CN110628651A - A liquid submerged fermentation method for improving spore activity of Paecilomyces lilacinus - Google Patents

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

A liquid submerged fermentation method for improving spore activity of Paecilomyces lilacinus

technical field

The invention relates to a liquid submerged fermentation method for improving the vigor of Paecilomyces lilacinus spores, belonging to the field of microbial fermentation.

Background technique

Paecilomyces lilacinus belongs to the subphylum Deuteromycotina, Solomycotina, Mycospora, Polyporaceae, and Paecilomyces. It has outstanding performance in the treatment and control of plant root-knot nematode diseases and is the main Applied strains.

The main feature of Paecilomyces lilacinus is that the conidiophores are upright, and there are often 2-4 sporophytes arranged in a round shape on the phialides, which are bottle-shaped or nearly spherical, and the conidia are monospore clusters, which are spindle-shaped or Elliptic, the spore wall is smooth or slightly rough, transparent and colorless individually, and purple when agglomerated.

Paecilomyces lilacinus (Thom.) Samson is an endoparasitic fungus that can parasitize a variety of plant parasitic nematodes. Domestic and foreign studies have shown that the plant-parasitic nematodes that Paecilomyces lilacinus can parasitize mainly include Meloidogyne spp, Heterodera spp, Ditylenchus clestructor , and Globoderarostochiensis .

At present, many nematode experts in the world are using Paecilomyces lilacinus to control root-knot nematodes, cyst nematodes and other plant parasitic nematodes, and have achieved good control effects. Studies have shown that Paecilomyces lilacinus has effects on soybeans, wheat, cotton, jute, fanberry, pumpkin, tea seedlings, peppers, chrysanthemums, grapes, hemp, potatoes, idiots, oranges, sweet potatoes, tobacco, peanuts, bananas, Radish, potato, watermelon, melon, guava, mangosteen, kiwi, cucumber and other crops have control effects on parasitic nematodes.

Since 1961, the Czech scholar Sasinakova used submerged liquid fermentation to produce blastospores of Beauveria bassiana for the first time, which has aroused widespread interest. Although liquid fermentation technology has the advantages of easy control of conditions, strong repeatability, less pollution, large output, and short production cycle in fungal sporulation fermentation, it cannot be widely used because of the low rate of viable spores produced and the quality of spores. promote.

At present, there are few studies on the control conditions of Paecilomyces lilacinus liquid submerged fermentation spore production technology at home and abroad. Based on the different dissolved oxygen and aeration requirements in the growth stage of the bacteria and the spore formation and maturity stages, it is applied to the control of fermentation parameters to control the fermentation parameters. Improve the survival rate of spores, there is no report in this regard.

Contents of the invention

In order to solve the problem of low viable spore rate of spores produced by Paecilomyces lilacinus liquid fermentation, the invention provides a liquid submerged fermentation method for improving the vigor of Paecilomyces lilacinus spores; The aeration and dissolved oxygen in the medium are controlled in stages. In the early stage of fermentation, Paecilomyces lilacinus is in the stage of mycelial growth. At this time, the biomass is low and the oxygen demand is low, so low aeration and 45-50% higher dissolved oxygen are used to Promote the steady growth of mycelia. As the fermentation progresses, the dissolved oxygen drops to about 15%, and the hypoxic stress begins to form. Paecilomyces lilacinus enters the sporulation stage. In order to meet the dissolved oxygen demand for sporulation and spore maturation, high aeration is provided Enhance the oxygen intake of bacteria, and maintain the dissolved oxygen at 5-10% in order not to break the environmental factors of hypoxic stress, thereby increasing the live spore rate of spores during the process of promoting spore production and spore maturation; this method produces The Paecilomyces lilacinus spores can be used to prevent and control root-knot nematode diseases of crops, and can also be used as microbial fertilizers to improve plant growth.

The inventive method comprises the following specific steps:

A. Preparation of strain plates

Inoculate the Paecilomyces lilacinus species on the PDA solid medium, put it in a 28°C incubator and cultivate it for 3 to 5 days, and wait for the mycelium to cover the plate and produce spores;

B. Liquid shake flask seed culture

Put the liquid medium into the conical flask, and sterilize it under high pressure at 121°C for 20 minutes. After cooling to room temperature, use a puncher to punch out the bacteria block and put it into the liquid medium, and place it in a constant temperature incubator at 28°C. , Cultivate for 24h-30h under the condition of 200r/min to obtain Paecilomyces lilacinus seed liquid;

C. Fermentation tank fermentation

Fill the fermenter with liquid culture medium, after autoclaving at 121°C for 20 minutes, cool to 26-28°C, inoculate the Paecilomyces lilacinus seed solution in the fermenter, and carry out submerged fermentation at 26-28°C for 36 ~48h, harvest spores after fermentation is completed;

The two-stage control method of aeration and dissolved oxygen is adopted in the above-mentioned liquid submerged fermentation process. During the growth stage of bacteria from 0 to 24 hours, the aeration ratio is 3:4 to 4:4 L/(L min), and the fermentation is controlled by controlling the rotation speed. The initial dissolved oxygen value of the solution is as high as 45-50%, which promotes the steady growth of mycelium; as the fermentation continues, the dissolved oxygen level drops to about 15% and begins to enter the sporulation stage, from 24h to 36-48h. In the mature stage, the dissolved oxygen value of the fermentation broth is controlled at 5-10%, and the aeration ratio is 2:1-2.5:1 L/(L·min), so as to promote sporulation and spore maturation, and increase the rate of viable spores.

