloride, 0.015g of magnesium sulfate heptahydrate, 0.00185g of cobalt chloride hexahydrate, 0.0025g of iron sulfate heptahydrate, 0.0008g of manganese sulfate monohydrate, 0.0007g of zinc sulfate heptahydrate in 50mL of culture medium;
(3) fermenting in a fermentation tank
Filling 2L of liquid culture medium into a 5L fermentation tank, sterilizing at 121 ℃ for 20min under high pressure, cooling to 28 ℃, inoculating the paecilomyces lilacinus seed liquid into the fermentation tank according to the proportion of 1.5% (v/v), wherein the fermentation control parameter of the fermentation tank is that the temperature is controlled to be 28 ℃, the aeration and dissolved oxygen are controlled by two stages, the fermentation period is 0-24h, the initial dissolved oxygen value is 48%, the aeration ratio is 1: 1L/(L.min), the fermentation period is 24-48h, the dissolved oxygen is maintained at 5%, and the aeration ratio is 2.5: 1L/(L.min); controlling fermentation parameters as shown in figures 1 and 2, and harvesting spores through fermentation for about 48 hours; the formula of the liquid culture medium in the fermentation tank is as follows: 45g/L of sucrose, 20g/L of soybean meal, 2g/L of monopotassium phosphate, 0.3g/L of anhydrous calcium chloride, 0.3g/L of magnesium sulfate heptahydrate, 0.037g/L of cobalt chloride hexahydrate, 0.05g/L of ferric sulfate heptahydrate, 0.016g/L of manganese sulfate monohydrate and 0.014g/L of zinc sulfate heptahydrate;
in the fermentation process, the hypha growth stage is 0-24h, the hypha rapidly grows, hypoxia stress is formed after dissolved oxygen gradually decreases for 24h, the paecilomyces lilacinus enters the sporulation stage, and the bacterium is enhanced to obtain oxygen from gas phase by providing high ventilation to 5L/min to promote sporulation and improve the spore activity rate.
Under the same culture medium formula conditions, the method of the embodiment is compared with the tank fermentation effect of other control processes (the control parameters are that the temperature is 28 ℃, the aeration rate ratio is 1:1L/(L/min) in 0-60h, and the initial dissolved oxygen value is 60%), the results are shown in figures 3 and 4, and the viable count of the paecilomyces lilacinus in the method of the invention is 12.35X 108The activity rate is 86% in 36h and 71% in 48 h; the method is obviously superior to the comparison process, and further single-factor analysis of variance shows that the significance of the single-factor analysis of variance of viable count of 36h and 48h in fermentation of the method and the comparison process is P<0.009，P<0.006。
Example 2: the liquid submerged fermentation method for improving the activity of paecilomyces lilacinus spores comprises the following steps:
(1) preparation of seed plates the same as in example 1, step (1);
(2) liquid shake flask seed culture same as example 1 step (2);
(3) fermenting in a fermentation tank
Placing 2L liquid culture medium into 5L fermentation tank, autoclaving at 121 deg.C for 20min, cooling to 26 deg.C, and mixing with Paecilomyces lilacinus seed solution at a ratio of 1.5% (v/v)Inoculating into a fermentation tank, wherein fermentation control parameters of the fermentation tank are that the temperature is controlled to be 27 ℃, aeration and dissolved oxygen are controlled by two stages, the growth stage of thallus is 0-24h, the initial dissolved oxygen value is 45%, the aeration ratio is 3: 4L/(L.min), the sporulation and maturation stage is 24-40h, the dissolved oxygen is maintained at 10%, and the aeration ratio is 2: 1L/(L.min); harvesting spores through fermentation for 40 hours; the liquid medium in the fermenter was the same as in example 1; as a result, the number of viable bacteria in Paecilomyces lilacinus was 8.8X 108The activity rate of the fertilizer per mL is 81% in 36h and 72% in 40 h.
Example 3: detection of activity of spore root-knot nematode killing produced by deep fermentation of paecilomyces lilacinus liquid
(1) Preparation of meloidogyne incognita egg suspension
Cutting diseased roots of the southern root knot nematode disease into pieces, putting the pieces into a container, adding a NaClO solution with the mass concentration of 2%, uniformly mixing and shaking for 2min to enable the broken egg sacs to suspend the eggs; injecting the worm egg suspension into a set sieve formed by combining 200 meshes, 400 meshes and 500 meshes, slowly flushing the set sieve by using sterile water, and after sieving, slowly flushing residues of the 500 meshes into a sterile container by using sterile water to obtain the collected worm egg suspension, wherein the concentration of the worm egg suspension is 2000 eggs/mL.
(2) Infection experimental process
Aspirate 500. mu.l of egg suspension and 50. mu.l of 2X 108Uniformly mixing spore/mL paecilomyces lilacinus spore suspension on a water agar plate, slightly spreading, and culturing in an incubator at 28 ℃; randomly punching sample blocks by using a puncher with the diameter of 1cm every 12h to observe the egg infection condition;
(3) results
Before the worm egg is not infected, the surface is smooth, the shape is regular, the in-egg structure is normal, hypha attachment cells are formed on the surface at the initial stage of infection, the surface of the worm egg becomes wrinkled along with the gradual increase of infection, hypha grows in the egg and grows out of the egg until the last egg infected with the hypha is full of the hypha, and the egg has no in-egg structure (figure 5, figure 6, figure 7 and figure 8); the results show that the spores harvested by liquid submerged fermentation have good infection activity.
Claims (1)
1. A liquid submerged fermentation method for improving the activity of paecilomyces lilacinus spores is characterized by comprising the following steps: the paecilomyces lilacinus seed liquid is inoculated in a liquid fermentation culture medium for fermentation, the fermentation time is 36-48 h, the temperature is 26-28 ℃, a two-stage control method of aeration and dissolved oxygen is adopted in the fermentation process, the aeration ratio is 3: 4-4: 4L/(L.min) in the thallus growth stage of 0-24h, the dissolved oxygen value of the fermentation liquid is 45-50% by controlling the rotating speed, the stable growth of hyphae is promoted, the spore formation and maturation stage is 24-36-48 h, the dissolved oxygen value of the fermentation liquid is controlled to be 5-10%, the aeration ratio is 2: 1-2.5: 1L/(L.min), the maturation of spores is promoted, and the activity rate is improved.
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