CN113215003A - Solid fermentation culture method of paecilomyces lilacinus - Google Patents
Solid fermentation culture method of paecilomyces lilacinus Download PDFInfo
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Abstract
The invention discloses a solid fermentation culture method of paecilomyces lilacinus, belonging to the technical field of biological engineering. The invention discloses a solid fermentation culture method of paecilomyces lilacinus, which comprises the steps of test tube activation of paecilomyces lilacinus, preparation of a first-stage seed solution of the paecilomyces lilacinus, preparation of a second-stage seed solution of the paecilomyces lilacinus and solid fermentation culture of the paecilomyces lilacinus. The method utilizes millet and rice as main culture materials, adjusts the acidity and nitrogen source of a fermentation culture medium through the acidic material of corn steep liquor, and reduces the viscosity of the material in the fermentation culture process by adding the water-retaining material of the superfine matter of white carbon black and silicon dioxide; the paecilomyces lilacinus cultured in the foam box has high viable bacteria content, the purity of the prepared microbial inoculum is high, the subsequent preservation survival rate of the microbial inoculum is high, and the field application effect is very good for years.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a solid fermentation culture method of paecilomyces lilacinus.
Background
Plant parasitic nematodes can parasitize more than 2000 plants, in recent years, diseases spread rapidly, vegetable root-knot nematodes are generated in areas over 2000 ten thousand acres in China, and the harm is very serious in all places in China. Root knot nematodes can puncture roots of hosts by using oral needles to absorb nutrition, cut off the source of plant life, cause yield and quality reduction, and simultaneously inject secretion by using the oral needles to stimulate parasitic cells near feeding points to proliferate, enlarge and excessively divide, cause malformation of diseased parts or formation of nodules with different sizes, commonly called root knots, and cause that root systems of crops cannot normally absorb nutrition, and crop leaves wither or even die when the root knots are serious; it also makes plants more susceptible to other fungal and bacterial diseases.
Aiming at the harm caused by root-knot nematodes, the existing chemical pesticides for preventing and treating the root-knot nematodes in the world are 21 in total, and are divided into four main classes: (1) the action mechanism of the halogenated hydrocarbon compound nematicide paralyzes the nematodes, causes the nematodes to be poisoned and die, and can directly kill the root-knot nematodes. (2) The methyl isothiocyanate is used for paralyzing the nematodes by carbamoyl compounds, and the activity of the nematodes is weakened. (3) The organophosphorus and carbamate nematocides are not used for killing nematodes, but rather are used for paralyzing the nematodes, weakening the mobility of the nematodes, reducing the number of plants initially invaded by the nematodes and reducing the ability of the nematodes to take food. However, because the chemical pesticide mainly acts on paralytic nematodes, the nematodes which are not dead enter a dormant period after poisoning, recover normal activities after passing adverse environments and when the conditions are suitable for propagation and growth, re-infect and damage crops. Meanwhile, after the root-knot nematodes are poisoned, the parts without death can generate drug resistance and drug resistance to chemical pesticides, the drug resistance and the drug resistance can be gradually increased along with the continuous generation and variation of the root-knot nematodes, and the drug resistance is strong. The chemical residue is serious, and the long-term use destroys polluted soil, causes soil hardening, acidification and other soil problems. And chemical pesticide residue causes the quality of the planted vegetables to be reduced. The egg killing effect is poor, only the nematodes in the soil can be killed, and the nematodes in the roots of crops cannot be killed. Beneficial bacteria in soil are killed while the nematodes are killed. Most chemicals are acidic, whereas nematodes and harmful bacteria prefer an acidic environment. The effective period is over, but the effective period provides a favorable living environment for the nematodes and the harmful bacteria, so that the nematodes and the harmful bacteria are more vigorously propagated, and the cycle is a vicious circle.
There are many fungi reported in recent years to be effective against root-knot nematodes, the most notable of which is paecilomyces lilacinus. According to the literature report, paecilomyces lilacinus has obvious inhibition effect on the root-knot nematode, and the paecilomyces lilacinus is used for preventing and treating plant parasitic nematodes such as the root-knot nematode and cyst nematode, so that good prevention effect is achieved. One of the research hotspots is the large-scale fermentation production and culture medium preparation aiming at the paecilomyces lilacinus. The paecilomyces lilacinus spores have relatively low production conditions and can be produced in various solid culture media. Various agricultural and sideline products such as rice, millet, wheat bran, food waste, agricultural and sideline products, waste residues, plant leaching liquor and the like can be used as a solid culture medium of the paecilomyces lilacinus to produce spores. However, the problems of low viable bacteria, serious infectious microbe pollution, short shelf life and the like commonly existing in the paecilomyces lilacinus strain products sold in China at present restrict the further field application effect of the paecilomyces lilacinus strain products.