The inventive method has following characteristics:

In the fermentation process control, aeration and dissolved oxygen are controlled in two stages. In the early stage of fermentation, Paecilomyces lilacinus is in the mycelial growth stage. At this time, the biomass is low and the oxygen demand is low, so low aeration and 45-50% higher Dissolved oxygen to promote the steady growth of mycelium. As the dissolved oxygen drops to about 15%, hypoxic stress begins to form, and Paecilomyces lilacinus enters the sporulation stage. In order to meet the dissolved oxygen demand for sporulation and spore maturation, high ventilation is provided Enhance the oxygen intake of bacteria, and maintain the dissolved oxygen at 5-10% in order not to break the low-oxygen stress environment, thereby improving the live spore rate of spores in the process of promoting spore production and spore maturation; the method of the present invention is simple and easy Yes, the obtained spores have a high survival rate and are suitable for industrial production and market promotion.

Description of drawings

Fig. 1 is the fermentation parameter control chart in embodiment 1;

Fig. 2 is a figure of control of ventilation in the fermentation process in embodiment 1;

Fig. 3 is the result comparison figure of the technique of the present invention and other control technique viable counts;

Fig. 4 is the comparison chart of the result of the live spore rate of the technique of the present invention and other control techniques;

Figure 5 is an uninfected normal worm egg;

Fig. 6 is that the hyphae of Paecilomyces lilacinus infect and enter the eggs;

Fig. 7 is the figure that the mycelium of Paecilomyces lilacinus protrudes from the ovum;

Figure 8 is a schematic diagram of the disintegration of eggs infected by Paecilomyces lilacinus.

Detailed ways

The technical scheme of the present invention will be described in further detail below through the examples, but the content of the present invention is not limited thereto. The method in the present embodiment is a conventional method if there is no special instructions, and the materials, reagents, etc. are used if no special instructions. obtained commercially.

Embodiment 1: the liquid submerged fermentation method that this improves Paecilomyces lilacinus spore vigor is as follows:

(1) Preparation of strain plates

Take 100g of peeled potatoes and boil them with 500mL of water for 20min, take the juice, add 10g of glucose, 10g of agar and water to the juice to make a 500mL solution, adjust the pH to 6.8-7.0, put it into a 2L conical flask, and extinguish After bacteria, pour the plate; then inoculate the Paecilomyces lilacinus species on the PDA plate, put it in a 28°C incubator and cultivate it for 5 days, and wait until the mycelium is full of the plate to produce spores;

(2) Liquid shake flask seed culture

Put 100mL of liquid medium into a 500mL conical flask, autoclave at 121°C for 20 minutes, and after cooling to room temperature, use a puncher to punch out the bacteria block into the liquid medium, and place it in a constant temperature incubator Cultivate at 28°C and 200r/min for 25 hours to obtain Paecilomyces lilacinus seed liquid; the liquid medium formula is: 2.25g glucose, 1g soybean powder, 0.1g potassium dihydrogen phosphate, 0.015g Anhydrous calcium chloride, 0.015g magnesium sulfate heptahydrate, 0.00185g cobalt chloride hexahydrate, 0.0025g iron sulfate heptahydrate, 0.0008g manganese sulfate monohydrate, 0.0007g zinc sulfate heptahydrate;

(3) Fermentation tank fermentation

Put 2L of liquid medium into a 5L fermenter, sterilize it under high pressure at 121°C for 20min, cool to 28°C, and inoculate the Paecilomyces lilacinus seed liquid in the fermenter at a ratio of 1.5% (v/v). The fermentation control parameters of the fermenter are temperature controlled at 28°C, aeration and dissolved oxygen are controlled in two stages, 0-24h is the growth stage of bacteria, the initial dissolved oxygen value is 48%, and the aeration ratio is 1:1 L/(L min ), 24-48h is the spore formation and maturation stage, the dissolved oxygen is maintained at 5%, and the aeration ratio is 2.5:1 L/(L min); the parameters of the fermentation are controlled as shown in Figures 1 and 2. After about 48 hours of fermentation, Harvest the spores; the formula of the liquid medium in the fermenter is: 45g/L sucrose, 20g/L soybean powder, 2g/L potassium dihydrogen phosphate, 0.3g/L anhydrous calcium chloride, 0.3g/L magnesium sulfate heptahydrate , 0.037g/L cobalt chloride hexahydrate, 0.05g/L iron sulfate heptahydrate, 0.016g/L manganese sulfate monohydrate, 0.014g/L zinc sulfate heptahydrate;

During the fermentation process, 0-24h is the mycelial growth stage, and the mycelium grows rapidly. As the dissolved oxygen gradually decreases for 24 hours, hypoxia stress is formed. Obtaining oxygen from the gas phase promotes sporulation and increases the rate of spore survival.

