CN114107097B - Solid fermentation process of streptomyces avermitilis active spores - Google Patents
Solid fermentation process of streptomyces avermitilis active spores Download PDFInfo
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- 239000007787 solid Substances 0.000 title claims abstract description 85
- 238000000855 fermentation Methods 0.000 title claims abstract description 70
- 230000004151 fermentation Effects 0.000 title claims abstract description 70
- 241001468227 Streptomyces avermitilis Species 0.000 title claims abstract description 31
- 239000001963 growth medium Substances 0.000 claims abstract description 56
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000000725 suspension Substances 0.000 claims abstract description 36
- 239000002609 medium Substances 0.000 claims abstract description 23
- 230000001954 sterilising effect Effects 0.000 claims abstract description 23
- 244000061458 Solanum melongena Species 0.000 claims abstract description 21
- 235000002597 Solanum melongena Nutrition 0.000 claims abstract description 21
- 239000008223 sterile water Substances 0.000 claims abstract description 20
- 239000000463 material Substances 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims description 27
- 230000001580 bacterial effect Effects 0.000 claims description 24
- 239000001888 Peptone Substances 0.000 claims description 18
- 108010080698 Peptones Proteins 0.000 claims description 18
- 235000019319 peptone Nutrition 0.000 claims description 18
- 238000010790 dilution Methods 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 14
- 239000002994 raw material Substances 0.000 claims description 14
- 229920001817 Agar Polymers 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 11
- 239000008272 agar Substances 0.000 claims description 11
- 235000015278 beef Nutrition 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 238000010998 test method Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 6
- 244000105624 Arachis hypogaea Species 0.000 claims description 6
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 6
- 235000018262 Arachis monticola Nutrition 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 235000020232 peanut Nutrition 0.000 claims description 6
- 238000009423 ventilation Methods 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 5
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 5
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 5
- 238000013329 compounding Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 235000013312 flour Nutrition 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 5
- 239000005660 Abamectin Substances 0.000 description 11
- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000575 pesticide Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 230000000853 biopesticidal effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 239000000243 solution Substances 0.000 description 1
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- 230000009261 transgenic effect Effects 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
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- 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
- C12N1/20—Bacteria; Culture media therefor
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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
- C12N3/00—Spore forming or isolating processes
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Abstract
The invention relates to a solid fermentation process of streptomyces avermitilis active spores, which comprises the following steps: step 1, preparing strains of streptomyces avermitilis: inoculating the frozen Streptomyces avermitilis strain on an eggplant bottle slant culture medium, and culturing for 7 days at 28-30 ℃ for later use; step 2, solid fermentation culture: step 2-1, preparing a solid fermentation medium, and sterilizing; step 2-2, preparing a strain suspension by using sterile water from the eggplant bottle inclined plane strain prepared in the step 1; step 2-3, uniformly inoculating the strain suspension into the solid fermentation medium prepared in the step 2-1; and 2-4, performing solid fermentation culture in a horizontal solid-state bioreactor for 9-11 days until the number of viable spores in each gram of material is more than or equal to 50 hundred million, and stopping fermentation. The invention can produce a large amount of streptomyces avermitilis active spores and lays a foundation for the industrialized development of the streptomyces avermitilis active spores.
Description
Technical Field
The invention belongs to the technical field of fermentation production, and particularly relates to a solid fermentation process of streptomyces avermitilis active spores.
Background
Biopesticides refer to agents that kill or inhibit agricultural pests using living organisms (fungi, bacteria, insect viruses, transgenic organisms, natural enemies, etc.) or their metabolites (pheromones, auxins, naphthylacetic acid, 2,4-D, etc.).
Avermectins (AVM), also known as Avermectins, are a group of macrolide antibiotics produced by fermentation of streptomyces avermitilis. The novel spectrum insecticidal green biological pesticide is widely applied to people since birth. However, in the development of avermectin industry, the drug resistance is gradually increased year by year, and the waste residue and waste water generated by liquid fermentation and the use of a large amount of organic solvents in the manufacturing process bring about bottleneck problems such as great harm to animals, plants, soil and ecological environment, and become an important problem for restricting the healthy development of the biopesticide industry.