Therefore, the problem to be solved by the technical personnel in the field is to provide a solid fermentation culture method of paecilomyces lilacinus.
Disclosure of Invention
In view of the above, the invention provides a solid fermentation culture method of paecilomyces lilacinus, and the cultured paecilomyces lilacinus has high viable bacteria content, high purity, high subsequent preservation survival rate and very good field application effect; solves a series of problems of serious mixed bacteria pollution, low spore content and the like in the production and application of the paecilomyces lilacinus, improves the yield and purity of the spores of the microbial inoculum, and improves the field application effect of the microbial inoculum.
In order to achieve the purpose, the invention adopts the following technical scheme:
a solid fermentation culture method of paecilomyces lilacinus comprises the following specific steps:
(1) preparing a test tube slant PDA culture medium suitable for growth of the paecilomyces lilacinus, inoculating pure paecilomyces lilacinus strains, culturing at 26-29 ℃, and taking the pure paecilomyces lilacinus strains as first-level test tube slant strains for later use after the pure paecilomyces lilacinus strains appear in 5-7 days of culture;
(2) preparing a triangular flask liquid culture medium, wherein the liquid filling amount of a 500ml triangular flask is 50-100 ml; inoculating 2 pieces of 1 square centimeter of first-stage test tube slant strains in the step (1), placing on a constant-temperature shaking table at 26-29 ℃, stirring and culturing at 150-200 r/min, sampling and inspecting at regular intervals, and culturing for 36-48 h after confirming that no other mixed bacteria are polluted, wherein the first-stage test tube slant strains are used as first-stage seed liquid for later use;
(3) preparing a 50L secondary fermentation tank culture medium suitable for growth of paecilomyces lilacinus, inoculating the primary seed solution in the step (2) into a fermentation tank through aseptic operation according to the inoculation amount of 5-10%, continuously introducing aseptic air by using an air compressor and an air filter, stirring and culturing, wherein the ratio of the air flow to the feed liquid is 1: 0.7-1.2, and the stirring speed is 100-150 r/min; keeping the pressure of 0.02-0.05 MPa in the fermentation tank, culturing at the constant temperature of 26-29 ℃ for 68-75 h, sampling, checking and avoiding other bacteria pollution, and using the obtained product as a secondary seed solution for standby use for solid culture inoculation;
(4) inoculating the secondary seed liquid obtained in the step (3) into a paecilomyces lilacinus solid fermentation culture medium for solid culture, and specifically comprising the following steps:
A. preparation of paecilomyces lilacinus solid fermentation culture medium
Pouring 1000g of rice or millet into a basin, injecting boiled clear water into the basin to ensure that the rice or millet is completely immersed in the clear water to ensure that the rice or millet fully absorbs water, simultaneously adding 5-10 g of potassium nitrate, 20-40 g of corn paste and 100-200 g of white carbon black or silicon dioxide, uniformly mixing to form a cluster by hand, and obtaining the paecilomyces lilacinus solid culture medium with the pH value of 5.0-6.0 after the mixture is scattered by touch; controlling the steam pressure to be 0.12-0.15 MPa, the temperature to be 120-125 ℃, cooking for 30-60 min, and cooling to 25-30 ℃ to obtain a paecilomyces lilacinus solid fermentation culture medium;
B. paecilomyces lilacinus solid fermentation medium inoculation culture
Uniformly spraying the secondary seed liquid obtained in the step (3) on a solid fermentation culture medium in an aseptic chamber in an inoculation amount of 5-10%, mixing and stirring to fully and uniformly mix the secondary seed liquid and the solid fermentation culture medium, and putting the mixture into a foam box for fermentation culture for 5-6 days to obtain a paecilomyces lilacinus culture material; flatly paving the compost in a foam box, paving the foam box with the thickness of 5-10 cm, covering a non-woven fabric, compacting the periphery of the foam box, and placing the foam box on a culture rack of a 10-ten-thousand-level clean culture room for solid culture, wherein the temperature and humidity are controlled in the solid culture process; when the paecilomyces lilacinus spores are mature, the fermentation is finished when the paecilomyces lilacinus spores begin to fall off; turning the material once for heat dissipation when the solid culture is carried out for 28-35 h, and circulating fresh air in the whole room for 3 times per hour; naturally airing and crushing the culture material after the fermentation is finished to obtain the paecilomyces lilacinus microbial inoculum.