Under the same medium formulation conditions, the method in this example is the same as other control processes (the control parameters are temperature 28°C, 0-60h ventilation ratio is 1:1L/(L/min), and the initial dissolved oxygen value is 60%) Upper tank fermentation effect contrast, the results are shown in Fig. 3, 4, the figure shows that the number of viable bacteria of the inventive method Paecilomyces lilacinus is 12.35 * 10 ⁸ /mL, and the viable spore rate is 86% for 36h, and is 71% for 48h; obviously It is better than the comparison process, and further one-way analysis of variance to it shows that the single-factor analysis of variance significance of the number of viable bacteria in the method of the present invention and the comparison process at fermentation 36h and 48h is P<0.009, P<0.006.

Embodiment 2: the liquid submerged fermentation method that this improves Paecilomyces lilacinus spore vigor is as follows:

(1) The preparation of the strain plate is the same as step (1) of Example 1;

(2) Liquid shake flask seed cultivation is the same as step (2) of Example 1;

(3) Fermentation tank fermentation

Put 2L of liquid culture medium into a 5L fermenter, sterilize it under high pressure at 121°C for 20min, cool to 26°C, and inoculate the Paecilomyces lilacinus seed liquid in the fermenter at a ratio of 1.5% (v/v). The fermentation control parameters of the fermenter are temperature controlled at 27°C, aeration and dissolved oxygen are controlled in two stages, 0-24h is the growth stage of bacteria, the initial dissolved oxygen value is 45%, and the aeration ratio is 3:4 L/(L min ), 24-40h is the spore formation and maturity stage, the dissolved oxygen is maintained at 10%, and the aeration ratio is 2:1 L/(L min); after 40 hours of fermentation, the spores are harvested; the liquid medium in the fermenter is the same as the implementation Example 1: The result was that the viable count of Paecilomyces lilacinus was 8.8×10 ⁸ /mL, and the viable spore rate was 81% at 36 hours and 72% at 40 hours.

Embodiment 3: the inspection of the spore killing root-knot nematode activity of Paecilomyces lilacinus liquid submerged fermentation output

(1) Preparation of root-knot nematode egg suspension

Cut the diseased roots of root-knot nematode plants into pieces and put them in the container, then add NaClO solution with a mass concentration of 2%, mix and shake for 2 minutes, so that the oocysts are ruptured and the eggs are suspended; inject the egg suspension into 200 mesh, 400 mesh 500 mesh and 500 mesh sieves, rinse the sieves slowly with sterile water, after sieving, slowly rinse the residue of the 500 mesh sieves with sterile water into a sterile container, which is the collected egg suspension, The concentration of the egg suspension was 2000 eggs/mL.

(2) Infection experiment process

Take 500 μl of egg suspension and 50 μl of Paecilomyces lilacinus spore suspension with a concentration of 2×10 ⁸ spores/mL, mix them evenly on the water agar plate, spread them lightly, and place them in a 28°C incubator Cultivate; use a 1cm diameter puncher to randomly punch sample blocks every 12 hours to observe the egg infection under the microscope;

(3) Results

Before the eggs are infected, the surface is smooth, the shape is regular, and the internal structure of the eggs is normal. In the early stage of infection, hyphae adherent cells are formed on the surface. As the infection gradually deepens, the surface of the eggs becomes wrinkled, and there are Mycelium grows and mycelia grows from the egg until the egg is filled with mycelium at the end of the infection, and there is no structure in the egg (Fig. 5, Fig. 6, Fig. 7, Fig. 8); the above results show that the liquid submerged fermentation of the present invention Harvested spores had good infective activity.

Claims (1)

1. A liquid submerged fermentation method improving spore vigor of Paecilomyces lilacinus, is characterized in that: Paecilomyces lilacinus seed liquid is inoculated in the liquid fermentation medium and fermented, and the fermentation time is 36～48h, and temperature is 26 ~28°C, the fermentation process adopts the two-stage control method of aeration and dissolved oxygen. During the growth stage of the bacteria from 0 to 24 hours, the aeration ratio is 3:4 to 4:4 L/(L min), and the fermentation is made by controlling the rotation speed. The dissolved oxygen value of the fermentation liquid is 45-50%, which promotes the stable growth of mycelia. From 24h to 36-48h, the spore formation and maturation stage, the dissolved oxygen value of the fermentation liquid is controlled at 5-10%, and the aeration ratio is 2:1- 2.5:1 L/(L min), promote spore production and spore maturation, and increase the rate of viable spores.