The novel safe and efficient biological pesticide variety is created by taking living microorganisms and active metabolites as active ingredients, and has the advantages of green and efficient, low cost, low pollution manufacturing process and the like, and gradually becomes the development research direction of the biological pesticide in the future, so that the development of the avermectin industry is balanced, and a solid fermentation process of the active spores of streptomyces avermitilis is developed to solve some problems brought by the avermectin industry, and the method is very important.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a solid fermentation process of Streptomyces avermitilis active spores, which is green, efficient, low in manufacturing cost and low in manufacturing process pollution.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a solid fermentation process of Streptomyces avermitilis active spores comprises the following steps:
step 1, preparing strains of streptomyces avermitilis:
inoculating the frozen Streptomyces avermitilis strain on an eggplant bottle slant culture medium, and culturing for 7 days at 28-30 ℃ for later use;
step 2, solid fermentation culture:
step 2-1, preparing a solid fermentation medium, and sterilizing;
step 2-2, preparing a strain suspension by using sterile water from the eggplant bottle inclined plane strain prepared in the step 1;
step 2-3, uniformly inoculating the strain suspension into the solid fermentation medium prepared in the step 2-1;
2-4, performing solid fermentation culture in a horizontal solid-state bioreactor for 9-11 days until the number of viable spores in each gram of material is more than or equal to 50 hundred million, and stopping fermentation; the culture conditions of the solid fermentation culture are as follows: sterile air ventilation 1:0.2-0.5v/v/min (sterile air ventilation refers to the volume ratio of medium to sterile air introduced per minute); reactor pressure: 0.005-0.01MPa, culture temperature of 28-30deg.C, and culture humidity of 50-60%.
Further, the solid fermentation process of the streptomyces avermitilis active spore further comprises the steps of 3 and preparation of active spore powder:
and (3) transferring the solid culture after fermentation in the step (2) into a biconical rotary blast dryer for drying until the water content of the material is less than or equal to 10%, and crushing the solid culture into powder with 60-80 meshes to obtain the avermectin spore powder.
Further, the eggplant bottle slant culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.03%, agar 1.8-2.2%, water 100mL, pH6.5-7.0.
Furthermore, the eggplant bottle slant culture medium also needs to be sterilized before inoculation, and the sterilization conditions of the eggplant bottle slant culture medium are as follows: sterilizing at 120deg.C under 0.11-0.12MPa for 30min.
Further, the solid fermentation medium: firstly, compounding the following raw materials in percentage by mass: 60-70% of bran, 12-15% of bran, 3-5% of bean cake, 3-5% of peanut cake powder, 8-10% of corn flour, 0.5% of calcium carbonate, 0.5% of ammonium sulfate, 0.1-0.2% of peptone, and natural pH, so that the total percentage of each raw material is ensured to be 100%; then adding water to adjust the humidity to 50-60%.
Further, the solid fermentation medium needs to be sterilized before inoculation, and the sterilization conditions of the solid fermentation medium are as follows: sterilizing at 120deg.C under 0.11-0.12MPa for 30min.
Further, the counting and detecting method of the viable spores comprises the following steps: the double-layer plate culture counting test method is adopted, 4 repeats are carried out, and the average value is taken.
Further, the double-layer plate culture counting test method specifically comprises the following steps:
1. preparation of the culture medium:
solid medium: beef extract 0.3%, peptone 0.5%, glucose 0.5%, agar 2.0-2.2%, water 100mL, pH6.5-7.0;
semi-solid medium: beef extract 0.3%, peptone 0.5%, glucose 0.5%, agar 0.8-1.2%, water 100mL; respectively filling the solid culture medium and the semisolid culture medium into 250mL triangular flasks, sterilizing 100mL each flask at 115-120 ℃ for 30min under the pressure of 0.11-0.12MPa for later use;
2. and (3) checking:
1) Taking 1g of sample to be detected, carrying out gradient dilution by using sterile water, and then adding 1mL of bacterial suspension subjected to gradient dilution into a semisolid culture medium to prepare a bacteria-containing semisolid culture medium;
2) Heating the solid culture medium to be molten, pouring the solid culture medium into a culture dish, pouring the bacteria-containing semisolid culture medium into the culture dish after the solid culture medium is cooled and solidified, standing the culture dish, culturing the culture dish in a constant temperature box at 28-30 ℃ after the solid culture medium is solidified, and recording the number of single viable spore colonies in each culture dish;
number of viable spores per spore powder = number of single viable spore colonies x dilution.
Further, the gradient dilution adopts 4-stage gradient dilution, and each stage of dilution is 100 times; the specific operation is as follows: 1g of the sample to be detected is added into 100mL of sterile water to obtain 10 -2 Order of magnitude bacterial suspension, then 1mL10 is taken -2 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -4 Order of magnitude bacterial suspension, then 1mL10 is taken -4 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -6 Bacterial suspensions of the order of magnitude; 1mL10 -6 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -8 Order of magnitude bacterial suspension.