Further, the triangular flask liquid culture medium in the step (2) contains the following components: 20g/L of glucose, 3g/L of sodium chloride, 10g/L of peptone, 5g/L of sodium nitrate, 2g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 0.05g/L of ferric sulfate and the balance of water, wherein the initial pH value of the culture medium is 6.0-7.0.
Further, the secondary fermentation tank culture medium in the step (3) contains the following components: 3g/L of ammonium chloride, 10g/L of corn paste, 10g/L of peptone, 6g/L of corn starch, 14g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of potassium dihydrogen phosphate, 3g/L of calcium carbonate, 0.05-5 g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric sulfate, 0.8g/L of sodium hydroxide and the balance of water; the initial pH value of the culture medium is 6.0-7.0.
Further, the temperature and humidity control program in the step (4) B is specifically: growing white villi on the raw material of the solid fermentation medium, and covering the solid fermentation medium completely before bacteria blocks begin to form on the surface of the fermentation material, wherein the cover of the foam box is tightly covered and moisturized, and the environmental temperature is 20-30 ℃; white fluff on the solid fermentation medium material is gradually changed into light purple, and in the process of completely forming bacterial blocks, a box cover of the foam box is gradually opened, the opening degree is increased along with the time extension, the temperature of the bacterial material is controlled to be 25-30 ℃, and the ambient temperature is 20-30 ℃; the fluff formed on the solid fermentation medium material is completely changed into light purple, the foam box cover is completely opened, ventilation and drying are increased, the sporulation is promoted, and the environmental temperature is 25-30 ℃.
According to the technical scheme, compared with the prior art, the solid fermentation culture method of paecilomyces lilacinus disclosed by the invention has the following beneficial effects:
(1) the invention takes rice and millet as the raw materials of the solid fermentation medium, is very suitable for the mass growth and reproduction of paecilomyces lilacinus, adds potassium nitrate and corn paste to supplement a nitrogen source required by the growth of the thalli, simultaneously the corn paste is acidic, the pH value of the culture material can be reduced to be below 6.0 after the addition, the growth of bacteria can be inhibited, the pure culture rate of the obtained thalli is high, the pure culture rate reaches more than 95%, and the thalli grow vigorously.
(2) According to the invention, the problem of viscosity of the fermentation material in the fermentation process can be well solved by adding very fine materials with very good water retention such as silicon dioxide and white carbon black, so that the fermentation material is very loose when the plate is turned over, oxygen is sufficient, and the fermentation effect is further improved; and the preservation effect of the well fermented microbial inoculum is further greatly improved.
(3) The invention solves the problems of moisture preservation and promotion of thallus germination in the early stage and reduction of humidity and promotion of sporulation in the later growth stage in the fungus culture process. The moisture of the solid culture material is generally low in the large-scale production and culture process of the fungi, the problem of early stage moisture preservation cannot be solved, the fungi cannot germinate, the germination speed is slow, and the culture fails. The adoption of a humidity controller increases the cost on the one hand and is easy to cause pollution on the other hand. The foam box adopted by the invention has good heat preservation and moisture preservation effects and replaces a tray for culture; solves the humidity problem of the fungus in the culture process.
(4) The invention innovating culture medium and culture method, the fermentation production speed of Paecilomyces lilacinus is finished 1-2 days earlier than that reported before, and the culture medium has high content, high product purity and prolonged storage life.
(5) The culture method of the invention is reliable, has high efficiency and is suitable for large-scale fermentation production.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A solid fermentation culture method of paecilomyces lilacinus comprises the following specific steps:
(1) paecilomyces lilacinus test tube activation: preparing a test tube slant PDA culture medium suitable for growth of the paecilomyces lilacinus, inoculating a pure paecilomyces lilacinus strain ACCC 30667, culturing at 28 ℃, and taking the pure paecilomyces lilacinus strain as a first-level test tube slant strain for later use after culturing for 7 days to generate lilac powdery conidia;
(2) preparing a paecilomyces lilacinus primary seed solution: preparing a triangular flask liquid culture medium, wherein the liquid filling amount of a 500ml triangular flask is 100 ml; inoculating 2 pieces of first-stage test tube slant strains with the square centimeter being 1 square, placing on a constant temperature shaking table at 28 ℃, stirring and culturing at 180 r/min, sampling and inspecting at regular intervals, and culturing for 36h after confirming that no other mixed bacteria is polluted, wherein the first-stage test tube slant strains are used as first-stage seed liquid for standby; the paecilomyces lilacinus triangular flask liquid culture medium contains the following components: 20g/L of glucose, 3g/L of sodium chloride, 10g/L of peptone, 5g/L of sodium nitrate, 2g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 0.05g/L of ferric sulfate and the balance of water, wherein the initial pH value of the culture medium is 6.0-7.0.
(3) Preparing paecilomyces lilacinus secondary seed liquid: preparing 50L of secondary fermentation tank culture medium, inoculating the obtained paecilomyces lilacinus primary seed liquid into a fermentation tank by 10% inoculation amount through aseptic operation, continuously introducing aseptic air by using an air compressor and an air filter, stirring and culturing, wherein the ratio of the ventilation amount to the feed liquid is 1: 0.7, stirring at 120r/min, maintaining the pressure of the tank at 0.03 MPa, culturing at 28 deg.C for 75h, sampling, checking to remove other bacteria, and using as secondary seed liquid for solid culture and inoculation. The secondary fermentation tank culture medium contains the following components: 3g/L of ammonium chloride, 10g/L of corn paste, 10g/L of peptone, 6g/L of corn starch, 14g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of potassium dihydrogen phosphate, 3g/L of calcium carbonate, 0.05-5 g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric sulfate, 0.8g/L of sodium hydroxide and the balance of water; the initial pH value of the culture medium is 6.0-7.0.
(4) Solid fermentation culture of paecilomyces lilacinus:
A. preparation of paecilomyces lilacinus solid fermentation culture medium
Pouring 1000g of rice into a basin, injecting boiled clear water into the basin to ensure that the rice is completely immersed in the clear water, soaking the rice to ensure that the rice completely absorbs water, simultaneously adding 10g of potassium nitrate, 20g of corn steep liquor and 150g of white carbon black, uniformly mixing the materials to reach the effect of hand-holding and conglobation, and obtaining the paecilomyces lilacinus solid culture medium with the pH value of 5.9; controlling the steam pressure to be 0.12MPa, the temperature to be 120 ℃, cooking for 60min, and cooling to 25-30 ℃ to obtain a paecilomyces lilacinus solid fermentation culture medium;
B. paecilomyces lilacinus solid fermentation medium inoculation culture
Uniformly spraying the secondary seed liquid on a solid fermentation culture medium in an aseptic chamber in an inoculation amount of 10%, mixing, fully and uniformly mixing the secondary seed liquid and the solid fermentation culture medium, and putting into a foam box for fermentation culture for 6 days to obtain a paecilomyces lilacinus culture material; flatly paving the compost in a foam box, paving the foam box with the thickness of 8 cm, covering non-woven fabrics, compacting the periphery, placing the foam box on a culture rack of a 10 ten thousand-level clean culture room for solid culture, and controlling the temperature and the humidity in the solid culture process;
the temperature and humidity control program is specifically: growing white villi on the raw material of the solid fermentation medium for 1-2 days, and tightly covering the foam box cover to keep moisture before bacteria blocks begin to form on the surface of the fermentation material, wherein the environmental temperature is 20-30 ℃; the plate turnover was carried out at 31h of cultivation. The white fluff on the solid fermentation medium material gradually changes into light purple, and the foam box cover is gradually opened in the process of completely forming the bacterial block, the opening degree is increased along with the time extension, the temperature of the bacterial material is controlled to be 25-30 ℃, and the ambient temperature is 20-30 ℃; the fluff formed on the solid fermentation medium material is completely changed into light purple within 5-6d, the foam box cover is completely opened, ventilation and drying are increased, the spore generation is promoted, and the environmental temperature is 25-30 ℃; when the paecilomyces lilacinus spores are mature, the fermentation is finished when the paecilomyces lilacinus spores begin to fall off; turning the material once for heat dissipation when the solid culture is carried out for 28-35 h, and circulating fresh air in the whole room for 3 times per hour; naturally airing and crushing the culture material after the fermentation is finished to obtain the paecilomyces lilacinus microbial inoculum. The viable count of the flat plate is detected, the viable count reaches 86 hundred million/g, and the rate of mixed bacteria is less than or equal to 2 percent.