Compared with the prior art, the invention has the beneficial effects that:
the streptomyces avermitilis active spore is prepared by a solid culture medium fermentation mode, the preparation method is green and efficient, the cost is low, high-pollution reagents such as organic solvents and the like are not used in the preparation process, the method is safe and environment-friendly, the problem of waste liquid discharge is avoided, and some harm to the environment in the avermectin industry is avoided.
In the solid fermentation process, soybean cake powder, peanut cake powder, corn flour and peptone are added as a carbon source and a nitrogen source, ammonium sulfate is used as a rapid carbon source, bran and wheat bran are added for adjusting the water content, in addition, the addition of the peptone in the culture medium provides a plurality of microelements for the growth and propagation of streptomyces avermitilis, and the components of each culture medium are reasonably matched to act together, so that the growth and propagation of streptomyces avermitilis and the generation of viable spores are promoted.
Detailed Description
The technical solutions of the present invention will be clearly and fully described below with reference to specific embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
1. the formula of the culture medium comprises:
eggplant bottle slant culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.03%, agar 2.0%, water 100mL, pH6.8; sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
solid fermentation medium: firstly, compounding the following raw materials in percentage by mass: 68.8% of bran, 13% of bran, 4% of bean cake, 4% of peanut cake powder, 9% of corn powder, 0.5% of calcium carbonate, 0.5% of ammonium sulfate, 0.2% of peptone and natural pH, and then adding water to adjust the humidity to 55%; sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
2. the fermentation process comprises the following steps:
a solid fermentation process of Streptomyces avermitilis active spores comprises the following steps:
step 1, preparing strains of streptomyces avermitilis:
inoculating the frozen Streptomyces avermitilis strain on an eggplant bottle slant culture medium, and culturing for 7 days at 28-30 ℃ for later use; the Streptomyces avermitilis strain is preserved in China agricultural university strain collection, address: beijing, china; the strain preservation number is M2021-001.
Step 2, solid fermentation culture:
step 2-1, preparing a solid fermentation medium, and sterilizing;
step 2-2, preparing a strain suspension by using sterile water from the eggplant bottle inclined plane strain prepared in the step 1;
step 2-3, uniformly inoculating the strain suspension into the solid fermentation medium prepared in the step 2-1;
2-4, performing solid fermentation culture in a horizontal solid-state bioreactor for 9-11 days until the number of viable spores in each gram of material is more than or equal to 50 hundred million, and stopping fermentation; the culture conditions of the solid fermentation culture are as follows: sterile air ventilation 1:0.35v/v/min; reactor pressure: 0.008MPa, culture temperature of 28-30 ℃ and culture humidity of 50-60%.
Step 3, preparation of viable spore powder:
and (3) transferring the solid culture after fermentation in the step (2) into a biconical rotary blast dryer for drying until the water content of the material is less than or equal to 10%, and crushing the solid culture into 70-mesh powder to obtain the avermectin spore powder.
Wherein, the method adopted in the step 2-4 for counting the number of the viable spores is a double-layer flat-plate culture counting test method, and 4 repetitions are carried out during counting, and an average value is obtained.
The double-layer plate culture counting test method comprises the following steps:
1. preparation of the culture medium:
the solid culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, agar 2.1%, water 100mL, pH6.5-7.0;
the semi-solid culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, agar 1.0%, water 100mL;
respectively filling the solid culture medium and the semisolid culture medium into 250mL triangular flasks, sterilizing 100mL each flask at 115-120 ℃ for 30min under the pressure of 0.11-0.12MPa for later use;
2. and (3) checking:
1) Taking 1g of sample to be detected, adding the sample into 100mL of sterile water to obtain 10 -2 Order of magnitude bacterial suspension, then 1mL10 is taken -2 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 < -4 > order bacterial suspension, and then 1mL of 10 is taken -4 Adding the order-of-magnitude bacterial suspension into 99mL of sterile water to obtain 10-6 order-of-magnitude bacterial suspension; 1mL10 -6 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -8 Bacterial suspensions of the order of magnitude; 1mL10 is taken -8 Adding the order-of-magnitude bacterial suspension into a semi-solid culture medium to prepare a bacteria-containing semi-solid culture medium;
2) Heating the solid culture medium to be molten, pouring the solid culture medium into a culture dish, pouring the bacteria-containing semisolid culture medium into the culture dish after the solid culture medium is cooled and solidified, standing the culture dish, culturing the culture dish in a constant temperature box at 28-30 ℃ after the solid culture medium is solidified, and recording the number of single viable spore colonies in each culture dish;
number of viable spores per spore powder = number of single viable spore colonies x dilution.