Example 2
A solid fermentation culture method of paecilomyces lilacinus comprises the following specific steps:
(1) paecilomyces lilacinus test tube activation: preparing a test tube slant PDA culture medium suitable for growth of the paecilomyces lilacinus, inoculating a pure paecilomyces lilacinus strain ACCC 30667, culturing at 26 ℃, and taking the pure paecilomyces lilacinus strain as a first-level test tube slant strain for later use after culturing for 6 days to generate lilac powdery conidia;
(2) preparing a paecilomyces lilacinus primary seed solution: preparing a triangular flask liquid culture medium, wherein the liquid filling amount of a 500ml triangular flask is 80 ml; inoculating 2 pieces of first-stage test tube slant strains with the square centimeter being 1 square, placing on a constant temperature shaking table at 27 ℃, stirring and culturing at 150r/min, sampling and inspecting at regular intervals, and culturing for 40h after confirming that no other mixed bacteria is polluted, wherein the first-stage test tube slant strains are used as first-stage seed liquid for standby; the paecilomyces lilacinus triangular flask liquid culture medium contains the following components: 20g/L of glucose, 3g/L of sodium chloride, 10g/L of peptone, 5g/L of sodium nitrate, 2g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 0.05g/L of ferric sulfate and the balance of water, wherein the initial pH value of the culture medium is 6.0-7.0.
(3) Preparing paecilomyces lilacinus secondary seed liquid: preparing 50L of secondary fermentation tank culture medium, inoculating the obtained paecilomyces lilacinus primary seed liquid into a fermentation tank by 8% inoculation amount through aseptic operation, continuously introducing aseptic air by using an air compressor and an air filter, stirring and culturing, wherein the ratio of the ventilation amount to the feed liquid is 1: 0.8, stirring at 150r/min, maintaining the pressure of the tank at 0.04 MPa, culturing at 29 deg.C for 70h, sampling, and testing to obtain a second-stage seed solution for use in solid-state culture and inoculation. The secondary fermentation tank culture medium contains the following components: 3g/L of ammonium chloride, 10g/L of corn paste, 10g/L of peptone, 6g/L of corn starch, 14g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of potassium dihydrogen phosphate, 3g/L of calcium carbonate, 0.05-5 g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric sulfate, 0.8g/L of sodium hydroxide and the balance of water; the initial pH value of the culture medium is 6.0-7.0.
(4) Solid fermentation culture of paecilomyces lilacinus:
A. preparation of paecilomyces lilacinus solid fermentation culture medium
Pouring 1000g of rice into a basin, injecting boiled clear water into the basin to ensure that the rice is completely immersed in the clear water, soaking the rice to ensure that the rice completely absorbs water, simultaneously adding 5g of potassium nitrate, 30g of corn steep liquor and 150g of silicon dioxide, uniformly mixing the materials to reach the effect of hand-holding and conglobation, and obtaining the paecilomyces lilacinus solid culture medium with the pH value of the culture medium being 5.4; controlling the steam pressure to be 0.15MPa, the temperature to be 125 ℃, cooking for 30min, and cooling to 25-30 ℃ to obtain a paecilomyces lilacinus solid fermentation culture medium;
B. paecilomyces lilacinus solid fermentation medium inoculation culture
Uniformly spraying the secondary seed liquid on a solid fermentation culture medium in an aseptic chamber by using the inoculation amount of 8%, mixing and stirring to fully and uniformly mix the secondary seed liquid and the solid fermentation culture medium, and putting the mixture into a foam box for fermentation culture for 5 days to obtain a paecilomyces lilacinus culture material; flatly paving the compost in a foam box, paving the foam box with the thickness of 5 cm, covering non-woven fabrics, compacting the periphery, placing the foam box on a culture rack of a 10 ten thousand-level clean culture room for solid culture, and controlling the temperature and the humidity in the solid culture process;
the temperature and humidity control program is specifically: growing white villi on the raw material of the solid fermentation medium for 1-2 days, and tightly covering the foam box cover to keep moisture before bacteria blocks begin to form on the surface of the fermentation material, wherein the environmental temperature is 20-30 ℃; the plate was turned over at 34h of cultivation. The white fluff on the solid fermentation medium material gradually changes into light purple, and the foam box cover is gradually opened in the process of completely forming the bacterial block, the opening degree is increased along with the time extension, the temperature of the bacterial material is controlled to be 25-30 ℃, and the ambient temperature is 20-30 ℃; the fluff formed on the solid fermentation medium material is completely changed into light purple within 5-6d, the foam box cover is completely opened, ventilation and drying are increased, the spore generation is promoted, and the environmental temperature is 25-30 ℃; when the paecilomyces lilacinus spores are mature, the fermentation is finished when the paecilomyces lilacinus spores begin to fall off; turning the material once for heat dissipation when the solid culture is carried out for 28-35 h, and circulating fresh air in the whole room for 3 times per hour; naturally airing and crushing the culture material after the fermentation is finished to obtain the paecilomyces lilacinus microbial inoculum. The viable count of the flat plate is detected, the viable count reaches 75 hundred million/g, and the rate of mixed bacteria is less than or equal to 2 percent.
Example 3
A solid fermentation culture method of paecilomyces lilacinus comprises the following specific steps:
(1) paecilomyces lilacinus test tube activation: preparing a test tube slant PDA culture medium suitable for growth of the paecilomyces lilacinus, inoculating a pure paecilomyces lilacinus strain ACCC 30667, culturing at 29 ℃, and taking the pure paecilomyces lilacinus strain as a first-level test tube slant strain for later use after culturing for 5 days to generate lilac powdery conidia;
(2) preparing a paecilomyces lilacinus primary seed solution: preparing a triangular flask liquid culture medium, wherein the liquid loading capacity of a 500ml triangular flask is 60 ml; inoculating 2 pieces of first-stage test tube slant strains with the square centimeter being 1, placing on a constant temperature shaking table at 27 ℃, stirring and culturing at 200 r/min, sampling and inspecting at regular intervals, and culturing for 36h after confirming no other mixed bacteria pollution, wherein the first-stage test tube slant strains are used as first-stage seed liquid for standby; the paecilomyces lilacinus triangular flask liquid culture medium contains the following components: 20g/L of glucose, 3g/L of sodium chloride, 10g/L of peptone, 5g/L of sodium nitrate, 2g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 0.05g/L of ferric sulfate and the balance of water, wherein the initial pH value of the culture medium is 6.0-7.0.
(3) Preparing paecilomyces lilacinus secondary seed liquid: preparing 50L of secondary fermentation tank culture medium, inoculating the obtained paecilomyces lilacinus primary seed liquid into a fermentation tank by a 5% inoculation amount through aseptic operation, continuously introducing aseptic air by using an air compressor and an air filter, stirring and culturing, wherein the ratio of the ventilation amount to the feed liquid is 1: 0.7, stirring speed of 170r/min, maintaining tank pressure of 0.03 MPa, culturing at 27 deg.C for 75h, sampling, checking, and taking it as secondary seed liquid for use in solid culture and inoculation. The secondary fermentation tank culture medium contains the following components: 3g/L of ammonium chloride, 10g/L of corn paste, 10g/L of peptone, 6g/L of corn starch, 14g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of potassium dihydrogen phosphate, 3g/L of calcium carbonate, 0.05-5 g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric sulfate, 0.8g/L of sodium hydroxide and the balance of water; the initial pH value of the culture medium is 6.0-7.0.
(4) Solid fermentation culture of paecilomyces lilacinus:
A. preparation of paecilomyces lilacinus solid fermentation culture medium
Pouring 1000g of millet into a basin, injecting boiled clear water into the millet to ensure that the millet is completely immersed in the clear water, soaking the millet to ensure that the millet completely absorbs water, simultaneously adding 5g of potassium nitrate, 30g of corn steep liquor and 120g of silicon dioxide, uniformly mixing the mixture to reach the effect of hand-holding and conglobation, and obtaining the paecilomyces lilacinus solid culture medium with the pH value of 5.5 after the mixture is scattered by touch; controlling the steam pressure to be 0.12MPa, the temperature to be 121 ℃, cooking for 60min, and cooling to 25-30 ℃ to obtain a paecilomyces lilacinus solid fermentation culture medium;
B. paecilomyces lilacinus solid fermentation medium inoculation culture
Uniformly spraying the secondary seed liquid on a solid fermentation culture medium in an aseptic chamber by using the inoculation amount of 5%, mixing and stirring to fully and uniformly mix the secondary seed liquid and the solid fermentation culture medium, and putting the mixture into a foam box for fermentation culture for 6 days to obtain a paecilomyces lilacinus culture material; flatly paving the compost in a foam box, paving the foam box with the thickness of 10 cm, covering non-woven fabrics, compacting the periphery, placing the foam box on a culture rack of a 10 ten thousand-level clean culture room for solid culture, and controlling the temperature and the humidity in the solid culture process;
the temperature and humidity control program is specifically: growing white villi on the raw material of the solid fermentation medium for 1-2 days, and tightly covering the foam box cover to keep moisture before bacteria blocks begin to form on the surface of the fermentation material, wherein the environmental temperature is 20-30 ℃; the plate turning is carried out when the culture is carried out for 30 h. The white fluff on the solid fermentation medium material gradually changes into light purple, and the foam box cover is gradually opened in the process of completely forming the bacterial block, the opening degree is increased along with the time extension, the temperature of the bacterial material is controlled to be 25-30 ℃, and