In this example, the dilution factor is 10 8 Multiple times.
Example 2:
1. the formula of the culture medium comprises:
eggplant bottle slant culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.03%, agar 1.8%, water 100mL, pH6.5; sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
solid fermentation medium: firstly, compounding the following raw materials in percentage by mass: 68.9% of bran, 12% of bran, 5% of bean cake, 3% of peanut cake powder, 10% of corn flour, 0.5% of calcium carbonate, 0.5% of ammonium sulfate, 0.1% of peptone and natural pH, and then adding water to adjust the humidity to 52%; sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
2. the fermentation process comprises the following steps:
a solid fermentation process of Streptomyces avermitilis active spores comprises the following steps:
step 1, preparing strains of streptomyces avermitilis:
inoculating the frozen Streptomyces avermitilis strain on an eggplant bottle slant culture medium, and culturing for 7 days at 28-30 ℃ for later use;
step 2, solid fermentation culture:
step 2-1, preparing a solid fermentation medium, and sterilizing;
step 2-2, preparing a strain suspension by using sterile water from the eggplant bottle inclined plane strain prepared in the step 1;
step 2-3, uniformly inoculating the strain suspension into the solid fermentation medium prepared in the step 2-1;
2-4, performing solid fermentation culture in a horizontal solid-state bioreactor for 9 days until the number of viable spores in each gram of material is more than or equal to 50 hundred million, and stopping fermentation; the culture conditions of the solid fermentation culture are as follows: sterile air ventilation 1:0.2v/v/min; reactor pressure: 0.005MPa, the culture temperature is 28-30 ℃, and the culture humidity is 50-60%.
Step 3, preparation of viable spore powder:
and (3) transferring the solid culture after fermentation in the step (2) into a biconical rotary blast dryer for drying until the water content of the material is less than or equal to 10%, and crushing the solid culture into powder of 80 meshes to obtain the avermectin spore powder.
Wherein, the method adopted in the step 2-4 for counting the number of the viable spores is a double-layer flat-plate culture counting test method, and 4 repetitions are carried out during counting, and an average value is obtained.
The double-layer plate culture counting test method comprises the following steps: as in example 1.
Example 3
1. The formula of the culture medium comprises:
eggplant bottle slant culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.03%, agar 2.2%, water 100mL, pH7.0; sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
solid fermentation medium: firstly, compounding the following raw materials in percentage by mass: 65.8% of bran, 15% of bran, 3% of bean cake, 5% of peanut cake powder, 10% of corn powder, 0.5% of calcium carbonate, 0.5% of ammonium sulfate, 0.2% of peptone, and natural pH, and then adding water to adjust the humidity to 60%; sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
2. the fermentation process comprises the following steps:
a solid fermentation process of Streptomyces avermitilis active spores comprises the following steps:
step 1, preparing strains of streptomyces avermitilis:
inoculating the frozen Streptomyces avermitilis strain on an eggplant bottle slant culture medium, and culturing for 7 days at 28-30 ℃ for later use;
step 2, solid fermentation culture:
step 2-1, preparing a solid fermentation medium, and sterilizing;
step 2-2, preparing a strain suspension by using sterile water from the eggplant bottle inclined plane strain prepared in the step 1;
step 2-3, uniformly inoculating the strain suspension into the solid fermentation medium prepared in the step 2-1;
2-4, performing solid fermentation culture in a horizontal solid-state bioreactor for 11 days until the number of viable spores in each gram of material is more than or equal to 50 hundred million, and stopping fermentation; the culture conditions of the solid fermentation culture are as follows: sterile air ventilation 1:0.5v/v/min; reactor pressure: 0.01MPa, the culture temperature is 28-30 ℃, and the culture humidity is 50-60%.
Step 3, preparation of viable spore powder:
and (3) transferring the solid culture after fermentation in the step (2) into a biconical rotary blast dryer for drying until the water content of the material is less than or equal to 10%, and crushing the solid culture into 60-mesh powder to obtain the avermectin spore powder.
Wherein, the method adopted in the step 2-4 for counting the number of the viable spores is a double-layer flat-plate culture counting test method, and 4 repetitions are carried out during counting, and an average value is obtained.
The double-layer plate culture counting test method comprises the following steps: as in example 1.