the ambient temperature is 20-30 ℃; the fluff formed on the solid fermentation medium material is completely changed into light purple within 5-6d, the foam box cover is completely opened, ventilation and drying are increased, the spore generation is promoted, and the environmental temperature is 25-30 ℃; when the paecilomyces lilacinus spores are mature, the fermentation is finished when the paecilomyces lilacinus spores begin to fall off; turning the material once for heat dissipation when the solid culture is carried out for 28-35 h, and circulating fresh air in the whole room for 3 times per hour; naturally airing and crushing the culture material after the fermentation is finished to obtain the paecilomyces lilacinus microbial inoculum. The viable count of the flat plate is detected, the viable count reaches 64 hundred million/g, and the rate of mixed bacteria is less than or equal to 2 percent.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. A solid fermentation culture method of paecilomyces lilacinus is characterized by comprising the following specific steps:
(1) preparing a test tube slant PDA culture medium, inoculating a pure paecilomyces lilacinus strain, culturing at 26-29 ℃, and taking the pure paecilomyces lilacinus strain as a first-level test tube slant strain for later use after culturing for 5-7 days to generate lilac powdery conidia;
(2) preparing a triangular flask liquid culture medium, wherein the liquid filling amount of a 500ml triangular flask is 50-100 ml; inoculating 2 pieces of 1 square centimeter of first-stage test tube slant strains in the step (1), placing on a constant-temperature shaking table at 26-29 ℃, stirring and culturing at 150-200 r/min, sampling and inspecting at regular intervals, and culturing for 36-48 h after confirming that no other mixed bacteria are polluted, wherein the first-stage test tube slant strains are used as first-stage seed liquid for later use;
(3) preparing a 50L secondary fermentation tank culture medium, inoculating the primary seed liquid in the step (2) into a fermentation tank through aseptic operation according to the inoculation amount of 5-10%, continuously introducing aseptic air by using an air compressor and an air filter, stirring and culturing, wherein the ratio of the ventilation volume to the material liquid is 1: 0.7-1.2, and the stirring speed is 100-150 r/min; keeping the pressure of 0.02-0.05 MPa in the fermentation tank, culturing at the constant temperature of 26-29 ℃ for 68-75 h, sampling, checking and avoiding other bacteria pollution, and using the obtained product as a secondary seed solution for standby use for solid culture inoculation;
(4) inoculating the secondary seed liquid obtained in the step (3) into a paecilomyces lilacinus solid fermentation culture medium for solid culture, and specifically comprising the following steps:
A. preparation of paecilomyces lilacinus solid fermentation culture medium
Pouring 1000g of rice or millet into a basin, injecting boiled clear water into the basin to ensure that the rice or millet is completely immersed in the clear water to ensure that the rice or millet fully absorbs water, simultaneously adding 5-10 g of potassium nitrate, 20-40 g of corn paste and 100-200 g of white carbon black or silicon dioxide, uniformly mixing to form a cluster by hand, and obtaining the paecilomyces lilacinus solid culture medium with the pH value of 5.0-6.0 after the mixture is scattered by touch; controlling the steam pressure to be 0.12-0.15 MPa, the temperature to be 120-125 ℃, cooking for 30-60 min, and cooling to 25-30 ℃ to obtain a paecilomyces lilacinus solid fermentation culture medium;
B. paecilomyces lilacinus solid fermentation medium inoculation culture
Uniformly spraying the secondary seed liquid obtained in the step (3) on a solid fermentation culture medium in an aseptic chamber in an inoculation amount of 5-10%, mixing and stirring to fully and uniformly mix the secondary seed liquid and the solid fermentation culture medium, and putting the mixture into a foam box for fermentation culture for 5-6 days to obtain a paecilomyces lilacinus culture material; flatly paving the compost in a foam box, paving the foam box with the thickness of 5-10 cm, covering a non-woven fabric, compacting the periphery of the foam box, and placing the foam box on a culture rack of a 10-ten-thousand-level clean culture room for solid culture, wherein the temperature and humidity are controlled in the solid culture process; when the paecilomyces lilacinus spores are mature, the fermentation is finished when the paecilomyces lilacinus spores begin to fall off; turning the material once for heat dissipation when the solid culture is carried out for 28-35 h, and circulating fresh air in the whole room for 3 times per hour; naturally airing and crushing the culture material after the fermentation is finished to obtain the paecilomyces lilacinus microbial inoculum.