The above described embodiments are only preferred examples of the invention and are not exhaustive of the possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (2)
1. The solid fermentation process of the streptomyces avermitilis active spore is characterized by comprising the following steps of:
step 1, preparing strains of streptomyces avermitilis:
inoculating the frozen Streptomyces avermitilis strain on an eggplant bottle slant culture medium, and culturing for 7 days at 28-30 ℃ for later use;
step 2, solid fermentation culture:
step 2-1, preparing a solid fermentation medium, and sterilizing;
step 2-2, preparing a strain suspension by using sterile water from the eggplant bottle inclined plane strain prepared in the step 1;
step 2-3, uniformly inoculating the strain suspension into the solid fermentation medium prepared in the step 2-1;
2-4, performing solid fermentation culture in a horizontal solid-state bioreactor for 9-11 days until the number of viable spores in each gram of material is more than or equal to 50 hundred million, and stopping fermentation; the culture conditions of the solid fermentation culture are as follows: sterile air ventilation 1:0.2-0.5v/v/min; reactor pressure: 0.005-0.01MPa, culture temperature of 28-30deg.C, and culture humidity of 50-60%;
step 3, preparation of viable spore powder:
transferring the solid culture after fermentation in the step 2 into a biconical rotary blast dryer for drying until the water content of the material is less than or equal to 10%, and pulverizing into 60-80 mesh powder to obtain avermans viable spore powder;
wherein, the eggplant bottle slant culture medium comprises the following raw materials in percentage by mass: beef extract 0.3%, peptone 0.5%, glucose 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.03%, agar 1.8-2.2%, water 100mL, pH6.5-7.0; the eggplant bottle slant culture medium also needs to be sterilized before inoculation, and the sterilization conditions of the eggplant bottle slant culture medium are as follows: sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
the solid fermentation culture medium is prepared by compounding the following raw materials in percentage by mass: 60-70% of bran, 12-15% of bran, 3-5% of bean cake, 3-5% of peanut cake powder, 8-10% of corn flour, 0.5% of calcium carbonate, 0.5% of ammonium sulfate, 0.1-0.2% of peptone, natural pH, ensuring that the total percentage of each raw material is 100%, and adding water to adjust the humidity to 50-60%; the solid fermentation culture medium also needs to be sterilized before inoculation, and the sterilization conditions of the solid fermentation culture medium are as follows: sterilizing at 120deg.C under 0.11-0.12MPa for 30min;
in the step 2-4, the counting and detecting method of the viable spores is a double-layer flat-plate culture counting and detecting method, the detection is repeated for 4 times, and an average value is obtained; the double-layer plate culture counting test method comprises the following steps:
(1) Preparation of the culture medium:
solid medium: beef extract 0.3%, peptone 0.5%, glucose 0.5%, agar 2.0-2.2%, water 100mL, pH6.5-7.0;
semi-solid medium: beef extract 0.3%, peptone 0.5%, glucose 0.5%, agar 0.8-1.2%, water 100mL;
respectively filling the solid culture medium and the semisolid culture medium into 250mL triangular flasks, sterilizing 100mL each flask at 115-120 ℃ for 30min under the pressure of 0.11-0.12MPa for later use;
(2) And (3) checking:
taking 1g of sample to be detected, carrying out gradient dilution by using sterile water, and then adding 1mL of bacterial suspension subjected to gradient dilution into a semisolid culture medium to prepare a bacteria-containing semisolid culture medium;
heating a solid culture medium to be molten, pouring the solid culture medium into a culture dish, pouring a fungus-containing semisolid culture medium into the culture dish after the solid culture medium is cooled and solidified, standing the culture dish, culturing the culture dish in a constant temperature oven at 28-30 ℃ after the solid culture medium is solidified, and recording the number of single viable spore colonies in each culture dish;
number of viable spores per spore powder = number of individual viable spore colonies x dilution.
2. The solid fermentation process according to claim 1, wherein the gradient dilution is performed by 4-stage gradient dilution, each stage dilution being 100 times, and the specific operation is as follows:
1g of the sample to be detected is added into 100mL of sterile water to obtain 10 -2 Order of magnitude bacterial suspension, then 1mL10 is taken -2 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -4 Order of magnitude bacterial suspension, then 1mL10 is taken -4 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -6 Bacterial suspensions of the order of magnitude; 1mL10 -6 The order of magnitude bacterial suspension is added into 99mL of sterile water to obtain 10 -8 Order of magnitude bacterial suspension.
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