2. The method for culturing by solid fermentation paecilomyces lilacinus according to claim 1, wherein the liquid culture medium in the triangular flask in the step (2) comprises the following components: 20g/L of glucose, 3g/L of sodium chloride, 10g/L of peptone, 5g/L of sodium nitrate, 2g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 0.3g/L of magnesium sulfate, 0.05g/L of ferric sulfate and the balance of water, wherein the initial pH value of the culture medium is 6.0-7.0.
3. The method for culturing Paecilomyces lilacinus by solid fermentation according to claim 1, wherein the secondary fermentor medium of step (3) contains the following components: 3g/L of ammonium chloride, 10g/L of corn paste, 10g/L of peptone, 6g/L of corn starch, 14g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of potassium dihydrogen phosphate, 3g/L of calcium carbonate, 0.05-5 g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.05g/L of ferric sulfate, 0.8g/L of sodium hydroxide and the balance of water; the initial pH value of the culture medium is 6.0-7.0.
4. The method for culturing paecilomyces lilacinus by solid fermentation according to claim 1, wherein the temperature and humidity control program in step (4) B comprises: growing white villi on the raw material of the solid fermentation medium, and covering the solid fermentation medium completely before bacteria blocks begin to form on the surface of the fermentation material, wherein the cover of the foam box is tightly covered and moisturized, and the environmental temperature is 20-30 ℃; white fluff on the solid fermentation medium material is gradually changed into light purple, and in the process of completely forming bacterial blocks, a box cover of the foam box is gradually opened, the opening degree is increased along with the time extension, the temperature of the bacterial material is controlled to be 25-30 ℃, and the ambient temperature is 20-30 ℃; the fluff formed on the solid fermentation medium material is completely changed into light purple, the foam box cover is completely opened, and the ambient temperature is 25-30 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480245A (en) * | 2022-01-18 | 2022-05-13 | 华南农业大学 | Penicillium sclerotiorum solid fermentation spore production method and application thereof |
| CN115466736A (en) * | 2022-10-28 | 2022-12-13 | 广东博沃特生物技术有限公司 | Feather-carbon double-base double-layer microcapsule preparation of paecilomyces lilacinus and preparation method thereof |
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| CN104928186A (en) * | 2015-05-08 | 2015-09-23 | 北京依科曼生物技术股份有限公司 | Paecilomyces lilacinus and scale preparation method thereof |
| CN111454847A (en) * | 2020-04-13 | 2020-07-28 | 陕西赛恩农业科技股份有限公司 | Solid fermentation method and solid fermentation culture medium for paecilomyces lilacinus |
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| CN104928186A (en) * | 2015-05-08 | 2015-09-23 | 北京依科曼生物技术股份有限公司 | Paecilomyces lilacinus and scale preparation method thereof |
| CN111454847A (en) * | 2020-04-13 | 2020-07-28 | 陕西赛恩农业科技股份有限公司 | Solid fermentation method and solid fermentation culture medium for paecilomyces lilacinus |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480245A (en) * | 2022-01-18 | 2022-05-13 | 华南农业大学 | Penicillium sclerotiorum solid fermentation spore production method and application thereof |
| CN114480245B (en) * | 2022-01-18 | 2023-09-05 | 华南农业大学 | Solid fermentation spore production method of penicillium sclerotium and application thereof |
| CN115466736A (en) * | 2022-10-28 | 2022-12-13 | 广东博沃特生物技术有限公司 | Feather-carbon double-base double-layer microcapsule preparation of paecilomyces lilacinus and preparation method thereof |
| CN115466736B (en) * | 2022-10-28 | 2023-06-09 | 广东博沃特生物技术有限公司 | Paecilomyces lilacinus feather carbon double-base double-layer microcapsule preparation and preparation method thereof |
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